Introduction

Scorzonera L. (Asteraceae, Cichorieae, Scorzonerinae) with 160–175 species, depending on the circumscription and species concepts, has a distribution spanning Europe and the Mediterranean region, N Africa and SW and C Asia, with a particularly high concentration of species in SW Asia (Bremer 1994; Rechinger 1977; Mabberley 2008; Norouzi & al. 2016; Kilian & al. 2009b+). In its widest circumscription, Scorzonera is characterized by simple or pinnatifid leaves, multiseriate and unequal phyllaries, glabrous or entirely lanate achenes, and plumose pappus bristles with soft and interwoven fimbriae (Lipschitz 1964; Rechinger 1977; Mavrodiev & al. 2004; Kilian & al. 2009a). The other unanimously recognized genera of subtribe Scorzonerinae are readily distinguished from Scorzonera in its widest sense: Tragopogon L., the only other large genus of the subtribe (c. 150 species), by its uniseriate involucre; Koelpinia Pall. by its scorpioid achenes without a pappus but with hooked projections; Epilasia (Bunge) Benth. & Hook. f. by its leafy outer series of phyllaries usually as long as or longer than the inner series; the monotypic Tourneuxia Coss. by its pappus laterally situated at the achene apex; and the monotypic Pterachaenia Benth. by its winged achenes (Mavrodiev & al. 2004; Kilian & al. 2009a).

Starting with Candolle (1805), who described the genus Podospermum DC., attempts have been made to segregate Scorzonera into separate genera; the most recent was by Nazarova (1990), who distinguished the monotypic Takhtajaniantha Nazarova from all other species in the genus. Podospermum was characterized by Candolle (1805) at generic rank by the combination of pinnatifid leaves and a well-expressed carpopodium, which is the sterile abscission zone at the base of the achene, attached to the receptacle and composed of one or more rows of cells that are distinct from the rest of the achene wall (Mukherjee & Nordenstam 2004). Due to this attractive diagnosis, the recognition of Podospermum at generic rank has been widely accepted (Candolle 1805, 1838; Cassini 1827; Dumortier 1827; Lessing 1832; Endlicher 1841; Grossheim 1949; Kuthatheladze 1978; Pignatti 1982; Tzvelev 1988; Nazarova 1997; Mavrodiev & al. 2004; Winfield & al. 2006; Greuter 2006+; Kilian & al. 2009a, 2009b+; Makbul & al. 2016). Instead of splitting Scorzonera into different genera, some workers (Lipschitz 1964; Rechinger 1977; Safavi 2013; Coşkunçelebı & al. 2015) recognized three subgenera: S. subg. Scorzonera, S. subg. Podospermum (DC.) Lipsch. and S. subg. Pseudopodospermum (Lipsch. & Krasch.) Lipsch., a treatment first established by Lipschitz (1935–1939) in his monograph of the genus. Lipschitz (1964) described the three subgenera based on the presence of a carpopodium with simple leaves (S. subg. Pseudopodospermum), the presence of a carpopodium with pinnatifid leaves (S. subg. Podospermum) and the absence of a carpopodium with simple leaves (S. subg. Scorzonera). Rechinger (1977) recognized that the carpopodium is sometimes inconspicuous in species of S. subg. Pseudopodospermum. He therefore considered other morphological characters, including a tuberous root, simple leaves, and a sculptured achene surface (lamellate, muricate, tuberculate or verrucose) to separate S. subg. Pseudopodospermum from S. subg. Scorzonera and S. subg. Podospermum. Morphological studies of achene features by Coşkunçelebı & al. (2016) confirmed that achene surface patterns are valuable for distinguishing between S. subg. Pseudopodospermum and S. subg. Podospermum (Coşkunçelebı & al. 2016). Those studies focused on Turkish species and they only sampled one and three species of S. subg. Pseudopodospermum and S. subg. Podospermum from Iran, respectively.

Previous molecular phylogenetic studies have sampled broadly across subtribe Scorzonerinae. The genus Scorzonera was resolved as polyphyletic, based on the nuclear ribosomal Internal Transcribed Spacer (nrITS; Mavrodiev & al. 2004) and a combined nrITS and external transcribed spacer (ETS) analysis and Amplified Fragment Polymorphisms (AFLPs) by Winfield & al. (2006). Intergeneric nodes in those analyses were, however, statistically unsupported. Both studies revealed that the “Lasiospora clade”, named after the Scorzonera segregate Lasiospora Cass. based on S. hirsuta (Gouan) L., represents a lineage that is far from the core of Scorzonera, in the sense of its type S. humilis L. Mavrodiev & al. (2004) showed that the Lasiospora clade can also be distinguished from Scorzonera based on chromosome number (6 and 7, respectively; see also Nazarova 1977; Diaz De La Guardia & Blanca 1987; Martin & al. 2012). The morphological distinction of Lasiospora species from Scorzonera is, however, unclear. Importantly, both studies confirmed that S. subg. Podospermum is monophyletic, but with S. purpurea L. resolved as sister to the Podospermum clade.

Zaika & al. (2020) recently provided a taxonomic reassessment of Scorzonera s.l. based on broad taxonomic sampling, carpological (including anatomical) data, and nrITS and two plastid markers (partial rbcL and matK) molecular phylogenetic analyses (Zaika & al. 2020). That study confirmed the polyphyly of Scorzonera and proposed a revised classification of the subtribe. As a result of their analyses, the following seven genera were confirmed: Gelasia Cass.; Pseudopodospermum (Lipsch. & Krasch.) Kuth.; Pterachaenia (including S. codringtonii Rech. f.); Scorzonera (including four major clades: Podospermum, Scorzonera s. str., S. albicaulis Bunge and S. purpurea); Takhtajaniantha; and the newly described Lipschitzia Zaika & al. (S. divaricata Turcz. clade) and Ramaliella Zaika & al. (S. polyclada Rech. f. & Köie clade). Therefore, the authors proposed a narrow circumscription of Scorzonera (containing the Podospermum clade) and accepted Pseudopodospermum as a separate genus. At present, we are uncertain of the most appropriate taxonomic concept for the clades Podospermum and Pseudopodospermum. Therefore, for the purpose of this study, we follow the wider circumscription of genus Scorzonera that recognizes these lineages at subgeneric rank: S. subg. Podospermum, and S. subg. Pseudopodospermum. This taxonomic concept is in accordance with the following studies: Lipschitz (1964), Rechinger (1977), Safavi (2013), and Coşkunçelebı & al. (2015). In contrast to Zaika & al. (2020), who investigated generic-level relationships within Scorzonera s.l. Our study focuses on the shallower taxonomic levels and aims to clarify the morphological delimitation among Iranian species within S. subg. Podospermum and S. subg. Pseudopodospermum and compare it with the nrITS tree.

