Interspecific Delimitation and Phylogenetic Origin of Pugionium

Journal of Systematics and Evolution 48 (3): 195–206 (2010) doi: 10.1111/j.1759-6831.2010.00078.x

Interspeciﬁc delimitation and phylogenetic origin of Pugionium (Brassicaceae)

∗ 1,2,3Qiu-Shi YU 1Qian WANG 1Ai-Lan WANG 1Gui-Li WU 3Jian-Quan LIU 1(Division of Molecular Ecology, Key Laboratory of Arid and Grassland Ecology, School of Life Science, Lanzhou University, Lanzhou 730000, China) 2(Minqin National Studies Station for Desert Steppe Ecosystem, Minqin 733300, China) 3(Gansu Desert Control Research Institute, Lanzhou 730070, China)

Abstract Pugionium (Brassicaceae) is a small genus that occurs in central Asian deserts. The interspecific delimitation and taxonomic treatments of this genus are disputed and its phylogenetic origin remains unknown. In the present study, we examined these issues based on morphological and molecular data obtained for the first time. We used statistical methods to examine inter- and intraspecific morphological variations. The results suggest that only two species, namely P. dolabratum and P. cornutum, can be warranted for all examined populations and specimens, whereas three species (P. calcaratum, P. cristatum, and P. pterocarpum) should be incorporated into P. dolabratum. This delimitation was further supported by the molecular data: all populations of P. dolabratum, P. calcaratum, P. cristatum, and P. pterocarpum shared the same internal transcribed spacer genotype, whereas those from P. cornutum had another type. Phylogenetic analyses of Pugionium and representative genera of Brassicaceae based on ndhF sequences suggest that this genus is sister to the genus Megacarpaea, which, together, comprise a well-supported lineage with Farsetia, Lobularia, Iberis, and Ionopsidium, whereas the two other genera that were previously suggested to be closely related to this genus (Isatis and Bunias) were placed in the other lineages. We further discuss the origin of Pugionium and suggest that it probably originated in central Asia when the climate became drier from the late Miocene. Key words internal transcribed spacer (ITS), interspecific delimitation, morphological variation, ndhF, phylogenetic origin, Pugionium.

Pugionium Gaertn. is a small genus of Brassicaceae not been completely acknowledged by later researchers that occurs in central Asia and is mainly distributed (e.g. Liu, 1987; Zhou et al., 1987; Zhang, 1995; Cheo in China (Illarionova, 1999). This genus was separated et al., 2001). from Bunias based on its peculiar fruits (Zhao, 1999): In addition to these taxonomic disputes, the phy- the silicles are indehiscent with dagger-like or wing- logenetic origin of Pugionium remains unknown. This like lateral appendages and the bilocular are one-seeded genus was included in and is similar to Bunias in terms (with the second rudimentary locule). Plants of this of floral morphology, but its psammophyte habit and genus grow in sandy soil, have a biennial habit and desert habitats are more similar to Isatis (Illarionova, are typical psammophytes. The stems usually produce 1999). However, Zhou et al. (1987) placed Pugionium nearly globose bunches that transform into tumbleweeds in the tribe Lepidieae, close to Iberis. None of these when the short growth season starts to come to an end assumptions has been tested. In the present study, we in the arid deserts. Since the establishment of Pugio- aimed to address these issues based on morphological nium in 1791, only two species, P. cornutum and P. and molecular data. First, we examined a large number dolabratum, were known until Komarov (1932) pub- of specimens in herbaria and then investigated morpho- lished another three species. Until now, five species and logical variations at the population level in the field. one variety have been described under this genus, specif- Second, we used statistical methods to compare these ically P. cornutum (L.) Gaertn., P. dolabratum Maxim., variations among and within the putative species. Based P. dolabratum var. platypterum, P. calcaratum Kom., on morphological variations and stability, we conducted P. cristatum Kom., and P. pterocarpum Kom. (Yang, a preliminary species delimitation. Third, we used nu- 1981; Zhou & Lan, 1998). However, these taxa have clear internal transcribed spacer (ITS) variations to test whether interspecific delimitation based on morphological variations is warranted at the molecular and population levels. Because of the rapid rate of mutations and Received: 11 January 2010 Accepted: 17 March 2010 concerted evolution, the ITS region has been widely ∗ Author for correspondence. E-mail: [email protected]; Tel.: 86-931-891- 4305; Fax: 86-931-891-4288. used to delimitate species boundaries and construct

