Scorzonera sensu lato (Asteraceae, Cichorieae) – taxonomic reassessment in the light of new molecular phylogenetic and carpological analyses

Abstract Scorzonera comprises 180–190 species and belongs to the subtribe Scorzonerinae. Its circumscription has long been the subject of debate and available molecular phylogenetic analyses affirmed the polyphyly of Scorzonera in its wide sense. We provide a re-evaluation of Scorzonera and other related genera, based on carpological (including anatomical) and extended molecular phylogenetic analyses. We present, for the first time, a comprehensive sampling, including Scorzonera in its widest sense and all other genera recognised in the Scorzonerinae. We conducted phylogenetic analyses using Maximum Parsimony, Maximum Likelihood and Bayesian analyses, based on sequences of the nuclear ribosomal ITS and of two plastid markers (partial rbcL and matK) and Maximum Parsimony for reconstructing the carpological character states at ancestral nodes. Achene characters, especially related to pericarp anatomy, such as general topography of the tissue types, disposition of the mechanical tissue and direction of its fibres, presence or absence of air cavities, provide, in certain cases, support for the phylogenetic lineages revealed. Confirming the polyphyly of Scorzonera, we propose a revised classification of the subtribe, accepting the genera Scorzonera (including four major clades: Scorzonera s. str., S. purpurea, S. albicaulis and Podospermum), Gelasia, Lipschitziagen. nov. (for the Scorzonera divaricata clade), Pseudopodospermum, Pterachaenia (also including Scorzonera codringtonii), Ramaliellagen. nov. (for the S. polyclada clade) and Takhtajaniantha. A key to the revised genera and a characterisation of the genera and major clades are provided.


Introduction
Scorzonera L. with some 180-190 species is the largest and name-giving genus of the subtribe Scorzonerinae of the Cichorieae (Kamelin and Tagaev 1986). It is widespread in the temperate and subtropical regions of Eurasia and N Africa, with the major centre of diversity in the arid and montane Irano-Turanian region. The genus is represented by perennial herbs often having a caudex or tuber, rarely by biennials or dwarf subshrubs, with linear to oblong, entire to pinnatisect leaves. The capitula have an involucre of imbricate phyllaries in several series. The achenes are cylindrical, with or without a tubular carpopodium and apically truncate or more rarely attenuate to beaked, ribbed or terete, hairy or glabrous, smooth or sculptured with stout conglomerations and their pappus is almost always of softly plumous bristles.
The Scorzonerinae are a morphologically clearly delimited subtribe, characterised by a unique plumose pappus (very rarely absent or non-plumose) of bristles with long soft and often intertwining fimbriae (Kilian et al. 2009a) and a pollen type of its own having colpori with only two (in all other members of the tribe with three) lacunae (Blackmore 1986) and forms a well-supported clade in phylogenetic analyses, based on morphological and molecular data (Bremer 1994;Whitton et al. 1995;Mavrodiev et al. 2004;Kilian et al. 2009a;Tremetsberger et al. 2013). The circumscription of Scorzonera, in contrast, has long been the subject of debate. Differences in morphological characters, such as leaf shape or achene shape and pubescence were, soon after the establishment of the genus, used to separate some taxa into the genera Podospermum (Candolle 1805), Gelasia (Cassini 1818) and Lasiospora (Cassini 1822), but only Scorzonera and Podospermum were accepted in the first comprehensive works on Asteraceae systematics by Lessing (1832) and Candolle (1838). Subsequent classification systems accepted Scorzonera again in a broader sense, with more or less elaborated infrageneric classification (Bentham in Bentham and Hooker 1873;Boissier 1875;Hoffmann 1890Hoffmann -1894Lipschitz 1935Lipschitz , 1939Lipschitz , 1964. Three major entities were mostly accepted in the 20 th century: Scorzonera s.str., Podospermum and Pseudopodospermum, either at subgeneric rank (Lipschitz 1964;Chamberlain 1975;Chater 1976;Rechinger 1977) or, more rarely, at generic rank (Kuthatheladze 1978). First molecular phylogenetic analyses, using the nuclear ribosomal Internal Transcribed Spacer (nrITS: Mavrodiev et al. 2004;Kilian et al. 2009a) and nrITS + the External Transcribed Spacer (ETS) sequences (Winfield et al. 2006), revealed that Scorzonera is actually a polyphyletic assemblage. Its different clades were resolved intermixed with the clades formed by the traditionally accepted genera Epilasia, Koelpinia, Pterachaenia, Tourneuxia and Tragopogon, as well as by Geropogon. However, the number of Scorzonera species included in molecular analyses is so far fairly limited and the deeper nodes in these analyses frequently received only weak or no statistical support. Molecular phylogenetic analyses with a denser sampling of Scorzonera s.l. and also including plastid DNA markers is, therefore, inevitable for shedding more light on the phylogeny of this genus.
Whereas the traditionally recognised genera of the Scorzonerinae are morphologically well delimited, characterisation of the phylogenetic lineages of Scorzonera s.l., so far resolved, appears difficult. There is strong indication that the Scorzonerinae (as well as Scorzonera s.l.) are separated into lineages with different basic chromosome numbers (x = 6 and 7: Mavrodiev et al. 2004;Winfield et al. 2006;Altinordu et al. 2015) and that the Scorzonera clades differ in pollen morphology (Askerova 1970;Blackmore 1981Blackmore , 1982Blackmore , 1986Díaz de la Guardia and Blanca 1985;Nazarova 1997;Mavrodiev et al. 2004;Türkmen et al. 2010;Pinar et al. 2016), but both features seem to be homoplastic. Achene and pappus morphology, but also achene anatomy, are significant in the systematics of the Asteraceae in general and the Cichorieae in particular (e.g. Becker 1913;Pak and Kawano 1990a, b;Kilian 1997;Illarionova 2001, 2002;Zarembo and Boyko 2008;Sukhorukov and Nilova 2015). A detailed recent survey of the achene morphology for 59 species of Scorzonera s.l. in Turkey, representing almost one third of all species, has been provided by Coşkunçelebı et al. (2016) but achene anatomy is known so far from five different members only (Boyko 2000;Makbul et al. 2012). Carpological studies, including anatomy and broad taxonomic sampling could, therefore, be informative for delimiting lineages within Scorzonera s.l.
The aims of the present study are (1) to provide a molecular phylogenetic analysis, using both nuclear and plastid DNA markers and a sampling that spans the entire subtribe, as well as the various groups of Scorzonera s.l.; (2) to investigate the variation in achene and pappus morphology and anatomy across the entire subtribe, to define carpological characters and states and to optimise them on to the new molecular phylogenetic tree, in order to assess the correspondence of carpological features with the principal clades and to gain insights into the evolution of carpological characters in the subtribe; (3) to characterise clades using carpological and, where available, further morphological and cytological characters, to review and, where sufficient evidence is provided, revise the current generic classification of the subtribe.

Sampling and material studied
In our carpological and molecular phylogenetic analyses, we included samples of the genus Scorzonera in its widest sense, as well as representatives of all other genera of the subtribe Scorzonerinae (Kilian et al. 2009b), to account for the previous findings that Scorzonera in all traditional circumscriptions is apparently polyphyletic. Leaf samples for DNA isolation and achene samples for carpological analysis were taken from live plants documented by herbarium specimens deposited in MW and from herbarium specimens of the herbaria B, BM, BR, E, FRU, G, HUJ, LE, MHA and MW with the permission of the curators. All samples used are listed in Table 1.

