astragalus (fabaceae): a molecular phylogenetic perspective · 2015. 4. 9. · key words:...

Astragalus (Fabaceae): A molecular phylogenetic perspective

M A R T I N 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 transcribed spacer region were sampled from Astragalus L. (Fabaceae), and its closest relatives within tribe Galegeae, 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 species 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 composed 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 substitution 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 previously published work and other recent developments on the phylogenetic relationships and diversification of Astragalus are reviewed.

Key words: Astragalus, diversification, Fabaceae, Neo-Astragalus.

The l egume genus Astragalus L., with an es t imated 2500 species of herbaceous peren- nial and annual species in the subfami ly Pap- i l i o n o i d e a e o f the F a b a c e a e ( M a b b e r l e y , 1997; Lewis et al., 2005) and the largest of 19 or so genera of ang iosperms with more than 1000 species, is an example o f an adaptive radia t ion on a global scale. Astragalus is d is t r ibuted main ly in cool to w a r m arid and semiar id regions of the northern hemisphere , South Amer ica , t ropical East Africa, and is especia l ly diverse in southwestern and Sino- H i m a l a y a n regions of As ia (ca. 1500-2000 spp.), western North A m e r i c a (ca. 4 0 0 - 4 5 0

spp.), and the Andes of South A m e r i c a (ca. 100 spp.). The geographic center o f divers i ty and p re sumed origin of Astragalus, l ike most o f its c lose relat ives in the tr ibe Ga legeae (Polhill , 1981a, 1981b), is Eurasia, and spe- cif ical ly the steppes and mounta ins o f southwestern to south-central As ia and the Hima- layan plateau. Of the two Ga legean genera with dis tr ibut ions in the New World , the other being Oxytropis DC. with ca. 330 spp. in the northern hemisphere (Bobrov et al., 1972; Welsh, 2001), only Astragalus has undergone extens ive diversif icat ions in both North and South Amer ica .

Brittonia, 57(4), 2005, pp. 382-396. ISSUED: 28 December 2005 �9 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 widespread distribution, it is hardly surpris- ing that no comprehensive monographic in- vestigation of Astragalus has been attempt- ed since the taxonomic treatments of the late 19 th century (Bunge, 1868, 1869; Taub- ert, 1894). With the exception of Rydberg (1929), who recognized 28 genera within North American Astragalus, in a revision not supported by subsequent authors (Bar- neby, 1964; Isely, 1998), and the segregation of the Eurasian subgenus Tragacantha as Astracantha (Podlech, 1983, but later re- versed by Zarre & Podlech, 1997), the circumscription of Astragalus remains relatively unchanged since that proposed by Bunge. During the 20 th century, taxonomic work has been more floristic and regional in scope, such as Johnston's (1938, 1947) compilations of South American species, Gillett's (1963) treatment of the few species that extend into tropical East Africa, Bar- neby's (1964) comprehensive revision of the more than 400 North American species, Goncharov's (1965) treatment for the Flora of the [former] USSR, and Lock and Simp- son's (1991) checklist of legumes from west Asia. Recent taxonomic studies, largely limited to Old World taxa, have emphasized primarily revisionary work at the sectional level and anatomical/morphological inves- tigations (e.g., Podlech, 1982, 1983, 1984, 1990, 1994; Engel, 1992; Wenninger, 1991; Zarre & Podlech, 1997). In South America, species-level and floristic work has contin- ued (G6mez-Sosa, 1979, 1981, 1997), but has not yet resulted in an infrageneric-level classification comparable to that of Barne- by's (! 964) monograph.

In addition to the lack of comprehensive monographic studies, the relationship of As- tragalus to certain morphologically similar genera in Galegeae, especially Oxytropis, but also Sphaerophysa DC., and Swainsona Salisb., has always been problematic. In- deed, both Ledingham (1957, 1960), on the basis of cytogenetic evidence, and Barneby (1952) on the basis of a number of morphological similarities, suggested that Oxy- tropis was derived from within Eurasian Astragalus. More recently, Podlech (1982) also implied that Oxytropis was nested within Old World Astragalus. Similarly, the

two current species of the Asian Sphaero- physa were originally included in the Lin- naean genus Phaca, but Gray (1864) and Bunge (1868, 1869) among others subsequently treated these as a subgenus of As- tragalus. While these taxa share a number of important morphological characters, Bar- neby (1964; p. 1162) and Isely (1998) treated this genus separate from Astragalus (later corroborated by molecular phylogenetic analysis; Sanderson & Wojciechowski, 1996). Since then, a number of other "morphologically isolated" Eurasian Astragalus species have been segregated as monotypic genera, largely on the basis of pod mor- phology (e.g., Barnebyella, Podlech, 1994; Ophiocarpus (Bunge) Ikonn.; Ikonnikov, 1977). Perhaps the most obvious example of recent taxonomic uncertainty regarding the circumscription of Astragalus was the elevation of the entire subgenus Tragacan- tha (sensu Bunge, 1868, 1869) to generic rank. This taxon, consisting of more than 200 species with striking spinescent habit and reduced inflorescences and fruits, was first recognized at the generic rank as Tra- gacantha Mill. (Borissova, 1937) and more recently as Astracantha by Podlech (1983), which was later rejected (Zarre & Podlech, 1997). Given these uncertainties surround- ing the circumscription of the genus, the question of the monophyly of Astragalus itself has remained open. Worse still, the view of Astragalus as a paraphyletic assem- blage or "wastebasket," basal within Gal- egeae with other genera (and other tribes) potentially nested within it (e.g., Polhill, 1981b), has prevailed well into the late 20 ~h century.