C 2010 Institute of Botany, Chinese Academy of Sciences 196 Journal of Systematics and Evolution Vol. 48 No. 3 2010 interspecific relationships (Baldwin et al., 1995). Fi- are sharply dilated from the base and broader than long nally, we used ndhF (a chloroplast gene) sequences to (Zhang, 1995). Zhou & Lan (1998) found that a few examine the phylogenetic origin of Pugionium. This specimens collected from Dengkou (Inner Mongolia, fragment was demonstrated to have a low mutation rate China) also have this character and proposed that this and it is more suitable for phylogenetic studies at high population could be ascribed to P. pterocarpum. Zhang taxonomic levels (Kim & Jansen, 1995; Li, 2008). The (1995) followed this suggestion, although Yang (1981) intergeneric relationships of Brassicaceae have been described it as a variety of P. dolabratum; that is, var. constructed successfully using ndhF sequences and the platypterum. Therefore, in the present study we assumed tribal delimitations have been discussed (Beilstein et al., that this population could tentatively represent P.ptero- 2006). Specifically, the aims of the present study were: carpum. Overall, we ascribed each individual and popu- (i) to determine how many species should be delimited lations to the corresponding species tentatively accord- in Pugionium; and (ii) to construct phylogenetic rela- ing to their taxonomic definitions (Zhou et al., 1987; tionships of this genus with other representative genera Zhang, 1995; Zhou & Lan, 1998; Illarionova, 1999; of Brassicaceae and to provide basic information to- Zhao, 1999; Cheo et al., 2001). wards an understanding its origin. We first checked morphological variations within populations and compared these variations against the identifying descriptions for each species. In order to 1 Material and methods test consistent difference among species, seven characteristics of silicles that have been used traditionally for 1.1 Plant samples for morphological variation and species delineation were measured. A total of 123 in- DNA sequence analyses dividuals from 16 populations (Table 1) were sampled All previous species of Pugionium were established for silicle collection (n = 3–15 individuals per popula- according to differences in silicle wings and the shape tion). Five silicles from each individual were randomly of the lobes of the cauline and radical leaves (Zhou selected to measure the corresponding silicle variables et al., 1987; Zhang, 1995; Zhou & Lan, 1998; Illari- (valve length, valve width, length of silicle wing, width onova, 1999; Zhao, 1999; Cheo et al., 2001). There- of silicle wing, the angle between the two wings, the fore, we chose typical locations of each species to in- length of the valve prickle, and the number of valve vestigate morphological variations among and within prickles). To provide a graphical representation of these species. These populations were ascribed to the five as- variables among species, a principal component anal- sumed species according to the specimen records in the ysis (PCA) was performed (Bitner-Mathe´ & Klaczko, herbaria, in combination with morphological characters 1999) using Multivariate Statistics Package (MVSP 3.1; observed in the field. Pugionium pterocarpum differs http://www.kovcomp.com/, accessed March 2008) and from P. dolabratum mainly in its silicle wings, which the PCA case scores were further used to group all

Table 1 Locations of each Pugionium species and the individuals analyzed for morphological and molecular (i.e. internal transcribed spacer genotype; A versus B) variations P. Population Latitude Longitude Alt. Voucher n ITS Tentative (m) specimens A B species 1 Zhongwei, NX 37◦27.860 104◦59.874 1303 Q.S. Yu, 6003 9 2 0 P.calcaratum 2 Lingwu, NX 38◦01.348 106◦41.000 1334 Q.S. Yu, 6005 7 2 0 P.dolabratum 3 Zuoqi, IM 38◦57.013 105◦39.511 1487 Q.S. Yu, 6011 15 2 0 P.cristatum 4 Dalateqi, IM 38◦49.687 106◦48.954 1166 Q.S. Yu, 6049 6 0 3 P.cornutum 5 Wuhai, IM 39◦43.817 106◦48.636 1083 Q.S. Yu, 6021 12 3 0 P.cristatum 6 Dengkou, IM 40◦14.014 106◦57.251 1047 Q.S. Yu, 6026 15 3 0 P.pterocarpum 7 Hohmudu, IM 40◦37.258 107◦22.749 1033 Q.S. Yu, 6030 6 2 0 P.dolabratum 8 Jixiang, IM 40◦49.502 107◦53.830 1031 Q.S. Yu, 6038 9 3 0 P.cristatum 9 Dalatu, IM 40◦41.206 108◦32.638 1048 Q.S. Yu, 6042 8 3 0 P.dolabratum 10 Sharizhao, IM 40◦29.899 108◦40.813 1062 Q.S. Yu, 6046 3 0 3 P.cornutum 11 Wushenqi, IM 40◦18.334 109◦42.807 1075 Q.S. Yu, 6065 3 0 3 P.cornutum 12 Zhungaerqi, IM 40◦07.931 110◦59.584 1124 Q.S. Yu, 6050 3 0 3 P.cornutum 13 Yulin, SX 38◦23.497 109◦40.263 1122 Q.S. Yu, 6061 3 0 3 P.cornutum 14 Aolezhao, IM 38◦07.486 107◦30.591 1348 Q.S. Yu, 6066 6 0 3 P.cornutum 15 Linze, GS 39◦20.836 100◦08.602 1387 Q.S. Yu, 6069 9 3 0 P.calcaratum 16 Gaotai, GS 39◦18.559 99◦43.751 1384 Q.S. Yu, 6071 9 2 0 P.dolabratum P., population code; Alt., Altitude; GS, Gansu; NX, Ningxia; IM, Inner Mongolia; SX, Shanxi; n, number of individuals sampled for morphological measurements; ITS, internal transcribed spacer.

