Proc. Natl. Acad. Sci. USA 77 (1980) 1229

Correction. In the article "Organelle DNA variation and sys-tematic relationships in the genus Zea: Teosinte" by D. H.Timothy, C. S. Levings III, D. R. Pring, M. F. Conde, and J. L.Kermicle, which appeared in the September 1979 issue of Proc.Natl. Acad. Sci. USA (76, 4220-4224), the authors request thatthe following corrections be noted. On p. 4220 in the Abstract,the fourth sentence should read "Separation of the teosinte andmaize organelle DNAs into six groups (Guatemala; perennialteosinte; Balsas and Huehuetenango; Central Plateau andChalco; Nobogame; and maize) approximated the biosystematicrelationships of the taxa." On p. 4222, in the right-hand columnunder the heading "Organelle DNA Evolution in Parallel withthe Nuclear System," the third sentence should read, "Thearrangement of these distinct groups into one hierarchicalstructure results in the delineation of six classes which corre-spond to conventionally determined taxonomic entities as fol-lows: (i) race Guatemala, (ii) perennial teosinte, (iii) racesHuehuetenango and Balsas, (iv) races Central Plateau andChalco, (v) race Nobogame, and (vi) several stocks of normalmaize of the U.S. Corn Belt type." On p. 4223 in line 14 of theright-hand column, delete "and maize," and in lines 3 and 4from the bottom, delete "as its ctDNA resembles maize,".

Correction. In the article "Triplet states in photosystem I ofspinach chloroplasts and subehloroplast particles" by Harry A.Frank, Mary B. McLean, and Kenneth Sauer, which appearedin the October 1979 issue of Proc. Natl. Acad. Sci. USA (76,5124-5128), an error occurred in the Proceedings editorialoffice. On p. 5125 in Fig. 2 and on p. 5126 in Fig. 3, the scaleof magnetic field strength, which is now shown to span the re-gion of 3000-3500 G in 100-G increments, should be correctedto span the region of 2750-3750 G in 200-G increments.

Correction. In the article "Effect of interchain disulfide bondon hapten binding properties of light chain dimer of protein315" by Raphael Zidovetzki, Arieh Licht, and Israel Pecht,which appeared in the November 1979 issue of Proc. Natl.Acad. Sci. USA (76,5848-5852), an undetected printer's errorresulted in the misspelling of the first author's surname. Itshould have appeared as "Zidovetzki."

Correction. In the article "Cloning of integrated Moloneysarcoma proviral DNA sequences in bacteriophage A" by G.F. Vande Woude, M. Oskarsson, L. W. Enquist, S. Nomura, M.Sullivan, and P. J. Fischinger, which appeared in the September1979 issue of Proc. Natl. Acad. Sci. USA (76, 4464-4468), theauthors request that the following correction be noted. On page4468 under Note Added in Proof, in line 2 "140-kb" should bechanged to "15.0-kb."

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Proc. Natl. Acad. Sci. USA 77 (1980) 1229

Correction. In the article "Organelle DNA variation and sys-tematic relationships in the genus Zea: Teosinte" by D. H.Timothy, C. S. Levings III, D. R. Pring, M. F. Conde, and J. L.Kermicle, which appeared in the September 1979 issue of Proc.Natl. Acad. Sci. USA (76, 4220-4224), the authors request thatthe following corrections be noted. On p. 4220 in the Abstract,the fourth sentence should read "Separation of the teosinte andmaize organelle DNAs into six groups (Guatemala; perennialteosinte; Balsas and Huehuetenango; Central Plateau andChalco; Nobogame; and maize) approximated the biosystematicrelationships of the taxa." On p. 4222, in the right-hand columnunder the heading "Organelle DNA Evolution in Parallel withthe Nuclear System," the third sentence should read, "Thearrangement of these distinct groups into one hierarchicalstructure results in the delineation of six classes which corre-spond to conventionally determined taxonomic entities as fol-lows: (i) race Guatemala, (ii) perennial teosinte, (iii) racesHuehuetenango and Balsas, (iv) races Central Plateau andChalco, (v) race Nobogame, and (vi) several stocks of normalmaize of the U.S. Corn Belt type." On p. 4223 in line 14 of theright-hand column, delete "and maize," and in lines 3 and 4from the bottom, delete "as its ctDNA resembles maize,".

