Nordic Journal of Botany 27: 425
436, 2009 doi: 10.1111/j.1756-1051.2009.00285.x, # The Authors. Journal compilation # Nordic Journal of Botany 2009 Subject Editor: Petra Korall. Accepted 10 February 2009 Molecular phylogeny of selected Old World Astragalus (Fabaceae): incongruence among chloroplast trnL-F, ndhF and nuclear ribosomal DNA ITS sequences Marzie Kazemi, Shahrokh Kazempour Osaloo, Ali Asghar Maassoumi and Eskandar Rastegar Pouyani M. Kazemi and S. Kazempour Osaloo (skosaloo@modares.ac.ir), Dept of Plant Biology, Faculty of Basic Sciences, Tarbiat Modares Univ., IR
14115-175 Tehran, Iran.
A. A. Maassoumi, Research Inst. of Forests and Rangelands, IR
13185-116 Tehran, Iran.
E. R. Pouyani, Dept of Biology, Tarbiat Moallem Univ. of Sabzevar, IR
397 Sabzevar, Iran. This study reports maximum parsimony and Bayesian phylogenetic analyses of selected Old World Astragalus using two chloroplast fragments including trnL-F and ndhF and the nuclear ribosomal internal transcribed spacer (nrDNA ITS). A total of 52 taxa including 34 euploid Old World and New World Astragalus, one aneuploid species from the NeoAstragalus clade as a representative and 14 other Astragalean taxa, plus Cheseneya astragalina and two species of Caragana as outgroups were analyzed for both trnL-F and nrDNA ITS regions. ndhF was analyzed in 30 taxa and the same number for the combination of these three datasets were examined. In general, the trnL-F dataset and the ndhF and nrDNA ITS datasets generated more or less the same clades within Astragalus. However, in the trnL-F and ndhF phylogenies, Astragalus species are not gathered in a single clade, the so-called Astragalus s.s., as indicated by the nrDNA ITS tree. Visual inspection of these three phylogenies revealed that they were inconsistent regarding the position and relationships of Astragalus hemsleyi, A. ophiocarpus, A. annularis
A. epiglottis/Astragalus pelecinus, A. echinatus and A. arizonicus. Incongruence length difference test suggested that the trnL-F, ndhF and nrDNA ITS datasets were incongruent. In spite of this, phylogenetic analyses of the combined datasets as one unit or as three partitions generated trees that were topologically similar as a mix of the cpDNA and the nrDNA ITS trees. However, the combined dataset provided more resolved and statistically supported clades. The recently described A. memoriosus appeared closely related to A. stocksii (both from sect. Caraganella) based on both trnL-F and nrDNA ITS sequences. Astragalus is a member of the polyphyletic tribe Galegeae (Fabaceae) which itself belongs to the chloroplast inverted repeat lacking clade (IRLC) (Wojciechowski et al. 1999, 2000, 2004). Based upon matK and nrDNA ITS phylogenies, this superclade contains 45 genera in 5 clades, namely the Astragalean clade, the Hedysaroid clade, the Vicioid clade, the Chesneya
Caragana clade and the Glycyrrhiza clade, plus three millettoid genera (Wojciechowski et al. 2004, Lock and Schrire 2005, Kazempour Osaloo 2007). The Astragalaean clade is further divided into several distinct subclades. One, the Astragalus s.s. clade, is composed of the vast majority of Astragalus species (Wojciechowski et al. 1999, Wojciechowski 2005, Kazempour Osaloo et al. 2003, 2005). As a supplement to the large number of nrDNA ITS sequences available (115 Astragalus species), cpDNA trnL intron data for 23 Astragalus species, mainly from the New World, was used by Wojciechowski et al. (1999). However, the trnL intron sequences did not retrieve Astragalus s. s. as a clade. The parsimony reconstruction of the Old World Astragalus based on nrDNA ITS and cpDNA ndhF for 143 and 43 species, respectively, conducted by Kazempour Osaloo et al. (2003), resulted in topologically incongruent trees regarding the position of some Astragalus species including A. epiglottis, A. annularis, A. grammocalyx, A. coelicolor, A. capito, A. hemsleyi, A. ophiocarpus, A. echinatus and A. oophorus (as a representative of the Neo-Astragalus clade). In this study, the trnL intron and trnL-trnF intergenic spacer along with ndhF and nrDNA ITS were analyzed to achieve the following goals: 1) comparing phylogenies derived from these three datasets regarding the position of conflicting Astragalus species, 2) assessing the combinability of these datasets, 3) evaluating the phylogenetic utility of the trnL-F region and the trnL intron indels for reconstructing relationships within Astragalus and allies, and 4) determining the phylogenetic status of the newly established species Astragalus memoriosus from north east of Iran (Pakravan et al. 1994). 425 Material and methods Sequence alignment Taxon sampling Sequences of trnL-F and nrDNA ITS were aligned manually using Clustal W (Thompson et al. 1994). Alignment of both regions required the introduction of numerous single and multiple-base indels (insertions/ deletions). In the case of the trnL-F, 8 unambiguous indels (the numbers 1 and 0 as insertion and deletion events, respectively) were added at the end of the dataset. In contrast, indels were treated as missing data for the nrDNA ITS dataset. The ndhF sequences were aligned manually and only a gap of six base pairs was detected. A total of 35 species of Astragalus (32 from the Old World Astragalus) plus 14 other related taxa of the Astragalean clade (Wojciechowski 2003, 2005), and Chesneya astragalina as well as two Caragana species as outgroups, were included in phylogenetic analyses based on both trnL-F and nrDNA ITS sequences. The trnL-F was newly sequenced for 24 Astragalus species in the present study. The trnL-F sequences for the remaining taxa included in this study have been sequenced by Wojciechowski et al. (1999) and Kazempour Osaloo et al. (2006) and were obtained from Genbank. The nrDNA ITS sequences