C 2010 Institute of Botany, Chinese Academy of Sciences YU et al.: Species delimitation and origin of Pugionium 197 individuals evaluated. In addition, one-way ANOVA viduals that likely represented four tentative species (i.e. was used to compare differences in silicle variables P.cornutum, P.dolabratum, P.calcaratum, and P.crista- among groups. tum) to identify the systematic position of the genus The molecular samples used to delimit species at based on the ndhF sequence. Pugionium pterocarpum the population level consisted of 43 individuals from was not included in the systematic analyses because: the 16 populations subjected to morphological investi- (i) the four assumed species represented the genus Pu- gation, representing all five tentative species occurring gionium; (ii) the sequences of the four individuals from in China (Table 2; n = 2–3 individuals in each pop- the four species showed no interspecific variations; and ulation). The internal transcribed spacer (ITS) varia- (iii) all individuals classified as P.pterocarpum have the tions (deposited under accession numbers EU999971– same ITS sequence as those classified as P. dolabra- EU999972) were investigated against the background of tum, P. calcaratum, and P. cristatum. The same DNA morphological variations. We further selected four indi- fragment was sequenced for the first time from another

Table 2 Sources of four tentative Pugionium species, list of taxa, and probable distributional range of representative genera analyzed in the present study, as well as GenBank sequence accession numbers for ndhF Taxon Distribution Voucher Accession no. Pugionium cornutum North China Q.S.Yu, 2006061∗ FJ183791 Pugionium calcaratum Northwestern China Q.S.Yu, 2006002∗ FJ183791 Pugionium cristatum Northwestern China Q.S.Yu, 2006076∗ FJ183791 Pugionium dolabratum North China/Mongolia/Russia Q.S.Yu, 2006072∗ FJ183791 Rorippa elata Temperate zones in the Northern Hemisphere LiuJQ-08XZ-212∗ GU174513 Megacarpaea delavayi Central Asia/Himalayas/Europe DuanWY-2009001∗ GU174514 Cardaria chalepensis Europe/Asia LiuJQ-08KLS-150∗ GU174515 Coelonema draboides Gansu, China LJQ-QLS-2008-0211∗ GU174516 Aethionema saxatile Algeria From GenBank DQ288726 Alliaria petiolata Xinjiang, China From GenBank DQ288727 Anelsonia eurycarpa California From GenBank DQ288729 Arabidopsis thaliana Central Asia/China/Africa From GenBank NC000932 Arabis alpina North China/Europe From GenBank DQ288731 Barbarea vulgaris Xinjiang, China/Europe From GenBank DQ288737 Brassica oleracea Mediterranean From GenBank DQ288742 Braya rosea Xinjiang, China/Siberia From GenBank DQ288743 Bunias orientalis Northeast China/Russia/Europe From GenBank DQ288744 Camelina microcarpa Xinjiang, China/central Asia/Europe From GenBank DQ288746 Capsella bursa-pastoris Temperate zones worldwide From GenBank DQ288748 Chorispora tenella Xinjiang, China/Europe/Mongolia From GenBank DQ288753 Christolea crassifolia Xinjiang, China/central Asia From GenBank DQ288754 Cleome rutidosperma French Guiana (outgroup) From GenBank DQ288755 Conringia persica Iran From GenBank DQ288756 Descurainia Sophia Asia/Europe/North America From GenBank DQ288759 Dilophia salsa Xinjiang, China/central Asia From GenBank DQ288761 Diptychocarpus strictus Xinjiang, China/central Asia From GenBank DQ288762 Dontostemon senilis Northwestern China From GenBank DQ288764 Euclidium syriacum Xinjiang, China/central Asia From GenBank DQ288767 Farsetia aegyptiaca Egypt From GenBank DQ288769 Goldbachia laevigata Xinjiang, China/Europe From GenBank DQ288771 Hedinia tibetica Gansu/Xingjiang/Tibet, China From GenBank DQ288774 Hesperis matronalis Central Asia/Europe/Italy From GenBank DQ288776 Iberis sempervirens Europe From GenBank DQ288781 Ionopsidium acaule Southwestern Europe From GenBank DQ288785 Isatis tinctoria Europe/central Asia/Mediterranean From GenBank DQ288786 Lepidium draba USA From GenBank DQ288790 Lobularia maritima Mediterranean From GenBank DQ288791 Polanisia dodecandra USA (outgroup) From GenBank DQ288815 Physaria ﬂoribunda USA From GenBank DQ288813 Pennellia longifolia Mexico From GenBank DQ288810 Malcolmia africana Xinjiang, China/north of Asia From GenBank DQ288793 Sisymbrium altissimum Xinjiang, China/central Asia From GenBank DQ288826 Sophiopsis annua Central Asia From GenBank DQ288831 Thlaspi arvense Asia From GenBank DQ288839 Turritis glabra Xinjiang, China/Europe/North America From GenBank DQ288840 Taphrospermum altaicum Northwest China/central Asia From GenBank DQ288836 Sterigmostemum acanthocarpum Iran From GenBank DQ288834 ∗Vouchers have been deposited in the Herbarium of School of Life Science, Lanzhou University, Lanzhou, China (LZU).