Correction. In the article "Triplet states in photosystem I ofspinach chloroplasts and subehloroplast particles" by Harry A.Frank, Mary B. McLean, and Kenneth Sauer, which appearedin the October 1979 issue of Proc. Natl. Acad. Sci. USA (76,5124-5128), an error occurred in the Proceedings editorialoffice. On p. 5125 in Fig. 2 and on p. 5126 in Fig. 3, the scaleof magnetic field strength, which is now shown to span the re-gion of 3000-3500 G in 100-G increments, should be correctedto span the region of 2750-3750 G in 200-G increments.

Correction. In the article "Effect of interchain disulfide bondon hapten binding properties of light chain dimer of protein315" by Raphael Zidovetzki, Arieh Licht, and Israel Pecht,which appeared in the November 1979 issue of Proc. Natl.Acad. Sci. USA (76,5848-5852), an undetected printer's errorresulted in the misspelling of the first author's surname. Itshould have appeared as "Zidovetzki."

Correction. In the article "Cloning of integrated Moloneysarcoma proviral DNA sequences in bacteriophage A" by G.F. Vande Woude, M. Oskarsson, L. W. Enquist, S. Nomura, M.Sullivan, and P. J. Fischinger, which appeared in the September1979 issue of Proc. Natl. Acad. Sci. USA (76, 4464-4468), theauthors request that the following correction be noted. On page4468 under Note Added in Proof, in line 2 "140-kb" should bechanged to "15.0-kb."

Corrections

Proc. Nadl. Acad. Sci. USAVol. 76, No. 9 p)P 4220-4224, September 1979Applied Biology

Organelle DNA variation and systematic relationships inthe genus Zea: Teosinte

(chloroplast DNA/mitochondrial DNA/maize relatives/teosinte races/restriction endonuclease fragment analysis)

D. H. TINMOTHY*, C. S. LE}VINGS III*, D. R. PRINGt, N4. F. CONDEt, AND J. L. KERMIICLE1*Departmllenls of Crop Science and Genetics, North Carolina State University, Raleigh, North Carolina 27650; tU. S. I)epartment of Agricuilture, SEA-AR, andDepartment of Plant P'athology, University of Florida. Gainesville, Florida 3f2611;1 and Laboratory of Genetics.U'niversity of Wisconsin. \adison, Wis(onsin 537()06

Coninrunicated by Stanley G. Stephens, June 12, 1979

ABSTRACT Chloroplast and mitochondrial DNAs from sixraces of annual teosinte (Guatemala, Huehuetenango, Balsas,Central Plateau, Chalco, and Nobogame), perennial teosinte,and maize were compared and grouped by restriction endonu-clease fragment analyses. Three groups of chloroplast DNAswere detected: (i) perennial teosinte and Guatemala; (ii) Balsasand Huehuetenango; and (iii) all other teosintes. Four groups

of mitochondrial DNAs were separated: (i) perennial teosinte;(ii) Guatemala; (iii) Nobogame; and (iv) all other teosintes.Separation of the teosinte and maize organelle DNAs into fivegroups (Guatemala; perennial teosinte; Balsas and Iluehuete-nango; Central Plateau and Chalco; Nobogame and maize) ap-

proximated the biosystematic relationships of the taxa. It wassuggested that the evolutions of the chloroplast and mitochon-drial DNAs may be independent of each other, that variationof organelle DNA within a species complex of an organism maybe the common condition, and that the DNAs of the organelleand nuclear systems evolve in reasonable harmony.

Evolutionary studies of higher plants have generally disre-garded the chloroplast and mitochondrial genomes. An ex-

ception has been the significant work with ribulose-bisphos-phate carboxylase, the large subunit of which is encoded by thechloroplast DNA (ctDNA) (1). Largely, this neglect hasstemmed from lack of appropriate methods. Recent develop-ments in molecular biology, especially the advent of restrictionendlontucleases, have provided new strategies for studying theevolution of organelle genomes.

Restriction endonucleases cleave double-stranded DNAs atspecific nucleotide sequence sites. When the DNA is of lowcomplexity, fractionation of the cleaved fragments by gelelectrophoresis produces a distinctive fragment pattern. Thispattern serves as a fingerprint of the original DNA moleculein a manner analogous to the trypsin digestion fingerprints ofproteins. The restriction fragment pattern of the DNA is a

fuFnction of the number and position of the cleavage sites rec-

ognized and digested by the endonuclease. Sequence distinc-tions among related DNAs can be resolved if they involvechanges in the number or position of cleavage sites. This pro-

vides a method for contrasting organelle DNA molecules ofhigher plants.The evolution and classification of maize. Zea mays L.

