TAXON 66 (3) • June 2017: 675–703 Kilian & al. • Diversification of the Lactucinae (Compositae)

Sub-Paratethyan origin and Middle to Late Miocene principal diversification of the Lactucinae (Compositae: ) inferred from molecular phylogenetics, divergence-dating and biogeographic analysis Norbert Kilian,1* Alexander Sennikov,2 Ze-Huan Wang,3 Birgit Gemeinholzer4 & Jian-Wen Zhang3 1 Botanic Garden and Botanical Museum Berlin, Freie Universität Berlin, Königin-Luise-Str. 6–8, 14195 Berlin, Germany 2 Botanical Museum, Finnish Museum of Natural History, P.O. Box 7, 00014 University of Helsinki, Finland; and Herbarium, Komarov Botanical Institute of Russian Academy of Sciences, Prof. Popov str. 2, 197376 St. Petersburg, Russia 3 Key Laboratory for Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, Yunnan, People’s Republic of China 4 AG Spezielle Botanik, Justus-Liebig-Universität Giessen, Heinrich-Buff-Ring 38, 35392 Giessen, Germany * Sequence of authors determines credit Author for correspondence: Norbert Kilian, [email protected] ORCID AS, http://orcid.org/0000-0001-6664-7657

DOI https://doi.org/10.12705/663.9

Abstract The Lactucinae or alliance include approximately 200 species distributed across the Northern Hemisphere and Africa. They were not recognised as a separate lineage until the late 20th century and their circumscription is still not fully settled. The generic classification of no other group of the Cichorieae has faced as many controversies as the Lactuca alliance and competing taxonomies coexist. This paper provides the first molecular phylogeny of the subtribe on a global scale as a major step towards its revised systematics. The sampling includes almost 60% of the species-level diversity and spans all species groups. Two datasets were created, one including the nrDNA ITS region, the other five concatenated non-coding plastid DNA loci. Maximum parsimony, maximum likelihood and Bayesian inference were used to produce a robust phylogenetic backbone. The diversification and expansion of the Lactucinae in a geohistorical context was reconstructed by estimating the age of their lineages using relaxed molecular clock dating and by inferring the ancestral areas using Bayesian binary analysis. The redelimited monophyletic Lactucinae are composed of seven lineages that also include , which is confirmed to have a single species, P. purpurea. The positions of two further lineages shift between Lactucinae and Crepidinae in the nuclear and plastid DNA phylogenies. Incongruence between the phylogenies suggests events of ancient reticulation or incomplete lineage sorting in the formation of these latter two lineages and in two of the seven other Lactucinae lineages. The phyloge- netic results show a dilemma for Lactucinae systematics: most generic concepts proposed to date are highly artificial but the resolved phylogenetic lineages do not constitute practicable taxonomic entities with our current knowledge. Diversification of the subtribe is inferred to have taken place since the Middle Miocene. Biogeographic analysis proposes that the clade originated in the mountains of the landmass mediating between the European and Asian continents and delimited in the south by the Tethys Sea and in the north by the Paratethys Sea. Several independent migrations have occurred into various parts of Asia, Europe, tropical Africa and North America.

Keywords ; biogeography; Lactuca alliance; Miocene; phylogenetics; uncorrelated relaxed molecular clock

Supplementary Material DNA sequence alignment files are available in the Supplementary Data section of the online version of this article at http://ingentaconnect.com/content/iapt/tax

INTRODUCTION Cichorium L. represent distantly related lineages, which are classified as subtribes Lactucinae and Cichoriinae, respectively, The Asteraceae as the largest family of flowering in the recent of the tribe by Kilian & al. (2009a), who provide only a handful of important food crops (Dempewolf recognised 11 subtribes. The Lactucinae include approximately & al., 2008). Among them, the Cichorieae are the tribe with 200 species (Kilian & al., 2009b–), form the fourth-largest the strongest representation, contributing (Lactuca subtribe and are distributed across the Northern Hemisphere sativa L.), endive (Cichorium endivia L.) and chicory (Cichorium and Africa. With respect to systematics, the Lactucinae are one intybus L.). In this tribe, the two genera Lactuca L. and of the least known subtribes of Cichorieae.

Received: 27 Jun 2016 | returned for (first) revision: 5 Oct 2016 | (last) revision received: 6 Feb 2017 | accepted: 7 Feb 2017 || publication date(s): online fast track, n/a; in print and online issues, 23 Jun 2017 || © International Association for Plant Taxonomy (IAPT) 2017

