1

Phylogenetic Origin of Phyllolobium with a Further

Implication for Diversifiction of Astragalus in China

Mingli Zhang 1*, Yun Kang 2*, Lihua Zhou 3 , Dietrich Podlech 4

(1 Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China; 2 Pharmacy School, Fudan University, Shanghai 200032, China;

3 Department of Botany, California Academy of Sciences, San Francisco, CA 94118-4503, USA; 4 Faculty of Biology, Münich University, Münich D-80336, Germany)

*Email <zhangml@ibcas.ac.cn, ykang123@yahoo.com.cn>

Abstract

Astragalus is a species-rich genus occurring in western arid habitats in China. However,

its diversification and infrageneric relationships in this region remain unclear. In this

study, based on the molecualr data, we aim to (1) test whether Phyllolobium (previously

treated as a subgenus Pogonophace in Astragalus) should be warranted and (2) date the

origin of Phyllolobium and probable diversification of Astragalus sensu stricto (s.s.). We

seqeunced five species from Phyllolobium firstly and collected all related sequences from

this genus,Astragalus s.s and their close relatives (Oxytropis and Caragana etc.). Our

phylogenetic analyses suggested that all species of Phyllolobium comprise a

monophyletic group sister to genera of subtribe Coluteinae. Molecular dating suggested

that Phyllolobium and Astragalus s.s. originated around 8 and 10 million years ago (Ma).

These two estimations are highly consistent with the intense uplifts of the Qinghai-

Tibetan Plateau (QTP) inferred from the geological evidence. In addition, one section of

Pogonophace (Sect.Robusti) was estimated to originate 2.50 Ma and this section with a

preferencing of dry habitats seems a trace of Asian intensified aridity resulting from the

intense uplift of the QTP.

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Key words: Astragalus, Phyllolobium, Sino-Himalayan flora, phylogeny, dating,

diversification, distribution pattern, Qinghai-Xizang (Tibet) Plateau (QTP).

As one of the largest genera of angiosperms, Astragalus comprises about 1800-2500

species worldwide (Polhill, 1981; Sanderson, 1991; Podlech, 1999; Lock, Schrire, 2005).

There are three diversification centers in the world, namely Iran-Turkey center,

Himalayan center and western North American center (Sanderson, 1991). As one of three

centers, Himalaya and adjacent regions, locating in southwestern China, is named the

Sino-Himalayan floristic region (Wu & Wu, 1999). Here has a rich biodiversity among

the Northern Temperate flora (Wu, 1988).

According to Fu et al. (1993), there are ca. 290 species belong to eight subgenera sensu

Bunge (1868, 1869, whole genus includes eleven subgenera) in China. Most species in

the genus have the characteristic of northern temperate herbaceous, appearing in the

southwestern cold and humid/drought area, and northwestern drought and semi-drought

area. For instance, subgenus Pogonophace, with a taxonomical diagnostic of the bearded

stigma, mainly occurs in southwestern cold and humid high mountains (Wenninger, 1991;

Zhang, 2000).

Astragalus molecular systematics in legume and the subfamily Faboideae

(Papilionoideae) (see Kajita et al., 2001; Dong et al. 2003; Wojciechowski, 2003, 2005;

Wojciechowski et al., 2004) shows that this genus should be regarded as one of

representative large groups in Galageae. Galageae, Vicieae and other temperate

herbaceous groups compose most of a young clade in legumes, to been named IRLC

(inverted-repeat-lacking clade) which is one of eleven subclades of the subfamily

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Faboideae (Wojciechowski et al., 2004). Liston & Wheeler (1994), Sanderson & Liston

(1995), Sanderson & Wojciechowski (1996), and Wojciechowski et al. (1999) discussed

the monophyly of Astragalus based on the sequence data. As a larger world-cosmopolitan

group, non-monophyly at subgeneric rank has been presented based on the molecular

phylogeny from Eurasian taxa (Kang et al., 2003; Kazempour et al., 2003, 2005;

Wojciechowski 2005). Two new genera, Phyllolobium and Podlechiella have been

proposed to be segregated from Astragalus (Kang et al., 2003; Kazempour et al., 2003,

2005). For the historical biogeography, Sanderson & Wojciechowski (1996) used ITS

data indicated that the diversification rates in Astragalus were higher than its closest

relatives. Wojciechowski (2005) used matK and ITS data to estimate the ages of

Astragalus and relatives; the results shown that Astragalus and its sister genus Oxytropis

shared a crown clade time ca. 16.1 Ma. The estimated age of Astragalus was ca. 12.4 Ma,

but the so-named Neo-Astragalus (New World especially North America aneuploid,

chromosome number n=11-15) diverged ca. 4.4 Ma. The Old World groups are generally

euploid (n=8, 16) and probably older than that of New World. Lavin et al. (2005) recently

used fossils as the calibrations to date legumes.They inferred a rapid diversification of

lineages during the Tertiary. Astragalus was demonstrated to be a rapidly evolving group

in Tertiary, and the divergence time of Coluteinae and Astragalinae was estimated at ca.

