Distribution and molecular diversity of arborescent Gossypium species

Distribution and molecular diversity of arborescentGossypium species

Chunda Feng, Mauricio Ulloa, Claudia Perez-M., and James McD. Stewart

Abstract: Mexico is a center of diversity of Gossypium. As currently circumscribed, arborescent Gossypium species (subsec-tion Erioxylum) are widely distributed in dry deciduous forests located from Sinaloa in the north of its range to Oaxaca inthe south of its range. However, extensive morphological variation exists among accessions from these different geographicregions. The objective of this work was to determine whether the observed morphological variation is reflected at the molec-ular level. Molecular diversity and phylogenetic relationships were estimated with 210 randomly amplified polymorphicDNA fragments and 766 amplified fragment length polymorphism fragments among 33 accessions of arborescent Gos-sypium, including 23 of Gossypium aridum, the most widely distributed of the arborescent Mexican diploid Gossypium spe-cies. Over 90% of the fragments were polymorphic; however, each accession contained only between 32% and 46% of thetotal loci. Two thirds of the loci among the G. aridum accessions had allelic frequencies lower than 80%. The genetic dis-tance between Gossypium gossypioides (subsection Selera) and species of subsection Erioxylum ranged between 0.64 and0.84. The genetic distance between two recognized species, Gossypium lobatum and Gossypium schwendimanii, within sub-section Erioxylum was 0.32. Most molecular data support the traditional classification of Gossypium species and the geo-graphical ecotypes of the G. aridum accessions. A newly collected accession, US-72, of subsection Erioxylum wasgenetically distant (range, 0.42–0.54) from the other species of the subsection. Molecular data support the recognition ofthis taxon as a new species. The molecular diversity among accessions of G. aridum was greater than that among the spe-cies of subsection Erioxylum. The results indicate this subsection deserves additional study to establish a defensible taxo-nomic treatment of the various taxa and to resolve genetically distant geographical ecotypes.

Key words: Gossypium, cotton, D genome, genetic resource, molecular diversity, phylogenetic relationship.

Résumé : Le Mexique constitue un centre de diversité du genre Gossypium. Tel que présentement circonscrit, les espècesarborescentes du genre Gossypium (Section Erioxylum) connaissent une distribution étendue dans les forêts sèches décidueslocalisées de Sinola dans le nord de son aire jusqu’à Oaxaca dans le sud de son aire. Cependant, il existe une large variationmorphologique au sein des accessions provenant de ces différentes régions géographiques. L’objectif du travail consistait àdéterminer si la variation morphologique observée se reflète à l’échelle moléculaire. Les auteurs ont estimé la diversité mo-léculaire et les relations phylogénétiques en utilisant 210 fragments d’ADN polymorphique amplifié aléatoirement (RAPD)et le polymorphisme de la longueur des fragments amplifiés (AFLP) chez 33 accessions de Gossypium arborescents incluant23 G. aridum, l’espèce la plus répandue des espèces de Gossypium diploïdes arborescentes du Mexique. Plus de 90 % desfragments sont polymorphiques; cependant, chaque accession ne contient que 32–46 % du total des lieux. Deux tiers deslieux parmi les accessions du G. aridum possèdent des fréquences alléliques inférieures à 80 %. La distance génétique entrele G. gossypioides (sous-section Selera) et les espèces de la sous-section Erioxylum va de 0,64 à 0,84. La distance génétiqueentre deux espèces reconnues, les G. lobatum et G. schwendimanii, au sein de la sous-section Erioxylum, est de 0,32. Laplupart des données moléculaires supportent la classification traditionnelle des espèces de Gossypium et les écotypes géogra-phiques des accessions du G. aridum. Une nouvelle accession récemment récoltée, US-72, de la sous-section Erioxylum s’a-vère génétiquement distante (allant de 0,42 à 0,54) des autres espèces de la sous-section. Les données moléculairessupportent la reconnaissance de ce taxon comme nouvelle espèce. La diversité moléculaire au sein des accessions du G. ari-dum est plus large qu’entre les espèces de la sous-section Erioxylum. Les résultats indiquent que cette sous-section méritede nouvelles études pour établir un traitement taxonomique défendable des différents taxons et résoudre les écotypes géogra-phiques génétiquement distants.

Mots‐clés : Gossypium, coton, génome D, ressource génétique, diversité moléculaire, relation phylogénétique.

[Traduit par la Rédaction]

Received 1 January 2010. Accepted 1 January 2011. Published at www.nrcresearchpress.com/cjb on 2 September 2011.

C. Feng* and J.M. Stewart. Department of Crop, Soil, and Environmental Sciences, University of Arkansas, Fayetteville, AR 72701,USA.M. Ulloa. USDA Agricultural Research Service, Western Integrated Cropping Systems Research Unit, Shafter, CA 93263, USA.C. Perez-M. Campo Experimental Iguala, Centro de Investigaciones Pacific sur-INIFAP, C.P. 40000, Iguala, Guerrero, México.

Corresponding author: James McD. Stewart (e-mail: [email protected]).

*Present address: Department of Plant Pathology, University of Arkansas, Fayetteville, AR 72701, USA.