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Recognition of Lactuca and its allies as a lineage of their (e.g., Cicerbita Wallr. in the sense of Wallroth, 1822). The other own is a rather recent achievement, gained by Bremer (1994) is diagnosed by a very limited set of one or a few characters through morphological phylogenetics. Throughout the 19th and (e.g., Cicerbita in the sense of Beauverd, 1910). In this way 20th centuries, the genera of the Lactuca alliance were seen and along with the progressive inventorying of the subtribe’s as parts of a large subtribe Crepidinae, alternatively named species diversity in its wide distribution area, the number of de- as Lactucinae, or of a corresponding entity (Candolle, 1838; scribed (heterotypic) genera rose from 4 in 1800, to 11 in 1900 Hoffmann, 1890–94; Stebbins, 1953; Jeffrey, 1966). Within up to 18 in 2015 (Kilian & al., 2009b–). During this time, com- this subtribe, especially Lactuca in the tradition of Bentham peting classifications have coexisted. Consequently, current (1873) and Hoffmann (1890–94) embraced an extraordinarily taxonomic treatments, exclusively available in the frame of, or wide range of taxa, spanning members of several of the modern preparatory for, Floras, usually greatly vary in the number and subtribes. Bremer (1994) inferred this large subtribe to be poly- concepts of the genera recognised. Taxonomic syntheses on a phyletic, with the three alliances of Lactuca, L. and global scale, even for single genera, are lacking altogether and Hieracium L. not being closely related to Crepis L. and its rendered almost impossible as long as the generic delimitation allies. Consequently, he reclassified all of them as separate is entirely unsettled. subtribes. His morphology-based results were confirmed by A first molecular phylogenetic analysis spanning the a phylogenetic analysis using chloroplast DNA restriction site Lactucinae but focussed on its Sino-Himalayan centre of di- variation (Whitton & al., 1995) and subsequently by analy- versity (Wang & al., 2013) has preceded this paper. Based on ses based on the nuclear ribosomal DNA internal transcribed both nuclear and plastid DNA datasets, Wang & al. (2013) pro- spacer (nrITS) region (Kilian & al., 2009a; Tremetsberger & vided evidence that the monophyletic core of the subtribe is al., 2012; Fernández-Mazuecos & al., 2016). The last analyses composed of six lineages with strong statistical support, which revealed that over 80% of the tribe’s species diversity is found they recognised at generic rank: one then unnamed, the others in two of the five main clades of the Cichorieae. The larger of being Cicerbita, Lactuca, Melanoseris Less., Notoseris C.Shih the two, home of two-thirds of the species diversity, includes, and Paraprenanthes C.Shih. Two early diverging lineages were besides the Lactucinae, the Crepidinae (s.str.), Chondrillinae, incongruent between nuclear and plastid data: Prenanthes pur- Hyoseridinae and Hypochaeridinae, and is addressed in the purea L. and Faberia, which is of hybrid origin. They were following as the “C2H2L clade”. only resolved within Lactucinae in the plastid DNA tree or the While delimitation of the Lactucinae from the other sub- nrITS tree, respectively. tribes of the C2H2L clade is rather straightforward, disentan- The case of the isolated Prenanthes purpurea, which is gling Lactucinae and Crepidinae morphologically has proven sister to the core Lactucinae in the plastid DNA phylogeny more difficult. Accordingly, the classification of the Lactucinae (Wang & al., 2013), appears puzzling. While this sister rela- provided by Bremer (1994) and largely maintained by Lack tionship is as expected, its clustering with representatives of (2006) has since received emendations chiefly concerning the Hypochaeridinae in the nrITS phylogenies (Kilian & al., the prenanthoid (Kilian & Gemeinholzer, 2007; Kilian & al., 2009a; Wang & al., 2013; Fernández-Mazuecos & al., 2016) 2009a) and habitually convergent alpine taxa (Kilian & al., strongly contradicts the morphological and cytological data 2009a; Zhang & al., 2011a, b). The scarcity of non-homoplastic (Wang & al., 2013). It arouses suspicion that the latter results morphological synapomorphies for the lineages classified as may be an artefact, perhaps being caused by long-branch attrac- subtribes (Bremer, 1994) is assumed to have a cause in the tion (Bergsten, 2015) due to a sketchy sampling of taxa of the sudden and rapid diversification of the two largest main clades early-​diverging lineages of the C2H2L clade in general, in of the Cichorieae in the Miocene, which frequently led to par- particular of the Lactucinae. allel character evolution in the diversifying lineages (Kilian This paper, continuing the investigation with the same set & al., 2009a; Tremetsberger & al., 2012). Ancient reticulation of markers as used by Wang & al. (2013), for the first time pro- between the diversifying subtribal lineages also appears to have vides a global perspective of the Lactucinae phylogeny and the played a role but may not usually be as unambiguously detecta- delimitation of the subtribe. Our sampling includes almost 60% ble as in the case of Faberia Hemsl., where it is accompanied by of the Lactucinae species worldwide, covers all species groups genome duplication (Liu & al., 2013; Liu & Yang, 2014; Wang and includes all species providing types of generic names of & al., 2014). Neither morphology nor molecular phylogeny so the subtribe in its widest sense. In particular, we include the far could provide unequivocal evidence for the classification of large SW to Central Asian portion of the subtribe, together with Faberia in either the Lactucinae or Crepidinae, its placement in the tropical African members. The latter were recently also the Lactucinae by Wang & al. (2013) therefore being inferred addressed in a molecular phylogenetic investigation of Lactuca chiefly from its position in the nuclear DNA tree considered by Wei & al. (2016, 2017), but with rather limited sampling. as the more reliable indication of the species tree. Wang & al. (2013) strikingly illuminated that morpholog- Shallowness of morphological discontinuities hampers ical features believed to be diagnostic for generic entities are even more the generic classification of the Lactuca alliance. homoplastic, with the effect that species or species groups in Supraspecific taxonomy of the Lactuca alliance in the last 250 one of the lineages often share more similarities with taxa in years has oscillated between two contrary kinds of generic other lineages than in the same lineage. The global perspec- concepts. One recognises intuitive gestalt or appearance types, tive in this paper will conclude the reconstruction of the phy- which are rather difficult to define by diagnostic characters logenetic lineages within the Lactucinae. We have, however,

