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Biosystems - An International Journal Dealing with all Aspects of Plant Biology Official Journal of the Societa Botanica Italiana

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Phylogeny and divergence times of the Coluteoid clade with special reference to Colutea () inferred from nrDNA ITS and two cpDNAs, matK and rpl32-trnL(UAG) sequences data

M. Moghaddam, S. Kazempour Osaloo, H. Hosseiny & F. Azimi

To cite this article: M. Moghaddam, S. Kazempour Osaloo, H. Hosseiny & F. Azimi (2016): Phylogeny and divergence times of the Coluteoid clade with special reference to Colutea (Fabaceae) inferred from nrDNA ITS and two cpDNAs, matK and rpl32-trnL(UAG) sequences data, Plant Biosystems - An International Journal Dealing with all Aspects of Plant Biology, DOI: 10.1080/11263504.2016.1244120

To link to this article: http://dx.doi.org/10.1080/11263504.2016.1244120

Published online: 19 Oct 2016.

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Download by: [Cornell University Library] Date: 30 October 2016, At: 10:43 Plant Biosystems, 2016 http://dx.doi.org/10.1080/11263504.2016.1244120

Phylogeny and divergence times of the Coluteoid clade with special reference to Colutea (Fabaceae) inferred from nrDNA ITS and two cpDNAs, matK and rpl32-trnL(UAG) sequences data

M. MOGHADDAM1, S. KAZEMPOUR OSALOO1, H. HOSSEINY1, & F. AZIMI2

1Department of Plant Biology, Faculty of Biological Sciences, Tarbiat Modares University, Iran and 2Natural Resource Research Center of Ardabil Province, Iran

Abstract

This study reconstructed the phylogeny of the Coluteoid clade using nrDNA ITS and plastid matK and rpl32-trnL(UAG) sequences data. The analyses resolve a well-supported Coluteoid clade, as sister to s.str. + Oxytropis, nested within the larger, strongly supported Astragalean clade. The Coluteoid clade is now composed of 12 genera including Podlechiella, , , , , Phyllolobium, Lessertia, Sutherlandia, , Smirnowia, Eremosparton and Colutea. Within this clade, Podlechiella is the first diverging lineage followed by successive subclades of Carmichaelia + Clianthus + Swainsona, Phyllolobium, Lessertia + Sutherlandia, Sphaerophysa + Smirnowia + Eremosparton, and Colutea. We assigned the formal tribal name to this clade and redefined the tribe Coluteae. A diagnostic key to the genera of the tribe is presented. Astragalus cysticalyx and A. sinicus have no relationship with the Coluteoid clade, instead, they are nested in Astragalus s. str. Resolution within Colutea is rather low, but several smaller subclades with low to high supports are found in the . None of the large sections in Colutea are monophyletic. Divergence time estimates revealed that the Coluteoid clade originated in the Early Miocene (20.4 Mya). Most of its members were diverged during the Late Miocene to Pliocene. Colutea and Podlechiella form the youngest lineages where the diversification occurred in the Pliocene- Pleistocene.

Keywords: Coluteae, divergence time, nrDNA ITS, phylogeny, plastid DNA, the Coluteoid clade

Introduction ciliate style (Lavin & Delgado 1990). All members of the clade except the annual Podlechiella are The Coluteoid clade is a heterogeneous group of perennial herbs, subshrubs, or /small trees. morphologically and ecologically distinct genera Based on the phylogenetic analysis of the combined and , which distributed throughout Africa to Australasia. Currently, it contains 12 genera, nrDNA ITS and matK sequences, Wojciechowski i.e. Colutea (including Oreophysa, see Kazempour (2005) indicated that Podlechiella is the first Osaloo et al. 2006), Smirnowia, Eremosparton, diverging taxon as sister to the other representatives Sphaerophysa, Lessertia, Sutherlandia, Carmichaelia, of the clade. Carmichaelia and allies formed own Clianthus, Montigena, Swainsona, Phyllolobium and subclade, as for Lessertia and Sutherlandia. In this Podlechiella as well as the two Astragalus species, study, Phyllolobium chinense Fisch. (=A. complanatus A. sinicus L. and A. cyticalalyx Ledeb. (Wojciechowski Bunge) formed a common clade with A. sinicus, and et al. 1999; Wojciechowski 2005; Lock & Schrire Colutea, represented by C. arborescense L., was sister 2005). Colutea and its allied genera were previously to A. cysticalyx. Furthermore, in the earlier study of treated in tribe subtribe Coluteinae Wojciechowski et al. (1999), Smirnowia and allies (Polhill 1981), while Carmichaelia and its allies (including A. cysticalyx) formed a distinct subclade formed subtribe Carmichaelinae (Heenan 1998a, within the Coluteoid clade. Kazempour Osaloo 1998b; Wagstaff et al. 1999) or carmichaelioid group et al. (2003) using molecular data (nrDNA ITS and (Lock & Schrire 2005). Phyllolobium and Podlechiella plastid ndhF) clearly indicated that A. sinicus is more have been recently segregated from Astragalus closely related to members of Astragalus s.str. than (Kazempour Osaloo et al. 2003; Zhang & Podlech to the Coluteoid clade. However, the taxonomic 2006). Most members of the clade are characterized composition of the Coluteoid clade and the exact by non-interlocking wing and keel petals and a phylogenetic status of A. cyticalyx are still unclear.

