P1. Syst. Evol. 252: 199-214 (2005) Plant Systematics DO1 SO. 1007/~00606-004-0294-0 and Evo1utiua Printed in Austria Phylogeny of the New World diploid cottons (Gossypium L., Malvaceae) based on sequences of three low-copy nuclear genes I. ~lvarez',R. cronn2, and J. F. wende13 '~ealJardin Botiinico de Madrid, CSIC, Madrid, Spain 2 Pacific Northwest Research Station, USDA Forest Service, Corvallis, Oregon, USA 3 Department of Ecology, Evolution, and Organismal Biology, Iowa State University, Ames, Iowa, USA Received October 4, 2004; accepted December 15, 2004 Published online: May 9, 2005 O Springer-Verlag 2005 Abstract. American diploid cottons (Gossypium L., Introduction subgenus Houzingenia Fryxell) form a monophy- letic group of 13 species distributed mainly in New World, diploid Gossypium species com- western Mexico, extending into Arizona, Baja prise a morphological and cytogenetic California, and with one disjunct species each in (D-genome) assemblage (Cronn et al. 2002, the Galapagos Islands and Peru. Prior phylogenetic Endrizzi et al. 1985, Wendel 1995, Wendel and analyses based on an alcohol dehydrogenase gene Cronn 2003) that taxonomically is recognized (AdhA) and nuclear ribosomal DNA indicated the as subgenus Houzingenia (Fryxell 1969, 1979, need for additional data from other molecular 1992). This group of plants includes 11 species markers to resolve phylogenetic relationships with- distributed primarily in SW Mexico and in this subgenus. Toward this end, we sequenced extending northward into Arizona, in addition three nuclear genes, the anonymous locus A1341, to two other species with disjunct distributions an alcohol dehydrogenase gene (AdhC), and a cellulose synthase gene (CesA 1b). Independent and in Peru and the Galapagos Islands (Fryxell combined analyses resolved clades that are congru- 1992). Although none of these species pro- ent with current taxonomy and previous phyloge- duces commercially important cotton fiber, the nies. Our analyses diagnose at least two long fact that one of the parental lineages of distance dispersal events from the Mexican main- allotetraploid cultivated cotton (G. hirsutum land to Baja California, following a rapid radiation L. and G. barbadense L.) belongs to this group of the primary lineages early in the diversification (Cronn et al. 1999, Endrizzi et al. 1985, Small of the subgenus. Molecular data support the et al. 1998, Small and Wendel 2000a) gives proposed recognition of a new species closely special relevance to the understanding of their related to Gossypium laxurn that was recently systematics and evolutionary relationships. collected in Mexico. Numerous molecular phylogenetic analyses have demonstrated that the subgenus is mono- Key words: Gossypium, Houzingenia, cotton, phy- phyletic (reviewed in Wendel and Cronn 2003). logeny, low-copy nuclear genes, alcohol dehy- However, while the circumscription of the drogenase, cellulose synthase. subgenus and species boundaries within this 200 I. Alvarez et al.: Phylogeny of Gossypium subgenus Houzingenia clade are reasonably well-understood (Fryxell loci can be resolved for Adh in Gossypium 1979, 1992), phylogenetic relationships among (Wendel, unpublished), the gene family is species remain unclear, despite numerous stud- much larger, including up to seven discrete ies (Cronn et al. 1996, DeJoode 1992, Fryxell loci in some diploid cotton species (Small and 1971, Liu et al. 2001, Seelanan et al. 1997, Wendel 2000a). One of these loci, AdhA, was Small and Wendel 2000b, Wendel 1995, Wen- selected for phylogenetic applications (Small del and Albert 1992). Phylogenetic analysis and Wendel 2000b) based on its homologous based on chloroplast restriction site analysis chromosome location in three genetic maps and chloroplast DNA sequences (Cronn et al. including diploid and allotetraploid cottons 2003, DeJoode 1992, Seelanan et al. 1997, (Brubaker et al. 1999), and on the results of Wendel and Albert 1992) have led to a number Southern hybridization analyses (Cronn et al. of phylogenetic conclusions that are at odds 1999) that indicate the existence of only one with numerous, unlinked nuclear markers and copy per genome in two species of the subge- morphological trends in the genus. Results nus (G. raimondii Ulbrich and G. trilobum from nuclear ribosomal sequences (Cronn (DC.) Skovsted). The phylogeny based on et al. 1996, Seelanan et al. 1997) have provided AdhA (Small and Wendel 2000b) supports equally controversial resolutions (e.g. Wendel the taxonomically recognized subsections and et al. 1995a,b), and resolution of these closely is generally congruent with previous analyses related species appears hampered by the pres- in the subgenus (Cronn et al. 1996, DeJoode ence of intraindividual polymorphism, some of 1992, Seelanan et al. 1997, Wendel and Albert which appears trans-specific. 