Origins of the African Yam bean (Sphenostylis stenocarpa, leguminosae): evidence from morphology, isozymes, chloroplast DNA, and linguistics

ORIGINS OF THE AFRICAN YAM BEAN ( SPHENOSTYLIS STENOCARPA, LEGUMINOSAE): EVIDENCE FROM MORPHOLOGY, ISOZYMES, CHLOROPLAST D N A , AND LINGUISTICS l

DANIEL POTTER AND JEFF J. DOYLE

Potter, D. and Jeff J. Doyle (L. H. Bailey Hortorium, Cornell University, Ithaca, N Y 14853). ORIGINS OF THE AF~CAN YAM BEAN (SeHE~OS~'US SrE~OCaReA, LEGtrM~OSAE): Evidence from Morphology, Isozymes, Chloroplast DNA, and Linguistics. Economic Botany 46(3):276- 292. 1992. Cladistic and phenetic analyses of morphological, chloroplast DNA, and isozyme variation were used to examine relationships among multiple accessions o f Sphenostylis stenocarpa, representing wild and cultivated populations from throughout the range of the species. In morphometric and isozyme analyses, greater variability was detected among wild than among cultivated populations, and no differentiation was found between races cultivated for tubers and those cultivated for seeds, cpDNA data, however, revealed five groups of plastomes within S. stenocarpa: one in accessions cultivated for tubers, one in accessions cultivated for seeds, and three among wild accessions. Linguistic evidence and observations on the uses o f the species in its two main areas of cultivation suggest independent origins of tuber- and seed- cultivated races. The data support two alternative explanations for the distribution of extant cultivated accessions ofS. stenocarpa. The first hypothesis is that the species was domesticated independently in western and central Africa, but that domestication events involved selection from a single restricted gene pool. The second hypothesis is that a single domestication event occurred in one of the two areas, but that human dispersal to the second area occurred prior to dispersal within either area.

Origenes del "frijol yam" de la Africa (Sphenostylis stenocarpa, LEGLrMINOS~): Evidencia de morfologia, isoenzimas, ADN del cloroplasto, y la lingiiistica. Se llevaron a cabo analisis cladfsticos y fen~ticos de la variaci6n morfol6gica, del ADN del cloroplasto y de las isoenzimas, para examinar las relaciones entre m~ltiples especfmenes de Sphenostylis stenocarpa, que re- presentan poblaciones silvestres y cultivadas de todo el rango de distribuci6n de la especie. En los analisis morfomdtricos y de isoenzimas, se detect6 mayor variabilidad entre las poblaciones silvestres que entre las cultivadas, y no se encontraron diferencias entre las razas cultivadas para tub~rculo y las cultivadas para semillas. El ADN del cloroplasto, sin embargo, revel6 cinco grupos de plastomas: uno en los especfmenes cultivados para tubdrculo, uno en los cultivados para semillas, y tres en los silvestres. Por otra parte, evidencias lingiifsticas y observaciones de los usos de la especie en sus dos principales areas de cultivo, sugieren un origen independiente de las razas cultivadas para tub~rculo y de los cultivadas para semillas. Los datos soportan dos expli- caciones alternativas de la distribuci6n de las formas cultivadas existentes de S. stenocarpa. La primera hip6tesis es que la especie fue domesticada independientemente en el centro yen el oeste de Africa, pero ambos eventos involucraron selecci6n a partir de un germoplasma ~nico y res- tringido. La segunda hip6tesis es que hubo un ~nico evento de domesticaci6n en una de las dos reas, pero que la dispersi6n pot humanos hacia la segunda tea ocurri6 antes que la dispersi6n dentro de la primera.

Key Words: Sphenostylis stenocarpa, multiple domestications, phylogeny, chloroplast DNA, morphometrics, isozymes.

1 Received 23 September 1991; accepted 20 March

Economic Botany 46(3) pp. 276-292. 1992 �9 1992, by The New York Botanical Garden, Bronx, NY 10458 U.S.A.

1992] POTTER & DOYLE: AFRICAN YAM BEAN 277

Sphenostylis stenocarpa (Hochst. ex A. Rich) Harms (Leguminosae: Phaseoleae) is grown as a food crop in both West and Central Africa. In West Africa, especially Nigeria, the plant is grown only for its edible seeds, while in Central Africa, pr imari ly Zaire, it is grown mostly for its edible tubers, although the seeds also may be con- sumed. In addit ion, plants o f this species are harvested from the wild in areas of central and East Africa, again pr imari ly for the tubers (Potter 1992). The species is morphologically variable and there is a number of consistent differences between wild and cultivated races, presumably the consequences of human selection. As a result, several proposals have been put forward concerning the origins o f extant cult ivated races of S. stenocarpa (Table 1).

One possibili ty is that a single domest icat ion event occurred, in either west or Central Africa, and that domest icated races at tained their present distr ibution as a result of human dispersal. Sites that previously have been proposed as the center of domest icat ion of S. stenocarpa include Ethiopia (Dalziel 1937; Kay 1973) and West Af- rica (Murdock 1959; Purseglove 1976), but Cen- tral Africa, specifically the region that is now Zaire and Congo, also must be considered as a possible site of domestication. A second hypothesis is that two independent domestication events produced seed- and tuber-cultivated races in West and Central Africa, respectively, as suggested by Okigbo (1973). A final possibil i ty is that mult iple domesticat ions occurred throughout West and Central Africa, with cult ivation for tubers and cult ivation for seeds each having originated more than once. This hypothesis is consistent with the idea that African agriculture is noncentric and that African crops, including S. stenocarpa, are not assignable to particular sites o f origin, but should be classified according to the ecological zones in which they were domest icated (Harlan 1971; Harlan et al. 1976).

In order to test these hypotheses, variat ion within S. stenocarpa at three levels was examined. Morphological variat ion was assessed using principal component analysis o f morphometr ic characters, including leaf and seed dimensions. Isozyme and chloroplast D N A (cpDNA) varia- t ion were studied across wild and cult ivated populations from throughout the range of the species. All of these methods have proven useful in systematic studies of other legume crop species (e.g., morphometr ics: Broich and Palmer 1980;

TABLE 1. HYPOTHESES CONCERNING THE ORIGINS

OF CULTIVATED SPHENOSTYLIS STENOCARPA.