for all 52 species except Astragalus memoriosus, published previously (Wojciechowski et al. 1999, Kazempour Osaloo et al. 2003, 2005, 2006), were obtained from Genbank. Also, all 30 ndhF sequences published in our previous study (Kazempour Osaloo et al. 2003) were taken from Genbank (Table 1). DNA extraction Total DNA was extracted from herbarium specimens deposited in the herbarium of the Res. Inst. of Forests and Rangelands (TARI), Tehran, Iran. The extraction method was based on the modified CTAB procedure of Doyle and Doyle (1987). In some cases, we used previously extracted DNA (Kazempour Osaloo et al. 2003) as a template for trn-F amplification and sequencing (Table 1). PCR and DNA sequencing The trnL-intron and the adjacent trnL-trnF intergenic spacer were amplified using the primers trn-c and trn-f of Taberlet et al. (1991). The nrDNA ITS region of A. memoriosus was amplified using the primers ITS4 and ITS5 of White et al. (1990). The total volume of amplification reactions was 50 ml, made up of 36 ml deionized water, 5 ml of 10 GeneTaq universal buffer, 5 ml of 2.5 mM dNTPs, 1 ml of each primer (5 pmol ml1), 0.6 ml (5 units) of Taq polymerase and 1.5 ml of template DNA. The PCR cycles consisted of 4 min at 958C for predenaturation followed by 36 cycles of 1 min at 958C for denaturation, 50 sec at 538C for primer annealing and 2 min at 728C for primer extension, followed by a final primer extension of 8 min at 728C. Purification of PCR products was performed using NucleoSpin extract kits (Machery-Nagel Apr 2004/rev.01) and directly sequenced using the ‘Big dye terminator cycle sequencing ready reaction kit’ with the same trn-c, trn-f, ITS4 and ITS5 primers. Sequencing of the fragments was performed in an ABI Prism 377 DNA sequencer. All sequences and voucher information of taxa included in the present study have been deposited in Genbank (see Table 1 for accession numbers). 426 Phylogenetic analyses Maximum parsimony Phylogenetic analyses were performed on each of the three single region datasets. Initially phylogenies were inferred from all datasets using the maximum parsimony method (MP) as implemented in PAUP* (Swofford 2002). Due to high levels of homoplasy in the trnL-F dataset, we could not determine the number of equally most parsimonious trees. So the following heuristic search strategies were employed (sensu Catalan et al. 1997, Downie et al. 1998, Kazempour Osaloo et al. 2003). One thousand replications of random addition sequence with tree
bisection
reconnection (TBR) branch swapping were initiated, but no more than five of the shortest trees from each replication were saved. These equally most parsimonious trees were then used as starting trees for TBR branch swapping (with ‘MulTrees’ on and ‘Steepest descent’ off). In all analyses, the maximum number of trees saved was set to 15 000 and these trees were allowed to swap to completion. The strict consensus of these 15 000 shortest trees was subsequently used as topological constraint in another round of 1000 replications of random addition sequence, but in this analysis, only those trees that did not fit the constraint tree were saved. No additional trees were found at the length of the initial 15 000 trees, which suggests that the strict consensus tree adequately summarizes the available evidence, even though the exact number of trees with that length is not known. For both the nrDNA ITS and ndhF datasets, a finite number of shortest trees was obtained using the heuristic search option with 100 replications of random sequence addition and TBR branch swapping (trees not shown here). To obtain confidence limit for various clades in the trees a bootstrap analysis (Felsenstein 1985) was conducted. Bootstrap values were calculated from 1000 replicates, simple sequence addition and TBR branch swapping with a set ‘Maxtrees’ limit of 100 trees per bootstrap replicate. The values were shown on the corresponding clades of a 50% majority rule consensus tree of Bayesian inference analysis (Fig. 1
3). To assess combinability of these three datasets, the incongruent length difference (ILD, Farris et al. 1995) test was conducted using PAUP* (Swofford 2002). Bayesian method Models of sequence evolution were selected using the program MrModeltest (Nylander 2004) as implemented in MrMTgui (Nuin 2005) based on the Akaike information Table 1. Samples included in cpDNA trnL-F, cpDNA ndhF and nrDNA ITS phylogenetic analyses. Species Astragalus adsurgens Pallas A. annularis Forsskal A. arizonicus Gray A. asterias Hohen. A. australis (L.) Lam. A. boeticus L. A. A. A. A. A. boeticus L. canadensis L. capito Boiss. caraganae Fisch. and Mey. coelicolor Sirj. and Rech. f. A. curvipes Trautv A. deickianus Bornm. A. A. A. A. A. A. A. A. A. A. A. A. A. echinatus Murray epiglottis L. eucosmus Robinson grammocalyx Boiss. and Hohen. hamosus L. hemsleyi Aitch. and Baker horridus Boiss. jessenii Bunge memoriosus Pakravan, Nasseh and Maass. migpo R. Kam. oophorus Wats. ophiocarpus Bunge paradoxus Bunge A. pelecinus L. A. peristereus Boiss. and Hausskn. A. pseudorhacodes Gontsch. A. robbinsii (Oakes) A. Gray var. minor (Hook.) Barneby A. schmalhausenii Bunge A. sinicus L. 427 A. A. A. A. A. squarrosus Bunge stocksii Benth. ex Bunge submitis Boiss. subsecundus Boiss. tribuloides Delile DNA source (location, voucher) China: USDA 462310, W and S 267 (ARIZ) Iran: Maass. and Abou. 51921 (TARI) USA: Sanderson 968 (DAV) Iran: Runemark and Mozaf. 30957 (TARI) NV: Eureka Co. Tiehm 11985 and Williams (RM) France: USDA 414243, W and S 300 (ARIZ) Iran: Maass. and Abou. 51949 (TARI) NV: Nye Co.W and S 302 (ARIZ) Iran: Foroughi 2913 (TARI) Iran: Mozaf. and Maass. 48076 (TARI) Iran: Wendelbo and Assadi 29725 (TARI) Iran: Maass. 