C 2010 Institute of Botany, Chinese Academy of Sciences 198 Journal of Systematics and Evolution Vol. 48 No. 3 2010 four Brassicaceae species/genera distributed in China, sisted of one cycle at 94◦C for 4 min, followed by 37 namely Megacarpaea delavayi, Cardaria chalepensis, cycles of 94◦C for 50 s, 48◦C for 60 s, and 72◦Cfor Rorippa elata, and Coelonema draboides. Leaves were 1 min 50 s, with a final extension for 8 min at 72◦C. collected in the field and dried with silica gel. The ori- The PCR products were purified using a TIAN- gin of the material used in the present study is given in quick Midi Purification Kit (Tiangen Biotech, Beijing, Table 1. Corresponding voucher specimens have been China) according to the manufacturer’sinstructions. The deposited in the Herbarium of School of Life Science, primers used for sequencing the ITS region were the Lanzhou University, Lanzhou, China (LZU). same as described above. Apart from the primer pair Beilstein et al. (2006) reconstructed the phylogeny of 5F and 3R described above, another four primers of Brassicaceae based on an ndhF sequence dataset were designed especially for sequencing the ndhF re- based on samples from 114 species of 101 of 350 genera, gion: 919F (5-ACT TTA GCT CTT GCT CAA-3), belonging to 17 of 19 tribes of the family. Phylogenetic 919R (5-TTG AGC AAG AGC TAA AGT-3), 1347F analyses based on molecular and morphological com- (5-TAT TTA CTT ACT TTT GAA GG-3), and 1347R binations identified 21 clades comprising 97 species. (5-CCT TCA AAA GTA GTA AAT A-3). Sequencing The remainder are closely related to the assumed clades reactions were performed on an ABI 3130 sequencer us- or remain isolated. We selected one species/genus from ing an ABI Prism Bigdye Terminator Cycle Sequencing each clade or genera isolated. All genera distributed in Ready Reaction Kit (Applied Biosystems, Foster City, the Mediterranean region and in northern China were CA, USA). Both DNA strands were sequenced using included in the study, especially those that were mor- forward and reverse primers. Sequences were recorded phologically similar to Pugionium, for example, Isatis, in both strands with an overlap of at least 90%. Bunias, and Megacarpaea. As previously (Koch et al., 2001; Hall et al., 2002), two species (i.e. Cleome ru- 1.3 Phylogenetic analyses of DNA datasets tidosperma and Polanisia dodecandra) were used to Alignments of sequences were conducted using group all the species in the present study. The eight Clustal X (Thompson et al., 1997) and refined man- new sequences obtained were deposited under accession ually. The boundaries of the ndhF sequences were de- numbers FJ183791 and GU174513–GU174516 and all termined by comparison with published sequences of other ndhF sequences were downloaded from GenBank the other Brassicaceae genera selected in the present (Table 2). study. The ndhF dataset was subjected to maximum parsimony (MP) and maximum likelihood (ML) using 1.2 DNA extraction, amplification, and sequencing PAUP ∗4.0b10 (Swofford, 2000) and Bayesian analyses Total genomic DNA was extracted from silica gel- using MrBayes 3.0 (Huelsenbeck & Ronquist, 2001). dried leaves using the 2× cetyltrimethylammonium Modeltest 3.06 (Posada & Crandall, 1998) was used bromide (CTAB) extraction protocol (Doyle & Doyle, to select parameters and assumptions for ML analy- 1987). The external primer pair ITS1/ITS4 (White et al., ses. The ML heuristic search parameters were sim- 1990) was used for the amplification and sequencing of ple addition of sequences of taxa with tree bisection– the ITS region. Following the methods of Zhang et al. reconnection (TBR) branch swapping, MULTREES, (2005), the polymerase chain reaction (PCR) amplifica- and COLLAPSE. The MP analyses (equally weighted tion for this region was performed in a volume of 25 μL, characters and nucleotide transformations) involved a containing 2.5 μLof10× PCR buffer, 0.2 μLTaqDNA heuristic search strategy with 100 replicates of the polymerase (5 U/μL; TakaRa Biotech, Dalian, China), random addition of sequences, in combination with 0.5 mmol/L dNTPs, 1.5 mmol/L MgCl2,2μmol/L each ACCTRAN character optimization, MULPARS+TBR primer, and 1.0 μL unquantified genomic DNA extract. branch swapping, and STEEPEST DESCENT options The PCR reaction profile consisted of one cycle at 94◦C on. Gaps were treated as missing characters. The boot- for 5 min, followed by 36 cycles of 94◦C for 45 s, 56◦C strap values (BP) were calculated from 1000 replicates for 45 s, and 72◦C for 1 min and 45 s, with a final using a heuristic search with simple addition with TBR extension for 7 min at 72◦C. and MULPARS options on (Felsenstein, 1985). For The ndhF region was amplified with primers 5F and Bayesian analyses, the best fitting model for the gen- 3R (Liu et al., 2002). The 25 μL volume of the mixture eral time reversible (GTR) model with a proportion of used in the PCR amplification of the ndhF region con- invariant characters (I) and rate variation as described tained 2.5 μLof10× PCR buffer, 0.25 μLTaqDNA by the shape parameter (G) was used and four simul- polymerase (5 U/μL; TakaRa Biotech), 0.5 mmol/L taneous Monte Carlo Markov chains (MCMC) were run dNTPs, 1.5 mmol/L MgCl2,2μmol/L each primer, and for 5 000 000 generations, with trees saved every 1000 12–60 ng plant genomic DNA. The PCR reaction con- generations. Base frequencies were derived empirically.

