Astragalus (): A molecular phylogenetic perspective

MARTIN F. WOJCIECHOWSKI

Wojciechowski, M. E (School of Life Sciences, Arizona State University, Tem- pe, AZ, 85287-4501, U.S.A.; e-mail: [email protected]). Astragalus (Fa- baceae): A molecular phylogenetic perspective. Brittonia 57: 382-396. 2005.-- Nucleotide sequences of the plastid mark gene and nuclear rDNA internal tran- scribed spacer region were sampled from Astragalus L. (Fabaceae), and its closest relatives within tribe , to infer phylogenetic relationships and estimate ages of diversification. Consistent with previous studies that emphasized sampling for nrDNA ITS primarily within either New World or Old World groups, Astragalus, with the exception of a few morphologically distinct species, is strongly supported as monophyletic based on maximum parsimony and Bayesian analyses of matK sequences as well as a combined sequence dataset. The matK data provides better resolution and stronger clade support for relationships among Astragalus and traditionally related genera than nrDNA ITS. Astragalus sensu stricto plus the genus Oxytropis are strongly supported as sister to a clade com- posed of strictly Old World (African, Australasian) genera such as Colutea, Suth- erlandia, Lessertia, Swainsona, and Carrnichaelia, plus several morphologically distinct segregates of Eurasian Astragalus. Ages of these clades and rates of nucleotide substitution estimated from a fossil-constrained, rate-smoothed, Bayes- ian analysis of matK sequences sampled from Hologalegina indicate Astragalus diverged from its sister group, Oxytropis, 12-16 Ma, with divergence of Neo- Astragalus beginning ca. 4.4 Ma. Estimates of absolute rates of nucleotide sub- stitution for Astragalus and sister groups, which range from 8.9 to 10.2 x 10 -~0 substitutions per site per year, are not unusual when compared to those estimated for other, mainly temperate groups of papilionoid legumes. The results of previ- ously published work and other recent developments on the phylogenetic rela- tionships and diversification of Astragalus are reviewed. Key words: Astragalus, diversification, Fabaceae, Neo-Astragalus.

The legume genus Astragalus L., with an spp.), and the Andes of South America (ca. estimated 2500 species of herbaceous peren- 100 spp.). The geographic center of diversity nial and annual species in the subfamily Pap- and presumed origin of Astragalus, like most ilionoideae of the Fabaceae (Mabberley, of its close relatives in the tribe Galegeae 1997; Lewis et al., 2005) and the largest of (Polhill, 1981a, 1981b), is Eurasia, and spe- 19 or so genera of angiosperms with more cifically the steppes and mountains of south- than 1000 species, is an example of an adap- western to south-central Asia and the Hima- tive radiation on a global scale. Astragalus is layan plateau. Of the two Galegean genera distributed mainly in cool to warm arid and with distributions in the New World, the other semiarid regions of the northern hemisphere, being Oxytropis DC. with ca. 330 spp. in the South America, tropical East Africa, and is northern hemisphere (Bobrov et al., 1972; especially diverse in southwestern and Sino- Welsh, 2001), only Astragalus has undergone Himalayan regions of Asia (ca. 1500-2000 extensive diversifications in both North and spp.), western (ca. 400-450 South America.

Brittonia, 57(4), 2005, pp. 382-396. ISSUED: 28 December 2005 2005, by The New York Botanical Garden Press, Bronx, NY 10458-5126 U.S.A. 2005] WOJCIECHOWSKI:MOLECULAR PHYLOGENETIC OF ASTRAGALUS 383

Given its huge species diversity and two current species of the Asian Sphaero- widespread distribution, it is hardly surpris- physa were originally included in the Lin- ing that no comprehensive monographic in- naean genus Phaca, but Gray (1864) and vestigation of Astragalus has been attempt- Bunge (1868, 1869) among others subse- ed since the taxonomic treatments of the quently treated these as a subgenus of As- late 19th century (Bunge, 1868, 1869; Taub- tragalus. While these taxa share a number ert, 1894). With the exception of Rydberg of important morphological characters, Bar- (1929), who recognized 28 genera within neby (1964; p. 1162) and Isely (1998) treat- North American Astragalus, in a revision ed this genus separate from Astragalus (lat- not supported by subsequent authors (Bar- er corroborated by molecular phylogenetic neby, 1964; Isely, 1998), and the segrega- analysis; Sanderson & Wojciechowski, tion of the Eurasian subgenus Tragacantha 1996). Since then, a number of other "mor- as Astracantha (Podlech, 1983, but later re- phologically isolated" Eurasian Astragalus versed by Zarre & Podlech, 1997), the cir- species have been segregated as monotypic cumscription of Astragalus remains rela- genera, largely on the basis of pod mor- tively unchanged since that proposed by phology (e.g., Barnebyella, Podlech, 1994; Bunge. During the 20 th century, taxonomic Ophiocarpus (Bunge) Ikonn.; Ikonnikov, work has been more floristic and regional 1977). Perhaps the most obvious example in scope, such as Johnston's (1938, 1947) of recent taxonomic uncertainty regarding compilations of South American species, the circumscription of Astragalus was the Gillett's (1963) treatment of the few species elevation of the entire subgenus Tragacan- that extend into tropical East Africa, Bar- tha (sensu Bunge, 1868, 1869) to generic neby's (1964) comprehensive revision of rank. This taxon, consisting of more than the more than 400 North American species, 200 species with striking spinescent habit Goncharov's (1965) treatment for the Flora and reduced inflorescences and fruits, was of the [former] USSR, and Lock and Simp- first recognized at the generic rank as Tra- son's (1991) checklist of legumes from west gacantha Mill. (Borissova, 1937) and more Asia. Recent taxonomic studies, largely recently as Astracantha by Podlech (1983), limited to Old World taxa, have emphasized which was later rejected (Zarre & Podlech, primarily revisionary work at the sectional 1997). Given these uncertainties surround- level and anatomical/morphological inves- ing the circumscription of the genus, the tigations (e.g., Podlech, 1982, 1983, 1984, question of the monophyly of Astragalus 1990, 1994; Engel, 1992; Wenninger, 1991; itself has remained open. Worse still, the Zarre & Podlech, 1997). In South America, view of Astragalus as a paraphyletic assem- species-level and floristic work has contin- blage or "wastebasket," basal within Gal- ued (G6mez-Sosa, 1979, 1981, 1997), but egeae with other genera (and other tribes) has not yet resulted in an infrageneric-level potentially nested within it (e.g., Polhill, classification comparable to that of Barne- 1981b), has prevailed well into the late 20 ~h by's (! 964) monograph. century. In addition to the lack of comprehensive In this context then, the first cladistic monographic studies, the relationship of As- studies on Astragalus began with analyses tragalus to certain morphologically similar of North American species using morpho- genera in Galegeae, especially Oxytropis, logical characters (Sanderson, 1991) and but also Sphaerophysa DC., and Swainsona molecular sequence and length polymor- Salisb., has always been problematic. In- phism data (Liston, 1992; Sanderson & deed, both Ledingham (1957, 1960), on the Doyle, 1993; Wojciechowski et al., 1993) basis of cytogenetic evidence, and Barneby in order to identify monophyletic groups (1952) on the basis of a number of mor- and begin to resolve their higher-level (in- phological similarities, suggested that Oxy- frageneric) relationships to Eurasian and tropis was derived from within Eurasian South America taxa. This was followed by Astragalus. More recently, Podlech (1982) broader-scale analyses of molecular data also implied that Oxytropis was nested from representative taxa of Galegeae and within Old World Astragalus. Similarly, the related tribes (Liston & Wheeler, 1994; 384 BR1TTONIA [VOL. 57