In this context then, the first cladistic studies on Astragalus began with analyses of North American species using morphological characters (Sanderson, 1991) and molecular sequence and length polymor- phism data (Liston, 1992; Sanderson & Doyle, 1993; Wojciechowski et al., 1993) in order to identify monophyletic groups and begin to resolve their higher-level (infrageneric) relationships to Eurasian and South America taxa. This was followed by broader-scale analyses of molecular data from representative taxa of Galegeae and related tribes (Liston & Wheeler, 1994;

384 BR1TTONIA [VOL. 57

Sanderson & Wojciechowski, 1996) to resolve the relationships of Astragalus to Ox- ytropis, and certain other genera in Gale- geae, within the larger " tempera te herbaceous g roup" of papilionoids circumscribed by Polhill (1981a). The most recent molecular phylogenetic analyses (Wojciechowski et al., 1999; Kazempour Osaloo et al., 2003, 2005), though still sparse in terms of sampling ( < 1 0 % of species), have focused more on a selection of Eurasian and North American species, based on morphological and cytogenetic diversity or the current sectional classification, to test the monophyly of infrageneric level groups as well as of the genus itself.

In this paper, I review how our ideas of Astragalus, both with respect to that of its phylogenetic relationships, at the infrageneric level and among closely related genera, and its diversification within the papilionoid legumes, have progressed in the past decade with the advent o f cladistic methods and molecular data. I argue that such approaches have had a dramatic im- pact on our understanding of Astragalus, but because of the unwieldiness of the genus, in terms of size and morphological complexity, and persistent lack of comprehensive sampling for most groups, this will remain a progress report.

Materials and Me t hods

TAXON SAMPLING

The study presents an analysis of new plastid matK gene and nuclear rDNA internal t r ansc r ibed spacer 1&2/5 .8S gene (nrDNA ITS) sequences f rom a number of taxa (see Appendix for source information), in combinat ion with previously published nrDNA ITS (Sanderson & Wojciechowski, 1996; Wojciechowski et al., 1999; Hu et al., 2002) and matK gene sequences (Wojcie- chowski et al., 2004). These studies should be consulted for relevant voucher information. Genomic DNAs were isolated f rom field-collected, g r eenhouse -g rown plants, silica-dried and herbarium material as described previously (Wojciechowski et al., 2004). Polymerase chain reaction amplifi- cation and sequencing of the n rDNA ITS and matK regions were per formed as de-

scribed previously (Wojciechowski et al., 1999, 2004). DNA sequencing was per- formed on an Applied Biosystems 3100 sequencer at the Arizona State University (DNA Laboratory, Tempe, Arizona, USA). Sequencer output files were assembled into contigs and edited using the program Se- q u e n c h e r 4.1 ( G e n e C o d e s , Ann Arbor , Michigan, USA) before alignment.

PHYLOGENETIC ANALYSES

Phylogenetic analyses of the combined matK plus nrDNA ITS sequence data set, consisting of 44 terminal taxa, utilized maximum pars imony (MP) and Bayesian approaches. Parsimony analyses were per- f o r m e d us ing PAUP* (ve r s ion 4 .0b10; Swofford, 2002) on Macintosh or Linux computers. Multiple tree searches were conducted using heuristic search options that included SIMPLE, CLOSEST, or RAN- D O M addition sequences (1000 replicates) holding five trees per replicate, and tree-bi- section-reconnection (TBR) branch swap- ping, with retention of multiple parsimonious trees (e.g., M A X T R E E S = 1000). Bayesian analyses were per formed using MrBayes version 3.0 (Huelsenbeck & Ron- quist, 2001) as described previously (Wo- jc iechowski et al., 2004; Lavin et al., 2005). Clade support was assessed using both non- parametric bootstrap resampling (Felsen- stein, 1985) and Bayesian posterior probabilities (Huelsenbeck et al., 2002). Non- parametric bootstrap proportions were esti- ma ted f r o m 500 b o o t s t r a p rep l ica tes incorporating heuristic pars imony searches using addition sequence and branch swap- ping options as in our standard pars imony analyses (as described above). Bayesian posterior probabilities were est imated as the proportion of trees sampled after 'burn- in ' that contained each of the observed bipar- titions. Combinabil i ty of the matK and n rDNA ITS data sets was assessed using the partition homogenei ty test ( ILD test) as implemented in PAUP*, using identical search options. Based on results f rom several previous studies of relationships within the inverted repeat-lacking clade (IRLC, see below), a large, well supported clade that is 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 chloroplast DNA (Lavin et al., 1990: Lis- ton, 1995), Glyo'rrhi=a lepidota was designated as the outgroup in this analysis.

Age estimates for Astragalus and the As- tragalean clade are based on analyses of evolutionary rates and divergence times of the family-wide plastid matK (335 taxa; Wojciechowski et al., 2004) and rbcL gene (241 taxa: Kajita el al., 2001) phylogenies presented in a recent study (Lavin et al., 2005). In that study, age constraints derived from the abundant, temporally continuous and worldwide Tertiary macrofossil record (Herendeen et al., 1992), were imposed on specilic clades in the matK (and rbcI,) gene trees, including the ages of the root node of legumes (tixed at 6 0 - 7 0 Ma) and 12 internal nodes (as minimum ages). The node closest to Astragalus with an assigned minimum age constraint was that of the Robinia stem clade (34 Ma: Laven et al., 2003), which is nested within the sister group to the inverted-repeat-lacking clade (see below). A matK data set containing 97 sequences (aligned length of 1521 characters) from Astragalus and papilionoid relatives in Hologalegina, which included 80 from the family wide analysis (Wojciechowski et al., 2004) plus 17 new sequences, was used in the present rates analysis. A nucleotide substitution model was selected using AIC implemented in Modeltest (version 3.06: Po- sada & Crandall, 1998). Branch lengths were estimated using a Bayesian approach that incorporated a GTR + I" + I model of nucleot ide subst i tut ion. One hundred Bayesian trees sampled at stationarity were then systematically analyzed using the penalized likelihood (PL) method of Sander- son (2002) in the program r8s (vers. 1.6: Sanderson, 2003), as described previously (Lavin et al., 2005). In addition to the age constraint imposed on the Robinia stem clade, the age of the Hologalegina crown clade (" root" node) was tixed at 50.6 Ma for the presen! analysis. The latter age constraint is based on the estimated age for this clade fi'om the results of Lavin et al. (2005).