Currently, the genus Scorzonera is represented by 57 species in Iran; of which 33% (20 species) are considered endemic to the country (Rechinger 1977; Safavi 2013; Safavi 2016; Safavi 2019). Of the 57 species in Iran, 36 belong to S. subg. Scorzonera (137 worldwide), nine to S. subg. Pseudopodospermum (20 worldwide) and 12 (21 worldwide) to S. subg. Podospermum (Kamelin & Tagaev 1986; Rechinger 1977; Safavi 2013; Safavi 2016; Safavi 2019). In Iran, S. subg. Podospermum and S. subg. Pseudopodospermum contain closely related species that are currently difficult to distinguish based on morphological characters, which limits species identification. With the exception of leaf-anatomical studies of Iranian species of S. subg. Pseudopodospermum and S. subg. Podospermum (Norouzi & al. 2016), and karyological analyses of a limited number of species from Iran (Safavi 1999; Bordbar & al. 2019; Hatami & al. 2019), no comprehensive study has been carried out to date to clarify the nomenclature and intergeneric and interspecific diagnostic characters of these subgenera in Iran. In order to clarify the circumscription of S. subg. Podospermum and S. subg. Pseudopodospermum and give stronger insight into these lineages in Iran, we conducted extensive sampling of the two subgenera in Iran for detailed morphological studies. We also sampled representatives of all major clades across Scorzonerinae for phylogenetic analysis based on nrITS. Therefore, the aims of the present study are to:

Material and methods

Sampling — For nrITS analyses, the sampling approach for Scorzonera subg. Podospermum and S. subg. Pseudopodospermum was guided by treatments in Flora iranica (Rechinger 1977). Refer to Appendix 1 for a list of all samples included in the nrITS analyses with voucher information and GenBank numbers for newly generated sequences in this study. See Table 1 for classifications of Scorzonera species based on previous studies. In order to include representatives from the major clades across Scorzonerinae in the molecular analyses, we also incorporated already published nrITS sequence data of members outside of the focus subgenera. Therefore, the ingroup comprised extensive sampling from Iranian S. subg. Podospermum and S. subg. Pseudopodospermum (Rechinger 1977), and sampling outside of those subgenera: species from S. subg. Scorzonera and from the genera Epilasia, Koelpinia, Pterachaenia and Tragopogon guided by the clades in subtribe Scorzonerinae in the nrITS analyses of Mavrodiev & al. (2004; Appendix 1). Sequences of nrITS for 29 accessions were newly generated in this study: 11 from species of S. subg. Podospermum, seven from species of S. subg. Pseudopodospermum and one species of the Lasiospora clade distributed in Iran. In some cases, there were multiple accessions per species. It was not possible to generate nrITS data for S. turkeviczii Krasch. & Lipsch. or S. syriaca Boiss. & Blanche (S. subg. Pseudopodospermum); however, these species were included in the morphological analyses and taxonomic treatment (see below; Table 1). Scorzonera lachnostegia (Woronow) Lipsch. (S. subg. Podospermum) is very rare in Iran (Safavi 2013); therefore, it was not possible to locate specimens for morphological or molecular studies. Sequences of 22 species from NCBI were included, corresponding to the three subgenera of Scorzonera and other related genera in subtribe Scorzonerinae. In total, nrITS sequences for 44 species of Scorzonerinae were included in the phylogenetic analyses. Sequences for Cichorium intybus L., Lactuca sativa L. and Scolymus maculatus L. from NCBI were included as outgroup species to Scorzonerinae (Appendix 1).

Phylogenetic reconstruction — Genomic DNA was extracted from leaf material using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. For amplification of the nrITS region, primers ITS-A and ITS-B were used (Blattner 1999). The PCR reaction mixture consisted of 20 µl deionized water, 7 µl 2×Taq DNA polymerase master mix Red (Amplicon, Cat. No.180301), 0.75 µl of each primer (50 pmol/µl), and 1.5 µl template DNA. PCR amplification consisted of an initial denaturation step of 3 minutes at 94°C, followed by 38 cycles of 30 seconds denaturation at 94°C, 40 seconds annealing at 53°C and 1-minute extension at 68°C, and a final extension step at 70°C for 10 minutes. Sequencing reactions were performed at Macrogen Inc. (Seoul, Korea) using the same PCR primers.

