Genetic characterization of three varieties of Astragalus lentiginosus (Fabaceae) BRIANJ. KNAus,' RICHC. CRONN,AND AARONLISTON I Knaus, B. J. (Oregon State University, Department of Botany and Plant Pa- thology, 2082 Cordley Hall, Corvallis, OR, 97331-2902, U.S.A.; e-mail: [email protected]), R. C. Cronn (USDA Forest Service Pacific Northwest Research Station, 3200 SW Jefferson Way, Corvallis, OR, 97331, U.S .A.; e-mail: [email protected]) & A. Liston (Oregon State University, Depart- ment of Botany and Plant Pathology, 2082 Cordley Hall, Corvallis, OR, 97331- 2902, U.S.A.; e-mail: [email protected]).Genetic characterization of three varieties of Astragalus lentiginosus (Fabaceae). Brittonia 57: 334-344. 2005.-Astragalus lentiginosus is a polymorphic species that occurs in geologi- cally young habitats and whose varietal circumscription implies active morpho- logical and genetic differentiation. In this preliminary study, we evaluate the po- tential of amplified fragment length polymorphism (AFLP) markers to resolve infraspecific taxa in three varieties of Astragalus lentiginosus. Distance-based principle coordinate and neighbor-joining analyses result in clustering of individ- uals that is congruent with population origin and varietal circumscription. Anal- ysis of molecular variance of two Oregon varieties demonstrates that varietal categories account for 11% of the total variance; in contrast, geographic proximity does not contribute to the total variance. AFLPs demonstrate an ability to dis- criminate varieties of A. lentiginosus despite a potentially confounding geographic pattern, and may prove effective at inferring relationships throughout the group. Key words: AFLP, amplified fragment length polymorphism, Astragalus lenti- ginosus, genetic differentiation, infraspecific taxa. Astragalus lentiginosus Dougl. ex Hook. populations and varieties may have oc- is a polymorphic species consisting of 40 curred as recently as the late Pleistocene1 varieties (Isely, 1998) distributed through- early Holocene period. This study of A. len- out the Intermountain and Desert Southwest tiginosus represents an investigation of a di- regions of North America. The current va- verse group whose circumscription reflects rietal circumscription reflects Barneby's dramatic morphological variation and im- (1945) view that A. lentiginosus comprises plies genetic differentiation. multiple lines of evolutionary radiation, as Jones (1923) provided the first revision well as occasional reticulation, both of of Astragalus during the twentieth century. which may explain the morphological in- Among his innovations was the reduction termediates between varieties. Many of the of the section Diphysi to a single species, habitats where extant populations occur Astragalus lentiginosus. He characterized (e.g., inland dune systems, desert seeps, this species as the "most variable of all As- mountain ridges) were profoundly different tragali" (Jones, 1923, p. 123). As defined during the Pleistocene (Grayson, 1993). Oc- by Jones (1923), this species consisted of currence on these geologically young hab- 18 varieties, many of which were originally itats suggests that the separation of current described as species. Within his varieties Jones included "forms" (never validly pub- lished) that had originally been recognized To whom correspondence should be addressed. as species or varieties. Brittonia, 57(4), 2005, pp. 334-344. ISSUED: 28 December 2005 O 2005, by The New York Botanical Garden Press, Bronx, NY 10458-5126 U.S.A. 20051 KNAUS ET AL.: ASTRAGALUS LENTIGINOSUS 335 Applying a very different taxonomic ap- 1976), but phylogenetic inference was lim- proach, Rydberg (1929) split Astragalus ited by the large size of the group and ap- into 28 genera (summarized by Barneby, parent homoplasy for this character. Molec- 1964). In doing so, he divided A. lentigi- ular phylogenetic studies of nuclear ribo- nosus between the genera Cystium (33 spe- somal DNA internal transcribed spacers cies, all currently known as A. lentiginosus) (Wojciechowski et al., 1993), chloroplast and Tium (39 species, only three of which DNA (Liston, 1992; Sanderson & Doyle, are currently known as A. lentiginosus). 