Hypothesis Amhor

Single domestication event:

Abyssinia (Ethiopia) Dalziel 1937 Nuclear Mande area of

western Sudan Murdock 1959 West Africa Purseglove 1976 Central Africa Potter 1991

Two or more domestication events: Independent domestication,

West and Central Africa Okigbo 1973

Multiple domestications, savanna-forest ecotone Harlan et al. 1976

cpDNA: Palmer et al. 1985; isozymes: Weeden 1984).

Evidence conceming the origins o f cultivated S. stenocarpa was also sought in an examinat ion of the common names for this species in the areas where it is grown. Linguistic evidence has been used to support hypotheses concerning the origins and history of other African crops (e.g., Shaw 1976; Wrigley 1960).

MATERIALS

A major goal of this study was to examine relationships among populat ions of S. stenocarpa from three sources: wild, seed-cultivated, and tuber-cult ivated. The available collections com- prised a mixture of herbar ium specimens, seeds from germplasm collections, and field collections of pressed specimens and seeds (Tables 2, 3, 4). Determinat ion o f the origin of each collection as wild, cult ivated for seeds, or cultivated for tubers presented several problems (Potter 1992). Each collection was nonetheless assigned to one of four groups: "wild ," "seed" (cultivated for edible seeds), " tuber" (cultivated for edible tubers), or "unknown," according to the following criteria: l) The "unknown" category was used only for herbar ium specimens lacking any clear indica- t ion of origin. 2) Seeds obtained from germplasm collections were classified based on information provided by the supervisors of the collections. 3) A few herbar ium specimens were clearly labeled as collected from the wild or from cultivation, while those with notat ions such as "forest" or "roadside bush" for habitat of origin were assumed to be wild. 4) Field observations, dis- cussions with African researchers, and the lit-

278 ECONOMIC BOTANY [VOL. 46

TABLE 2. COLLECTIONS USED IN MORPHOMETRIC STUDY OF LEAF (1--60) AND OF FLORAL AND LEAF (1--34) DIMENSIONS.

Collector Number Herb. Locality Group

01 Batten-Poole 284 K Nigeria: Jos Plateau Seed 02 Lamb 57 K Nigeria: Zaria Seed 03 Dept. Agric. L601 K Nigeria: Samaru Seed 04 Breteler 1925 BR Cameroon: Nkolbisson Unknown 05 deWilde 3755 BR Cameroon: Nkolbisson Unknown 06 Jacques-Felix 8173 K Cameroon: Mt. Nangue Unknown 07 LeTestu s.n. BM C.A.R. : Haute-Kotto Unknown 08 LeTestu 5144 BR Gabon Unknown 09 Gillardin 196 BR Zaire: Luputapata Wild l0 Pauwels 6242 BR Zaire: Kimbanseke Unknown 11 Quarre 2468 BR Zaire: Shaba, Ututuy Unknown 12 Homble 1185 BR Zaire: Vallee Kapiri Unknown 13 Kalenda 151 BR Zaire: Univ. E'ville Unknown 14 Michel & Reed 1497 BR Burundi: Kharo-Kofli Unknown 15 Malaisse 4010 BR Zaire: Campus Kasapa Unknown 16 Compere 1139 BR Zaire: Kiazi, Mvuazi Unknown 17 DeWitte 241 BR Zaire: Kiambi Unknown 18 Reekmans 6311 BR Burundi: Butare-Dunga Wild 19 Schmitz 2297 BR Zaire: Keyberg Wild 20 Potter 900515-01 BH Zaire: Matari Tuber 21 Reekmans 5985 BR Burundi: Ruyigi Wild 22 Pirozynski 5651 BR Tanzania: Gombe Sir. Wild 23 Schlieben 2024 BR Tanzania: Mahenge Wild 24 Exp. Lac Tang. 1655 BR Zaire: Fizi Wild 25 Schlieben 6129 BR Tanzania: Lindi Wild 26 Milne-Redhead & Taylor 9215 BR Tanzania: Songea Wild 27 Polhill & Paulo 1352 BR Tanzania: Iringa Wild 28 Potter 870329-02 BH Tanzania: Iringa Wild 29 Potter 870611-04 BH Tanzania: Sakura Wild 30 Archbold 808B K Tanzania: Korogwe Wild 31 Richards 4730 BR Zambia: near Abercorn Wild 32 Richards 11012 BR Tanzania: Kasangu Wild 33 Lejoly 82/746 BR Zaire: Bationge Unknown 34 Trapnell 1763 BR Zambia: Abercorn Unknown 35 Potter 880706-01 BH Zaire: Menkao Wild 36 Potter 900515-06 BH Zaire: Pungwe Tuber 37 Wild 4720 BR Zimbabwe: Harare Wild 38 Potter 880629-03 BH Zaire: Kafubu Tuber 39 Potter 880706-03 BH Zaire: Menkao Tuber 40 Potter 880614-03 BH Zaire: Yungu Tuber 41 Potter 880614-02 BH Zaire: Zomfi Tuber 42 Potter 900605-04 BH Nigeria (IITA 87) Seed 43 Potter 900605-05 BH Nigeria (IITA 111) Seed 44 Potter 900605-03 BH Nigeria (IITA 115) Seed 45 Potter 900605-01 BH Nigeria: Alifekede Seed 46 Potter 900605-02 BH Nigeria: Arugu Seed 47 Irvine 1649 K Ghana: Achimoto Seed 48 Potter 900605-06 BH Nigeria: Atam Seed 49 Potter 900515-03 BH Nigeria: Ohuno Seed 50 Fanshawe 1080 K Zambia: Ndola Unknown 51 Loveridge 144 K Uganda: Budongo For. Wild 52 Fanshawe 817 BR Zambia: Kitwe Unknown 53 Rogers 5804 K Zimbabwe: Harare Wild

1992] 279 POTTER & DOYLE: AFRICAN YAM BEAN

TABLE 2. CONTINUED.