Correspondence: S. Kazempour Osaloo, Department of Plant Biology, Faculty of Biological Sciences, Tarbiat Modares University, Tehran 14115-154, Iran. Email: [email protected] © 2016 Società Botanica Italiana 2 M. Moghaddam et al.

After Swainsona (about 80 spp.) and Lessertia fied using the primers ITS5 m of Sang et al. (1995) (about 50 spp), Colutea is the third large genus of and ITS4 of White et al. (1990) or AB101F and the clade with about 30 species. Colutea is distribut- AB102R (Douzery et al. 1999). The matK gene was ed from Mediterranean region to China, Himalaya, amplified using the primer of trnK685F (Steele & and east–northeast Africa. In Iran, Colutea has nine ­Wojciechowski 2003) and matK1320R (designed in species in 3–5 sections (Browicz 1984; Pooyan et al. this study). The rpl32-trnL(UAG) region was amplified 2014). Among the species-rich genera of the Colu- using primer rpl32-F and trnL(UAG) described in Shaw teoid clade, Carmichaelia and Swainsona (Wagstaff et al. (2007). The PCR amplification was carried out et al. 1999), Phyllolobium (Kang et al. 2003; Zhang in the volume of 20 μL, containing 8.2-μL deionized et al. 2012) and Colutea (Mirzaei et al. 2015) have water, 10 ml of the 2 × Taq DNA polymerase master been subjects of phylogenetic analyses using nrDNA mix Red (Amplicon, Cat. No. 180301, 150-mMTris- H ITS data. HCl P 8.5, 40-Mm (NH4)2SO4, 3.0-Mm MgCl2, By employing penalized likelihood estimates 0.4-Mm dNTPs, 0.05 units μl−1AmpliconTaq DNA of ages and rates of nucleotide substitutions using polymerase, inert red dye and a stabilizer) 0.5 μl matK sequences, Wojciechowski (2005) provided the of each primer (10 pmol/μl), and 1 μl of template estimated mean age of 13.7 (±1.70) Mya, the middle DNA (20 ng/μl). PCR procedures for nrDNA ITS Miocene, and mean rate of 0.000894 (±0.000080) region were 2 min 30 s at 94°C for predenaturation for the Coluteoid Clade. followed by 30 cycles of 45 s at 94°C for denatura- Hitherto, no detailed phylogenetic treatment tion, 40 s at 56°C for primer annealing and 40 s at and divergence time estimates using multiple 72°C for primer extension, followed by a final ­primer DNA sequence­ data have been conducted on the ­extension of 7 min at 72°C. for matK region, the ­Coluteoid clade in general and Colutea in particular. PCR condition was 2 min 30 s at 94°C followed by In this study, the nuclear ribosomal DNA internal-­ 32–35 cycles of 40 s at 94°C, 40 s at 53°C, and 1 min transcribed spacer (nrDNA ITS) and two plastid 20 s at 72°C, and final extension of 7 min at 72°C fragments, matK gene and rpl32-trnL(UAG) intergenic was performed. For rpl32-trnL(UAG)region, the PCR spacer, were sequenced for phylogenetic reconstruc- condition was 5 min at 80°C followed by 28 cycles of tions and estimation of ages and rates. 50 s at 95°C, 45 s at 54°C, and 50 s at 65°C, and the

The main goals of present study are: (1) to evalu- final extension of 5 min at 65°C was performed. The ate the taxonomic status of the Coluteoid clade and ensuring PCR fragments were separated by electro- to elucidate relationships within it, (2) to test the phoresis in 1% agarose gels in 1 × TAE (pH = 8) monophyly of Colutea and its major sections, (3) to buffer, stained with ethidium bromide and were assess the accurate taxonomic position of Astragalus photographed with a UV gel documentation ­system cysticalyx, and (4) to estimate molecular divergence (UVItec, Cambridge, UK). Purification of PCR times and ages of the Coluteoid clade and its major products was performed using NucleoSpin Extract subclades. kits (MACHERY-NAGEL April 2004/Rev.01). Each region was directly sequenced using the Big Dye Terminator Cycle Sequencing Ready Reaction Materials and methods kit with the same primers in an ABI 3730xl DNA Taxon sampling Analyzer (Applied Biosystems, USA) through ­Pishgam Inc. Forty-three accessions representing 29 species ­belonging to Coluteoid clade and 12 species of the related clade (Astragalus + Oxytropis) were included Sequence alignment in the analyses. One species of Caragana and one species of Eversmania were selected as outgroups. Each of the individual data-sets was edited ­using In addition to the newly generated nrDNA ITS ­BioEdit ver. 7.0.9.0 (Hall 1999) and aligned with (23), matK (21) and rpl32-trnL (28) ­sequences, web-based version of MUSCLE (Edgar 2004; (UAG) at http://www.ebi.ac.uk/Tools/msa/muscle/) and 20 nrDNA ITS, 16 matK, and 1 rpl32-trnL(UAG) ­sequences were retrieved from GenBank. All taxa ­adjusted manually. The alignment of the data-sets together with their origin, voucher information, and required for the introduction of numerous single- GenBank accession numbers are listed in Table I. and multiple-base indels (insertions/deletions). The indels were treated as missing data for all data-sets.