Resul ts

COMBINED MATK/NRDNA ITS DATA

The combined matK + nrDNA ITS dataset included a total 2253 aligned positions

(matK, 1551; nrl)NA ITS, 702), with 508 potentially parsimony-informative characters (24%, which excludes 137 indel and missing characters). The partition homoge- neity test results showed the two data sets were not incongruent (p = 0.080). Maxi- mum parsimony analyses of the combined dataset (2116 characters included) resulted in a single most parsimonious tree of 2001 steps (CI -- 0.5857: RI = 0.7182). This tree, with non-parametric bootstrap and Bayesian support values, is shown in Figure 1. Compared to the parsimony tree, minor incongruencies, such as the position of A. sinicus/A, complanatus (syn. Phyllolobium chinense Fisch.) lineage in the Coluteoid clade, were observed in the Bayesian con- sensus, but these do not conflict in terms of clades with high branch supports (e.g., parsimony bootstrap values over 60%).

The results shown in Figure 1 are gen- erally consistent with results based on previous, separate analyses of nrDNA ITS sequences (Wojciechowski et al., 1999) and marK gene sequences (Wojciechowski et al., 2000, 2004), in resolving a monophyletic Astragalus (in the strict sense) nested within the larger, strongly supported Astra- galean clade (Sanderson & Wojciechowski, 1996). The combined analysis of nrDNA ITS with matK data presented here, how- even provides greater support for the Col- uteoid clade (and slightly better support of others such as O,vytropis) as well as a more completely resolved tree with taxa such as Oxvtrotffs, Astragalus vogelii, Astragalus epiglottis, and Astragalus pelecinus (syn. Biserrula pelecinus L.; Barneby, 1964, p. 26) whose relationships within the Astra- galean clade were not resolved unequivocally by previous analyses of nrDNA ITS sequences alone (Wojc iechowsk i et al., 1999: Kazempour Osaloo et al,, 2003). The tinding that A. epiglottis plus A. pelecinus forms the sister group to Astragalus sensu stricto as defined previously (Wojciechows- ki et al., 1999) is not unexpected but this relationship has not been unequivocally resolved, or well supported, in analyses of nrl)NA ITS or plastid ndhF sequences alone (Wojciechowski et al., 1999; Kazem- pour Osaloo et al., 2003). Moreover, results based on the combined analyses of matK

386 BRITTONIA [VOL. 57

98

Glycyrrhiza lepidota Caileryaatropurpurea

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Parochetus communis

F ~ - - 4

99i

Vicioid c l a d e / ~ ~ , 100 ,~,---~ ~. . . . . . . . . . . . . .

,00L Caragana arborescens

Galega orientalis Cice r arietin urn

Medicago sativa Melilotus alba

Trigonella foenu m-g raecum Trifo liu m n an u m

Pisum sativu m Viciafaba

Lens culinaris

"~ i00 [

Hedysaroid ~'1 clade

93

I i00 Astragalean clade "~

74

i00

Alhagi maurorum Onobrychis montana

Hedysaru m boreate Podlechiellavogelii

~ . Swainsona pterostylis Clianthus puniceus

Carmich aelia williamsii ~ L 100 [ Astragalus sinicus

L Phyllolobium chinense Astragalus cysticalyx Coluteaarborescens

essertia herbacea utherlandia frutescens

xytropis lambertii I Oxytropis pilosa Oxytropis Oxytropis deflexa

t ~ Astragalus epiglottis Astragalus pelecinus

Coluteoid clade

87

J i L

A s t r a g a l u s s s "r 80L.-

73

- - Astragalus corrugatus - - - - Astragalus amedcanus

- - Astragalus atropilosulus 0~________~_l ~st rag alu s adsurgens

Astragalus alpinus stragalus cerasocrenus

- - 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. Glycyrrhiza is designated as the outgroup for all analyses.

2005] WOJCIECHOWSKI: MOLECULAR PHYLOGENET1C OF ASTRAGALUS 387

and nrDNA ITS sequences presented here (Fig. 1) concur with Wojciechowski et al. (2004) which first showed significant support (both bootstrap values and Bayesian posterior probabilities) for placement of the genus Oxytropis as the sister group to As- tragalus sensu stricto plus A. epiglottis/A. pelecinus, within the Astragalean clade. Previous analyses have suggested Oxytropis is more closely related to the Coluteoid clade (Liston & Wheeler, 1994; Sanderson & Wojciechowski, 1996; Kazempour Osa- loo et al., 2003), forms the basally branch- ing lineage within the Astragalean clade (Wojciechowski et al., 2000), or was unresolved (Wojciechowski et al., 1999).