14.8 Ma (see Fig. 2 and node 78 of Table 2, Lavin et al., 2005). The divergence time of

Astragalus should certainly be later of that time. However, for such a large genus, only

four species of Astragalus were sampled in their dataset (Lavin et al., 2005). Recently, a

South American Astragalus phylogenetic dating based on ITS, trnD-trnT and trnfM-trnS1

sequences, showed a recent and rapid diversification in this area (Scherson et al, 2008).

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Therefore, we so far can investigate Astragalus diversification in China in the light of

previous molecular framework of phylogenetics and biogeography. Meanwhile, a

stimulating motivation for this paper also came from the Astragalus diversification in

Sino-Himalayan region/China. As the well-known, the intense uplifting of QTP during

the Late Tertiary tremendously changed the environment and ecology in Asia and China,

organic evolution must be influenced by this geological event. The Astragalus

distribution pattern of southwestern cold and dry alpine area and northwestern dry area in

China, should be rationally analyzed by joining the background of historical geological

processes in Sino-Himalayan region and China.

Now we turn our attention to the diversification of Astragalus in China, 1) to test

whether Phyllolobium should be warranted; 2) to date the origin of Phyllolobium and

probable diversification of Astragalus in China; 3) to understand the cause of Astragalus/

Phyllolobium diversification by linking it to the uplifting of QTP

Materials and Methods

Taxa sampling

All species of Astragalus in Sino-Himalaya/China, with nrDNA ITS 5.8S and

contributed to Genbank, and five species in Pogonophace to be sequenced firstly by us in

this paper, were sampled, see appendix 1. The outgroup genera of Astragalus, such

Oxytropis, Caragana, Hedysarum, Colutea, Swainsona, were also analyzed using data

from Genbank. Other related legume groups which have fossil records, such as Sophora,

Dalbergia, Pueraria, Cercis, Acacia, and so on, were also sampled so that use them as

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the calibration constraint for the phylogenetic dating. thus, a total 101 species were

evaluated, see appendix 1.

DNA sequence data

Five species in Pogonophace were collected from wild populations in southwestern

China, see appendix 1. The leaves were dried by silica gel for DNA extraction. The

sequencing procedure of ITS 5.8S utilized was that of Kang et al. (2002). All other

sequence data for putatively related taxa were downloaded from the Genbank, see

appendix 1.

Phylogenetic analysis

ITS sequence data for all 101 species were transferred to BioEdit ver.5.0.9 (Hall, 1999);

the alignment was done by using Clustal X 1.83 (Jeanmougin et al., 1998). The alignment

data then was moved to BioEdit v.5.0.9 for manual improvement. An original nexus form

of the dataset was exported for phylogenetic analysis.

Maximum parsimony analysis

Parsimony analyses were implemented by employing PAUP ver. 4.0 (Swofford, 2002).

Multiple tree searches were conducted using heuristic search options, including random

additional sequences (1000 replicates) holding ten trees per replicate, and tree-bisection-

reconnection (TBR) branch swapping, with retention of multiple parsimonious trees.

These parsimonious trees then were used to calculate the consensus tree.

Bootstrap analyses were used to determine clade support. Non-parametric bootstrap

resampling proportions (Felsentein, 1985) were estimated from 1000 bootstrap replicates

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incorporating heuristic parsimony searches using addition sequence and branch swapping

options, and TBR and maxtrees.

Modeltest

For the Maximum Likelihood (ML) and MrBayes analyses (MB), the best fit of DNA

substitution model should be found. The Akaike information criterion (AIC) and

hierarchical likelihood ratio test (hLRT) were calculated based on the log likelihood

scores of 56 models using Modeltest 3.5 (Posada & Crandall, 1998). In general, AIC is

chosen (Posada & Buckley, 2004).

For ITS spacer dataset of this paper, the GTR+I+G model by AIC was selected as the

most appreciated model in Modeltest 3.5. It was with the nucleotide frequencies A =

0.2039, C = 0.3075, G = 02741, T = 0.2145, a gamma shape parameter of 1.8037 and an

assumed proportion of invariable sites of 0.2904.

Maximum likelihood analysis

Maximum likelihood search was performed on the basis of the result of Modeltest in

PAUP. The parameters of best model, such as the base frequency, the mean relative

substitution rates, proportion of invariable sites, Gamma distribution shape, were all

employed. The heuristic search and bootstrap were implemented as in parsimony analysis

in PAUP above mentioned.

Bayesian inference

Bayesian inference of phylogenetic trees was analyzed by employing MrBayes version

3.0 (Huelsenbeck & Ronquist, 2001). Some parameters from the Modeltest, was also

included in the analysis. The option was set up using 1,000,000 generations of Markov

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Chain Monte Carlo (MCMC) searches and a sample frequency of 1000. Saturation was

reached after a burn-in of 1000 generations.

The clade support was assessed using Bayesian posterior probabilities (Huelsenbeck et

al., 2002), these probabilities were estimated as the proportion of trees sampled after

‘burn-in’ that contained each of the observed bipartitions.

