The phylogenetic position of the genus Astragalus (fabaceae): Evidence from the chloroplast genes rpoC1 and rpoC2

BiochemicalSystematicsand Ecology,Vol. 22, No. 4, pp. 377-388, 1994 ElsevierScience Ltd Printed in Great Britain

Pergarnon 0305-1978(93)E0022-1

The Phylogenetic Position of the Genus Asfragalus (Fabaceae): Evidence from the Chloroplast Genes rpoC1 and rpoC2 AARON LISTON and JOHN A. WHEELER Department

of Botany and Plant Pathology, Cordley Hall 2082, Oregon State University, Corvallis, OR 97331-2992, U.S.A.

Key Word Index--Astmgaus; Galegeae; Fabaceae; chloroplast-DNA; rpoC1; reaction; restriction-site mapping; molecular phylogeny; weighted parsimony.

rpoC2;

polymerase

chain

Abstract-A molecular phylogeny was inferred from the analysis of mapped restriction site variation in 51 species representing the genus Asttzgaus and putatively related genera in the legume tribes Galegeae, Millettieae, Trifolieae and Vicieae. The resulting hypothesis of phylogenetic relationships suggests: (1) the majority of Astigahs species share a common ancestor within the Galegeae; (2) the recognition of the segregate genera Astracantha and BiserruIa makes Astragaus paraphyletic; (3) aneupoloidy and the annual habit have been derived on multiple occasions within the genus Astragalus; and (4) Astragalus as currently circumscribed is polyphyletic since the species A. conplanatus, A. cy&a/yx, A. sinicus and A. vogeliishare a common ancestor with members of the subtribe Coluteinae.

Introduction L. (Fabaceae: Faboideae) is generally considered the largest genus of vascular plants with an estimated 2500-3000 species (Podlech, 1986; Lock and Simpson, 1991). It is a member of the tribe Galegeae (Polhill, 1981) and belongs to the clade of papilionoid legumes which have lost the chloroplast DNA inverted repeat (Lavin et al., 1990; Liston, in press). Astragalus is widely distributed in temperature regions of the Northern Hemisphere. The greatest numbers of species are found in the arid, continental regions of western North America (400 species) and Central Asia (2000-2500 species). An additional 150 species are known from temperate South America and one species extends along the East African mountains to Transvaal, South Africa. Molecular data has been previously used to formulate hypotheses of phylogenetic relationships among the North American species (Liston, 1992; Sanderson and Doyle, 1993) and to test the hypothesis that the North American species represent a monophyletic lineage (Wojciechowski et al., 1993). The goal of the present study is to develop a hypothesis for the phylogenetic position of the genus Asttzgaus within the tribe Galegeae based on mapped restriction sites in the chloroplast genes rpoC1 and rpoC2. The taxonomic history of the genus Astragahs has been recently reviewed (Sanderson and Liston, in press). The genus is morphologically diverse, and several schemes of generic segregation have been proposed in the past. Hutchinson (1964) lists 96 synonyms of Astragalus. However, most systematists now recognize a single large genus. Only the Eurasian segregate Astmcantha Podl. ( = subgenus Tragacantha Bunge; Podlech, 1982, 1983) is commonly accepted (cf; Lock and Simpson, 1991; Engel, 1991). Astracantha is recognized not on the basis of any unique morphological feature, but rather a set of correlated characters including rachis thorns, cushion growth form, sessile turbinate calyces and unilocular, one or two seeded fruits (Podlech, 1982). This “tragacanthic” habit is also found in species of several section Astragahs

