KEW BULLETIN VOL. 68: 1 Y 9 (2013) ISSN: 0075-5974 (PRINT) ISSN: 1874-933X (ELECTRONIC) A cryptic taxon rather than a hybrid species of Tragopogon (Asteraceae) from the Czech Republic Evgeny V. Mavrodiev1,2, Frantisek Krahulec3, Douglas E. Soltis1,2 & Pamela S. Soltis2 Summary. Tragopogon ×mirabilis Rouy is described as a diploid hybrid between T. porrifolius and T. pratensis. A population of T. ×mirabilis from Central Bohemia, Czech Republic, was recently investigated and, unlike previous reports of T. ×mirabilis, was found to be highly fertile. This fertile diploid hybrid population was considered to represent an alternative evolutionary pathway to polyploidy in Tragopogon. To determine the parentage of the plants from Bohemia, we investigated 12 samples of T. ×mirabilis with ITS, ETS, LFY and plastid (rpL16 gene, intron 1, tRNA-Leu (trnL) gene, intron, trnL-trnF intergenic spacer, psbA-trnH intergenic spacer, and trnG-trnT intergenic spacer) sequence data. None of the Bohemian plants have sequences that are consistent with a hybrid origin between T. porrifolius (incl. T. australis) and T. pratensis. Our data suggest that this fertile population of “T. ×mirabilis” may represent an unrecognised diploid species from the Angustissimi clade sensu Mavrodiev et al. (Int. J. Pl. Sci. 164: 1 – 19, 2005), a clade with a centre of distribution in the Caucasus, and hybrids of this unknown species with T. orientalis or T. hayekii (0 T. orientalis L. var. hayekii Soó), a species closely related to T. pratensis and native to Bohemia. The Bohemian population of “Tragopogon ×mirabilis” clearly requires more investigation, but based on our data it does not appear to represent T. porrifolius × T. pratensis. Key Words. Hybridisation, LFY, plant invasions, plastid sequence data, polyploidy, rDNA, Tragopogon ×mirabilis Rouy. Introduction The first artificial interspecific plant hybrid for scientific study was generated by Linnaeus in 1760 and involved Tragopogon porrifolius L. and T. pratensis L. (reviewed in Ownbey 1950). Many years later, naturally occurring hybrids between these two diploid species were named T. ×mirabilis Rouy (Rouy 1890, reviewed in Krahulec et al. 2005), and have been widely reported from Europe (France, Germany, England) and North America (Farwell 1930; Ownbey 1950; Novak et al. 1991; Krahulec et al. 2005). Artificial hybrids between T. porrifolius and T. pratensis are reported to have low fertility (Fahselt et al. 1976). Recently, Krahulec et al. (2005) investigated a particularly interesting natural population of what has been referred to as Tragopogon ×mirabilis. This population of T. ×mirabilis from Central Bohemia, Czech Republic, was originally discovered in 1921 – 1922 (Novak 1922 in Krahulec et al. 2005) and today exhibits a diverse range of morphology and fertility among plants. Most plants are partially or fully fertile, but fully sterile plants are also noted. Krahulec et al. (2005) reported that the germination rate of achenes collected in the field varied from zero to 100% (depending on the plant), with an average of almost 60% (for achenes collected in 2002). Unusual root-borne shoots (ramets) were present on mature plants of this population of Tragopogon ×mirabilis from Bohemia, facilitating perenniality and vegetative reproduction (Krahulec et al. 2005). These structures are not found in other populations of T. ×mirabilis, or in T. pratensis or T. porrifolius, and are also rare in Tragopogon (Mavrodiev et al. 2008a). Krahulec et al. (2005) postulated that these fertile plants of T. ×mirabilis might represent the early stages of a stabilised hybrid species in Tragopogon. If this hypothesis is true, hybrid speciation would represent an additional reticulate speciation mechanism in Tragopogon to polyploidy, which has played an important role in the genus. To evaluate this hypothesis of diploid hybrid speciation in Tragopogon, it is imperative to demonstrate that the putative population of T. ×mirabilis from Accepted for publication 12 December 2012. 