Sanderson & Wojciechowski, 1996) to re- scribed previously (Wojciechowski et al., solve the relationships of Astragalus to Ox- 1999, 2004). DNA sequencing was per- ytropis, and certain other genera in Gale- formed on an Applied Biosystems 3100 se- geae, within the larger "temperate herba- quencer at the Arizona State University ceous group" of papilionoids circumscribed (DNA Laboratory, Tempe, Arizona, USA). by Polhill (1981a). The most recent molec- Sequencer output files were assembled into ular phylogenetic analyses (Wojciechowski contigs and edited using the program Se- et al., 1999; Kazempour Osaloo et al., 2003, quencher 4.1 (GeneCodes, Ann Arbor, 2005), though still sparse in terms of sam- Michigan, USA) before alignment. pling (<10% of species), have focused more on a selection of Eurasian and North PHYLOGENETIC ANALYSES American species, based on morphological and cytogenetic diversity or the current sec- Phylogenetic analyses of the combined tional classification, to test the monophyly matK plus nrDNA ITS sequence data set, of infrageneric level groups as well as of consisting of 44 terminal taxa, utilized max- the genus itself. imum parsimony (MP) and Bayesian ap- In this paper, I review how our ideas of proaches. Parsimony analyses were per- Astragalus, both with respect to that of its formed using PAUP* (version 4.0b10; phylogenetic relationships, at the infrage- Swofford, 2002) on Macintosh or Linux neric level and among closely related gen- computers. Multiple tree searches were con- era, and its diversification within the papi- ducted using heuristic search options that lionoid legumes, have progressed in the included SIMPLE, CLOSEST, or RAN- past decade with the advent of cladistic DOM addition sequences (1000 replicates) methods and molecular data. I argue that holding five trees per replicate, and tree-bi- such approaches have had a dramatic im- section-reconnection (TBR) branch swap- pact on our understanding of Astragalus, ping, with retention of multiple parsimoni- but because of the unwieldiness of the ge- ous trees (e.g., MAXTREES = 1000). nus, in terms of size and morphological Bayesian analyses were performed using complexity, and persistent lack of compre- MrBayes version 3.0 (Huelsenbeck & Ron- hensive sampling for most groups, this will quist, 2001) as described previously (Wo- remain a progress report. jciechowski et al., 2004; Lavin et al., 2005). Clade support was assessed using both non- Materials and Methods parametric bootstrap resampling (Felsen- stein, 1985) and Bayesian posterior proba- TAXON SAMPLING bilities (Huelsenbeck et al., 2002). Non- The study presents an analysis of new parametric bootstrap proportions were esti- plastid matK gene and nuclear rDNA inter- mated from 500 bootstrap replicates nal transcribed spacer 1&2/5.8S gene incorporating heuristic parsimony searches (nrDNA ITS) sequences from a number of using addition sequence and branch swap- taxa (see Appendix for source information), ping options as in our standard parsimony in combination with previously published analyses (as described above). Bayesian nrDNA ITS (Sanderson & Wojciechowski, posterior probabilities were estimated as the 1996; Wojciechowski et al., 1999; Hu et al., proportion of trees sampled after 'burn-in' 2002) and matK gene sequences (Wojcie- that contained each of the observed bipar- chowski et al., 2004). These studies should titions. Combinability of the matK and be consulted for relevant voucher informa- nrDNA ITS data sets was assessed using tion. Genomic DNAs were isolated from the partition homogeneity test (ILD test) as field-collected, greenhouse-grown , implemented in PAUP*, using identical silica-dried and herbarium material as de- search options. Based on results from sev- scribed previously (Wojciechowski et al., eral previous studies of relationships within 2004). Polymerase chain reaction amplifi- the inverted repeat-lacking clade (IRLC, see cation and sequencing of the nrDNA ITS below), a large, well supported clade that is and matK regions were performed as de- distinguished at the molecular level by loss 2005 ] WOJCIECHOWSKI: MOI,ECULAR PIIYLOGENE'I'IC OF ASTRAGAI,US 385 of one copy of the large inverted repeat in (matK, 1551; nrl)NA ITS, 702), with 508 chloroplast DNA (Lavin et al., 1990: Lis- potentially