Dating

Reference fossils

Legumes are well represented in the fossil record (Heredeen et al., 1992). But there are

no fossils assignable to Astragalus and allied genera, such as Oxytropis, Caragana,

Hedysarum, Colutea, and Swainsona, etc. According to Tao (1992), Tao et al. (2000),

Institute of Botany & Nanjing Institute of Geology and Palaeontology (IB & NJGP),

Academia Sinica (1978), Li et al. (1995), and Guo & Zhou (1992) more than 20 legume

genera are recorded from fossil records from China. Most of these are dated to the

Miocene, several approach to Eocene and Paleocene, but only Bauhinia and Cercis

extend to the Late Cretaceous. Sophora occurring in Oligocene-Miocene, is credible,

since fossils are known from the Eocene of eastern Siberia and North America (IB &

NJGP, 1978; Tao et al., 2000). In China, Sophora fossils were found in Oligocene from

Heilongjiang, Miocene from Shandong and Yunnan, and Pliocene from Shanxi (Tao et al.,

2000). Acacia in Eocene is also well represented in museum collections. Dalbergia

fossils including leaves and fruit were recorded Eocene-Miocene boundary in North

America, and Oligocene-Miocene boundary in Europe, Miocene in Yunnan Province,

China. Fossil leaves of Pueraria were appeared in Miocene in Shandong and Yunnan

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provinces, China (IB & NJGP, 1978; Tao et al., 2000). These genera were used as

outgroups and the constraint minimum ages, i.e., the maximum fossil times were selected

as the generic minimum age MRCA (most recent common ancestor) in dating. For details

of the fossil references refer to Table 1.

Table 1. Fossil references of five genera use for dating ____________________________________________________________________________________________ Taxa Time Ma Location Reference Cercis 60-11 Late Cretaceous-Miocene China Tao, 1992; Tao et al.,2000 Eocene American Lavin et al., 2005 Acacia 47-42 Eocene Liaoning, China; Tanzania Tao,et al.,2000; Heredeen et al.,2000 15 Miocene Dominican Lavin et al.,2005 Sophora 35-9 Oligocene-Miocene China, Siberia, N America Tao et al.,2000; IB & NJGP,1978 Pueraria 17-5 Miocene Yunnan, Shandong, China Tao,1992,2000 Dalbergia 19.5-5 Miocene China, N America, Europe Tao,1992,2000; IB & NJGP,1978 _____________________________________________________________________________________________

In terms of the ancient fossil record and phylogenetic tree, the most root should be

selected Cercis. In southern China Guangdong province, Cercis fossil found in Late

Cretaceous to Eocene, at Oligocene in Yunnan, and Miocene in Shandong and Qinghai.

So, both genera were used to root taxa for constraint ages, and root age was determined

as 60 Ma.

Dating implement

Phylogenetic dating was conducted by three approaches, r8s, PAML and BEAST. In r8s

version 1.7 (http://ginger.ucdavis.edu/r8s/) based on the Bayesian tree, Penalized

likelihood PL (semi-parametric rate smoothing, Sanderson, 2002) and NPRS (non-

parametric rate smoothing, Sanderson, 1997) methods were used. However, PL is

generally in favor with and many applications (e.g. Bremer et al., 2004; Renner & Zhang,

2004; Near & Sanderson, 2004; Renner & Givnish eds., 2004; Anderson et al., 2005; Bell

& Donoghue, 2005; Lavin et al., 2005). And the smoothing parameter of Cross

Validation option in r8s were estimated for using in PL and NPRS.

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PAML (Phylogenetic Analysis by Maximum Likelihood, Yang, 2007) was

implemented by PAML 4.2 (http://abacus.gene.ucl.ac.uk/software/paml.html). PAML

belongs to relaxed molecular clock method, Bayesian tree was used as BASEML, dating

was using MCMCTREE in PAML. The independent rate model (Rannala & Yang, 2007)

with HKY85+Γ5 model for nucleotide substitutions, and multiple fossil calibrations were

used. Two separate MCMC analyses were run for 1,000,000 generations (burnin 10%)

with parameters sample size every 1000 steps. The mean and 95% confidential internals

of MRCA (most recent common ancestor) nodes were yielded.

BEAST v1.46 (http://beast.bio.ed.ac.uk/) was also used to estimate divergence times

(Drummond & Rambaut, 2007). Best-fit models of nucleotide substitution parameters for

the priors in BEAST were yielded by Modeltest. A Yule process speciation prior and an

uncorrelated lognormal model of rate variation were accepted. And normal distribution

was chosen. Tracer v1.4 was used to measure the efficient sample size of each parameter

and mean and 95% confidential internals. Two separate MCMC analyses were run for

10,000,000 generations (burnin 10%) with parameters sample size every 1000 steps. The

mean and 95% confidential internals of MRCA nodes were produced.

Results

Phylogeny

In the maximum parsimony analysis, ITS sequence dataset consist of total 719 aligned

positions, with 385 potential parsimony-informative characters. Parsimonious trees was

with length=1730, CI=0.4965, RI=0.8756, a consensus tree of the most parsimonious

trees was yielded. ML resulted in one best tree. Bayesian analysis presented 10001 burn-

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in trees, and a consensus tree was conducted with the support of posterior probabilities to

clades.