54 Malaisse 55 Broadhurst 56 Potter 57 Potter 58 Potter 59 Potter 60 Potter

10585 BR Zaire: Luiswishi Unknown 450 K Kenya: Kipkarren Wild 900615-01 BH Zaire: Mbemba-Matendi Tuber 900615-02 BH Nigeria: Uzuakoli Seed 900615-04 BH Nigeria: Elimgbu Seed 900615-05 BH Zaire: Mupenda Tuber 900615-06 BH Nigeria: Awgu Seed

erature all indicate that S. stenocarpa is cultivated pr imari ly for its tubers in Central Africa and for its seeds in West Africa; thus, geographical origin was used to assign herbarium specimens of cultivated status to the " tuber" or "seed" groups. 5) Field collections were classified based on collection locality (Potter 1992).

When all collections were assigned to groups, the herbarium material included more wild than cult ivated specimens, while the seed material included more cult ivated accessions. Isozyme and cpDNA studies were l imited to those accessions for which viable seeds were available. In order to increase the amount of cult ivated material in the morphometr ic study, plants grown in the greenhouse from seeds collected in Africa were included. The possible effects o f growth in the greenhouse must be considered, but evidence from plants for which both seed and vegetative material were collected indicates that leaflet dimensions are pr imari ly determined genetically, not environmentally. For example, greenhouse- grown plants of the "Sakura" accession from Tanzania had long narrow leaflets similar to those of the wild plants from which the seeds were collected.

For all analyses, accessions were chosen to represent as broad a geographical range within each group as possible. Thus, seed accessions from Ghana and from six of the ten states in Nigeria where they are cultivated, and tuber accessions from the four major areas in Zaire where they are cult ivated (Potter 1992) were included. Her- bar ium specimens included plants o f wild and unknown origins from the following countries: Cameroon, Gabon, Central African Republic, Zaire, Uganda, Burundi, Kenya, Tanzania, Zam- bia, and Zimbabwe (Tables 2, 3). Only five wild accessions were available for the isozyme and c p D N A studies, but these represented a wide geographical range: eastern and western Tanza-

nia, Zimbabwe, Gabon, and western Zaire (Table 4).

M E T H O D S

LINGUISTIC EVIDENCE

A list o f common names for S. stenocarpa in several areas of tropical Africa, arranged by language group according to the classification system of Fivaz and Scott (1977), is presented in the accompanying paper (Potter 1992). These were compiled from a variety o f sources, including several publications, herbar ium specimens, and field observations. Vicki Carstens, o f the De- par tment o f Modern Languages and Linguistics, Cornell Universi ty, was consulted regarding possible relationships among the names from different language groups and geographical areas.

MORPHOMETRIC STUDIES

An initial morphometr ic analysis was carried out using 24 vegetative and floral dimensions (Table 5) measured from each o f 34 herbar ium specimens (numbers 1-34 in Table 2). Because flowers were available for only a l imited number o f specimens of cult ivated origin (three seed and one tuber), a second analysis was performed, using only six leaf dimensions (numbers 1-6; Table 5) measured from each of 60 herbar ium specimens (Table 2). For each specimen, the largest flower or the largest leaf on the sheet was measured. A separate analysis was carried out using seeds from fifty-one collections (Table 4); three measurements were taken from each seed (Table 3).

Morphometr ic data were analyzed using the m u l t i g r o u p p r i n c i p a l c o m p o n e n t ana lys i s (MPCA) option of Biostat II (Pimentel and Smith 1986). All accessions were included as a single group. Data were log,o transformed, a procedure that emphasizes differences in shape rather than


TABLE 3. COLLECTIONS USED FOR SEED MEASUREMENTS.


O1 02 03 04 05 06 07 O8 09 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51

IITA TSS 1 BH Nigeria: Obudu Seed IITA TSS 7 BH Nigeria: Umuchile Seed IITA TSS l0 BH Nigeria: Obuduiogoja Seed IITA TSS 4 BH Nigeria: Enugu Seed IITA TSS 47 BH Nigeria Seed IITA TSS 65 BH Zaire Tuber IITA TSS 73 BH Ghana Seed IITA TSS 74 BH Ghana Seed IITA TSS 76 BH Ghana Seed IITA TSS 77 BH Ghana Seed IITA TSS 83 BH Nigeria: Ikot-Ekpene Seed IITA TSS 87 BH Nigeria: Ikot-Ekpene Seed IITA TSS 89 BH Nigeria: Ikot-Ekpene Seed IITA TSS 90 BH Nigeria: Ikot-Ekpene Seed IITA TSS 95 BH Nigeria: Ikot-Ekpene Seed IITA TSS 94 BH Nigeria: Ikot-Ekpene Seed IITA TSS 100 BH Nigeria: Ikot-Ekpene Seed IITA TSS 106 BH Nigeria: Ikot-Ekpene Seed IITA TSS 111 BH Nigeria Seed IITA TSS 115 BH Nigeria Seed Ghana C. R.S. s.n. BH Ghana Seed Potter 880706-03 BH Zaire: Menkao Tuber Kitenge s.n. BH Zaire: Menkao Wild Potter 900701-01 BH Zaire: Bibwanga Tuber Potter s.n. BH Zaire: Kikwit Tuber Potter s.n. BH Zaire: Kingungi Tuber Potter 900515-01 BH Zaire: Mahari Tuber Potter 900615-01 BH Zaire: Mbemba-Matendi Tuber Potter 900615-05 BH Zaire: Mupenda Tuber Potter 900515-06 BH Zaire: Pungwe Tuber Potter s.n. BH Zaire: Yungu Tuber Potter 900605-01 BH Nigeria: Alifekede Seed Potter 900605-02 BH Nigeria: Arugu Seed Potter 900605-06 BH Nigeria: Atam Seed Potter 900615-06 BH Nigeria: Awgu Seed Potter 900615-04 BH Nigeria: Elimgbu Seed Potter 860115-01 BH Nigeria: Enugu Seed Potter 900615-03 BH Nigeria: Nimwoye Seed Potter 900515-03 BH Nigeria: Ohuno Seed Potter 860114-02 BH Nigeria: Owerre-Ezeorba Seed Potter 900515-04 BH Nigeria: Uzuakoli Seed Padulosi PS 89-163 BH Gabon Wild Potter 870605-01 BH Tanzania: Iringa Wild Potter 870611-04 BH Tanzania: Sakura Wild Mithen 650 BH Zimbabwe: Dombashawa Wild Archbold 808b K Tanzania: Korogwe Wild Robyns 2086 BR Zaire: Pweto Wild Pawek 8704 K Malawi: Mzuzu Wild Fanshawe 1080 BR Zambia: Ndola Wild Gillett 413 BR Zaire: Kisantu Wild Faulkner 1224 K Tanzania: Magenga Wild

1992] 281 POTTER & DOYLE: AFRICAN YAM BEAN

TABLE 4. ACCESSIONS INCLUDED IN ISOZYME AND cPDNA STUDIES.