47553 (TARI) Iran: Maass. and Mirhosseini 59381 (TARI) Morocco: Podlech 46718 (TARI) Morocco: Podlech 45851 (TARI) AK: Walker 81-86 (COLO) Iran: Maass. 55123 (TARI) Iran: Maass. 47586 (TARI) Iran: Zarre 69578 (TARI) Iran: Mozaf. 54874 (TARI) Iran: Mozaf. and Maass. 48062 (TARI) Iran: Maass. et al. 72315 (TARI) Iran: Rechinger 51029 (TARI) USA: Tiehm 12045 (KYO) Iran: Maass. 55143 (TARI) Iran: Wendelbo and Assadi 19281 (TARI) Australia (adventive): USDA186284, Wand S 294 (ARIZ) Iran: DLEG 880051 (ARIZ) Iran: Assadi and Mozaf. 35472 (TARI) WY: Lincoln Co. Holmgren and Holmgren 9065 (RM) Iran: Maass. 55146 (TARI) China: USDA 150557, W and S 408 (ARIZ) Iran: Maass. and Abou. 52026 (TARI) Iran: Foroughi 10802 (TARI) Iran: Mozaf. and Maass. 47960 (TARI) Iran: Maass. 55105 (TARI) Iran: Maass. and Abou. 52003 (TARI) Genbank accession no. cpDNA trnL-F cpDNA ndhF nrDNA ITSc AF126980 * AF121674 AB485924 AF126973 AF126989 AB052043 AB052063b * AB051912 AF121690 AB051917 AF126985 * AF121676 AF126982 * * * AF126981 AB485925a AB485944a AB485927a * * AB052075 AB052052 AB052076 AB051937 U50496,U50497 AB051966 AB051942 AB051995 AB485938a AB485928a AB052049 * AB051955 AB051992 AB287411 AB485923a AF126987 AB485926a AB485945a AB485940a AB485933a AB485939a AB485943a AB485930a AB051938 AB051910 AF121684 AB051994 AB051936 AB052003 AB052002 AB051950 AB485947 AB051928 AB485941a AB485934a AB052062 AB052042 * AB052077 AB052055 AB052064 AB052065 AB052051 * AB052039 AB052063 AB052040 AB052074 AF126995 * AF126984 AB485932a AF126986 * AB052061 AB485946a AF127001 AB052046 * AB485931a AB485942a AB485935a AB485936a AB485929a AB052058 AB052044 AB052068 AB052056 AB052057 U50518, U50519 U50494,U50495 AB051979 U50490,U50 491 AB051933 U50502, U50503 AB051987 AB051966 AB052009 AB051985 AB051922 a AB051927 AB052001 428 Table 1 (Continued) Species A. umbellatus Bunge A. verus Olivier Caragana grandiflora (M. B.) DC. Caragana korshinskii Kom. Carmichaelia williamsii Kirk Chesneya astragalina Jaub.and Spach. Clianthus puniceus (G. Don) Lindley Colutea arborescens L. Colutea persica Bioss. Colutea triphylla Bunge ex. Bioss. Lessertia herbacea DC. Oxytropis aucheri Bioss. Oxytropis lambertii Pursh Podlechiella vogelii subsp. fatimensis (Chiov.) Maassoumi and Kazempour Osaloo Sphaerophysa salsula (Pallas) DC. Sutherlandia frutescens L. Swainsona pterostylis (DC.) Bakh. f. DNA source (location, voucher) Genbank accession no. cpDNA trnL-F cpDNA ndhF nrDNA ITSc USA: Parker 88-78 (COLO) Iran: Mozaf. and Maass. 47797 (TARI) Iran: Assadi and Shahsavari 65834 (TARI) China: Y. Z. Zhao9205 (PE) New Zealand: Sanderson 1550 (DAV) Iran: Assadi and Maass. 55503 (TARI) New Zealand: Tand M 7140 (Liston, 960, OSC) [former] USSR: USDA 369222 W and S 406 (ARIZ) Iran: Foroughi 17434 (TARI) Iran: Foroughi et al. 12312 (TARI) South Africa: W and S 299 (ARIZ) Iran: Maass. 55104 (TARI) AZ: Coconino Co: Wojciechowski 155 (ARIZ) Iran: Mozaf. et al. 39103 (TARI) AF121683 AB485937a AB287412 AB052073 AB052035 AF126988 AB052023 AB051905 AY633700 AF127000 AB287413 AF126998 * * AB052036 * AY626914 U50520,U50521 AB051906 L10800,L10801 AF126993 * U56009,U56010 AB287414 AB287415 AF126997 AB287416 AF126991 AB052037 * * AB052038 * AB051907 AB287410 AF121752 AB051908 AF121753 AB287417 AB052041 AB051911 Asia (adventive): Yoder-Williams 78-120A-1 (RENO) Mexico: W and S 266 (ARIZ) Ausralia: DLEG 900185, W and S 296 (ARIZ) AF126996 * U56011,U56012 AF126994 AF126999 * * U50516,U50517 U56007,U56008 Abbreviations used in plant accession information: A, Arnold Arboretum/ Gray herbarium, Harvard Univ., Cambridge; Abou., Abouhamzeh; ARIZ, Univ. of Arizona herbarium, Tucson; COLO, Univ. of Colorado herbarium, Boulder; DAV, Univ. of California, Davis herbarium, Davis; DELEP/ DLEG, Desert Legume Program (Univ. of Arizona), Tucson; KYO, Kyoto Univ. herbarium, Kyoto, Japan; Maass., Maassoumi; Mozaf., Mozaffarian; PE, herbarium of Inst. of Botany, the Chinse Acad. of Sci., Beijing; RENO, Univ. of Nevada herbarium, Reno; RM, Rocky Mountain herbarium, Univ. of Wyoming, Laramie; W and S, Wojciechowski and Sanderson; TARI, herbarium of the Res. Inst. of Forests and Rangelands, Tehran, Iran; USDA, US. Dept of Agricult. Plant Introduction Station. a trnL intron/trnL-F sequences for these taxa were newly sequenced in this study, and the remainder was retrieved from Genbank. b In the case of ndhF sequences, A. arizonicus was used instead of A. oophorus. c All nrDNA ITS sequences, except for Astragalus memoriosus, were retrieved from Genbank. The two accession numbers for nrDNA ITS of some taxa represent ITS1 and ITS2 regions, respectively. Astragalus grammocalyx Astragalus coelicolor Astragalus capito Astragalus migpo 0.96 Astragalus tribuloides 6 Astragalus asterias 0.52 Astragalus deickianus F 1.00 0.51 Astragalus squarrosus 6 55 Astragalus adsurgens 1 Astragalus pseudorhacodes 0.55 Astragalus arizonicus* 0.56 4 Astragalus echinatus Astragalus verus Astragalus peristerus 1.00 Astragalus submitis 0.70 Astragalus paradoxus Astragalus horridus Astragalus hamosus 0.89 Astragalus boeticus 0.69 62 H Astragalus subsecundus 4 Astragalus canadensis Astragalus curvipes 0.99 0.56 Astragalus caraganae 88 Astragalus jessenii 0.97 78 A Astragalus stocksii 1.00 0.96 94 Astragalus memoriosus 67 Astragalus umbellatus 0.83 Astragalus robbinsii 0.61 Astragalus eucosmus 0.68 61 B 1.00 Astragalus australis 3 85 8 Astragalus schmalhausenii 3 Astragalus epiglottis 1.00 97 Astragalus annularis 1.00 84 Astragalus pelecinus Colutea triphylla 0.51 Colutea persica 0.98 68 Colutea arborescens Lessertia herbacea 1.00 0.56 Sutherlandia frutescens 67 Sphaerophysa salsula Podlechiella vogelii Carmichaelia williamsii 0.92 Clianthus puniceus 1.00 94 7 Swainsona pterostylis Oxytropis lambertii 1.00 Oxytropis aucheri 81 0.99 0.97 60 61 Astragalean clade 1.00 98 Astragalus hemsleyi 1.00 2 100 5 Astragalus ophiocarpus Phyllolobium sp. (Astragalus sinicus) 1.00 Caragana grandiflora Caragana korshinskii Chesneya astragalina 0.1 substitutions site-1 Figure 1. Fifty percent majority rule consensus tree resulting from Bayesian analyses of the cpDNA trnL-F data set. Numbers above branches are posterior probabilities and MP bootstrap values are below them, values B50% were not shown. Clades within Astragalus s.s. are identified by letters (A