C 2010 Institute of Botany, Chinese Academy of Sciences YU et al.: Species delimitation and origin of Pugionium 199

Burn-in, the generation time for each parameter to reach thermore, the lobes of cauline leaves in P.cornutum vary the stationary state, was determined by visual inspec- greatly from wide lanceolate to oblong within or among tion of log-likelihood values. We discarded the first 499 individuals in each population observed, whereas in the trees to avoid trees that may have been sampled prior to other four species they vary slightly from narrow lance- convergence of the Markov chains. The last 4501 of the olate to line. However, there is no significant difference 5000 trees were sampled to construct a 50% majority in leaf shape among or within populations of P.calcara- rule consensus tree with PAUP∗. The posterior proba- tum, P.cristatum, P.pterocarpum, and P.dolabratum. bility (PP) of each topological bipartition was estimated The silicles of all populations ascribed to P. cor- by its frequency across all 4501 trees sampled. nutum are stable, with narrow wings and acuminate at The molecular clock hypothesis was tested with the apex, although the number of valve prickles varies a likelihood-ratio test (LRT; Huelsenbeck & Rannala, greatly (Fig. 1: A). However, shape, length, and diverg- 1997) using PAUP∗, comparing the log-likelihood of the ing angles of the silicle wings varied considerably in ML trees with and without an assumption of a molecular each population (even among different silicles on the clock. A global molecular clock was rejected because of same individual) of P. calcaratum, P. cristatum, P. pte- significant differences between the constrained and un- rocarpum, and P. dolabratum (Fig. 1: B–F). The silicle constrained analyses, so semiparametric rate smoothing wings dilated sharply or slightly from the base, were with a penalized likelihood (PL) approach was used to longer than broad (lanceolate to oblong) or broader than produce an ultrametric tree with the aid of r8s (Sander- long, and the apex was truncate or acuminate. These sil- son, 2002). The PL approach combines the likelihood icle variations were not closely correlated to other char- term for a saturated model with a different rate on ev- acteristics (e.g. leaf shape) among individuals. In fact, in ery branch and the non-parametric penalty function that each population a few individuals were found that were keeps those rate estimates from varying excessively indicative of each of the four described species on the across the tree. The relative contribution of the two basis of their silicle characters. Despite the considerable terms is controlled by a smoothing parameter, which difference in the flowering and fruiting stages among in- was determined based on a data-driven cross-validation dividuals in the same population, these silicle variations procedure implemented in r8s (Sanderson, 2002). To ob- did not exhibit any correlation with such growth stages. tain standard deviations for estimated divergence times, Therefore, we conducted PCA analyses of seven silicle the dataset was bootstrapped 100 times using the SEQ- characters (i.e. valve length, valve width, length of the BOOT module from PHYLIP (Felsenstein, 1989) and silicle wing, the width of the silicle wing, the angle be- branch lengths were re-estimated for each node under tween two wings, the length of the valve prickle, and the constrained initial topology in PAUP∗. The dating the number of valve prickles). Two principal compo- analyses were then repeated for each tree and node nents (PC1 and PC2) accounted for 50.6% and 22.5%, statistics were summarized using the PROFILE com- respectively, of the total normalized variance of these mand of r8s (Sanderson, 2002). Brassicaceae was es- parameters (Table 3). The scatterplots (Fig. 2) based on timated to diverge from a closely related family 50 the PCA case scores identified two distinct groups. The mya (Koch et al., 2001) and this stem age was used first group consisted of 99 individuals sampled from to calibrate the divergence between the total family and 10 populations of the four species (P. calcaratum, P. outgroups. cristatum, P. pterocarpum, and P. dolabratum). Within this group, the individuals sampled were plotted as a continuous distribution without distinct subgroups cor- 2 Results responding to the four putative species or geographical distributions of the 10 populations (Table 1; Popula- 2.1 Morphological variation within and between tions 1–3, 5–9, and 15–16). The second group, being species clearly isolated from the first, comprised 24 individuals Most leaves of all putative species in each popu- sampled from six populations ascribed to P. cornutum lation examined are pinnatisect or pinnatisect-like, al- (Table 1; Populations 4 and 10–14). Because only a few though a few cauline leaves are entire and line-like of the seven silicle characters were demonstrated to con- within the four species (P. calcaratum, P. cristatum, P. tributed greatly to two principal components (PC1 and pterocarpum, and P. dolabratum; Fig. 1). Leave lobes PC2; Table 3), we further tested whether these seven are obviously wider in P. cornutum (mean 2 cm) than quantitative characters were significantly distinct be- the other four putative species (mean 0.3 cm), but the tween the two groups using one-way ANOVA (Fig. 3; lobe number of a single leaf of the former (mean 11) is here we tentatively treated the first group as P.dolabra- fewer than that of the latter four species (mean 21). Fur- tum). Values for valve length, valve width, the length