Collector Number Herb. Origin Acronym cpDNA Isozyme

S. zimbabweensis

Mithen

S. stenocarpa

640 BH

Potter 870605-01 BH Potter 870611-04 BH Mithen 650 BH Padulosi PS89-163 BH Kitenge s.n. BH

Potter 880706-03 BH IITA TSS 65 BH Potter 900701-01 BH Potter 900515-01 BH Potter 8806 BH Potter 880614-06 BH Potter 900515-06 BH Potter 880629-03 BH Potter s.n. BH Potter s.n. BH Potter s.n. BH

Potter 900615-03 BH Potter 900515-03 BH Potter 900615-06 BH Potter 860114-02 BH Potter 900615 -04 BH Potter 900515-04 BH IITA TSS 56 BH IITA TSS 73 BH IITA TSS 86 BH IITA TSS 74 BH IITA TSS 47 BH IITA TSS 114 BH Ghana s.n. BH

Zimbabwe: Chimanimani ZIM Yes Yes

Wild:

Tanzania: Iringa IRI Yes Yes Tanzania: Sakura SAK Yes Yes Zimbabwe: Dombashawa DOM Yes Yes Gabon GAB Yes Yes Zaire: Menkao MEW Yes Yes

Tuber:

Zaire: Mankao MEC Yes Yes Zaire 065 Yes Yes Zaire: Bibwanga BIB Yes Yes Zaire: Matari MAT Yes Yes Zaire: Mupenda MUP Yes Yes Zaire: Mbemba-Matendi MBE Yes Yes Zaire: Pungwe PUN Yes Yes Zaire: Kafubu KAF No Yes Zaire: Kikwit KIK Yes No Zaire: Kingungi KIN Yes No Zaire: Yungu YUN Yes No

Seed:

Nigeria: Nimwoye NIM Yes Yes Nigeria: Ohuno OHU Yes Yes Nigeria: Awgu AWG Yes Yes Nigeria: Owerre-Ezeorba OWE Yes Yes Nigeria: Elimgbu ELI Yes Yes Nigeria: Uzuakoli UZU Yes Yes Nigeria: Okigwe 056 No Yes Ghana 073 No Yes Nigeria: Ikot-Ekpene 086 No Yes Ghana 074 Yes No Nigeria 047 Yes No Nigeria: Ikot-Ekpene 114 Yes No Ghana CRS Yes No

in size (Pimentel and Smith 1986). Significance of the differences in PCA scores between groups o f accessions was tested using the analysis of variance for unequal sample sizes (AOVU) option of Biostat II (Pimentel and Smith 1986).

CHLOROPLAST D N A STUDY

Total D N A was extracted from leaves or entire seedlings using the method of Doyle and Doyle (1987). Isolation of D N A was followed by di- gestion with restriction enzymes and electrophoresis in agarose gels as described by Doyle (1988), except that ethidium bromide was included in

the gels, rather than used to stain them after electrophoresis. Transfer o f D N A fragments to nylon membranes followed the methods of Southern (1975) and Wahl et al. (1979) with mi- nor modifications (Potter 1991).

Specific sequences of the cpDNA molecule were detected using radioactive probes prepared from the cloned cpDNA library o f mung bean [ Vigna radiata (L.) R. Wilczek] (Palmer and Thompson 1981), provided courtesy o f J. Palmer, Indiana University. Filters were sequentially hybridized with ten cloned fragments, representing the entire mung bean plastome. D N A fragments were


TABLE 5. MORPHOMETRIC CHARACTERS.

Floral and leaflet characters: 1) Petiole length 2) Rachis length 3) Terminal leaflet length 4) Terminal leaflet width at 1/4 length from base 5) Terminal leaflet width at midpoint 6) Terminal leaflet width at 1/4 length from apex 7) Calyx length 8) Standard petal claw length 9) Standard petal length to emargination

10) Length of emargination of standard petal 11) Standard petal width 12) Wing petal spur length 13) Wing petal claw length 14) Wing petal length 15) Wing petal width at narrowest point 16) Wing petal width at widest point 17) Wider keel petal length 18) Wider keel petal width at base 19) Wider keel petal width at center 20) Wider keel petal width at apex 21) Narrower keel petal length 22) Narrower keel petal width at base 23) Narrower keel petal width at center 24) Narrower keel petal width at apex

Seed characters:

1) Seed length 2) Seed width, between lateral surfaces 3) Seed height, hilum to abaxial surface

labeled with 32p using either the method o f nick translat ion (Maniatis et al. 1982) or the method o f random priming (Feinberg and Vogelstein 1983, 1984) with the Amersham mult ipr ime D N A labeling system, as specified by the man- ufacturer. Hybridizat ion followed the procedure

of Ladin et al. (1984), except that dextran sulfate was omit ted from the hybridizat ion buffer. Au- toradiography and stripping of filters for reprob- ing followed the methods o f Doyle et al. (1990).

Fi lms were developed and restriction fragment patterns were analyzed for presence or absence o f restriction enzyme cleavage sites, inversions, and size variations. Characters were defined as differences in restriction fragment pattern that could be at tr ibuted to restriction site gains or losses.

The analysis o f cpDNA variat ion within S. stenocarpa was part of a larger study of cpDNA phylogeny in the entire genus. Seven accessions of S. stenocarpa, chosen to represent the geographical range o f that species and to include wild and both types of cultivated accessions, were included in an initial survey using 21 restriction enzymes and ten probes. These were included in a cladistic analysis of restriction enzyme site variabil i ty in the entire genus (Potter 1991), in which four characters united the accessions o f S. stenocarpa. Addit ional accessions from each group (wild, seed, and tuber; Table 4) were analyzed using a subset o f the original enzyme- probe combinat ions to check several key characters (listed in Table 6): characters 2, 4, 5, and 6 to verify placement within S. stenocarpa, plus all restriction sites shown to be variable within the species in the original analysis (characters 1, 3, 7, 8, 9, and 10). Characters were polarized using the outgroup method (Watrous and Whee- ler 198 l) with one accession ofSphenostylis zimbabweensis Mithen as the outgroup. The data matr ix for cladistic analysis appears in Table 7. Cladistic analyses were carried out using the computer programs HENNIG86 (by J. S. Farris,

TABLE 6. CHARACTERS USED IN CLADISTIC ANALYSIS OF cPDNA VARIATION WITHIN SPHENOSTYLIS

STENOCARPA.