Sequences were initially aligned using MAFFT v. 6.0 (Katoh & Toh 2008) and checked manually using the program PhyDE v. 0.9971 (Müller & al. 2005). Indels were coded as binary characters using the simple indel coding approach, according to Simmons & Ochoterena (2000) in SeqState v. 1.4.1 (Müller 2005). Phylogenetic analyses were conducted using Maximum Parsimony (MP), Maximum Likelihood (ML) and Bayesian Inference (BI). Maximum Parsimony analyses were performed using heuristic searches in PAUP* v. 4.0b10 (Swofford 2003) in combination with parsimony Ratchet (Nixon 1999) in PRAP (Müller 2004). Ratchet settings included 200 iterations with 25% of the positions randomly unweighted (weight = 2), and 100 random additional cycles. Jackknife (JK) support was estimated in PAUP by conducting a single heuristic search within each 10 000 replicates using the Tree Bisection and Re-connection branch-swapping algorithm and a deletion of 36.79% characters in each replicate. A strict-consensus tree was constructed from all saved trees. The symmetrical (SYM) nucleotide substitution model was selected as the best-fit model using JModelTest v.2.1.6 (Darriba & al. 2012), according to the Akaike Information Criterion (AIC). The neighbour-joining algorithm BIONJ was used for the initial tree search (Gascuel 1997; Saitou & Nei 1987). Maximum Likelihood analyses were conducted using the graphical user interface of RAxML v. 1.5b1 (Silvestro & Michalak 2012). Bootstrap support was estimated based on the majority-rule consensus tree from 1000 replicates with 200 searches and the final tree topology was evaluated under the GTR GAMMA algorithm because the SYM model is not available in RAxML. Bayesian Inference was conducted in MrBayes v. 3.2 (Ronquist & Huelsenbeck 2003) on the CIPRES science gateway (Miller & al. 2010). Four runs each with four chains were performed for 30 million generations, sampling every 2000 generations. After removing 10% of the sampled trees as burn-in, a 50% majority-rule consensus tree was constructed. Final tree visualization was carried out using TreeGraph v. 2.13.0-748 beta (Stöver & Müller 2010). Final DNA sequences were submitted to ENA ( https://www.ebi.ac.uk/ena) using the software tool annonex2embl (Gruenstaeudl 2019).

Table 1.

Scorzonera species included in this study and their subgeneric classification based on previous literature (in column 2: Lipschitz 1964; Rechinger 1977; Kamelin & Tagaev 1986; Mavrodiev & al. 2014; Safavi 2013; Zaika & al. 2020) compared to results of phylogenetic analyses (Fig. 1) and the taxonomic treatment of this study (in column 3).

Morphological and taxonomic studies — Collections of Scorzonera subg. Podospermum and S. subg. Pseudopodospermum were made between 2015 and 2018 across a broad geographic range in Iran in order to sample the maximum diversity of each taxon. Specimens from those field trips were deposited at MIR (see Appendix 2 for a list of specimens examined for the morphological and taxonomic studies). Live plants in the field and herbarium specimens in B, FMUH, HSHU, IRAN, JE, MIR and W were examined (herbarium codes according to Thiers 2019+; Appendix 2). Digitized specimens were examined via virtual herbarium catalogues at E ( https://data.rbge.org.uk/search/herbarium/), G ( http://www.ville-ge.ch/musinfo/bd/cjb/chg/), LINN ( http://linnean-online.org/linnaean_herbarium.html), P ( https://science.mnhn.fr/institution/mnhn/search) and via JSTOR Global Plants ( https://plants.jstor.org/). Where possible, type specimens were examined by E.H. in the herbaria B, JE and W, from images provided by LE for types in that herbarium, and via virtual herbarium catalogues and JSTOR Global Plants for types in G and LINN. Descriptions of morphological characters were based on our observations and measurements (life cycle, plant height, root, stem, leaves, flowering capitula, fruiting capitula, achenes, pappus; Appendix 4) with comparisons to previous relevant studies (Lipschitz 1964; Rechinger 1977; Safavi 2013). Terminology for vegetative and reproductive morphological characters follows Beentje (2010). For each species the general distribution, updated nomenclature, and synonyms are given based on Flora iranica, Flora of Iran, and the Cichorieae portal (Rechinger 1977; Safavi 2013; Kilian & al. 2009+).

Macro photographs of achenes from species belonging to Scorzonera subg. Podospermum and S. subg. Pseudopodospermum were made using a stereomicroscope (Olympus SZX16) equipped with DP72 (a 12.5 megapixel digital colour camera), connected to cellSens Standard programme with an extended focus imaging function, at the Botanic Garden and Botanical Museum Berlin.

Results

Molecular analyses — The aligned nrITS data matrix comprised of 55 sequences and 826 characters including 126 coded indels, 291 parsimony informative sites and 156 parsimony uninformative sites (see alignment in Appendix 3). Maximum Parsimony analyses resulted in 16 most parsimonious trees with a length of 1096, a consistency index of 0.615, and a retention index of 0.863.

Bayesian Inference, ML, and MP analyses of the nrITS dataset produced identical topologies. The MrBayes 50% majority-rule consensus tree is presented in Fig. 1. Here, we report statistical support values that are well-supported (>0.95 posterior probability [PP], and >80% BS and JK) in parentheses (Fig. 1). Subtribe Scorzonerinae received full statistical support (1 PP, 100 JK, 100 BS; Fig. 1). The Lasiospora clade was resolved as monophyletic (1 PP, 100 JK, 100 BS) and as sister to a clade (1 PP, 99 JK, 97 BS) containing a polytomy that consisted of the rest of subtribe Scorzonerinae. This polytomy comprised five clades; one clade (1 PP, 92 BS) included in two subclades that contained the genera Epilasia (1 PP, 100 JK, 100 BS) and Tragopogon (1 PP, 100 JK, 100 BS), respectively. A second lineage in the polytomy corresponded to a single accession representing the monotypic genus Pterachaenia. The third clade in the polytomy (1 PP, 93 JK, 94 BS) contained the genus Koelpinia, sister to a clade consisting of Scorzonera intricata Boiss, and S. tortuosissima Boiss. (S. intricata clade; 1 PP, 100 JK, 100 BS). In a fourth clade (1 PP, 91 JK, 84 BS), S. humilis and S. aristata Ramond ex DC. (S. humilis clade; 1 PP, 99 JK, 99 BS) and a single accession of S. purpurea were resolved as consecutive sisters to a clade uniting the members of S. subg. Podospermum (1 PP, 100 JK, 100 BS). The relationships within the S. subg. Podospermum clade were largely unresolved, with the exception of the S. songorica (Kar. & Kir.) Lipsch. & Vassilcz. clade (1 PP, 100 JK, 100 BS) and the S. cana (C. A. Mey.) O. Hoffm. clade (0.99 PP, 91 JK, 80 BS). The fifth clade included S. subg. Pseudopodospermum (1 PP, 100 JK, 100 BS). Within S. subg. Pseudopodospermum, an accession of S. calyculata Boiss. was resolved as sister to S. papposa DC., which was sister to the S. mollis M. Bieb. clade (13 samples of nine species; 1 PP, 87 JK, 95 BS). The S. mollis clade contained a polytomy consisting of an accession of S. mollis, one clade (0.99 PP, 64 JK, 71 BS) containing three accessions of S. szowitzii DC. and another clade (1 PP) with the three samples of S. raddeana C. Winkl. (1 PP, 93 JK, 94 BS) in one subclade that was sister to a subclade containing S. phaeopappa (Boiss.) Boiss., S. semicana DC., S. mucida Rech. f. & al., and S. tunicata Rech. f. & Köie (0.93 PP).