DNA isolation, PCR and sequencing Phylogenetic analyses Total genomic DNA was isolated from materi- als using the modified CTAB method of Doyle and Parsimony method. Maximum parsimony (MP) Doyle (1987). The nrDNA ITS region was ampli- analyses were conducted using PAUP* version Phylogeny and divergence times of the Coluteoid clade 3

Table I. Taxa included in the nrDNA ITS, matK and rpl32-trnL(UAG) analyses. GenBank accession no.

Species DNA source (location, voucher) nrDNA ITS matK rpl32-trnL(UAG) Colutea cilicica Boiss. & Bal. Turkey: Ozbek 1569 (GAZI) LC164570/ LC164592/ LC164609 C. gracilis Freyn & Sint. Iran: Faghihnia & Zangui 18724 (FUMH) LC164572/-/ LC164611 C. porphyrogramma Rech.f. Iran: Ghahreman et al. 22335 (TUH) LC164577/ LC164596/ LC164616 C. persica Boiss. Iran: Manucheri et al. 277(TARI) LC164576/ LC164595/ LC164615 C. gifana Parsa Iran: Memariani & Zangui 39615 (FUMH) LC164571/ LC085325/ LC164610 C. paulsenii Freyn Tajikistan: Pekovo 223 (TARI) LC164575/ LC164594/ LC164614 C. armena Boiss.& Huet. Turkey: Duzenli 1210 (ANK) LC164568/ LC164591/ LC164607 C. melanocalyx Boiss.& Heldr. Turkey: Pesermen & Guner 1522 (ANK) LC164574/ LC164593/ LC164613 C. komarovii Takht. Iran: Ghahremani & Imani 7815 (TMUH) LC164573/ LC085324/ LC164612 C. triphylla Bunge ex Bioss. Iran: Assadi & Mozaffarian 33325 (TARI) LC164578/ LC085323/ LC164617 C. arborescens L. GenBank U56009, U56010b /AY386874b /- C. uniflora G. Beck Iran:Charkhchian, 1687 (TMUH) LC164579/-/ LC164618 C. buhsei (Boiss.) Shap. Iran: Hosseiny 2008-5-8 (TMUH) LC164569/ LC085326/ LC164608 (Pall.) DC. Iran: Azimi & Talaii 3687 (TMUH) LC164588/ LC164603/ LC164630 S. kotschyana Boiss. Turkey: Aytac et al. 6585 (GAZI) LC164587/ LC164602/ LC164629 Lessertia capitata E. Mey South Africa: Goldblatt 2220 (MSB) LC164583/ LC164598/ LC164622 L. benguellensis Baler South Africa: Giessjun 55 (MSB) LC164582/ LC085328/ LC164621 L. annularis Burch. South Africa: Merxmuller & Giessjun 28236 LC164581/ LC085327/ LC164620 (MSB) Eremosparton flaccidum Litw. Turkmanistan: Beljanina et al. 9353 (MSB) LC164580/ LC164597/ LC164619 Smirnowia turkestana Bunge Iran: Maddah & Moradi 3981 (TARI) LC164586/ LC164601/ LC164628 Sutherlandia frutescens (L.) R. Br. South Africa: Mummenhoff 851 (TMUH) LC164589/ LC164604/ LC164631 Podlechiella vogelii ssp. vogelii (Webb) Algeria: Podlech 36700 (TARI) LC164585/ LC164600/ LC164627 P.vogelii ssp. fatimensis (Chiov) Iran: Mozaffarian et al. 39103 (TARI) AB051911b/ LC164599/ LC164626 Oxytropis aucheri Boiss. Iran: Maassoumi 55104 (TARI) AB051908b/-/ LC164623 O. lambertii Pursh. GenBank L10807b/ AY386915b/- O. pilosa (L.)DC. GenBank AF121759b/ AY920452b/- O. deflexa (Pallas) DC. GenBank L10804b/ AY386878b/- O. szovitsii Boiss. & Buhse Iran: Assadi & Maassoumi, (TARI) AB051909b/-/LC164624 Astragalus sinicus L. Japan: Kazempour Osaloo 1999-01 (TARI) AB051985b/-/AB908471b A. canadensis L. GenBank U50496b/ AY386875b/- A. patagonicus Speg. GenBank AF121746b/AY920446b/- A. nothoxys A.Gray GenBank AF121688b/AY386877b/-