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Botany 89: 615–624 (2011) doi:10.1139/B11-042 Published by NRC Research Press

Introduction

The genetic diversity among modern upland cotton culti-vars in the United States (Iqbal et al. 2001; Abdalla et al.2001; Lu and Myers 2002), China (Xu et al. 2002), Australia(Multani and Lyon 1995), Pakistan (Iqbal et al. 1997; Rah-man et al. 2002), and Greece (Linos et al. 2002) is very low.This low or narrow genetic diversity in cotton cultivars hasbeen suggested as a contributor to an apparent plateau inbreeding progress (Meredith 1992; Ulloa 2006) and poten-tially could limit future cotton improvement (Ulloa et al.2009). One solution to this problem is to collect, evaluate,and utilize a broad range of cotton germplasm, with specialemphasis on diploid species of the Gossypium genus.The Gossypium genus contains 45 diploid and 5 allotetra-

ploid species (Fryxell et al. 1992; Ulloa et al. 2006) groupedinto nine genomic types (x = 2n = 26 or n = 13) with thefollowing designations: AD, A, B, C, D, E, F, G, and K (Per-cival et al. 1999). Mexico, along with Australia and EasternAfrica, is a center of diversity of the genus Gossypium. Themost widely planted tetraploid, Gossypium hirsutum L., origi-nated in Mexico, and 11 of the 13 known diploid Gossypiumspecies of the Western Hemisphere are endemic to that coun-try. However, not much of the Mexican diploid Gossypiumgermplasm is exploited. Although none of these Mexicandiploid species produces cotton fibers, a closely related D ge-nome species, Gossypium raimondii Ulb., is a putative pro-genitor of allotetraploid cotton. In recent years, the USDepartment of Agriculture and the Mexican Instituto Nacio-nal de Investigaciónes Forestales Agricolas y Pecuarias (INI-FAP) have sponsored joint Gossypium germplasm collectiontrips by US and Mexican cotton scientists. As a result ofthese efforts, a significant number of additional Gossypiumaccessions from various parts of Mexico are now availablefor evaluation, including several accessions of each of the ar-borescent species (Ulloa et al. 2006).Traditional taxonomy of Gossypium is based on morphol-

ogy (plant shape and height, leaf and capsule shapes, flowers,seed size, etc.). The most recent classification of Gossypiumspecies was made by Fryxell (1992) and Fryxell et al. (1992).Morphological characteristics between two species may notbe distinctive, and intermediate phenotypes are occasionallyobserved, especially in subsection Erioxylum, which makethe classification difficult. Molecular methods provide an al-ternative solution to distinguish species when morphologicalcharacteristics are not well defined. They not only provide ameasure of molecular diversity, but also offer an additionaltool to measure relatedness among the species and at thesame time provide insights into phylogenetic relationships ofspecies and evolution of the genus. In other crops, for in-stance rice (Ganesh Ram et al. 2007) and maize (Vigourouxet al. 2008), molecular markers such as microsatellites havebeen used to reveal genetic diversity and to distinguish spe-cies or wild relatives. In cotton, Gossypium species havebeen evaluated by a single gene or a few loci (e.g., the Adhgene (Small et al. 1998; Cronn et al. 1999; Small and Wen-del 2000a, 2000b), FAD2-1 gene (Liu et al. 2001), Ces A1gene (Wendel et al. 2002), internal transcribed spacer (ITS)of ribosomal DNA (Wendel et al. 1995; Pillay and Myers1999), ribosomal DNA (Wendel et al. 1995; Buckler et al.1997), and chloroplast DNA (Small et al. 1998)), or by repet-

itive DNA (Zhao et al. 1998; Hanson et al. 1998, 1999) andgenome size (Wendel et al. 2002). Álvarez et al. (2005) ana-lyzed the phylogeny of the New World diploid Gossypiumbased on the sequences of three nuclear genes, and recentlybased on AFLP markers. Álvarez and Wendel (2006) re-ported on the introgression of genetic material from sectionIntegrifolia into a taxon of subsection Erioxylum. Most mo-lecular data are concordant with traditional taxonomic divi-sions. Until recently (Ulloa et al. 2006), evaluation of the D-genome species of Gossypium, especially those in sectionErioxylum, has been limited by the lack of resource materialfor ex situ evaluation, as the report of Álvarez and Wendel(2006) has indicated.Gossypium aridum (Rose & Standl.) Skovst., as currently

taxonomically circumscribed (Fryxell 1992), is the mostwidely distributed of the Mexican diploid Gossypium species,with populations located from central Sinaloa in the north ofits range to eastern Oaxaca in the south of its range. Morpho-logical variations, such as leaf shape and size, petal spot, de-foliation time, and vestiture, have been observed amongaccessions of G. aridum growing under greenhouse condi-tions. Moreover, these morphological variations have alsobeen observed among herbarium specimens housed at theMexican National Herbarium and collected from their naturalhabitat in different geographical locations. Álvarez and Wen-del (2006) found extensive variation among populations ofG. aridum, especially those originating from the state of Col-ima. They have presented evidence that G. aridum from cen-tral Colima experienced hybridization with a Gossypiumspecies related to section Integrifolia that resulted in cyto-plasmic substitution, as well as acquisition of some novel ge-netic material. The extent of diversity of the G. aridum“ecotypes” is not well documented and suggested phyloge-netic relationships with other arborescent Gossypium speciesmay be questioned because of inadequacy of sampling depth.The objective of this study was to provide additional in-

sight into the molecular genetic diversity and phylogenetic re-lationships within the collections of Gossypium representingsection Erioxylum (collected from Sinaloa to Oaxaca) basedon extensive sampling of randomly amplified polymorphicDNA (RAPD) and amplified fragment length polymorphism(AFLP) markers.