(Received 7 January 1993) 377

378

A. LISTON AND J. A. WHEELER

retained in Astragalus. Engel (1991) provides anatomical evidence for the distinction of the tragacanthic Astragalus from Astracantha. The subgeneric classification of Astragalus has developed independently in the Old and New Worlds (Barneby, 1964; Podlech, 1990 and references therein). Bunge (1868, 1880) recognizes 10 subgenera for the Old World Astragalus, while Barneby recognizes seven informal "phalanxes" in North America. Four phalanxes representing 15 species are equivalent to four of Bunge's subgenera, while the remaining 350 species are accommodated in three endemic phalanxes (Barneby, 1964). The Old World species can be placed in an estimated 150 sections (Podlech, 1986). A total of 93 sections are recognized for the North American species (Barneby, 1964). No subgeneric or sectional classification exists for the South American species (Johnston, 1947). A fundamental cytological distinction separates the Old and New World species of Astragalus (reviewed in Spellenberg, 1976). The three endemic New World phalanxes are aneuploid (n = 11-15) while the majority of Old World species have euploid chromosome numbers of n = 8, 16, 24 . . . . However, approximately 10% (35 out of 312) of the Old World chromosome counts can be interpreted as aneuploid (Ashraf and Gohil, 1988). Recent phylogenetic analyses of chloroplast DNA (cpDNA) restriction site and nuclear ribosomal sequence data have provided molecular evidence for the monophyly of this aneuploid New World clade (Liston, 1992; Sanderson and Doyle, 1993; Wojciechowski et al., 1993). Nevertheless, no diagnostic morphological features have been associated with this difference. Likewise, no obvious morphological synapomorphies support the monophyly of the entire genus (Sanderson and Liston, in press). The large number of species, as well as the edaphic and morphological specializations of many of them, could be interpreted as evidence for a derived position of the genus within the Galegeae. Alternatively, the genus might be considered an artificial (paraphyletic or polyphyletic) assemblage which has given rise to other genera of the Galegeae. The fact that several apparently plesiomorphic characters are found in some Astraga/us species (e.g. reduced pulvini is some shrubby species, cf. Polhill, 1981) would support this second hypothesis. The elucidation of phylogenetic relationships within Astragalus and the Galegeae should resolve these conflicting alternatives. Restriction site analysis of PCR-amplified genomic regions has been applied in a number of recent studies (Arnold et al., 1991; Cohan et al., 1991; Scholfield, et al., 1991; Chen et al., 1992; Liston 1992; Rieseberg et al., 1992) and has several appealing features. These include (1) reduced time, expense and requirements of equipment and space; (2) ability to use reduced amounts of DNA (particularly important when isolating from small organisms or valuable/limited specimens or cultures); (3) robustness of the polymerase reaction (crude total genomic DNA isolations are satisfactory; (4) non-radioactive detection of restriction fragments; and (5) ability to easily target specific regions of the genome to suit the application. This technique was used to construct restriction site maps of the chloroplast genes RNA polymerase C1 (rpoC1) and RNA polymerase C2 (rpoC2). These genes are located in the large single copy region of the chloroplast genome. The gene rpoC1 contains an intron (absent in rice and maize) and is separated from rpoC2 by an intergenic space (IGS). The utility of these genes in the phylogenetic analysis of Astragalus has been previously demonstrated (Liston, 1992). Materials and Methods Taxonomicsampling. A total of 51 species representing the Galegeae and related tribes was sampled (Table 1), Astragalus was represented by 28 species from 23 sections of the genus. Nine of the 10 Eurasian subgenera and five of the seven North American phalanxes were sampled. Considering the vast size of Astragalus,

CHLOROPLASTGENESOF ASTRAGALUS

379

approximately 1% of its species could be included in the present study. An effort was made to include a broad morphological and geographic representation of the genus. In addition, fifteen genera of the Galegeae (including all four subtribes) and six members of the Trifolieae, Vicieae and Millettieae were included (Table 1). The sampled Astragalus species incuded six North American (4, 17, 25, 26, 28, 31), two South American (24, 27) and three Eurasian aneuploids (15, 18, 22). With the exception of three circumboreal species (5, 7, 10), the remaining euploids are restricted to Eurasia or the mountains of East Africa (9 only). Among the euploids, multiple species of subgenus Phaca (7, 10, 11, 21, 23) and Pogonophace (9, 13, 29) were included. These subgeners have been considered to represent basal lineages in Astragalus (Barneby, 1964; Podlech, 1986; Wenninger, 1991). Fourteen of the sampled Astragalus are herbaceous perennials (5, 6, 7, 9, 10, 11, 12, 13, 16, 17, 21, 24, 26, 27), thirteen are annuals (4, 8, 14, 15, 18, 19, 20, 22, 25, 26, 29, 30, 31) and one species shares the tragacanthic habit with Astracantha (23). rpoCl-rpoC2 analysis. The PCR amplification of the plastid genes rpoC1 and rpoC2 followed the procedure of Arnold et al. (1991) as modified by Liston (1992). Total DNAs were isolated following the protocol of Doyle and Doyle (1987). The amplification reactions were conducted for 1 min at 94°C, followed by 35 cycles of 1 min at 94°C (denaturation), 1 min at 55°C (annealing) and 3 min at 72=C (primer extension) with a final 7 min at 72°C. The oligonucleotide primers 5'-AAGCGGAATTTGTGCTTGTG-3' (rpoC1-195) and 5"-TAGACATCGGTACTCCAGTGC-3" (rpoC2-1364) were used (Liston, 1992). The addition of 10 p.g bovine serum albumin to each 100 Id reaction increased amplification reliability and yield. The amplification products were digested with the following 19 restriction enzymes: Haelll, Hhal, (4 bp recognition sites); BsrnAI (5 bp recognition site); Asel, BarnHI, BstBI, BstXI, Clal, Dral, EcoRI, EcoRV, Kpnl, Mlul, Ndel, Pstl, Sspl, Xbal, Xhol and Xmnl (6 bp recognition sites). The digested PCR products were electrophoresed in 1.4% agarose gels, stained with ethidium bromide and photographed. Double digests were performed to determine the linear order of restriction sites. Site mapping was aided by comparisons to published rpoC1 and rpoC2 sequences (Ohyama et al., 1986; Shinozaki et al., 1986; Hudson et al., 1988; Shimada eta/., 1990; Igloi etaL, 1990). Data analysis. Nucleotide divergence was estimated by the maximum likelihood method of Nei and Tajima (1983). Wagner parsimony analysis was carried out on a Sun 6/670 computer with the UNIX version of PAUP 3.0s (Swofford, 1991). Restriction sites were scored as binary characters. Two genera of the Millettieae (Callerya, Wisteria) were designated outgroups. The heuristic search options CLOSEST and SIMPLE addition sequences were used with TBR (tree bisection-reconnection) swapping. In addition, 500 replicates of RANDOM addition sequences, followed by TBR swapping on the resulting trees, was performed in order to uncover potential islands of equally parsimonious trees (Maddison, 1991; Wojciechowski et al., 1993). Bootstrap resampling (Felsenstein, 1985; Sanderson, 1989) was performed using 1000 replicates of heuristic searches; each replicate consisted of 500 random addition sequences. Parsimony analysis with a priori weighting based on the relative probabilities of restriction site gains and losses was also conducted. Restriction site gains were weighted 1.3 and 1.1 times site losses (Albert et aL, 1992).