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Fig. 1. Gross morphology and silicles of Pugionium cornutum and P. dolabratum. A, six silicles of P. cornutum from six populations (Table 1; Populations 4 and 10–14). B, Silicles collected from 16 population (one to two silicles from each population) of P. dolabratum showing continuous variations of silicle forms. C–F, silicles collected from the Population 9 (Dalatu, IM; C), Population 3 (Zuoqi, IM; D), the Population 1 (Zhongwei, NX; E), and Population 6 (Dengkou, IM; F). These four populations were considered as the type origins of P.dolabratum (L.) Gaertn., P.cristatum Kom., P. calcaratum Kom., and P. pterocarpum Kom., respectively, in China. G, A young individual of P. cornutum showing basal leaves with broadly rhombic lobes. H, A mature individual of P.cornutum showing a distinct stem. I, A young individual of P.dolabratum showing basal leaves with linear lobes. J, A mature individual of P.dolabratum growing on the ﬁxed sandy land in the ﬁeld with many basal branches.

C 2010 Institute of Botany, Chinese Academy of Sciences YU et al.: Species delimitation and origin of Pugionium 201

Table 3 Principal component analysis variable loadings of seven silicle sis (consisting of P. calcaratum, P. cristatum, P. ptero- measurements across 16 populations carpum, and P.dolabratum) have the same ITS-A geno- Variable PC1 PC2 type, whereas all individuals in the second group (P. A1: Valve length 0.393 −0.382 cornutum) share the ITS-B genotype. A2: Valve width 0.123 −0.686 A3: Length of silicle wing 0.481 0.019 A4: Width of silicle wing −0.386 −0.043 2.3 Phylogenetic analysis of the ndhF dataset A5: Angle between two silicle wings −0.461 0.130 A6: Length of valve prickle 0.261 0.504 The four individuals of Pugionium species eval- A7: Number of valve prickles 0.412 0.332 uated had completely identical ndhF sequences. The Eigenvalue 3.544 1.572 aligned ndhF matrix of 47 species treated as the in- Ratio of contribution (%) 50.622.5 Cumulative ratio of contribution (%) 50.673.1 group contained 2068 sites, of which 303 were variable but phylogenetically uninformative and 352 were variable and informative (gaps excluded). Six indels/gaps of the silicle wing, the length of the valve prickle, and (1–9 bp) were restricted to single species and therefore the number of valve prickles in the second group were showed no phylogenetic information. Only one indel significantly higher than in the first group (P < 0.05), (6 bp) shared by two species was phylogenetically infor- whereas the width of silicle wing (A4) and the angles mative. Parsimony analysis identified 323 equally parsi- between two silicle wings (A5) in the second group were monious trees with 1377 steps, a consistency index (CI) significantly lower than in the first group (P < 0.001). of 0.617, and a retention index (RI) of 0.578. The strict These analyses obviously suggest that it is better to treat MP consensus tree and the ML tree (−lnL = 10965.55, all described species of this genus as two taxonomical the best-fit model GTR+I+G) were mostly congruent species (i.e. P.dolabratum and P.cornutum). in topology with the 50% majority rule consensus tree derived from Bayesian analysis (under the GTR+I+G 2.2 Divergence of ITS sequence model; Fig. 4). These phylogenetic analyses suggested The aligned ITS matrix of Pugionium plants is that Pugionium clustered as a monophyletic lineage 710 bp in length and two different sequences, A and B (BP = 83%; PP = 99%) with Megacarpaea, Farse- (Table 1), were identified in the 43 individuals sampled tia, Lobularia, Iberis, and Ionopsidium. Within this lin- due to one nucleotide substitution at site 120 (A→C). eage, Pugionium is sister to Megacarpaea with strong The split of the sampled ITS sequences was highly con- supports (BP = 98%; PP = 100%). If we assumed that gruent with the divergence of morphological characters Brassicaceae originated 50 mya (Koch et al., 2001), the between the two groups suggested by PCA analyses. divergences within this lineage were estimated to be ca. All individuals in the first group in the PCA analy- 3.2 mya with a 95% highest posterior density (HPD)

Fig. 2. Scatterplots based on principal component analysis case scores for each individual evaluated. Each individual was tentatively named based on gross morphology in the ﬁeld as Pugionium calcaratum (), P.cristatum (), P.dolabratum (), P.pterocarpum (), and P.cornutum (•).