H). Astragalus arizonicus as a representative of New World aneuploid Astragalus species is indicated by an asterisk. Clades supported by indels in trnL intron are indicated by a bar ( ) with the indel number below it. criterion (AIC) (Posada and Buckley 2004). On the basis of this analysis, datasets were analyzed using the GTR IG and TVMIG models for trnL-F and ndhF sequences, respectively, and the SYMIG model for nrDNA ITS. trnL-F indel characters were included as a separate partition along with trnL-F sequences only and a 429 Astragalus verus Astragalus paradoxus 0.97 G 77 Astragalus submitis 1.00 91 Astragalus horridus 1.00 95 Astragalus echinatus 1.00 0.55 85 Astragalus oophorus* H Astragalus subsecundus Astragalus migpo 0.94 D F 1.00 Astragalus tribuloides 62 0.99 56 Astragalus squarrosus 1.00 79 C Astragalus pseudorhacodes Astragalus hamosus 1.00 99 1.00 Astragalus boeticus 98 Astragalus caraganae 1.00 95 Astagalus jessenii 0.89 A 62 Astragalus curvipes 0.99 1.00 84 97 Astragalus stocksii 0.70 B Astragalus schmalhausenii Astragalus epiglottis 1.00 100 Astragalean clade Astragalus annularis 1.00 Asrtagalus grammocalyx 100 1.00 A. ophiocarpus- A. grammocalyx clade 1.00 Astragalus capito 100 Astragalus coelicolor 92 Astragalus ophiocarpus 1.00 100 0.54 Astragalus hemsleyi Podlechiella vogelii 0.90 Oxytropis aucheri Colutea persica Chesneya astragalina Caragana grandiflora 0.01 substitutions site -1 Figure 2. Fifty percent majority rule consensus tree resulting from Bayesian analyses of the cpDNA ndhF. Posterior probabilities are presented above branches and MP bootstrap values are below them, values B50% were not shown. Clades within Astragalus s.s. are identified by letters (A
H). Astragalus oophorus as a representative of New World aneuploid Astragalus species is indicated by an asterisk. standard (morphology) discrete state model (lset coding variable, (nst 1)G) was applied to this partition. The combined sequences for 30 taxa were analyzed as a single partition with the GTRIG model or in three partitions using the same models combined. The program MrBayes (Ronquist and Huelsenbeck 2003) was used for the Bayesian phylogenetic analyses. For the partitioned 430 analysis, the substitution and branch length estimates were allowed to vary independently in each partition. Posteriors on the model parameters were estimated from the data, using the default priors. Both the single and the combined dataset analyses were run for 5 million generations, except for the ndhF dataset for which the analysis was run for 3 million generations, using Markov chain Monte Carlo Astragalus verus Astragalus peristerus G Astragalus paradoxus 1.00 0.62 Astragalus submitis 95 Astragalus hemsleyi 0.85 0.70 51 87 Astragalus horridus H Astragalus subsecundus Astragalus grammocalyx 1.00 1.00 96 Astragalus coelicolor 70 Astragalus capito 0.76 Astragalus asterias 0.98 Astragalus tribuloides 67 1.00 Astragalus deickianus 51 Astragalus arizonicus* 0.89 58 Astragalus echinatus Astragalus adsurgens 0.78 54 Astragalus pseudorhacodes Astragalus ophiocarpus Astragalus migpo Astragalus squarrosus Astragalus s.s. Astragalus jessenii 1.00 88 Astragalus caraganae 1.00 80 0.99 Astragalus memoriosus 1.00 72 100 Astragalus stocksii 1.00 A Astragalus curvipes 100 Astragalus umbellatus Astragalus eucosmus 1.00 1.00 99 Astragalus robbinsii 100 B 1.00 Astragalus australis 95 Astragalus schmalhausenii Astragalus hamosus 1.00 96 C 1.00 Astragalus boeticus 99 Astragalus canadensis Colutea triphylla 1.00 1.00 89 Colutea persica 1.00 91 Colutea arborescens 100 1.00 Sutherlandia frutescens 1.00 82 99 Lessertia herbacea 0.93 Sphaerophysa salsula 75 Phyllolobium sp. 0.99 Coluteoid clade Carmichaelia williamsii 1.00 0.96 97 1.00 Clianthus puniceus 53 87 Swainsona pterostylis 0.55 Podlechiella vogelii 53 Astragalus annularis 1.00 1.00 Astragalus epiglottis 98 99 Astragalus pelecinus Oxytropis aucheri 1.00 100 Oxytropis lambertii Caragana grandiflora 1.00 Caragana korshinskii Chesneya astragalina 1.00 0.90 Astragalean clade 0.1 93 substitutions site-1 Figure 3. Fifty percent majority rule consensus tree resulting from Bayesian analyses of the nrDNA ITS data set. Numbers above branches are posterior probabilities and the numbers below them indicate MP bootstrap values. Values B50% were not shown. Clades within Astragalus s.s. are identified by letters (A
H). Astragalus arizonicus as a representative of New World aneuploid Astragalus species is indicated by an asterisk. search. MrBayes performed two simultaneous analyses starting from different random trees (Nruns 2) each with four Markov chains and trees sampled at every 100 generations. The following criteria were considered for adjusting the number of generations to ensure stationarity of the Markov chain: 1) a stable value of the log likelihood of the cold chain in two separate runs, 2) a value approaching zero for the standard deviation of split frequencies (0.005 for both runs) and, 3) a value approaching 1.0 for the potential scale reduction factor 431 (PSRF) for each parameter in the model. The trees sampled after reaching the stationary phase were collected and used to build a 50% majority rule consensus tree accompanied with posterior probability (PP) values using Treeview (Page 1996). Results trnL-F sequence data Alignment