Analysis of phylogenetic relationships of Brassicaceae species based on Chs sequences

Biochemical Systematics and Ecology 38 (2010) 731–739

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Analysis of phylogenetic relationships of Brassicaceae species based on Chs sequences Bo Zhao a, Lei Liu a, Dunyan Tan b, Jianbo Wang a, * a b

Key Laboratory of the MOE for Plant Development Biology, College of Life Sciences, Wuhan University, Wuhan 430072, China College of Forestry Science, Xinjiang Agricultural University, Orumqi 830052, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 8 December 2009 Accepted 12 June 2010

Sequences of nuclear chalcone synthase gene (Chs) were analyzed for species of the Brassicaceae family to reconstruct phylogenetic relationships. The phylogeny for 106 species of 60 genera was reconstructed, and assigned to 24 tribes, using maximum parsimony, maximum likelihood, and neighbor-joining methods. Most of the tribes can be assigned to the major lineages (Lineages I–III) suggested by Beilstein et al. (2006). The tribe Camelineae was not monophyletic. Conringia planisiliqua together with Orychophragmus violaceus would not be recognized as a new tribe proposed by the previous studies, and C. planisiliqua should be a member of tribe Isatideae. The genera delimitation and monophyly of the expanded Solms-laubachia were also confirmed by our data. Furthermore, one parent of inter-tribal allopolyploid Pachycladon appeared to be most closely associated with Crucihimalaya, Transberingia and tribes Boechereae and Halimolobeae, another parent was proved to be in tribe Smelowskieae. Ó 2010 Elsevier Ltd. All rights reserved.

Keywords: Brassicaceae Chalcone synthase gene (Chs) Lineages I–III Pachycladon Allopolyploidy

1. Introduction The Brassicaceae, a cosmopolitan family, which comprises approximately 338 genera and some 3700 species (Al-Shehbaz et al., 2006), is always recognized as a rather natural monophyletic group based on the characters of six stamens in a tetradynamous pattern (two short and four long), cruciform corolla, distinct capsular fruit and pungency (Zhou et al., 2001). Of those currently recognized, 225 genera (66% of the total) are either monotypic or oligotypic (with 2–4 spp.). Traditionally, this family is subdivided into a tribal system by Schulz (1936) followed morphological characters: fruit shape, position of the embryo, cotyledons, and hair shape, for instance (Hayek, 1911; Schulz, 1936; Janchen, 1942; Al-Shehbaz, 1984). With the application of molecular markers, many morphological characters on which traditional systematic relationships based are proved homoplasious, consequently, the Schulz’s taxonomic systems has proven to be highly artificial (Price et al., 1994; Warwick and Black, 1994, 1997a,b; Koch et al., 1999, 2000, 2001, 2003, 2007; Bailey et al., 2002; Warwick and Sauder, 2005; Khosravi et al., 2009; Mummenhoff et al., 2009 and references therein). The publication of family-wide molecular phylogenetic studies based on nuclear PHYA (Beilstein et al., 2008), ITS (Warwick et al., 2010; Bailey et al., 2006), the chloroplast ndhF (Beilstein et al., 2006, 2008), the mitochondrial nad4 intron (Couvreur et al., 2010; Franzke et al., 2009) and supermatrix including the combination of eight loci analyses (Couvreur et al., 2010) provide us a new look for our understanding of phylogenetic relationships in the Brassicaceae. Forty-four tribes are recognized in these recent taxonomic treatments (Warwick et al., 2010). Among these tribes, tribe Aethionemeae is separated from the rest of the Brassicaceae family with high bootstrap value, while the remainders are divided into three larger moderately

* Corresponding author. Tel./fax: þ86 27 68752213. E-mail address: [email protected] (J. Wang). 0305-1978/$ – see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2010.06.003