Three phylogenetic analyses resulted in almost identical topology of trees, especially at

the generic level, only exceptionally, Astragalus cysticalyx nested in the clade of

Sphaerophysa and Colutea, and Calophaca tianshanica nested in Caragana clade. The

clades were with high bootstrap and posterior probability values, most over 90%

bootstrap and 95% posterior probability. A Bayesian inference tree was shown as the

phylogenetic relationships in Fig. 1.

Dating

The dating provided estimated ages of Astragalus and relatives, see Table 2. In r8s, the

cross-validation value of all methods is identical, smoothing value 1. All fidelities of used

methods were shown “good”. So, the alternate estimated ages should be in favor of PL-

TN, likes most applications (e.g. Bremer et al., 2004; Anderson et al., 2005; Near &

Sanderson, 2004; Lavin et al., 2005; Wojciechowski, 2005; Near & Sanderson, 2004;

Bell & Donoghue, 2005). The dating results of Astragalus and relatives in r8s were

presented in Fig. 2 and Table 2.

The estimated ages of Astragalus and relatives in PAML and BEAST for related nodes

refer to Table 2, were offered in Table 2. Three approaches provide roughly congruent

estimated ages to the nodes. However, the estimated ages in PAML and BEAST are

higher than that of r8s especially those in BEAST, for instance, node 8 Phyllolobium 8.39

Ma in PL, 6.39 Ma in PAML, 12.43 in BEAST, and node 10 Astragalus 10.34 Ma in PL,

12.78 Ma in PAML, 18.90 in BEAST etc. The estimated ages from r8s and PAML are

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resemble and available. So, in following discussion, we will prefer the estimated ages

from r8s and PAML.

Table 2. The estimated ages using different methods and algorithms, in PAMLand BEAST mean and 95% confidential intervals of the nodes are shown. The classification system following Lock & Schrire (2005), the subtribes following Polhill (1981), the number in front of taxa refer to the nodes in Fig. 2. ______________________________________________________________________________________________ r8s PAML4.2 BEAST

PL-TN PL-Powell NPRS-Powell MCMCTREE 1 Tribe Hedysareae 28.65 29.27 32.01 29.35 (20.29-39.75) 25.68 (24.76-40.56) 2 Caragana 12.22 12.77 20.57 9.28 (4.01-17.20) 14.02 (5.16-32.49) 3 Hedysarum 13.29 13.85 16.80 12.22 (6.20-20.84) 16.13 (6.32-27.46) 4 Tribe Galageae 17.87 18.68 21.04 18.88 (13.01-26.29) 25.56 (16.07-34.18) 4 Subtribe Astragaleae s.str. 17.87 18.68 21.04 18.88 (13.01-26.29) 25.56 (16.07-34.18) 5 Oxytropis 9.63 11.65 15.97 3.66 (1.41-7.86) 9.27 (2.2-20.2)

6 Subtribe Coluteoid s.str. 13.80 14.51 16.34 12.31 (7.85-17.25) 20.96 (12.12-27.93) 7 Phyllolobium+Swainsona 13.30 14.04 15.69 11.00 (7.14-16.10) 19.21 (10.85-25.61) 8 Phyllolobium 8.39 8.82 9.68 6.39 (3.73-10.50) 12.43 (6.74-19.88),

9 Swainsona 9.62 10.21 12.07 7.64 (5.04-11.22) 15.02 (7.99-21.12) 10 Astragalus 10.34 10.92 13.83 12.78 (8.59-18.66) 18.90 (10.4-27.05)

11 Subgen. Astragalus 6.25 6.60 8.63 5.79 (2.72-9.75) 9.44 (3.46-16.79) 12 Subgen. Phaca 3.29 3.51 5.80 3.80 (1.60-7.11) 8.28 (1.91-14.82) 13 Subgen. Pogonophace 2.50 2.68 4.41 1.53 (0.45-3.43) 3.28 (2.07-14.4) ______________________________________________________________________________________________

Discussions

Phyllolobium, a credible monophylenetic genus, and related remarkable to Coluteae

changes the monophyly of Astragalus

From Fig. 1, we determined that Phyllolobium was phylogenetically far away from

Astragalus in contrast to previous presentations (Kang et al., 2003; Wojciechowski &

Sanderson, 2004; Wojciechowski, 2005), which did not sample sufficient taxa from

Phyllolobium. For instance, Wojciechowski & Sanderson (2004) and Wojciechowski

(2005) only sampled one species, A. complanatus (=Phyllolobium chinense. Kang et al.

(2002) selected 7 species from Phyllolobium. Thus it is not surprising that their result

based on a few species is not affirmed or somewhat suspect. In this paper Fig. 1 and Fig.

2, we smapled a total of 15 species including 13 from Phyllolobium (Ph. sinicus in

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Genbank is perhaps Ph. chinense) and two A. hoatchy and A. dshimensis from Astragalus,

were sampled from previous Pogonophace sensu Zhang (2002). In Phyllolobium, Ph.

chinense (=A. complanatus) belongs to sect. Phyllolobium, Ph. hendensornii to sect.