1 Department of Biology, University of Florida, Gainesville, FL 32611, USA. e-mail: evgeny@ufl.edu (author for correspondence), dsoltis@botany.ufl.edu 2 Florida Museum of Natural History, University of Florida, Gainesville, FL 32611, USA. e-mail: psoltis@flmnh.ufl.edu 3 Institute of Botany, Academy of Sciences of the Czech Republic, Prùhonice, CZ 252 43, Czech Republic. e-mail: krahulec@ibot.cas.cz © The Board of Trustees of the Royal Botanic Gardens, Kew, 2013 KEW BULLETIN VOL. 68(1) the Czech Republic is indeed of hybrid origin involving T. pratensis and T. porrifolius as parents. The previous identification of the population from central Bohemia as representing T. ×mirabilis was based solely on morphology. One of the diploid parents, T. pratensis (yellow ligules), has been reported from the same general area, but T. porrifolius (purple ligules) has not (Krahulec et al. 2005), although it has been widely, albeit sporadically, cultivated in Europe for hundreds of years. Its contribution to the putative population of T. ×mirabilis from the Czech Republic seemed obvious from the flower colour (yellow ligulae with some purple) of the hybrids (Krahulec et al. 2005), although other examples of this hybrid combination produce purple to rust-coloured ligules (Novak et al. 1991). The population is diploid with 2n 0 12 (Krahulec et al. 2005). To evaluate the proposed diploid hybrid origin of the Bohemian population of T. ×mirabilis from T. pratensis × T. porrifolius, we employed plastid and nuclear molecular markers and made additional chromosome counts. Material and Methods Mitotic chromosomes were studied using root meristems obtained from adult plants. These roots were pretreated with 8-hydroxyquinoline for four hours, fixed in ethyl alcohol-acetic acid (3:1), digested in glusulase (Soltis 1981), stained in an acetic orcein solution, squashed, and viewed using light microscopy. Methods of rDNA amplification and sequencing, alignment, cloning and phylogenetic analyses were described earlier (Mavrodiev et al. 2005, 2007, 2008b, 2013). Pairwise distances were calculated using PAUP* 4.0b10 (Swofford 2002). The ITS (5.8S rRNA gene, internal transcribed spacer 1 (ITS 1) and 2 (ITS 2)) + ETS data set for phylogenetic analyses consisted of 55 ITS and ETS sequences of Tragopogon from Mavrodiev et al. (2005, 2007, 2008b). For this study we obtained sequences for five plastid regions from four different individuals of Tragopogon ×mirabilis. These four individuals, and an additional eight individuals of T. ×mirabilis, were sequenced for ITS and ETS. We also sequenced one accession each of T. elatior Steven (ITS), T. cretaceus S. A. Nikitin (ITS and ETS), T. porrifolius (ITS and ETS) from Slovenia (cultivated in the Botanical Garden of the University of Ljubljana), and T. pratensis (ITS and ETS) from the Czech Republic (Appendix 1). With these additions, our data matrix included all species, broadly defined, from Flora Europaea (Richardson 1976), except Tragopogon lassiticus Rech. f., a narrow endemic from Crete. Sequences of Lactuca and Podospermum served as outgroups for both the plastid and rDNA trees (see also Mavrodiev et al. 2005). Scorzonera tortuosissima was the outgroup in the LFY analysis. © The Board of Trustees of the Royal Botanic Gardens, Kew, 2013 In some polyploids of Tragopogon, only one parental ITS/ETS contribution is seen in the raw chromatogram. Cloning and sequencing, however, often reveal both parental types (one in very low abundance). Therefore, we also included sequences of 40 ITS and 28 ETS clones from three of the 12 samples of T. ×mirabilis analysed here. As in previous papers (Mavrodiev et al. 2005, 2007), ITS and ETS data sets were combined and analysed. The raw ITS and ETS sequences from individual samples of Tragopogon ×mirabilis were also combined and added to the ITS + ETS matrix. We did not combine ITS and ETS clone sequences; the ITS and ETS clone sequences of T. ×mirabilis were added