parsimony-informative charac- ton, 1995), Glyo'rrhi=a lepidota was des- ters (24%, which excludes 137 indel and ignated as the outgroup in this analysis. missing characters). The partition homoge- Age estimates for Astragalus and the As- neity test results showed the two data sets tragalean clade are based on analyses of were not incongruent (p = 0.080). Maxi- evolutionary rates and divergence times of mum parsimony analyses of the combined the family-wide plastid matK (335 taxa; dataset (2116 characters included) resulted Wojciechowski et al., 2004) and rbcL gene in a single most parsimonious tree of 2001 (241 taxa: Kajita el al., 2001) phylogenies steps (CI -- 0.5857: RI = 0.7182). This presented in a recent study (Lavin et al., tree, with non-parametric bootstrap and 2005). In that study, age constraints derived Bayesian support values, is shown in Figure from the abundant, temporally continuous 1. Compared to the parsimony tree, minor and worldwide Tertiary macrofossil record incongruencies, such as the position of A. (Herendeen et al., 1992), were imposed on sinicus/A, complanatus (syn. Phyllolobium specilic clades in the matK (and rbcI,) gene chinense Fisch.) lineage in the Coluteoid trees, including the ages of the root node of clade, were observed in the Bayesian con- legumes (tixed at 60-70 Ma) and 12 inter- sensus, but these do not conflict in terms of nal nodes (as minimum ages). The node clades with high branch supports (e.g., par- closest to Astragalus with an assigned min- simony bootstrap values over 60%). imum age constraint was that of the Robinia The results shown in Figure 1 are gen- stem clade (34 Ma: Laven et al., 2003), erally consistent with results based on pre- which is nested within the sister group to vious, separate analyses of nrDNA ITS se- the inverted-repeat-lacking clade (see be- quences (Wojciechowski et al., 1999) and low). A matK data set containing 97 se- marK gene sequences (Wojciechowski et quences (aligned length of 1521 characters) al., 2000, 2004), in resolving a monophy- from Astragalus and papilionoid relatives in letic (in the strict sense) nested Hologalegina, which included 80 from the Astragalus within the larger, strongly supported Astra- family wide analysis (Wojciechowski et al., 2004) plus 17 new sequences, was used in galean clade (Sanderson & Wojciechowski, the present rates analysis. A nucleotide sub- 1996). The combined analysis of nrDNA stitution model was selected using AIC im- ITS with matK data presented here, how- plemented in Modeltest (version 3.06: Po- even provides greater support for the Col- sada & Crandall, 1998). Branch lengths uteoid clade (and slightly better support of were estimated using a Bayesian approach others such as O,vytropis) as well as a more that incorporated a GTR + I" + I model of completely resolved tree with taxa such as nucleotide substitution. One hundred Oxvtrotffs, Astragalus vogelii, Astragalus Bayesian trees sampled at stationarity were epiglottis, and Astragalus pelecinus (syn. then systematically analyzed using the pe- Biserrula pelecinus L.; Barneby, 1964, p. nalized likelihood (PL) method of Sander- 26) whose relationships within the Astra- son (2002) in the program r8s (vers. 1.6: galean clade were not resolved unequivo- Sanderson, 2003), as described previously cally by previous analyses of nrDNA ITS (Lavin et al., 2005). In addition to the age sequences alone (Wojciechowski et al., constraint imposed on the Robinia stem 1999: Kazempour Osaloo et al,, 2003). The clade, the age of the Hologalegina crown tinding that A. epiglottis plus A. pelecinus clade ("root" node) was tixed at 50.6 Ma forms the sister group to Astragalus sensu for the presen! analysis. The latter age con- stricto as defined previously (Wojciechows- straint is based on the estimated age for this ki et al., 1999) is not unexpected but this clade fi'om the results of Lavin et al. (2005). relationship has not been unequivocally re- solved, or well supported, in analyses of Results nrl)NA ITS or plastid ndhF sequences COMBINED MATK/NRDNA ITS DATA alone (Wojciechowski et al., 1999; Kazem- The combined matK + nrDNA ITS da- pour Osaloo et al., 2003). Moreover, results taset included a total 2253 aligned positions based on the combined analyses of matK 386 BRITTONIA [VOL. 57