Materials and methods

Plant material and DNA extractionThe 33 taxa examined were collected from seven Mexican

states (Table 1). The identification codes, species designation(based on the currently accepted classification scheme (Fryx-ell 1992)), state of origin, geographical information on germ-plasm collection sites (including latitude, longitude, andaltitude), and number of polymorphic fragments observedare also presented in this table. The samples classified asG. aridum (D4) are arranged by the latitude, from north tosouth, at which the original accession was collected. Twenty-four of the species accessions (US numbers) were collectedin 2002. US designation was assigned to each accession us-ing the first letter of each collector’s last name, Ulloa andStewart, respectively (Ulloa et al. 2006). The remaining nineaccessions were previously collected by Hugo Cota-Sanchez(HC) or Dan DeJoode (DJD). With the exception of DJD

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179 from the first six accessions in Table 1, DNA samples ofthe remaining five (DJD and HC) were kindly donated by thelaboratory of Jonathan F. Wendel, Iowa State University,Ames, Iowa. These accessions were from the northern partof the range of G. aridum, including material (HC10) col-lected in Sinaloa. The other 28 accessions were grown inpots in a greenhouse at the University of Arkansas, Fayette-ville, Ark. The two accessions designated DJD168-1 andDJD168-2 are plants of seeds from the same collection byDan DeJoode in Colima and grown at Iowa State Universityand the University of Arkansas, respectively. Since the mainobjective of the work was not to detect evidence of introgres-sion, but rather to obtain a broad measure of diversity withinthe section, and no obvious variation was observed amongplants from the same accession, only one plant per accessionwas examined.The donated DNA was extracted using a DNeasy plant

mini kit (Qiagen, Valencia, Calif.). Otherwise, young leaveswere picked from each genotype, and DNA was extractedwith the cetyltrimethylammonium bromide (CTAB) method:fresh leaves were ground with liquid nitrogen and 0.25 g offine powder was placed in a 1.5 mL tube, followed by theaddition of 30 µL b-mercaptoethanol and 750 µL extractionbuffer (0.1 mol/L Tris–HCl (pH 8.0), 1.4 mol/L NaCl,0.02 mol/L Na2-EDTA, 2% (m/v) CTAB, 0.1% (m/v) diethyl-dithiocarbamic acid, and 2% (m/v) polyvinylpyrrolidone).The suspended powder was incubated at 65 °C for 30 minbefore 750 µL chloroform – isoamyl alcohol (24:1) wasadded, and the tubes were mixed well. The samples werecentrifuged for 10 min at 20 800g, and the aqueous phasewas transferred to new tubes. Each tube received 2 µL of1 mg/mL RNase A and was incubated at 37 °C for 30 min.Then 750 µL chloroform – isoamyl alcohol (24:1) was addedto each tube, mixed well, and again centrifuged at 20 800g

Table 1. Accession identification, species, genome, origin, and number of polymorphic fragments obtained from each arborescent Gossy-pium species examined.

Geographical informationa

Acc.No.

AccessionID Species Genome Origin

No. of polymorphicfragments Lat. (N) Long. (W)

Elevation(m)

1 HC4 G. aridium D4 Sinaloa 253 24°50′ 107°23′ 332 HC10 G. aridium D4 Sinaloa 253 24°50′ 107°23′ 333 DJD185 G. aridium D4 Jalisco U.E.b 290 20°53′ 103°44′ 9964 DJD172 G. aridium D4 Jalisco C.E.c 319 19°35′ 105°06′ 145 DJD179 G. aridium D4 Jalisco C.E.c 327 19°35′ 105°06′ 146 DJD168-1 G. aridium D4 Colima 282 19°11′ 104°32′ 287 DJD168-2 G. aridium D4 Colima 301 19°11′ 104°32′ 288 US-83 G. aridium D4 Michoacán 328 18°20′41″ 101°53′51″ 2079 DJD123 G. aridium D4 Michoacán 275 18°20′ 101°53′ 20710 US-4 G. aridium D4 Puebla 307 18°08′13″ 98°18′10″ 95811 US-5 G. aridium D4 Puebla 327 18°08′26″ 98°17′59″ 101612 US-81 G. aridium D4 Guerrero 322 17°59′37″ 101°46′35″ 12713 US-80 G. aridium D4 Guerrero 337 17°59′05″ 101°47′54″ 7014 US-78 G. aridium D4 Guerrero 336 17°16′07″ 101°01′48″ 2915 DJD122 G. aridium D4 Guerrero 279 17°16′ 101°01′ 3116 US-76 G. aridium D4 Guerrero 337 17°09′59″ 100°33′37″ 3017 US-10 G. aridium D4 Oaxaca 330 16°37′10″ 94°58′7″ 19018 US-11 G. aridium D4 Oaxaca 335 16°36′06″ 94°56′52″ 8919 US-17 G. aridium D4 Oaxaca 337 16°34′52″ 94°48′59″ 26920 US-41 G. aridium D4 Oaxaca 339 16°22′32″ 95°23′06″ 20021 US-12 G. aridium D4 Oaxaca 337 16°06′45″ 95°19′13″ 14322 US-15 G. aridium D4 Oaxaca 349 16°02′50″ 95°40′24″ 12523 US-13 G. aridium D4 Oaxaca 330 15°59′02″ 95°31′11″ 40024 US-86 G. lobatum D7 Michoacán 283 19°04′44″ 102°04′01″ 37425 US-84 G. schwendimanii D11 Michoacán 277 18°21′39″ 101°53′58″ 34026 US-65 G. laxum D9 Guerrero 307 17°48′41″ 99°33′40″ 70827 US-66 G. laxum D9 Guerrero 307 17°53′51″ 99°34′46″ 57528 US-67 G. laxum D9 Guerrero 312 17°55′22″ 99°37′02″ 56029 US-68 G. laxum D9 Guerrero 309 17°57′48″ 99°31′59″ 72430 US-70 G. laxum D9 Guerrero 315 17°54′38″ 99°22′55″ 81031 US-72 Gossypium sp. nov. Dx Guerrero 314 17°57′58″ 98°59′07″ 64232 US-43 G. gossypioides D6 Oaxaca 211 16°31′28″ 95°55′43″ 113033 US-46 G. gossypioides D6 Oaxaca 217 16°41′57″ 96°11′29″ 1050