of the trnL intron, trnL 3? exon and trnL-F intergenic spacer plus indels resulted in a data matrix of 1097 nucleotides, of which 129 sites were potentially parsimony informative. The length of the trnL intron in the taxa examined varied from 333 bp in Astragalus eucosmus and A. robbinsii to 563 bp in A. squarrosus. Similarly, the length of trnL-F intergenic spacer ranged from 98 bp in Astragalus jessenii to 372 bp in Caragana korshinskii. Within the Astragalean clade, the length of this region ranged from 98 (in Astragalus jessenii) to 115 bp (in A. ophiocarpus). The length of trnL 3? exon is 50 bp for all the taxa examined. The Bayesian analysis of the trnL-F dataset resulted in 37 501 trees after discarding 12 500 initial trees as burn in. The 50% majority rule consensus of these trees with posterior probabilities and bootstrap values is shown in Fig. 1. Clades within Astragalus s.s. identified by letters (A
H) are based on Kazempour Osaloo et al. (2003, 2005). The topology of this tree is almost the same as that of the MP tree (not shown) except for resolution and monophyly of some sub-clades within the Astragalean clade. The Astragalean clade is a well supported large polytomy composed of 5 sub-clades and one branch leading to Phyllolobium sp. The largest, but weakly supported (PP 0.83), sub-clade comprised most Astragalus species except Astragalus hemsleyi and A. ophiocarpus, which made their own sub-clade. This large sub-clade was not seen on the MP tree. The three remaining sub-clades consisted of Oxytropis, Carmichaelia
Clianthus
Swainsona, and Colutea through Podlechiella vogelii. The two latter ones were united as a weakly supported polytomy (BP B50%) as the socalled Coluteoid clade in the MP tree. ndhF sequence data The aligned ndhF sequences of 30 taxa produced a matrix of 2103 bp of which 150 bp were parsimony informative characters. Based on this data, the Astragalean clade is a well supported (PP 1) assemblage composed of two clades: one of them, as weakly supported, comprises eight species. Three of these, viz Podlechiella vogelii, Oxytropis aucheri and Colutea persica, form a moderately supported (PP 0.9) subclade, and the other five species, from A. grammocalyx through A. hemsleyi, form the so-called A. ophiocarpus
A. grammocalyx clade (Kazempour Osaloo et al. 2003). The next large clade contains the remaining Astragalus species supported by a posterior probability of 1 and a bootstrap value of 97% (Fig. 2). 432 nrDNA ITS sequence data The length of the nrDNA ITS dataset for 52 taxa was 645 nucleotide sites, of which 169 sites were potentially parsimony informative characters. The Bayesian tree with posterior probabilities and bootstrap values is presented in Fig. 3. In this tree, as well as in the MP tree (not shown), the Astragalean clade is again a well supported assemblage comprising three sub-clades: 1) a monophyletic Oxytropis, 2) a weakly supported clade composed of the sister group relationship between the Coluteoid clade (sensu Wojciechowski 2005) and a sub-clade including Astragalus annularis, A. epiglottis and Astragalus pelecinus, and 3) the so-called Astragalus s.s. comprising the vast majority of the species of the genus (Wojciechowski et al. 1999, Kazempour Osaloo et al. 2003, 2005). The combined sequence data ILD test suggested that the trnL-F, ndhF and nrDNA ITS datasets were incongruent (p B0.01). Following the suggestions of several authors (Seelanan et al. 1997, Wiens 1998, Reeves et al. 2001, Yoder et al. 2001) that the ILD test may be unreliable, we still decided to combine these datasets directly. The combined data matrix was 3792 nucleotide sites long, of which 370 were parsimony informative. Bayesian analysis of the combined dataset as a single partition generated a tree that is roughly the same as that obtained by the MP method (Fig. 4). This Bayesian tree is topologically almost identical to the tree resulting from Bayesian analysis of the data as three partitions (tree not shown), except that the relationship between Astragalus subsecundus and the two sub-clades of 15 species from A. echinatus through A. grammocalyx within Astragalus s.s. was resolved while, in the three partition analysis, this relationship was collapsed as a trichotomy. The general topology of the combined tree is a mixed topology of both the cpDNA and, in particular, the nrDNA ITS trees. Discussion Incongruence among the phylogenies Molecular trees based on nuclear, chloroplast or mitochondrial sequences (gene trees) do not necessarily show the evolutionary pathways of the taxa under study (species trees). Several biological factors such as hybridization, introgression, unequal rates of molecular evolution, lineage sorting and technical errors such long branch attraction and insufficient taxonomic sampling may result in erroneous phylogenies (Riesberg and Soltis 1991, Soltis and Kuzoff 1995, Wendel and Doyle 1998, Kazempour Osaloo et al. 2003). The combined dataset, like the nrDNA ITS one, retrieved the well supported so-called Astragalus s.s. clade (BP 82% and PP 1 in Fig. 4). In contrast, the combined phylogeny, as well as both the trnL-F and the ndhF phylogenies were markedly different from the nrDNA ITS phylogeny regarding the relationships of some species. These three phylogenies place two morphologically distant species, A. hemsleyi and A. ophiocarpus, as sister taxa (Fig. 1, 2, 4), whereas the nrDNA ITS phylogeny did not unite Astragalus grammocalyx 1.00 97 1.00 1.00 A. ophiocarpus- A. grammocalyx clade Astragalus coelicolor Astragalus capito 1.00 86 Astragalus ophiocarpus 1.00 0.77 F 1.00 Astragalus hemsleyi 0.98 Astragalus tribuloides Astragalus migpo 1.00 59 0.99 Astragalus pseudorhacodes 66 Astragalus squarrosus Astragalus paradoxus 0.59 1.00 93 1.00 87 