Results The primers rpoC1-195 and rpoC2-1364 amplified an approximately 4100-4150 bp product in all 51 species. Digestion with 19 endonucleases revealed a total of 99 restriction sites, 89 of which were variable (Fig. 1). Restriction site mapping revealed two regions of length mutation. In the species Astragalus contortuplicatus, the nontranscribed spacer between rpoC1 and rpoC2 is approximately 10 bp longer than in any other surveyed taxon. More extensive length differences were observed in the intron. Small length variations which occur between closely related species, although of potential phylogenetic information (Liston, 1992), were not quantified in this study. The intron in the genera Alhagi, Callerya, Calophaca, Caragana, Galega, Glycyrrhiza, Halimodendron and Mille~a is larger than the other taxa by approximately 50 bp. However, the homology of this mutation is uncertain, and it was not included in subsequent phylogenetic analysis. Percentage cpDNA divergence, as calculated from restriction site differences, ranged from 0 to 5.1%. The following pairs and trios of taxa had identical haplotypes for the surveyed endonucleases: As~acantha amblolepis, Ac. eriocephala and Astragalus icmadophilus; As~ragalus asterias and A. cicer; A. cyrnbicarpos, A. filicaulis and A. hamosus; A. acutirostris, A. leptocarpus and A. rattanii; A. wrightii and an unidentified species from Argentina (KEW 1988-1222); A. cornplanatus and A. sinicus; A. cysticalyx and Sphaerophysa salsula ; Glycyrrhiza echinata and G lepidota. Only the first species in each group was included in subsequent phylogenetic analyses. Of the 157 restriction site changes in the weighted parsimony analysis, 36 (22.9%) were

18. 19. 20. 21. 22 23. 24. 25. 26 27. 28.

17.

16.

15.

12. 13. 14.

11.

6. 7. 8. 9. 10.

1. 2. 3. 4. 5.

Alhag[ maurorum Med. Astracantha amblolepis (Fisch.) Podl. Astracantha er/ocepha/a (Willd.t Podl. AstragalusacutirostrisWats. Astragalus adsurgens Pallas Astragalus alopecias Pallas Astragalusa/pinusL Astragalus asterias Ledeb. Astragalus atropilosulus (Hoc.) Bunge Astragalus australis (L.) Lam. Astragalus chlnensis L.f. Astragalus cicerL. Astragatus complanatus R.Br. Astragalus contortuplicatus L Astraga/us cymbicarpos Brot. Astragalus cysticalyx Ledeb. Astra~/us doME/as~~(T. & G.) Gray Astragalus echinatus Murray Astraga/usepiglottis L. Astragalus filicaulis Fish. & Mey AstraEalus glycyphyllos L. Astragalus hamosus L. Astragalus icmadophHus Hand.-Mazz. Astragalus indet. Astraalus leptocarpusT. & G. Astragalus oophorus Wats. Astragalus pehuenches Niederlein Astragalus rattaniiGray