1992), although relationships among sections The use of low-copy nuclear genes to infer and subsections remained unresolved. plant phylogenies is rapidly increasing, due in More recently, Cronn et al. (2003) used part to the recent accessibility of many nuclear four chloroplast genes and eight low-copy genes (characterized in gene discovery and nuclear genes to reevaluate the evolutionary genome sequencing projects) and their higher history of G. gossypioides (Ulbrich) Standley. resolution, as has been demonstrated for Among the nuclear genes were two alcohol various groups (reviewed in Sang 2002, Small dehydrogenase genes (AdhA, AdhC), two cel- et al. 2004). In Gossypium, the characterization lulose synthase genes (CesAl, CesAl b), a fatty of numerous low-copy nuclear genes (Cronn acid desaturase intron (FAD2-1 intron), and et al. 2002, Cronn et al. 1999, Senchina et al. the anonymous genes A1341, G1121, and 2003, Small and Wendel 2000a) has yielded a G1262. In this study, six of the 13 D-genome wealth of nuclear gene markers for studying species were included. The individual molecu- Gossypium (Cronn et al. 2002, Small et al. lar markers used in this study revealed levels of 2004, Small et al. 1998, Small and Wendel variation that provided modest resolution of 2000b) and related genera (Wendel et al. New World species; however, results from this 2002). To date, these markers have been study indicated that combining the most applied primarily to just a few representatives informative genes might have the potential to of subgenus Houzingenia, as studies to date resolve phylogenetic relationships among all have focused on higher-level phylogenetic 13 species in the subgenus. With this objective relationships (Cronn et al. 2002, 2003; Wendel in mind, we sequenced the three most infor- et al. 2002). mative nuclear genes (A1341, CesAlb, and The most comprehensive molecular sys- AdhC) that showed variation at different levels tematic study of New World cottons to date (Cronn et al. 2002, Seelanan et al. 1999, Sen- was performed by Small and Wendel (2000b) china et al. 2003, Small et al. 1998) from using a member of the Adh gene family in representatives of all species. To these data Gossypium (Cronn et al. 1999, Small and we added previously generated sequences of Wendel 2000a). While two to three allozyme the AdhA gene (Small and Wendel 2000b). I. ~lvarezet al.: Phylogeny of Gossypium subgenus Houzingenia 20 1 Insights from these four nuclear genes are and CesAlb) as molecular markers. This selection compared to results obtained with nuclear was based on copy number of each gene (inferred ribosomal DNA, and the most recent taxo- to be single-copy from earlier work) and on the nomic treatment of Fryxell (1992) is evaluated knowledge of their orthology across different in light of these combined results. genomes in cotton (Brubaker et al. 1999, Cronn and Wendel 1998, Small and Wendel 2000a). Additionally, we selected genes that in previous Materials and methods analyses (Cronn et al. 2002) showed a relatively high ratio of phylogenetically informative sites (PI) Plant materials. Sampling included the 13 Ameri- compared to other low-copy nuclear genes. The can diploid cottons: Gossypium aridum (Rose & A1341 locus (0.7 kb) is an anonymous gene that Standley) Skovsted, G. armourianum Kearny, corresponds to a PstI mapping probe (Brubaker G. davidsonii Kellogg, G. gossypioides (Ulbrich) and Wendel 1994, Cronn and Wendel 1998, Cronn Standley, G. harknessii Brandegee, G. klotzschianum et al. 1999); this gene has a PI ratio slightly higher Andersson, G. laxum Phillips, G. lobatum Gentry, G. than the AdhA gene used in the previous phyloge- raimondii Ulbrich, G. schwendimanii Fryxell & netic analysis of the subgenus (Small and Wendel Koch, G. thurberi Todaro, G. trilobum, and 2000b). A region of the CesAl b gene (Cronn et al. G. turneri Fryxell. Besides, we included one specimen 2002, Cronn et al. 1999) has a similar PI ratio (Gossypium sp.) that is suggested to be a new species to plastid genes, although this low ratio is related to G. aridum (Ulloa et al., unpublished). In compensated by its length (1.15 kb). From the some cases, more than one accession per species was Adh gene family, we sequenced a portion (0.94 kb) sampled, based on our assessments of variability of the AdhC gene that has a high PI ratio similar to within each species (some species are narrowly some chloroplast spacers, and higher than other distributed and relatively invariable morphologically nuclear genes in a previous analysis (Cronn et al. and with respect to allozyme markers, whereas 2002). others are more widespread and/or exhibit greater Amplification primers were those used previ- variation). Six species that belong to different ously (Cronn et al. 2002), namely A1 341 F and cytogenetic and taxonomic groups: G. anomalum A1 341 R for the A1341 locus, CelAF and CelAR Wawra ex Wawra & Peyritch, G. bickii Prokhanov, for the CesAlb partial gene, and ADHx4-3 and G. longicalyx J. B. Hutchinson & Lee, G. robinsonii ADH-P2 for the AdhC partial gene. For accessions F. von Mueller, and Gossypioides kirkii (Mast.) J. of G. lobatum, a new set of internal forward and B. Hutchinson and Kokia drynarioides (Seemann) reverse primers (GTG AGG CTT CTA GGA TCA Lewton were included as an outgroup (Table I).