The combined analysis of matK and nrDNA ITS data also provides measurable support for a sister group relationship of Caragana plus the hedysaroid clade to the Astragalean clade, a relationship that has been variously, but mainly weakly, supported in analyses of matK (Wojciechowski et al., 2000, 2004) or n rDNA ITS alone (Sanderson & Wojciechowski, 1996; Hu et al., 2002). This sister group relationship of Caragana plus the hedysaroid clade to the Astragalean clade, rather than sister to the Vicioid clade, is consistent with suggestions (e.g., Polhill, 1981b) that Caragana and allies as well as tribe Hedysareae, which are p r edominan t l y Euras ian Nor th A m e r i c a n groups, were derived f rom the "astragaloid p a r t " o f G a l e g e a e (Astragalus and its woody relatives) in Asia.

Relationships within Astragalus are also consistent with previous results based on analyses of nrDNA ITS data alone (Wojcie- chowski et al., 1999; Kazempour Osaloo et al., 2003). For example, the North Ameri- can euploid species, represented in this analysis by A. adsurgens, A. atpinus, A. americanus, and A. canadensis, are more closely related to Old World taxa than to the Nor th A m e r i c a n aneup lo id species . While sampling for the combined data set is more limited, species f rom each of the " m a j o r " Old World groups ( " A " - " E " ) described in Wojciechowski et al. (1999) are represented here, and Neo-Astragalus is resolved as a moderate ly supported clade nested within one of these Old World groups ( " E " ; Wojciechowski et al., 1999).

In the present analysis, the three South American species fo rm a well-supported monophylet ic group sister to the two North American aneuploids, but in other analyses with greater sampling of North and South American species overall (Wojciechowski et al., 1999; Scherson et al., 2005) the South American species comprise at least two, independently derived clades within Neo-Astragalus.

ESTIMATED AGES OF ASTRAGALEAN AND ASTRAGALUS CROWN GLADES

Lavin et al. (2005) provides estimated ages of many of the larger clades of legumes that can serve as calibration points for rates analyses of groups that lack a fossil record, such as the Astragalean clade. Fossil evidence for taxa within the IRLC is minimal at best; Vicia-like leaves with ten- drils and inflated, Phaca-like pods have been described f rom the Florissant Beds flora of Colorado, USA (Oligocene, ca. 30 Ma; MacGinitie, 1953). Because these have not been definitively identified and there- fore cannot be unequivocal ly assigned to any specific node amongst the clades of interest here, they provide no useful t ime calibration. For this reason, a rates analysis of matK sequences f rom the larger Hologale- gina clade, with both estimated and fossil- calibrated nodes (Lavin et al., 2005), was conducted.

The mean PL substitution rates across 100 Bayesian trees for nodes identified in the matK phylogeny derived f rom analyses of the 97-taxon data set (not shown) range f rom an average of 8.3 X 10 10 substitutions per site per year across the IRLC to 10.6 X 10 -1~ substitutions per site per year for the Vicioid clade (Table I; rates ex- pressed as s/s/Ma). The age estimated for the Astragalean clade here is comparable to that presented in Lavin et al. (2005), although slightly older (16.1 Ma -+ 1.82 versus 14.8 Ma). The est imated ages for As- tragalus (12.4 + 1.45 Ma) and Neo-Astrag- alus (4.4 _+ 0.78 Ma) are also similar to an earlier estimate (11 Ma, 4 - 5 Ma, respectively) based on the simplistic assumption of a molecular clock for n rDNA ITS se-


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)

H o l o g a l e g i n a Sesbania vesicaria- 50 ,6* . . . . . ( " r o o t " ) Astragalus patagonicus

R o b i n i e a e Robinia pseudoacacia- 34 ,0** - - 34 .0 - - - - - - Coursetia axillaris

I R L C Glycyrrhiza lepidota 36 .0 2 . 4 0 30 .9 43 .7 0 . 0 0 0 8 2 5 0 . 0 0 0 0 6 8 -Astragalus patagonicus

A s t r a g a l e a n Sutherlandia frutescens- 16.1 1 .82 12.1 20 .6 0 . 0 0 0 9 1 0 . 0 0 0 0 7 8 Astragalus patagonicus

Astragalus Astragalus pelecinus 12.4 1.45 9.3 17.7 0 . 0 0 0 9 4 9 0 . 0 0 0 0 8 3 -Astragalus patagonicus

N e o - A s t r a g a l u s Astraga/us lonchocarpus- 4 .4 0 .78 2 .8 6 .8 0 . 0 0 1 0 2 1 0 . 0 0 0 0 9 4 Astragalus patagonicus

Oxytropis Oxytropis deflexa- 4 .6 0 .92 2 .4 7.1 0 . 0 0 0 8 9 9 0 . 0 0 0 0 8 8 Oxvtropis pilosa

C o l u t e o i d c l a d e Carmichaelia williamsii- 13.7 1 .70 10.1 19.4 0 . 0 0 0 8 9 4 0 . 0 0 0 0 8 0 Sutherlandia frutescens

H e d y s a r o i d c l a d e Alhagi rnaurorum- 21 .4 2 .43 16.3 26 .9 0 . 0 0 0 9 3 5 0 . 0 0 0 0 7 9 Hedysarum boreale

Vic io id c l a d e Galega orientalis- 26.8 2 .20 22 .5 33 .4 0 . 0 0 1 0 5 9 0 . 0 0 0 0 8 3 Vicia sativa

P L a n a l y s e s u sed " T N " a l g o r i t h m , s m o o t h i n g v a l u e S = 6 3 . 1 0 (10t s). T h e first n o d e , H o l o g a l e g i n a * , is a s s i g n e d a f ixed a g e (50 .6 M a ) a n d the s econd , R o b i n i e a e * * , is a s s i g n e d a m i n i m u m a g e ( 3 4 . 0 Ma) . M a = m i l l i o n y e a r s ; S D = s t a n d a r d d e v i a t i o n ; s /s = subs t i t u t i ons p e r site.

quence evolution (Wojciechowski et al., 1999).