Astragalus submitis G 1.00 Astragalus verus 98 0.98 1.00 Astragalus horridus 91 Astragalus arizonicus* 1.00 99 1.00 60 0.72 Astragalus echinatus H Astragalus subsecundus Astragalus hamosus C 1.00 100 Astragalus boeticus Astragalus s.s. B Astragalus schmalhausenii 1.00 82 Astragalus jessenii 1.00 98 0.54 53 1.00 69 Astragalus caraganae A1.00 Astragalus curvipes 100 Astragalus stocksii Astragalean clade Astragalus annularis 1.00 100 1.00 Astragalus epiglottis 100 Coluteoid clade 1.00 77 Colutea persica Podlechiella vogelii Oxytropis aucheri Chesneya astragalina Caragana grandiflora 0.1 substitutions site -1 Figure 4. Fifty percent majority rule consensus tree resulting from Bayesian analyses of the combined dataset (cpDNA trnL-F, ndhF and nrDNA ITS). Posterior probabilities are above branches and MP bootstrap values are presented below them, values B50% were not shown. Clades within Astragalus s.s. are identified by letters (A
H). Astragalus arizonicus as a representative of New World aneuploid Astragalus species is indicated by an asterisk. these two taxa (Fig. 3). However, the derived position of these two species within the Astragalus s.s. clade is the same on the combined and nrDNA ITS trees. Hybridization and introgression are thought, however, to be rare or non- existing within the genus Astragalus (Liston 1992, Sanderson and Doyle 1993, Judd et al. 2008). Parallel evolution and long branch attraction (Felsenstein 1978, Anderson and Swofford 2004) may be a more admissible cause for this 433 discordance. In all combined, trnL-F and ndhF trees, the two species are on long branches. As noted in Kazempour Osaloo et al. (2003), A. hemsleyi and A. ophiocarpus are commonly considered to belong to two different sections, namely Acanthophace (Maassoumi 1998, Zarre and Podlech 2001) and Ophiocarpus (Maassoumi 1986), respectively, and differ from each other in gross morphology including thorny woody perennial habit, paripinnate leaves, solitary flowers and ovoid bilocular pods in A. hemsleyi versus annual habit, imparipinnate leaves, raceme inflorescence and linear unilocular pods in A. ophiocarpus. The same situation was found concerning the position of the three closely related species A. epiglottis, A. annularis and A. pelecinus. These taxa are placed outside the Astragalus s.s. as weakly allied with the Coluteoid clade on the nrDNA ITS tree, but on the combined, trnL-F and ndhF are sister to the large clade of Astragalus species. In previous works based on restriction site analysis of chloroplast rpoC1 and rpoC2 genes (Liston and Wheeler 1994) and a combination of matK and nrDNA ITS data (Wojciechowski 2005), A. epiglottis, plus Astragalus pelecinus (and likely A. annularis) form the sister group to Astragalus s.s. The sister group relationship between A. echinatus
A. arizonicus/A. oophorus (a representative of Neo-Astragalus) and the thorny cushion forming Astragalus species (clade G, Fig. 2
4) is another difference between the combined, trnL-F and ndhF phylogenies and the nrDNA ITS one. This sister group relationship was also found in previous studies using chloroplast rpoC genes (Liston and Wheeler 1994), trnL intron (Wojciechowski et al. 1999), ndhF (Kazempour Osaloo et al. 2003) and matK-nrDNA ITS (Wojciechowski 2005). At present, no obvious morphological synapomorphy links thorny cushion forming Astragalus with A. echinatus
Neo-Astragalus. Position of the three closest species A. capito, A. grammocalyx and A. coelicolor is the same in all of the combined, trnL-F and nrDNA ITS trees within the Astragalus s. s. clade. However, in the ndhF tree, they were positioned outside the bulk of Astragalus species forming a sister clade with A. ophiocarpus
A. hemsleyi. These species in the ndhF tree are also found on long branches and may suffer from long branch attraction. Based on the results of the present study and other studies (Reeves et al. 2001, Yoder et al. 2001), analyzing a combined dataset, despite some incongruence between distinct datasets, seems to be useful to deduce phylogenetic relationships with more resolution. Hence, it may be useful to study more Astragalus species using other nuclear single copy sequences with higher evolutionary rate, such as CNGC4 (Scherson et al. 2005), the second intron of the LEAFY gene or intron of Mendel’s stem length gene Le, for inferring species phylogeny among the Old World Astragalus. Phylogenetic efficiency of the trnL intron and the trnL-F intergenic spacer as well as trnL intron indels By designing universal primers for amplification of trnL intron and trnL-F intergenic spacer, Taberlet et al. (1991) showed that they could be useful for inferring relationships at the infra-generic level in various plants. However, our phylogenetic analysis of these fragments for Astragalus and 434 related genera revealed that they are much less informative than nrDNA ITS sequences for the same taxa, a result similar to those of several other authors (Gielly et al. 1996, Small et al. 1998, Wojciechowski et al. 1999). The lower rate of nucleotide substitutions in the trnL intron is due to its catalytic properties and forming secondary structures (Kuhsel et al. 1990, Taberlet et al. 1991). For a sub-set of 30 taxa including 27 species of Astragalus and allied genera plus three outgroup species, the aligned length of the trnL-F intergenic spacer was 403 bp, vs 685 bp for the trnL intron, and only 22 parsimony informative sites were detected in the trnL-F intergenic spacer vs 88 in the trnL intron. In other legumes such as the tribe Fabeae (Kenicer et al. 2005, Oskoeiyan et al. unpubl.), a shorter length and a lower number of parsimony informative characters in the trnL-F intergenic spacer has also been found, indicating that the spacer alone is not useful for inferring phylogeny, at least among these group of legumes. However, our data are obviously different from previous findings that the trnL-F intergenic spacer is much more variable, despite its shorter length, than the trnL intron (Shaw et al. 2005). Meanwhile, the trnL intron ranges from 388 to 613 bp in our taxa, which is the same as in other angiosperms examined, but the length of the trnL-F intergenic spacer, ranging from 98 to 115 bp in Astragalus and related genera, is much shorter than the previously reported range of 207 to 474 bp (Shaw et al. 2005). Several authors (Bayer and Starr 1998, Wojciechowski et al. 1999) indicated that the trnL intron indels are phylogenetically informative. At the end of our trnL intron dataset, we scored 8 indels ranging from 3