A. peristereus Boiss. & Hausskn. GenBank U50494b/AY920449b/- A. cysticalyx Ledeb.a GenBank AF121682b/AY920443b/- A. cysticalyx Ledeb. China: Xu 87146 (MSB) LC164567/ LC164590/ LC164605 A. adsurgens Pallas GenBank AF121674b/ HM049537b/- Phyllolobium chinense Fisch. GenBank GU217602b/ HM049530b/- P. balfourianum (N. D. Simpson) M. L. China: Dickore 14239 (MSB) LC164584/-/ LC164625 Zhang & Podlech Eversmania subspinosa (Fisch) B. Fedtsch. Iran: Freitag & Mozaffarian 28397 (TARI) AB329692b/AB854573b/LC137102b Caragana grandiflora (M.B.) DC. Iran: Assadi & Shahsavari 65834 (TARI) AB051905b/AB854564b/ LC164606 Kirk GenBank U50520b/AY386873b/- (G.Don) Sol. Ex Lindl. GenBank L10800b/AY386914b/- Swainsona pterostylis (DC.) Bakh. f. GenBank U56007b/AF142735b/- aThe voucher specimen of A. cysticalyx determined in the previous works (e.g., Liston & Wheeler 1994) definitely belongs to Sphaerophysa salsula. bSequences from GenBank. (-) not available in GenBank. Abbreviations used in plant accession information: ANK, Ankara Universitesi Herbarium, Ankara, Turkey, FMUH, Ferdowsi University of Mashhad Herbarium, Mashhad, Iran, GAZI, Gazi Universitesi Herbarium, Ankara, Turkey, MSB Herbarium of Ludwig-Maximil- ians-Universitat, Munchen, Germany, TARI Herbarium of the Research Institute of Forests and Rangelands, Tehran, Iran, TMUH Tarbiat Modares University Herbarium, Tehran, Iran, TUH Tehran University Herbarium.

4.0b10 (Swofford 2002). The heuristic search option program MrModeltest version 2.3 (Nylander 2004) was employed for each of the data-sets, using tree based on the Akaike information criterion (AIC) bisection-reconnection (TBR) branch swapping, (Posada & Buckley 2004). The data-set was analyzed with 1000 replication of random addition sequence as a single partition with GTR + I + G model using and an automatic increase in the maximum number the program MrBayes version 3.1.2 (Ronquist of trees. Uninformative characters were excluded & Huelsenbeck 2003). Posteriors on the model from the analyses. Branch support values were parameters were estimated from the data, using the calculated using a full heuristic search with 1000 default priors. The analysis was done with 5 million bootstrap replicates (Felsenstein 1985) each with generation, using Markov chain Monte Carlo search. simple addition sequence. MrBayes performed two simultaneous analyses starting from different random trees (Nruns = 2) Bayesian method. Model of sequence evolution each with for Markov chain and trees sampled at for the combined data-set was selected using the every 100 generations. The first 25% of trees were 4 M. Moghaddam et al. discarded as the burn-in. The remaining trees were BEAST ver. 1.7 (Drummond et al. 2012). Since then used to build a 50% majority rule consensus the reliable fossil records for the Coluteoid clade tree accompanied with posterior probability (PP) are rather poor, the clock was calibrated using the values. Tree visualization was carried out using Tree estimate of mean age 33.0 ± 2 Mya for a node, View version 1.6.6 (Page 2001). designated as node 76, encompassing from Caragana arborescens through Astragalus americanus based upon matK sequence data of Lavin et al. (2005). Likelihood method. Maximum likelihood (ML) A likelihood-ratio test implemented in MEGA 6 analyses were performed for the data-sets in the (Tamura et al. 2013) was used to test if the different program raxmlGUI (Silvestro & Michalak 2012). partitions included in dating analysis were evolving The model of evolution employed for each data-set clock-like. This information was used to choose is the same as that of Bayesian analyses. Parametric between the strict-clock and the relaxed uncorrelated bootstrap values for ML were calculated in raxmlGUI lognormal clock priors implemented in BEAST. In based on 1000 replicates with one search replicate this study, strict clock prior was selected. Analyses per bootstrap replicate. were run for 10 × 106 generations, with a burn-in of one million generations. Incongruent length difference test. Combinability of nuclear and plastid data-sets was assessed using Results the partition homogeneity test [the incongruence length difference test (Incongruent length difference Phylogenetic analyses test (ILD)) of Farris et al. 1995] as implemented in The visual inspection showed that all of the clades PAUP* (Swofford 2002). The test was conducted with present in the nrDNA ITS tree were almost the exclusion of invariant characters (Cunningham ­recovered in the plastid (matK + rpl32-trnL(UAG)) 1997) using the heuristic search option involving tree (­Figure 1). Also, the partition homogeneity­ test simple addition sequence and TBR branch swapping ­suggested that the trees obtained from the ­nrDNA with 1000 homogeneity replicates. ITS and the plastid data-sets were congruent­