Discussion

Rupert C. Barneby, in his monumental Atlas of North American Astragalus (1964), discussed in detail the ecological specialization and evolut ionary significance of many of the morphological characters he had observed in his extensive study of As- tragalus, among them the development of the bilocular pod, the independent origins of annual forms, and graduation of the pet- als. He envisioned that multiple invasions, representing different lineages, of astragali from Asia into the Americas had occurred without an obvious reciprocal exchange, and that the endemic or "autochthonous" species groups (aneuploid American As- tragalus) had their origins in subtropical arid regions rather than in the colder, high latitudes they would passage during migra- tion from eastern Asia via the Bering land bridge. Furthermore, he suggested the long separate history of the 'endemic' New World and presumably more recent immi-

grant Old World groups, as implied by dif- ferences in apparent dispersal ability and the cytogenetic evidence then available, would justify their taxonomic separation (section level and above) in the American flora even if morphological evidence for that separation was lacking (Barneby, 1964; p. 30-31). Beyond the recognition that the North American species represented seven major phylogenetic groups (his "phalanx- es"), Barneby did not venture an opinion on their relationships, suggesting that such speculation rested on the assumption that evolution and specialization proceeded in concert, the evidence for which he considered "contradictory and at best controver- sial" in Astragalus (Barneby, 1964; p. 21). Indeed, his skepticism about the validity of a one-dimensional sequence to ever accu- rately reflect the true evolutionary history of Astragalus led him to the conclusion (Barneby, 1964; p. 21) that "the so-called phylogenies, which current botanical fash- ion seems to require of the taxonomic worker, are for the most part flimsy struc- tures, compounded of nine parts speculation


to one of observed fact; many of them col- lapse under logical scrutiny and serve no useful purpose."

While Barneby may have doubted the usefulness of phylogenies forty years ago, the molecular phylogenetic studies that began in the 1990s have had a significant im- pact on our understanding of the relationships and diversification of Astragalus, and their findings vindicate some of his ideas about the genus. Early studies (Liston & Wheeler, 1994; Sanderson & Wojciechows- ki, 1996) indicated that Astragalus is nested within a clade now referred to as the IRLC. This clade, consisting of all members of the traditionally circumscribed tribes Carmi- chaelieae (or Carmichaelinae, Wagstaff et al., 1999), Cicereae, Galegeae, Hedysareae, Trifolieae, and Vicieae (Fabeae), and at least three genera of Millettieae (Callerya Endlicher, sampled here, Afgekia Craib, Wisteria Nutt.), is a vast, cosmopoli tan as- semblage (>4200 spp.) that corresponds closely to Polhill 's (1981 a) " temperate herbaceous group," and one of two subclades of Hologalegina, the largest clade of papi- l ionoid l egumes ( W o j c i e c h o w s k i et al., 2000). The monophyly of the IRLC has been consistently and strongly supported in all subsequent cladistic analyses of molecular sequence data (Doyle et al., 1997; Hu et al., 2002; Wojciechowski et al., 2000, 2004; Kajita et al., 2001) as well as plastid D N A structural evidence (Lavin et al., 1990; Liston, 1995; M. E Wojciechowski, unpubl, data). Nested within this radiation of predominantly herbaceous and temperate papilionoids, Astragalus sensu lato together with Oxytropis, Galegeae subtribes Colutei- nae and Carmichaelinae, comprise a well- supported group we have previously referred to as the Astragalean clade (Sander- son & Wojciechowski, 1996). The Astra- galean clade (Fig. 1) had consistently been resolved with high statistical support (e.g., 100% bootstrap proportions) and shown to be composed of two main subclades, one essentially corresponding to the subtribe Coluteinae plus Carmichael inae (the "Col- uteoid" clade) while Astragalus and Oxy- tropis comprise the other.

Scattered throughout the African-Austra- lasian Coluteoid clade is a heterogeneous

assortment of ecologically and morpholog- ica l ly d i s t inc t ive spec ies of Astragalus, which now appear based on molecular evidence to be more closely related to members of Coluteinae than to Astragalus. This includes the annual Astragalus vogelii, recently raised to generic rank as Podlechiel- la Maassoumi and Kazempour Osaloo (Ka- zempour Osaloo et al., 2003), Astragalus cysticalyx, Astragalus complanatus (syn. Phyllolobium chinense Fisch.), and Astrag- alus sinicus (Liston & Wheeler, 1994; Wo- jc iechowski et al., 1999). The latter two species, from Bunge 's subgenus Pogono- phace, possess morphological features such as pubescent styles (Kang & Zhang, 2004) that are diagnostic of other genera in subtribe Coluteinae (e.g., Colutea) and thus consistent with their p lacement in this clade (Wojciechowski et al., 1999; this study). In fact, these and other morphological similarities led Barneby (1964; p. 1164) to con- jecture that A. cornplanatus (sect. Phyllo- lobium) and other "pr imit ively barbistyled Astragali referred by Bunge to the subgenus Pogonophace" might be more appropriate- ly treated outside Astragalus sensu lato. The phylogenetic position of subgenus Po- gonophace within the Coluteoid clade has been further substantiated by recent analyses of n rDNA ITS data (Kang et al., 2003; Kazempour Osaloo et al., 2005), which show that a number of species sampled f rom this subgenus fo rm a well-supported monophylet ic group within the Coluteoid subclade that includes the Asian-African genera Colutea, Lessertia, and Sutherlandia plus Astragalus cysticalyx, a result that has prompted the suggestion that they be treated as the genus Phyllolobium (Kang et al., 2004). However, at least three other members of this subgenus (e.g., Astragalus atropilosulus, sect. Chlorostachys) are nested within Astragalus sensu stricto.