DNA extraction, amplification and sequencing
Total DNA was extracted from 30 mg of dried plant material. Samples were manually homogenised in a paper envelope and a modification of the CTAB-method of Doyle and Doyle (1987) by Krinitsyna et al. (2015) was used. After lysis with CTAB-lysis buffer (2% CTAB, 0.1 M Tris-HCl (pH 8.0), 1.4 M NaCl, 20 mM EDTA (pH 8.0), 1% PVP, 0.2% 2-mercaptoethanol, 0.1 mM DTT) for 1 hour at 60 °C, the mixture was centrifuged for 5 min at 13,400 rpm (12.080 g) and the supernatant was transferred to a new tube. An equal volume of propyl alcohol was added to the lysate and the samples were incubated at room temperature for 10 min and centrifuged again for 10 min as above. The supernatant was discarded and the precipitate was washed twice using 80% ethanol, dried for 3-5 min and dissolved in 30 µl of deionised water. The stock solution of magnetic particles (Agencourt AMPure XP, Beckman Coulter) was diluted and thoroughly mixed with the buffer (18% PEG-8000, 1M NaCl, 10mM Tris-HCl ph 8.0, 1mM EDTA pH 8.0) in the ratio of 1 to 3 (v/v). Then DNA samples were purified with the suspension of magnetic beads at a ratio of 1:1 (v/v). Concentration and purity of DNA samples were assessed by OD 260/280 and OD 260/230 ratios on the NanoPhotometer N60-Touch (Implen, Germany). DNA samples were normalised to 10 ng/µl before sample preparation. Then 5 µl (50 ng) of each normalised sample was used for PCR and library preparation. Sequences of three markers were used for the molecular phylogenetic analyses: (1) the nrITS region (including ITS1, 5.8S rRNA gene and ITS2); (2) a ~570 bp fragment of the plastid DNA ribulose 1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene; (3) a 750 bp fragment of the plastid DNA maturase K gene (matK).
The nrITS region was sequenced on the MiSeq (Illumina, USA). A two-step PCR method was used for library preparation: first-stage PCR using fusion primers containing the primer sequences from Baldwin (1992) and White et al. (1990) and Illumina adaptor tails: ITS5-Illu-F and ITS2-Illu-R; ITS3-Illu-F and ITS4-Illu-R. PCRs were carried out in Thermal Cycler T100 (Bio-Rad, USA) under the programme: 95° -10 min; 30 cycles each for 95° -5 s, 50° -20 s, 72° -20 s; finally 72° -5 min. PCR products (expected size of 400 bp) were checked on 1.2% agarose gels and purified with the suspension of magnetic beads Agencourt AMPure XP (Beckman Coulter, USA) in the ratio of 1:1.4 (v/v) and mixed in equimolar proportions for each mix. Second-stage PCR performed with 50 ng of mixed products and Nextera index primers (Illumina, USA), according to manufacturer's instructions. Both PCR stages were conducted using Taq DNA polymerase (New England Biolabs, USA). Illumina libraries were sequenced on the MiSeq with MiSeq Reagent Kit v2 for 500 cycles (Illumina, USA).
The rbcL marker was sequenced on the 454 platform (GS Junior, Roche, Switzerland). The three-step PCR method was used for library preparation. All PCR stages were conducted using Phusion high fidelity DNA-polymerase (New England Biolabs, USA). The first-stage PCR was performed using a reaction mixture of a total volume of 20 µl: 4 µl 5× buffer for Phusion high fidelity DNA-polymerase, 250 µM dNTP (Thermo Scientific, USA), 0.2 µl Phusion high fidelity DNA-polymerase and 0.8 pM each primers (rbcLa-F and rbcLa-R, Hollingsworth et al. 2009).
The PCR conditions were as follows: 98 °C -3 min; 7 cycles of 98 °C -5 s, 50 °C -30 s, and 72 °C -30 s; finally 72 °C -5 min. Amplification products were used without purification for the second round of PCR which was performed using primers rbcLa-454-F and rbcLa-454-R. PCR was performed using a reaction mixture of a total volume of 20 µl: 4 µl 5× buffer of Phusion high fidelity DNA-polymerase, 250 µM dNTP (Thermo Scientific, USA), 0.2 µl of Phusion high fidelity DNApolymerase, 0.3 pM of each primer, 4 µl of amplification products of the previous stage. PCR conditions: 98 °C -1 min; 18 cycles each for 98 °C -5 s, 55 °C -30 s and 72 °C -30 s; finally 72 °C -5 min. PCR products (expected size of 600 bp) were checked on 1.2% agarose gels and without purification used for third-stage PCR with primers MIDh-454-F and MIDh-454-R, containing barcodes (MIDx) and adapter sequences for sequencing on the 454 platform (GS Junior Systems, Roche, Switzerland). Adapter sequences are described in Gholami et al. (2012). Sample preparation was carried out with a protocol for the sequencing of amplicons Lib-A. Sequencing of the 454 platform (GS Junior, Roche, Switzerland) was performed according to the manufacturer's instructions.
In addition to the 72 nrITS, 65 matK and 65 rbcL sequences generated in the present study, some published sequences (see Table 1) were taken from the INSDC (International Nucleotide Sequence Database Collaboration).

Sequence alignment and phylogenetic analyses
Sequences were aligned with MAFFT version 7 using default parameters (Katoh et al. 2017) and the alignment was adjusted manually using PhyDe (version 0.9971; Müller et al. 2010). Simple indel coding, according to Simmons and Ochoterena (2000) implemented in SeqState v.1.40 (Müller 2005), was applied to the ITS matrix which showed length mutational variation in contrast to the plastid DNA matrix. Phylogenetic relationships were reconstructed separately for the nuclear and plastid DNA datasets using maximum parsimony (MP), Bayesian inference (BI) and maximum likelihood (ML). The MP analyses were performed applying the parsimony ratchet (Nixon 1999) with PRAP v.2.0 (Müller 2004) in combination with PAUP v.4.0b10 (Swofford 2003), using 200 ratchet iterations with 25% of the positions randomly upweighted (weight = 2) during each replicate and 10 random addition cycles. Jackknife (JK) support values were calculated in PAUP with 10,000 jackknife replicates and the TBR branch swapping algorithm with 36.788% of characters deleted and one tree held during each replicate. The ML analyses were done with RaxML (Stamatakis 2014) in the version of RAxMLHPC v.8 on XSEDE on the CIPRES Science Gateway (Miller et al. 2010). Four partitions were separated in the nrITS dataset (three DNA: ITS1, 5.8s, ITS2, one binary), whereas the plastid DNA dataset with exclusively coding sequences was left unpartitioned. A rapid bootstrap analysis and search for the best-scoring ML tree in one single programme run with 100 bootstrap replicates was carried out using the general time-reversible (GTR) + Γ model as the standard predefined substitution model in RAxML. The BI analyses were performed with the MPI version of MrBayes (Ronquist et al. 2012) on the high-performance computing system of the Freie Universität Berlin. Instead of a priori testing of the optimal substitution model, sampling across the entire general time reversible (GTR) model space in the Bayesian MCMC analysis was applied (Huelsenbeck et al. 2004). Four partitions were separated in the nrITS dataset (as in ML) and two partitions in the plastid DNA dataset (matK, rbcL). Two simultaneous runs of four parallel chains each were performed for 3 × 10 7 generations with a sample frequency of 1 tree per 2000 generations. Convergence of the runs was checked by making sure that the average standard deviation of split frequencies of the post-burn-in runs was below 0.01 and the effective sampling size (ESS) well above 200 in either run for all parameters. TreeGraph v.2 (Stöver and Müller 2010) was used to visualise the trees with statistical node support.
Only marginal achenes of a capitulum were selected for the analysis. The cross-sections were made by hand in three topographical zones: in the basal third of the achene body, in the median third and in the apical third (or beak if present). The medium portion of the outer achenes is the most informative part, where all features are fully developed and was used for the carpological descriptions and phylogenetic reconstruction. Longitudinal sections were prepared in some cases to detect the peculiarities in the parenchymatous structures of the mesocarp. Prior to sectioning, the achenes were soaked in a mixture of ethyl alcohol, water and glycerol in equal proportions for a few days at 30-40 o C. The sections were stained with 2% carbol fuchsin, then with 0.4% picroindigocarmine and 100% ethanol (preparation of the reagents after Barykina et al. 2004).
The morphology of the achenes was documented using an Olympus SZ61 camera; the cross-sections were studied using Nikon Eclipse Ci microscopes and documented with a Nikon DS-Vi1 camera (Nikon Corporation, Japan) at the Department of Higher Plants (Moscow State University). The ultrasculpture of the achene surface was investigated using an SEM (JSM-6380 LA) at the Laboratory of Electron Microscopy Center at Moscow M.V. Lomonosov State University.
Characterisation of diaspore morphology and anatomy, character coding and analysis of character distribution and evolution Describing carpological features, we applied the tripartite descriptive model (see, for example, Henning et al. 2018), where a feature comprises a character, which is, for its definition, decomposed into a "structure" (a physical component of an organism, for example, a carpophore) and a "property" (e.g. presence) and they were coded with binary or multiple unordered categorical states. Based on our carpological analysis, we selected and defined 16 achene characters potentially relevant for the reconstruction of character evolution. The character data matrix was built and analysed with Mesquite version 3.5 (Maddison and Maddison 2014;Mesquite Project Team 2014). Reconstruction of the character states at ancestral nodes was analysed with MP using the nrDNA tree as hypothesis of phylogenetic relationships and the 'Trace character history' and 'Trace all characters' tools as display methods. The character definitions and their state coding are provided under Results.