separately to the combined ITS + ETS data set. The ITS and ETS regions are well known for complex molecular evolutionary processes that may confound the utility of these regions in phylogeny reconstruction (e.g., Bailey et al. 2003). In addition, PCR artifacts and recombinants (e.g., Buckler et al. 1997; Kovarik et al. 2005) further complicate the use of these loci. Following the lead of other workers (Campbell et al. 1997; Andreasen & Baldwin 2003), we employed a cautious approach in evaluating ITS and ETS data from the putative hybrid T. ×mirabilis. In the case of non-identical clones, we aligned the clones together and with the overall data set and then examined by eye each clone carefully to detect all possible examples of Taq error (any apomorphic sites found in a single clone, but not present in the raw sequence) or possible recombinants. Five plastid regions (rpL16 gene, intron 1, tRNALeu (trnL) gene, intron, trnL-trnF intergenic spacer, psbA-trnH intergenic spacer, and trnG-trnT intergenic spacer) were amplified, sequenced, and analysed following Mavrodiev et al. (2008b). Previous studies have shown the utility of LFY intron 2 for low-level phylogenetic analysis (e.g., Grob et al. 2004, reviewed in Howarth & Baum 2005). The LFY data set used here consisted of 50 species of Tragopogon from Mavrodiev et al. (2013); to this data set we added two LFY clone sequences from one plant of T. ×mirabilis # 813 (the only sequences that could be obtained) (Appendix 1). Results Krahulec et al. (2005) indicated that the chromosome number of the Bohemian plants of Tragopogon ×mirabilis is diploid with 2n 0 12. Our cytological studies confirm that these plants are diploid. The aligned ITS + ETS matrix consists of 1,268 characters (288 in ITS-1; 168 in 5.8S; 239 in ITS-2; 573 in ETS). The electropherograms obtained via ITS and ETS sequencing of PCR products derived from genomic DNA of 11 of 12 samples of Tragopogon ×mirabilis revealed no more than two polymorphic sites A CRYPTIC TAXON OF TRAGOPOGON FROM THE CZECH REPUBLIC each. For ITS, seven samples of T. ×mirabilis showed no polymorphic sites, two samples had one polymorphic site, and two samples had two polymorphic sites. Four samples of T. ×mirabilis exhibited no polymorphic sites for ETS, six samples had one polymorphic site, and one sample had two polymorphic sites. Generally, the polymorphic sites were not the same in the different plants. In contrast, the 12th sample of T. ×mirabilis possessed 13 polymorphic sites in ETS and five polymorphic sites in ITS. Clones of ITS and ETS from two samples of Tragopogon ×mirabilis having no polymorphic sites were either identical or differed from one another by only one or two substitutions. Cloning from sample # 2 – 14 with five polymorphic ITS and 13 polymorphic ETS sites provided 16 ITS sequences with minimal differences (Fig. 1). In contrast, cloning of ETS from the same sample revealed several distinct clone types. Six of 15 clones were of two distinct types, and as expected from “jumping PCR” (e.g. Buckler et al. 1997), the remaining clones are putative recombinants. We added six non-recombinant clones from sample # 2 – 14, as well as all other raw sequences and clones of Tragopogon ×mirabilis to the combined ITS + ETS data set. This data set contained 793 constant, 300 variable but parsimony-uninformative, and 175 potentially parsimony-informative characters. Phylogenetic analyses of this matrix yielded 7,623 equally most parsimonious trees of length 722, CI 0 0.75, RI 0 0.83. Most ITS and ETS clones and all combined raw ITS and ETS sequences of T. ×mirabilis appear in a moderately supported (BS 0 72%) Angustissimi clade together with T. graminifolius DC., T. pusillus M. Bieb., T. segetum Kuth., T. sosnowskyi Kuth., T. latifolius Boiss. (2n 0 12), T. filifolius Rehmann ex Boiss., and T. serotinus Sosn. apud Kuth. (Fig. 1). Two ETS clones, however, appear in the Tragopogon clade (BS 0 70%) as part of a well-supported subclade (BS 0 80%) together with