aThe original geographical information from HC and DJD accessions were not available. The GPS locations in Table 1 for these accessions are the nearestknown city from where these accessions were taken or are based on similar locations where J. Stewart and M. Ulloa collected germplasm during 2004–2006explorations in Mexico (unpublished data).

bJalisco upland ecotype.cJalisco coastal ecotype.

Feng et al. 617


for 10 min. The aqueous layers were transferred to newtubes and mixed with two volumes of cold (4 °C) absoluteethanol to precipitate DNA. The DNA pellets were trans-ferred to new tubes with pipette tips, soaked in 70% ethanolfor 2 h, air dried, and then dissolved in 200 µL ddH2O. TheDNA was quantified spectrophotometrically (Hitachi U-2000,East Lyme, Conn.) and diluted to 20 or 100 ng/µL with waterfor RAPD or AFLP analysis, respectively.

RAPD and AFLP analysisRAPD analysis was conducted with twenty-six 10-mer ran-

dom primers (The University of British Columbia: UBC 1–9,12, 13, 301, 302, 304–310, 317–320, 601, 603) in a 25 µLaqueous volume for PCR, which was performed as follows:94 °C for 2 min, followed by 40 cycles of 94 °C for 30 s,40 °C for 30 s, and 72 °C for 45 s, with a final 5 min exten-sion at 72 °C (MJ Research PTC thermal cycler, Hercules,Calif.). PCR products were separated by electrophoresisthrough a 1% agarose gel containing 100 mg ethidium bro-mide per 100 mL of gel. The image of the resolved gel underUV light was recorded with a Bio-Rad Gel Doc 2000 System(Bio-Rad Laboratories, Hercules, Calif.).An AFLP Analysis System I kit (Invitrogen, Carlsbad,

Calif.) was used to generate the AFLP fragments following themanufacturer’s manual, with some modification. For each sam-ple, 125 ng of genomic DNA was digested with EcoRI andMseI at 37 °C for 2 h in 2.5 µL (0.5 µL of 5× Reaction buffer,1.25 µL of 100 ng/µL DNA, 0.25 µL of EcoRI–MseI, and0.5 µL ddH2O). The restriction enzymes were denatured byheating the tubes at 70 °C for 10 min. To ligate adapters tothe ends of the digested DNA, 2.6 µL of 2× ligation bufferand 0.1 µL T4 ligase were added to each tube. The tubes wereincubated at 20 °C for 2 h and then diluted 10-fold with TEbuffer. The preamplification reactions were run for 20 cyclesat 94 °C for 30 s, 56 °C for 60 s, and 72 °C for 60 s in a totalvolume of 10.5 µL containing 1.35 µL ligated DNA, 8 µLpreamplification primer mixture, 1.05 µL of 10× Taq poly-merase buffer with Mg2+, and 0.1 µL of 5 U/µL Taq DNApolymerase (Promega Corporation, Madison, Wis.). The pre-amplification products were diluted 50-fold and used as theselective amplification templates. The selective amplificationreactions were conducted in 8 µL consisting of 3.30 µLddH2O, 0.8 µL of 10× polymerase buffer, 0.06 µL EcoRIprimer, 1.8 µL MseI primer, 0.04 µL of 5 U/µL TaqDNA polymerase (Promega Corporation), and 2 µL preampli-fication templates. Selective amplification conditions were94 °C for 30 s, 65 °C for 30 s, and 72 °C for 45 s with touch-down at –0.7 °C per cycle for 12 cycles, and then 94 °C for30 s, 56 °C for 30 s, and 72 °C for 45 s for 28 cycles. Sixteenprimer combinations (Table 2) were used in the selective am-plification reactions. For each set of primer combinations theamplified products were separated electrophoretically in a 6%denaturing polyacrylamide gel that was then stained with sil-ver for visualization (Promega Corporation 2004).Because high diversity was observed in US-72 and the two

G. gossypioides accessions (US-43 and US-46), a new set ofpreamplifications for these three samples was made fromDNA and compared with the previous preamplifications usingfive primer combinations. The AFLP profiles of each genotypewere identical when amplified with the same primer combina-tion. Then two AFLP primer combinations (E-AAC–M-CTG

and E-AGG–M-CTT) and two 10-mer RAPD primers (301and 302) were used in two replicate amplifications for the33 genotypes. For each primer or primer combination, thestrong RAPD fragments and the clear AFLP fragments in the100–1000 bp size range of each accession were highly repro-ducible from two replicate amplifications. Thus strong RAPDfragments and clear AFLP fragments ranging in size from 100to 1000 bp were scored for further data analysis.