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postponed drawing taxonomic conclusions, in favour of our as well as material received from collections and/or the per- in-depth study of the character evolution in the subtribe to be sonal herbaria by courtesy of Hossein Akhani (Tehran, Iran), completed prior to, and fundamental for, a generic reclassifi- Robert von Blittersdorff (Frankfurt a. M., Germany), Ralf cation and species inventory of the subtribe. Hand (Berlin, Germany), Magda Bou Dagher Kharrat (Beirut, Wang & al. (2013) detected cases of hard topological incon- Lebanon), Georg and Sabine Miehe (Marburg, Germany), gruence within the core Lactucinae (Wendel & Doyle, 1998) Marjan Niketić and Gordana Tomović (Belgrade, Serbia), between phylogenetic reconstructions based on nrITS and plas- Gerald Parolly (Berlin, Germany), Eckhard von Raab-Straube tid DNA, for which non-biological factors could be excluded as (Berlin, Germany), Michael Ristow (Potsdam, Germany) and an explanation. To what extent either hybridisation or incom- Alexander Sukhorukov (Moscow, Russia). Our sampling for plete lineage sorting are the causes, has not been analysed yet the molecular analyses presented here comprises samples of and will be the subject of another paper. 137 species, of which 116 are species of subtribe Lactucinae In this paper, we use the phylogenetic reconstruction of and 21 form the outgroup. More than 700 individual marker the subtribe to investigate for the first time the biogeographic sequences were newly generated and are first published in this history of the Lactucinae. The key to this is the knowledge study (see Appendix 1). The 116 ingroup species represent all of the age of the lineage. Age estimations are challenging as known species groups of the subtribe worldwide, altogether such (Renner, 2005; Ho & Phillips, 2009; Duchêne & al., 2014; almost 60% of the estimated total number of approximately Hipsley & Müller, 2014) but notoriously difficult in groups 200 species (including all types of generic names) of the sub- such as Asteraceae where fossil records are scarce (Funk & al., tribe (Kilian & al., 2009b–). The outgroup taxa were selected 2009). Particular care is required if secondary calibrations are to represent the related subtribes Crepidinae, Hyoseridinae the last resort (Schenk, 2016). Summarising the available data, and Hypochaeridinae of the C2H2L clade of the Cichorieae Funk & al. (2009) concluded that all tribes of the Asteraceae according to molecular analyses by Kilian & al. (2009a) and are estimated to have evolved around 25–35 million years ago Tremetsberger & al. (2012), plus a representative (Scorzonera (mya) and the family around 41–50 mya. Tremetsberger & al. hispanica L.) of the earlier diverging Scorzonerinae clade. (2012) provided the first estimates of the age of the Cichorieae The last was used to root the tree. To account for the still and their subtribes based on pollen fossil records, the phy- unsettled taxonomic delimitation between Lactucinae and logenetic placements of which were thoroughly discussed. Crepidinae, we selected representatives of the major lineages Other works on Cichorieae provided age estimates focussing of the Crepidinae based on Zhang & al. (2011a) and our own on selected generic lineages and using largely the same fossil unpublished studies. records (Zhang & al., 2011a; Fernández-Mazuecos & al., 2016) DNA isolation, amplification and sequencing. — Extraction or secondary calibration based on them (Tremetsberger & al., of DNA and amplification of markers followed the protocols 2016). The divergence of the Lactucinae has so far been esti- described by Wang & al. (2013). One nuclear locus and five mated to have taken place 4–15 mya, but they were represented non-coding loci of the chloroplast genome were used as mark- in these studies by a few taxa only. Within the Cichorieae as a ers. The nrITS region (ITS1, 5.8S rDNA, ITS2) was amplified tribe mainly distributed in the temperate zone of the Northern using either the primer combinations ITS4/ITS5 (White & al., Hemisphere, the Lactucinae have a wide, Eurasian, African 1990) or ITSA/ITSB (Blattner, 1999). The chloroplast markers and North American distribution (Kilian & al., 2009a). An were amplified using the following primers: (1) the petB-petD ancestral area analysis has been carried out to date neither for spacer plus petD intron were co-amplified with the universal the tribe nor for the Lactucinae. primers PIpetB1411F/PIpetD738R (Löhne & Borsch, 2005); The aims of this paper are therefore threefold: (1) to recon- (2) the psbA-trnH spacer with the universal primers psbAF/ struct the phylogeny of the Lactucinae based on a worldwide trnHR (Sang & al., 1997); (3) the 5′trnL(uaa)-trnF spacer with sampling covering all species groups based on nuclear and the universal primers trnC/trnF (Taberlet & al., 1991); (4) the plastid DNA datasets; (2) to reassess the phylogenetic position rpl32-trnL(uag) spacer with the primers rpl32-F/trnL(UAG) of the isolated Prenanthes purpurea and other prenanthoid taxa and (5) the 5′rps16-trnQ(uug) spacer with the primers rps16x1/ of unclear subtribal affinity; (3) to gain first insights into the trnQ(UUG) (Shaw & al., 2007). The purified amplification diversification and expansion of the Lactucinae in a geohis- products were earlier directly sequenced on a Beckman- torical context by divergence time estimation for the lineages Coulter CEQ 8000 sequencer or more recently sequenced of the subtribe through relaxed molecular clock dating and by via StarSeq (Mainz, Germany) or Macrogen (Seoul, South ancestral area estimation, analysing nuclear and plastid DNA Korea) with the same primers as used for amplification. The datasets. newly generated sequences were submitted to the International Nucleotide Sequence Database Collaboration (INSDC, includ- ing GenBank/EMBL/DDBJ), see Appendix 1. MATERIALS AND METHODS Sequence alignment and coding of length mutational events. — The boundaries of the nrITS region (ITS1, 5.8S Plant material and sampling. — Our study is based on rDNA, ITS2) and the petD marker (petD intron, 5′petB-petD herbarium material from the herbaria B, BEO, BEOU, BR, spacer) were defined according to Goertzen & al. (2003) CAS, E, GAT, GFW, H, IRAN, KUN, L, LE, MW, PRC, US, and Löhne & Borsch (2005), respectively. The boundaries W and WAG, including personal collections by the authors, of the other markers were taken from, and their designation