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supported monophyletic groups (Lineages I–III) and some small monophyletic groups. But the deep phylogenetic relationships within the lineages are still unsolved, and in particular with regard to the deeper nodes. The lack of resolution of phylogenetic relationships of Brassicaceae is probably due to the incomplete samples and the limited molecular markers. The broad-based phylogenetic framework for Brassicaceae is still in its initial stage. The representatives of each genera and new tribe in previous phylogenetic framework study are often rare, thus the present monophyletic tribes and genera actually may be not monophyletic and need to be revised. In addition, that the gene trees may be fundamentally incongruent with the true species phylogeny, due to various biological phenomena such as introgression, lineage sorting, or mistaken orthology. For example, the strong support for the monophyly of the tribes in phylogeny generated from ndhF data contrasted with the failure of Camelineae to form a monophyletic group in PHYA and ITS phylogenies (Beilstein et al., 2008). Hence, a complete molecular phylogeny based on a combination of several nuclear, plastidic, and mitochondrial markers for more species are necessary for inferring phylogenetic relationships of this family (German et al., 2009). Al-Shehbaz et al. (2006), Bailey et al. (2006), Couvreur et al. (2010) and Franzke et al. (2009) argue that the lack of resolution could be related to a rapid radiation triggered by a combination of genome duplication and climate change. Moreover, succeeding parallel, convergent and reticulate evolution yield more difficulties in assessing phylogenies. For example, genus Pachycladon consists of nine species, eight species are endemic to New Zealand, the other one is endemic to Tasmania. These species form a monophyletic lineage and have obtained different morphologies (leaf, inflorescence, growth habit) and occupy different habitats (different elevations and soil substrates). Recently, an inter-tribal allopolyploidization event was detected in this genus, but the parents of its two genomes are unclear. So further sampling is necessary to be able to comment more precisely on the origin of two genomes to resolve the phylogenetic positions of Pachycladon (Joly et al., 2009). Chalcone synthase gene (Chs) is a nuclear gene that plays a central role in secondary metabolism of flavonoid biosynthesis (Durbin et al., 1995). It has been shown to be extremely useful for phylogenetic analysis among cruciferous plants (Koch et al., 2000, 2001; Yue et al., 2006) and has provided high confidence in reconstructions, particularly at deeper nodes. Furthermore, due to its biparental inheritance, the Chs sequence has been successfully employed to identify progenitors of hybrids, to investigate the origin of polyploid species (Lihová et al., 2006; Joly et al., 2009). In the present study, we sequenced and analyzed the Chs sequence to elucidate: (i) the phylogenetic relationships of taxa within the Brassicaceae family, and (ii) the origin of allopolyploid genus Pachycladon. 2. Materials and methods 2.1. Taxon sampling and outgroup selection The species of Brassicaceae used in this study were listed in Table 1. The present study included 44 new Chs Brassicaceae sequences, as well as 76 Chs Brassicaceae sequences taken from GenBank. Leaf material of the majority of species were collected and desiccated in silica gel, with collecting trips in Xinjiang, Hubei, Henan, Neimenggu and Yunnan province in China. Several species were grown from seeds obtained from the Xinjiang Agricultural University, China. Aethionema grandiflora served as outgroup as usual. This species has already been shown on the molecular level to be only distantly related to other crucifers (Galloway et al., 1998; Zunk et al., 1999; Koch et al., 2001; Hall et al., 2002; Bailey et al., 2006; Beilstein et al., 2006), although, traditionally Aethionema is placed in tribe Lepidieae (Hayek, 1911; Schulz, 1936; Janchen, 1942).