Phylogenetic reconstruction based on the nrITS region
The alignment of the nrITS region had a length of 707 characters; together with the coded indels, the matrix included a total of 832 characters, of which 422 were parsimony-informative. The MP analysis resulted in 697 most parsimonious trees (L = 2202, CI = 0.416, RI = 0.848, RC = 0.353, HI = 0.584). The 50% majority consensus tree was largely congruent in topology with the 50% majority consensus trees of the BI and ML analyses. Fig. 1 shows the BI cladogram with the MP jackknife (JK) support values above and the BI posterior probabilities (PP) and ML bootstrap (BS) support values below the branches.
The Scorzonerinae are resolved as monophyletic with strong statistical support (clade 1; JK = 97, PP= 1, BS = 100). The subtribe includes three major clades (1A, 1B, 1C), which received strong (1C; JK = 95, PP = 1, BS = 98) or full statistical support (1A, 1B). Their relationships to each other are unresolved. Clade 1A solely comprises the genus Tourneuxia. Clade 1B includes one part of Scorzonera s.l. We designate it as Gelasia clade, because it includes Scorzonera villosa, which provides the type of Gelasia Cass., being the oldest available generic name for members of this clade. Clade 1C includes the large remainder of the subtribe separated in two well-supported subclades. The 50% majority consensus tree was largely congruent in topology with the 50% majority consensus trees of the BI and ML analyses. Fig. 2 shows the BI cladogram with the MP jackknife (JK) support values above and the BI posterior probabilities (PP) and ML bootstrap (BS) support values below the branches.
The Scorzonerinae are resolved as monophyletic and Tourneuxia as sister to the remainder of the subtribe with strong support (clade 1; JK = 96, PP = 1, BS = 95). The latter clade 2 is a polytomy: clade 2A with almost full support includes the Gelasia clade; clade 2B with strong support only in MP includes Geropogon, Tragopogon and the Scorzonera divaricata clade; clade 2C with low support (JK = 65, PP = 0.97, BS = 61) Incongruences between nrDNA and plastid DNA phylogenies Fewer of the deep nodes are resolved in the plastid DNA tree compared to the nrITS tree, but both reconstructions revealed largely the same major terminal clades. Statistically significant topological differences are few. They mainly concern the relationships of three clades: (1) Epilasia appears as sister to a clade combining Geropogon and Tragopogon in the nrITS tree, whereas as sister to Pseudopodospermum in the plastid DNA tree; (2) Scorzonera divaricata is sister to the Tragopogon-Geropogon clade in the plastid tree, but nested in a polytomy in the nrITS tree without the latter clade; (3) the Scorzonera purpurea-S. renzii clades are nested in the nrITS tree in a polytomy with S. angustifolia and the S. albicaulis clade, but in the plastid DNA tree, these two species are found in a polytomy with the Podospermum clade.

Morphological and anatomical characters of the achene in the Scorzonerinae
The capitula of the Scorzonerinae are, in principle, homocarpic. Minor differentiations corresponding to the centripetal development of the florets and thus also achenes in the Asteraceae capitula may, however, occur. The marginal achenes are considered as the most representative type. Achene wall anatomy and morphology are fully developed in the middle third of the achene as in all Asteraceae. Correspondingly, all features refer to the middle third of the marginal achenes. The achene wall ( Fig. 3) is composed of five segments corresponding to the five principal vascular bundles of the achene wall (plesiomorphic state in all Cichorieae and all Compositae) that may be barely noticeable. The principal vascular bundles of the achene wall can be seen often well below the principal ribs even if the latter are not clearly expressed. In the Scorzonerinae, each segment forms one main rib and, usually, two secondary ribs, the latter shared with the contiguous segments, resulting in a pattern of 10 ribs, either differentiated or not into more prominent principal and less prominent secondary ribs. The achene epidermis is the outer cell layer of the achene wall and can be glabrous, hairy or papillose. The achene surface can also exhibit emergences: stout cell conglomerations, hooked spines or tubercles with verrucose ribs consisting of pore parenchyma. Below the epidermis, the achene wall is composed of sclerenchyma-   ). Abbreviations in pericarp: oe -outer epidermis, ac -air cavity, p -parenchyma, scl -sclerenchyma, vbvascular bundles, ie -inner epidermis; abbreviations in seed: sc -seed coat, en -endosperm, sh -seed hollow (embryo not shown).
tous and parenchymatous layers of various arrangements and thickness. Sometimes, air cavities are present, resulting from rupture of thin-walled parenchyma. The endocarp is often obliterated. The seed coat is ± adjoining to the endocarp and consists of two or several layers, sometimes with the vestiges of vascular bundles. The endosperm is twolayered and the vertical embryo occupies almost all volume in the achene corpus.
The morphological characters of the pappus, as part of the diaspore, are also included in the carpological analysis.

Coding of carpological characters and states in Scorzonerinae
The carpologial analysis across the subtribe revealed a variety of features, which were found to be diagnostic for species or groups of them. In the following, these carpological features are coded in characters (composed of a structure and a property) and states.
In the character designation, structures and substructures are separated by colons and these are separated from the respective property by a semicolon. 1. Achene: carpopodium (formed by the tube-like protruding achene wall); presence 0: absent 1: present Carpopodia as a protrusion of the basal achene wall surrounding the stipe-like structure in which the vascular bundles of the achene enter the receptacle, are known throughout the family and the tribe (Haque and Godward 1984). A particular conspicuous tube-like protrusion of the achene wall is present in some members of Scorzonera s.l. (entirely omitted by Haque and Godward 1984) and has been employed in the past as a diagnostic character to delimit taxa at infrageneric or generic rank (S. subg. Podospermum, S. sect. Purpurea and Pseudopodospermum).  6. Achene wall: sclerenchyma; arrangement 0: Continuous sclerenchymatous layers (sheath) equal in thickness present 1: Discontinuous sclerenchymatous layers with a gap in the principal ribs 2: Continuous sclerenchymatous layers with a narrow or wider hunch on either side of the principal rib 3: Continuous or slightly discontinuous sclerenchyma with well-expressed invaginations (depressions).
Sclerenchyma as stabilising element develops in the achene wall above the vascular bundles into different patterns and formations as defined in the states coding. 7. Achene wall: sclerenchyma; orientation 0: Sclerenchyma entirely with parallel orientation to achene axis 1: Sclerenchyma differentiated: outer layer with parallel, inner with oblique to perpendicular orientation to achene axis.
2: Sclerenchyma only in emergences or rib areas perpendicular (or oblique) to achene axis.
8. Achene wall: parenchyma; arrangement (Fig. 6) 0: Present as subepidermal continuous layer(s) 1: Continuous parenchyma layers above and below the sclerenchyma 2: Insular in principal ribs below sclerenchyma and in secondary ribs above sclerenchyma 3: Only insular in principal ribs below sclerenchyma 4: Insular above sclerenchymatous invaginations and below sclerenchyma in the principal ribs 5: Only insular above sclerenchymatous invaginations 6: Absent 7: Parenchyma continuous above sclerenchyma and below sclerenchyma insular in principal ribs 8: Only insular in second ribs above sclerenchyma Besides sclerenchyma, the main element of the achene wall is parenchyma, but often the parenchyma elements are left behind in quantity by the sclerenchyma. The distribution of the parenchyma elements in the achene wall is taxon specific and several different arrangements have been found as outlined in the character state coding. The various ways the parenchyma can be arranged in the achene wall is schematically shown in Fig. 6. As in the other cases, we have refrained from speculations about evolutionary pathways for these arrangements and have coded them as unordered states. 9. Achene wall: parenchyma; differentiation 0: Parenchyma only as subepidermal mechanical parenchyma 1: Subepidermal collenchyma-like layers differentiated besides parenchyma 2: Parenchyma of thin-walled cells only 3: Parenchyma of two types present, mechanical parenchyma and such with thinwalled cells The unusual collenchyma-like layers are built of thin-walled cells with prominent intercellular spaces of triangular shape. The parenchyma in character states 0 and 2-3 lack prominent intercellular spaces.  10. Achene wall: air cavities; occurrence 0: Absent 1: Present 11. Achene wall; ribbing pattern 0: Each segment with a principal rib and 2 secondary ribs, the latter shared with the contiguous segments, achene middle third, thus with 5 principal and 5 secondary ribs 1: Each segment with a principal rib only, achene middle third, thus with 5 ribs 2: No distinct ribs developed, middle third ± terete (roundish) 3: Two or more ribs enlarged to wings 12. Achene wall: tannins; presence 0: Absent 1: Present in cell protoplast 2: Present in cell walls only 13. Achene beak; presence 0: Absent 1: Present The beak is defined as the more or less abrupt attenuation of the achene apex into a stipe-like structure carrying the pappus disc.
14. Pappus; presence 0: Absent 1: Present 15. Pappus; structure 0: Of entirely softly plumose bristles (with long soft fimbriae all along the bristle) 1: Of entirely softly plumose bristles and scabrid awns 2: Of bristles softly plumose in lower and scabrid in upper half or third (at least 5-10 longer bristles have unequal lateral fimbriae) 4: Bristles scabrid completely or for the most part The setaceous pappus in the Cichorieae consists of bristles with lateral projection (= fimbriae) not or little exceeding the diameter of the bristles, which make the bristles rough, thus "scabrid". Alternatively, the lateral projection may be many times longer than the bristle diameter and the bristles thus featherlike or "plumose". Plumose pappi in the Cichorieae have evolved as three different types, each specific for one subtribe (Kilian et al. 2009a). The plumose bristles in the Scorzonerinae are characterised by soft and often intertwining fimbriae pointing in all directions and consisting of a row of flattened cells. Taxon specific variation of the pappus structure concerns the composition of the pappus by plumose and scabrid elements or the differentiation of the fimbriae development in one and the same pappus element. 16. Pappus; colour 0: white 1: dirty white 2: fulvous (at least in basal part) 3: yellowish 4: grey or blackish