Data analysisThe fragments were scored visually from agarose gels with

ethidium bromide (RAPDs) and silver-stained polyacrylamidegel plates (AFLPs). Presence or absence of a PCR productwas recorded as 1 or 0, respectively. The Numerical Taxon-omy and Multivariate Analysis System (NTSYS-pc) version2.1 (Rohlf 2002) was used to calculate the genetic distances(Nei 1972) and to analyze phylogenetic relationships of arbor-escent Gossypium species by the unweighted pair groupmethod with arithmetic mean (Saitou and Nei 1987) clusteringanalysis and principal component analysis. Computations forthe statistical analysis of the data were performed with Phylo-genetic Analysis Using Parsimony (PAUP* 4.0 beta) (Swof-ford 1998) to compare results generated by different methods(parsimony, neighbor joining, and unweighted pair groupmethod with arithmetic mean) and for bootstrapping (Felsen-stein 1985). Similar results were observed when RAPD orAFLP molecular data were analyzed separately. Herein, wepresent the combined RAPD and AFLP molecular data.

Results

RAPD and AFLP fragment analysesA total of 976 fragments were generated by RAPD and

AFLP methodologies; 90% of these fragments were polymor-phic, indicating abundant diversity among the 33 arborescent

Table 2. EcoRI and MseI primer terminal selectivenucleotides used to detect amplified fragment lengthpolymorphism among 33 Gossypium accessions.

No. EcoRIa MseIb

1 -AAC-3′ -CTC-3′2 -AAC-3′ -CTT-3′3 -AAC-3′ -CTG-3′4 -AGG-3′ -CAC-3′5 -AGG-3′ -CAA-3′6 -AGG-3′ -CTT-3′7 -AGG-3′ -CTG-3′8 -ACC-3′ -CAA-3′9 -ACT-3′ -CAC-3′10 -ACT-3′ -CAA-3′11 -ACT-3′ -CTT-3′12 -ACT-3′ -CTG-3′13 -ACA-3′ -CAC-3′14 -ACA-3′ -CAA-3′15 -ACA-3′ -CTT-3′16 -ACA-3′ -CTG-3′Note: The 3-nt sequences represent the selective nucleo-

tides for EcoRI and MseI primers, respectively.aThe sequence of the EcoRI primers was 5′-GACTGCG-

TACCAATTCA+XXX-3′.bThe sequence of the MseI primers was 5′-GATGAGTC-

CTGAGTAAC+XXX-3′.

618 Botany, Vol. 89, 2011


Gossypium accessions (Table 3). Since a fragment is ampli-fied from one specific location of the genome, we assumedthat one fragment equaled one locus. Ninety-four fragments(<10% of the loci) were common to all entries (i.e., mono-morphic for section Erioxylum, excluding G. raimondii,which was not examined). The percentage of polymorphicfragments among the accessions was high (>90%), and eachaccession contained about one third of the total loci exam-ined. The distribution of allelic frequencies revealed the plen-tiful diversity among all accessions and separately among all23 taxa designated G. aridum (Fig. 1). Among all accessions,332 fragments (34% of loci) were rare, with a frequencylower than 10%, while over 50% of the loci had a frequencylower than 30%. A total of 292 fragments were unique to aspecies, or in the case of G. aridum, to an ecotype. Amongthe 23 G. aridum accessions, 717 fragments were amplified,of which 536 (75%) were polymorphic within the species.The mean number of polymorphic fragments amplified in

an accession was 306, with a range of 211–349 (Table 1). In-terestingly, the monospecific subsection Selera, representedby G. gossypioides, yielded the lowest number of polymor-phic fragments among all taxa. Within a taxonomic speciesgroup, or ecotype in the case of G. aridum, the number ofpolymorphic fragments from each accession was similar. Theseven accessions of the Oaxaca ecotype had the highest num-bers of polymorphic fragments.

Many of the fragments were specific to a species or eco-type. Subsection Selera contained nearly three times as manysynapomorphic loci as subsection Erioxylum (99 vs. 36; Ta-ble 4) among the 211–217 loci that were polymorphic be-tween these two subsections of Erioxylum (Table 1). Thetwo subsections are tethered by the fact that nearly one thirdof all fragments (31%) amplified from G. gossypioides weremonomorphic among all representatives of both subsections.The number of synapomorphic fragments varied among

the species G. laxum, G. lobatum, G. schwendimanii, the pur-ported new species (US-72) (Feng et al. 2003; Ulloa et al.2006), and the G. aridum accessions representing the eco-types from Colima and Oaxaca. Accession US-72 was nota-ble in that it yielded the highest number of synapomorphicfragments (59). In addition, 11 fragments were uniquely ab-sent from this taxon. Among those accessions recognized asG. aridum under current circumscription (Fryxell 1992),grouping them by their geographical origin (or ecotype) wasinformative. The G. aridum accessions from Colima yielded31 synapomorphic fragments, while the accessions of thisspecies from Oaxaca gave 12 such fragments (Table 4).