(p = 0.4); and thus, we combined these data-sets (­Figure 2). Both nrDNA ITS and, in particular Divergence time estimation. Divergence times were ­plastid (matK + rpl32-­trnL ) topologies have, estimated from the combined phylogeny using (UAG)

Figure 1. A comparison of A. nrDNA ITS and B. matK+ rpl32-trnL(UAG) datasets for Coluteoid clade and Astragalus s.str. obtained from Bayesian inference. Phylogeny and divergence times of the Coluteoid clade 5 however, generally ­lower resolution and node sup- Maximum parsimony, maximum likelihood, and port than the combined data-set. We described and Bayesian analyses of the combined data-set gave interpreted the results mainly based on the com- very similar results. We here show only Bayesian tree bined nrDNA ITS and plastid data-set. along with posterior probabilities and ML and MP

Figure 2. Fifty percent majority rule consensus tree resulting from Bayesian analysis of the combined nrDNA ITS, matK and rpl32- trnL(UAG) dataset. Numbers above branches are posterior probability and likelihood as well as parsimony bootstrap values, respectively. Values <50 % were not shown. 6 M. Moghaddam et al. bootstrap values (Figure 2). The length and compo- (MRCA) of the Coluteoid clade was dated to the sition of each DNA region sequenced, as well as the Early Miocene (20.4 Mya), most genera of which tree statistics from the nuclear and plastid as well as originated in the Late Miocene through Pliocene to combined analyses are summarized in Table II. As Pleistocene (Figure 3). When two molecular clock shown in Figure 2, Oxytropis and Astragalus s.str. are rate variation models (strict vs. relaxed clock) were sister taxa and, in turn, united with the Coluteoid tested, the mean rate of the individual clock models clade. This clade was composed of six well-support- did not show any variation. The fastest and the slow- ed, successive sub-clades (indicated by “I” through est rate were found in the Astragalean clade (node 1) “VI”). Podlechiella was the first diverging lineage (I) and the Coluteoid clade (node 3), respectively. The as sister to the remaining subclades. Three Australian rate of evolution in node 1 was 0.0026 substitu- / taxa (Carmichaelia, Clianthus and tions per site per million years (s/s/My) (95% HPD: Swainsona), constituted the subclade “II,” followed 0.0002–0.0051) and in node 3, 0.0008 s/s/Ma (95% by Phyllolobium (the subclade “III”). Next clade HPD: 0.0001–0.0026). was the South African subclade (“IV”) comprising Lessertia and Sutherlandia, followed by the Central Asian subclade (“V”) of Sphaerophysa (represented Discussion by three accessions of two species), Smirnowia and Taxonomic status of and relationships within the Eremosparton and the subclade Colutea (“VI”). With- Coluteoid clade in Colutea subclade, C. cilicica Boiss. & Bal. are sister to the remaining species. The present work in agreement with previous stud- ies (Wojciechowski et al. 1999; Lock & Schrire 2005; Wojciechowski 2005) retrieved the Coluteoid clade Divergence time estimates as a well-supported lineage. According to the data presented here, the clade is now only composed Our BEAST chronogram is largely consistent with of 12 genera including Podlechiella, ­Swainsona, those resulting from Bayesian analyses (Figure 2). ­Carmichaelia, Clianthus, Montigena (not ­analyzed The overall estimated mean ages and mean rates herein), Phyllolobium, Lessertia, Sutherlandia, of evolution are presented in Table III. Our mean

Sphaerophysa, Smirnowia, Eremosparton, and ­Colutea nodal Bayesian divergence time estimates indicated (including the monotypic Oreophysa, Kazempour that stem–node (the root) is estimated to be Early Osaloo et al. 2006). The members of the clade ­Oligocene, ~33 Mya. Furthermore, the analysis sug- are characterized by having pubescent styles and gested an Oligocene-Miocene origin of the ­Astragalean ­mainly unilocular pods. Unlike previous studies clade (Figure 3). The most recent common ancestor

Table II. Dataset and tree statistics from separate and combined analyses of the nuclear and chloroplast regions.

nrDNA ITS plastid (matK + rpl32-trnL(UAG)) Combined Number of sequences 43 43 43 Nucleotide sites 681 2711 3392 Informative characters 146 243 389 Uninformative characters 535 2468 3003 CI of MPTs 0.647 0.711 0.676 RI of MPTs 0.828 0.808 0.812 Number of MPTs 322 >200000 136584 Length of MPTs (steps) 326 418 752 Evolutionary model selected (under AIC) SYM+G GTR+G GTR+I+G

Table III. The node mean ages and rates and range (95% HPD) of evolution with the posterior probability are shown.