Glycyrrhiza lepidota Caileryaatropurpurea

...... Parochetuscommunis Galegaorientalis F~--4 Cicer arietinurn 99i Medicago sativa Melilotus alba Vicioid c lade/~ ~, 100 ~.,~,---~...... Trigonella foenu m-g raecum Trifo liu m nan um Pisum sativum 98 ,00L Viciafaba Lens culinaris Caraganaarborescens "~ i00 [ Alhagi maurorum i00 Onobrychis montana Hedysaroid~'1 Hedysarum boreate clade Podlechiellavogelii ~. Swainsonapterostylis 93 puniceus williamsii ~L 100 [ Astragalussinicus Coluteoid L Phyllolobiumchinense clade Astragaluscysticalyx I i00 Coluteaarborescens essertia herbacea Astragalean clade"~ utherlandiafrutescens xytropis lambertii I Oxytropis pilosa Oxytropis Oxytropis deflexa 74 t ~ Astragalusepiglottis Astragalus pelecinus

87 -- Astragaluscorrugatus ---- Astragalus amedcanus

-- Astragalusatropilosulus J i 0~______~_l~st ragalu s adsurgens L Astragalusalpinus stragaluscerasocrenus Astragalus s s "r 80L.- 73

-- I 0 changes

Neo-Astragalus FIG. 1. Phylogeny of IR-lacking ctade based on parsimony analyses of combined plastid matK gene and nrDNA ITS data. The tree shown is the single most parsimonious tree (length = 2001 steps; CI = 0.5857; RI = 0.7182) derived from heuristic search analyses of sequences from 44 taxa (2116 included characters). Non- parametric bootstrap proportions are indicated above or immediately to the left of appropriate nodes tbr which support values were greater than 50%. The heavy lines designate branches with Bayesian posterior probabilities of 95-100% (0.95-1.00); the gray boxes delimit Astragalus sensu stricto and Neo-Astragalus (Wojciechowski et al,, 1999). Estimated branch lengths are shown; scale is indicated at lower left. is designated as the outgroup for all analyses. 2005] WOJCIECHOWSKI: MOLECULAR PHYLOGENET1C OF ASTRAGALUS 387

and nrDNA ITS sequences presented here In the present analysis, the three South (Fig. 1) concur with Wojciechowski et al. American species form a well-supported (2004) which first showed significant sup- monophyletic group sister to the two North port (both bootstrap values and Bayesian American aneuploids, but in other analyses posterior probabilities) for placement of the with greater sampling of North and South genus Oxytropis as the sister group to As- American species overall (Wojciechowski tragalus sensu stricto plus A. epiglottis/A. et al., 1999; Scherson et al., 2005) the pelecinus, within the Astragalean clade. South American species comprise at least Previous analyses have suggested Oxytropis two, independently derived clades within is more closely related to the Coluteoid Neo-Astragalus. clade (Liston & Wheeler, 1994; Sanderson & Wojciechowski, 1996; Kazempour Osa- ESTIMATED AGES OF ASTRAGALEAN AND loo et al., 2003), forms the basally branch- ASTRAGALUS CROWN GLADES ing lineage within the Astragalean clade (Wojciechowski et al., 2000), or was unre- Lavin et al. (2005) provides estimated solved (Wojciechowski et al., 1999). ages of many of the larger clades of le- The combined analysis of matK and gumes that can serve as calibration points nrDNA ITS data also provides measurable for rates analyses of groups that lack a fos- support for a sister group relationship of sil record, such as the Astragalean clade. plus the hedysaroid clade to the Caragana Fossil evidence for taxa within the IRLC is Astragalean clade, a relationship that has minimal at best; Vicia-like leaves with ten- been variously, but mainly weakly, sup- drils and inflated, Phaca-like pods have ported in analyses of matK (Wojciechowski been described from the Florissant Beds et al., 2000, 2004) or nrDNA ITS alone flora of Colorado, USA (Oligocene, ca. 30 (Sanderson & Wojciechowski, 1996; Hu et Ma; MacGinitie, 1953). Because these have al., 2002). This sister group relationship of not been definitively identified and there- Caragana plus the hedysaroid clade to the fore cannot be unequivocally assigned to Astragalean clade, rather than sister to the any specific node amongst the clades of in- Vicioid clade, is consistent with suggestions (e.g., Polhill, 1981b) that Caragana and al- terest here, they provide no useful time cal- lies as well as tribe Hedysareae, which are ibration. For this reason, a rates analysis of predominantly Eurasian North American matK sequences from the larger Hologale- groups, were derived from the "astragaloid gina clade, with both estimated and fossil- part" of Galegeae (Astragalus and its calibrated nodes (Lavin et al., 2005), was woody relatives) in Asia. conducted. Relationships within Astragalus are also The mean PL substitution rates across consistent with previous results based on 100 Bayesian trees for nodes identified in analyses of nrDNA ITS data alone (Wojcie- the matK phylogeny derived from analyses chowski et al., 1999; Kazempour Osaloo et of the 97-taxon data set (not shown) range al., 2003). For example, the North Ameri- from an average of 8.3 X 10 10 substitu- can euploid species, represented in this tions per site per year across the IRLC to analysis by A. adsurgens, A. atpinus, A. 10.6 X 10 -1~ substitutions per site per year americanus, and A. canadensis, are more for the Vicioid clade (Table I; rates ex- closely related to Old World taxa than to pressed as s/s/Ma). The age estimated for the North American aneuploid species. the Astragalean clade here is comparable to While sampling for the combined data set that presented in Lavin et al. (2005), al- is more limited, species from each of the though slightly older (16.1 Ma -+ 1.82 ver- "major" Old World groups ("A"-"E") de- sus 14.8 Ma). The estimated ages for As- scribed in Wojciechowski et al. (1999) are tragalus (12.4 + 1.45 Ma) and Neo-Astrag- represented here, and Neo-Astragalus is re- alus (4.4 _+ 0.78 Ma) are also similar to an solved as a moderately supported clade earlier estimate (11 Ma, 4-5 Ma, respec- nested within one of these Old World tively) based on the simplistic assumption groups ("E"; Wojciechowski et al., 1999). of a molecular clock for nrDNA ITS se- 388 BRITTONIA [VOL. 57

TABLE I PENALIZED LIKELIHOOD (PL) ESTIMATES OF AGES AND RATES OF NUCLEOTIDE SUBSTITUTION FOR DESIGNATED NODES ACROSS 100 MATK TREES DERIVED FROM BAYESIAN ANALYSES (97-TAXON DATA SET), SAMPLED AT STATIONARITY

Mean age Mean rate Node Node definition, MRCA of (Ma) SD (age) Min (Ma) Max (Ma) (s/s/Ma) SD (rate) Hologalegina Sesbania vesicaria- 50,6* ..... ("root") Astragalus patagonicus Robinieae Robinia pseudoacacia- 34,0** -- 34.0 ------Coursetia axillaris IRLC Glycyrrhiza lepidota 36.0 2.40 30.9 43.7 0.000825 0.000068 -Astragalus patagonicus Astragalean Sutherlandiafrutescens- 16.1 1.82 12.1 20.6 0.00091 0.000078 Astragalus patagonicus Astragalus Astragalus pelecinus 12.4 1.45 9.3 17.7 0.000949 0.000083 -Astragalus patagonicus Neo-Astragalus Astraga/us lonchocarpus- 4.4 0.78 2.8 6.8 0.001021 0.000094 Astragalus patagonicus Oxytropis Oxytropis deflexa- 4.6 0.92 2.4 7.1 0.000899 0.000088 Oxvtropis pilosa Coluteoid clade Carmichaeliawilliamsii- 13.7 1.70 10.1 19.4 0.000894 0.000080 Sutherlandia frutescens Hedysaroid clade Alhagi rnaurorum- 21.4 2.43 16.3 26.9 0.000935 0.000079 Hedysarum boreale Vicioid clade Galega orientalis- 26.8 2.20 22.5 33.4 0.001059 0.000083 Vicia sativa PL analyses used "TN" algorithm, smoothing value S = 63.10 (10t s). The first node, Hologalegina*, is assigned a fixed age (50.6 Ma) and the second, Robinieae**, is assigned a minimum age (34.0 Ma). Ma = million years; SD = standard deviation; s/s = substitutions per site.