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corresponds to, the annotated complete chloroplast genome consensus tree was calculated from the most parsimonious sequence of Lactuca sativa (INSDC: DQ383816) by Timme trees obtained. Jackknife (JK) support values for the nodes & al. (2007). found by the MP analysis were calculated in PAUP applying the The ITS sequences were aligned automatically using optimal jackknife parameters according to Farris & al. (1996) Muscle v.3.8.31 (Edgar, 2004) and checked in PhyDE v.0.9971 and Müller (2005b; 10,000 jackknife replicates using the TBR (Müller & al., 2010). No sequence part needed to be excluded branch swapping algorithm with 36.788% of characters deleted from the analysis because of alignment uncertainties. The and one tree held during each replicate). alignment had a length of 687 characters. Bayesian analyses were performed using the MPI ver- The plastid sequences were first automatically aligned us- sion of MrBayes v.3.2 (Ronquist & al., 2012) installed on the ing Muscle, then adjusted manually to a motif-based alignment Soroban high-performance computing system at the Scientific in PhyDE following the criteria outlined by Kelchner (2000), Computing Service of the Freie Universität Berlin. Optimal nu- Borsch & al. (2003) and Löhne & Borsch (2005). Length varia- cleotide substitution models that best fit the data were searched ble mononucleotide stretches (microsatellites) and hypervaria- with MrModeltest v.2.3 (Nylander, 2004) separately for each ble sections were excluded from the final alignment because of of the three loci ITS1, 5.8S, ITS2 of the nrITS dataset and their homology uncertainties, and inversions were re-inverted each of the five plastid loci, and selected following the Akaike prior to phylogenetic reconstruction. The final alignment had information criterion (AIC). The best-fit models chosen were a length of 5795 characters. GTR + I + G for ITS1, GTR + G for ITS2, SYM + I + G for 5.8S, Two separate datasets were built: one of the nuclear rDNA GTR + I for the petD region, and GTR + G for the other four ITS region, the other of the five concatenated non-coding plastid loci. The F81 model implemented for restriction sites chloroplast DNA regions petD, psbA-trnH, 5′trnL(uaa)-trnF, and other binary data was applied to the coded indels and inver- rpl32-trnL(uag) and 5′rps16-trnQ(uug). In ca. 7 % of the samples sions, with the coding bias option “variable” selected. The data­ of the plastid DNA matrix not all five loci were successfully sets were partitioned into three partitions for the nuclear loci, sequenced. Such incomplete data for single samples were toler- five partitions for the plastid loci and one partition including ated, as this is usually not detrimental for phylogenetic analyses the coded indels and inversions. The analyses were performed (Wiens & Morrill, 2011). Indels were coded as binary inform- with two simultaneous runs of four parallel Metropolis-coupled ative characters according to the simple indel coding (SIC) Markov Chains Monte Carlo (MCMCMC) each for 50 mil- method (Simmons & Ochoterena, 2000) implemented in the lion generations and, starting with a random tree, one tree was program SeqState v.1.40 (Müller, 2005a). SIC performs about as saved every 1000th generation. Since in initial runs the default good as the modified complex indel coding (MCIC) (Simmons temperature of 0.1 resulted in a low proportion of successful & al., 2007) but has the advantage that the SIC matrix can also state exchanges between cold and heated chains, heating tem- be analysed with most programs for phylogenetic analyses. Five perature was reduced to 0.03, which successfully increased the reinverted inversions in the plastid matrix were coded manually efficiency of the Metropolis coupling. For other parameters as a single binary character (0 = absent, 1 = present). Indels and the default settings were left unchanged. Convergence of the inversions added 145 binary characters to the nrITS matrix, runs onto the stationary distribution was checked by examin- resulting in a total of 833 characters, and 602 to the plastid ing the average standard deviation of split frequencies and by DNA matrix, resulting in a total of 6398 characters. ensuring that the post-burn-in effective sampling size (ESS) as Phylogenetic reconstruction. — The nuclear and plastid measured with Tracer v.1.6 (Drummond & Rambaut, 2007) was datasets were analysed separately. Our previous study on the well above 200 in either run for all parameters. The first 2500 phylogeny of subtribe Lactucinae revealed several hard top- trees were discarded as burn-in, by which the average standard ological incongruences between the nuclear and the plastid deviation of split frequencies of the two runs had dropped to trees (Wang & al., 2013); therefore no analyses of a combined less than and remained below 0.01. The remaining 47,500 trees dataset were performed. were used to generate a majority-rule consensus tree. Phylogenetic relationships were reconstructed using Maximum likelihood analyses were performed using maximum parsimony (MP), Bayesian analysis (BA) and RAxML (Stamatakis, 2014) in the v.8.2.4 installed on the maximum likelihood (ML). Maximum parsimony analyses CIPRES Science Gateway (Miller & al., 2010) as RAxML- were performed using the parsimony ratchet (Nixon, 1999) HPC v.8 on XSEDE. The nrITS dataset was partitioned into two with PRAP v.2.0 (Müller, 2004) in combination with PAUP partitions, one for the DNA and one binary for the coded indels. v.4.0b10 (Swofford, 2003). Standard ratchet settings were used: The plastid DNA dataset was partitioned as in the Bayesian 200 ratchet iterations with 25% of the positions randomly up- analysis into five partitions for the plastid loci and one binary weighted (weight = 2) during each replicate and 10 random partition for the coded indels and inversions. Analyses were addition cycles. The generated command files also including done with the partitioned matrices reduced by RAxML from the nexus data matrix were run in PAUP using the heuristic identical sequences. Rapid bootstrapping (with the maximum search option with the following parameters: all characters set of 1000 replicates) integrated with a thorough ML search for have equal weight, simple addition of sequences, TBR branch- the optimal tree was carried out using the CAT approximation ing swapping, maxtrees setting to 100 and auto-increased by (Stamatakis, 2006) of the general time reversible (GTR) model 100, one non-binary starting tree arbitrarily dichotomised be- of nucleotide substitution under the gamma model of rate het- fore branch swapping, only one tree saved. A majority-rule erogeneity for all DNA partitions and BINCAT for the binary

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partitions. Bootstrap support (BS) values were written by the & al., 2012), because other pollen finds are associated with program on the best maximum likelihood tree. much greater calibration uncertainty and are peripheral to our TreeGraph v.2 (Stöver & Müller, 2010) was used to visual- sampling. We implemented a prior using each the lowermost ise the trees with statistical node support and to assess tree and uppermost estimates of the 95% HPDs in the two calcu- topologies. lations by Tremetsberger & al. (2012). This resulted in a wide Topological hypothesis testing with the stepping-stone range, with a lower bound at 3.6 mya and an upper bound at sampling approach (Bergsten & al., 2013) implemented in 14.7 mya. Taking the uncertainty of the age of this clade into MrBayes was employed to provide additional evidence whether consideration and following the recommendation by Schenk Prenanthes purpurea and the Lactucinae are sister clades in the (2016), we gave this prior a uniform distribution, so that every nrITS phylogeny or not. The two hypotheses were set as hard age between the hard upper and lower bounds has equal prior topological constraint and corresponding negative constraint, probability. The following other settings were applied: DNA respectively, and tested each with four parallel runs with two partitions and substitution models as used for the phylogenetic simultaneous chains of 50 steps for 196,000 generations (392 analyses and a binary partition for inversion and indels, apply- samples) within each step; a total of 9,996,000 generations ing a simple (HKY) substitution model; a single lognormal (19,992 samples) were collected while the first 196,000 gen- relaxed uncorrelated clock with estimated rate for each dataset; erations (392 samples) were discarded as initial burn-in; addi- Yule Process as tree prior with a random starting tree; an expo- tionally, at the beginning of each step 49,000 generations (98 nential distribution for the ucld.mean hyperparameter with the samples) were discarded as burn-in. The optimal substitution median placed centrally in the right order of magnitude (10−3) model was not specified a priori but sampling was done across of the expected substitution rate. The nrITS tree topology was the entire general time reversible (GTR) model space in the constrained to ensure inclusion of Prenanthes purpurea in the Bayesian MCMC analysis (Huelsenbeck & al., 2004). The cal- Lactucinae lineage. Four independent Monte Carlo Markov culations were also done with the MPI version of MrBayes on chains (MCMC) were run for 108 generations and a tree was the Soroban high-performance computing system of the Freie sampled every 10,000th generation. Tracer was used to judge Universität Berlin. the appropriate burn-in, to check that the independent MCMC Divergence time estimation. — An estimation of the age runs had converged on the same distribution and that the ESS of the subtribe and its different lineages was achieved with the for all parameters was well above 200 in the individual runs. uncorrelated relaxed molecular clock approach, which accounts The trees of the single runs were combined with LogCombiner, for lineage-specific substitution rate heterogeneity (Drummond using a burn-in of 10% and TreeAnnotator was used to obtain & al., 2006) and which is implemented in a Bayesian frame- and FigTree v.1.4.2 (Rambaut, 2014) to visualise the maximum work in BEAST (Drummond & Rambaut, 2007). We used a clade credibility tree of the 36,000 trees saved for either dataset. local installation of version 1.8.2 of BEAST for setting up and Biogeography and ancestral area estimation. — Ancestral testing runs and the version 1.8.0 installed on the Soroban high-​ area ranges were estimated using a Bayesian binary MCMC performance computing system of the Freie Universität Berlin (BBM) analysis as implemented in RASP (Reconstruct for the calculations. Ancestral State in Phylogenies; Yu & al., 2015). BBM calcu- We made calculations independently for the nrITS and lates the average probability of the presence and absence of an the plastid DNA datasets, using reduced matrices with a cor- ancestral area state and of all its possible combinations with responding sampling and usually with a single sample per other area states (the area range) over all trees sampled for a taxon only. Two nodes were calibrated: (1) One was calibrated given node in a phylogenetic hypothesis which is enforced as with the oldest reported Cichorieae fossil (early Miocene, a topological constraint in the analysis. As a result, the prob- 22–28.4 mya), the Cichorium intybus type pollen described abilities are given for each area range for each selected node, by Hochuli (1978). Two options exist for its placement which represent the ancestral state of that node. (Tremetsberger & al., 2012: fig. 1). Since, however, all taxa but The dated trees obtained with BEAST from the nrITS and one of our sampling belong to the C2H2L clade and the single the plastid DNA datasets were used as input for the BBM anal- exception is Scorzonera hispida, being sister to the remainder, yses. The area unit coding was based on the TDWG scheme calibration of the stem node of the C2H2L subtree with this (Brummitt, 2001), using combinations of the area units of hi- fossil is in line with both hypotheses. The age range of the erarchy level 2 (see Fig. 3 and its caption for the details). The fossil’s deposit given, we used an exponential calibration prior Cichorieae Systematics Portal (Kilian & al., 2009b–) was used with a hard lower bound of 22 mya. An exponential distribution as the data source for TDWG scheme-based presumed native of the prior probability with an upper bound much beyond the distribution ranges of the sampled taxa. The BBM analyses upper age of the fossil’s deposit is thought to account better were run independently for the two datasets each for 107 gen- for the uncertainty of the actual upper age of the clade than the erations with 10 simultaneous chains, the temperature was set lognormal distribution chosen by Tremetsberger & al. (2012). as 0.1, one tree was sampled every 1000th generation, the first The mean was set to 9.24 mya, which places the median of 20% of the 10,000 sampled trees were discarded as burn-in, the prior probability at the upper age of the fossil’s deposit at the maximum number of area units for ancestral distribution 28.4 mya and the upper limit of the 95% confidence interval ranges was limited to 3 considering the large size of the areas at 49.68 mya. (2) A secondary calibration prior was set for the chosen, and the fixed JC + G (Jukes-Cantor + Gamma) model crown node of the core Lactucinae (following Tremetsberger was applied.