2.2. DNA extraction, amplification and sequencing Genomic DNA was extracted and purified from leaves desiccated in silica gel following the protocol described by Doyle and Doyle (1990) with little modifications to improve the DNA quality. PCR amplification of the entire Chs gene including the promoter region was performed using the primers CHS-FOR1 (50 -cttcatctgcccgtccatctaacc-30 ) (promoter specific) and CHSREV5 (50 -ggaacgctgtgcaagac-30 ) in exon 2 designed by Koch et al. (2000). The PCR amplification was carried out in a total reaction volume of 25 mL containing 1 reaction buffer, 1.5 mM MgCl2, 0.5 mM of each primers, 200 mM dNTPs (TaKaRa Inc.), 20–30 ng template DNA, 1 unit of Taq Polymerase (TaKaRa Inc.), with an addition of 0.2 mg mL1 BSA (Bovine Serum Albumin) and distillation water up to the final volume. The thermocycling profile consisted of an initial denaturation step at 94  C, for 5 min, followed by 35 cycles of 1 min at 94  C, 1 min at 54–60  C (depending on primer combination), 1 min at 72  C, and final extension step of 10 min at 72  C. The amplification products were analyzed on 1.5% agarose gel and excised. Then the PCR products were purified using a DNA gel extraction kit (V-gene Biotechnology Co.) and linked into the PCR 2.1 vector (Invitrogen Life Technologies Company). Up to 10 cloned PCR products were sequenced to maximize the chance of obtaining all the Chs sequences from the donor species, but we only isolated two different Chs genes from Dontostemon senilis. The difference between the two sequences was just different length of intron, so we treated them as one in data analysis. The sequencing was carried out using ABI BigDye terminators according to the manufacture instructions, and run on an ABI 3730 sequencer (Applied Biosystems). For multiple identical sequences resulted from cloned PCR products of one accession, only one sequence was included in the data set.

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Table 1 Species names and accession numbers of Chs sequences used for phylogenetic analysis. Asterisks (*) indicate that the sequences were obtained from GenBank. The taxonomy followed the recent family-wide Brassicaceae species checklist (Warwick and Al-Shehbaz, 2006). Genus

Species

Aethionema Alliaria Arabidopsis

A. grandiflora A. petiolata A. halleri A. lyrata A. thaliana A. hirsute A. alpina A. deltoidea B. pulvinata B. vulgaris B. incana B. holboellii B. fendleri B. lyallii B. parishii B. stricta B. rapa B. oleracea B. nigra B. napus B. juncea B. carinata C. microcarpa C. bursa-pastoris C. impatiens C. microzyga C. flexuosa C. draba C. crassifolia C. niyaensis C. danica C. planisiliqua C. didymus C. mollissima 1 C. mollissima 2 C. mollissima 3 C. himalaica D. Sophia D. baiogoinensis D. himalayensis D. linearis D. stewartii D. elegans D. senilis E. handel-mazzettii E. cheiranthoides E. siliculosum E. sisymbrioides E. edwardsii F. alpina H. perplexa H. taxkorganica H. trichosepala I. tinctoria I. abulense L. lehmannii L. exscapa L. pamirica L. campestre L. apetalum L. perfoliatum L. filifolium L. maritime M. africana M. incana M. perfoliatum N. officinale

Arabis Aubrieta Baimashania Barbarea Berteroa Boechera

Brassica

Camelina Capsella Cardamine

Cardaria Christolea Cochlearia Conringia Coronopus Crucihimalaya

Descurainia Desideria

Dontostemon Erysimum

Eutrema Fourraea Halimolobos Hedinia Hesperis Isatis Ionopsidium Lachnoloma Leiospora Lepidium

Leptaleum Lobularia Malcolmia Matthiola Microthlaspi Nasturtium

Voucher

wangyong2005062

wh02 wh04 wh03 wh01 wh05 wh06 xj03 wh08 wh07 ligs09 xj05 ks02 xj04 wh09

hn02

wangyong2005468 xj28 hn05 xj06 xj30 xj31

xj32 sc01 wh11 xj27

hn01 xj02 wangyong2005089 wh12 xj33

wh14

GenBank accession no. AF112082* AF144537* AF112095* AF112103* AF112086* AF112096* AF112084* AF112109* DQ409227* AF112108* GQ983047 FJ645079* AF112090* AF112099* AF112101* AY612784* GQ983031 GQ983032 GQ983033 GQ983005 GQ983004 GQ983034 GQ983008 GQ983009 GQ983021 GQ983022 AY623911 GQ983015 DQ409232* GQ983006 AF144532* GQ983028 GQ983017 FJ645084* FJ645085* FJ645086* AY612786* GQ983013 DQ409228* DQ409242* DQ409230* DQ409241* GQ983045 GQ983046 DQ409238* GQ983011 GQ983010 GQ983012 GQ983024 AF112102* AF112094* GQ983014 GQ983044 GQ983026 AF144542* GQ983040 DQ409239* DQ409231* AF144534* GQ983018 GQ983016 GQ983036 GQ983048 GQ983038 X17577* AF144536* GQ983023 (continued on next page)

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Table 1 (continued) Genus

Species

Voucher

GenBank accession no.