Genetic diversity among arborescent Gossypium speciesThe mean genetic distances among the arborescent Gos-

sypium species clearly showed the relatedness of these spe-cies (Table 5). The two G. gossypioides accessions

Table 3. Number of scored fragments generated by two marker systems (randomly amplified polymorphic DNA (RAPD) andamplified fragment length polymorphism (AFLP)) used to assess the genetic relatedness of 33 accessions of arborescentGossypium.

Fragments

Fragment typeNo. primers orcombinations Mean no./primer

No. ofmonomorphic

No. ofpolymorphic % polymorphic Total no.

RAPD 26 8 (4–16)a 38 172 82 210AFLP 16 48 (23–86)a 60 706 92 766Total no. loci 98 878 90 976

aRange in the number of fragments appears in parenthesis.

Fig. 1. Allelic frequency among 33 arborescent Gossypium accessions and among 23 Gossypium aridum accessions.

Feng et al. 619


(subsection Selera) were quite similar to each other, with agenetic distance of only 0.01, but they were genetically dis-tant from species of subsection Erioxylum (range 0.64–0.84).The proposed taxon (US-72) was genetically distant from allspecies examined. The smallest genetic distance between US-72 and any other arborescent species was 0.42 with G. laxum.Gossypium schwendimanii was genetically closer to G. laxum(0.12 to 0.16) than to other taxa. The five G. laxum acces-sions were quite similar, with very little diversity within thetaxon (genetic distance range 0.02–0.04). The genetic distan-ces between G. laxum accessions and G. aridum accessionsranged from 0.32 to 0.48 depending on the G. aridum eco-type. The genetic distances among accessions of G. aridum,as currently circumscribed, were greater than within anyother group (range, 0.02–0.54).Because of the extensive genetic diversity found among

the 23 G. aridum accessions, these were analyzed separately.These accessions were collected from seven Mexican statesand clustered into distinct ecotypes that, for the most part, re-flected their origin (Fig. 2). The genetic distances among ac-cessions within states and between states supported theclustering results (Table 6). The accessions from Colimawere the most divergent of these taxa and appear to representa distinct lineage. The second most divergent group consistedof the accessions originating in Oaxaca. While genetic diver-sity among the Oaxaca accessions was low, this group wasdistinct from the other accessions of G. aridum, particularlythose from Colima and Sinaloa. The genetic diversity among

the G. aridum populations from Jalisco was related to thesites of origin of the accessions. DJD172 and DJD179 werecollected from coastal foothills, whereas DJD185 was col-lected at an upland site with an elevation of approximately1000 m on the rim of the canyon of Rio Grande de Santiago.Likewise, the accessions from Guerrero and Michoacán rep-resented coastal accessions as well as accessions that werefrom inland areas sympatric with G. lobatum, and especiallywith G. schwendimanii. In these cases, molecular diversityreflected the differences in habitats of the various accessions.Some of the accessions (e.g., US-81, US-83) displayed mor-phological features in the capsules that were similar to thoseof G. schwendimanii (J. Stewart, personal observation),which may be reflective of introgression and could accountfor their distinctive positions in the cladogram (Fig. 2).

Phylogenic relationships of arborescent GossypiumIdentical or very similar consensus phylogenetic trees of

the arborescent Gossypium accessions were generated byNTSYS 2.1 and PAUP* 4.0 utilizing different methods, in-cluding neighbor joining, unweighted pair group methodwith arithmetic mean, and parsimony. Only a dendrogrambased on the most parsimonious branch assembly with boot-strap values is presented (Fig. 2). Subsection Selera acces-sions were clearly divergent from the other taxa and were setas an outgroup for subsection Erioxylum. Within subsectionErioxylum, G. schwendimanii formed a clade with G. laxumwith strong bootstrap support. Separate clades for G. lobatum

Table 4. Selected Gossypium taxa with numerous randomly amplified polymorphic DNA and amplified frag-ment length polymorphism alleles uniquely present and absent.

Fragments

Acc.No. Accession ID

Total no.present

Uniquely present inall representativesa

Uniquely absent fromall representativesb

All Section Erioxylum 976 94 —32–33 Subsection Selera (D6) 99 361–31 Subsection Erioxylum 36 991–23 D4 (G. aridum) 3 06–7 D4 (G. aridum) (Colima) 31 317–23 D4 (G. aridum) (Oaxaca) 12 024 D7 (G. lobatum) 26 426–30 D9 (G. laxum) 18 025 D11 (G. schwendimanii) 12 031 Dx (Gossypium sp. nov. US-72) 59 11

aFragments can only be found in a given ecotype, but not in other ecotypes.bFragments can be found in all except one ecotype.

Table 5. Ranges in genetic distance coefficients among the species represented in 33 acces-sions of Gossypium section Erioxylum.

D4 US-72 D7 D9 D11 D6

D4 0.02–0.54US-72 0.43–0.51D7 0.26–0.39 0.49D9 0.32–0.50 0.42–0.43 0.33–0.35 0.02–0.04D11 0.32–0.48 0.48 0.32 0.12–0.16D6 0.72–0.82 0.83–0.84 0.71–0.73 0.67–0.71 0.64–0.65 0.01

Note: Genome designations: D4, G. aridum; D6, G. gossypioides; D7, G. lobatum; D9, G. laxum; D11,G. schwendimanii; US-72, Gossypium sp. nov. A single number indicates the genetic distance betweentwo accessions.