4 to over 260 base pairs. These were mapped on appropriate branches on the tree using ACCTRAN optimization (Fig. 1) showing that 5 indels, i.e. no. 1, 2, 5, 7 and 8, have evolved once as true synapomorphies, whereas the remaining three, i.e. no. 3, 4 and 6, evolved two times each. Phylogenetic position and biogeography of Astragalus memoriosus (sect. Caraganella) Astragalus memoriosus was first reported from northeast of Iran, Golestan National Park (1000 m a.s.l.), as a new species of the section Caraganella by Pakravan et al. (1994). Members of this section are characterized as shrublets with yellowish petals, stipitate pods and medifixed hairs. Astragalus stocksii, A. reshadensis and A. koschukensis are three other species of this section of which only A. stocksii is distributed from Afghanistan and Pakistan to south and eastern Iran whereas the others are exclusively found in Afghanistan and Pakistan (Podlech 1975). Results based on the analyses of trnL-F and nrDNA ITS sequences presented here (Fig. 1, 3) show a close relationship of A. memoriosus and A. stocksii and strongly supported the monophyly of this section that along with basifixed hair sections is nested in clade ‘A’, being consistent with previous studies based on the analyses of nrDNA ITS and ndhF data (Kazempour Osaloo et al. 2003, 2005). Animals (e.g. birds, herbivorous mammals), ocean and air currents are important factors contributing to the distribution of flowering plants across geological barriers. Although most birds can not retain seeds for long periods of time, it has been reported that viable seeds may be transported in the gizzard or the intestinal tract of some migratory birds as long as 200
300 h, which is long enough for a bird to cover several thousands of kilometers (Fukuda et al. 2001). Considering the high dispersability of legumes in general (Raven and Polhill 1981) and assuming the high viability of seeds in Fabaceae and Astragalus, the disjunction in geographical distribution of A. memoriosus relative to its closest allies in the section is possibly due to factors such as bird-disperal and air currents during million years of evolution. Acknowledgements
This research was supported by a research fund of Tarbiat Modares Univ. This work represents partial fulfillment of the requirement for obtaining an MS degree by M. Kazemi from Tarbiat Modares Univ. References Anderson, F. E. and Swofford, D. L. 2004. Should we be worried about long-branch attraction in real datasets? Investigation using metazoan 18S rDNA.
Mol. Phylogenet. Evol. 33: 440
451. Bayer, R. J. and Starr, J. R. 1998. Tribal phylogeny of the Asteraceae based on two non-coding chloroplast sequences, the trnL intron and trnL/trnF intergenic spacer.
Ann. Miss. Bot. Gard. 85: 242
256. Catalan, P. et al. 1997. Phylogeny of Poaceae subfamily Pooideae based on by chloroplast ndhF gene DNA sequences.
Mol. Phylogenet. Evol. 8: 150
166. Downie, S. R. et al. 1998. Molecular systematics of Apiaceae subfamily Apioideae: phylogenetic analyses of nuclear ribosomal DNA internal transcribed spacer and plastid rpoC1 intron sequences.
Am. J. Bot. 85: 563
591. Doyle, J. J. and Doyle, J. L. 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue.
Phytochem. Bull. 19: 11
15. Farris, J. S. et al. 1995. Testing significance of incongruence.
Cladistics 10: 315
319. Felsenstein, J. 1978. Cases in which parsinomy and compatibility methods will be positively misleading.
Syst. Zool. 27: 401
410. Felsenstein, J. 1985. Confidence limits on phylogenies: an approach using the bootstrap.
Evolution 39: 783
791. Fukuda, T. et al. 2001. Phylogeny and biogeography of the genus Lycium (Solanaceae): inferences from chloroplast DNA sequences.
Mol. Phylogenet. Evol. 19: 246
258. Gielly, L. et al. 1996. Phylogenetic use of noncoding regions in the genus Gentiana L.: chloroplast trnL (UAA) intron versus nuclear ribosomal internal transcribed spacer sequences.
Mol. Phylogenet. Evol. 5: 460
466. Judd, W. S. et al. 2008. Plant systematics: a phylogenetic approach (3rd ed.).
Sinauer. Kazempour Osaloo, S. 2007. Phylogenetic relationships in the inverted repeat lacking clade (IRLC) of papilionoid legumes based on nrDNA ITS sequences.
1st Natl Plant Taxonomy Conference of Iran, Tehran, pp. 106
107. Kazempour Osaloo, S. et al. 2003. Molecular systematics of the genus Astragalus L. (Fabaceae): phylogenetic analyses of nuclear ribosomal DNA internal transcribed spacers and chloroplast gene ndhF sequences.
Plant Syst. Evol. 242: 1
32. Kazempour Osaloo, S. et al. 2005. Molecular systematics of the Old World Astragalus (Fabaceae) as inferred from nrDNA ITS sequence data.
Brittonia 57: 367
381. Kazempour Osaloo, S. et al. 2006. Phylogenetic status of Oreophysa microphylla (Fabaceae
Galegeae) based on nrDNA (ITS region) and cpDNA (trnL intron/ trnL-trnF intergenic spacer) sequences.
Rostaniha 7: 177
188. Kenicer, G. J. et al. 2005. Systematics and biogeography of Lathyrus (Leguminoseae) based on internal transcribed spacer and cpDNA sequence data.