Neotorularia Olimarabidopsis

N. korolkowii O. pumila 1 O. pumila 2 O. cabulica O. violaceum O. violaceus P. cheesemanii 1 P. cheesemanii 2 P. enysii 1 P. enysii 2 P. exilis 1 P. exilis 2 P. fastigiata 1 P. fastigiata 2 P. latisiliqua 1 P. latisiliqua 2 P. novae-zelandiae 1 P. novae-zelandiae 2 P. stellata 1 P. stellata 2 P. wallii 1 P. wallii 2 P. multicaule P. nudicaulis P. jafrii R. sativus R. indica R. cantoniensis R. amphbia S. alba S. irio S. altissimum S. eurycarpa 1 S. eurycarpa 2 S. lanata S. linearifolia S. minor S. platycarpa S. pulcherrima S. retropilosa S. sp. indet. 1 S. sp. indet. 2 S. xerophyta 1 S. xerophyta 2 S. zhongdianensis S. annua S. matthioloides T. lasiocarpa T. quadricornis T. salsuginea T. arvense T. bursifolia T. glabra

xj20

GQ983039 AF112092* AF112093* AF144533* GQ983042 GQ983035 FJ645095* FJ645096* FJ645105* FJ645106* FJ645093* FJ645094* FJ645091* FJ645092* FJ645097* FJ645098* FJ645099* FJ645100* FJ645101* FJ645102* FJ645103* FJ645104* GQ983027 DQ409229* DQ409237* EF408924* GQ983019 GQ983020 AF144530* X14314* AF144541* GQ983029 DQ409240* DQ409219* DQ409222* DQ409225* DQ409233* DQ409221* DQ409223* DQ409224* DQ409234* DQ409236* DQ409220* DQ409235* DQ409226* GQ983007 GQ983041 GQ983030 GQ983037 GQ983025 AF144535* FJ645087* AF112091*

Oreoloma Orychophragmus Pachycladon

Pachypterygium Parrya Phaeonychium Raphanus Rorippa

Sinapis Sisymbrium Solms-laubachia

Sophiopsis Sterigmostemum Tauscheria Tetracme Thellungiella Thlaspi Transberingia Turritis

xj34 wh16

xj07

wh19 wh18 wh20 xj35

xj36 xj25 xj17 xj14 hn04

2.3. Data analysis The limits of the introns and promoter regions of the Chs gene were determined by comparing with published data (Koch et al., 1999), and these regions were removed manually. The remaining coding sequences were aligned using CLUSTAL X (Thompson et al., 1997). The alignment was further examined and slightly edited manually if necessary. The basic sequence statistics, including conserved sites, variable sites, parsimony-informative sites, singleton sites, and transition/transversion (ns:nv) ratio were analyzed with MEGA version 3.1 (Kumar et al., 2004). Maximum parsimony analyses were executed in PAUP* version 4.0b10 (Swofford, 2003). Insertions/deletions (Indels) were treated as separated characters for phylogenetic analysis using Seqstate* (Müller, 2005) with SIC algorithm. All the characters were equally weighted. Heuristic searches were replicated 1000 times with random taxon-addition sequences, tree Bisection–Reconnection (TBR) branch swapping and with the options MULPARS in effect and STEEPEST DESCENT off. Bootstrapping with 1000 resamplings identified relative support value for clades (Felsenstein, 1985).