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Fig. 3. Tests of differences in the seven silicle parameters of the recircumscribed Pugionium dolabratum and P.cornutum. A1–A7 indicate valve length (in cm), valve width (in cm), wing length (in cm), wing width (in cm), angle between the wings, the length of valve prickles (in cm), and the number of valve prickles, respectively (see Table 1). of 1.5–4.9 mya between Pugionium and Megacarpaea, or more different taxa for three reasons. First, morpho- 6.4 mya (95% HPD: 5.2–9.7 mya) among the three logical variations between two or more species usually clades comprising Iberis, Farsetia, and Lobularia, and occur among individuals and these variations are usually 8.7 mya (95% HPD: 7.8–12.5 mya) between Ionopsid- correlated with some special mechanism/s responsible ium and the remaining genera. for reproductive isolations. However, silicle shape, the most important characteristic used to define Pugionium species, was revealed to vary among silicles from the 3 Discussion same individual in each of the four species examined. We also failed to find individuals with contrasting char- 3.1 Interspecific delimitation acteristics (e.g. silicles) that differed in the flowering and Intraspecific variations in morphology have long fruiting stages. Both early and late-flowering individuals been acknowledged to lead to difficulties in species de- had diverse silicle shapes. Furthermore, individuals with limitation and following taxonomic definitions (Grant, the same silicle shapes showed a continuous spectrum 1981). In addition, sympatric distributions of the closely of small lobes of the cauline leaves, from line to narrow related species confound taxonomic delimitation of re- lanceolate. Second, because of lineage sorting and di- productively isolated lineages. Due to gene flow, two vergent selections, any reproductively isolated species parental species and their hybrids may together con- will acquire a different niche from its sister species and stitute a continuous morphological variation as found form pure populations (Rundle & Nosil, 2005). How- within species (Thorsson´ et al., 2007). Therefore, it ever, we failed to find any population that is typical of is difficult to discern these two scenarios, namely in- each of the four species. The morphological continuum traspecific variations versus morphological mixtures of in each population of the four species may reflect ran- two species in a few species-complexes or sympatrically dom mating among individuals within the same species, distributed locations. In the present study, we examined which results in continuous character variations and in- morphological variations of representative populations distinct correlations among different traits. Our PCA of four species (P. calcaratum, P. cristatum, P. ptero- analyses confirmed that none of four species could be carpum, and P.dolabratum) and found that in each pop- distinguished on the basis of the seven silicle variables ulation both taxonomic characters (silicle and leaf) show evaluated (Fig. 2). Third, ITS mutations are sensitive considerable variation among individuals (Fig. 1: B–F). to the divergence of closely related species and the hy- We believe that this morphological continuum repre- brids of the diverged lineages usually have additive nu- sents intraspecific variations rather than mixtures of two cleotides (e.g. Sang et al., 1995; Wendel et al., 1995;

C 2010 Institute of Botany, Chinese Academy of Sciences YU et al.: Species delimitation and origin of Pugionium 203

Fig. 4. The 50% majority rule consensus tree derived from Bayesian analysis of the ndhF dataset (left) and the maximum likelihood (ML) tree (right) constructed from the ndhF dataset with gaps excluded. Posterior probabilities are noted above the branches, whereas bootstrap support values (>50%) are given below the branches. Pugionium and genera assumed to be closely related are indicated by the gray shading. The indicated timescales show the calibration point (50 mya) and the estimated divergence times.

Mummenhoff et al., 2004). However, we failed to reveal cauline leaves simple, oblong-lanceolate or lanceolate, any mutations among the 25 individuals from the four sinuate- or erose-dentate” (Illarionova, 1999). The same species sampled. All these lines of evidence suggested diagnostic characteristics were suggested by Komarov that the continuous character variation in the popula- (1932) when this species was first described. Our field tions of the four nominal species (i.e. P. calcaratum, P. investigations, as well as examinations of specimens cristatum, P. pterocarpum, and P. dolabratum) are in- from the herbaria, suggested that a few individuals in traspecific rather than interspecific variations and these China, especially in Dengkou (Inner Mongolia), are to- four nominal species should be treated as one species, P. tally consistent with these descriptions and delimita- dolabratum. However, Illarionova (1999) suggested that tions. Therefore, the populations occurring in Mongo- P.pterocarpum is restricted to those populations occur- lia and Russia actually result from the further northern ring in Mongolia and Russia. He further pointed out extension of the integrated P.dolabratum. that this species differs from P. dolabratum in “silicle The currently circumscribed P. dolabratum is dis- wings sharply dilated from the base, broader than long; tinctly different from the other species, P. cornutum. radical leaves pinnatisect, with broadly rhombic lobes, Our PCA analysis of the silicle variables suggested that