Carpological characterisation of the clades and ancestral character reconstruction
The matrix of the 16 carpological characters is given in Table 2. Representative achene cross sections photographs and schemes representative for the clades are presented in  The detailed carpological descriptions of the principal terminal clades are presented in the concluding part "Taxonomy", below. Here, we summarise the anatomical features for the individual clades.  [-1, -3] 0 n/a 0 0 n/a 0 2    Tourneuxia clade Fig. 7A, B Achenes without carpopodium, with two ribs elongated into small wings; achene epidermis glabrous; emergences absent; subepidermal parenchyma almost continuous, more prominent in the winged areas and much thinner (1-3 layers) between them, of thin-walled cells only; sclerenchyma continuous, its fibres of parallel orientation, sometimes slightly obliquely orientated in the region of the wings; no air cavities; tannins absent.  Fig. 7C, D Achenes without carpopodium; achene epidermis often densely covered with soft multicellular hairs making a long woolly indumentum; emergences mostly absent; subepidermal parenchyma usually insular, of thin-walled cells only; sclerenchyma continuous with an invagination on either side of the principal rib, parallel; no air cavities; tannins absent. Fig. 8A, B

Podospermum clade
The results of the ancestral character reconstruction, based on the nrITS tree, are presented in Suppl. material 1: Fig. S1. For further details, see the corresponding section in the Discussion.

Phylogenetic reconstruction of the Scorzonerinae and its significance for the generic classification of the subtribe
The molecular phylogenetic study, presented here, is the one with the broadest sampling across the subtribe to date and the first comparing reconstructions, based on nuclear ribosomal and plastid DNA markers. It fully confirms previous studies (Mavrodiev et al. 2004;Winfield et al. 2006) in that Scorzonera is polyphyletic in all traditional circumscriptions, based on morphological data. Our broader sampling, however, elucidates an even higher extent of polyphyly than assumed so far. Actually, Scorzonera s.l. comprises at least six independent lineages.
Several deeper nodes of the Scorzonerinae are not resolved in either reconstruction and in previous analyses. However, our nuclear and plastid DNA trees are topologically largely congruent and supplement each other to some extent, allowing a first hypothesis on the major lineages of the Scorzonerinae and their relationships, which are discussed following the structure of the ITS tree (Fig. 1).

The Tourneuxia and Gelasia lineages
The nrITS tree (Fig. 1) separates three well-supported clades of the subtribe in a basal polytomy: (1) the Tourneuxia clade, (2) the Gelasia clade and (3) the remainder. In our plastid DNA tree (Fig. 2), the monospecific N African Tourneuxia is resolved with strong support as sister to all other members of the subtribe which are placed in a large polytomy, whereas the Gelasia clade is resolved with almost full support as one of the six clades of this polytomy. A sister group relationship of the monospecific N African Tourneuxia to all other members is the hitherto best indication for its systematic position and is not in contradiction to the nrITS tree. Taking the evidence from both trees, we may further hypothesise, thus, that Tourneuxia and Gelasia are successive sisters to the remainder of the subtribe. The nrDNA-based reconstructions by Mavrodiev et al. (2004) and Winfield et al. (2006), showed a sister group relationship of Tourneuxia with the Gelasia (as Lasiospora) clade or the Gelasia and Tourneuxia clades as successive sisters to the remainder of the subtribe, but without statistical support.
The N African distribution of the early diverging Tourneuxia lineage corroborates this region being part of the ancestral area of the tribe Cichorieae (compare Kilian et al. 2009b). This clade comprises the genus Tourneuxia, consisting of T. variifolia only, which is characterised by the annual life form and heteromorphic (entire to pinnatisect) leaves. The pollen structure of T. variifolia, consisting of 9 (6 abporal and 3 equatorial) lacunae, is not unique amongst Scorzonerinae and found also in Pterachaenia and different groups of Scorzonera s.l. (Blackmore 1982;Nazarova 1997). Additionally, Tourneuxia shares the basic chromosome number x = 7 with many other Scorzonerinae (Watanabe 2018), its characterisation by Mavrodiev et al. (2004: fig. 4) as having x = 6 being erroneous. Tourneuxia has a carpological type of its own with the following com-bination of the features: achenes somewhat flattened, with two small wing-like ribs; pappus inserted obliquely to the achene body.
The sizable Gelasia lineage has full statistical support in both our trees. Including a considerable number of species of some sections of Scorzonera subg. Scorzonera (S. sect. Anatolia, S. sect. Infrarosulares, S. sect. Nervosae, S. sect. Pulvinares, S. sect. Subaphyllae, S. sect. Trachyactis, S. sect. Tuberosae, S. sect. Vierhapperia), it is resolved far remote from the core of Scorzonera in both our trees. This illustrates a surprisingly strong discrepancy between the traditional classification of the genus and molecular phylogenetic results. The lineage has already been resolved by Mavrodiev et al. (2004) and Winfield et al. (2006) as the "Lasiospora clade", after Scorzonera subg. Lasiospora (Cass.) Tzvelev and the genus Lasiospora Cass. (Cassini 1822), respectively, lectotypified by S. hirsuta L. (Tzvelev 1989). As we now have shown for the first time that also Scorzonera villosa Scop. is a member of this clade, the oldest available generic name for this lineage is Gelasia Cass. (Cassini 1818). Gelasia was separated by Cassini (1818), mostly based on the pappus structure ("aigrette irrégulière", viz. pappus plumose in lower portion and scabrid in upper part) and Lasiospora was described in having pubescent achenes (Cassini 1822). As our character state reconstructions show, the heteromorphic pappus bristles are, however, a synapomorphy of the entire subtribe with only single shifts to homomorphic bristles of either type (Suppl. material 1: Fig. S1, character 15). Hairy achenes, in contrast, seem to be a synapomorphy of the Gelasia lineage but (a) shifts to glabrous achenes occur and (b) variously hairy achenes have been developed in three other clades of the subtribe: in the Podospermum clade, Pseudopodospermum (S. turcomanica, not included in the character reconstruction tree) and the Takhtajaniantha clade (Suppl. material 1: Fig. S1; character 3), but the pubescence of the achenes in these clades is rather scattered, indistinctly expressed or glabrescent in contrast to lanate achenes in Gelasia. The lineage has further carpological peculiarities (Fig. 7C, D): all species belong to the same carpological type with no carpopodium, ± continuous sclerenchyma layers with well-expressed invaginations, which in a similar way is otherwise only present in the S. polyclada and Pterachaenia clades (Suppl. material 1: Fig. S1, character 6) and, besides, with insular parenchyma above the sclerenchyma in significant thickness (Suppl. material 1: Fig.  S1, character 8), similarly to that also found in other clades. The basic chromosome number for the Gelasia lineage seems to be x = 6 (De Santis et al. 1976;Humphries et al. 1978;Nazarova 1984;Magulaev 1986;Constantinidis et al. 2002), judging from the vast majority of chromosome number reports available (Watanabe 2018), but in a few cases x = 7 has also been reported (Sz.-Borsos 1970;Nazarova 1980;Vogt and Oberprieler 2008;Watanabe 2018). Further studies will have to show whether the deviating numbers are due to erroneous identifications or countings or whether two different basic numbers are present in this lineage, as is the case, for example, in the Pterachaenia lineage, see below. As no other, more distinct morphological features characterise the Gelasia lineage, it is no surprise that it has not been recognised as a separate genus for almost 200 years.