T. orientalis L., T. longifolius Heldr. & Sart., T. hayeki (Soó) I. Richardson and T. tommasinii Sch. Bip. (Fig. 1). ITS and ETS sequences of T. pratensis from the Czech Republic generated here formed a clade with the sequences of T. pratensis generated in Mavrodiev et al. (2005). ITS and ETS sequences of T. porrifolius from the Czech Republic are also identical to sequences of T. porrifolius “salsify” and form a clade with T. lamottei Rouy as in our earlier studies (Mavrodiev et al. 2005, 2007) (Fig. 1). The aligned plastid data set consists of 3,240 characters (540 in the trnL intron, 495 in the trnL-trnF spacer, 525 in the psbA-trnH spacer 1,140 in rpL16, intron 1 and 540 in the trnG-trnT spacer). The plastid matrix contained 2,877 constant, 181 variable but parsimony-uninformative, and 182 potentially parsimony-informative characters. Phylogenetic analyses of this matrix yielded 91 equally most parsimonious trees of length 474, CI 0 0.83, RI 0 0.83. Four individuals of Tragopogon ×mirabilis from Central Bohemia were analysed for these five plastid regions. Individuals # 815 and # 816 were identical in sequence, as were individuals # 813 and # 814, but the two pairs of samples differ by a few substitutions and by a 43-bp deletion in rpL16, intron 1. No other species analysed have this deletion in rpL16. Despite their differences, all four samples of T. ×mirabilis appear to be a part of clade A with T. filifolius, T. graminifolius, T. latifolius (2n 0 12), T. pusillus, T. segetum, T. serotinus and T. sosnowskyi (Fig. 2). These species constitute the Angustissimi clade in the ITS + ETS tree (Mavrodiev et al. 2005). The aligned LFY data set consists of 1,161 characters (790 constant, 233 variable but parsimony-uninformative, and 138 potentially parsimony-informative characters). We added two clones from sample # 813 to the LFY data set. Phylogenetic analyses of this matrix yielded 63 equally most parsimonious trees of length 530, CI 0 0.79, RI 0 0.90. Both clones of the analysed sample of Tragopogon ×mirabilis (# 813) appear in a strongly supported (BS 0 92%) clade, Tragopogon I, with T. minor, T. orientalis, T. pratensis and T. porrifolius “salsify” (Fig. 3). Discussion A population of Tragopogon ×mirabilis from Central Bohemia was proposed as a fertile diploid hybrid between T. porrifolius and T. pratensis (Krahulec et al. 2005). Our data, however, exclude both T. porrifolius and T. pratensis as parents of this unusual population from Bohemia. Instead, our results place most samples from Bohemia in the Angustissimi clade. T. porrifolius is polyphyletic, but no accessions of T. porrifolius belong to the Angustissimi clade (Mavrodiev et al. 2007; see also Fig. 1). The ITS and ETS sequences of T. porrifolius “salsify” (which had been considered to be T. porrifolius subsp. porrifolius sensu Richardson (1976)) and T. porrifolius from the Czech Republic are sister to T. lamottei (Fig. 1). Likewise, the plastid sequences of T. porrifolius and “T. ×mirabilis” fall in separate clades (Fig. 2). The LFY sequences of T. pratensis, T. orientalis and T. minor are very similar to the cloned sequences from the putative T. ×mirabilis (Fig. 3); from the LFY tree it is clear that T. porrifolius “salsify” is not the most likely parent of Bohemian T. ×mirabilis (Fig. 3). Hence, there is no molecular evidence to support the proposal that plants that have been named “T. ×mirabilis” from Bohemia have a contribution from T. porrifolius. Tragopogon pratensis is also non-monophyletic (Mavrodiev et al., unpublished). Plants identified as T. pratensis appear in either the Tragopogon or Brevirostres clades (data not shown), but never in the Angustissimi clade sensu Mavrodiev et al. (2005). Plastid sequences likewise place “T. ×mirabilis” and © The Board of Trustees of the Royal Botanic Gardens, Kew, 2013 KEW BULLETIN VOL. 68(1) ITS + ETS 1 0 1 64 1 61 1 2 5 1 5 2 70 57 82 1 6 2 3 4 0 0 7 1 2 86 7 2 62 68 7 73 2 4 74 5 2 3 5 4 7 92 6 1 4 10 0 0 0 0 1 3 1 1 63 7 1 