Am. J. Bot. 92: 1199
1209. Kuhsel, M. G. et al. 1990. A ancient group I intron shared by Eubacteria and chloroplasts.
Science 250: 1570
1573. Liston, A. 1992. Isozyme systematics of Astragalus L. sect. Leptocarpi subsect. Californici (Fabaceae).
Syst. Bot. 17: 367
379. Liston, A. and Wheeler, J. A. 1994. The phylogenetic position of the genus Astragalus (Fabaceae): evidence from the chloroplast genes rpoC1 and rpoC2.
Biochem. Syst. Ecol. 22: 377
388. Lock, J. M. and Schrire, B. D. 2005. The tribe Galegeae.
In: Lewis, G. et al. (eds), Legumes of the world. R. Bot. Gard. Kew, pp. 475
487. Maassoumi, A. A. 1986. Astragalus L. Vol. 1. Annuals.
Res. Inst. For. and Rangelands, Tehran. Maassoumi, A. A. 1998. Astragalus in the Old World: check-list.
Res. Inst. For. and Rangelands, Tehran. Nuin, P. 2005. MrMTgui 1.0, ver. 1.6. Program distributed by the author at /<http://www.genedrift.org/mtgui.php/>. Nylander, J. A. A. 2004. MrModeltest, ver. 2. Program distributed by the author.
Evol. Biol. Centre, Uppsala Univ. Page, R. D. M. 1996. TREEVIEW: an application to display phylogenetic trees on personal computers.
Computer Appl. Biosci. 12: 357
358. Pakravan, M. et al. 1994. A new species of Astragalus L. sect. Caraganella (Papilionaceae) from Iran.
Iran. J. Bot. 6: 257
259. Podlech, D. 1975. Revision der Sektion Caraganella Bunge der Gattung Astragalus L.
Mitt. Bot. Staatssamml. Munch. 12: 153
166. Posada, D. and Buckley, T. 2004. Model selection and model averaging in phylognetics: advantages of Akaike information criterion and Bayesian approaches over likelihood ratio tests.
Syst. Biol. 53: 793
808. Raven, P. H. and Polhill, R. M. 1981. Biogeography of the Leguminosae.
In: Polhill, R. M. and Raven, P. H. (eds), Advances in legume systematics, part 1. R. Bot. Gard. Kew, pp. 27
34. Reeves, G. et al. 2001. Molecular systematics of Iridaceae: evidence from four plastid DNA regions.
Am. J. Bot. 88: 2074
2087. Rieseberg, L. H. and Soltis, D. E. 1991. Phylogenetic consequences of cytoplasmic gene flow in plants.
Evol. Trends Plants 5: 65
84. Ronquist, F. and Huelsenbeck, J. P. 2003. MrBayes 3: Bayesian phylogenetic inference under mixed models.
Bioinformatics 19: 1572
1574. Sanderson, M. J. and Doyle, J. J. 1993. Phylogenetic relationships in North American Astragalus (Fabaceae) based on chloroplast DNA restriction site variation.
Syst. Bot. 18: 395
408. Scherson, R. A. et al. 2005. Phylogenetics of New World Astragalus: screening of novel nuclear loci for the reconstruction of phylogenies at low taxonomic levels.
Brittonia 57: 354
366. Seelanan, T. et al. 1997. Congruence and consensus in the cotton tribe (Malvaceae).
Syst. Bot. 22: 259
290. Shaw, J. et al. 2005. The tortoise and the hare II: relative utility of 21 noncoding chloroplast DNA sequences for phylogenetic analysis.
Am. J. Bot. 92: 142
166. Small, R. L. et al. 1998. The tortoise and hare: choosing between noncoding plastome and nuclear Adh sequences for phylogeny reconstruction in a recently diverged plant group.
Am. J. Bot. 85: 1301
1315. 435 Soltis, D. E. and Kuzoff, R. K. 1995. Discordance between nuclear and chloroplast phylogenies in the Heuchera group (Saxifragaceae).
Evolution 49: 727
742. Swofford, D. L. 2002. PAUP*: Phylogenetic analysis using parsimony (*and other methods), ver. 4.0b10.
Sinauer. Taberlet, P. et al. 1991. Universal primers for amplification of three non-coding regions of chloroplast DNA.
Plant Mol. Biol. 17: 1105
1109. Thompson, J. D. et al. 1994. Clustal W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position, specific gap penalties, and weight matrix choice.
Nucl. Acids Res. 22: 4673
4680. Wendel, J. F. and Doyle, J. J. 1998. Phylogenetic incongruence: window into genome history and molecular evolution.
In: Soltis, D. E. et al. (eds), Molecular systematics of plants II, DNA sequencing. Kluwer, pp. 265
296. White, T. J. et al. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics.
In: Innis, M. A. et al. (eds), PCR protocols: a guide to methods and applications. Academic Press, pp. 315
322. Wiens, J. J. 1998. Combining data sets with different phylogenetic histories.
Syst. Biol. 47: 568
581. Wojciechowski, M. F. 2003. Reconstructing the phylogeny of legumes (Leguminosae): an early 21st century perspective. 436
In: Klitgaard, B. B. and Bruneau, A. (eds), Advances in legume systematics, part 10. Higher level systematics. R. Bot. Gard. Kew, pp. 5
35. Wojciechowski, M. F. 2005. Astragalus (Fabaceae): a molecular phylogenetic perspective.
Brittonia 57: 382
396. Wojciechowski, M. F. et al. 1999. Evidence on the monophyly of Astragalus (Fabaceae) and its major subgroups based on nuclear ribosomal DNA ITS and chloroplast DNA trnL intron data.
Syst. Bot. 24: 409
437. Wojciechowski, M. F. et al. 2000. Molecular phylogeny of the ‘temperate herbaceous tribes’ of papilionoid legumes: a supertree approach.
In: Herendeen, P. S. and Bruneau, A. (eds), Advances in legume systematics, part 9. R. Bot. Gard. Kew, pp. 277
298. Wojciechowski, M. F. et al. 2004. A phylogeny of legumes (Leguminoseae) based on analysis of the plastid matK gene resolves many well
supported sub-clades within the family.
Am. J. Bot. 9: 1846
1862. Yoder, A. D. et al. 2001. Failure of the ILD to determine data combinability for slow loris phylogeny.
Syst. Biol. 50: 408
424. Zarre, S. and Podlech, D. 2001. Taxonomic revision of Astragalus sect. Acanthophace (Fabaceae).
Sendtnera 7: 233
255.