GALEGEAE ASTRAGALINAE

TRIBE SUBTRIBE Species

(Piptolobi): Leptocarpi

(Piptolobi): Leptocarpi (Piptolobi): Megacarpi

Tragacantha: Rhacophorus Tragaeantha: Adiaspastus (Piptolobi): Leptocarpi Cercidothrix: Onobrychoidei Calycophysa: Alopecias Phaca: Komaroviella Trimenieeus: Oxyglottis Pogonophace: Chlorostachys Phaca: Hemiphragmium Phaca: Nuculiella Hypoglottis: Hypoglottis Pogonophace: Phyllolobium Trimeniaeus: Cycloglottis Epiglottis: Edodimus Calyocystis: Cysticalyx (Piptolobi): Inflati Trimeniaeus: Pentaglottis Epiglottis: Epiglottis Trimeniaeus: Oxyglottis Phaca: Glycyphyllos Epiglottis: Buceras Phaca: Acidodes

Subgenus (Phalanx): Section*

TABLE 1. TAXA EXAMINED FOR rpoC RESTRICTIONSITE VARIATION

USSR Turkey Turkey California, USA China Iran Wyoming (OW) Israel Kenya Washington (OW) cultivated cultivated China China Spain USSR California, USA Morocco Israel Afghanistan Turkey Israel Turkey Argentina Texas, USA Nevada, USA Argentina California, USA

Location?

966 972 973 718 911 967 968 885 887 Kaye 526 970 Halse 4285 889 816. 899 961 772 897 892 none 903 881 971 none 893 851 none 703

Vouchers

KEW 1991-634

USDA 516498 Danin et aL 70156 USDA 220769 USDA 121096 Liston 70310 Engel 117 KEW 1988-1222 Orzelt & Bridges 6537

USDA 318944 USDA 440146

DEEP 90-0279

USDA 89399

USDA 464864 Tehran Bot. Garden 226 USDA 232534 Danin 70110 USDA 192953

USDA 502281 Engel 167 Enget 098

Source

o

Glycyrrh~a echinata L. Glycyrrhizalepidopta Pursh

none

none none

877 879 878

978 979

882

960 964 902 813 962 884

906 890 895 898 none 974 908 883

R.O. Hampton

R.O. Hampton R.O. Hampton

Monrovia Nursery USDA 2774 Corvallis, OR

Tehran Bot. Garden 258 USDA 215214

USDA 325341

Dean 2.2.89 Henderson 512

Thompson & Morgan 7140 DELEP 89-0385 Dean 580

USDA 150557 Shmida 3.2.80 Turner 6601 Parag 70164 KEW 699-69-06597 USDA 310390 Morton Arboretum DELEP 90-0305

*Sectional classification follows Barneby (1964) for North America and Lock and Simpson (1991) for Eurasia and Africa. No sectional classification exits for the South American species of Astragalus, tOW designates North American collections of euploid Astragalus species. All cultivated plants are of Eurasian origin. :~Collection numbers of A. Liston unless indicated otherwise. All vouchers are deposites at OSC.

cultivated

V~CIEAE 51. Pisumsadvum L. 'Sparkle'

China North America cultivated (China) cultivated cultivated

Callerya redculata (Benth.) Schot Wisteriafrutescens (L.) Poiret Wisteriasinensis (Sims) DC.

Iran Nebraska, USA

TRIFOLIEAE 49. Medicago sadva L. 'DuPuRs' Trifolium repens L. 'New Zealand White' 50.

48.

47.

46,

MILLETTIEAE

45.

44.

GLYCYRRHIZINAE

USSR

China Egypt Texas, USA Israel Russia USSR Eurasia Colorado, USA

GALEGINAE 43. Galegaorientalis L.

Pogonophace: Lotidium Epiglottis: Herpocaulos Trimeniaeus: Oxyglottis

New Zealand Israel S. Africa China S, Africa Australia

Astragalus sinicus L. Astragalus vogelii(Webb) Bornm. Astragalus wrightiiGray Biserrula pelecinus L. Calophacawolgarica (L.f.) DC. Caraganaarborescens Lam. Ha/imodendron ha/odendron (Pallas) Vos Oxytropis sericea Nutt,

COLUTEINAE 37. Clianthuapuniceus (G. Don) Lindley 38. Coluteaisttia Mill. 39. Lesserda annularis Burch, 40. Sphaerophysasalsula (Pallas) DC. 41. Sutherlandia frutescens (L.) R.Br. 42. Swainsona macculochiana I£ MuelL

35. 36.

34.

29. 30. 31, 32. 33.