No. Enzyme Probe Plesiomorphic Apomorphic Marker for:.

1 BamHI 8, 9 11.5 7.8, 3.7 STS 2 BgllI 8, 9 2.15 1.7, 0.45 all stenocarpa 3 BgllI 13 3.6 2.5, 1.1 STS, STT, STW 4 DraI 3 5.7 5.2, 0.5 all stenocarpa 5 EcoO109 3 2.4, 1.6 4.0 all stenocarpa 6 EcoRI 8, 9 4.1 3.3, 0.8 all stenocarpa 7 EcoRI 12 2.1, 0.9 3.0 STS 8 EcoRI 3 3.9 2.6, 1.3 STT 9 EcoRI 3 1.45 0.89, 0.56 STT

10 HindllI 5 5.8 3.0, 2.8 STE, STW, STT, STS


version 1.5) and CLADOS (by K. C. Nixon, version 0.9).

ISOZYME STUDY

Population samples were not available for any of the accessions included in this study. Seeds of wild plants are difficult to find in the field because pods dehisce at maturi ty; thus, very l imited material of wild accessions was available. Seeds of cult ivated plants were obtained from farms, mar- kets, or insti tutional collections; in none of these cases had seeds been collected separately from different plants. Prel iminary isozyme studies showed very little variat ion across and within cult ivated accessions. For these reasons, a single seed was used to represent each accession in the present study (Table 4).

Six enzyme systems were assayed: aconitase (EC 4.2.1.3; ACO), alcohol dehydrogenase (EC 1.1.1.1 ; ADH); glucosephosphate isomerase (EC 5.3.1.9; GPI); i soc i t ra te dehydrogenase (EC 1.1. 1.42; IDH); phosphoglucomutase (EC 2.7.5.1; PGM); and 6-phosphogluconate dehydrogenase (EC 1.1.1.44; PGD). Isozymes were named as proposed by Myers and Weeden (1988) for Pha- seolus vulgaris L., with loci numbered beginning with the most anodal one. Alleles at each puta- t ive locus were designated as A (most mobile or anodal), B (intermediate mobility), or C (least mobile) (Garvin et al. 1989).

Procedures for starch gel electrophoresis and visualization of enzyme activity followed the methods o f Wendel and Weeden (1989), with minor modifications (Potter 1991). Seeds were soaked for 12-36 hours in distilled water, then macerated in 0.1 M Tris-HC1, pH 8.0 with 0.1% (v/v) 2-mercaptoethanol. The seed extracts were absorbed onto filter paper wicks that were then inserted into 11.5% starch gels. Two gel systems were run: Histidine-Citrate, pH 6.5 (modified from Stuber et. al. 1977), used to resolve ACO, ADH, PGD, and PGM, and Lithium-Borate, pH 8.3 (Ashton and Braden 1961), used to resolve GPI and IDH. Following electrophoresis at 4~ gels were sliced and stained for enzyme activity using standard recipes (Potter 199 t).

Allele frequencies were calculated for each accession or group of accessions. These were used to calculate Nei 's genetic distance (D; Nei 1972), between individual accessions. A dendrogram was constructed based on genetic distance calcula- tions using the unweighted pair group method (UPGMA; Sneath and Sokal 1973).

TABLE 7. DATA MATRIX FOR CLADISTIC ANALYSIS OF cPDNA IN S. STENOCARPA.

ZIM 00000000000 STZ 01011110000 STE 01011110001 STW 01111110001 STT 01111110111 STS 11111111001

RESULTS

LINGUISTIC EVIDENCE

Most of the languages in which common names for S. stenocarpa were available (Potter 1992) belong to the Niger-Congo subfamily of the Con- go-Kordofanian family o f languages. Within this large family, most of the west African languages listed belong to Gur or Kwa groups, while most o f the languages listed from Zaire belong to one of the Bantu groups. While there is considerable similari ty of common names within most language groups, and some apparent borrowing of names between groups within major families, there is no evidence of direct borrowing of a name for S. stenocarpa from a central African language by a west African language or the reverse (V. Carstens, pers. comm.). Thus, the names "kutreku," "aki tereku," and "apet reku" are found in three languages of the Western Group of Kwa languages spoken in Ghana, and variants of the name "pempo" ("mpempo," "semfn," "masemfu," "semvu") and of the name "mal- ubwe" ( " k i l u m b w e l u m b w e , " " m a l u m b w e l - umbwe," "dilombwe") are both used in languages belonging to two or more Bantu groups in central Africa, but no similari ty was seen between any of the Bantu names and any of the west African names.

MORPHOMETRIC STUDIES

A plot of the first and second axes from the principal component analysis of 24 leaf and floral characters from 34 specimens appears in Fig. 1, where tuber, seed, wild, and unknown specimens are indicated by different symbols. Wild and unknown accessions were scattered over the entire plot, while the four cultivated accessions formed a loose cluster on the left. The fact that only one tuber accession was included in this analysis made it impossible to draw any conclusions concerning the distinctiveness of tuber and seed groups. The three largest contributions to the first component


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3 4

Fig. 1-4. Plots of first and second components from principal component analysis of specimens of Sphe- nostylis stenocarpa using leaf, floral, and seed dimensions. Triangles = wild accessions; squares = accessions cultivated for tubers; circles = accessions cultivated for seeds; inverted triangles = accessions of unknown origin. Fig. 1. Plot from analysis of 34 specimens using 24 leaf and floral dimensions. First and second principal components accounted for 53% and 20% of the total variance, respectively. Fig. 2. Plot from analysis of 34 specimens using six leaf dimensions. First and second principal components accounted for 72% and 15% of the total variance, respectively. Fig. 3. Plot from analysis of 60 specimens using six leaf dimensions. First and second principal components accounted for 76% and 14% of the total variance, respectively. Fig. 4. Plot from analysis of 51 accessions of Sphenostylis stenocarpa using 3 seed dimensions. First and second principal components accounted for 96% and 3% of the total variance, respectively.