Mean age Min Max Mean rate Node Node definition (Ma) (Ma) (Ma) (s/s/Ma) 95% HPD (Ma) Root Caragana grandiflora –Colutea uniflora 33 – – – – 1 Astragalus patagonicus – Coluteauniflora 24.5 15.9 34.1 0.0026 0.0002–0.0051 2 Oxytropis aucheri – Astragalus patagonicus 20.8 11.7 34.2 0.0017 0.0002–0.0049 3 Podlechiella vogelii –Colutea uniflora 20.4 11.8 32.2 0.0008 0.0001–0.0026 4 Clianthus puniceus – Swainsona pterostylis 8.5 3.2 16.3 0.0013 0.0004–0.0034 5 Phyllolobium balfourianum – P. chinense 6.2 1.6 13.09 0.0015 0.0004–0.0035 6 Sphaerophysa salsula– Smirnowia turkestana 6.3 2.7 11.4 0.0022 0.0006–0.006 7 Colutea cilicica – C. uniflora 3.2 1.3 6.6 0.0013 0.0003–0.0023 8 Lessertia capitata – Sutherlandia frutescens 4.5 1.5 9.5 0.0013 0.0003–0.0023 9 Podlechiella vogelii ssp. fatimensis – P. vogelii ssp. vogelii 3.2 0.7 8.1 0.0016 0.0007–0.0028 Notes: Node numbers refer to Figure 3. Mean ages and mean rates and 95% highest posterior density (HPD) intervals of divergence times are in millions of years before the present. Ma = million years; s/s/Ma = substitutions per site per million years. Phylogeny and divergence times of the Coluteoid clade 7

Figure 3. Chronogram inferred from BEAST. Each node represents the mean divergence time estimate and grey bars represent the 95% highest posterior density intervals around mean nodal ages. Numbers corresponding to dated groups shown in Table III are written above the nodes.

(Liston & Wheeler 1994; Sanderson & Liston 1995; clusion. In contrast, as mentioned above, our molec- ­Wojciechowski et al. 1999; Wojciechowski 2005), ular data clearly revealed that Astragalus cysticalyx, results of our study indicated that neither Astragalus (the herbarium specimen used for this species pre- sinicus L. nor A. cysticalyx Ledeb. is closely related to served in MSB and confirmed by D. Podlech) has no the Coluteoid clade, instead nested within Astragalus­ relationship with the Coluteoid clade, instead, it is s.str. (Figures 1 and 2). Kazempour Osaloo et al. well united with A. adsurgens Pall. within Astragalus (2003, 2005) have already revealed the accurate s. str. clade. The identical rpoC, matK, and nrDNA position of A. sinicus as a member of Astragalus ITS sequences of A. cysticalyx with S. salsula indi- s.str. and well united with the Taiwanese endemic cates that the voucher specimen of A. cysticalyx used A. ­nankotaizanensis Sasaki. Liston and Wheeler as a source of DNA in the previous studies definitely (1994), based on parsimony analysis of restriction belongs to S. salsula. Therefore, the Coluteoid clade site data of plastid gene rpoC, firstly pointed out that has no species of Astragalus. The present study sug- A. cysticalyx was placed within the Coluteoid clade. gests that the Coluteoid clade should be redefined to They noted that: “although this species has an iden- encompass all members of the tribe Coluteae men- tical haplotype with Sphaerophysa salsula (Pall.) DC., tioned above (see “Taxonomic treatment”). no obvious morphological characters link these two Within the Coluteoid clade, Podlechiella (subclade taxa.” The subsequent studies using nrDNA ITS and “I”) formed the most basal branch. This conclusion matK from the same aliquot (Wojciechowski et al. was first reached by Wojciechowski (2005). It is a 1999; Wojciechowski 2005), reached the same con- monotypic genus with two subspecies, P. vogelii (Webb) 8 M. Moghaddam et al.