quence evolution (Wojciechowski et al., grant Old World groups, as implied by dif- 1999). ferences in apparent dispersal ability and the cytogenetic evidence then available, Discussion would justify their taxonomic separation (section level and above) in the American Rupert C. Barneby, in his monumental flora even if morphological evidence for Atlas of North American Astragalus (1964), that separation was lacking (Barneby, 1964; discussed in detail the ecological speciali- p. 30-31). Beyond the recognition that the zation and evolutionary significance of North American species represented seven many of the morphological characters he had observed in his extensive study of As- major phylogenetic groups (his "phalanx- tragalus, among them the development of es"), Barneby did not venture an opinion the bilocular pod, the independent origins on their relationships, suggesting that such of annual forms, and graduation of the pet- speculation rested on the assumption that als. He envisioned that multiple invasions, evolution and specialization proceeded in representing different lineages, of astragali concert, the evidence for which he consid- from Asia into the Americas had occurred ered "contradictory and at best controver- without an obvious reciprocal exchange, sial" in Astragalus (Barneby, 1964; p. 21). and that the endemic or "autochthonous" Indeed, his skepticism about the validity of species groups (aneuploid American As- a one-dimensional sequence to ever accu- tragalus) had their origins in subtropical rately reflect the true evolutionary history arid regions rather than in the colder, high of Astragalus led him to the conclusion latitudes they would passage during migra- (Barneby, 1964; p. 21) that "the so-called tion from eastern Asia via the Bering land phylogenies, which current botanical fash- bridge. Furthermore, he suggested the long ion seems to require of the taxonomic separate history of the 'endemic' New worker, are for the most part flimsy struc- World and presumably more recent immi- tures, compounded of nine parts speculation 2005] WOJCIECHOWSKI: MOLECULAR PHYLOGENETIC OF ASTRAGALUS 389 to one of observed fact; many of them col- assortment of ecologically and morpholog- lapse under logical scrutiny and serve no ically distinctive species of Astragalus, useful purpose." which now appear based on molecular ev- While Barneby may have doubted the idence to be more closely related to mem- usefulness of phylogenies forty years ago, bers of Coluteinae than to Astragalus. This the molecular phylogenetic studies that be- includes the annual Astragalus vogelii, re- gan in the 1990s have had a significant im- cently raised to generic rank as Podlechiel- pact on our understanding of the relation- la Maassoumi and Kazempour Osaloo (Ka- ships and diversification of Astragalus, and zempour Osaloo et al., 2003), Astragalus their findings vindicate some of his ideas cysticalyx, Astragalus complanatus (syn. about the genus. Early studies (Liston & Phyllolobium chinense Fisch.), and Astrag- Wheeler, 1994; Sanderson & Wojciechows- alus sinicus (Liston & Wheeler, 1994; Wo- ki, 1996) indicated that Astragalus is nested jciechowski et al., 1999). The latter two within a clade now referred to as the IRLC. species, from Bunge's subgenus Pogono- This clade, consisting of all members of the phace, possess morphological features such traditionally circumscribed tribes Carmi- as pubescent styles (Kang & Zhang, 2004) chaelieae (or Carmichaelinae, Wagstaff et that are diagnostic of other genera in sub- al., 1999), Cicereae, Galegeae, Hedysareae, tribe Coluteinae (e.g., Colutea) and thus Trifolieae, and Vicieae (Fabeae), and at consistent with their placement in this clade least three genera of Millettieae (Callerya (Wojciechowski et al., 1999; this study). In Endlicher, sampled here, Afgekia Craib, fact, these and other morphological similar- Wisteria Nutt.), is a vast, cosmopolitan as- ities led Barneby (1964; p. 1164) to con- semblage (>4200 spp.) that corresponds jecture that A. cornplanatus (sect. Phyllo- closely to Polhill's (1981 a) "temperate her- lobium) and other "primitively barbistyled baceous group," and one of two subclades Astragali referred by Bunge to the subgenus of Hologalegina, the largest clade of papi- Pogonophace" might be more appropriate- lionoid legumes (Wojciechowski et al., ly treated outside Astragalus sensu lato. 2000). The monophyly of the IRLC has The phylogenetic position of subgenus Po- been consistently and strongly supported in gonophace within the Coluteoid clade has all subsequent cladistic analyses of molec- been further substantiated by recent analy- ular sequence data (Doyle et al., 1997; Hu ses of nrDNA ITS data (Kang et al., 2003; et al., 2002; Wojciechowski et al., 2000, Kazempour Osaloo et al., 2005), which 2004; Kajita et al., 2001) as well as plastid show that a number of species sampled DNA structural evidence (Lavin et al., from this subgenus form a well-supported 1990; Liston, 1995; M. E Wojciechowski, monophyletic group within the Coluteoid unpubl, data). Nested within this radiation subclade that includes the Asian-African of predominantly herbaceous and temperate genera Colutea, Lessertia, and Sutherlandia papilionoids, Astragalus sensu lato together plus Astragalus cysticalyx, a result that has with Oxytropis, Galegeae subtribes Colutei- prompted the suggestion that they be treat- nae and Carmichaelinae, comprise a well- ed as the genus Phyllolobium (Kang et al., supported group we have previously re- 2004). However, at least three other mem- ferred to as the Astragalean clade (Sander- bers of this subgenus (e.g., Astragalus atro- son & Wojciechowski, 1996). The Astra- pilosulus, sect. Chlorostachys) are nested galean clade (Fig. 1) had consistently been within Astragalus sensu stricto. resolved with high statistical support (e.g., "Taxonomically isolated species are 100% bootstrap proportions) and shown to characteristic ofAstragalus" Barneby noted be composed of two main subclades, one (1964; p. 32), and a number of primarily essentially corresponding to the subtribe annual Eurasian species of the genus at one Coluteinae plus Carmichaelinae (the "Col- time or another placed in monotypic sec- uteoid" clade) while Astragalus and Oxy- tions have been segregated as new genera tropis comprise the other. whose phylogenetic positions are for the Scattered throughout the African-Austra- most part unknown. Astragalus migpo, seg- lasian Coluteoid clade is a heterogeneous regated as Barnebyella calycina, Astragalus 390 BRITTON1A [VOL. 57