The Geropogon and Tragopogon lineages
The monospecific Geropogon and the sizable Tragopogon are consistently and with strong support resolved as sister groups in both trees. Like the previous analyses by Mavrodiev et al. (2004), Winfield et al. (2006) and Mavrodiev et al. (2012), our analysis revealed Tragopogon as monophyletic. The different topology by Mavrodiev et al. (2004), with Geropogon as sister to the Podospermum clade, has no statistical support and Mavrodiev et al. (2012) also revealed Geropogon and Tragopogon as sister groups with high support, based on nuclear ribosomal and nuclear markers. In the past, Geropogon was sometimes merged with Tragopogon (e.g. Richardson 1976), but more frequently accepted in its generic status, mostly based on different life form and pappus characters (annual vs. biennial or perennial and awned pappus vs. plumose pappus in outer achenes, respectively (Boissier 1875;Borisova 1964;Rechinger 1977;Meikle 1985). Pollen structure, chromosome numbers (Díaz de la Blanca 1986, 1988;Blackmore and Barnes 1987) and anatomy of the inner achenes (Sukhorukov and Nilova 2015), moreover, distinguish both genera.
Both trees are incongruent, however, with respect to the relationships of the Geropogon-Tragopogon clade: in the nrITS tree, the Epilasia lineage is the sister group to the former with strong statistical support as was also found by Mavrodiev et al. (2004) and the three are sister to the other members of the subtribe, Tourneuxia and Gelasia excluded (Fig. 1). In contrast, in the plastid DNA tree, the Scorzonera divaricata clade is sister to the Geropogon-Tragopogon clade, while their relationship is not resolved and Epilasia is sister to Podospermum with strong support (Fig. 2).

The Epilasia lineage
This lineage is restricted to SW and W Central Asia, is in our analysis represented by two of its three annual species and received full support in the nrITS tree. Epilasia was first recognised as a section of Scorzonera by Bunge (1852) because of its annual life form, outer leaf-like phyllaries and the pappus arising from a flat or caplike pappus disk. Later, it was established at generic rank by Bentham (in Bentham and Hooker 1873) and has since been unanimously accepted as a separate genus (e.g. Lipschitz 1939;Rechinger 1977;Tagaev 1993;Mavrodiev et al. 2004;Tzvelev 2008). Epilasia is characterised by the unique combination of pollen structure (18 lacunae: 6 abporal, 6 paraporal, 6 polar: Blackmore 1986), the basic chromosome number x = 6 with a diploid or tetraploid chromosome complement (Watanabe 2018) and a distinct achene anatomy (see Taxonomy).

The lineages outside the Scorzonera-Pseudopodospermum-Takhtajaniantha clade
In the nrITS tree ( Fig. 1) four lineages, which are also found in the plastid DNA tree, are resolved with full support, although with partly incongruent relationships.
The Scorzonera divaricata lineage so far only contains the name-giving species, a subspinescent divaricately-branched perennial herb or subshrub, restricted to N and Central China and Mongolia. In the nrITS tree, its relationship is unresolved, in the plastid DNA tree it is resolved as sister to the Geropogon-Tragopogon clade. Previously, S. divaricata was included in S. sect. Polyclada (Lipschitz 1939(Lipschitz , 1964, which mostly contained the species having capitula with a few florets, a frequently homoplastic character state. Notably, the habitually most similar S. pseudodivaricata is found entirely unrelated, being nested in the Takhtajaniantha clade. The basic chromosome number of S. divaricata is x = 7 (Khatoon and Ali 1988). Carpologically, it is characterised by the peculiar combination of 10 ribs, subepidermal parenchyma divided into transparent cell layers and layers with tanniniferous cell walls followed by sclerenchyma equal in thickness. We recognise this lineage as a new genus Lipschitzia (see Taxonomy).
The Pterachaenia lineage was first established as Scorzonera sect. Pterachaenia by Bentham (in Bentham and Hooker 1873) and it was first Lipschitz (1939) who assumed that the section may deserve generic rank. Pterachaenia includes a single species, P. stewartii from E Iran, Afghanistan and Pakistan, which is peculiar by its winged achenes and annual life form. Unexpectedly, Scorzonera codringtonii is resolved as sister to P. stewartii with full support in the nrITS tree and moderate support in the plastid DNA tree. These taxa were never before considered as relatives. Both have a similar life form (annual in P. stewartii and annual to short-lived perennial in S. codringtonii) and habit, a similar achene anatomy apart from the wings in P. stewartii and a similar distribution area. However, following the literature, they differ in pollen structure (9 [6 abporal and 3 equatorial] lacunae in P. stewartii and 18 [6 abporal, 6 equatorial and 6 interporal] lacunae in S. codringtonii (Blackmore 1982) and chromosome numbers (P. stewartii having x = 6; S. codringtonii x = 7: Watanabe 2018).
The Scorzonera polyclada lineage is sister to Koelpinia in the nrITS tree, whereas the deeper nodes are unresolved (Fig. 1). In the plastid DNA tree, it is sister to the Pterachaenia lineage and both, in turn, are sister to Koelpinia (Fig. 2). In both trees, the S. polyclada lineage is shown here for the first time to constitute a lineage separate from the core of Scorzonera. The suffruticose, divaricately branching species S. polyclada and S. longipapposa of Afghanistan, as well as the more widely distributed SW Asian S. intricata were considered as closely related already by Rechinger (1977). Considering morphological resemblance, we assume that species, such as S. tortuosissima (having pollen with 24 lacunae, Askerova 1987), also belong to this lineage. Chromosome numbers x = 7, diploid (data for S. koelpinioides and S. tortuosissima: Razaq et al. 1994). We recognise this lineage as a new genus Ramaliella (see Taxonomy).
The Koelpinia lineage includes only the small genus Koelpinia (five annual species) with an Irano-Turanian distribution, extending into the S Mediterranean area. The plastid DNA tree places Koelpinia in closer relationship to both the S. polyclada and Pterachaenia lineages (with only moderate support) and the nrITS tree only to the former (but with strong support). A closer relationship between Koelpinia and Pterachaenia has already been revealed by Mavrodiev et al. (2004), although without statistical support. Its pollen with 15 (6 abporal, 3 equatorial, 6 interporal) lacunae is rather similar to Tragopogon (Blackmore 1982;Nazarova, 1997). The achenes are distinct from all other Scorzonerinae due to the columnar-scorpioid shape, the surface covered with retrorse subulate spine-like emergences suitable for epizoochory and peculiar, elongated, more or less stout papillae resembling glandular hairs. This lineage is another example for the co-occurrence of both basic numbers 6 and 7; moreover, it forms polyploids up to the octaploid level in K. linearis.