Morphology — Images of achenes of 22 species are presented in this study (Fig. 2; Fig. 3). A summary of the achene surface pattern, pubescence, carpopodium and pappus bristles of species in Scorzonera subg. Podospermum and S. subg. Pseudopodospermum from Iran is provided in Table 2. Our observations revealed that, in S. subg. Podospermum, outer achenes are smooth, subterete to sulcate, glabrous or lanate, with a conspicuous carpopodium, and the pappi are apically scabrous with plumose bristles for most of the length. In contrast, in S. subg. Pseudopodospermum, the outer achenes are lamellate, muricate, or verrucose, with or without a conspicuous carpopodium, glabrous, and the pappi either consist of bristles that are plumose for the entire length or for most of the length, and are apically scabrous, sometimes with five obvious longer scabrous bristles. More detailed characteristics of achenes for each species are given in Table 2.

Fig. 1.

Bayesian 50% majority-rule consensus tree inferred from the nuclear Internal Transcribed Spacer dataset. Values above nodes indicate posterior probability (bold) and jack-knife support (italic), and values below nodes indicate bootstrap support. Tip names correspond to species names and GenBank numbers or DNA numbers for newly generated sequences; see Appendix 1 for specimen details and Appendix 3 for the alignment). Clade names are indicated to the right of the phylogenetic tree. The star corresponds to the monophyletic Scorzonera subg. Podospermum and the circle corresponds to S. subg. Pseudopodospermum. Squares next to two tip names correspond to accessions of species names that are newly synonymized with S. szowitzii in this study (LAC540 S. leptophylla; LAC529 S. stenocephala; see the taxonomic treatment for details).

In addition to achene morphology, a range of morphological features was examined for the identification key and taxonomic treatment below. Voucher information for specimens examined are provided for all species in Appendix 3; and the summary of morphological features (life cycle, plant height, root, stem, leaves, flowering capitula, fruiting capitula, achenes, pappus) for selected species in Scorzonera subg. Podospermum and S. subg. Pseudopodospermum are provided in Appendix 4.

Table 2.

Comparisons of achene features (achene surface, pubescence, carpopodium and pappus features) of representative species within Scorzonera subg. Podospermum and S. subg. Pseudopodospermum.

Discussion

Relationships within subtribe Scorzonerinae — The topology inferred in our phylogenetic analyses (Fig. 1) confirms the results of previous studies that showed Scorzonera s.l. is polyphyletic (Mavrodiev & al. 2004; Winfield & al. 2006; Zaika & al. 2020). Based on our molecular analysis, the Lasiospora clade is well-resolved as a lineage that is remote from the core of Scorzonera, in the sense of its type S. humilis, in accordance with Mavrodiev & al. (2004), Winfield & al. (2006) and Zaika & al. (2020). However, Zaika & al. revealed that S. villosa Scop. is included in this lineage, and they proposed the oldest generic name: Gelasia. Our molecular analysis showed a sister-group relationship between the genus Koelpinia (K. linearis Pall. and K. macrantha C. Winkl.) and the S. intricata clade (S. intricata and S. tortuosissima here) from S. sect. Intricatae (Boiss.) Lipsch. within S. subg. Scorzonera. Our findings are in agreement with Zaika & al (2019), who revealed a sister relationship between the S. polyclada clade (including S. intricata, S. longipapposa Rech. f. and S. polyclada from S. sect. Intricatae of S. subg. Scorzonera) and Koelpinia proposing the generic name Ramaliella for S. polyclada. The sister relationship between Epilasia and Tragopogon in Fig. 1 is also congruent with previous studies (Mavrodiev & al. 2006; Zaika & al. 2020).

The remainder of the ingroup forms two major well-supported clades: one including the Scorzonera humilis clade, S. purpurea and S. subg. Podospermum and the other including S. subg. Pseudopodospermum. Our phylogenetic analyses incorporated some rare species of S. subg. Podospermum (S. kandavanica Rech. f., S. persepolitana Boiss.) and S. subg. Pseudopodospermum (S. szowitzii, S. tunicata) that had never been sampled before. Scorzonera subg. Podospermum and S. subg. Pseudopodospermum are monophyletic according to our nrITS tree (Fig. 1). Based on our nrITS phylogenetic tree, the S. humilis clade containing S. humilis, the type of Scorzonera, was resolved as sister to a clade containing S. purpurea and S. subg. Podospermum. This supports the treatment of S. subg. Podospermum as a subgenus within the genus Scorzonera (Lipschitz 1964; Rechinger 1977; Kamelin & Tagaev 1986) in contrast to previous treatments that considered it as an independent genus (Candolle 1805; 1838; Cassini 1827; Dumortier 1827; Lessing 1832; Endlicher 1841; Grossheim 1949; Kuthatheladze 1978; Pignatti 1982; Tzvelev 1988; Nazarova 1997; Mavrodiev & al. 2004; Winfield & al. 2006; Greuter 2006+; Kilian & al. 2009a, 2009b+; Makbul & al. 2016). Scorzonera humilis, S. purpurea and species of S. subg. Podospermum are morphologically distinct. However, based on morphological observations by E.H., they share the following morphological characters: woody cylindric root (non-tuberous) and the presence of basal leaves with few and small cauline leaves.