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Phylogenetic trees were also constructed by neighbor-joining (NJ) method, used the MEGA* v. The datasets were operated with the Complete Deletion option on, Kimura 2-parameter nucleotide substitution model. Bootstrap tests with 1000 replicates were applied giving the relative robustness of each clade. PHYML (PHYlogenetic inferences using Maximum-Likelihood) (www.atgc-montpellier.fr/phyml/; Guindon and Gascuel, 2003) was used to construct a maximum-likelihood (ML) tree by selecting the general time-reversible (GTR)-based substitution matrix and gamma distribution. Model parameters were set to those estimated by Modeltest 3.7 (Posada and Crandall, 1998), and the TrN þ I þ G models were selected for Chs ML tree. The BOOTPHYML program was used to perform 1000 bootstrap replicates. 3. Results 3.1. Variation in Chs sequences Forty-four Chs sequences were newly gathered in this study, and additional 76 Chs sequences were obtained from GenBank and included in this study (Table 1). The limits of the introns and promoter regions of the Chs gene were determined by comparison with published data, and these regions were removed manually. Total datasets obtained by multiple alignments consisted of 1128 sites with 522 invariable sites (46.3%), while 429 sites (38.0%) of the remaining 606 variable sites were potentially parsimony informative. 3.2. The Chs-phylogenetic analysis The maximum parsimony analysis of Chs data matrix resulted in twenty-four equally most parsimonious trees with tree length ¼ 3722 (CI ¼ 0.2751; RI ¼ 0.7247). The trees obtained from NJ method and ML method shared similar topology with that from MP method, and the strict consensus tree with the bootstrap values acquired by MP analysis was shown in Fig. 1. The tree topology was consistent with the molecular phylogenies published to date (Koch et al., 2003; Bailey et al., 2006; Beilstein et al., 2006; Warwick et al., 2010; Khosravi et al., 2009). Tribe Aethionema was sister to an unresolved basal polytomy including the rest of the family. Most of the tribes could be assigned to the major lineages (Lineages I–III) of Beilstein et al. (2006) which are also supported by data from other markers (Koch et al., 2007; Beilstein et al., 2008; Franzke et al., 2009; Lysak et al., 2009). The other small monophyletic clades not included in three major lineages were Thlaspideae, Eutremeae, Noccaeeae, Cochlearieae, Alysseae, Arabideae, Anastaticeae clade and Fourraea alpina. Lineage I comprised tribes Halimolobeae, Boechereae, Camelineae, Cardamineae, Erysimeae, Descurainiea, Lepidieae and Smelowskiea. Two distinct types of sequences were identified at a Chs locus for all Pachycladon species. Each type of sequence formed a monophyletic Pachycladon clade, one monophyletic Pachycladon clade, Boechereae clade and Halimolobeae clade formed a clade, and then together with Crucihimalaya clade nested within Cardamineae clade; another monophyletic Pachycladon clade nested within Smelowskiea clade. In both of the Pachycladon clades, Pachycladon eesemanii and Pachycladon exilis were sister species to a clade that includes all other species of Pachycladon as previous studies. Crucihimalaya clade comprised Transberingia bursifolia and Crucihimalaya. The other tribes of lineage I each formed a single clade with moderate bootstrap value. Lineage II clade with well supported could be divided into three subclades: (i) Orychophragmus subclade; (ii) Brassiceae subclade; (iii) Isatideae and Sisymbrieae subclade. Orychophragmus violaceus and Sinapis alba formed Orychophragmus subclade. In Brassiceae subclade, seven species of two genera (Brassica and Raphanus) formed a well support clade. Brassica was divided into two evolutionary lineages as before: the nigra lineage and the rapa/oleracea lineage (Song et al., 1990). Raphanus was more closely related to Brassica rapa/oleracea than that to Brassica nigra with high bootstrap values. In Isatideae and Sisymbrieae subclade, Sisymbrium altissimum and Sisymbrium irio formed a monophyletic clade and then together with Conringia planisiliqua, Isatis alba and Pachypterygium multicaule shared a sister relationship with Tauscheria lasiocarpa. Lineage III was a primarily Asian radiation (Beilstein et al., 2008). This lineage consisted of two main clades: (i) Anchonieae, Euclidieae and Chorisporeae clade; (ii) Dontostemoneae and Hesperideae clade. In the first clade, Parrya nudicaulis which belonged to tribe Chorisporeae was strongly supported as a sister to Euclidieae clade and Anchonieae clade. Then, the Anchonieae clade containing Sterigmostemum matthioloides, Oreoloma violaceum and Matthiola incana, was a sister group to the Euclidieae clade which included 10 genera: Leiospora, Lachnoloma, Malcolmia, Leptaleum, Tetracme, Christolea, Neotorularia, Phaeonychium, Desideria and Solms-laubachia. The Dontostemoneae and Hesperideae subclade only included Dontostemon elegans, D. senilis and Hesperis trichosepala. 4. Discussion 4.1. Phylogeny relationship of Brassicaceae species Based on current knowledge, 44 tribes are recognized in the most recent taxonomic treatments (Al-Shehbaz et al., 2006; Bailey et al., 2006; Beilstein et al., 2006, 2008; Warwick et al., 2007, 2010; Couvreur et al., 2010; Franzke et al., 2009). Except for the well-supported sister group relationship of tribe Aethionemeae to the rest of the family, most of the rest tribes fall into one of three larger monophyletic groups (Lineages I–III). Lineage I comprises the tribes Camelineae, Boechereae,