(Gramineae) 2n = 20, and its relatives have been studied ex-

tensively. rhe closest living relative of maize is the dliploid (2n= 20) annual teosinte [Z. mayis L. ssp. mexicana (Schrad.) Iltisand Z. mays L. ssp. luxurigns (Duriet) 1Itis]. In adldition tomorphological and cytological (listinctions, the latter two sub-species differ in that ssp. nmexicana, consisting of races Nobo-game, Central Plateau, Chalco, Balsas, and Huehuetenango (2),

The p)ubl)lication costs of this article were (lefrayed in part by pagec(harg( payment. This article must therefore be hereby marked "ad-verlisernent in accor(lance with 18 U. S. C. §1734 solely to indicatethis fact.

hybridizes readily with maize tinder natural conditions whereasthe more primitive ssp. luxurians, race Guatemala (2), does not.The tetraploid (2n = 40) perennial teosinte [Zea perennis(Hitch.) Reeves and Niangelsdorf has recently been rediscov-ered near the area of its original collection (3). From this sameregion, a perennial cliploid, Zea diploperennis Iltis, Doebley,and Guzman, that is interfertile with maize has just been re-ported (4).

Four (Nobogame, Central Plateau, Chalco, and Balsas) of thesix described races of annual teosinte are from Mexico; Hue-huetenango is from northern Guatemala and Guatemala is fromsouthern Guatemala (3). The race Guatemala is genetically themost distinct (5). The least maize-like annual teosinte in Mexicois Balsas. It is similar in several respects to Huehuetenango. OnlyGuatemala, with its basal tillering, a perennial-like growthhabit, is more distinct from maize than is Balsas (5). The mostmaize-like is Chalco, a maize-mimetic.The patterns of variation reported so far, and the interpre-

tations based upon them, have been considered almost exclu-sively in terms of nuclear events. A priori one might sup-pose-in the long term-that nuclear and cytoplasmic diversityshould develop in harmony with each other. However, theevidence currently available concerning short-term eventssuggests that cytoplasmic diversity is unaffected by the im-mediate male parent-i.e., cytop)lasmic inheritance is unipar-ental (6). It is clear that biparental inheritance in the nucleusand uniparental inheritance in the cytoplasm might producerather different patterns of diversity. Nonetheless, the inter-relationships of the two systems may be of fundamental im-portance to understanding evolution.The objectives of this studly were to assess teosinte organelle

DNA restriction patterns as expressions of evolutionary elementsand to devise an organellar hierarchy based on restrictionfragment patterns for comparison with the model system de-rived by conventional taxonomic methlos. This would permitsome insight into changes in organelle DNAs and their evolu-tionary role.

MATERIALS AND METHODSOrganelle DNAs were studied from the maize and teosinte racesor varieties listed in Table 1. Mlost of the annual teosinte ma-terials trace to collections maile by H. Garrison Wilkes; El Sa-lado of the Balsas race was collected by George NV. Beadle. Amaize stock carrying the cytoplasm of perennial teosinte wasextracted byv J. B. Beckett from material tracing to the originalcollection of Collins and Kempton. To facilitate handling andto increase seed yields, the annual and perennial teosintecytoplasnis were routinely transferred to maize nuclear back-

Abbreviations: mtDNA, rnitochod rial DNA; Otl)N A, chloroplastDNA.

4220

Proc. Natl. Acad. Sci. USA 76 (1979) 4221

Table 1. Cytoplasmic sources of teosinte organelle DNAs

Cytoplasm Entry Collector and site of original source

Nobogame 74-47 Wilkes, Nobogame, Tarahumare Valley, Chihuahua, Mexico, PI 34324970-4 Hernandez-X., Nobogame, Guadalupe y Calvo, Chihuahua, Mexico

Chalco P 450 Wilkes, Amecameca, Mexico, MexicoCentral Plateau P 444 Wilkes, Col. 45121 Guanajuato, MexicoBalsas P 447 Wilkes, Col. 47890, Michoacan, Mexico