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RESULTS sister-​group relationship of the terminal clade B (Prenanthes purpurea) to clade 2 is strong in BI but weak in ML and MP Phylogenetic analyses. — Phylogenetic reconstruction (PP = 0.98, BS = 72, JK = 57.8). The support for the successive based on the nrITS dataset on the one hand and on the concat- sister-group relationships of the terminal clades E and F to the enated plastid DNA marker dataset on the other hand each re- corresponding remainder of the subtribe is only weak (PP = sulted in 50% consensus trees of almost identical topology. We 1, BS = 75, JK = 62.8 and PP = 0.98, BS = 57, respectively). present the BI phylograms (Figs. 1, 2), with Bayesian posterior The polytomous clade 4 also received weak support only (PP probability (PP) and MP Jackknife support (JK) values above, = 0.81, BS = 61, JK = 51.7). and ML Bootstrap support (BS) values below the branches. Testing of the topological hypothesis whether Prenanthes Sequence statistics and phylogenetic performance of the nrITS purpurea and the core Lactucinae are sister clades in the ITS region and the non-coding plastid markers have been addressed phylogeny or not revealed strong evidence for their sister-group​ in our previous paper (Wang & al., 2013). relationship since the 2 × loge Bayes factor much exceeds the Nuclear ribosomal DNA phylogeny. — Tree optimisations threshold of >10 for very strong support (according to Kass & based on the nrITS dataset revealed a clade 1 (Fig. 1) with rea- Raftery, 1995) of the topology of hypothesis 1 (see Table 1). sonable support in BI and ML but no support in MP (PP = 0.98, Plastid DNA phylogeny. — Tree optimisations based on the BS = 72, JK = 57.8). Its further relationship is not resolved in concatenated plastid DNA dataset revealed a clade 1 (Fig. 2) either analysis, being part of unsupported basal polytomies with strong support (PP = 1, BS = 96, JK = 95.8). It includes of different topologies. BI (Fig. 1) reconstructed clade 1 in a the core Lactucinae as defined by Wang & al. (2013) and, sis- trichotomy with Prenanthes abietina (Boiss. & Balansa) Kirp. ter to them with strong support (PP = 1, BS = 96, JK = 95.8) and the clade of subtribe Hypochaeridinae, and this trichotomy the Prenanthes purpurea clade B. The immediate sister to in turn is part of another one with the clades representing the clade 1 is the Crepidinae clade (clade support PP = 1, BS 96, subtribes Crepidinae and Hyoseridinae. Clade 1 represents JK = 69.1; sister-group relationship support PP = 0.92, BS = subtribe Lactucinae as defined by Wang & al. (2013), including 74, JK = 53.1) followed, but without statistical support, by the Prenanthes purpurea + clade 2. Clade 2 is a trichotomy of the Hypochaeridinae and Hyoseridinae clades. Deeply nested in terminal clades C, D and clade 3, the last forming the core the Crepidinae clade are (1) the Prenanthes abietina clade A, Lactucinae (in the sense of Wang & al., 2013). This clade 3 (2) the Lactuca triquetra clade C and (3) the Faberia clade D. consists of the major terminal clades E to J. Clades G to J Clade 1 includes, in consecutive sister-group​ relationships, five form a large polytomy (clade 4), to which clades E and F are major clades as already identified by Wang & al. (2013): apart successive sister groups. Clades B to C are monospecific rep- from the Prenanthes purpurea clade B these are the Cicerbita resenting the chiefly European Prenanthes purpurea and the clade E (full support in all three optimisation methods both E Mediterranean Lactuca triquetra (Labill.) Boiss. The others, for the clade and the sister-​group relationship), the Notoseris- all with strong or very strong statistical support, represent the Paraprenanthes clade G/H (clade support PP = 1, BS = 100, major phylogenetic lineages of the subtribe as already identi- JK = 99.6; sister-​group relationship support PP = 1, BS = 100, fied by Wang & al. (2013). These are the Faberia lineage (clade JK = 99.9), the Kovalevskiella (= “Cicerbita II” sensu Wang & D; PP = 1, BS = 100, JK = 99.9), the Cicerbita lineage (clade al., 2013) clade F (clade support PP = 1, BS = 100, JK = 100; E; PP = 1, BS = 97, JK = 92.7), the Kovalevskiella Kamelin sister-​group relationship support PP = 1, BS = 100, JK = 99.9) lineage (“Cicerbita II” sensu Wang & al., 2013; clade F; PP and the Lactuca-Melanoseris clade I/J (PP = 0.99, BS = 83, = 1, BS = 96, JK = 96.5), the Notoseris lineage (clade G; PP JK = 62.6). The Notoseris-Paraprenanthes clade G/H and the = 1, BS = 100, JK = 99.9), the Paraprenanthes lineage (clade Lactuca-Melanoseris clade I/J merge the respective clades G H; PP = 1, BS = 95, JK = 92.1), the Lactuca lineage (clade and H, and I and J, as revealed in the ITS tree, and also show I; PP = 1, BS = 96, JK = 89.7) and the Melanoseris lineage a different internal topology. (clade J; PP = 1, BS = 97, JK = 88.2). Statistical support for the Incongruences between nuclear and plastid DNA phylog- eny. — Topological incongruences between the nrDNA and the plastid DNA phylogenies are manifold as evidenced by Table 1. Marginal log likelihood for hypothesis 1 (H1, sister-group rela- a comparison of the corresponding phylograms (Figs. 1, 2). tionship of Prenanthes purpurea L. and clade 2 Lactucinae, see Fig. 1) The vast majority of the incongruences have strong to very and hypothesis 2 (H2 , P. purpurea not sister to clade 2), and two times strong statistical support. Exceptions are several incongru- the difference of their mean resulting in the 2×loge Bayes factor (Kass & Raftery, 1995). ences regarding deeper nodes, which make up the phylogenetic backbone of the subtribe as well as its relationship among the Marginal log Marginal log 2×loge Bayes factor representatives of the C2H2L clade of the Cichorieae (Kilian Runs likelihood H1 likelihood H2 of H1 versus H2 & al., 2009a; Tremetsberger & al., 2012). The hard topological 1 −17482.21 −17541.92 incongruences (Wendel & Doyle, 1998) concern the circum- 2 −17475.49 −17543.31 scription of the subtribe and relationships between and within 3 −17462.24 −17528.86 major lineages. Some of these corroborate the findings in our 4 −17475.16 −17537.71 previous paper focussing on the subtribe in its Chinese centre of diversity (Wang & al., 2013), but others are new. Mean −17463.63 −17530.25 133.24 These new hard topological incongruences are:

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Paraprenanthes oligolepis LAC 022 Fig. 1A–C. Majority consensus phylo- Notoseris henryi LAC 056 1 / 88 gram of the Lactucinae from the 81.2 Notoseris henryi LAC 062 Notoseris macilenta LAC 064 Bayesian analysis (support values: 1 / 97 93.7 Notoseris macilenta LAC 065 Bayesian posterior probability / maxi- 1 / 98 Notoseris macilenta LAC 143 mum likelhood bootstrap, second line: 89.2 Notoseris macilenta LAC 145 1 / 100 Notoseris khasiana LAC 066 maximum parsimony jackknife) based 99.4 0.94 / 69 Notoseris khasiana LAC 068 on the nrITS dataset. Branch colours 92.1 Notoseris triflora × khasiana LAC 151 refer to the phylogenetic lineages, G 1 / 100 Notoseris triflora LAC 059 lineages A to H having a unique colour 1 / 100 100 Notoseris triflora LAC 061 99.9 Notoseris yakoensis LAC 054 each, colours of clades of lineage I 1 / 100 Notoseris scandens LAC 052 ranging from yellowish orange to 100 Notoseris scandens LAC 147 Kovalevskiella rosea LAC 232 red, those of lineage J from blue to 3 0.99 / 74 62.7 Kovalevskiella rosea LAC 233 blue-green; A, Prenanthes abietina; 1 / 75 0.97 / 70 Kovalevskiella kovalevskiana LAC 241 62.8 B, Prenanthes purpurea; C, Lactuca 1 / 99 Kovalevskiella kovalevskiana LAC 207 98.1 triquetra; D, Faberia; E, Cicerbita; 1 / 94 Kovalevskiella zerawschanica LAC 270 F, Kovalevskiella; G, Notoseris; 92.8 Kovalevskiella zerawschanica LAC 239 Kovalevskiella zerawschanica LAC 238 H, Paraprenanthes; I, Lactuca; J, Lactuca mira LAC 267 Melanoseris. 0.99 / 82 Chaetoseris roborowskii LAC 216 Chaetoseris roborowskii LAC 019 F 1 / 95 1 / 96 Chaetoseris roborowskii LAC 018 98.8 96.5 1 / 100 Chaetoseris roborowskii yellow LAC 222 99.8 0.58 1 / 100 Stenoseris auriculiformis LAC 016 A 100 Stenoseris auriculiformis LAC 017 Lactuca alaica LAC 268 1 / 100 Cicerbita azurea LAC 014 100 Cicerbita azurea LAC 015 1 / 89 90.5 0.96 Cephalorrhynchus microcephalus LAC 326 1 / 95 Cephalorrhynchus hispidus LAC 011 91.7 Cephalorrhynchus kirpicznikovii LAC 235 2 0.95 60.6 Cephalorrhynchus hispidus LAC 240 1 / 99 Cephalorrhynchus hispidus LAC 275 0.89 98.9 1 / 100 Cephalorrhynchus cypricus LAC 215 0.98 / 83 100 86.6 Cephalorrhynchus subplumosus LAC 226 Cephalorrhynchus hispidus LAC 214 Mycelis muralis LAC 138 1 / 100 Mycelis muralis LAC 139 E 100 1 / 97 0.99 / 95 Mycelis muralis LAC 274 92.7 95.8 1 / 100 Cicerbita petiolata LAC 271 100 Cicerbita petiolata LAC 136 1 / 75 1 / 100 Cicerbita pancicii LAC 272 87.1 1 100 Cicerbita pancicii LAC 273 1 / 100 Cicerbita alpina LAC 133 0.98 / 72 98.1 57.8 Cicerbita alpina LAC 012 Cicerbita alpina LAC 137 D 1 / 88 Faberia sinensis LAC 006 1 / 100 87.6 Faberia faberi LAC 010 99.9 Faberia nanchuanensis LAC 005 0.8 / 75 Lactuca triquetra LAC 278 C 63 Lactuca triquetra LAC 277 1 / 100 Lactuca triquetra LAC 276 0.71 100 0.96 / 83 Lactuca triquetra LAC 285 63.5 Lactuca triquetra LAC 286 Prenanthes purpurea HQ161931 B 1 / 100 Prenanthes purpurea LAC 141 100 Prenanthes purpurea LAC 142 A Prenanthes purpurea LAC 013 Prenanthes abietina LAC 294 Hypochaeridinae Leontodon tuberosus LAC 134 1 / 100 Hypochaeris radicata LAC 283 99.6 Hypochaeris maculata LAC 287 1 / 100 Soroseris erysimoides LAC 004 0.8 100 Soroseris glomerata LAC 297 // 0.5 Sonchella dentata LAC 300 0.9 1 / 100 Nabalus trifoliolatus LAC 288 99.9 Nabalus altissimus LAC 299 1 / 98 Nabalus sagittatus HQ161964 98.7 0.55 / 51 0.52 Dubyaea hispida LAC 298 Nabalus tatarinowii LAC 289 Ixeris chinensis subsp. versicolor LAC 291 Crepidinae 1 / 100 0.99 / 51 Crepis multicaulis LAC 003 67.8 100 Crepis sancta LAC 293 Lagoseriopsis popovii LAC 290 Youngia cineripappa LAC 292 Crepidiastrum tenuifolium LAC 295 Hyoseridinae 1 / 100 Launaea sarmentosa LAC 001 100 Reichardia dichotoma LAC 181 // Scorzonera hispanica LAC 296