Fig. 1. The strict consensus tree based on a maximum-parsimony (MP) analysis of Chs sequences. Tree length ¼ 3722, consistency index (CI) ¼ 0.2751, retention index (RI) ¼ 0.7247. Numbers above and below the branches indicate bootstrap values >50% by MP/ML/NJ analysis, respectively. The lineages of the family are indicated I–III. Information about tribal assignments according to Warwick et al. (2010) is given on the right margin.

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Halimolobeae, Physarieae, Cardamineae, Lepidieae, Descurainieae and Smelowskieae. In Lineage II, Conringieae, Schizopetaleae, Sisymbrieae, Brassiceae, and Isatideae are somewhat closely related, most probably together with Iberideae, Eutremeae, and Thlaspideae. Euclidieae, Anchonieae and, most probably, Hesperideae form lineage III. Data from the nuclear marker Chs increased confidence in the three larger tribal groups (called Lineages I–III, Fig. 1) and tribes inferred from nuclear markers ndhF, PHYA, ITS and the mitochondrial marker nad4 intron analyses (Bailey et al., 2006; Beilstein et al., 2006, 2008; Couvreur et al., 2010; Franzke et al., 2009). However, the phylogenetic relationships among the larger tribal groups were either undetected or with poor support, with only Lineage II received consensus bootstrap support >80% (Fig. 1), as the earlier findings. The monophyletic clades not included in three major lineages were Thlaspideae, Eutremeae, Noccaeeae, Cochlearieae, Alysseae, Arabideae, Anastaticeae clade and F. alpina. Fourraea alpine has not yet been assigned to any tribes, because it fell in single clade distinct from the recognized tribes as previous study by Couvreur et al. (2010). 4.1.1. Lineage I Lineage I sensu Beilstein et al. (2006, 2008), comprises tribes Camelineae, Boechereae, Halimolobeae, Physarieae, Cardamineae, Lepidieae, Descurainieae, and Smelowskieae. This lineage also appeared in the phylogenetic analysis of our data (Fig.1). Two distinct types of sequences were identified at a Chs locus for all Pachycladon species. Each type of sequence formed a monophyletic Pachycladon clade, one monophyletic Pachycladon clade, Boechereae clade and Halimolobeae clade, together with Crucihimalaya clade nested within tribe Cardamineae, and all together formed a moderate supported clade (Fig. 1). This result was in full agreement with the previous phylogenetic studies based on the nuclear PHYA locus (Beilstein et al., 2008). Crucihimalaya clade, Pachycladon clade and tribe Boechereae also nested within tribe Camelineae based on five single-copy nuclear genes (Joly et al., 2009). These results all showed that tribe Camelineae was paraphyletic. Furthermore in supermatrix analyses including the combination of eight loci (Couvreur et al., 2010), tribes Boechereae, Halimolobeae, Crucihimalaya clade and Australia-New Zealand clades nested within tribe Camelineae. So, we disposed to expanded tribe Camelineae more widely including species of Crucihimalaya clade, the Australia-New Zealand clade, tribe Boechereae, Halimolobeae and Camelineae. The expanded tribe Camelineae needed further morphological and phylogenetic studies of more samples. Seven species of three genera (Cardaria, Lepidium and Coronopus) were included in tribe Lepidieae (Fig. 1). Traditionally, the classification to separate Coronopus and Cardaria from Lepidium was the thick fruits with indehiscent silicle (Mummenhoff et al., 2001). Al-Shehbaz et al. (2002) suggested that