('3 -r

r-.

z Go o

Go

O

5

382

A . L I S T E N A N D J. A . W H E E L E R

Site: 1111111111222222222233333333334444444444555•5555•56•6••66a•6777777777•••888•8•88•9999•99•9 Taxon 1 2 3 4 5 6 7 • • • 1 2 3 4 5 • 7 • 9 • 1 2 3 4 5 • 7 8 9 • 1 2 3 4 5 6 7 8 • • 1 2 3 4 5 • 7 • • • 1 2 3 4 • 6 7 • • • 1 2 3 4 • • 7 • • • 1 2 3 4 5 6 7 • 9 • 1 2 3 4 5 • 7 8 9 • 1 2 3 4 5 • 7 8 9 1. 2. 4, 5, 6. 7. 8. 9. 10. 11. 13. 14, 15. 16. 17. 18. 19. 21. 26. 27, 30. 31. 32. 33, 34. 35. 36. 37. 38. 39. 41. 42. 43. 44. 46. 47. 48. 49. 50. 51.

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1.

2. 3. 4. 5, 6.

7. 8. 8.

10, 11. 12. 13, 14, 15. 16.

17. 18. 19. 20. 21. 22. 23, 24, 25.

380 470 660 Asel - 680 Asel - 1380 Aset - 1450 Asel - 2310 Asel ~ 2600 Asel - 2 8 2 0 Asel - 3200 Ase] - 3470 Asel - 3560 BarnHI - 935 BarnHt - 1185 BarnHI - 2095 BsmAI - 310 BsmAI - 480 BsmAI - 2 8 0 BsmAI - 1000 BsmAI - 1340 BsmAI - 1570 BsmAI - 1630 BsmAI - 2090 BstBI - 710 BstBI - 880 Asel

-

Asel

-

Asel

-

FIG. 1. D A T A M A T R I X

FOR THE

26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36 37.

38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50.

1010 1080 - 1770 BsrB{ - 2480 BstBI - 2940 BstBI - 3550 Bst81 - 3 5 9 0 BstBI - 4050 BstXt - 950 BsrXl 1815 BstX] - 1950 Clal 1445 C l a l - 17OO C/al - 2 6 3 0 C/at - 3560 Dral - 2 6 4 0 Dral - 2815 Dral 3575 Dral 3600 EcoRI - 360 EcoRI - 450 EcoRI - 640 EcoRI - 1220 EcoRI - 1475 EcoRI - 1560 BstBI

-

BstBI

-

BstBI

rpoCl-rpoC2

scored for each of the 40 species included

PHYLOGENETIC

51. 52, 53. 54, 55. 56. 57. 58. 59. 60. 61. 62.

63. 64, 65. 66. 67, 68. 69. 70. 71 72. 73. 74. 75.

- 2650 - 110 - 510 EcoRV - 560 EcoRV - 640 EcoRV - 2565 EcoRV - 2625 EcoRV - 3345 EcoRV - 3870 Haelll - 815 Haell} - 965 Haelll * 1555 Haelll - 1860 Haelll - 2 1 3 5 Haelll - 2490 Haelll - 2545 Haelll - 2755 Haelll - 3650 H h a ] - 1630 Hhal - 1710 Hhal - 1880 Hhal - 2580 Hhal - 3290 Hhal - 4010 Kpnl - 2405 EcoRI

EcoRV

EcoRV

ANALYSIS,

The

mapped

76 77. 78. 79. 80. 81,

82. 83. 84, 85. e6, 87. 88. 59. 90. 91, 92. 93. 94. 95. 96. 97. 98, 99.

restriction

- 2565 - 3405 - 3350 Ndel - 2555 Psrl - 3 7 8 0 Sspl - 425 Sspl - 675 Sspl - 1080 Sspl 1440 Sspi - 3070 Sspl . 3585 Xbal 1525 Xbal - 3530 Xhol - 2530 Xhol - 3150 Xhol - 3800 Xmn[ . 680 Xmnl . 1835 Xmnl - 2215 Xmnl - 2650 Xmnl - 2685 Xmnl - 3370 Xmnl - 3790 Xmnl 4050 Kpnl

Kpnl Mlul

sites ( l i s t e d b e l o w )

in t h e a n a l y s i s (see T a b l e 1). S p e c i e s w i t h i d e n t i c a l h a p l o t y p e s

are excluded

are (see

t e x t ) . S i t e p r e s e n c e = 1; site a b s e n c e = 0. T h e p o s i t i o n o f e a c h r e s t r i c t i o n site is g i v e n in b a s e p a i r s ( b p ) r e l a t i v e t o t h e p r i m e r s rp0C1-195

(zero) a n d

rp0C2-t364

(4100 bp).

restricted to five highly divergent taxa: Astragalus vogelii, A. epiglottis, Alhagi maurorum, Trifolium repens and the genus Glycyrrhiza. Conversely, low levels of restriction site variation were detected within the large clade composed of most Astragalus and the Astracantha species.