El Salado 76-70 Beadle, El Salado, Mazatlfin, Guerrero, MexicoEl Salado P 455 Beadle, El Salado, Mazatla'n, Guerrero, Mexico

Huehuetenango P 452 Wilkes, San Antonio Huista, Huehuetenango, GuatemalaGuatemala P 453 Wilkes, Col. 51186, Jutiapa, GuatemalaPerennial P 457 Collins and Kempton, Ciudad Guzman, Jalisco, Mexico

grounds by backerossing procedures, except that entry 76-70El Salado of the Balsas race was a sib-mated increase, in Mexico,of the original collection. In all backcrosses, teosinte cytoplasmsalways participated as the female (ear) parent. Strict maternalinheritance of organelle genomes has been demonstrated pre-viously in the genus Zea (6-8). Each stock, except El Salado76-70, traces to a single teosinte seed. The thrust of the inves-tigation was to survey a broad sample of the species complexrather than to sample within races or individual populations.However, in those case in which different samples of a taxawere used, no differences were found within comparisons oftwo different collections of Nobogame, two bulked samplesgathered in different years of El Salado, and two differentplants in the collection of Guatemala.

Mitochondrial DNA (mtDNA) was isolated from etiolatedmesocotyl and coleoptile tissue of 5- to 7-day-old seedlings.Chloroplast ctDNA was isolated from plants held in darknessfor 48 hr before the 20- to 30-day-old leaves were harvested.The isolation of organelles, preparation of organelle DNAs, andrestriction endonuclease fragment analyses were as described(7-9). The restriction endonucleases used were BamHI, EcoRI,HindIII, Sal I, and Xho I (Bethesda Research Labs; MilesLaboratories, Inc.; or New England Biolabs). Visually identicaldigestion patterns of organelle DNAs from the various taxa weregrouped. Each group was arbitrarily numbered as 1, 2, 3, or 4.Nobogame mitochondrial DNA was not analyzed with Sal Iand HindIll because of inadequate seed supply.

RESULTSTeosinte ctDNA Digestions. Restriction endonuclease

fragment analysis of ctDNAs isolated from the eight teosintecollections revealed three different groups: perennial teosinteand Guatemala; Huehuetenango and Balsas; Chalco, CentralPlateau, Nobogame, and El Salado (Table 2). Irrespective ofthe endonuclease used, the pattern characteristic of perennialteosinte was identical to that of Guatemala and different from.that of the remaining annual teosintes and maize (Fig. 1, lanesA, C, and F). With all three enzymes tested, the patterns pro-duced by digestion of Huehuetenango and Balsas ctDNAs wereindistinguishable from each other (Fig. 1, lanes B, D, and E) andfrom those characteristic of most maize (7). Chalco, CentralPlateau, Nobogame, and El Salado ctDNAs digested by HindIIIand BamHI were indistinguishable from most maize ctDNAs(7). However, EcoRI digests of these four annual teosintes re-vealed a pattern different from the characteristic of Hue-huetenango and Balsas annual teosintes (Fig. 1, lanes E and G)and maize (7).

Although the endonuclease digestion fragment patternsshowed a great deal of similarity when the perennial teosinteand Guatemala were compared to the other annual teosintes,the differences that characterize each pattern were highly re-producible.

Teosinte mtDNA Digestions. The complexity of themtDNA restriction patterns, in contrast to those from ctDNAs,does not permit an unambiguous band-by-band comparisonamong low molecular weight fragments in the lower third ofthe gel. However, comparisons among high molecular weightfragments are clearly managed. Four groups were distinguishedby restriction endonuclease fragment analysis of mtDNA: pe-rennial teosinte; Guatemala; Nobogame; and all other teosintes(Table 2). Except for Nobogame, the same groupings weregenerated by four different restriction endonucleases-BamHI,Sal I, Xho I, and HindIII-even though these four enzymeshave distinctive site-specific restriction sites (Fig. 2; HindIIInot shown).

A LB (c U (iFIC. 1. Agarose gel electrophoretic patterns of restriction en-

donuclease digests of ctDNAs isolated from perennial and annualteosinte collections. Lanes: A and B, BamHI digests of ctDNAs iso-lated from Zea perennis (A) and Huehuetenango (B); C and D,HindIII digests of ctDNAs isolated from Zea perennis (C) and Cen-tral Plateau (D); E, F, and G, EcoRI digests of ctDNAs isolated fromBalsas (E), Zea perennis (F), and Chalco (G).