Lipschitz and Krascheninnikov (Lipschitz 1935) described Scorzonera subg. Pseudopodospermum as a section within the genus Scorzonera containing 14 species. Later, Lipschitz (1964) changed its taxonomic rank to subgenus, which is widely accepted in the following floras: Flora URSS (Lipschitz 1964), Flora iranica (Rechinger 1977), and Flora of Iran (Safavi 2013). We took the descriptions of species within this subgenus from different regional Floras into account: Flora URSS (Lipschitz 1964), Flora iranica (Rechinger 1977), Flora of Iran (Safavi 2013) and from recent studies (Coşkunçelebı & al. 2015; Coşkunçelebı & al. 2016; Norouzi & al. 2016; Hatami & al. 2019), which included additional species that were not considered members of S. subg. Pseudopodospermum by Lipschitz (1964): S. aksekiensis A. Duran & M. Öztürk, S. elata Boiss., S. inaequiscapa Boiss., S. mucida, S. pachycephala Podlech & Rech. f., S. syriaca and S. tunicata. The topology inferred by our molecular analyses and morphological observations, in particular of the achene surface, supports a broader circumscription of S. subg. Pseudopodospermum, to include S. calyculata and S. papposa. Zaika & al. (2020) also included members of S. sect. Incisae Lipsch. and S. sect. Papposae Lipsch. & Krasch. in S. subg. Pseudopodospermum, which was treated at generic level, based on their analyses of nrITS and plastid data and carpological features.

The monophyly of clades corresponding to Scorzonera subg. Podospermum and S. subg. Pseudopodospermum was confirmed based on broad taxonomic sampling in Zaika & al. (2020) similar to previous studies that had less sampling (Mavrodiev & al. 2004). In the nrITS and plastid phylogenetic trees in Zaika & al. (2020), there is limited phylogenetic resolution within the Podospermum and Pseudopodospermum clades. Therefore, the fact that we did not include all species that were sampled in the phylogenetic analyses in Zaika & al. (2020) does not cause a bias in our phylogenetic analyses. Furthermore, we broadened the taxonomic sampling compared to Zaika & al. (2020): three species in S. subg. Pseudopodospermum (S. semicana, S. szowitzii and S. tunicata) and two species in Podospermum (S. kandavanica and S. persepolitana). Below, we discuss the results of our morphological analyses of Iranian species of S. subg. Podospermum and S. subg. Pseudopodospermum in the context of our nrITS tree (Fig. 1).

Scorzonera subg. Podospermum — The monophyly of Scorzonera subg. Podospermum (Fig. 1) morphologically corresponds to a combination of morphological characters containing pinnatifid leaves, phyllaries with a black corniculate projection (Fig. 4A), and the presence of a swollen carpopodium (Fig. 2A–J; Fig. 4B). Species of S. subg. Podospermum are mostly distributed in the Euro-Siberian region in the north of Iran, and in the Irano-Turanian region, particularly the Kurdo-Zagrosian zone in the west of Iran (Zohary 1973; Rechinger 1977; Safavi 2013). This subgenus is most common in highland regions (>1000 m a.s.l) of more or less wet meadows, ruderal sandy or gravelly soils, stony slopes, or wet corners of agricultural fields (based on observations by E.H. and M.M.; Fig. 4D–F).

Fig. 2.

Macro photographs of achenes of species within Scorzonera subg. Podospermum (A–J), S. purpurea (K, L), and S. subg. Pseudopodospermum (M–T). – Scale bar lengths are given in parentheses after figure letters. – Voucher locality, collection date, collector(s) and number, and herbarium code for each sample are given in parentheses after species names. – Scorzonera subg. Podospermum: A (2 mm) and B (1 mm): S. cana (Greece, Grevena, 2.1 km NE of Paliouria, 31 May 1990, Laubwald & al. 8941, B). – C (2 mm) and D (1 mm): S. kandavanica (Iran, N Iran, 6 km from Lowshan to Jirandeh, 20 May 2016, Mirtadzadini 2212, MIR). – E (2 mm) and F (2 mm): S. laciniata (Iran, Zanjan to Bijar, 22 May 2016, Mirtadzadini 2216, MIR). – G (5 mm) and H (1 mm): S. persepolitana (Iran, Esfahan, near Delijan on clay hill, 20 May 2010, Mirtadzadini 2218, MIR). – I (2 mm) and J (1 mm): S. songorica (Iran, Kerman, Sardu, 24 Jun 2017, Hatami & al. 2247, MIR). – K (2 mm) and L (1 mm): S. purpurea (Germany, Frankenhausen, Jun 1994, W. Becker s.n., B). – S. subg. Pseudopodospermum: M (5 mm) and N (1 mm): S. papposa (Palestine, Shefela, 23 Mar 2010, Ristow 34/10, B). – O (5 mm) and P (2 mm): S. phaeopappa (Iran, Kordestan, Marivan, Mirtadzadini 2162, MIR). – Q (5 mm) and R (2 mm): S. raddeana (Iran, Fars, between Estahban and Niriz, 3 May 2016, Mirtadzadini & al. 2157, MIR). – S (5 mm) and T (2 mm): S. calyculata, Iran, Between Karaj and Qazvin, 22 Jun 2011, Mirtadzadini 2219, MIR). – Macro photographs produced using a stereomicroscope (Olympus SZX16) equipped with DP72 (a 12.5 megapixel cooled digital colour camera) at the Botanic Garden and Botanical Museum Berlin.

All species of Scorzonera subg. Podospermum that are distributed in Iran (Rechinger 1977; Safavi 2013) are included in our phylogenetic analyses, with the exception of S. lachnostegia, a rare species, which was sampled by Zaika & al. (2020) and resolved in a polytomy within the Podospermum clade in their nrITS tree. In spite of sampling multiple accessions of different species in our phylogenetic studies, the interspecific relationships were poorly resolved based on nrITS sequences, similar to Zaika & al. (2020) (Fig. 1). However, morphological characters, particularly achene features, were informative for clarifying the interspecific circumscription of this subgenus; see taxonomic treatment below (Table 2; Fig. 2A–J).