Maassoumi & Kaz. Osaloo ssp. vogelii and P. vogelii the placements of these taxa may be the result of ssp. fatimensis (Chiov.) Maassoumi & Kaz. Osaloo hybridization/introgression event and subsequent distributed in subtropical deserts of North Africa chloroplast capture or lineage sorting. The inter- and southwest Asia (Kazempour Osaloo et al. 2003). specific relationships are not sufficiently resolved Among the members of the Coluteoid clade, this is the within the bulk of Colutea. Three smaller subclades sole genus having annual life cycle, medifixed hairs, (C. persica Boiss./C. komarovii Takht.; C. gifana very small (c. 3 mm), and autogamy (Podlech Parsa, C. uniflora G. Beck/C. paulsenii Freyn and 1984, 1999, personal observation). The current C. arborescens L./C. melanocalyx Boiss.& Heldr.) with hypothesis of phylogenetic relationships suggests that low to high supports are, however, found in the these features are plesiomorphic in the Coluteoid combined tree. The present data suggest that the calde. The findings of the previous studies (Wagstaff multi-specific sections Colutea, Armata, and Rostrata et al. 1999; Wojciechowski et al. 1999; Wojciechowski are not monophyletic and that their representatives 2005) are in accordance with our results in placing analyzed here are intermixed. This contradicts with the Australian-New Zealand lineage (subclade “II”) the conclusions of Mirzaei et al. (2015). Hence, as the next subclade . Another subclade is Himalayan the delimitation of the sections based upon gross Phyllolobium (subclade “III”) as successive sister morphological characters (Browicz 1963a, 1984; to the remaining taxa. All Phyllolobium species are Pooyan et al. 2014) seems to be artificial. perennial and occur mainly in the southwestern high The low phylogenetic resolution within Colutea mountains of China (Zhang 2003; Zhang & Podlech reflects the low sequence divergence values across it, 2006). As next, Lessertia and Sutherlandia (subclade i.e. less than 0.024 (0.000–0.023) substitutions per “IV”) form a monophyletic group restricted to South site for ITS and less than 0.007 (0.000–0.006) for Africa. The well-supported subclade “V” is composed plastid data-set. The low nucleotide variability dur- of Eremosparton flaccidum Litw. together with the ing speciation might be an indicator of rapid radia- monotypic Smirnowia (S. turkestana Bunge) and two tion in a lineage’s evolutionary history (Baldwin & species of Sphaerophysa. In this subclade, Eremosparton Sanderson 1998; Richardson et al. 2001; Hughes & and Smirnowia are restricted to central Asian deserts, Eastwood 2006; Sun et al. 2012). The lower nucle- Iran and Afghanistan, and Sphaerophyas has wide otide substitution rate among Colutea species might distribution in Central Asia, Iran, Afghanistan, and also be explained by the generation time (GT; Graur Turkey (Rechinger 1984). The last subclade (“VI”) & Li 2000; Gaut et al. 2011) and the rate of mitosis comprises only the species of Colutea. The monophyly (ROM; Lanfear et al. 2013) hypotheses. There is now of this genus was also confirmed by the recent nrDNA a consensus that evolutionary rate is negatively cor- ITS study of Mirzaei et al. (2015). related with both GT and ROM. Both ­phenomena in Colutea have to be longer and slower, respectively, because of the woody (shrubby/tree) habit. Shrubs/ Taxon relationships within Colutea trees are consistently evolving more slowly than Colutea is a medium-sized genus of ca. 30 species ­related herbaceous (Smith & Donoghue 2008; with shrubby to sub-shrubby habit or small tree Gaut et al. 2011; Lanfear et al. 2013). having inflated pods (Browicz1963a, 1968, 1984; Mabberley 2008; Pooyan et al. 2014). The genus Divergence time estimation was first subdivided into two sections, Colutea (as Eucolutea) and Oreophysa Bunge ex Boiss. by Based on an evolutionary rates analysis of the matK Boissier (1872). In a monographic work, Browicz phylogeny, Wojciechowski (2005) indicated that the (1963a) classified the genus into four sections, divergence time of the Astragalean clade is in the Armata Browicz, Colutea, Multiflora Browicz, and Early Miocene (16.1 Mya). Our dating analysis of Rostrata Browicz. Simultaneously, Browicz (1963b) the combined data estimates, however, the age of resurrected Oreophysa (Bunge ex Boiss.) Bornm. this clade 24.5 Mya (Oligocene-Miocene bounda- (Bornmuller 1905) as a monotypic genus, restricted ry), with the split between Oxytropis/Astragalus s.str. to northern Iran in Elburz Mountains. In a recent and the Coluteoid clade to be 20.8 and 20.4 Mya, molecular phylogenetic study, Kazempour Osaloo et respectively. This finding is in agreement with Axel- al. (2006), however, reduced it to the sectional level, rod’s hypothesis (1992) which postulated that rep- as sect. Oreophysa within Colutea (C. triphylla Bunge ex resentatives of the Astragalean clade were widely Bioss.). Plastid and the combined analyses show that distributed in temperate regions in Oligocene. Our Colutea cilicica Boiss. & Bal. forms a sister-group to the estimated mean age indicates that the Coluteoid remaining species of Colutea. But, in the nrDNA ITS clade dates to the Early Miocene. This is more or tree, C. arborescence L. is the first diverging branch of less consistent with estimates from analysis of matK Colutea. This discrepancy between the trees regarding sequences of Wojciechowski (2005), who suggest- Phylogeny and divergence times of the Coluteoid clade 9 ed the mean age of 13.7 Mya (Middle Miocene). Conclusions Our dating analysis indicates that the diversification­ The present study using multiple sequence of some subclades of the Coluteoid