Applied Biology: Timothy et al.

4222 Applied Biology: Timothy et al.

Table 2. Classification of teosinte races and maize based on restriction endonuclease fragment analyses of organelle DNAsDNA groups*

Chloroplast MitochondrialEcoRI BamHI HindIII Sal I Xho I BamHI HindIII

Cytoplasm 1 2 3* 1 2 1 2 1 2 3 1 2 3 1 2 3 4 1 2 3

Maize:tB37andmostotherU.S. maize + + + - - - - - - +

Teosinte:Nobogame + + + No test + + No testChalco + + + + + + +Central Plateau + + + + + + +El Salado (Balsas) + + + + + + +Balsas + + + + + + +Huehuetenango + + + + + + +Guatemala + + + + + + +Perennial + + + + + + +

* The numbers under the various restriction enzymes represent the number of arbitrary groups required to include identical fragment patternsin separate categories. A + in any column indicates a fragment pattern unique to members in that category. A blank in a column indicatesthat no fragment pattern fits that category.

t A complete classification for maize mitochondrial DNA is not included because three of the enzymes, Sal I, Xho I, and HindIII, express sufficientvariability among stocks of maize that a single representative pattern is precluded. This is indicated by a - for each category.

The mtDNA BamHI restriction patterns of all the annualteosintes except Guatemala were similar to the maize pattern.Of these five, Nobogame showed no difference from maize; theremaining four annual races had one extra band not seen inmaize or Nobogame. In contrast, patterns of maize were dif-ferent (not shown) from those of annual teosintes when en-donucleases Xho I and Sal I were used. These distinctions weregenerally slight-e.g., comparisons of one maize inbred withthe annual teosintes, other than Guatemala, revealed a two-band difference with Xho I digestion. Organelle DNA diversitywithin maize and teosinte is recognized, and critical compari-sons of the extent of such variation would be premature. In anearlier study with commercial maize inbreds and hybrids,variations among mtDNA restriction patterns were obtainedwith Xho I (unpublished data), BamHI, Sal I, and HindIII (9).Interestingly, BamHI did not differentiate mtDNAs amongcommercial corns (9) that were distinguished by the other en-zymes. The fact that BamHI is less discriminatory may beimportant in establishing general groups but not fine con-trasts.

DISCUSSIONThe data presented in this paper are significant in that theyform a basis from which to begin formulating concepts of or-ganelle evolution. In this regard, three important phenomenaare suggested: (i) heterogeneity exists among organelle DNAsof a species complex, (ii) the mtDNA and ctDNA systems mayvary independently of each other, and (iii) the organelle DNAsevolve in concert with the nuclear system.,

Another important facet of this report is that it indicates howorganelle evolution can be traced within a species complex byutilizing endonuclease restriction patterns to visualize differ-ences in the organelle genomes. Thus, the data permit com-parative analyses of the putative relationships of the classifi-cations of teosinte in terms of the total organism (conventionalbiosystematic determinations by genetic, cytogenetic, andtaxonomic methods) versus the restriction fragment groupingsof the organelle genomes.

Heterogeneity among Organelle Genomes. It has becomeincreasingly apparent that variation of organelle DNA withinthe species complex of the organism may be a common condi-tion. In this paper, three groupings of mtDNA and three ofctDNA were found among the six races of annual teosinte.Moreover, previous work (9) suggested mtDNA diversity

among normal (fertile) maize cytoplasms-e.g., differencesamong restriction patterns ranged from one to four bands. Evengreater differences, involving as many as 17 bands (7, 8), werefound when normal maize cytoplasms were compared withmale-sterile cytoplasms. Minor differences in mtDNA havebeen reported in soybeans (10).

Although comparisons of restriction patterns indicate se-quence differences, the effect of this variation on gene ex-pression is not known. The distinctions could result fromgenomic rearrangements that do not alter expression, but itseems unlikely that no genetic differences could exist as a resultof sequence modifications. Differences in mtDNA restrictionpatterns and accompanying genetic expression have beendemonstrated between normal and cytoplasmic male-sterilemaize (7, 8) and wheat (11). Also, teosinte cytoplasm is differentfrom maize in that certain genetic characters-e.g., iojap, achlorophyl mutant-behave differently in teosinte cytoplasmthan in maize cytoplasm (12). This differential reaction mightnow be explained in terms of ctDNA.