The lineages of the Scorzonera-Pseudopodospermum-Takhtajaniantha clade
Its three major clades, which include the vast majority of Scorzonera in the wide traditional sense, were resolved in both trees and received strong to even full support in the nrITS tree and moderate to strong support in the plastid DNA tree. The sister group relationship of Pseudopodospermum and Epilasia revealed in the plastid DNA tree but incongruent to the nrITS topology has been mentioned already above. The relationship between the three major clades is only resolved in the nrITS tree, where the Pseudopodospermum and Takhtajaniantha clades are sister (with only moderate support: PP = 0.90, BS = 67, not resolved in MP tree) and both in turn are sister to the Scorzonera clade with fairly strong support (PP = 0.99; BS = 98, not resolved in MP tree).
The Takhtajaniantha  it also includes S. pusilla, the only member of S. sect. Pusillae, which was split from Scorzonera as the monotypic genus Takhtajaniantha by Nazarova (1990). Decisive for its separation was the peculiar combination of the presence of a tuber deeply sunken in the substrate, apically acuminate and somewhat coiled leaves, snow-white pappus, as well as is karyotype indicating allotetraploidy (2n = 28), there for the first time found in Scorzonera. The basic number of x = 7 is also shared by the other members of the lineage so far known. Later, Nazarova (1997) discovered the similarity of the pollen of T. pusilla, having only six abporal lacunae, to S. austriaca, S. ikonnikovii and S. tau-saghyz, which is a pollen type, however, also present in the Scorzonera s.str. clade (Blackmore 1982). Takhtajaniantha species possess two carpological types ("Takhtajaniantha" and "Pseudodivaricata") (see Taxonomy and Fig. 11A-D). Many species are characterised by a caudex with fibrous leaf sheath residues, a feature, however, not exclusive to this lineage, but also present in the S. purpurea clade, but only here combined with lanate leaf axiles; otherwise, the lineage seems to lack morpological features distinctly circumscribing it, although several species remarkably resemble each other. The centre of the distribution of Takhtajaniantha is Central Asia as a part of the Irano-Turanian region, with extensions into the E Mediterranean area and E Europe (S. pusilla) and into Central Europe and South Siberia (S. austriaca).
The Pseudopodospermum lineage has full support in the nrITS tree and moderately strong support (JS = 75, PP = 0.99, BS = 73) in the plastid DNA tree. The generic rank proposed by Kuthatheladze (1978) for S. subg. Pseudopodospermum, which is typified by Scorzonera mollis, is corroborated by the fact that this lineage is resolved in both our reconstructions, separate from the core of Scorzonera. The Pseudopodospermum lineage, however, also includes species previously placed in sections Incisae, Foliosae, Papposae and Hissaricae of the former subgenus Scorzonera (Lipschitz 1964). It is predominantly distributed in the Irano-Turanian and E Mediterranean regions. Morphologically, the species united here are highly variable in many characters, such as presence of tubers and achene carpopodium, floret colour, but they are all perennial herbs. All species, so far investigated, are characterised by the basic chromosome number x = 7 and pollen with 20 (6 abporal, 6 equatorial, 6 interporal, 2 polar) lacunae (Blackmore 1986;Nazarova 1997;Pinar et al. 2016). Our carpological results show that all representatives can be divided into three carpological types that differ from each other by anatomical characters (arrangement of sclerenchyma, presence of stout conglomerations, air cavities and tanniniferous cells; Figs 3, 12A-F).
The Scorzonera lineage received strong support in both trees (nrITS: JS = 76, PP = 1, BS = 98; plastid DNA: JS = 93, PP = 1, BS = 88). All members share a basic chromosome number of x = 7. It includes four major terminal clades: the Scorzonera s.str. clade, the S. purpurea clade, the S. albicaulis clade and the Podospermum clade. Besides, S. rupicola, an intricately branched shrublet and S. renzii, a linear-leafy perennial herb with peculiar involucre, both from Iran and Turkey, respectively, form separate clades. The latter, little known species was previously placed in S. sect. Turkestanicae (Rechinger 1977;Kamelin and Tagaev 1986;Coşkunçelebı et al. 2016). The relationships between the clades of the Scorzonera lineage, as well as within these clades, are not well resolved. Moreover, the relationships of S. renzii and the S. purpurea clade are incongruent between the nrITS and the plastid DNA tree.
Scorzonera s.str. clade: This clade unites S. humilis (providing the type of the name Scorzonera), S. aristata and S. parviflora. The close relationship of S. humilis with S. aristata and S. parviflora was assumed already by Díaz de la Guardia and Blanca (1987), whereas it was considered as the sole member of S. sect. Parviflorae by Kamelin and Tagaev (1986) or related with S. mongolica by Lipschitz (1964) due to the snow-white pappus and similar tolerance of saline habitats. The following features seem characteristic for this clade: capitula solitary or by a few; apex of phyllaries often with a red spot; small, echinate or echinolophate pollen with 6 (all abporal) or 18 lacunae (S. aristata; Halbritter and Berger 2017; erroneously as with 15 lacunae by Díaz de la Guardia and Blanca 1985); glabrous and beakless achenes with similar anatomy (present investigation). Scorzonera parviflora is distributed from Central Europe to Central Asia, whereas S. humilis is widespread in Europe and S. aristata restricted to SW Europe (Kilian et al. 2009b). Another species, S. radiata, distributed in Central and Far East Asia (Shih and Kilian 2011), may be related to these three species, based on general morphology and carpology, but this needs confirmation by molecular phylogenetics.
Scorzonera purpurea clade (S. purpurea, S. rosea, S. renzii): Scorzonera purpurea and its close relative S. rosea were often considered to belong to S. subg. Podospermum due to the presence of a carpopodium (Mavrodiev et al. 2004;Greuter and van Raab-Straube 2006), but our analysis clearly shows the homoplasy of this character state.
The main characteristics of this group are the fibrous leaf sheath residues (as otherwise present in Takhtajaniantha), combined with often graminoid leaves and purple or lilac florets, pollen with 18 (6 abporal, 6 equatorial, 6 interporal) lacunae, narrow achenes with the same carpological type (present investigation). Carpology corroborates the close affinity of S. rhodantha (SE Europe) to this clade and it has sometimes been considered as subspecies of S. purpurea (S. purpurea subsp. peristerica: Lack and Kilian in Strid and Tan 1991) or S. rosea (Greuter and van Raab-Straube 2006, sub Podospermum roseum subsp. peristericum).
Scorzonera albicaulis clade: This clade includes species of Scorzonera sect. Piptopogon, S. sect. Turkestanicae and S. sect. Polycladae. Morphologically, all species share the graminoid leaves, achenes attenuated into a more or less prominent beak, an easily caducous pappus and an achene surface slightly scabrid due to attenuate elongations of the epidermis cells. The presence of beaked achenes was the reason for Kamelin and Tagaev (1986) to reinstate the previously monotypic genus Achyroseris Sch.Bip., based on A. macrospermum Sch.Bip. (= Scorzonera albicaulis Bunge) and for the transfer of further species with beaked achenes from Scorzonera to Achyroseris by Tagaev (1993). The members of the S. albicaulis clade are predominantly distributed in Central Asia, extending to W Asia and the N Himalaya (S. virgata); one species (S. angustifolia) is confined to the Iberian Peninsula and Morocco. They share a pollen with 24 (6 abporal, 6 equatorial, 6 interporal, 6 polar) lacunae (Poddubnaya-Arnoldi et al. 1934, Nazarova 1997. Carpologically, the members of this clade are divided into two types. The main anatomical difference between them is the presence of remarkable subepidermal thick-walled parenchyma with well-visible intercellular spaces (Fig. 14A, B) in S. angustifolia, S. baldschuanica, S. bracteosa and S. tragopogonoides. The other members (S. acanthoclada, S. albicaulis, S. racemosa, S. turkestanica and S. virgata) have the same achene anatomy as the Scorzonera s.str. clade (Fig. 13A, B), but often with a significant reduction of the parenchyma portion and always with a yellowish, usually easily deciduous pappus (persistent and dirty white or white in the Scorzonera s.str. clade). Carpological and morphological characters suggest the close affinity of S. crassicaulis, S. franchetii, S. petrovii, S. rupicola (not studied by molecular phylogeny) to this clade.
Podospermum clade: This clade unites the majority of the members of Scorzonera subg. Podospermum sensu Kuthatheladze (1978) and the genus Podospermum sensu Kamelin and Tagaev (1986), respectively. Usually its members are characterised by pinnately lobed leaves, but sometimes not all leaves of an individual plant are divided and a few species (may) have only entire to sinuate leaves (S. hieraciifolia, S. songorica). Moreover, the pinnately lobed leaves are shown in our analysis to be a homoplastic state, because the pinnately leafy species of the former Podospermum sections Incisae (S. bicolor, S. calyculata, S. incisa, S. libanotica, S. reverchonii, S. troodea) and Brevicaulis (S. brevicaulis) are resolved as members of the Pseudopodospermum clade. All members of the Podospermum clade have a well-developed carpopodium, which is another homoplasy shared with the Pseudopodospermum clade and also with the S. purpurea clade. Peculiar for the members of the Podospermum clade are: (1) horn-like appendages on the outer phyllaries of the capitula and (2) the diversely orientated sclerenchymatous fibres in the mesocarp of the achene wall. A limited number of species, investigated palynologically, share pollen with 24 (6 abporal, 6 equatorial, 6 interporal, 6 polar) lacunae (Nazarova 1997). Most species are distributed in SW Asia, extending to W China (S. songorica), N Africa and S and Central Europe (S. laciniata).
Resolution within the clade is very poor and also, morphologically, its members are fairly uniform, indicating a young diversification age. Some samples of the widely distributed, variable species Scorzonera laciniata and S. cana occupy different positions in both the nrITS and plastid DNA trees (Figs 1, 2). This may be connected with the cryptic or poorly understood taxa described at species or varietal rank (e.g. Candolle 1838; Koch 1850, both as Podospermum ;Chamberlain 1975;Nazarova 1995) that are identified as S. laciniata and S. cana. Besides, the distinction of both taxa is somewhat subtle, relying on the life form (monocarpic S. laciniata and polycarpic in S. cana) and the length of the ligules in relation to the involucre.
Tourneuxia matches this ancestral type most closely, except for the presence of ribs enlarged to wings (character state 11/3, Suppl. material 1: Fig. S1: 11), a homoplastic state that occurs a second time in Pterachaenia and a third time in Scorzonera armeniaca (core Scorzonera-Podospermum clade).
Amongst the carpological characters analysed, non-homoplastic synapomorphies for clades of the Scorzonerinae are the rare exception (see below, character states 7/2, 12/1, Suppl. material 1: Fig. S1: 7, 12). Most states occur independently in several clades and, in larger clades, often more than one state is represented. These patterns may partly be explained by the fact that dispersal-related achene characters are strongly exposed and rapidly respond to selection pressure (e.g. Cody and Overton 1996;Cheptou et al. 2008) and, whenever different clades diverge under similar ecological conditions, also to parallel evolution. Achene wall anatomy seems to be less exposed to selection pressure, because the same functional result may be achieved by different anatomical constructions. Little surprisingly, amongst the least homoplastic states are anatomical ones particularly (with characters 6-10). In the following, we discuss which insights for the evolution of the carpological characters the analysis revealed.
The conspicuous tubular carpopodium (character 1, Suppl. material 1: Fig. S1: 1) in some members of the subtribe seems to have evolved at least three times, being present in the Epilasia, Pseudopodospermum and the Scorzonera purpurea and Podospermum clades. According to Haque and Godward (1984), the carpopodium as a rigid structure not contracting on drying when the achene matures, may build up tension in the cells of the abscission layer and thus facilitate abscission of the achene from the receptacle. These authors also suggested an evolution of the carpopodium in the Cichorieae from the dominating interrupted forms as basal rib outgrowths to uninterrupted ringlike forms with smooth surface, which are rare in the tribe. The aforementioned clades provide further examples of the uninterrupted form for the tribe in addition to Lactuca given by these authors. It seems remarkable in this context that, in the Scorzonerinae, interrupted carpopodia or a series from interrupted to more or less or fully uninterrupted ringlike, do not occur and carpopodia are either entirely ringlike or absent.
If a carpopodium is present, there is either some sort of border with respect to texture and shape between carpopodium and basal achene corpus or none (character 2, Suppl. material 1: Fig. S1: 2). It seems that the presence of such a border is plesiomorphic and, only in comparatively few topologically scattered cases, the border vanished.
The plesiomorphic state of an achene surface with unicellular papillae or mamillae (character 3, Suppl. material 1: Fig. S1: 3) seems to have shifted several times towards a glabrous surface, four times to a pubescence of multicellular eglandular hairs and one time each to drastically elongate (Koelpinia) and stout hair-like papillae (Epilasia).
Sclerenchyma in the achene wall is responsible for the stabilty of the structure and is derived from the xylem elements of the vascular bundles of the achene wall. The ancestral condition of a continuous sclerenchymatous sheath in the wall (character 6, Suppl. material 1: Fig. S1: 6) is predominant and present in all major clades. Hunch-like outgrowths of the scleremcyma sheath flanking the principal ribs are the predominant state in the Takhtajaniantha clade but are also present in the Pseudopodospermum and Scorzonera s.str. clades). In other members of these two clades and in the Gelasia clade, the sclerenchyma shows well-expressed depressions. A differentiation of the sclerenchyma orientation with the outer layers parallel and the inner perpendicular to the achene axis seems a non-homoplastic synapomorphy of the Podospermum clade (character 7, Suppl. material 1: Fig. S1: 7). Perpendicular or oblique sclerenchyma layers restricted to the ribs and emergences have evolved in the Tourneuxia, Epilasia and Koelpinia clades.
The parenchyma of the achene wall shows considerable diversity regarding arrangement (character 8, Suppl. material 1: Fig. S1: 8) and differentiation (character 9, Suppl. material 1: Fig. S1: 9). The ancestral state of a continuous subepidermal layer (8/0) has shifted to various types of insular presence, in particular in the Gelasia clade. Continuous layers above and below the sclerenchyma (8/1) are present in the Scorzonera clade; the distribution of this state in the Podospermum clade for which otherwise an arrangement continuous above and insular below the sclerenchyma (8/7) was revealed as synapomorphy, indicates a close relationship between these two states. Regarding its differentiation, the parenchyma has apparently been differentiated several times in the evolution of the subtribe to support stabilisation of the achene wall, either as mechanical or collenchymalike tissue or both (9/1-3).
Our reconstruction of evolution of the ribbing pattern (character 11, Suppl. material 1: Fig. S1: 11) revealed that, from the ancestral state with 5 prominent main ribs (11/1), shifts have occurred towards (11/2) reduction of the prominence of the ribs and a terete cross section shape, (11/3) enlargement of ribs as wings (in four terminal clades) and (11/0) differentiation of secondary ribs, whereby in the subtribe always adjacent secondary ribs are fused to a condition with 10 alternating principal and secondary ribs.
Occurrence of tannins (character 12, Suppl. material 1: Fig. S1: 12) has evolved in the cell walls (12/2) in the Epilasia and S. divaricata clades and in the protoplast (12/1) as an exclusive synapomorphy of in the Pseudopodospermum clade (but with reversions in several terminals).
The pappus has been lost once in the Koelpinia clade (character 14, Suppl. material 1: Fig. S1: 14; for parallels in other subtribes, see under Taxonomic conclusions). Its structure (character 15, Suppl. material 1: Fig. S1: 15) is almost uniform in the entire subtribe: it is mostly composed of basally softly plumose and apically scabrid bristles (15/2). Shifts to purely scabrid bristles (15/3) have occurred in Gelasia and, in the outer achenes only (15/1) in Geropogon, the opposite shift to purely plumose bristles (15/0) in species of Tragopogon. With respect to pappus colour (character 16, Suppl. material 1: Fig. S1: 16), the fulvous colour was revealed as ancestral and as a synapomorphy of Gelasia (and Tourneuxia). Shifts are indicated to have occurred to a dirty white colour at some state of uncertain position in the evolution of the subtribe, with further shifts to yellowish, pure white or grey and reversals to fulvous.