Trichostylus, the rest species is included in sect. Bibracteola. These three sections and

their members in fact present a taxonomical framework within Phyllolobium (Zhang &

Podlech, 2006). Therefore, Phyllolobium can be confirmed confidently to be a genus and

monophyletic. This is very different from the traditional circumscriptions of taxonomical

treatment to put Phyllolobium to Astragalus (Fu et al., 1993; Polhill, 1981). In addition,

Phyllolobium is allied to the subtribe Coluteinae rather than Astragaleae. Consequently,

the diversification of Phyllolobium extracting from Astragalus demonstrates that the

customary circumscription of Astragalus is not monophyletic. The Old World Astragalus

molecular systematics recently revealed that eight traditionally recognized Astragalus

subgenera Epiglottis, Trimeniaeus, Phaca, Hypoglottis, Calycophysa, Tragacantha,

Cercidothrix and Calycocystis have no monophyletic support (Kazempour et al., 2003,

2005). Clearly, Phyllolobium provides another non-monophyletic evidence to Astragalus,

and it jumped over the circumscription of Astragalus.

At the same time, in the light of Phyllolobium appearing concentrately in Hengduan

Mts. and eastern Himalaya (Zhang, 2000), the variation of Phyllolobium could be

considered as one of most notable diversification in Astragalus and in Sino-Himalayan

center of this genus.

Phylogenetic relationships of Astragalus/Phyllolobium and ralatives

The topology of phylogenetic tree concerning tribe Galegeae and tribe Hydesareae in the

present paper (see Fig. 1, Fig. 2), is in agreement with Wojciechowski et al (1999, 2000).

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The molecular phylogeny (Wojciechowski et al., 1999, 2000) obviously affected previous

tribe Galageae classification (Polhill, 1981), see recent delimitation of taxa (Lock &

Schrire, 2005). A remarkable change in recent system (Lock & Schrire, 2005) is that

Caragana and allied groups, Calophaca and Halimodendron were extracted from tribe

Galegeae and transferred into tribe Hedysareae. This change was also shown clearly in

Fig. 1, Fig. 2. Astragalus and Oxytropis are generally considered as a sister group and the

latter is considered to be dereived from the former (Pohill, 1981), but Oxytropis was in

front of Astragalus in Fig. 2.

Within Astragalus, according to Fu et al. (1993), five subgenera were involved in this

paper. In Fig. 2, three phylogenetic clades were clearly present. Subgenus Phaca was

monophyletic. A. hoatchy and A. dshimensis in subgenus Pogonophace also constituted a

monophyletic group. Subgenus Cercidothrix (with A. hamosus and A. adsurgens) and

subgenus Astragalus shared an ancestral node. Subgenus Astragalus, a large group

comprising more than 60 species in China, does not appear to be monophyletic. So, like

as previous studies (see Kazempour et al., 2005), most subgenera in Astragalus have less

monophyletic support.

Infra-Phyllolobium, there is no a clear taxonomic status to be shown, but both sect.

Phyllolobium with Ph. chinese (=A. complanatus) and sect. Trichostylus with A.

hendensornii respectively are distantly related to other species of sect. Bibracteola in

phylogenetic tree, it implies three sections are resolved (Fig. 2).

Diversification ages of Astragalus/Phyllolobium and relatives

As the outgroup of Astragalus, Hedysaroid clade had an estimated age ca. 28.65 Ma in

PL 29.35 Ma in PAML 25.68 Ma in BEAST, see Table 2, it approaches Lavin et al.

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(2005) estimated age ca. 29.3 (node 77). Caragana, Hedysarum, Oxytropis, and

Swainsona etc. had the estimated ages at Late Miocene ca. 13.85-7 Ma.

In the previous dating for Astragalus and relatives, Wojciechowski (2003) estimated

Astragalean crown age was 8-10 Ma (8.8-10 Ma use PL, 10.1 Ma use NPRS), or 16.4 Ma

(Wojciechowski, 2005), or ca. 10 Ma (Lavin et al., 2004), or ca. 14.8 Ma (Lavin et al.,

2005). All of these differences are perhaps mainly resulted from different taxa or

different sequence datasets, such as matK, rbcL and ITS sequence datasets. Our dating to

Astragalean clade was ca 17.87 Ma in PL (18.88 Ma in PAML), which is approach 16.4

Ma (Wojciechowski, 2005).

For Astragalus age, Wojciechowski (2005) estimated it as 12.4 Ma; our estimated age

based on Chinese species in Table 2, was 10.34 Ma in PL (12.78 Ma in PAML), which is

behind the world-wide Astragalus age and older than that of 4.4 Ma of the New World

“Neo-Astragalus” aneuploid group (Wojciechowski, 2005), it should be rational.

Meanwhile, this older age 10.34 (12.78) Ma near 12.4 Ma, implies that the Chinese

Astragalus groups should be ancient in the genus.