" T a x o n o m i c a l l y i so la ted spec ies are characteristic ofAstragalus" Barneby noted (1964; p. 32), and a number of primarily annual Eurasian species of the genus at one t ime or another placed in monotypic sections have been segregated as new genera whose phylogenetic positions are for the most part unknown. Astragalus migpo, segregated as Barnebyella calycina, Astragalus

390 BRITTON1A [VOL. 57

ophiocarpus as Ophiocarpus aitchisonii, Astragalus dipelta as Didymopelta turkestanica, Astragalus schmalhausenii as Sew- erzowia turkestanica, Astragalus vicarius as Sewerzowia vicaria, and Astragalus thlaspi Lipsky as Thlaspidium (Lipsky) Rassulova, are clearly nested within Astrag- alus sensu stricto based on molecular phylogenetic evidence (Kazempour Osaloo et al., 2003, 2005). Virtually all of these species possess unusual fruit morphologies, such as laterally compressed, unilocular, one- to two-seeded, sessile to stipitate pods, or unique combinations of features that have led to their segregation f rom Astrag- alus by various authors. The phylogenetic position of the Mediterranean annual As- tragalus pelecinus (L.) Barneby (=Biser- rula pelecinus L.), which Barneby (1964; p. 26) describes as a "perfect ly unexcep- tional Astragalus," has been variously supported as sister to Oxytropis, nested with Coluteoid clade, or unresolved with respect to Astragatus sensu stricto (Sanderson & Wojciechowski, 1996; Wojciechowski et al., 1999; Kazempour Osaloo et al., 2003). This species shares a number of relatively uncommon morphological features with As- tragalus epiglottis, ano the r w i d e s p r e a d Mediterranean annual, including flowers with only five fertile stamens and dorsiven- trally compressed pods (Matthews, 1970). Molecular studies have shown these two species to be strongly supported as sister taxa (Sanderson & Wojciechowski , 1996; Wojciechowski et al., 1999) or closely related to Astragalus annularis in a well-sup- por ted subc l ade within the A s t r a g a l e a n clade (Kazempour Osaloo et al., 2003, 2005). Based on molecular evidence Liston and Wheeler (1994; p. 385) suggested that these two taxa might represent the "deepest branches in the Astragalus clade." Results presented here strongly support this hypothesis, that A. pelecinus plus A. epiglottis (and likely A. annularis) form the sister group to Astragalus sensu stricto. In light of the con- gruence (and support) of the results presented here, a more explicit phylogenetic redefinition of the genus Astragalus, so as to include these two taxa plus the clade referred to as "Astragalus sensu stricto'" as i n f o r m a l l y def ined p r e v i o u s l y (Wojcie-

chowski et al., 1999), seems warranted. Thus, Astragalus is hereby defined as the clade delimited by the most recent common ancestor of Astragalus pelecinus and As- tragalus patagonicus.

Other subgroups of Astragalus possess- ing remarkable ecological specializations or un ique c o m b i n a t i o n s of m o r p h o l o g i c a l characters that have prompted their segregation as new genera, the Eurasian Astra- cantha Podlech (Astragalus subgenus Tra- gacantha) and the North Amer ican Oro- phaca (Torr. & Gray) Britton (phalanx Or- ophaca), have also been shown to be nested within Astragalus (Wojciechowski et al., 1999). Detailed analyses of morphological characters subsequently led Zarre and Pod- lech (1997) to question the monophyly of Astracantha (and return it to Astragalus). Now, more extensive molecular sampling of Tragacantha sections for both nrDNA ITS and plastid ndhF sequences confirm the paraphyly of this subgenus within Old World Astragatus with its members scattered among other thorny, cushion forming species representing other subgenera (Ka- zempour Osaloo et al., 2003, 2005).

In contrast, the genus Oxytropis, whose taxonomic history has been long inter- twined with, if not considered "organical ly part o f " , Astragalus (Barneby, 1964; p.21), is clearly not nested within Astragalus. Rather, there is now substantial support for Oxytropis as its sister group. This result substantiates Barneby 's (1952, 1964) long held contention that Oxytropis is a distinct group of Eurasian origin that is closely related to (but taxonomically separate from) Astragalus, and, has which subsequently fol lowed a parallel course of ecological specialization, mol-phological, and biogeographic diversification.

Prior to molecular phylogenetic work, the only solid evidence for infrageneric groups within Astragalus was a near perfect correlation between ch romosome number and geographic distribution. The vast majority of Old World species possess euploid ch romosome numbers (based on n = 8) while the vast majority of North American and all South American species surveyed possess numbers in an aneuploid series of n = 11 to n = 15 (reviewed in Wojcie-


chowski et al., 1993). The earliest molecular studies (Sanderson & Doyle, 1993; Wo- jc iechowski et al., 1993; Liston & Wheeler, 1994) suggested that the North American species with aneuploid chromosome numbers form a monophylet ic group derived f rom Eurasian euploid taxa. Subsequent s tudies (Sander son & W o j c i e c h o w s k i , 1996; Wojciechowski et al., 1999) suggested the monophyly of a group comprising all New World aneuploids (Neo-Astragalus) nested within one of several groups containing euploid and the few aneuploid Eur- asian species as well as all the euploid species found in North America. These findings fur ther c o r r o b o r a t e d L e d i n g h a m ' s (1957, 1960) hypothesis of a major division in the genus based on cytogenetic data and biogeographic separation (Old World versus New World). Although the identity of the Old World sister taxon to Neo-Astragalus remains uncertain, our results suggest that the diversification of Neo-Astragalus was exclusively confined to the New World. The relatively few euploid species found in North America are representatives of Old World groups that appear to have immi- grated (e.g., by dispersal or introductions) to the New World subsequent to and unre- lated to the diversification of Neo-Astraga- lus (Wojciechowski et al., 1999).