Taxonomic conclusions
We have shown above that morphology, even extended to include fruit anatomy, does not very well reflect the structure of the subtribe as revealed through molecular phylogenetics. Actually, most of the lineages resolved are difficult to characterise by morphology. Even more distant clades of the Scorzonerinae are often not well distinguished morphologically. Neither gross morphology nor fruit anatomy provides non-homoplastic synapomorphies for most of the major lineages. A prominent example is Gelasia, of which many species are nicely recognisable by the very conspicuous longlanate achene indumentum; in a number of species, however, a reversal to glabrous achenes has occurred and what remains is some overall similarity of the Gelasia species which cannot be appropriately expressed in a character-state matrix or an identification key. This situation is apparently responsible for the reluctant reception of any classification of Scorzonerinae with more than the few morphologically conspicuous elements and the perseverance of a polyphyletic taxonomic concept of Scorzonera in spite of contrary evidence. We assume, however, that practical taxonomic experience in the application of a phylogenetic classification will bring to light new means to distinguish the various entities.
Our molecular phylogenetic analysis, based on nrITS, shows three principal options for a revised classification, based on monophyletic generic concepts. The first is to separate generically the Gelasia clade and retain the remainder of the current Scorzonera, but also include in it the genera Pterachaenia and Koelpinia. In this option Scorzonera would encompass clade 1C2 (Fig. 1). Since a second taxon, of Scorzonera and without winged achenes, was resolved in the Pterachaenia clade, the winged achenes are no argument against its inclusion in Scorzonera. The inclusion of Koelpinia with epizoochorous achenes without pappus seems more unconventional, but there are other cases in the Cichorieae were taxa without pappus, all traditionally placed in genera of their own for this conspicuous feature, turn out in molecular phylogenetic analyses to be nested in larger pappus-bearing genera; examples are Lapsana and Rhagadiolus being nested in Crepis (Enke and Gemeinholzer 2008) and Lapsanastrum being nested in Youngia (Deng et al. 2014). However, the actual inconvenience and challenge is the diagnosing of Gelasia as a separate genus. This burden is inevitable -unless treating the subtribe as a single genus (or as two genera, if Tourneuxia is retained) which is definitely undesirable -and consequently shared by all three available options. The second option is to recognise Scorzonera in the circumscription of clade 2 (Fig. 1). This means to separate Gelasia, as well as the S. divariata and the S. polyclada clades. This option retains Koelpinia and Pterachaenia as separate genera and requires thus the establishing of two other small but rather easily recognisable genera besides Gelasia. The third option is to recognise Scorzonera in the sense of clade 3 ( Fig.  1), thus also generically separating Pseudopodospermum and Takhtajaniantha. This option is the one which is equally resolved in both the nrITS and plastid tree (Figs 1,2) and the one here proposed.