Within Astragalus, the estimated ages of three subgenera Phaca, Astragalus, and

Pogonophace, are shown in Table 2 and Fig. 2. Their divergence time was approximately

at 6.5-3.3 Ma in PL. Subgenus Phaca formed a natural group; its estimated age was ca.

3.29 Ma in PL (3.80 Ma in PAML). The clade age of subgenus Astragalus was older, ca.

6.25 Ma in PL (5.79 Ma in PAML). While subgenus Pogonophace (Fu et al., 1993;

Zhang, 2002), comprising section Robusti with two species A. hoantchy and A.

dshimensis and distributed in the arid area of northwestern China (Zhang, 2000), had an

young estimated age 2.50 Ma in PL (1.53 Ma in PAML)

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Phyllolobium has an estimated age 8.39 Ma in PL (6.39 Ma in PAML), indicating its

diversification is older than that of the subgenera within Astragalus mentioned just above,

not only this, considering the born age 13.30 Ma in PL (11 Ma in PAML) of

Phyllolobium and Swainosa, probably means these two groups respectively from Tibet

and Australia have an early diversification since this born age approaches the diversified

age 13.80 Ma in PL (12.31 Ma in PAML) of three groups respectively from South

Europe, Mediterranean, West and Central Asia (groups of Coluteae, such as Colutea,

Sphaerophysa), Tibet (Phyllolobium) and Australia (Swainosa). Obviously, these legume

groups present a case of three systematic and phytogeographical disjunctive

diversifications from Europe-Mediterranean-W & C Asia, Tibet and Australia.

Analyses of the distribution pattern from QTP uplift and the dating

According to the distribution pattern of Astragalus in Sino-Himalayan region/China, a

rational explanation of diversification should be linked to the geological process of the

QTP uplifting in Miocene. The ecological environment and organic evolution in

Himalayas and adjacent region are tremendously influenced by QTP uplifting, which has

been illustrated by many organisms, e.g. Nannoglottis (Asteraceae) (Liu et al., 2002),

Ligularia-Cremanthodium-Parasenecio complex (Asteraceae) (Liu et al., 2006), the

schiozothoracine fishes (He et al., 2003). Astragalus and allied genera, as mentioned

above, with the Miocene estimated ages, should be certainly explained under the

background of QTP intense uplifting in Miocene.

Based on the investigations, the QTP intense uplifting appears at about 8 Ma (Harrison

et al, 1992; Molnar et al., 1993) or early 14-15 Ma (Coleman & Hodges, 1995; Spicer et

al., 2003), or late 3.6 Ma (Li et al., 1979, 1998; Zhong et al., 1996; Zheng et al., 2000).

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Anyway, the period 8-3.6 Ma is very important yet since the evidence of 3.6 Ma of Asian

monsoon origin (An et al., 2001). The Asian monsoon was speculated to be resulted from

the QTP intense uplifting. Consequent climate change heavily influenced biological

evolution.

The estimated age ca. 10.34 Ma in PL (12.78 Ma in PAML)of Astragalus in the Sino-

Himalaya/China, was approximately at the beginning stage of the plateau intense uplift.

The divergence times 6.5-3.3 Ma in PL at subgeneric level in Astragalus, fall well into

time 8-3.6 Ma of the plateau intense uplift. It implies that Astragalus diversification in

Sino-Himalayan region was triggered by the uplifting, in other words, Astragalus and the

geology in this region are co-evolutionary.

The Phyllolobium estimated age was ca. 8.39 Ma in PL (6.39 Ma in PAML) just

approaching 8 Ma of the QTP intense uplifting, clearly it implies that Phyllolobium is a

product of the plateau intense uplifting. In particular, most Phyllolobium species densely

endemic to Hengduan Mts. and eastern Himalaya (Zhang, 2000), an observation that

tends to support our proposal of the relationship of geological uplift and biological

diversification.

Like the Asian monsoon formation, another striking result of QTP intense uplifting, is

the enhanced aridity in Asian interior land since Pliocene 3.6 Ma (Li et al., 1979, 1998;

Zhong & Jing, 1996; Cheng et al., 2000). The most strongly influenced area is

northwestern China, located just north of the plateau. To annotate this environment and

ecological process, section Robusti with A. hoantchy and A. dshimensis of subgenus

Pogonophace, and its estimated age, should be a well performer. These two species are

located at north of the plateau, Xinjiang and western Inner Mongolia provinces in China

17

(Zhang, 2000), and had an estimated age 2.50 in PL (1.53 Ma in PAML)Ma, later than

the intense time of plateau uplift of 3.6 Ma. So, it could be regarded as one of arid-

adapted lineages in Astragalus resulting from the plateau intense uplift.

Acknowledgments

Thanks to Peter W. Fritsch (San Francisco, CAS, USA), Joachim W. Kadereit (Mainz,

Mainz University, Germany), and Gordon C. Tucker (Eastern Illinois University, USA)

for their valuable comments to manuscript, especially G.C. Tucker’ English improvement.

Thanks also to Q.B. Wang and B.J. Zhong (Fudan Unversity, Shanghai) for their

calculating helps. Funding from the Chinese Academy of Sciences (KSCX2-YW-2-

069)，National Natural Science Foundation of China (NSFC 30500035), and China

Scholarship Council (CSC) supported M.L. Zhang’s visit to the CAS for this study.