C 2010 Institute of Botany, Chinese Academy of Sciences 204 Journal of Systematics and Evolution Vol. 48 No. 3 2010 all individuals of P. cornutum clustered together as the This suggests that trichomes in the Pugionium lineage other distinct group (Fig. 2). In fact, all silicle traits are more diverse, as suggested by Ancevˇ & Goranova measured in this species differ significantly from those (2006). In addition, Warwick et al. (2008) suggested of P. dolabratum (Fig. 3). These two species also differ that Farsetia and Lobularia should be assigned to Mal- from one another in leaf width (Fig. 1). In particular, colmieae, a recently described tribe, based on molecular all individuals of the two species have a unique ITS and morphological evidence (Al-Shehbaz & Warwick, mutation that is a characteristic barcode for them. The 2007). The present analyses suggest that Pugionium,as only mutation between them further suggests that these well as Megacarpaea, Iberis, and Ionopsidium, should two species diverged very recently. In addition, in the be included together in this new tribe. field we found that nearly every P. cornutum individ- It has long been assumed that the floristic elements ual has only one distinct stem >1.5 m high and looks distributed in central Asia diversified after the ancient like a small tree, whereas P. dolabratum has many ra- Mediterranean Sea retreated and central Asia started dial branches, which results in a globose bunch with to become arid (e.g. Liu, 1995). This rapid change of a maximum height of 80 cm (Fig. 1). Although both habitat has resulted in the extinction of most tree or species are distributed in desert habitats and share a few shrub species preferring a wet climate. However, a few overlapping distributions in the Maowusu Sandy Land herbal lineages, for example Brassicaceae, may have and Kubuqi Desert (Zhao, 1999), their microhabitats are survived and started to diversify (Al-Shehbaz et al., different. Pugionium dolabratum usually prefers fixed 2006). Pugionium and its five closely related genera sandy lands in desert steppes or semi-fixed dunes along (Megacarpaea, Farsetia, Lobularia, Iberis,andIonop- the fringes of deserts with more co-occurring species, sidium) are disjunctively distributed from central Asia whereas P. cornutum grows in the mobile dunes usu- to Europe, with a few species centered in the Mediter- ally without any other accompanying species. The dis- ranean region (Zhou et al., 1987; Al-Shehbaz et al., tinct stems of the latter species probably reflect its high 2006; Warwick et al., 2008). These distribution pat- adaptive evolution in order not to be buried by flow- terns also suggest that their origin and diversification ing sand. In terms of distributional regions, P.dolabra- are probably correlated with the formation of these tum is distributed mainly in Gansu, Ningxia, and Inner arid habitats. Furthermore, our estimates of the di- Mongolia, west of the Helan Mountains, whereas P.cor- vergences within the Pugionium lineage and the five nutum occurs in Shanxi, Ningxia, and Inner Mongolia, closely related genera fall within 3.2–8.7 mya. During east and/or southeast of the Helan Mountains. The for- this time, extensive uplifting of the Qinghai–Tibetan mer species further extends to adjacent Mongolia and Plateau led to climate change worldwide (e.g. Cerling Russia. et al., 1997), especially the formation of the winter mon- soon in northwestern China and the more arid habi- 3.2 Phylogenetic origin of the genus tats in central Asia (e.g. Quade et al., 1992). This cor- Although more genera are not included in the relation further enforces the idea that the geological present study, the genera selected for investigation com- and environmental changes since the late Miocene in prise most lineages of the family and genera presumed western China greatly promoted plant diversification to be close to Pugionium or having similar distri- in this region, especially those plants preferring arid butions with it. Our phylogenetic analyses based on habitats. ndhF sequences suggest that Pugionium is sister to Megacarpaea and, together, they comprise a monophyletic lineage with Farsetia, Lobularia, Iberis and 4 Concluding remarks Ionopsidium (Fig. 4). This result is partly consistent with the taxonomic treatment by Zhou et al. (1987), In the present study, we present a detailed inves- who placed this genus in the Lepidieae together with tigation of morphological variations within and among Iberis and Megacarpaea. However, two other genera, populations of Pugionium in China. Statistical analyses Isatis and Bunias, previously assumed to be similar and the molecular barcodes of individuals at the popula- to Pugionium (Zhao, 1999; Illarionova, 1999), were tion level suggest that only two species can be discerned placed in other lineages. Trichomes have been used as within the genus. This genus originated in central Asia an important characteristic in the classification of Bras- during the Pliocene. The relatively ancient origin of the sicaceae (e.g. Khalik, 2005). Farsetia and Lobularia genus and recent divergence between species provides have malpighiaceous trichomes (Beilstein et al., 2006), a good model for studying interspecific differentiation whereas all individuals of Pugionium sampled in the and speciation modes in arid habitats at the population present study have simple trichomes without branches. and genomic levels in the future.

C 2010 Institute of Botany, Chinese Academy of Sciences YU et al.: Species delimitation and origin of Pugionium 205

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C 2010 Institute of Botany, Chinese Academy of Sciences