2 84 14 1 1 2 1 2 2 1 85 60 4 74 4 2 70 2 2 6 0 0 1 63 1 2 0 1 2 1 0 2 0 1 0 0 1 0 0 3 10 72 2 9 2 1 1 2 7 3 100 80 0 3 2 2 2 50 1 2 2 70 68 1 60 1 5 6 0 0 9 2 68 99 3 2 5 4 0 11 2 5 1 4 59 8 73 84 3 83 12 167 104 T. major T. cretaceus T. dubius T. pterodes T. angustifolius T. stenophyllus T. coelesyriacus T. afghanicus T. capitatus T. coloratus T. longirostris T. australis T. crocifolius T. balcanicum T. samaritani T. porrifolius T. sinuatus T. olympicus T. elatior T. bornmuelleri T. collinus T. elongatus T. rechingeri T. ruber T. marginifolius T. marginatus T. montanus T. jesdianus T. albinerve T. armeniacus T. bakhtiaricus T. kotschyi T. porphyrocephalus T. fibrosum T. aureus T. graminifolius T. pusillus T. segetum T. sosnowskyi T. latifolius (2n = 12) T. filifolius T. x mirabilis * T. x mirabilis * T. x mirabilis * (8) T. x mirabilis (ITS) (1) T. x mirabilis (ITS) (16) T. x mirabilis (ITS) (7) T. x mirabilis (ITS) (14) T. x mirabilis (ETS) (18) T. x mirabilis (ETS) (6) T. x mirabilis (ETS) (4) T. x mirabilis (ITS) (2) T. serotinus T. kemulariae T. orientalis T. x mirabilis (ETS) (2) T. longifolius T. hayekii T. tommasinii T. pratensis T. pratensis (Czech Republic) T. minor T. trachycarpus T. porrifolius "salsify" T. porrifolius (Slovenia) T. lamottei T. brevirostris T. dasyrhynchus T. dubjanskyi T. heterospermus T. podolicus T. reticulatus T. ruthenicus T. undulatus T. kindingeri Lactuca sp. Podospermum jacquinianum Majores Chromopappus Hebecarpus Collini Profundisulcati Angustissimi Tragopogon Brevirostres Outgroups Fig. 1. One of 7,623 shortest trees (length 722, 722, CI 0 0.75, RI 0 0.83) resulting from maximum parsimony analyses of the ITS + ETS data set for Tragopogon. Arrows indicate branches that collapse in the strict consensus. Numbers above branches indicate branch lengths. Jackknife values for nodes receiving support greater than 50% are indicated in italics below the branches. Clones are in bold; the numbers of identical sequences are in parentheses. Clones and raw sequences of one sample of T. ×mirabilis with several polymorphic sites are in red. Asterisks indicate direct (raw) sequences of T. ×mirabilis obtained from different individuals. Clade names are based on names of sections of Borisova (1964) and are taken from Mavrodiev et al. (2005). © The Board of Trustees of the Royal Botanic Gardens, Kew, 2013 A CRYPTIC TAXON OF TRAGOPOGON FROM THE CZECH REPUBLIC 0 1 2 3 91 5 16 T. pusillus 3 2 T. graminifolius (2n = 12) T. serotinus 0 1 T. filifolius T. x mirabilis (2) 56 0 T. segetum 10 99 4 0 4 Clade A T. sosnovskyi T. latifofilus (2n = 12) 2 3 T. x mirabilis (2)* 7 2 T. dubius 2 6 12 77 0 1 0 3 5 2 2 0 68 T. crocifolius T. kemulariae T. dasyrhynchus T. undulatus T. orientalis T. trachycarpus 4 4 T. ruthenicus 2 T. pratensis 1 7 1 3 2 7 1 2 2 2 T. samaritani T. marginatus T. tomentulosus T. marginifolius 1 2 T. lamottei T. porrifolius 4 1 Clade B Clade C T. ruber 100 4 17 T. coloratus 5 T. collinus 0 4 89 0 2 2 1 63 51 2 2 76 3 T. australis T. sinuatus T. krascheninnikovii Clade D T. longirostris 101 15 T. rechingeri 2 2 1 8 1 T. tash - kala Clade E T. olympicus T albinerve 3 T. makaschwilii 64 35 Lactuca sp. Outgroups Podospermum jacquinianum Fig. 2. One of 91 shortest trees (length 233, CI 0 0.83, RI 0 0.83) resulting from maximum parsimony analyses of the plastid (rpL16 gene, intron 1, tRNA-Leu (trnL) gene, intron, trnL-trnF intergenic spacer, psbA-trnH intergenic spacer, and trnG-trnT intergenic spacer) data set for Tragopogon. Arrows indicate branches that collapse in the strict consensus. Numbers above branches indicate branch lengths. Jackknife values for nodes receiving support greater than 50% are indicated in italics below the branches. Samples of T. ×mirabilis are in red; the numbers of identical raw sequences are in parentheses. Asterisks indicate direct (raw) sequences of T. ×mirabilis with the deletion in rpL16, intron 1. © The Board of Trustees of the Royal Botanic Gardens, Kew, 2013 KEW BULLETIN VOL. 68(1) LFY 