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Appendix. Taxa samples, first five marking “*” are new reports of the ITS 5.8S from this paper,

the rest are downloaded from the GenBank. Total 101 species are listed.

*A.angustifoliolatus K.T. Fu Lijiang, Yunnna, China, Zhu S.X., Kang Y., No.43-2 (PE) FJ236887

*A.campdontoides Simps. Eryuan, Yunnan, Zhu S.X., Kang Y., No.39 (PE) FJ236888

*A.camptodontus Franch. Zhongdian, Yunnan, China, Zhu S.X., Kang Y., No.64 (PE) FJ236899

*A.flavovirens K.T. Fu Zhongdian, Yunnan, China, Zhu S.X., Kang Y., No.61 (PE) FJ236898

*A.pastorius Tsai et Yu Xiangchen, Sichuan,China, Zhu S.X., Kang Y., No.47 (PE) FJ236892

Astragalus adsurgens Pall. AF121674; A.aksuensis Bunge AF359753; A.alpinus L. ITS1 L10760+

ITS2 L10761; A.balfourianus Simps. AF521951; A.chinesis L. f. AF121681; A.complanatus R. Br. ex

Bunge AF521950; A.cysticalyx Ledeb. AF121682; A.dshimensis Gontsch. AF359755; A.hamosus L.

ITS1 L10778+ ITS2 L10779; A.hendersonii Baker AF521957; A.hoantchy Franch. AF521952;

A.lehmannianus Bunge AF359756; A.lepsensis Bunge AF359752; A.membranaceus Bunge

AF359749; A.milingensis Ni et P.C. Li AF521954; A.mongolicus Bunge AF359750;

A.nankotaizanensis Sasaki AF121680; A.polycladus Bur. et Franch. AF121676; A.propinquus B.

Schischk. AF359751; A.sinicus L. ITS1 U50502+ ITS2 U50503; A.tanguticus Batalin AF521956;

A.tribulifolius Benth. ex Bunge AF521953; A.yatungensis Ni et P.C. Li AF521955;

Acacia farnesiana Wall. AF360728; Ac.mearnsi De Will.AF360750; Ac.senegal AF360727;

Calophaca tianshanica (B. Fedtsch.) Boris. ITS1 U51220+ ITS2 U51221;

Cararagana korshinskii Kom. AY626914; Car.microphylla Lam. AY626915; Car.roboroviskyi Kom.

AF521958; Car.sibirica Fabr. (=Car. arborescens Lam.) AY626912;

Cercis canadensis L. AF390188; Ce.chinensis Bunge AF286351; Ce.chingii Chun AF286350;

Ce.chuniana Metcalf AF286349; Ce.gigantea AF390194; Ce.occidentalis Torr. AF286352;

Ce.racemosa Oliver AF288782; Ce.siliquastrum L. AF286353;

Colutea arborescens L. ITS1 U56009+ ITS2 U56010; Co.istria Mill. ITS1 U69544+ ITS2 U69545

Dalbergia congestiflora Pittier AF068140; D.foliolosa Benth. AF189002; D.sissoo Roxb. AF189023;

Hedysarum aculeolatum Michx AY772222; H.carnosulum Greene AY772224; H.coronarium L.

AY772225

H.flexuosum L. AY775312; H.humile L. AY772227; H.membranaceum Coss. et Bal. AY772228;

H.pallidum Desf. AY772229; H.spinosissimum L. subsp. capitatum AY772223;

Oxytropis besseyi (Rydb.) Blank. var. ventosa (Greene) Barneby AF121756; O.lamberti Pursh

AF121753; O.multiceps Nutt. AF121760; O.oreophila A. Gray AF121755; O.pilosa DC. AF121759;

23

O.sericea Schur AF121757; O.splendens Douql. AF121761; O.szovitsii Boiss. et BuhseAF121754;

O.viscida Nutt. ex Torr. et GrayAF121758

Pueraria lobata (Willd.) Ohwi (=P. montana var. lobata) AF338215; P.montana (Lour.) Merr.

AF338216; P.thomsonii Benth. (=P. montana var. thomsonii) AF338217;

Sophora affinis Torr. et Gary U59886; S.cassioides (Phil.) Sparre AY056081; S.chathamica Cockayne

AY046513; S.chrysophylla Seem. AY056070; S.davidii Kom. ex Pavol. AF467496; S.denudata Bory

AY056071; S.flavescens Ait. AF123452; S.fulvida (Allan) Heenan & de Lange AY056072; S.godleyi

Heenan & de Lange AY056073; S.howinsula (W. R. B. Oliver) P.S. Green AY046514; S.microphylla

Ait. var. longicarinata AY056074; S.molloyi Heenan & de Lange AY056076; S.prostrata Buchanan

AY056077; S.raivavaeensis H.St. John AY056080; S.secundiflora Laq. ex DC. AF174638;

S.tetraphylla (ex Ives) A. Gary AJ310734; S.tetraptera J.S. Mill. AY056078; S.tomentosa L.