Within the Scorzonera songorica clade (Fig. 1), S. meshhedensis (Rech. f.) Rech. f. and S. songorica are morphologically similar to each other in possessing basal or cauline simple leaves (see Fig. 4E for S. meshhedensis). The pappus of these two species can be easily detached; a characteristic that is unique to this lineage among all Scorzonera species sampled in this study. The type of S. subg. Podospermum is S. laciniata L. (Lipschitz 1964). It is morphologically distinctive in having the longest carpopodium (6–7 mm) with the highest ratio of carpopodium length to total achene length among all species of S. subg. Podospermum (Table 2; Fig. 2E, F). The S. cana clade received strong statistical support (0.99 PP, 91 JK, 80 BS), but its internal relationships are unresolved due to the low variation of the nrITS region. However, species within the S. cana clade can be distinguished based on diagnostic morphological characters (Fig. 4C, D; Appendix 4); see taxonomic treatment below.

Previous treatments placed Scorzonera purpurea in S. subg. Podospermum (Candolle 1838; Kamelin & Tagaev 1986), whereas others treated it as a member of S. subg. Scorzonera sect. Purpureae Lipsch. (Lipschitz 1964; Zaika & al. 2020). The achenes of S. purpurea possess pale and tube-like swollen bases (Lipschitz 1964; Chater 1976), which is morphologically similar to the carpopodium of species in S. subg. Podospermum (Fig. 2K, L). However, the swollen bases are entirely fertile in S. purpurea compared to infertile carpopodia in species of S. subg. Podospermum. Furthermore, members of S. subg. Podospermum differ from S. purpurea in having pinnatifid leaves and phyllaries with corniculate projections (Fig. 4A); in contrast, S. purpurea has simple leaves and no corniculate projections. Although a sister relationship between S. purpurea and S. subg. Podospermum is well-supported in our nrITS analyses (95 PP; Fig. 1), we propose to maintain the treatment of this species outside of S. subg. Podospermum based on morphological differences observed here and in accordance with previous studies (Lipschitz 1964; Zaika & al. 2020).

Scorzonera subg. Pseudopodospermum — Our nrITS analyses included all species of S. subg. Pseudopodospermum distributed in Iran (Rechinger 1977; Safavi 2013; except S. syriaca and S. turkeviczii); as well as S. mollis, which represents the type of this subgenus (Lipschitz 1964). Members of this subgenus are widely distributed in dry to humid areas of the Irano-Turanian region (Zohary 1973; Rechinger 1977; Safavi 2013) and mostly grow in highland regions (>1000 m a.s.l) on sandy, clay or gravelly steppe hills, stony and rocky slopes, limestone hills in grassy steppes, stony semi-deserts, and open grassland (based on observations by E.H. and M.M.).

Scorzonera subg. Pseudopodospermum is monophyletic based on our nrITS phylogeny (Fig. 1) and the morphological observations in this study suggest that a sculptured (as opposed to smooth) achene-wall surface provides an exclusive synapomorphy for this lineage (Table 2). Within S. subg. Pseudopodospermum, the type of sculpturing on the achene surface is variable among species, and can be muricate, tuberculate, lamellate or verrucose (Table 2; Fig. 2M–T). Moreover, we include sequences of species from Iran that also have sculptured achenes from S. sect. Incisae and S. sect. Papposae to clarify their phylogenetic position and compare their morphological characters with members of S. subg. Pseudopodospermum. The S. mollis clade in our nrITS tree (Fig. 1) represents S. subg. Pseudopodospermum in its traditional circumscription (Lipschitz 1964; Rechinger 1977; Fig. 1). Based on our nrITS tree, S. calyculata (S. subg. Scorzonera sect. Incisae) and S. papposa (S. subg. Scorzonera sect. Papposae) are sister to the S. mollis clade with full statistical support and together they form a monophyletic clade. In Iran, S. sect. Incisae is represented by S. calyculata, and S. sect. Papposae by S. ovata Trautv., S. papposa and S. paradoxa Fisch. & C. A. Mey. ex DC.; we include all species in our morphological studies and the taxonomic treatment (see below). Morphological comparisons revealed that the sculptured achene surface supports a close relationship between the S. mollis clade and S. calyculata and all members of S. sect. Papposae in Iran, in support of the close relationship observed in the nrITS tree (Table 2; Fig. 1). We therefore propose a broader circumscription of S. subg. Pseudopodospermum to include members of S. sect. Incisae and S. sect. Papposae (S. calyculata and S. papposa, S. ovata and S. paradoxa; Table 1). Members of S. sect. Incisae and S. sect. Papposae outside of Iran also have sculptured achene surfaces (Lipschitz 1964; Rechinger 1977); it would therefore be beneficial incorporate all members of these sections and S. subg. Pseudopodospermum into future phylogenetic studies. Based on previous treatments (Lipschitz 1964; Rechinger 1977), members of S. sect. Incisae and S. sect. Papposae were morphologically distinguishable within S. subg. Scorzonera according to the absence of a carpopodium. Furthermore, we found that the carpopodium is only sometimes swollen among members of the S. mollis clade. The newly circumscribed S. subg. Pseudopodospermum in our study contains species either without a carpopodium or with a carpopodium that may be conspicuous or inconspicuous. Our study therefore suggests that the presence or absence of carpopodia is not a diagnostic character for intersubgeneric classification within the genus Scorzonera, in accordance with Haque & Godward (1984) and Zaika & al. (2020).