clade might data demonstrated that the Coluteoid clade is a have occurred ­contemporaneously during the Late well-supported lineage and thus herein redefined ­Miocene. Among these subclades, the youngest age to interpret its own tribe Coluteae, which compris- ones (3.2 Mya) are Podlechiella vogelii and Colutea es 12 genera. Podlechiella vogelii is retrieved as the (­Figure 3). In the ­Coluteoid clade, the first branch, basal most taxon of the tribe. Colutea is monophy- P. vogelii, diverged from the rest of the Coluteoid letic as sister to the Central Asian desertic genera, clade 20.4 Mya, although­ it began to diversify to two although the resolution and relationships within subspecies (Kazempour Osaloo et al. 2003) in the it are not sufficiently resolved. Our data clearly Pliocene (3.2 Mya). Next is Australian–New Zealand demonstrated that A. cysticalyx and A. sinicus have subclade (carmichaeloid group), which comprises no relationship with the Coluteoid clade, but they ­Carmichaelia, Clianthus, Swainsona, and Montigena are belonging to Astragalus s. str. Based on this (not analyzed in this study), diverged by the Late study, the tribe is originated in early Miocene and ­Miocene (8.5 Mya).The third subclade is Phyllolobium, diversified during the late Miocene through the which diverged by the Late Miocene (6.2 Mya) Pliocene–Pleistocene. The additional taxon sam- ­according to our results. Zhang et al. (2012) based pling and DNA markers especially nuclear single on nrDNA ITS, however, estimated 3.96 Mya (95% copy genes are definitely necessary to be analyzed HPD: 1.84–6.59) for Phyllolobium and pointed out for getting a clear-cut picture of Colutea phyloge- the genus has undergone rapid diversification in ny. Moreover, it will be also merit to focus on the the Late Pliocene and the Early-to-Mid Pleistocene evolutionary history of Swainsona and Lessertia, as which matches with the beginning of the intense uplift the largest genera of the tribe. of the Qinghai-Tibetan Plateau during the Late ­Pliocene. The South African subclade (Sutherlandia+ Lessertia) diverged by the Pliocene (4.5 Mya). This is consistent with recent studies which suggesting a Taxonomic treatment recent burst speciation might be occurred in the flora of Southern Africa triggered by the climatic ­changes Tribe Coluteae (Benth.) Hutch., The genera of flow- near the Miocene–Pliocene boundary (Schnitzler­ ering plants, vol. 1: 404, 1964. Emend. Kaz. Osaloo et al. 2011). The South African endemics and & Moghaddam. ­Carmichaeloid group have independent northern Synonym: Tribe Galegeae subtribe Coluteinae­ hemisphere origin within the Coluteoid clade. This (as Coluteae) Benth., Gen. Pl. 1: 446, 1865; conclusion was first reached by Wagstaff et al. (1999). ­Advances in Legume Systematics 1: 359, 1981; Tribe Sphaerophysa, Smirnowia, Eremosparton, which are re- ­Carmichaeliae Hutch., The genera of ­flowering stricted to dry sandy deserts in Central Asia, began plants, vol. 1: 373, 1964, Advances in Legume to diversify in the Late Miocene (6.3 Mya). During ­Systematics 1: 364, 1981. the Middle–Late Miocene (17–5 Mya), the climate in Shrubs, subshrubs, and perennial or rarely ­annual Central Asia was drying to an increasing extent (Guo herbs, imparipinnate, rarely 1-­foliolate, ­rarely et al. 2008; Miao et al. 2012; Zhang et al. 2014). This reduced to scales; leaflets entire; stipels ­absent; harsh environmental condition accompanied by the ­Indumentum of basifixed and rarely medifixed hairs., special life form (subshrubby) and restrictions pro- flowers in axillary sometimes pendulous ­, vided by reproductive characteristics of these genera flowers larg rarely smaller (c. 3 mm); bracts and might explain the lower speciation in this subclade. It bracteoles small or absent; calyx-teeth subequal or appears that Colutea was diverged from Central Asiatic upper two shorter; corolla papilionaceous, standard relatives (Sphaerophysa, Smirnowia and Eremosparton) often reflexed, stamens free, anthers uniform 2 in the Late Miocene period (10 Mya), but its (1)-thecous, ovules two-numerous, style barbate, ­diversification occurred during the Pliocene and Pleis- rarely glabrous. Fruit often inflated or turgid, rarely tocene. Unlike the Central Asian desertic ­relatives, not inflated, mostly unilocular, rarely semi-bilocular, Colutea has a shrubby/tree habit and is ­mostly usually indehiscent. reniform. The type genus: ­distributed locally in drier mountainous ­regions. Colutea L. During the Pleistocene, the evolution of species­ in A key to the genera of the tribe Coluteae adopted the mountains was more strongly enhanced. A corre- and modified from Hutchinson (1964) and Polhill lation between the shift from herbaceous/subshrubby (1981). to shrubby/tree habit and the shift from lowland to 1. Annual herbs; North Africa, and southwest montane habitats is common among alpine species Asia……………………...... ……Podlechiella that might give rise to species radiations (Hughes & - Perennial herbs, shrubs, subshrubs, or small Atchison 2015; Schwery et al. 2015). trees …………………………………..…2 10 M. Moghaddam et al.

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Zhang ML, Kang Y, Zhong Y, Sanderson SC. 2012. Intense uplift Zhang ML, Hao XL, Sanderson SC, Vyacheslav BV, Sukhorukov of the Qinghai-Tibetan Plateau triggered rapid diversification AP, Zhang X. 2014. Spatiotemporal evolution of Reaumuria of Phyllolobium (Leguminosae) in the Late Cenozoic. Plant (Tamaricaceae) in Central Asia: insights from molecular Ecol Divers 5: 491–499. biogeography. Phytotaxa 167: 89–103. 本文献由“学霸图书馆-文献云下载”收集自网络，仅供学习交流使用。

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