An important outcome of all these molecular studies is a phylogenetic perspective f rom which to evaluate previous classifica- tions schemes of Astragalus, which were based largely on morphological criteria. Barneby came to the conclusion that the North American species "represented seven major phylogenetic branches of the genus" (1964; p. 33), four of which he considered equivalent to Asiatic subgenera (sensu Bun- ge, 1868, 1869) and included species representative of the most primitive as well as some of the more advanced sections of the Old World. Yet, he did not speculate on what relationships the North American pha- lanxes might have to each other, or to the Old World subgenera. He did regard the subgenus Phaca ( sensu Bunge , 1868, 1869), the largest in the Old World, which he defined more broadly to include North American species with partially to completely unilocular (inflated or not) pods, as

the "pr imit ive p h y l u m " in the genus, a con- cept not too different from that of the orig- inal Linnaean genus Phaca. But, as we point out elsewhere (Wojciechowski et al., 1999), even with taxon sampling too limited to address r igorously the question of its monophyly, it is evident from the molecular phylogenies that this phalanx (subgenus), like the others, is not monophyletic. It is also noteworthy that even with much greater sampling of Old World taxa than that study, Kazempour Osaloo et al. (2003, 2005) have come to essentially the same conclusions in showing that none of the traditionally recognized Old World subgenera, or most of the species-rich sections, is m o n o p h y l e t i c . T h e s e p h y l o g e n i e s a lso clearly refute a more recent and reductionist subgeneric classification of the Old World taxa based on a few, single characters (Pod- lech, 1982, 1991).

Fortunately, we are now beginning to see the broad outline of infrageneric relationships within the genus. The molecular phylogenies resulting f rom these studies (Wo- jc iechowski et al., 1999; Kazempour Osa- loo et al., 2003, 2005), including the results presented here, have identified several moderately to well-supported subclades within Astragalus sensu stricto (fig. 2 in Wojcie- chowski et al., 1999; Kazempour Osaloo et al., 2003; fig. 1 in Kazempour Osaloo et al., 2005). A highly simplified schematic diagram of Astragalus and its sister group Ox- ytropis based on results of phylogenetic analyses of nuclear and plastid sequence data derived f rom these studies is presented here in Figure 2. It is important to note this is meant to portray molecular phylogenetic data that is still very p re l imina ry - - the taxonomic composit ion and relationships of each of these subclades, here designated by letter names only for convenience, are for the most part still poorly resolved and lacking in statistical support or obvious diag- nosable characters, and are likely to change with more comprehensive sampling especially of Old World taxa and analyses of additional sources of data. Thus, for the foreseeable future these lettered subclades will primarily serve as landmarks to guide taxon sampling and evaluate the diagnostic potential of morphological characters that


- 4.6 Ma Oxytropis

12.4 M~

A. pelecinus/A, epiglottis

~ l clade A NA euploid(s) c~~r lade B

- - ~ NA euploid(s)

clade C

NA euploid(s)

Astragalus

clade D

~ clade F

NA euploid(s)

,,.~ ~J, N~ e o - A s t r a g a l u s

.... S A m e r i c a n 1

S American 2

ade I

lade G2

clade G1

FIG, 2. Simplified schematic diagram of Astragatus and sister group Oxytropis, showing phylogenetic relationships 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.

wil l u l t ima te ly f o r m the basis for a revised, in f ragener ic c lass i f icat ion o f the genus.

Based on an evo lu t iona ry rates analysis o f the rnatK p h y l o g e n y (Table I) the estimated age o f the As t raga lean c rown c lade is 16.1 + 1.8 Ma, with the spli t be tween Oxytropis and Astragalus to be be tween 16

and 12.4 Ma. The es t imated age o f the divers i f ica t ion o f the Astragalus (ca. 2500 spp.) c r o w n c lade is e s t ima ted at 12.4 Ma, whi le that o f the 500 or so species o f Neo- As t raga lus , in which the South A m e r i c a n species are nested and c o m p r i s e at least two independen t ly der ived subclades , is 4.4 +


0.78 Ma. Interestingly, the diversification of the Oxytropis (ca. 330 spp.) crown clade is 4.6 _+ 0.92 Ma, much later than that of As- tragalus, but similar in timing to that of Neo-Astragalus. This finding is consistent with Barneby's hypothesis of a separate origin for Oxytropis, compared to Astragalus, but parallel pattern of diversification.