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and all G. aridum accessions had moderate bootstrap support,while a separate clade of US-72 from all other species ofsubsection Erioxylum had strong bootstrap support. The Col-ima accessions formed a clade separate from other G. aridumaccessions with moderate bootstrap support. The remainingG. aridum accessions separated into three subgroups. Onesubgroup contained the Oaxaca accessions plus one unusual

accession (US-81) from Guerrero that morphologically ap-peared intermediate between G. aridum and G. schwendimanii(M. Ulloa and J. Stewart, personal observations); a secondclade consisted of accessions from Sinaloa, Jalisco, and Pue-bla; and the third consisted of the coastal accessions of Guer-rero and Michoacán (Fig. 2).The phylogenetic relationships among the 33 arborescent

Fig. 2. Dendrogram based on the most parsimonious branch assembly of 33 arborescent Gossypium accessions using 972 randomly amplifiedpolymorphic DNA and amplified fragment length polymorphism fragments. Arborescent Gossypium examined: D4, G. aridum; D6, G. gossy-pioides; D7, G. lobatum; D9, G. laxum; D11, G. schwendimanii; D12, Gossypium sp. nov. Bootstrap values of 50% or more are presented onbranching points.

Table 6. Ranges in genetic distances among G. aridum accessions collected from different states of Mexico.

Colima Jalisco Sinaloa Guerrero Puebla Mich. OaxacaColima 0.08a

Jalisco 0.28–0.40 0.10–0.23Sinaloa 0.35–0.38 0.22–0.26 0.05Guerrero 0.30–0.37 0.14–0.32 0.19–0.30 0.07–0.24Puebla 0.29–0.34 0.15–0.21 0.23–0.25 0.15–0.28 0.04Mich. 0.30–0.34 0.14–0.25 0.23–0.27 0.09–0.23 0.16–0.19 0.19Oaxaca 0.35–0.41 0.23–0.33 0.32–0.34 0.10–0.30 0.25–0.28 0.19–0.26 0.02–0.05

aThe single number is the genetic distance between two accessions.

Feng et al. 621


Gossypium accessions were also demonstrated by principalcomponents analysis of genetic distances, visualized by athree-dimensional plot of the genotypes based on componentscores (Fig. 3). The first principal component accounted for56% of the total variation. Gossypium gossypioides acces-sions (US-43 and US-46) were separated from subsectionErioxylum species mainly by this component. Also, US-72was partially separated from the other accessions by thiscomponent. The second principal component described 10%of the total variation. Gossypium laxum (US-65–US-70),G. schwendimanii (US-84), US-72, G. lobatum (US-86), andthe Colima accessions (DJD168-1 and DJD168-2) were wellseparated from other G. aridum accessions by this compo-nent. The third principal component represented 6% of thetotal variation by which G. aridum accessions were split intotwo groups; one group consisted of Oaxaca accessions (US-10–US-17 and US-41), while the other group consisted ofG. aridum accessions from Jalisco (DJD172, DJD179,DJD185), Puebla (US-4 and US-5), and Sinaloa (HC4 andHC10). The accessions of G. aridum from Guerrero (US-76–US-81 and DJD123) and Michoacán (US-83 and DJD123)were intermediate to these two groups.

DiscussionMolecular techniques have proved to be powerful tools in

the assessment of genetic variation and in the elucidation ofgenetic relationships between species. Conflicts have arisenwhen phylogenetic relationships were based on sequence dif-

ferences within single genes (Wendel et al. 1995; Liu et al.2001). In this study, we used RAPD and AFLP markers todetermine the genetic diversity and phylogenetic relationshipsamong 33 accessions of arborescent Gossypium, including 23G. aridum accessions endemic to Mexico. DNA fragmentsamplified by RAPD and AFLP techniques are mostly distrib-uted randomly throughout the genome, and close to 1000fragments were used herein for the estimation of genetic di-versity. Hence, the RAPD and AFLP markers detectedamong the accessions examined provide a robust measure-ment of the diversity among these taxa. Most of our molecu-lar data support the traditional classification of the knownGossypium species and distinguish the geographical ecotypesof G. aridum accessions. In addition, a newly collected ac-cession (US-72) from the subsection Erioxylum was recog-nized as a new species. However, these results also indicatethat the subsection Erioxylum deserves additional study todifferentiate various taxa and to resolve genetically distantgeographical ecotypes.Genetic diversity can be measured in three different ways

with molecular markers. First, it is measured as the percent-age of polymorphic fragments; second, as the allelic fre-quency among all entries; and third, as the percentage of theloci present in each entry. In this investigation, over 90% ofthe 972 fragments examined were polymorphic and, of these,more than 75% of the loci among all accessions and twothirds of the loci among the 23 G. aridum accessions had al-lele frequencies less than 80%. The percentage of the totalloci found in any one accession ranged between 31% and

Fig. 3. Phylogenetic relationship among 33 arborescent Gossypium accessions visualized by principal components analysis of genetic distancesbased on randomly amplified polymorphic DNA and amplified fragment length polymorphism analysis (see Table 1 for the description of ac-cessions). Subsection Selera is separated from subsection Erioxylum by the first principal component. Species of subsection Erioxylum are se-parated mainly by the second principal component. The G. aridum accessions were separated by the second and third principal components.