Key to the revised genera of the Scorzonerinae
The following key is a first attempt and provides some guidance rather than a truly reliable means for identification, for the reasons explained above. We expect that with the new classification at hand, taxonomic experience with the subtribe will lead to the construction of a more reliable key. Pollen: echinolophate, tricolporate and each colpus divided into 2 lacunae, with 9 (6 abporal and 3 equatorial) lacunae (Blackmore 1982).
Achenes: 3-6 mm, compressed, smooth, with two ribs elongated into small wings, without carpopodium; achene wall with continuous sclerenchymatous layer, of parallel orientation, sometimes the fibres of the sclerenchyma are slightly obliquely orientated in the region of the wings, parenchyma subepidermal, almost continuous, more prominent in the winged areas and much thinner (1-3 layers) between them, of thin-walled cells only, air cavities absent, tannins absent.
Pappus: 4-7 mm, obliquely inserted in the marginal achenes, bristles plumose almost entirely, but scabrid in upper portion, the fimbriae of which are soft and tangled with each other, proximally brownish, distally dirty white.
Achenes: 4-12 mm long, without carpopodium, often densely covered with long woolly indumentum, more rarely glabrous, straight or slightly curved, 5-ribbed or roundish, smooth or rarely with stout conglomerations (G. caespitosa, G. pygmaea and G. villosa), parenchyma usually insular above invaginations of the sclerenchyma and below the principal ribs or only insular above invaginations of sclerenchyma, air cavities absent, sclerenchymatous layers continuous or discontinuous in the main ribs, forming a sheath, with an invagination on either side of the principal rib, fibres orientated parallel to the fruit axis, tannins absent.
Pappus: 5-24 mm; bristles plumose in lower portion and scabrid in upper portion or more rarely, almost completely scabrid; dirty white, yellow or fulvous.
Achenes: 5-10 mm, terete or with 5-10 less distinct ribs, glabrous, with carpopodium and stout hair-like papillae; either with apical flat pappus disk and pappus (E. acrolasia, E. mirabilis) or with conic caplike pappus disk and pappus covering the upper half of the achene (E. hemilasia); pericarp with thin-walled subepidermal parenchymatous sheath, the cell walls of which can be filled with the tannins and continuous sclerenchymatous layers orientated parallel to the fruit axis (but sometimes perpendicular in the ribs of E. mirabilis), air cavities absent.
Pappus: 5-8 mm, dense, ash-grey or rusty, five of its bristles (rarely more) fragile, barbellate at tip, other bristles long-plumose or rarely bristles plumose in lower portion and scabrid in upper portion.
Pollen: data n/a. Achenes: 6-10 mm, cylindrical, with 10 ribs, smooth only apically with cylindrical papillae, without emergences and carpopodium; achene wall with both thin-and thickwalled cells (in latter case, the walls are be filled with the tannins) and continuous sclerenchymatous layers which cells orientated parallel to the achene axis, air cavities absent.
Pappus :  Note. Usually Lipschitz (1939: 31) is given as the place of publication for the generic name Pterachaenia. His statement "I think this section will also turn out to be a separate genus (gen. proprium Pterachaenia)" [translated from Russian] qualifies it, however, as a provisional genus name not formally accepted by its author. Diagnostic features. Flowering stems several or many, unbranched, leafless (scapes); phyllaries lanceolate, acute; florets yellow with red veins; achenes with 2-3 wings or without.
Description. Habit, life form, subterranean parts: perennial with caudex or annual herbs, with taproot.
Stem, synflorescence: stems leafless, unbranched (scapes) several to many, pubescent in lower part and glabrous in upper part, with terminal capitulum.
Achenes: 12-15 mm, straight, without carpopodium; with 2-3 elongated ribs forming wings denticulate in upper part (Pterachaenia stewartii) or without wings (P. codringtonii), papillate and with small emergences (spinulae) (P. stewartii) or with smooth surface (P. codringtonii), with five principal ribs, parenchyma of two types, with thick-walled cells and with thin-walled cells, discontinuous (located above sclerenchyma between the principal ribs and below sclerenchyma in the rib areas) or only insular above sclerenchyma, air cavities absent, sclerenchyma continuous, equal in thickness, fibres orientated parallel to the fruit axis, tannins absent.
Achenes: 8-28 mm, straight or slightly curved, with or without carpopodium, usually with five, rarely with ten ribs or without, surface papillate and/or usually with stout conglomerations or the outer strongly rugose or tuberculate-squamate; achene wall with parenchyma subepidermal, continuous (or very rarely sometimes irregularly discontinuous), often reduced to one-to several layers consisting of thin-and/or thick-walled cells, sometimes Type species. Takhtajaniantha pusilla (Pall.) Nazarova Diagnostic features. Perennials; caudex often with dark brown fibrous leaf sheath residues and base of the rosulate leaves often lanate; pollen with 6 lacunae.
Description. Habit, life form, subterranean parts: perennial herbs or subshrublets with a taproot or a tuber.
Leaves: rosulate or crowded in the basal part of the stem, rarely dispersed along the stem, sessile or petiolate, filiform or ovate, apically sometimes hooked (T. pusilla), margin flat or undulate, entire or denticulate.
Stem, synflorescence: stem solitary or several, usually leafy but bracteate in S. acanthoclada and S. racemosa, capitula terminal and solitary or numerous, in spiciform or corymbiform synflorescence.
Capitula: pubescent and often glabrescent, phyllaries in several series, outer phyllaries 1/2-1/3 as long as inner ones, triangular-ovate, the inner phyllaries triangular to lanceolate, receptacle glabrous, capitula with 4-12 florets, yellow, pink or orange (S. transiliensis), 1.5-2 times exceeding the involucre. Achenes: 7-45 mm, straight, without carpopodium, with more or less expressed beak, 10-or rarely 5-ribbed, papillate; achene wall with parenchyma well-expressed and represented by collenchyma-like cells, then present only as subepidermal continuous layers or sometimes parenchyma absent or discontinuous in the rib areas, insular in principal ribs below sclerenchyma and in secondary ribs above sclerenchyma or absent, air cavities absent, sclerenchyma present as layers with a gap in the principal ribs or continuous sclerenchymatous layers, its fibres orientated parallel to the fruit axis, tannins absent.

Species of unclear position
Scorzonera renzii (not carpologically studied) is sister to the Scorzonera purpurea clade in our nrITS tree and forms a polytomy with the Scorzonera purpurea and Podospermum clades in the plastid DNA tree. The spiciform synflorescences and other morphological features (tall stem, graminoid rosulate leaves without residues; pollen with 24 lacunae) rather indicate an affinity to the S. albicaulis clade. Scorzonera angustifolia (pollen with 24 lacunae) and S.rupicola show some affinity to the S. albicaulis clade, but the second is resolved in the nrITS tree as sister to the polytomy including the S. purpurea and S. albicaulis clades, whereas the first is the third element of that polytomy.

Excluded names
Scorzonera sect. Pentachlamys DC., Prodr. 7: 125. 1838, including two species from Nepal, S. bupleuroides D.Don, Prodr. Fl. Nepal.: 162. 1825 and S. roylei DC., Prodr. 7: 125. 1838, do not refer to Scorzonera or one of the genera segregated here. The holotype of S. roylei in G-DC (G00498633) is a species of Tragopogon. Original material of S. bupleuroides seems to be lost and the species is hardly a member of Scorzonera or one of its segregates as was already concluded by Lipschitz (1939).