ophiocarpus as Ophiocarpus aitchisonii, chowski et al., 1999), seems warranted. Astragalus dipelta as Didymopelta turkes- Thus, Astragalus is hereby defined as the tanica, Astragalus schmalhausenii as Sew- clade delimited by the most recent common erzowia turkestanica, Astragalus vicarius ancestor of Astragalus pelecinus and As- as Sewerzowia vicaria, and Astragalus tragalus patagonicus. thlaspi Lipsky as Thlaspidium (Lipsky) Other subgroups of Astragalus possess- Rassulova, are clearly nested within Astrag- ing remarkable ecological specializations or alus sensu stricto based on molecular phy- unique combinations of morphological logenetic evidence (Kazempour Osaloo et characters that have prompted their segre- al., 2003, 2005). Virtually all of these spe- gation as new genera, the Eurasian Astra- cies possess unusual fruit morphologies, cantha Podlech (Astragalus subgenus Tra- such as laterally compressed, unilocular, gacantha) and the North American Oro- one- to two-seeded, sessile to stipitate pods, phaca (Torr. & Gray) Britton (phalanx Or- or unique combinations of features that ophaca), have also been shown to be nested have led to their segregation from Astrag- within Astragalus (Wojciechowski et al., alus by various authors. The phylogenetic 1999). Detailed analyses of morphological position of the Mediterranean annual As- characters subsequently led Zarre and Pod- tragalus pelecinus (L.) Barneby (=Biser- lech (1997) to question the monophyly of rula pelecinus L.), which Barneby (1964; Astracantha (and return it to Astragalus). p. 26) describes as a "perfectly unexcep- Now, more extensive molecular sampling tional Astragalus," has been variously sup- of Tragacantha sections for both nrDNA ported as sister to Oxytropis, nested with ITS and plastid ndhF sequences confirm the Coluteoid clade, or unresolved with respect paraphyly of this subgenus within Old to Astragatus sensu stricto (Sanderson & World Astragatus with its members scat- Wojciechowski, 1996; Wojciechowski et tered among other thorny, cushion forming al., 1999; Kazempour Osaloo et al., 2003). species representing other subgenera (Ka- This species shares a number of relatively zempour Osaloo et al., 2003, 2005). uncommon morphological features with As- In contrast, the genus Oxytropis, whose tragalus epiglottis, another widespread taxonomic history has been long inter- Mediterranean annual, including flowers twined with, if not considered "organically with only five fertile stamens and dorsiven- part of", Astragalus (Barneby, 1964; p.21), trally compressed pods (Matthews, 1970). is clearly not nested within Astragalus. Molecular studies have shown these two Rather, there is now substantial support for species to be strongly supported as sister Oxytropis as its sister group. This result taxa (Sanderson & Wojciechowski, 1996; substantiates Barneby's (1952, 1964) long Wojciechowski et al., 1999) or closely re- held contention that Oxytropis is a distinct lated to Astragalus annularis in a well-sup- group of Eurasian origin that is closely re- ported subclade within the Astragalean lated to (but taxonomically separate from) clade (Kazempour Osaloo et al., 2003, Astragalus, and, has which subsequently 2005). Based on molecular evidence Liston followed a parallel course of ecological and Wheeler (1994; p. 385) suggested that specialization, mol-phological, and biogeo- these two taxa might represent the "deepest graphic diversification. branches in the Astragalus clade." Results Prior to molecular phylogenetic work, presented here strongly support this hypoth- the only solid evidence for infrageneric esis, that A. pelecinus plus A. epiglottis (and groups within Astragalus was a near perfect likely A. annularis) form the sister group to correlation between chromosome number Astragalus sensu stricto. In light of the con- and geographic distribution. The vast ma- gruence (and support) of the results pre- jority of Old World species possess euploid sented here, a more explicit phylogenetic chromosome numbers (based on n = 8) redefinition of the genus Astragalus, so as while the vast majority of North American to include these two taxa plus the clade re- and all South American species surveyed ferred to as "Astragalus sensu stricto'" as possess numbers in an aneuploid series of informally defined previously (Wojcie- n = 11 to n = 15 (reviewed in Wojcie- 2005] WOJCIECHOWSKI: MOLECULAR PHYLOGENETIC OF ASTRAGALUS 391 chowski et al., 1993). The earliest molecu- the "primitive phylum" in the genus, a con- lar studies (Sanderson & Doyle, 1993; Wo- cept not too different from that of the orig- jciechowski et al., 1993; Liston & Wheeler, inal Linnaean genus Phaca. But, as we 1994) suggested that the North American point out elsewhere (Wojciechowski et al., species with aneuploid chromosome num- 1999), even with taxon sampling too lim- bers form a monophyletic group derived ited to address rigorously the question of its from Eurasian euploid taxa. Subsequent monophyly, it is evident from the molecular studies (Sanderson & Wojciechowski, phylogenies that this phalanx (subgenus), 1996; Wojciechowski et al., 1999) suggest- like the others, is not monophyletic. It is ed the monophyly of a group comprising all also noteworthy that even with much great- New World aneuploids (Neo-Astragalus) er sampling of Old World taxa than that nested within one of several groups con- study, Kazempour Osaloo et al. (2003, taining euploid and the few aneuploid Eur- 2005) have come to essentially the same asian species as well as all the euploid spe- conclusions in showing that none of the tra- cies found in North America. These find- ditionally recognized Old World subgenera, ings further corroborated Ledingham's or most of the species-rich sections, is (1957, 1960) hypothesis of a major division monophyletic. These phylogenies also in the genus based on cytogenetic data and clearly refute a more recent and reductionist biogeographic separation (Old World versus subgeneric classification of the Old World New World). Although the identity of the taxa based on a few, single characters (Pod- Old World sister taxon to Neo-Astragalus lech, 1982, 1991). remains uncertain, our results suggest that Fortunately, we are now beginning to see the diversification of Neo-Astragalus was the broad outline of infrageneric relation- exclusively confined to the New World. The ships within the genus. The molecular phy- relatively few euploid species found in logenies resulting from these studies (Wo- North America are representatives of Old jciechowski et al., 1999; Kazempour Osa- World groups that appear to have immi- loo et al., 2003, 2005), including the results grated (e.g., by dispersal or introductions) presented here, have identified several mod- to the New World subsequent to and unre- erately to well-supported subclades within lated to the diversification of Neo-Astraga- Astragalus sensu stricto (fig. 2 in Wojcie- lus (Wojciechowski et al., 1999). chowski et al., 1999; Kazempour Osaloo et An important outcome of all these mo- al., 2003; fig. 1 in Kazempour Osaloo et al., lecular studies is a phylogenetic perspective 2005). A highly simplified schematic dia- from which to evaluate previous classifica- gram of Astragalus and its sister group Ox- tions schemes of Astragalus, which were ytropis based on results of phylogenetic based largely on morphological criteria. analyses of nuclear and plastid sequence Barneby came to the conclusion that the data derived from these studies is presented North American species "represented seven here in Figure 2. It is important to note this major phylogenetic branches of the genus" is meant to portray molecular phylogenetic (1964; p. 33), four of which he considered data that is still very preliminary--the tax- equivalent to Asiatic subgenera (sensu Bun- onomic composition and relationships of ge, 1868, 1869) and included species rep- each of these subclades, here designated by resentative of the most primitive as well as letter names only for convenience, are for some of the more advanced sections of the the most part still poorly resolved and lack- Old World. Yet, he did not speculate on ing in statistical support or obvious diag- what relationships the North American pha- nosable characters, and are likely to change lanxes might have to each other, or to the with more comprehensive sampling espe- Old World subgenera. He did regard the cially of Old World taxa and analyses of subgenus Phaca (sensu Bunge, 1868, additional sources of data. Thus, for the 1869), the largest in the Old World, which foreseeable future these lettered subclades he defined more broadly to include North will primarily serve as landmarks to guide American species with partially to com- taxon sampling and evaluate the diagnostic pletely unilocular (inflated or not) pods, as potential of morphological characters that 392 BRITTONIA [VOL. 57