Independent Variations in ctDNA and mtDNA Systems.The groupings of the taxa by ctDNA-vs-mtDNA fragmentpattern analyses were different. Specifically, two taxa, perennialteosinte and Guatemala, with dissimilar mtDNAs were indis-tinguishable in ctDNAs. Furthermore, the races Balsas andHuehuetenango were identical to Central Plateau and Chalco,and nearly so to Nobogame, on the basis of mtDNA patterns.But, they were readily separated on the basis of ctDNA patterns.If evolutions of the two organelles could not occur indepen-dently, the four groups of mtDNAs would not be different from,or allow for easy separation on the basis of organelle DNAsfrom, the three groups of ctDNAs.

Organelle DNA Evolution in Parallel with the NuclearSystem. It is also suggested that the organelle DNAs evolve inreasonable concert with the nuclear system. Four groups ofmtDNAs and three groups of ctDNAs were detected. The ar-rangement of these distinct groups into one hierarchial structureresults in the delineation of five classes which correspond toconventionally determined taxonomic entities as follows: (i) raceGuatemala, (ii) perennial teosinte, (iii) races Huehuetenangoand Balsas, (iv) races Central Plateau and Chalco, and (v) raceNobogame and several stocks of normal maize of the U.S. CornBelt type. Thus, the maize relatives demonstrated here to bethe most distinct from maize in organelle DNA patterns-e.g.,Guatemala-are those most distinct from maize when evalu-

Proc. Natl. Acad. Sci. USA 76 (1979)

Proc. Natl. Acad. Sci. USA 76 (1979) 4223

A B C D E F G H J

FIG. 2. Agarose gel electrophoresis patterns of restriction en-donuclease digests of mtDNAs isolated from perennial and annualteosinte. Lanes: A-D, BamHI digests of mtDNAs isolated fromNobogame (A), Central Plateau (B), Guatemala (C), and perennialteosinte (D); E, F, and G, Sal I digests of mtDNAs isolated fromCentral Plateau (E), Guatemala (F), and perennial teosinte (G); H,I, and J, Xho I digests of mtDNAs isolated from Huehuetenango (H),Guatemala (I), and perennial teosinte (J).

ated by traditional genetic, cytogenetic, and taxonomic criteria.Those taxa considered most closely related to maize by con-ventional systematics-e.g., Chalco-did not differ apprecia-bly, or were indistinguishable, from maize in restriction patternanalyses. Consequently, our evidence suggests that the degreeof divergence in organelle DNAs conforms appreciably to thedegree of divergence in phylogenetic relationship of the or-ganism as determined by conventional methods.

Studies of certain mitochondrial and chloroplast enzymeshave established that the protein is made up of subunits codedfor by both the nuclear and organelle genomes (1, 13). Thissuggests a molecular basis for complementary evolution ofnuclear and organelle genomes. Similarly, nuclear genes inmaize, fertility restorers, can countermand the cytoplasmicmale-sterility trait and restore pollen fertility. The interactionof these genetic systems requires a harmonious evolution forsurvival.

Organelle DNAs and Relationships of Teosintes andMaize. The five classes of organelle DNAs noted above and thecharacteristics of their fragment band patterns reflect to a

reasonable degree the phylogenetic affinity among the taxa andtheir supposed amounts of hybridization and introgression (Figs.1 and 2). Studies of chromosome knobs and inversions have ledto the interpretations that annual teosinte populations are de-rivatives of a common ancestor (14) and that the Mexicanteosintes, rather than the Guatemalan teosintes, play a majorrole in the history of maize (15).Our data support these conclusions in part, in that only the

race Guatemala and perennial teosinte differ markedly frommaize in both mtDNA and ctDNA fragment patterns. Digestionpatterns of ctDNAs from Balsas (the least maize-like of theMexican teosintes) and Huehuetenango, from northwesternGuatemala, indicate that they differ somewhat from the otherMexican teosintes and maize. This infers a slightly differentorigin of these two races than that indicated by mtDNA di-gestion or that a change in the chloroplast occurred with, orafter, morphological differentiation.