features of the fruit valves are adaptative traits for seed dispersal, thus could not be as evidence in genus delimitation, and the typical indehiscent fruit type of Cardaria also occurs in Lepidium heterophyllum var. alatostylum (Thellung,1906). Thus, the common ancestor of Cardaria and Lepidium could have been characterized by indehiscent fruits and the species of section Lepidium evolved dehiscence. Furthermore, all molecular phylogenetic studies consistently indicated that many species with similar fruits may be only distantly related, and on the contrary, species with dramatically different fruits may be very closely related (Koch et al., 2003; Mühlhausen, 2005; Bailey et al., 2006; Al-Shehbaz et al., 2006, and references therein). Cardaria and Coronopus nested with Lepidium (Fig. 1) revealed that they should fall within Lepidium, although they have different types of fruit as previous molecular analysis. Therefore, our result supported that Cardaria draba and Cardaria didymus should be renamed as Lepidium draba and Lepidium didymu as suggested by Al-Shehbaz et al. (2002) and Zhou et al. (2001). 4.1.2. Lineage II Lineage II sensu Beilstein et al. (2006, 2008), is monophyletic and includes Brassiceae, Isatideae, Sisymbrieae and Schizopetaleae. The same lineage with most of the above tribes except Schizopetaleae was also established in the Chs phylogenetic tree with high bootstrap values (Fig. 1). In previous ITS studies, O. violaceus and C. planisiliqua were closely related to I. alba and T. lasiocarpa, then together formed a clade (Khosravi et al., 2009). A clade consisting of Boreava, Isatis, Myagrum, Sameraria, Tauscheria (from Isatideae) and C. planisiliqua was detected in the cpDNA study by Khosravi (2001). Thus the Orychophragmus clade (with C. planisiliqua and Orychophragmus) was suggested to be recognized as a new tribe, with terete, latiseptate (vs. angustiseptate) and linear dehiscent siliques (vs. indehiscent silicles), many (vs. 1 or 2) seeds per fruit (Khosravi et al., 2009). However, in our study, Orychophragmus clade which only included O. violaceus and S. alba, shared a sister relationship with Brassiceae, Isatideae and Sisymbrieae clade in Chs phylogenetic analysis, and C. planisiliqua shared a close relationship with I. alba and P. multicaule (Fig. 1). Similarly, O. violaceus did not share a close relationship with any clade but was a part of Lineage II (German et al., 2009; Couvreur et al., 2010). C. planisiliqua fell within a well-supported clade with I. alba and T. lasiocarpa in the trnF(GAA) analysis of Koch et al. (2007). Thus C. planisiliqua and O. violaceus should not be recognized as a separate tribe, and C. planisiliqua should be a member of tribe Isatideae. In fact, O. violaceus belongs to Chinese endemic O. violaceus complex which consists of Alliaria grandifolia, Cardamine limprichtiana, O. ziguiensis, Orychophragmus diffuses, Orychophragmus hupehensis, Orychophragmus taibaiensis and O. violaceus. The position of these seven species needed further studies with members of Brassiceae, Schizopetaleae and Sisymbrieae (Couvreur et al., 2010). 4.1.3. Lineage III The phylogenetic analyses showed that Lineage III contained 5 tribes, Anchonieae, Euclidieae, Chorisporeae, Dontostemoneae and Hesperideae. For Anchonieae, Chorisporeae, Dontostemoneae and Hesperideae, the samples of these four tribes were not enough, but each of them was monophyletic and still could be separated from each other.