3 T. x mirabilis (1) 0 2 T. x mirabilis (1) 1 T. orientalis 0 6 3 92 94 1 6 T. brevirostris 1 2 T. dubjanskyi 0 56 T. floccosus 0 17 1 2 57 85 1 1 1 0 11 55 3 100 3 4 70 T. dasyrhynchus T. jesdianus 5 T. albinerve 10 T. bakhtiaricus 5 1 0 55 1 3 1 65 2 52 3 7 19 7 T. marginatus T. marginifolius T. ruber Collini + Profundisulcati T. fibrosum 61 T. serotinus 10 T. makaschwilii 19 T. kemulariae 0 19 1 100 3 6 7 0 87 2 5 2 55 0 18 5 100 0 2 Chromopappus T. cupani T. sinuatus T. filifolius T. graminifolius T. rechingeri 7 T. kotschyi 6 20 T. balcanicum 0 98 1 0 53 1 1 63 2 6 T. dubius Majores + Angustissimi T. samaritani T. major 0 1 2 2 2 84 T. capitatus T. longirostris 0 93 T. coloratus var. floccosus T. pusillus 14 165 T. coloratus T. australis 2 99 91 T. daghestanicus T. segetum 10 17 Tragopogon II + Brevirostres T. tommasinii 13 4 T. undulatus T. trachycarpus 18 2 T. ruthenicus T. lamottei 7 3 T. podolicus T. heterospermus 2 7 Tragopogon I T. minor T. porrifolius "salsify" 0 98 T. pratensis 4 T. ptaerocarpus T. pterodes T. crocifolius T. rezaiyensis Scorzonera tortuosissima Outgroup Fig. 3. One of 63 shortest trees (length 530, CI 0 0.79, RI 0 0.90) resulting from maximum parsimony analyses of the LFY data set for Tragopogon. Arrows indicate branches that collapse in the strict consensus. Numbers above branches indicate branch lengths. Jackknife values for nodes receiving support greater than 50% are indicated in italics below the branches. Samples of T. ×mirabilis are in red; the numbers of identical raw sequences are in parentheses. Clade names are based on names of sections of Borisova (1964) and are taken from Mavrodiev et al. (2005). © The Board of Trustees of the Royal Botanic Gardens, Kew, 2013 A CRYPTIC TAXON OF TRAGOPOGON FROM THE CZECH REPUBLIC T. pratensis in separate clades (Fig. 2). Thus, “T. ×mirabilis” from the Czech Republic does not contain sequences of either T. porrifolius or T. pratensis. The origin of the population of “Tragopogon ×mirabilis” from Central Bohemia may be complex. Some samples of “T. ×mirabilis” may represent an undescribed diploid species from the Angustissimi clade sensu Mavrodiev et al. (2005). Diagnostic attributes of the Angustissimi clade are problematic (Mavrodiev et al. 2005), but most species of this clade have similar geographic distributions that centre on the Caucasus (Mavrodiev et al. 2005). The original determination of the Bohemian plants as Tragopogon ×mirabilis was based on the fact that T. pratensis is currently present at the locality, and the contribution of T. porrifolius seemed possible from the violet flower colour of the capitula on some plants (Krahulec et al. 2005). This character cannot exclude T. ×mirabilis from the Angustissimi clade, however, as some species from the clade are known to have violet ligulae (e. g., T. sosnowskyi). Comparisons with all known members of the Angustissimi clade (Tragopogon filifolius, T. graminifolius, T. latifolius (2n 0 12), T. pusillus, T. segetum, T. serotinus and T. sosnowskyi) given below indicate that T. ×mirabilis differs from all of these species morphologically. Tragopogon serotinus and T. sosnowskyi are biennial plants. These species have narrowly cylindrical capitula (up to 5 cm) and leaves that are narrowly linear to filiform, without an undulate margin and often with a folded lamina (Borisova 1964). All of these characters are absent from the Bohemian plants reported as T. ×mirabilis (Krahulec et al. 2005). Tragopogon pusillus has a long, vertical, slender root, swollen at some depth in a mostly oblong tuber. Plants of T. pusillus are relatively small (normally 5 – 15 cm high) and never form dense tussocks (e.g., Borisova 1964). In contrast, the Bohemian plants of T. ×mirabilis are relatively large (normally more then 15 cm in height), have a spread root system without tubers, and always form dense tussocks (Krahulec et al. 2005). Tragopogon serotinus differs from “T. ×mirabilis” in its very slender and very strongly branched