AY725482; S.toromira (R.A. Phil.) Skottsb. ST409921;

Sphaerophysa salsula DC. ITS1 U560011+ITS2 U560012;

Swainsona campylantha AF113855; Sw.cyclocarpa AF113856; Sw.decurrens AF113857

Sw.formosa F. Muell. AF113859; Sw.kingii F. Muell. AF113860; Sw.murrayana Wawra AF113861;

Sw.oroboides F. Muell. ex Benth. AF113862; Sw.parviflora Benth. AF113863; Sw.purpurea (A.T.

Lee) Joy Thomps. AF113864; Sw.stenodonta F. Muell. AF113865; Sw.swainsonioides AF113866;

Sw.villosa AF113867.

Fig. 1 Phylogenetic trees reconstruction were used three approaches, maximum

parsimony, maximum likelihood and Bayesian inference. Three phylogenetic analyses

resulted in almost identical topology of trees, especially at the generic level. The clades

were with high bootstrap to parsimony and posterior probability values to Bayesian,

most over 90% bootstrap and 95% posterior probability respectively. A Bayesian

inference tree Fig. 1 is used to show the phylogenetic relationships of legumes taxa and

circles at the nodes shown 95% posterior probability.

Fig. 2 Chronogram yielded by dating PL in r8s, focusing on Astragalus and relatives, the

estimated ages of the related taxa in r8s, PAML and BEAST refer to Table 2.

0.1

Acacia mearnsiiAc.farnesianaAc.senegal

Cercis giganteaCe.racemosa Ce.chinensis Ce.chingii Ce.chuniana Ce.canadensis Ce.occidentalis Ce.siliquastrum

Astragalus lehmannianusA.chinesis A.hoantchy A.dshimensis A.propinqus A.membranaceus A.mongolicusA.aksuensis A.lepsensis A.hamosus A.adsurgens

A.nankotaizanens A.alpinus A.polycladus

A.milingensis A.tanguticus A.tribulifoliusA.pastorius A.camptodontoides A.angustifoliolatusA.camptodontus A.balfourianusA.flavovirens

A.complanatusA.sinicus

A.hendersoniiA.yatungensisSwainsona swainsoniodesSw.murrayanaSw.purpurea Sw.parviflora Sw.campylantha

Sw.villosa Sw.oroboides Sw.formosa Sw.decurrens Sw.stenodonta Sw.cyclocarpa Swainsona kingii Sphaerophysa salsula Astragalus cysticalyxColutea istriaCo.arborescens

Oxytropis multiceps O.besseyi O.pilosa O.szovitsii O.splendens O.oreophila O.lambertii O.sericea O.viscida Hedysarum membranceum

H.spinosissimumH.pallidum H.humile H.aculeolatum H.carnosum H.coronarium Hedysarum flexuosum

Caragana roborovskii Calophaca tianshanicaCaragana microphylla Car.sibirica Car.korshinskii

Pueraria montana P.thomsonii P.lobata

Sophora affinis S.secundiflora S.flavescens

S.davidii S.raivavaeensis S.fulvida S.godleyi S.prostrata S.tetraptera

S.tomentosa S.chathamica S.howinsu S.tetraphylla S.toromira S.cassioides S.denudata S.molloyi S.chrysophylla S.microphylla

Dalbergia foliolosa D.congestiflora D.sissoo

o

o

o

o

o

o o

o

o

o

o

o

o

o

o

o

o

o

o

o o

o

o

o

o

o

o

o

o

1

A.lehmannianus A.chinesis A.hoantchy A.dshimensis A.propinqus A.membranaceus A.mongolicus A.aksuensis A.lepsensis A.hamosus A.adsurgens A.nankotaizanens A.alpinus A.polycladus Phyllolobium milingenum Ph.tanguticum Ph..tribulifolium Ph.pastorium Ph.campdontoides Ph.angustifoliolatum Ph.camptodontum Ph.balfourianum Ph.flavovirens Ph.chinese Ph.sinicus Ph.hendersonii Ph.donicumSwainsona swainsoniodes Sw.murrayana Sw.purpurea Sw.parviflora Sw.campylantha Sw.villosa Sw.oroboides Sw.formosa Sw.decurrens Sw.stenodonta Sw.cyclocarpa Sw.kingii Sphaerophysa salsula Astragalus cysticalyx Colutea istria Co.arborenscens Oxytropis multiceps O.besseyi O.polosa O.szovitsii O.splendens O.oreophila O.lambertii O.sericea O.viscida Hedysarum membranaceum H.spinosissimum H.pallidum H.humile H.aculeolatum H.carnosum H.coronarium H.flexuosum Caragana roborovskii Calophaca tianshanica Caragana microphylla Car.sibirica Car.korshinskii

25 10 15 0 5 20 30

subgen. Calycophysa

subgen. Astragalus

subgen. Pogonophace

subgen. Cercidoothrix

Phyllolobium

subgen. Phaca

subgen. Astragalus

1

2

11

6 9

5

7

8

13 12

4

3

10

Myr

Fig. 2