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100 Lactuca paradoxa LAC 198 0.81 / 88 84.4 Lactuca tuberosa yellow LAC 178 Lactuca tuberosa LAC 179 B 1 / 99 0.8 / 75 Lactuca tuberosa LAC 118 I9 100 67.9 Lactuca tuberosa LAC 180 1 / 100 Lactuca dissecta LAC 116 1 / 95 100 Lactuca dolichophylla LAC 117 68.2 Lactuca inermis LAC 119 I8 1 / 100 Lactuca inermis LAC 177 100 1 / 100 Lactuca inermis LAC 176 100 Lactuca tenerrima LAC 171 Lactuca ludoviciana LAC 153 0.79 / 67 1 / 100 HQ161956 53 99.3 Lactuca hirsuta HQ172901 0.98 / 82 1 / 100 Lactuca graminifolia LAC 155 55.5 I7 99.9 Lactuca biennis HQ161959 1 / 100 Lactuca floridana LAC 154 100 1 / 100 Cicerbita plumieri LAC 149 100 Cicerbita plumieri LAC 186 0.98 / 72 1 / 100 Lactuca perennis LAC 113 68.9 0.56 100 Lactuca perennis LAC 334 1 / 85 1 / 99 Lactuca intricata LAC 183 52.4 I6 1 / 100 99.6 Lactuca intricata LAC 182 100 Lactuca undulata LAC 115 1 / 99 97.8 Lactuca undulata LAC 114 I5 0.94 / 65 Lactuca glauciifolia LAC 184 63.5 Cephalorrhynchus picridiformis LAC 185 0.95 / 72 63.4 Cicerbita racemosa LAC 187 I4 Cicerbita racemosa LAC 188 1 / 100 Cicerbita macrophylla LAC 189 100 Cicerbita macrophylla LAC 190 1 / 97 95.1 LAC 146 1 / 100 Lactuca virosa LAC 246 0.99 / 70 0.7 / 84 100 Lactuca georgica LAC 164 66.2 62.5 Lactuca sativa LAC 130 4 I3d 1 / 100 LAC 144 0.81 / 61 1 / 98 100 Lactuca serriola LAC 247 51.7 99.4 Lactuca serriola LAC 163 1 / 88 1 / 100 Lactuca saligna LAC 148 94.1 I3c 100 Lactuca saligna LAC 281 Lactuca quercina LAC 165 I3 1 / 87 1 / 100 86.8 Lactuca tetrantha LAC 167 99.9 0.51 / 78 Lactuca alpestris LAC 168 I3b 1 / 100 Lactuca viminea LAC 127 0.96 / 81 100 Lactuca acanthifolia LAC 162 I 0.92 / 70 Lactuca orientalis LAC 126 1 / 96 52.7 I3a 1 Lactuca tatarica LAC 166 89.7 1 / 100 100 Lactuca tatarica LAC 248 99.9 Lactuca sibirica LAC 128 Lactuca indica LAC 169 1 / 100 Lactuca indica LAC 170 I2 99.3 1 / 100 Lactuca indica LAC 121 100 Lactuca formosana LAC 122 1 / 97 Lactuca raddeana LAC 124 88.9 1 / 84 1 / 98 Lactuca glareosa LAC 196 88.4 94.8 Lactuca glareosa LAC 195 I1 Lactuca oyukludaghensis LAC 191 1 / 100 Lactuca variabilis LAC 192 99.6 Lactuca variabilis LAC 265 Cicerbita deltoidea LAC 194 Lactuca aurea LAC 193 Paraprenanthes yunnanensis LAC 044 1 / 99 Paraprenanthes yunnanensis LAC 490 98.5 0.61 / 98 Paraprenanthes yunnanensis LAC 491 Paraprenanthes meridionalis LAC 036 1 / 99 Paraprenanthes prenanthoides LAC 035 99.6 Paraprenanthes sororia LAC 037

Paraprenanthes sororia LAC 038 1 / 98 0.98 / 98 Paraprenanthes diversifolia LAC 041 97.8 82.1 0.54 / 82 Paraprenanthes wilsonii LAC 049 1 / 98 Paraprenanthes wilsonii LAC 051 0.98 / 57 0.99 98.1 Paraprenanthes melanantha LAC 046 77.2 Paraprenanthes melanantha LAC 047 H 1 / 100 Paraprenanthes parishii LAC 028 1 / 95 100 Paraprenanthes parishii LAC 029 92.1 0.91 / 87 Paraprenanthes triflora LAC 025 1 / 99 Paraprenanthes triflora LAC 152 1 / 100 Paraprenanthes triflora LAC 266 99.9 1 Paraprenanthes oligolepis LAC 020 Paraprenanthes oligolepis LAC 022 Notoseris henryi LAC 056

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(1) The Caucasian Prenanthes abietina (clade A) ap- (2) The E Mediterranean Lactuca triquetra (clade C) groups pears in an unresolved position between the Lactucinae plus with the Lactucinae clade in the nrITS phylogeny, but is deeply Prenanthes purpurea and the Hypochaeridinae in the nrITS nested in the Crepidinae clade in the plastid DNA phylogeny. phylogeny, while being deeply nested in the Crepidinae clade (3) The well-supported Lactuca clade I (branches yel- in the plastid DNA phylogeny. lowish orange to red; PP = 1, BS = 96, JK = 89.7) and the