TABLE 8. PRESUMED GENOTYPES OF S. STENOCARPA ACCESSIONS FOR TWELVE PUTATIVE ISOZYME

LOCI.

DOM CC BB BB BB BB AA CC BB AA BB BB AA IRI BB AA BB BB AC AC CC BB AA BB AA BB SAK BB AB BB BB AA BB CC BB AA BB AB AA MEW BB BB BB BB AA AA CC BB AA BB AA AA GAB BB BB BB BB AA AA CC BB AA BB AA AA MEC BB BB AA BB CC AA CC BB AA BB AB AA KIK BB BB * BB CC AA CC BB AA BB BB AA PUN BB BB AA BB CC AA CC BB AA BB BB AA MBE BB BB BB BB CC AA AB BB AA BB BB APt MUP BB BB BB BB CC AA CC BB AA BB BB AA MAT BB BB * BB CC AA CC BB AA BB BB AA 065 BB BB AA BB CC AA CC BB AA BB BB AA KIN BB BB BB BB CC AA CC BB AA BB AB AA BIB BB BB AA BB CC AA CC BB AA BB BB AA YUN BB BB AA BB CC AA CC BB AA BB BB APt 074 BB BB BB BB CC AA CC BB AA BB BB AA CRS BB BB BB BB CC AA CC BB AA BB BB AA 047 BB BB BB BB CC AA CC BB AA BB BB AA 114 BB BB BB BB CC AA CC BB AA BB BB AA AWG AB BB BB BB CC AA CC BB AA BB BB AA ELI BB BC BB BB CC AA CC BB AA BB BB AA NIM BB BB BB BB CC AA CC BB AA BB BB AA OHU BB BB BB BB CC AA CC BB AA BB BB AA UZU BB BB BB BB CC AA CC BB AA BB BB AA OWE BB BB BB BB CC AA CC BB AA BB BB AA

* Missing dam.

in this analysis were from characters 4-6, all leaf characters; this component accounted for 53% of the variance across all characters in all specimens. The second component, which accounted for 20% of the total variance, included roughly equal contributions from characters 4-6 and five of the floral characters. When the analysis was repeated using the same 34 specimens but only the six leaf characters, the plot obtained (Fig. 2) differed in the precise placement o f many of the collections, but the general conclusions were the same as in the original analysis: wild accessions showed more variability than cultivated ones, but neither group formed a tight cluster.

These observations provided justification for an expanded study, using additional specimens for which flowers were not available. A plot o f the first and second axes from the principal com-

ponent analysis of leaf dimensions from 60 specimens appears in Fig. 3. No difference was detected between seed and tuber accessions. In contrast, while there is some overlap between wild accessions and cultivated accessions as a group, the cultivated plants are generally clus- tered toward the lower left of the plot, and wild

collections extend toward the upper right. When all wild collections were treated as one group and all cultivated plants (both tuber and seed) as a second, analysis o f variance showed highly sig- nificant differences between their average scores for both the first (F = 13.5; P = 0.001) and second (F = 18.4; P = 0.001) components.

Principal component analysis of seed dimensions produced three distinct groups, corre- sponding to seed, tuber, and wild accessions (Fig. 4). These reflect three discrete size classes: the smallest seeds were those o f wild accessions; the largest, those o f seed-cultivated accessions.

ISOZYMES

Twelve putative loci were consistently re- solved in the six enzyme systems examined. Pre- sumed genotypes for each accession at these loci are given in Table 8. A second, more cathodal, putative A D H locus was seen in all seed but in

only about half o f the wild and tuber accessions, and was not included in the analysis. A dupli-

cation o f the cytosolic gene for GPI is expressed Sphenostylis and some other papilionoid le- gumes (Weeden et al. 1989). Thus, three iso-


TABLE 9. N E I ' S STANDARD GENETIC DISTANCES BETWEEN ACCESSIONS OF S. STENOCARPA.

I 2 3 4 5 6 7 8 9 10 11

1 D O M 2 IRI .570 3 SAK .362 .258 4 GAB, MEW .288 .244 .139 5 MEC .324 .405 .341 .212 6 KIK, MAT .201 .404 .271 .244 .023 7 PUN, 065, BIB, YUN .288 .495 ,362 ,288 .023 8 MBE .266 .474 .341 ,266 .021 9 KIN .212 .280 .223 .112 .072

10 MUP, 8 seed* .182 .362 ,244 .182 .112 1 1 A W G .161 .405 .280 .212 .140 12 ELI .212 .341 . 251 .212 .140

.000

.072 .161

.023 .112 .091

.000 .087 .066 .021

.023 .112 .091 .044

.023 . l l 2 .091 .044 .021 .021 .044

* 8 seed = OWE, NIM, CRS, 074, 114, 047, UZU, OHU.

zymes, GPI-1 representing the plastid form and GPI-2 and GPI-3 representing cytosolic forms, were observed for this system. Each wild accession had a unique combinat ion ofgenotypes for the loci examined, as did some o f the cult ivated accessions. In contrast, many of the cultivated accessions had identical isozyme complements and there was no correlation between genotype and cult ivation for seeds or tubers.

The alleles observed in the cultivated accessions represented a subset o f those in the wild accessions. As with the principal component analysis of leaf dimensions, there were no fixed differences, such as presence or absence of an allele, between any groups of accessions that cor- responded to geographic origin or uses. Among the cult ivated accessions, in fact, one o f the tuber accessions (MUP) had an isozyme profile identical to eight of the seed accessions.

Nei 's genetic distances (Nei 1972) between all pairs o f accessions are presented in Table 9. The average genetic distance among accessions was greatest within the wild group (0.253), intermediate within the tuber group (0.048), and lowest within the seed group (0.008). The average genetic distance between seed and tuber accessions was 0.059; between wild and seed accessions, 0.234; and between wild and tuber accessions, 0.292.