Our results from mtDNA digestions do not reveal differencesamong the races Huehuetenango, Balsas, Central Plateau, andChalco. This indicates a common cytoplasm of these taxa and,hence, a common maternal ancestry. Nobogame differs fromthese only by exhibiting one less band when its mtDNA is di-gested by BamHI, in which case it does not differ from normalmaize (9). Therefore, the relationships of mitochondrial ge-nomes among these taxa are considerably closer to each otherthan to those of Guatemala and perennial teosinte.The relationship of perennial teosinte, a tetraploid, to the

annual and newly found perennial populations (4) is in doubt.Its chromosome morphology resembles that of maize ratherthan Guatemala teosinte (16), and it behaves cytologically asa newly induced experimental maize autotetraploid with 7 to9 quadrivalents (16).

Striking differences are noted with mtDNAs of perennialteosinte and Guatemala. Although the ctDNAs of these twoteosintes are indistinguishable, they differ from the others. Themost logical interpretation is that perennial teosinte and Gua-temala or their antecedents shared a common cytoplasm beforea change in the mitochondrial genome occurred. This couldmean that the genetic pool containing the common cytoplasmwas the original stock from which these two and perhaps theother teosintes evolved, or that perennial teosinte and Guate-mala were a segment of teosintes that have remained isolatedgenetically from the others. The two concepts are not mutuallyexclusive.

Refinements in determining the relationships or originswithin teosinte and maize must await further study. If appre-ciable organellar DNA variation is found within teosinte races,or between maize of meso-America and the U.S. Corn Belt,refinement of the racial relationships among teosintes, and ofteosinte to maize may be indicated. Nuclear events influencingsubjective judgments of taxonomy may also superficially maskmaternal inheritance (i.e., organelle diversity or material lin-eages), especially in taxa of broad geographical range andsubject to introgression-e.g., Balsas. Accounts from Mexicoof introgression by teosinte into maize and by maize into teo-sinte have been available for a long time; even in pre-Colom-bian times, the maize-mimetic Chalco teosinte was recognizedand referred to in the Florentine Codex (5). Teosinte hybridizeswith maize throughout its entire range but most frequently inthe races Chalco, Central Plateau, and Nobogame (2,5). Lesseramounts of maize hybridization occur with Balsas and Hue-huetenango (5, 17). The El Salado accession of Balsas fallsoutside the Balsas Huehuetenango organelle group, as its ctDNAresembles maize, although it is a good morphological specimenof the Balsas race. This accession may be the result of differentfemale lineage or of teosinte nuclear introgression into maize

Applied Biology: Timothy et al.

4224 Applied Biology: Timothy et al.

cytoplasm. Considerable maize-teosinte introgression has beendescribed with Balsas teosinte, particularly in conjunction withselection and the formation of seed-dormant teosinte (El Salado)in the Balsas River watershed during the last 50 years (5). Ad-ditional sampling between and within populations shouldclarify these phylogenies.

Seed of some of the stocks was graciously supplied by George W.Beadle (Chicago, IL), Pioneer Hi-Bred International, Inc. (Johnston,IA), Southern Regional Plant Introduction Station, (USDA, Experiment,GA), E. Hernandez-X. (Escuela Nacional de Postgraduados, Chapingo,Mexico). This has been a cooperative investigation of the North Car-olina Agricultural Research Service, U.S. Department of Agriculture,Science and Education Administration, Agricultural Research, andInstitute of Food and Agricultural Sciences, University of Florida, andthe Wisconsin Agricultural Experiment Station. This report is journalseries paper no. 5906 North Carolina Agricultural Research Service,Raleigh. This research was supported in part by grants from the Na-tional Science Foundation (DEB 78-00538 and PCM 78-05753) andPioneer Hi-Bred International, Inc.

1. Wildman, S. G., Kawashima, N., Bourgue, D. P., Wong, F., Singh,S., Chan, P. H., Kowk, S. Y., Sakano, K., Kung, S. D. & Thornber,J. P. (1973) in The Biochemistry of Gene Expression in HigherOrganisms, eds. Pollack, J. K. & Lee, J. W. (Australia and NewZealand Book Co., Artarmon, Australia), pp. 443-456.

2. Wilkes, H. G. (1967) Teosinte: the Closest Relative of Maize(Bussey Inst., Harvard Univ. Press, Cambridge, MA).

3. Guzman Mejia, R. (1978) Phytologia 38, 177.4. Iltis, H. M., Doebley, J. F., Guzman M., R. & Pazy, B. (1979)

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Proc. Natl. Acad. Sci. USA 76 (1979)