The Scorzonera mollis clade within S. subg. Pseudopodospermum (Fig. 1) contains a number of well-supported subclades, which we now discuss. In the nrITS analyses, we include sequences of an accession that we identified as S. szowitzii (LAC530) and accessions that corresponded of S. leptophylla (DC.) Krasch. & Lipsch. (LAC540) and S. stenocephala Boiss. (LAC529) according to species descriptions in Flora iranica (Fig. 1; Rechinger 1977). All three accessions were resolved in a strongly supported clade (0.99 PP; Fig. 1). We also studied morphological characters of multiple populations of S. leptophylla, S. stenocephala and S. szowitzii from several geographic regions in the field and herbaria (Appendix 1; Appendix 2). We observed extreme phenotypic plasticity among these species in vegetative characters including plant height, leaf width, possessing an entire or undulate leaf margin, and glabrous or tomentose indumentum (Appendix 4). All species are indistinguishable according to capitula length, number of florets in each capitulum, and achene and pappus features (Table 2; Appendix 4). Based on overlapping morphological characters and nomenclatural priority, we treat them as S. szowitzii (see taxonomic treatment below).

Three accessions of Scorzonera raddeana in the nrITS analyses show intraspecific variation, which is consistent with morphological variation; length and width of fruiting capitula and width of leaves are variable between individuals; LAC540 and LAC534 were distinct from LAC513 (see taxonomic treatment below). However, all individuals of S. raddeana samples in the nrITS tree have the typical S. raddeana achene and pappus type (Table 2). Scorzonera phaeopappa is resolved as sister to S. semicana with high statistical support (Fig. 1). Based on our observations, a combination of the following characters represents the synapomorphy for the S. phaeopappa and S. semicana subclade: achenes with a swollen carpopodium and five conspicuously long scabrous bristles in the pappus (Table 2; Fig. 2O; Fig. 3Q; Appendix 4). Another strongly supported clade (1 PP, 87 JK, 90 BS) contains S. mucida and S. tunicata with similar morphological characters (Table 2; Appendix 4). We consider them as separate species based on differences in achene and phyllary characters (see Notes under S. mucida). Hatami & al. (2019) recently found that chromosome number and ploidy are also different between these species: tetraploid and 2n = 28 in S. mucida in contrast to diploid and 2n = 14 in S. tunicata. The placement of the diploid S. tunicata in a clade with the tetraploid S. mucida in the nrITS tree may suggest that S. tunicata represents a parent of S. mucida. Further studies are required to determine if S. mucida is an auto- or allotetraploid. It was not possible to include other species from S. subg. Scorzonera that have sculptured achenes in our molecular studies because they are rare in Iran, including S. helodes Rech. f. and S. limnophila Boiss. (S. sect. Dimophopapposae Lipsch.) and S. nivalis Boiss. & Hausskn. (S. sect. Foliosae (Boiss.) Lipsch.) (Rechinger 1977; Kamelin & Tagaev 1986). It would be beneficial to include these rare Scorzonera species that also have sculptured achenes in future studies, in order to explore their relationships with members of S. subg. Pseudopodospermum.

Taxonomic treatment of Scorzonera subg. Podospermum and S. subg. Pseudopodospermum in Iran

Habit — Most taxa in Scorzonera subg. Podospermum and S. subg. Pseudopodospermum are perennial, possessing a taproot or tuberous root with developed lateral roots (Appendix 4). Morphology and placement of tubers are not used here as diagnostic features because they are highly variable within species depending on the ecological conditions. Tubers may be cylindric or spherical and can be deep underground or near to the surface. Some species within S. subg. Podospermum possess a caudex, which is the persisting woody axis of the (former) rosette shoot (Beentje 2010) that may be branched or unbranched, characterized by densely set leaf scars or leaf remains; it is often dark brown and transversely rough with numerous dry and membranous scales at the apex. With the exception of two species in S. subg. Podospermum, all species of S. subg. Podospermum and S. subg. Pseudopodospermum are perennial herbs with either a caespitose, subcaespitose or caulescent habit (Fig. 4: Fig. 5). In caespitose perennials, the flowering stems arise from radical rosettes and are often scape-like, thus leafless or bearing few reduced leaves or bracts (Fig. 4C, F; Fig. 5A, D, E). Caulescent perennials usually have developed cauline leaves and a branched flowering stem (Fig. 4D; Fig. 5B). Two species (S. laciniata and S. songorica from S. subg. Podospermum) are biennial with a thin taproot, typically lacking remains of previously withered leaves.

Leaves — Basal and cauline leaves vary from undivided to deeply pinnatisect among species of Scorzonera subg. Podospermum and S. subg. Pseudopodospermum (Fig. 4; Fig. 5). Undivided leaves may be linear, lanceolate or ovate and the leaf margin may be flat or undulate (Fig. 5F, G). Pinnatifid leaves are pinnately divided but not all the way down to the rachis, whereas pinnatisect leaves (Fig. 4C, D) are deeply divided reaching the rachis (Allaby 1992). Leaf segments may be linear, oval-lanceolate or orbicular. The leaves can be sessile or with a long petiole-like portion usually with an enlarged base.

Capitula and phyllaries — The length and width of full-flowering and fruiting capitula were examined from herbarium samples. Length of capitulum was measured from the base of the longest innermost phyllary to the apex (Appendix 4). Phyllaries are always herbaceous, usually with a scarious margin; the width of the margin varies depending on the species. Small black spiny appendages occur on the apex of phyllaries only in members of Scorzonera subg. Podospermum (Fig. 4A).

Achenes — Achenes are sometimes ribbed and the ribbing depth varies ranging from subterete to sulcate. The ribs are either smooth (Scorzonera subg. Podospermum; Fig. 2A–J) or with tuberculate, lamellate or verrucose sculptures (S. subg. Pseudopodospermum; Fig. 2M–T). Species in S. subg. Pseudopodospermum always have glabrous achenes, however in Podospermum they can be glabrous or hairy (Fig. 2). The achene features are often not fully expressed in the innermost achenes of a capitulum; therefore, we only examine the outermost achenes in this study.

Fig. 3.