CHLOROPLAST GENES OF ASTRAGALUS

383

Variation was restricted to a single species at 44 sites, leaving 45 sites of potential phylogenetic information. Wagner (unweighted) parsimony analysis resulted in 9016 most parsimonious 156-step trees (not shown). Values of the consistency index for informative characters, the retention index and the rescaled consistency index (Farris, 1989), were 0.402, 0.775 and 0.442, respectively. Identical sets of 228 equally parsimonious trees were obtained in the two weighted parsimony analyses. Tree lengths were 163.6 steps in the 1.1:1 gain:loss weighting, and 176.8 steps in the 1.3:1 gain:loss weighting. These trees are equivalent to a 157-step unweighted tree. A majority-rule consensus tree with compatible groups is illustrated (Fig. 2). Values of the consistency index for informative characters, the retention index and the rescaled consistency index, were 0.398, 0.772 and 0.438, respectively. Bootstrap resampling of the character matrix resulted in seven clades found in 50% or more of the replicates (Fig. 2). A comparison of the unweighted and weighted analyses based on their strict consensus trees (not shown) reveals the following two differences: (1) the clade consisting of subtribe Coluteinae and three species of Astragalus is monophyletic and sister to the remaining species of Astragalus, Astracantha and Biserulla in the weighted analyses. In the unweighted analysis, Swainsona is the sister taxon to the Coluteinae and the above three genera. (2) The genera Calophaca, Caragana and Halimodendron are monophyletic and sister to the remaining Astragalinae (excluding Alhagl) and Coluteinae in the weighted analyses. The genus Glycerrhiza belongs to the clade of Galega, Medicago, Pisum and Trifolium. In the unweighted analysis, Glycyrrhiza is also a potential sister group to the Astragalinae and Coluteinae, resulting in a trichotomy. The results of the weighted analysis are more congruent with data from nuclear ribosomal DNA internal transcribed spacer (ITS) sequences (Sanderson and Liston, in press) and an expanded rpoC analysis of the Coluteinae and Astragalinae (Liston and Schwar-zbach, in prep.). For this reason, the following discussion is based upon the weighted parsimony results. Discussion

Position of Astragalus in the Galegeae Most of the examined species of Astragalus belong to a monophyletic clade (Fig. 2). The inclusion of a large number of representative genera from the Galegeae strengthens this phylogenetic hypothesis. The significance of the hypothesized relationships among the Galegeae is discussed elsewhere (Sanderson and Liston, in press). In the following discussion, only the phylogenetic position of Astragalus itself is examined. According to the hypothesis of phylogenetic relationships presented here, the taxonomic recognition of Astracantha and Biserrula makes Astragalus paraphyletic. The two Astracantha species are associated with Astragalus icrnadophilus, a tragacanthic species. In fact, no sequence divergence was detected between the three. Morphological similarity between these two groups has been interpreted as resulting from convergent evolution (Podlech, 1982; Engle, 1991). Alternatively, the distinction between the two groups has been considered artificial (Chamberlain and Matthews, 1970). The results of this study would suggest a single origin of the tragacanthic habit in Astragalus. The monotypic annual, Biserrula pelecinus, has been considered distinct by most regional floras (but see Meikle, 1977). The single species of Biserrula was transferred to Astragalus by Barneby (1964) in a footnote to his monograph of the North American species. The rpoC results support Barneby's taxonomic treatment. The position of four Astragalus species [A. complanatus and A. sinicus (identical haplotypes), A. cysticalyx, A. vogelill in this hypothesis of phylogenetic relationships would suggest that the genus is polyphyletic (Fig. 2). The four species are associated

384

A. LISTONAND J. A. WHEELER Alhagi maurorum

amblolepis

Astracantha i Astragalus acutirostris - - Astragalus douglasii _ _ l - ~ Astragalus echinatus

~

~

Astragalus oophorus

I L Astragalus pehuenches Astragalus wrightii

AO A AO A A AO

~_j Astragalus adsurgens --

~[

1

r~J

/

I I t

97

I

1 L~

|

Astragalus cymbicarpos - - Astragalus alpinus

0 AO

- Astragalus australis - - Astragalus chinensis ----Astragalus

contortuplicatus

O

- - Astragalus alopecias - Astragalus atropilosulus

~ I Biserrufa pelecinus 63 L ~ _ _ Astragalus epiglottis

O O

I - -Astragalus complanatus ~ - Astragalus vogelii I L ~ - cOlutea istria ~ Astragalus cysticalyx

I I L 6~

I L Astragalus glycyphyllos ~ Astragatus asterias

O

-Oxytropissericea

I J

-C,anthus .uniceus

t__~

t

Swainsona macculochiana

I ~Lessethearl~i;af~i~tescens [--?~2 ?2 ~

Calophaca wolgarica Caragana arborescens Halimodendron halodendron Medicago sativa Pisum sativurn