The dendrogram constructed using Nei 's genetic distance values (Fig. 5) is presented as an illustration o f a phenetic analysis and is not in- tended as an hypothesis of ancestor--descendant relationships among these accessions (Throck- mor ton 1977). This dendrogram shows that, for the systems examined, the isozyme profiles of all cult ivated accessions were more similar to one

another than to those o f any wild accessions, and that some tuber accessions (e.g., KIN) were more similar to some seed accessions (e.g., ELI and AWG) than they were to other tuber accessions (e.g., BIB and YUN).

c P D N A

Cladistic analysis o f cpDNA restriction site variation within S. stenocarpa produced one most pars imonious cladogram, with consistency of 1.0 (Fig. 6). The plastomes of S. stenocarpa formed a monophylet ic group, united by four unique mutations. Five plastome types were detected within S. stenocarpa; three in wild accessions and two in cult ivated accessions. All tuber accessions shared one plastome type marked by two unique mutat ions and all seed accessions shared a distinct type also marked by two unique mutations. Neither o f these plastome types was found in any o f the five wild accessions examined. The plastome of the D O M accession from Zimbabwe was basal within the species, differing by at least one mutat ion from the plastomes o f all remaining accessions. The two accessions from Tanzania (IRI and SAK) had the same plastome type, as did the wild accessions from western central Af- rica (GAB and MEW). The last two shared one unique mutat ion with all cultivated accessions.

DISCUSSION

GENERAL OBSERVATIONS

The difference between the cult ivated and wild accessions observed in the PCA of leaf d imen- sions probably reflects human selection for ro- bust plants. None o f the cult ivated accessions are of the "'Sphenostylis congensis'" type, with


.386

.240

.218

.072

.081 i

.021 MUP,

.025 8 seed, KIK, MAT

m KIN

ELI

AWG

MBE

.023 " E BIB,

PUN, YUN, 065 MEC

DOM

.139 MEW, GAB

SAK

IRI

375 .550 .525 .300 .275 .250 .225 ,2QO .!75 .150 .125 .I00 .075 .050 .025 0

Fig. 5. Dendogram constructed from Nei's genetic distance values between accessions of Sphenostylis stenocarpa, based on isozyme data.

long, slender leaflets, and these are the wild accessions that were the least s imilar to the cultivated ones in the principal component analysis (numbers 2 l , 24, and 27; upper right hand corner o f Fig. 3).

The three seed size classes detected in S. s teno-

carpa are undoubtedly the result of human se-

lection. The largest seeds are those of the accessions that are grown for consumption o f the seeds themselves, but seeds larger than those of wild plants would probably also be selected in areas where tubers are eaten, because seeds are used to propagate the plants there.

None of the wild accessions included in the


ZIM

DOM

2 4 5 6

10

IRI, SAK 8 9

1 7

MEC, 065, BIB, MAT, MUP, MBE, PUN, KAF

MEW, GAB

NIM, OHU, AWG, OWE, ELI, UZU, 056, 073, I]86

Fig. 6. Single most parsimonious cladogram from cladistic analysis of cpDNA restriction site characters for accessions of Sphenostylis stenocarpa. Consistency index = 1.0.

cpDNA analysis appear to represent escapes from cultivation, since there was no case o f a cultivated plastome type appearing in a wild plant. Even the MEW accession, which came from the same field as the MEC accession, did not share its plastome type. The possibili ty that some of the wild collections included in the morphometric analysis o f leaflet dimensions represent escapes from cultivation cannot be ruled out.

The small sample sizes used here present problems in the interpretat ion of the isozyme data. Nei (1978) examined the use o f small sample sizes in the determinat ion of genetic distances and construction ofdendrograms from them, and concluded that samples as small as one individual per populat ion (as used here) may be ac- ceptable i f average heterozygosity is low (0.1 or less), i f a large number of loc i are examined, and i f differences in genetic distance between pairs o f populat ions are relatively high. The first of these criteria is met here, with an average observed heterozygosity across all accessions of 0.03. The other two criteria are not met. Only a relatively small number of loci were examined, and the differences in genetic distances between pairs o f cult ivated and pairs of wild accessions are low, although the distances between wild and cultivated accessions are quite high. Consequently, it would be unwise to place much confidence in the details o f the dendrogram based on isozyme data presented here. Nonetheless, three general conclusions may be drawn from these analyses: 1) The isozyme data do not show a sharp differ-

entiation between accessions cult ivated for tubers and those cultivated for seeds; in fact, iden- t ica l i soz yme p h e n o t y p e s were o b s e r v e d in accessions from both groups. 2) For the enzyme systems examined, there is greater genetic variabil i ty among the wild than among the cult ivated accessions studied. 3) For the enzyme systems examined, there is greater genetic diversity among the tuber than among the seed cult ivated groups.

None of the three conclusions ment ioned above, the first two of which are also supported by the morphometr ic analysis, is surprising. Sphenostylis stenocarpa is apparently a crop at an early stage of domestication. Only a few morphological changes, all presumably the direct results of human selection, distinguish cultivated from wild races, and the species has not been the subject o f intensive breeding or crop improvement programs that could generate a number o f divergent cultivated varieties. Thus, lower genetic diversi ty among cultivated than wild accessions is expected. Lower variabil i ty among seed than among tuber accessions could suggest that the former group was derived from the lat- ter, but it could also result from independent domesticat ions from a single wild species, with somewhat more variabil i ty having been included in the central African tuber domesticates, per- haps because domest icat ion took place over a broader range within that area. The lack of differentiation among cultivated races is similar to the results o f a study of cult ivated grain amaranths (Amaranthus spp.) in India (Jain et al.


1980), in which no al lozyme variat ion was found among samples from across a wide geographic range, in spite of considerable morphological variabil i ty among these accessions. The authors at tr ibuted this to the relatively recent introduc- tion and rapid spread of grain amaranths in In- dia, during which human selection had produced morphological diversi ty but mainta ined enzyme monomorphism. Low isozyme variation was also found in chickpea (Cicer arietinum L.) from a wide geographical range (Oram et al. 1987), but this contrasts with the results of studies of isozyme variat ion in other major crop plants with long histories under cultivation. For example, isozyme variat ion in maize (Zea rnays L.) has been shown to be correlated with alti tude in Mexico (Doebley et al. 1985) and Guatemala (Bretting et al. 1990). In a study of 90 cultivars of white seeded bean (Phaseolus vulgaris), Wee- den (1984) found that 58% of the cultivars tested had unique al lozyme phenotypes and the remaining 42% could be divided into groups of 2 - 5 cultivars, while among 21 culti~ears of Cucur- bita pepo L., Ignart and Weeden (1984) found that certain isozyme phenotypes characterized each of the five fruit types, while cultivars within fruit type were often isozymically indistinguishable.