Previous estimates of lineage diversification rates for Astragalus (Wojciechowski et al., 1999) suggested unusually high rates for the genus (sensu stricto) and especially Neo-Astragalus, compared to estimates for most continental plant families and other angiosperm clades (e.g., Magall6n & San- derson, 2001), and even often-cited exam- ples of insular adaptive radiations (e.g., Ha- waiian silverswords; Baldwin & Sanderson, 1998), although perhaps statistically not ex- ceptionally high compared to its closest relatives (Sanderson & Wojciechowski, 1996). Using the ages for Astragalus and Neo-As- tragalus estimated here (based on the matK phylogeny) , l ineage diversification rates based on standing diversities (r = In N/t; referred to in Wojciechowski et al., 1999) would be 0.63 spp./Ma for Astragalus and 1.41 spp./Ma for Neo-Astragalus, using their ages (t) and diversities (N) of 2500 and 500 species, respectively. These estimates are comparable to, and thus corrob- orate, previous estimates for these same clades (0.71 and 1.48 spp./Ma, respectively; Wojciechowski et al., 1999) based on nr- DNA ITS sequence evolution. Furthermore, these estimates are unusually high when compared against the average rates of 0.50 spp./Ma estimated for the Astragalean clade (N = 3070; Sanderson & Wojciechowski, 1996) and 0.23 spp./Ma estimated for the IRLC (N = 4250; Wojciechowski & San- derson, 1996). Yet, estimated rates of nucleotide substitution for Astragalus and its relatives in the Astragalean clade, based on fossi l-cal ibrated, f ami ly -wide molecular phylogenies (matK and rbcL, Lavin et al., 2005) like those reported here, indicate that absolute rates of molecular evolution for this group (9.1 • 10 m substitutions per site per year; Table I) are not at all unusual when compared to other, mainly temperate groups of papilionoid legumes (from Lavin et al., 2005): e.g., Crotalaria-Lupinus, 8.1-

9.0 • 10 10 substitutions per site per year; Amorpheae, 8.4 • 10-m; robinioids, 7.8 • 10-r~ vicioid clade, 8.4-10.8 • 10 10; Car- agana plus hedysaroids, 7.7 • 10 10. Such new estimates of rates of molecular evolution in this and other papilionoid groups, combined with newer, more rigorous, quan- titative methods now permit a more accu- rate assessment of lineage diversification rates for Astragalus (M. E Wojciechowski & M. J. Sanderson, unpubl, data), and un- doubtedly will help us to explain the remarkable pattern of continental diversification observed in this, unusually species-rich clade.

A c k n o w l e d g m e n t s

The author thanks R. Scherson and A. Liston for providing genomic DNAs of several South American and Asian Astragalus. The author thanks K. R Steele, A. Delgado- Salinas, and an anonymous reviewer for their many helpful comments and suggestions to improve earlier versions of this manuscript. Lastly, the author especially wishes to thank A. Liston for originally suggesting the idea of a symposium to hon- or the legacy of Rupert C. Barneby and his many contributions to legume systematics, and M. J. Sanderson for reinvigorating an earlier interest in this genus and for many memorable field excursions in search of the often-elusive Astragalus. This study was supported in part by funds provided by Ar- izona State University to the author.

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Appendix

S p e c i e s , c o l l e c t i o n l o c a l i t y , v o u c h e r spec imen, and G e n B a n k access ion number s for new sequences derived for this study ( l m a t K , 2nrDNA ITS) are provided, Data sets in N E X U S format are ava i lab le f rom the author, All other sequences used in this study have been published previously (San- derson & Wojc iechowsk i , 1996; Wojc iechowski et al., 1999, 2004; Hu el al., 2000) and are available from GenBank (http://www. ncbi.nlm.nih.gov/Entrez/index.html) and from TreeBASE (h t tp : / /www. t reebase .org / ) .

Astragalus adsurgens Pallas; China (USDA PI 462310); Wojciechowski & Sanderson 267 (ARIZ); JAY920437. Astragalux alpinus L.; USA, Wyoming (USDA PI 232536); Wojciechowski & Sanderson 183 (ARIZ); ]AY920438. Astragalus arnottianus (Gillies) Reiche; Argentina; Sanderson 2520 (DAV); ~AY920439. Astragalus atropilosulus (Hochst.) Bunge var. venosus (Hochst.) Gillett; Kenya (USDA Pl 193735); Wojciechowski & Sanderson 301 (ARIZ); ~AY920440. Astragalus cerasocrenus Bunge; Iran (DELEP 880043); IAY920444. Astragalus complanatus R. Br. (syn. Phyllolobium chmense Fisch.); China (DELEP 900279); Liston 889 (OSC); rAY920441. As-

tragalus corrugatus Bertol.; Iran (USDA PI 227441); Wojciechowski & Sanderson 164 (ARIZ); IAY920442. Astragalus cysticalyx Ledeb.; former USSR (USDA PI 440146); Liston 961 (OSC); ~AY920443. Astragalus epiglottis L.; Israel (Liston 892, OSC); Wojciechowski & Sanderson 405 (ARIZ); IAY920445. Astragalus patagonicus (Phil.) Spegazzini; Argentina; Sanderson 2515 (DAV); JAY920446. Astragalus pehuenehes Niederlein; Argentina; Sanderson 2518 (DAV); ~AY920447. Astragalus pelecinus (L.) Barneby (syn. Biserrula pelecinus L.); Australia (adventive, USDA PI 186284); Wojeieehowski & Sanderson 294 (ARIZ); JAY920448. AsCraga[~is peristereus Boiss. & Hausskn.; lran (DELEP 880051); rAY920449. Astragalus xinicus L.; China (USDA PI 150557); Wojciechowski & San- derson 408 (ARIZ); rAY920450. Astragalus vogelii (Webb.) Bornm. (syn. Podlechiella vogelii (Webb.) Maassoumi et Kazempour Osaloo); Egypt, Sinai; Lis'- ton 890 (OSC); 'AY920451. Galega orientalis Lain; former USSR (USDA PI 325337); Wojciechowski & Sanderson 399 (ASU); 2AY553709. Lessertia herbacea DC.; South Africa (USDA PI 207923); Wojcie- chowski & Sanderson 299 (ARIZ); ~AY920453. Oxy- tropis pilosa (L.) DC.; former USSR (USDA P1 420696); Wojciechowski & Sanderson 306 (ARIZ); ~AY920452. Parochetus communis Buch.-Ham. ex D. Don; cultivated (A. Liston); Wojciechowski 901 (ASU); 2AY553710.

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