stems, narrow leaves, densely pubescent peduncles, and small capitula (Borisova 1964). Tragopogon filifolius differs from “T. ×mirabilis” in possessing narrow leaves (normally filiform) and in having orange ligulae with a number of purplish stripes. Bohemian plants normally have yellow, paleyellow, whitish, or sometimes violet ligules (Krahulec et al. 2005). Tragopogon graminifolius may be the most similar member of the Angustissimi clade to “T. ×mirabilis” from Bohemia. Morphologically, T. graminifolius looks like T. orientalis and has sometimes been considered conspecific with it (reviewed in Borisova 1964), but Bohemian plants of “T. ×mirabilis” are unique in that they produce root-borne shoots (or “ramets” using the terms of Krahulec et al. 2005). These structures appear not only on the main root, but also on the numerous finer roots (Krahulec et al. 2005). As far as we know, there are two described species of Tragopogon that have root buds and root-borne shoots as found in the Bohemian plants considered to represent “T. ×mirabilis”. One of these species is a narrow endemic from South Belorussia, T. lithuanicus (DC.) Boriss. This species is known only from a few old collections and is morphologically very distinct from T. ×mirabilis (Borisova 1964). Another species that produces rootborne shoots is a recently described tetraploid, T. soltisiorum Mavrodiev (Mavrodiev et al. 2008a). This species, however, is known only from south Russia and is distinct from T. ×mirabilis both morphologically (Mavrodiev et al. 2008a) and molecularly (Mavrodiev et al., unpublished). DNA content data may also argue against Bohemian “Tragopogon ×mirabilis” being a recent hybrid between T. pratensis and T. porrifolius. The DNA content of T. ×mirabilis is about 18% lower than that for T. pratensis and 42% lower than that for T. porrifolius (Krahulec et al. 2005). Loss of DNA does occur in hybrids and polyploids (e.g., Tate et al. 2006, 2009; Buggs et al. 2009; Koh et al. 2010), but such a substantial loss seems unlikely in a recent hybrid (Kovarik et al. 2005). We propose that the samples of putative Tragopogon ×mirabilis represent an undescribed diploid species of Tragopogon from the Angustissimi clade sensu Mavrodiev et al. (2005). If this is correct, it is likely that the material from the Czech Republic was introduced into Bohemia from the Caucasus and nearby areas, where all other Angustissimi clade members occur. Because most of the plants of this population of “T. ×mirabilis” occurred in the vicinity of a small railway station (Krahulec et al. 2005), the plants may be the result of human-mediated introduction. Plant invasions are playing a significant role in the dynamics of floras worldwide, yet the initial introduction of a species into a new region may go unnoticed (Mack & Erneberg 2002). We also detected putative hybrid individuals in the Bohemian population. The ITS + ETS data suggest that one sample (# 2 – 14) is a hybrid (or introgressant) between the undescribed species from the Angustissimi clade and Tragopogon orientalis, T. hayekii (0 T. orientalis var. hayekii Soó), T. tommasinii, or T. longifolius. The LFY data also indicate a close relationship of “T. ×mirabilis” to T. orientalis (Fig. 3). Some hybrids involving T. orientalis are known: T. major × T. orientalis L., described as T. ×crantzii Dichtl, T. orientalis × T. pratensis, T. orientalis × T. tommasinii (Ownbey 1950); see also Wilson (1983). Plastid sequences of this sample of putative T. ×mirabilis appear to be © The Board of Trustees of the Royal Botanic Gardens, Kew, 2013 KEW BULLETIN VOL. 68(1) part of a weakly supported clade (BS 0 56%) with T. filifolius, T. graminifolius, T. pusillus, T. segetum, T. serotinus and T. sosnowskyi (Fig. 2). Several diploid species of Tragopogon (e.g., T. porrifolius subsp. porrifolius, T. brevirostres DC., T. bornmuelleri Ownbey & Rech. f., T. pterodes Pančić, T. serotinus, T. collinus DC.) have two pairs of chromosomes with satellites that correspond