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, phyloge- netic 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 morpholog- ical variations is warranted at the molecular and popu- lation 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 charac- teristics 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.