Macro photographs of achenes of species within Scorzonera subg. Podospermum and S. subg. Pseudopodospermum. – Scale bar lengths are given in parentheses after figure letters. – Voucher locality, collection date, collector(s) and number, and herbarium code for each sample are given in parentheses after species names. – A (2 mm) and B (1 mm): S. armeniaca (Iran, Azerbaijan, 36 km from Ahar to Tabriz, alt. 1554 m, 18 Jun 2015, Mirtadzadini 2239, MIR). – C (2 mm) and D (1 mm): S. grossheimii (Iran, Gorgan, Almeh, alt. 1500–1800 m, 8–10 Jun 1975, Rechinger 53108, B). – E (5 mm) and F (1 mm): S. luristanica (Iran, Kermanshah, between Quriqala and Paweh, 34°57′28.9″N, 46°26′46.5″E, alt. 1246 m, Mirtadzadini & al. 2213, MIR). – G (2 mm) and H (1 mm): S. meshhedensis (Iran, Kerman to Bam road, Golbaf, near Abolfazl mosque, 14 Apr 2016, Samareh 2244, MIR). – I (2 mm) and J (1 mm): S. meyeri (Iran, Khorassan, SW of Bojnurd, Salook mt., 11 Aug 1994, Zangooei & al. 24493, FUMH). – K (2 mm) and L (1 mm): S. ovata (Iran, Baluchistan, NE of Bazman, Siah Band mt. range, 28 Apr 2017, Mirtadzadini 2322, MIR). – M (5 mm) and N (2 mm): S. mucida (Iran, Kerman, Deh Bala, Bordbar 3000, MIR). – O (2 mm) and P (2 mm): S. mollis subsp. mollis (Greece, Kilkis, E of Polykastro 41°00′21″N, 22°38′12″E, 21 Apr 2006, Willing 153 107, B). – Q (5 mm) and R (2 mm): S. semicana (Turkey, Mardin, 5 km E of Mardin, 1100 m, 25 May 1957, Davis & Hedge D.28603, W). – S (2 mm) and T (2 mm): S. szowitzii (Iran, East Azerbaijan, N of Tabriz, Eynali mountain, 10 Jun 2013, Ebrahimi 2989, MIR). – U (5 mm) and V (2 mm): S. tunicata (Iran, Khorassan, 32 km from Birjand to Qa'en, alt. 1991 m, 7 May 2015, Mirtadzadini 2158, MIR). – W (5 mm) and X (2 mm): S. turkeviczii (Iran, 7 km NE of Karaj toward Tschalus, after Sarv-e Dar village, 18 May 2016, Mirtadzadini 2160, MIR). – Macro photographs produced using a stereomicroscope (Olympus SZX16) equipped with DP72 (a 12.5 megapixel cooled digital colour camera) at the Botanic Garden and Botanical Museum Berlin.

Fig. 4.

Photographs of representatives of Scorzonera subg. Podospermum in the field. For each image the locality and date of the photograph are given, and where applicable voucher data are given (collector and number, herbarium code). – A: black corniculate appendages on phyllaries (see voucher under E); B: swollen carpopodium on achenes (see voucher under D); C: S. armeniaca, NW Iran, Tabriz, Varzeqan, 5 Jun 2017; D: S. persepolitana, Iran, Esfahan, near Delijan on clay hill, 20 May 2010, Mirtadzadini 2218, MIR; E: S. meshhedensis, Iran, Kerman, Golbaf, 14 Apr 2016, Mirtadzadini 2244, MIR; F: S. meyeri, Iran, Zanjan to Abbar, May 2017. – Photographs by M. Mirtadzadini (A, B, D), A. Ebrahimi (C, F; Tehran university, Tehran, Iran), M. Samareh (E; Shahid Bahonar University of Kerman, Kerman, Iran).

Fig. 5.

Photographs of representatives of Scorzonera subg. Pseudopodospermum in the field. For each image the locality and date of the photograph are given. – A: S. raddeana, Iran, Kerman, Sirch, 22 Apr 2015; B: S. phaeopappa, Iran, Kordestan, Baneh, May 2019 (B1: habit; B2: inflorescence); C: S. calyculata, N Iran, Gilan, Lowshan, May 2016 (C1: habit; C2: inflorescence); D: S. ovata, SE Iran, Baluchistan, between Iranshahr and Sarbaz; E: S. paradoxa, Iran, Fars, Eqlid, Apr 2007; F: S. mucida, Iran, Kerman, Mahan, May 2019 (F1: habit; F2: inflorescence); G: S. mucida, Iran, Kerman, Kuhbanan, Jun 2017. – All photographs by M. Mirtadzadini.

Carpopodium — The carpopodium refers to the basal prolongation of the achene wall forming a hollow tube-like sterile foot, which may be swollen or not in comparison to the fertile portion (Mukherjee & Nordenstam 2004). Species within Scorzonera subg. Podospermum almost always have a conspicuous carpopodium (Fig. 2A–H). In contrast, within S. subg. Pseudopodospermum, the carpopodium may be absent (e.g. S. calyculata [Fig. 2S, T], S. ovata, S. papposa [Fig. 2M, N] and S. paradoxa), or inconspicuous to conspicuous (Fig. 2O–R; Fig. 3K, L). The size and presence of the carpopodium is important for interspecific delimitation but not informative at intersubgeneric level.

Pappus — Within Scorzonera subg. Podospermum and S. subg. Pseudopodospermum, the setaceous pappus is typically persistent, but in two cases (S. meshhedensis and S. songorica) the bristles can be easily detached (S. songorica in Fig. 2J). Pappus bristles are entirely plumose in S. ovata (Fig. 3K, L) and S. papposa (Fig. 2M), in contrast to pappus bristles that are plumose in the proximal part and naked or scabrous at the distal part for all other species in both subgenera (Table 2; Fig. 2; Fig. 3). The pappus bristles on a single achene can be of equal or unequal lengths. Some species have only five bristles that are conspicuously longer and darker than the others (e.g. S. phaeopappa; Fig. 2O).