to the 18S-26S rDNA loci (Nucleolar Organising Regions, NORs). This change in satellite number may be the result of translocation events (Ownbey & McCollum 1954) and has apparently occurred independently multiple times given that species with both one and two pairs of satellites are distributed throughout the phylogenetic tree (Mavrodiev et al. 2005). Two pairs of satellited chromosomes and corresponding NORs might be a source of ITS and ETS variation that could contribute added polymorphism in a diploid. However, only a single ITS and ETS sequence type was evident in raw chromatograms of samples with two pairs of satellite chromosomes noted above (Mavrodiev et al. 2005). The cloned sequences of the hybrid sample # 2 – 14 from the study population are not identical in sequence to any species in our analysis (Fig. 1) but form a clade (80% BS) in the ITS + ETS tree with Tragopogon orientalis, T. longifolius, T. hayeki and T. tommasinii. Using pairwise distances we found that the ETS clones of sample # 2 – 14 are most similar to the ETS sequences of T. hayekii or T. orientalis (0.00231 vs T. hayekii, 0.00373 vs T. orientalis, 0.00805 vs T. tommasinii, 0.01524 vs T. longifolius). The widely distributed Tragopogon orientalis is a part of the Czech flora (Krahulec et al. 2005), T. hayekii is known only from Central Romania and Macedonia, the rare T. tommasinii is found only in Slovenia and northern Italy, and T. longifolius is an endemic of Greece (Richardson 1976). Based on geographical distribution, T. orientalis appears to be the best candidate to be a parent of the “hybrid” individual we detected. Furthermore, some plants of T. ×mirabilis from Bohemia were even identified as T. orientalis, especially when only the lengths of the ligules and involucral bracts were considered (Krahulec et al. 2005). Our molecular analyses of Tragopogon to date (Mavrodiev et al. 2005, 2008b, 2013) indicate that T. tommasinii and T. hayekii are distinct from T. orientalis and T. pratensis, which agrees in part with Richardson (1976), who accepted T. tommasinii and T. hayekii as separate species. Although the Bohemian population of “Tragopogon ×mirabilis” clearly requires more investigation, our sampling does not show it to be derived from T. porrifolius × T. pratensis. We propose that hybridisation of this unrecognised species with Bohemian plants of T. orientalis has subsequently yielded additional variation in morphology and fertility. © The Board of Trustees of the Royal Botanic Gardens, Kew, 2013 Acknowledgements We thank Dr Alex A. Sennikov (University of Helsinki, Finland) for providing samples of leaf material. This work was support by US NSF grants MCB-0346437 and DEB-0614421. Appendix 1 Voucher data and the GenBank accession numbers for individuals sequenced in this study. Tragopogon cretaceus S. A. Nikitin: European Russia, det. Tzvelev N. N. (MW). 5.8S rRNA gene, internal transcribed spacer 1 (ITS 1) and 2 (ITS 2): HQ456271; external transcribed spacer: HQ456289. T. elatior Steven: Laspi, a mare ad altitudi 200 hexap., Taur. merid., Compere (H). Internal transcribed spacer 1 (ITS1): HQ456272. T. ×mirabilis Rouy: Roudnice nad Labem, 14 – 15E, 50 – 25 N, alt. c. 180 – 190 m. 15 Oct. 2005, Jan Novak. Field collections. 5.8S rRNA gene, internal transcribed spacer 1 (ITS 1) and 2 (ITS 2): HQ456256–68; LEAFY (LFY) gene, intron 2: HQ456290–91; psbA-trnH intergenic spacer, plastid: HQ456292–93; rpL16 gene, intron 1: HQ456294–95; trnG-trnT intergenic spacer: HQ456296– 97; trnL-trnF intergenic spacer: HQ456298–99; tRNALeu (trnL) gene, intron: HQ456300–01. T. porrifolius L.: Slovenia, Botanic Garden, Ljubljana, collection # 1007. 5.8S rRNA gene, internal transcribed spacer 1 (ITS 1) and 2 (ITS 2): HQ456269; external transcribed spacer: HQ456287. 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Karyotypes of Tragopogon (Compositae: Lactuceae). Brittonia 35: 341 – 350. © The Board of Trustees of the Royal Botanic Gardens, Kew, 2013 View publication stats