O

72 Trifolium repens - - Glycyrrhiza echinata Wisteria frutescens --Wisteria sinensis - -

i_ i 0

l ~ I i 5

Calterya reticulata

FIG. 2. ONE OF 288 EQUALLYPARSIMONIOUSTREES OF 157 STEPS PRODUCEDBY WEIGHTED PARSIMONYANALYSISOF rpoC RESTRCTION SITE MUTATION DATA FOR ASTRAGALUS, OTHER GENERA OF GALEGEAE AND OUTGROUPS. In this analysis, restriction site gains were weighted 1.3 times site losses.This tree is identical to the majority-rule consensus tree with compatible groups. Branch lengths in this figure are proportional to number of character state changes inferred under default ACCTRAN optimization in PAUP 3.0s. Nodes with bootstrap values above 50% are labelled. Note that the bootstrap analysis was carried out using unweighted characters, it could not be performed with weighted parsimony due to computational constraints. A = Aneuploid species of Astragalus. O = Annual species.

with six genera of the subtribe Coluteinae in a monophyletic "coluteoid" clade. Astragalus complanatus and A. sinicusare members of subgenus Pogonophace. The position of this species pair would suggest that their characteristic ciliate stigmas (Wenninger, 1991) are homologous with the diagnostic pollen brushes of the Coluteinae (Lavin and Delgado, 1990). The possible association of A. complanatus with the Coluteinae was previously commented upon by Barneby (1964). It would be

CHLOROPLASTGENES OF ASTRAGALUS

385

premature to remove subgenus Pogonophace from Astragalus before additional sources of phylogenetic evidence are compared. Subgenus Pogonophace itself is not monophyletic (cf. the position of A. atropilosulus) and additional members must be examined before the status of the subgenus is resolved. Astragalus vogelii is morphologically and geographically isolated in Astragalus (Podlech, 1984). It forms the monotypic section Herpocaulos and is characterized by a unique combination of medifixed hairs and unilocular fruits. It is the only Astragalus species widely distributed in the subtropical deserts of North Africa and Southwest Asia (Podlech, 1984). The findings presented here would suggest that rather than being an aberrant Astragalus, A. vogelii is rather a highly specialized member of the "coluteoid" clade exhibiting morphological convergence to Astragalus. Like the above three species, A. cysticalyx (the only representative of subgenus Calycocystis in the study) is placed amog the Coluteinae in the rpoC analysis. Although this species has an identical haplotype with Sphaerophysa salsula, no obvious morphological characters link these two taxa.

Aneuploid species The clade of New World aneuploid Astragalus is supported by only a single restriction site loss in this study (Fig. 2). However, strong evidence for its monophyly has been demonstrated in previous molecular phylogenies (Liston, 1992; Sanderson and Doyle, 1993; Wojciechowski eta/., 1993). A surprising result is the occurrence of the Old World aneuploid A. echinatus as a member of the above New World clade in the rpoC analysis. The potential status of this species as a sister group to the New World aneuploids awaits corroborative data from additional genetic loci. Astragalus echinatus (placed in the monotypic section Pentaglottis) is an annual species from the Mediterranean basin. Its large-seeded, coarsely tuberculate, indehiscent fruits are unparalleled in the genus. Thus morphological evidence does not support the possibility of this species being an introduced species in the Old World. Two additional Old World aneuploids, A. hamosus and A. cymbicarpos would appear to have an independent origin from the North American aneuploids, in agreement with the findings of Wojciechowski et al. (1993). The two sampled South American species are also included in the clade of New World aneuploids. This result is congruent with morphological (Johnston, 1947; Barneby, 1964) and cytogenetic evidence (Ledingham and Pepper, 1973). The haplotype identity between an unidentified South American Astragalus (KEW 19881222) and A. wrightii suggests that this species which is morphologically isolated in North America (Barneby, 1964; Liston, 1992) may be related to a South American lineage.

Annuals Podlech (1991; pers. comm.) has suggested that all Eurasian annuals (with the possible exception of A. boeEcus L.--not included here) share a common ancestor. The cladogram presented here hypothesizes several independent origins of the annual habit (Fig. 2). Interestingly, the annuals A. epiglo~'s and Biserulla represent the deepest branches in the Astragalus clade (Fig. 2). The scattered distribution of Astragalus epiglo~s in the Mediterranean basin has been described as "relictual" (Podlech, pers. comm.). Biserrulla is more widespread in the Mediterranean basin, but also extends into the mountains of Eastern Africa, where it is represented by an endemic subspecies (Wickens, 1976). In addition to the annual habit, these two species share several morphological features, including dorsiventrelly flattened pods and flowers with only five fertile anthers. The present hypothesis of phylogenetic relationships might suggest that these characters are plesiomorphic in Astragalus. A more likely scenario would be that these morphologically isolated annuals represent