622 Botany, Vol. 89, 2011


45%. All of these observations indicate a very diverse geneticbase among the 33 arborescent Gossypium accessions andalso among those accessions classified as G. aridum.Álvarez and Wendel (2006) found extensive variation

among populations of G. aridum, and accessions originatingin Colima were especially distinct from those originating atother locations. They presented evidence that the G. aridumfrom Colima experienced a hybridization event with a Gos-sypium taxon related to section Integrifolia that resulted inacquisition of the donor cytoplasm, as well as some nucleargenetic material. The results presented herein support the dis-tinctiveness of the Colima accessions from other G. aridumaccessions. We did not determine how much of the polymor-phism could be attributed to the unique cytoplasm or to thenuclear diversity.Numerous synapomorphic fragments (26 in G. lobatum

(US-86), 17 in G. laxum accessions, and 12 in G. schwendi-manii (US-84)) were detected among the recognized species.As stated previously, the most synapomorphic fragmentswere found in US-72. Some fragments were present in onlytwo or three species, or ecotypes, especially when US-72was involved. However, there were also fragments specific totwo or several accessions but absent in US-72. For example,15 fragments were unique to G. laxum and G. schwendinma-nii, 10 fragments were unique to US-81 and seven Oaxacaaccessions, and 3 fragments were specific to G. laxum andseven Oaxaca accessions. This was a surprising result, sinceG. schwendimanii and G. lobatum are sympatric in part oftheir range and are thought to form hybrids in their nativehabitat (note on voucher specimen for DeJoode and CalzadaNo. 146, Mexican National Herbarium). However, these twospecies did not share many specific fragments, and the ge-netic distance between them was not low. It is unknownwhether this is the result of different speciation times forthese two species. Gossypium laxum and G. schwendimaniiare not known to be sympatric in any part of their distribu-tion range.Existing geopolitical boundaries can incorporate more than

one ecotype of G. aridum. For example, accession DJD185was collected at a relatively high altitude northeast of Guada-lajara in the interior of Jalisco, while accessions DJD172 andDJD179 were collected near the Pacific coast. The geneticdistance between the upland and coastal accessions from Ja-lisco was more than twice that between the two coastal acces-sions. The upland ecotype formed a clade with accessionscollected from the northern parts of the G. aridum range (Si-naloa), while the coastal ecotypes formed a separate clade(Fig. 2).Álvarez and Wendel (2006) reported that G. aridum from

Colima possesses a cytoplasm donated from a taxon relatedto section Integrifolia. The results of this investigation sup-port the reported distinctiveness of the G. aridum of Colima.As stated previously, the two accessions from that state had31 synapomorphic fragments and formed a clade that sepa-rated them from all other taxa in subsection Erioxylum. Inspite of the molecular distinctiveness of the Colima acces-sions, Álvarez and Wendel (2006) have maintained the spe-cific epithet of these as G. aridum.Gossypium lobatum and G. schwendimanii are two mor-

phologically distinct species (Fryxell 1992) in which the ge-netic distance was determined to be 0.32. Considering the

genetic distances (0.12–0.16) between G. schwendimanii andanother distinct species, G. laxum, 0.32 can be regarded as aconservative estimate of genetic distance between two arbor-escent Gossypium species. The genetic distances between ac-cession US-72 and all other accessions were greater than0.32, thus supporting the conjecture that US-72 is an unde-scribed species. The conjecture is also supported by the factthat 59 fragments were synapomorphic in US-72, while 11fragments were uniquely absent from this accession. Also,the dendrogram (Fig. 2) that places US-72 basal to the fourcurrently recognized species (G. aridum, G. lobatum,G. laxum, and G. schwendimanii), and the clustering resultthat places it intermediate to these taxa, suggest that it maybe ancestral to the subsection.The mean genetic distances between the G. aridum acces-

sions from Colima and accessions from other Mexican statesranged from 0.32 to 0.38, that is, they were equal to orgreater than the genetic distances between two recognizedspecies. The genetic distance between Oaxaca accessionsand Sinaloa accessions (from the type locale of G. aridum)was 0.33, which was also greater than the maximum 0.32 be-tween the four well-recognized species. These data suggestthat the taxonomic treatment of subsection Erioxylum is in-complete. Based on the results reported here, additional com-parisons of morphological and cytogenetic characteristicsamong the taxa of subsection Erioxylum are justified to es-tablish a defensible taxonomic treatment of the various taxa.

AcknowledgementsThe authors would like to offer their appreciation and

heartfelt thanks to the many individuals, too numerous toname, residing in the various states who contributed invalu-able service, guidance, and help without which the collectionof some of these accessions would have been much less fruit-ful. This study was partially supported by a specific coopera-tive agreement between the USDA Agricultural ResearchService and the Mexican agency INIFAP (ARIS Log Nos.530-21220-001-10S and 5303-2-F159). Mention of tradenames or commercial products in this article is solely for thepurpose of providing specific information and does not implyrecommendation or endorsement by the US Department ofAgriculture. The US Department of Agriculture is an equalopportunity provider and employer.

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Distribution and molecular diversity of arborescent Gossypium species