- 4.6 Ma Oxytropis

A. pelecinus/A, epiglottis ~l clade A 12.4 M~ NA euploid(s)

c~~r-- ~ lade NAB euploid(s)

clade C

NA euploid(s)

~ clade F Astragalus NA euploid(s) ,,.~ ~J, N~eo-Astragalus clade D .... S American 1 S American 2

ade I lade G2

clade G1

FIG, 2. Simplified schematic diagram of Astragatus and sister group Oxytropis, showing phylogenetic re- lationships of moderately to well-supported subclades identified within Astragalus, based on cladistic analyses of molecular sequence data presented here and in studies by Wojciechowski et al. (1999) and Kazempour Osaloo et al. (2003, 2005). Triangles designated by letter names shown in light gray shading represent large groups within Old World Astragalus referred to in these studies. The aneuploid New World Astragalus clade, Neo- Astragalus, and the distribution of the North American euploid species (NA euploids) are also shown. Estimated ages (in millions of years) of selected clades are indicated. will ultimately form the basis for a revised, and 12.4 Ma. The estimated age of the di- infrageneric classification of the genus. versification of the Astragalus (ca. 2500 Based on an evolutionary rates analysis spp.) crown clade is estimated at 12.4 Ma, of the rnatK phylogeny (Table I) the esti- while that of the 500 or so species of Neo- mated age of the Astragalean crown clade Astragalus, in which the South American is 16.1 + 1.8 Ma, with the split between species are nested and comprise at least two Oxytropis and Astragalus to be between 16 independently derived subclades, is 4.4 + 2005] WOJCIECHOWSKI: MOLECULAR PHYLOGENETIC OF ASTRAGALUS 393