1993), and combined nuclear and chloro- This revision resulted in most of Jones's plast DNA datasets (Wojciechowski et al., (1923) varieties (and forms) being raised to 1999) have demonstrated the monophyly of species (Barneby, 1945). the New World aneuploid species of As- Barneby's treatments (1945, 1964, 1989) tragalus. However, none of these studies reduced the group back to a single species, has resolved interspecific relationships Astragalus lentiginosus, with ca. 40 varie- within this clade. ties. This classification came from the re- Amplified fragment length polymor- duction of many of Rydberg's (1929) spe- phism (AFLP) analysis (Vos et al., 1995) is cies to varieties, or similarly elevating an anonymous genetic fingerprinting tech- many of Jones's (1923) "forms" to varie- nique developed for plant breeding that has ties. Since Barneby 's 1945 treatment there been adapted to studies of natural popula- has been a reduction in the original number tions (Wolfe & Liston, 1998; Mueller & of names accepted as varieties in part due Wolfenbarger, 1999). The AFLP method to additional collecting that has blurred has been used to infer interspecific (Abdel- some intervarietal distinctions (Barneby, fattah et al., 2002; Beardsley et al., 2003) 1964, 1989). This trend has been partially and intraspecific (Brouat et al., 2004; Juan offset by the description of new varieties et al., 2004; Travis et al., 1996) relation- since 1964 (e-g., Barneby, 1977; Welsh, ships as well as population level dynamics 1981). While the currently recognized num- (He et al., 2004). This method was applied ber of varieties is 40 (Isely, 1998) there is by Travis et al. (1996) to evaluate popula- still an active debate as to what constitutes tion-level differentiation of Astragalus A. lentiginosus (Alexander, 2005) as well as cremnophylax var. cremnophylax occurring to the validity of the varieties. at the Grand Canyon, U.S.A. Results from The varieties of Astragalus lentiginosus that study showed strong differentiation of are morphologically distinct when observed north and south rim populations, suggesting at distant stations, yet when geographically that AFLPs are sufficiently sensitive to dis- proximal their distinctiveness may become cern genetic differences in recently di- obscured to the point where they are indis- verged lineages, such as A. lentiginosus. tinguishable (Barneby, 1964, p. 922). Most In this preliminary study we utilize of the group is characterized by an inflated AFLP analysis to test Barneby's (1945, bilocular pod with a false septum that in- 1964, 1989) taxonomic treatment of three trudes from the abaxial surface and is in- Astragalus lentiginosus varieties. We focus complete in the beak. Barneby (1945, 1964, on the question of whether the sampled 1989) created major divisions within the populations of A. lentiginosus varieties dis- group based on flower size (keel greater or play genetic relationships that can be attri- less than 8.5 mm), raceme length (greater buted to geographic proximity, or whether or less than 4 cm), and flower color (purple the varieties exhibit genetic cohesiveness. or white). Attempts to evaluate taxonomic interpre- Methods tations within Astragalus utilizing molecu- lar methods have been complicated by low levels of divergence among the New World Plants included in this study were select- species. Chromosome number variation was ed to determine whether genetic differenti- identified as potentially useful at the infra- ation could be discerned across the latitu- generic level (Barneby, 1964; Spellenberg, dinal extent of Astragalus lentiginosus. 336 BRITTONIA [VOL. 57 TABLEI COLLECTIONSOF Astragalus lentiginosus Altitude Population N Habitat Latitude Longitude (m) State County lentiginosus-1 8 Juniper woodland 42.76 - 118.74 1915 OR Harney lentiginosus-2 8 Ponderosa pine forest 42.27 - 121.30 1490 OR Klarnath salinus-1 8 Sage shrubland 42.43 -1 18.08 1325 OR Harney salinus-2 7 Sage shrubland 43.32 -121.06 1318 OR Lake variabilis-1 8 Disturbed desert 34.57 -1 17.41 873 CA San Bemardino variabilis-2 7 Dune system 36.65 -116.57 744 NV Nye Leaves were collected during the summer the Columbia Basin to northern California of 2004 (Table I), tissue was desiccated in and the northwestern Great Basin. Barneby silica gel and stored at 4OC until DNA ex- (1945, 1964, 1989) placed this taxon among traction. Collections were made from six lo- the A. lentiginosus possessing short flowers cations (Fig. I), with two locations per va- (keel length < 8.5 mm), short racemes (axis riety. Geographic distances between sample < 4 cm long in fruit), and white flowers. locations were calculated with ArcView Astragalus lentiginosus var. lentiginosus is GIs 3.2 (Environmental Systems Research distinctive among the varieties of A. lenti- Institute, Inc.). ginosus in having moderately inflated pods Three morphologically distinct varieties with a coarse texture (thick walls), de- were included in this study. Astragalus Zen- scribed as stiffly papery, leathery, or woody tiginosus var. lentiginosus is a slender, de- (as opposed to thinly papery or membra- cumbent to prostrate perennial occurring in naceous). Ponderosa pine or juniper woodland from Astragalus lentiginosus var. salinus (Howell) Barneby is a short-lived perennial consisting of diffuse and ascending