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A. LISTONAND J. A. WHEELER

the extant members of a basal Astragalus lineage of u n k n o w n g r o w t h form. A l t h o u g h the annual habit is sometimes considered an "evolutionary dead end", this hypothesis w o u l d suggest that characteristic features of A. epiglottis and Biserrula (self-pollination, highly modified fruit morphology) have allowed them to persist. Conclusions As noted above, several regions of the resulting cladograms lack robustness and phylogenetic interpretations must await more detailed analysis of unresolved clades. For example, the relationships a m o n g the majority of Astragalus species and a m o n g the genera of the Coluteinae are poorly resolved and are likely to change as additional taxa and mapped restriction sites are added to the analysis. W i t h o u t additional evidence, it is impossible to determine w h e t h e r ctades supported by a single step (such as the sister-group relationship of the tragacanthic species and the N e w World aneuploids) are artifactual or indicative of monophyly. A n additional source of potential error is the presence of several "long branches" (Fig. 2). Parsimony analysis can give erroneous results in this situation since the accumulation of homoplastic changes in highly divergent taxa can lead to artifactual associations (Felsenstein, 1978). Long branches may result from unequal rates of evolution (Lake, 1987) or insufficient sampling of taxa in these "sparse regions" (Swofford and Olsen, 1990). It should be noted that most of the highly divergent taxa are also morphologically isolated and w i t h o u t apparent close relatives ( e . g . A . contortuplicatus, A. epiglottis, A. vogelii, Glycyrrhiza). This w o u l d suggest that the branch pruning has occurred by extinction, and that these plants are evolutionary relicts. Comparisons of branch lengths reported here to results from sequencing of the nuclear ribosomal DNA internal transcribed spacer (Sanderson and Liston, in press; Sanderson, unpublished) suggests that some long branches may be gene (or genome) specific. Thus incorporating additional loci into the phylogenetic analysis could assist in the further resolution of the inter- and intrageneric relationships of Astragalus. Acknowledgements--We thank the following persons and institutions for providing seeds and plant material used in this study: Sue Dean (S. Africa), Thomas Engel (Germany), Jeff Doyle (U.S.A.), Richard Hampton (U.S.A.), George Ledingham (Canada), Joy Thompson (Australia), Desert Legume Program (and Matthew Johnson), Morton Arboretum (and Floyd Swink), Royal Botanic Gardens Kew (and Joan Barnham), University of Texas Herbarium and the USDA National Plant Germplasm System (and David Brenner and David Stout). Funding for this work was provided by an Oregon State University General Research Fund grant to A. Liston, Andrea Schwarzbachprovided helpful laboratory assistance. References Albert, V. A., Mishler, B. D. and Chase, M. W. (1992) Character state weighting for restriction site data in phylogenetic reconstruction, with an example from chloroplast DNA. In Molecular Systernatics of Plants, (Soltis, D., Soltis, P. and Doyle, J., eds), pp. 369-403. Chapman and Hall, New York. Arnold, M. L., Buckner, C. M. and Robinson, J. J. (1991) Pollen mediated introgression and hybrid speciation in Louisiana irises. Proc. Natn. Acad. Sci. U.S.A,88, 1398-1402. Ashraf, M. and Gohil, R. M. (1988) Studies on the cytology of legumes of Kashmir Himalaya. I. Cytology of Astragalus melanostachysBenth. ex Bunge with a new base number of the genus. Carylogia41, 61-67. Barneby, R. C. (1964)Atlas of North American Astragalus. Mem. New YorkBot. Card. 13, 1-1188. Bunge, A. (1868) Generis Astragali species gerontogeae. Mem. Acad. Imp. Sci. St. Petersbourg11, 1-140. Bunge, A. (1880) Astragaleae: In Fedtschenko: Reise in Turkestan III. Izv. Imp. Obsc. Ljubit. Eststv. Moskovsk. Univ. 26, 160-318. Chamberlain, D. F. and Matthews, V. A. (1970)Astragalus. In Flora of Turkeyandthe EastAegeanIslands(Davis, P. H., ed.), pp. 49-254. Edinburgh University Press, Edinburgh. Chen, W., Hoy, J. W. and Schneider, R. W. (1992) Species-specific polymorphisms in transcribed ribosomal DNA of five Pythium species. Exp. Mycology 16, 22-34. Cohan, F. M., Roberts, M. S. and King, E. C. (1991). The potential for genetic exchange by transformation within a natural population of Bacillus subtilis. Evolution45, 1383-1421.

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