0.78 Ma. Interestingly, the diversification of 9.0 • 10 10 substitutions per site per year; the Oxytropis (ca. 330 spp.) crown clade is Amorpheae, 8.4 • 10-m; robinioids, 7.8 • 4.6 _+ 0.92 Ma, much later than that of As- 10-r~ vicioid clade, 8.4-10.8 • 10 10; Car- tragalus, but similar in timing to that of agana plus hedysaroids, 7.7 • 10 10. Such Neo-Astragalus. This finding is consistent new estimates of rates of molecular evolu- with Barneby's hypothesis of a separate or- tion in this and other papilionoid groups, igin for Oxytropis, compared to Astragalus, combined with newer, more rigorous, quan- but parallel pattern of diversification. titative methods now permit a more accu- Previous estimates of lineage diversifi- rate assessment of lineage diversification cation rates for Astragalus (Wojciechowski rates for Astragalus (M. E Wojciechowski et al., 1999) suggested unusually high rates & M. J. Sanderson, unpubl, data), and un- for the genus (sensu stricto) and especially doubtedly will help us to explain the re- Neo-Astragalus, compared to estimates for markable pattern of continental diversifica- most continental families and other tion observed in this, unusually species-rich angiosperm clades (e.g., Magall6n & San- clade. derson, 2001), and even often-cited exam- ples of insular adaptive radiations (e.g., Ha- Acknowledgments waiian silverswords; Baldwin & Sanderson, The author thanks R. Scherson and A. 1998), although perhaps statistically not ex- Liston for providing genomic DNAs of sev- ceptionally high compared to its closest rel- eral South American and Asian Astragalus. atives (Sanderson & Wojciechowski, 1996). The author thanks K. R Steele, A. Delgado- Using the ages for Astragalus and Neo-As- Salinas, and an anonymous reviewer for tragalus estimated here (based on the matK their many helpful comments and sugges- phylogeny), lineage diversification rates tions to improve earlier versions of this based on standing diversities (r = In N/t; manuscript. Lastly, the author especially referred to in Wojciechowski et al., 1999) wishes to thank A. Liston for originally would be 0.63 spp./Ma for Astragalus and suggesting the idea of a symposium to hon- 1.41 spp./Ma for Neo-Astragalus, using or the legacy of Rupert C. Barneby and his their ages (t) and diversities (N) of 2500 many contributions to legume systematics, and 500 species, respectively. These esti- and M. J. Sanderson for reinvigorating an mates are comparable to, and thus corrob- earlier interest in this genus and for many orate, previous estimates for these same memorable field excursions in search of the clades (0.71 and 1.48 spp./Ma, respectively; often-elusive Astragalus. This study was Wojciechowski et al., 1999) based on nr- supported in part by funds provided by Ar- DNA ITS sequence evolution. Furthermore, izona State University to the author. these estimates are unusually high when compared against the average rates of 0.50 Literature Cited spp./Ma estimated for the Astragalean clade (N = 3070; Sanderson & Wojciechowski, Baldwin, B. G. & M. J. Sanderson. 1998. Age and 1996) and 0.23 spp./Ma estimated for the rate of diversification of the Hawaiian silversword IRLC (N = 4250; Wojciechowski & San- alliance (Compositae). Proceedings of the National Academy of Sciences of the 95: derson, 1996). Yet, estimated rates of nu- 9402-9406. cleotide substitution for Astragalus and its Barneby, R. C. 1952. A revision of the North Amer- relatives in the Astragalean clade, based on ican species of Oxytropis DC. Proceedings of the fossil-calibrated, family-wide molecular Academy of Sciences, Series IV, 27: phylogenies (matK and rbcL, Lavin et al., 177-312. --. 1964. Atlas of North American Astragalus. 2005) like those reported here, indicate that Memoirs of The New York Botanical Garden 13: absolute rates of molecular evolution for l-1188. this group (9.1 • 10 m substitutions per site Bobrov, E. G., A. 13. Borisova, B. A. Fedchenko, S. per year; Table I) are not at all unusual G. Gorshkova, Y. S. Grigor'ev, A. A. Grossgeim, A. N. Krishtofovich, I. A. Linchevskii, A. S. Loz- when compared to other, mainly temperate ina-Lozinskaya, L V. Palibin, K. K. Shaparenko, groups of papilionoid legumes (from Lavin B. K. Shishkin, I. T. Vasil'ehenko & V. N. et al., 2005): e.g., Crotalaria-Lupinus, 8.1- Vasil'ev. 1972. Leguminosae: Oxytropis, Hedysa- 394 BRITTONIA [VOL. 57

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Appendix

Species, collection locality, voucher tragalus corrugatus Bertol.; Iran (USDA PI 227441); specimen, and GenBank accession numbers Wojciechowski & Sanderson 164 (ARIZ); IAY920442. Astragalus cysticalyx Ledeb.; former USSR (USDA PI for new sequences derived for this study 440146); Liston 961 (OSC); ~AY920443. Astragalus (lmatK, 2nrDNA ITS) are provided, Data epiglottis L.; Israel (Liston 892, OSC); Wojciechowski sets in NEXUS format are available from & Sanderson 405 (ARIZ); IAY920445. Astragalus pa- the author, All other sequences used in this tagonicus (Phil.) Spegazzini; Argentina; Sanderson study have been published previously (San- 2515 (DAV); JAY920446. Astragalus pehuenehes Niederlein; Argentina; Sanderson 2518 (DAV); derson & Wojciechowski, 1996; Wojcie- ~AY920447. Astragalus pelecinus (L.) Barneby (syn. chowski et al., 1999, 2004; Hu el al., 2000) Biserrula pelecinus L.); Australia (adventive, USDA and are available from GenBank (http://www. PI 186284); Wojeieehowski & Sanderson 294 (ARIZ); ncbi.nlm.nih.gov/Entrez/index.html) and from JAY920448. AsCraga[~is peristereus Boiss. & Hausskn.; TreeBASE (http://www.treebase.org/). lran (DELEP 880051); rAY920449. Astragalus xinicus L.; China (USDA PI 150557); Wojciechowski & San- Astragalus adsurgens Pallas; China (USDA PI derson 408 (ARIZ); rAY920450. Astragalus vogelii 462310); Wojciechowski & Sanderson 267 (ARIZ); (Webb.) Bornm. (syn. Podlechiella vogelii (Webb.) JAY920437. Astragalux alpinus L.; USA, Wyoming Maassoumi et Kazempour Osaloo); Egypt, Sinai; Lis'- (USDA PI 232536); Wojciechowski & Sanderson 183 ton 890 (OSC); 'AY920451. Galega orientalis Lain; (ARIZ); ]AY920438. Astragalus arnottianus (Gillies) former USSR (USDA PI 325337); Wojciechowski & Reiche; Argentina; Sanderson 2520 (DAV); Sanderson 399 (ASU); 2AY553709. Lessertia herba- ~AY920439. Astragalus atropilosulus (Hochst.) Bunge cea DC.; South Africa (USDA PI 207923); Wojcie- var. venosus (Hochst.) Gillett; Kenya (USDA Pl chowski & Sanderson 299 (ARIZ); ~AY920453. Oxy- 193735); Wojciechowski & Sanderson 301 (ARIZ); tropis pilosa (L.) DC.; former USSR (USDA P1 ~AY920440. Astragalus cerasocrenus Bunge; Iran 420696); Wojciechowski & Sanderson 306 (ARIZ); (DELEP 880043); IAY920444. Astragalus complana- ~AY920452. Parochetus communis Buch.-Ham. ex D. tus R. Br. (syn. Phyllolobium chmense Fisch.); China Don; cultivated (A. Liston); Wojciechowski 901 (DELEP 900279); Liston 889 (OSC); rAY920441. As- (ASU); 2AY553710.