ORIGINS OF CULTIVATED RACES

The strongest evidence in support of the hypothesis of multiple origins of cultivated races (Table l) would have been the identification of two or more groups of accessions within each major area of cultivation, and the observation of a closer relationship between cultivated and wild accessions from the same geographical area than between cultivated accessions from different areas. This was not observed: the morphometric and isozyme analyses failed to identify any subgroups among cultivated accessions, while the cpDNA analysis revealed only two groups of plastomes among cultivated plants: those of accessions cultivated for seeds and those of accessions cultivated for tubers.

The cpDNA results, while favoring the hypothesis of two independent domestications, are not conclusive. Although there were no synapomorphies detected for the plastomes of seed and tuber accessions, it is possible that such a syn- apomorphy would be detected if addit ional restriction enzymes were used. Even if the two

groups o f accessions were derived from the same populat ion of wild progenitors, however, their plastomes would not necessarily share any unique mutations. The stronger evidence for two domestications is the fact that the mutat ions that distinguish seed and tuber groups were found in all accessions sampled within each area. This observation, however, is not inconsistent with the hypothesis o f a single domest icat ion event. An early dispersal from the area o f original domestication to the second area, followed by ac- cumulat ion of new mutat ions and subsequent dispersal within each area, could have produced the observed distr ibution of plastome types. I f this were true, central Africa would seem the more likely area for the original domesticat ion event, since wild plants with a plastome type close to that of the cultivated accessions occur there. Wild accessions orS. stenocarpa from west Africa, however, were not available. Field observations and examinat ion o f herbarium specimens indicate that the species is currently found only in cult ivation in west Africa, although wild races may have existed there previously, and might have been lost due to increased populat ion and cult ivation of land.

Further evidence for two independent domestications comes from linguistic data and obser- vat ions on the uses o f the plants from the two areas where it is cultivated. There is no evidence for the borrowing of a west African name for S. stenocarpa by a central African language, nor for the reverse, as might be observed i f domestication had occurred in one area followed by human dispersal to the second (Shaw 1976). In addit ion, the use of the tubers seems to be unknown in west Africa, and the use of the seeds is rare in Zaire.

Conversely, both the isozyme and morphometric data seem to point toward a single origin for all domest icated races examined, since, in both cases, the cult ivated accessions as a group showed considerably less variability than the wild accessions and no differentiation between seed and tuber groups. It is possible, however, that the restricted variabil i ty in both morphology and isozymes occurred in parallel in tuber and seed cultivated races as the result of evolut ionary bot- tlenecks during domesticat ion. More precisely, i f genetically similar populat ions were sampled in independent domest icat ion events in the two areas, thJe resulting domesticates might be phe- netically indistinguishable, especially for char-


acteristics such as isozyme profiles and leaf dimensions, which are not directly related to the different uses o f the plants in the two areas.

The hypothesis of a single domest icat ion event is further supported by the morphological differences between cultivated and wild collections of this species (Potter 1992). The fixed differences in plastome type and seed sizes observed between the tuber and seed groups of accessions provide justification for treating them as separate species under a phylogenetic species concept (Nixon and Wheeler 1990). A taxonomic sepa- ration of these groups is not recommended, since they cannot be readily distinguished on morphological grounds, but the existence of fixed differences between them means that relationships between the two groups of accessions are phylogenetic, rather than tokogenetic (Hennig 1966; Wheeler and Nixon 1990). The derived characters that all cultivated accessions share may therefore be treated as synapomorphies for the two groups. Characters such as delayed dehis- cence or increased size of pods must be considered unreliable as indicators of phylogenetic relationship, since these features have been selected independently during the domesticat ion of several legume species (Zohary 1984). There is no apparent reason, however, that loss of scuff on the seed surface should be observed in both seed and tuber groups unless they were derived from the same ancestral population. Nonetheless, the possibil i ty that glabrous seeds were selected independent ly in the two areas cannot be ruled out. It is possible, for example, that smooth seed surface is genetically correlated with another feature selected by humans, such as large seeds.

In summary, the hypothesis of a single domesticat ion event and that of two independent domest icat ions are each strongly supported by some of the data, but neither is inconsistent with any part of the data. Moreover, each of these hypotheses can be modified so that it is supported by all o f the data.

If two independent domest icat ion events occurred in the two main areas of cultivation, then it is likely that both domesticat ions involved selection from one somewhat restricted gene pool, resulting in the low levels of isozyme and morphological variat ion observed across all cultivated accessions. Alternatively, if a single domesticat ion event occurred in either west Africa or central Africa, then it is likely that there was

an early dispersal from this pr imary center to a secondary center of origin, and that dispersal within either area occurred somewhat later, after the mutat ions that mark either plastome type had occurred. Cultural and linguistic evidence for this early dispersal may have been obliter- ated. To the extent that we have sampled the variat ion in this species, we cannot distinguish between these two hypotheses.

ACNOWLEDGMENTS

We gratefully acknowledge financial assistance from the National Sci- ence Foundation (BSR-8701159 and BSR-8805630), the Wiegand Fund of Cornell University, the Comen University Graduate School Summer Research Travel Fund, and the Harold E. Moore, Jr. Endowment. We also thank J. i. Davis, D. Garvin, P. S. Manos, and N. F. Weeden for assistance with interpretation of isozyme data; J. L. Doyle for technical assistance with the cpDNA study; V. Carstens for assistance in interpretation of linguistic data; K. Mitchell and L. Richter for preparation of figures; Enrique Estrada for translating the abstract; and T. A. LaRue and F. A. Harrington for reviewing early drafts of this paper.

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Documents

Origins of the African Yam bean (Sphenostylis stenocarpa, leguminosae): evidence from morphology, isozymes, chloroplast DNA, and linguistics