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In tribe Euclidieae, an expanded Solms-laubachia with 26 species was proposed by Yue et al. (2006, 2008). All species of Desideria and Phaeonychium jafrii were transferred to Solms-laubachia based on phylogenitic studies of chloroplast sequences matK and trnL-F, the nuclear sequences Chs and ITS, as well as SEM survey which revealed that the seed epidermis micromorphology of Desideria baiogoinensis was most similar to that of Solms-laubachia lanata (Yue et al., 2008). But the expanded Solms-laubachia was only analyzed with other 3 members of Euclidieae, and the monophyly of this genera needed further research to confirm its delimitation. For this purpose, 24 different species of 10 genera of Euclidieae, including our data and the sequence from Yue et al. (2006), were examined in Chs phylogenetic study. The 10 genera, including Solms-laubachia together with Desideria, Phaeonychium, Neotorularia, Christolea, Tetracme, Leptaleum, Malcolmia, Lachnoloma and Leiospora, formed Euclidieae clade with high bootstrap value (Fig. 1). Furthermore, phylogenetic resolution was higher between genera but lower for interspecific relationships of expanded Solms-laubachia. Four Desideria species (D. baiogoinensis, Desideria himalayensis, Desideria linearis and Desideria stewartii), a species of Phaeonychium (P. jafrii) and Solms-laubachia formed a well-supported monophyletic subclade, with Neotorularia and Christolea as sister group, seperating from other members of Euclidieae. Both Solms-laubachia and Desideria were polyphyletic, as they appeared in more than two positions in subclade, within which P. jafrii was embedded. Therefore, our results indicated that Solms-laubachia should be expanded to include Desideria and P. jafrii as suggested by Yue et al. (2006, 2008). 4.2. The origin of the genus Pachycladon It has been proposed that hybridization is important for the evolution of certain floras such as that of New Zealand (Hair, 1966), which has produced many Late tertiary species radiations (Winkworth et al., 2005). The allopolyploid genus Pachycladon in New Zealand is one such example. The whole genus (2n ¼ 4 ¼ 20) is of allopolyploid origin from distant parents in the Brassicaceae family (Collins et al., 2008). Phylogenetic analyses of molecular markers have given us more confidence in identifying polyploid events and to determine the parents of hybrids (Straub et al., 2006). Phylogenies of the five nuclear genes reveal two distinct types of sequences at a gene locus for all Pachycladon species. Each type of sequence form a monophyletic Pachycladon clade, the phylogenetic positions of two monophyletic Pachycladon clades are highly similar across gene trees. Together, these observations support an allopolyploid origin for the genus (Joly et al., 2009). Crucihimalaya and Transberingia with a close relationship to one monophyletic Pachycladon clade are good candidates for one of the parental lines (Heenan et al., 2002). Because the chloroplast genome is maternally inherited in plants, the rbcL gene tree represents a maternal genealogy of plants that offers an opportunity to identify the maternal parents of allotetraploid species. Phylogenetic analysis of the chloroplast rbcL gene suggests that another monophyletic Pachycladon clade is close to the maternal parent, and tribe Smelowskieae was the putative maternal parent (Joly et al., 2009). Our Chs gene tree including all major tribes that could potentially contain the parents for the two genomes of Pachycladon revealed that two distinct types of sequences were identified at a Chs locus for all Pachycladon species. Each type of sequence formed a monophyletic Pachycladon clade, one monophyletic Pachycladon clade together with tribes Boechereae and Halimolobeae nested within tribe Cardamineae clade including Crucihimalaya clade, the other one nested within tribe Smelowskiea. In both of the Pachycladon clades, P. eesemanii and P. exilis were sister species to a clade that includes all other species of Pachycladon as previous studies (Fig. 1). Molecular studies of the whole mustard family indicated that New Zealand clade containing Pachycladon shared a sister relationship with Crucihimalaya clade, and then together with the Boechereae and Halimolobeae clade nested within tribe Cardamineae (Couvreur et al., 2010). Therefore, one parent of inter-tribal allopolyploid Pachycladon appeared to be most closely associated with Crucihimalaya and tribes Boechereae and Halimolobeae, the other parent was proved to be in the tribe Smelowskieae. 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