0.99 / 99 Melanoseris graciliflora LAC 259 83.1 Melanoseris graciliflora LAC 250 0.95 / 76 C 59.9 Melanoseris graciliflora LAC 249 1 / 88 Melanoseris graciliflora LAC 112 84 Melanoseris tenuis LAC 237 1 Melanoseris tenuis LAC 107 0.95 / 82 99.8 Melanoseris tenuis LAC 106 0.95 / 68 81.5 Melanoseris atropurpurea LAC 101 Melanoseris atropurpurea LAC 099 0.98 / 54 Melanoseris likiangensis LAC 258 0.99 / 62 58.8 Melanoseris likiangensis LAC 098 Melanoseris macrantha LAC 075 Melanoseris monocephala LAC 231 0.97 / 67 87.4 Melanosris henryi LAC 280 0.93 / 92 0.97 / 88 1 / 94 Melanoseris violifolia LAC 076 86.1 0.86 / 61 0.82 / 58 Melanoseris violifolia LAC 077 56.4 53.3 Melanoseris violifolia LAC 254 0.53 / 67 Melanoseris macrorhiza LAC 253 66.4 Melanoseris macrorhiza LAC 073 0.56 / 59 Melanoseris macrorhiza LAC 074

0.97 / 63 Melanoseris lessertiana LAC 071 63.5 Prenanthes sumatrana LAC 279 1 / 97 Melanoseris aff. cyanea LAC 257 1 / 95 98.7 Melanoseris cyanea LAC 089 95.3 Melanoseris cyanea LAC 084 1 / 92 Melanoseris cyanea LAC 080 90.7 0.98 / 75 J3 Melanoseris aff. cyanea LAC 257 74.1 1 / 98 Melanoseris qinghaica LAC 078 97.2 Melanoseris decipiens LAC 256 Melanoseris brunoniana LAC 255 1 / 100 Melanoseris souliei LAC 251 1 / 100 100 Melanoseris souliei LAC 252 100 1 / 100 Melanoseris souliei LAC 069 1 / 97 96.9 100 Melanoseris souliei LAC 070 0.84 / 87 1 / 100 Cephalorrhynchus brassicifolius LAC 225 63.2 99.7 Cephalorrhynchus brassicifolius LAC 227 0.97 / 95 97.7 Cephalorrhynchus brassicifolius LAC 218 J2g Cephalorrhynchus polycladus LAC 219 1 / 99 1 / 100 Cephalorrhynchus polycladus LAC 282 0.99 / 76 1 / 93 99.6 99.9 Cephalorrhynchus polycladus LAC 213 82 1 / 98 Cephalorrhynchus takhtadzhianii LAC 234 J2f 95.1 Cephalorrhynchus soongoricus LAC 208 0.78 / 51 1 / 100 Cephalorrhynchus soongoricus LAC 211 100 Cephalorrhynchus soongoricus LAC 210 1 / 99 Cicerbita bourgaei LAC 260 1 / 100 99.7 J2e Cicerbita bourgaei LAC 150 99.7 Cicerbita prenanthoides LAC 229 0.6 / 55 61.2 Cicerbita prenanthoides LAC 230 0.74 / 75 J2d Lactuca rosularis LAC 199 1 / 98 Steptorhamphus pumilus LAC 261 97 Lactuca hazaranensis LAC 262 J2c 1 / 100 Melanoseris bracteata LAC 244 1 / 75 1 / 100 100 72.8 Melanoseris bracteata LAC 221 100 J2b Melanoseris bracteata LAC 072 J 1 / 100 Cicerbita thianschanica LAC 263 1 / 97 J2 100 Cicerbita thianschanica LAC 264 88.2 Cephalorrhynchus kossinskyi LAC 220 1 / 63 Cephalorrhynchus kossinskyi LAC 212 99.5 1 / 100 Cephalorrhynchus kossinskyi LAC 217 J2a 99.1 1 / 92 0.64 / 82 Cephalorrhynchus kossinskyi LAC 243 88.9 57.5 Steptorhamphus crassicaulis LAC 224 1 / 98 Steptorhamphus crassicaulis LAC 223 88.6 Steptorhamphus persicus LAC 284 1 / 95 1 Lactuca praecox T2 LAC 203 98.6 99.3 Lactuca praecox T3 LAC 204 Lactuca calophylla LAC 200 0.6 / 53 1 / 100 Lactuca schulzeana LAC 205 99.7 Lactuca ugandensis LAC 206 1 / 100 Lactuca setosa LAC 201 J1 99.9 1 / 96 Lactuca lasiorhiza LAC 199 Lactuca praecox T1 LAC 202 93.3 1 / 100 Lactuca attenuata LAC 197 100 Lactuca paradoxa LAC 198 0.81 / 88

Version of Record 683 Kilian & al. • Diversification of the Lactucinae (Compositae) TAXON 66 (3) • June 2017: 675–703

74.1 Cicerbita azurea LAC 015 3 Kovalevskiella kovalevskiana LAC 207 A 3 1 / 100 Notoseris henryi LAC 056 1 / 62 99.9 63 Notoseris henryi LAC 062 1 / 92 Notoseris macilenta LAC 064 93.2 0.98 / 88 Notoseris macilenta LAC 065 63.7 Notoseris triflora LAC 059 1 / 74 Notoseris triflora LAC 061 89.6 Notoseris khasiana LAC 068 Notoseris khasiana LAC 066

1 / 99 Paraprenanthes meridionalis LAC 036 0.99 / 69 87.1 Paraprenanthes prenanthoides LAC 035 1 / 92 83.1 Paraprenanthes diversifolia LAC 041 Paraprenanthes wilsonii LAC 049 0.99 / 68 57.6 Paraprenanthes wilsonii LAC 051 0.96 / 67 Paraprenanthes yunnanensis LAC 044 67.5 Paraprenanthes yunnanensis NK172 1 / 100 1 / 100 Paraprenanthes sororia LAC 037 2 95.7 99.6 Paraprenanthes sororia LAC 038 1 / 100 Paraprenanthes melanantha LAC 046 1 / 100 99.7 Paraprenanthes melanantha LAC 047 100 G/H 1 / 100 Paraprenanthes parishii LAC 028 1 / 100 100 Paraprenanthes parishii LAC 030 99.6 1 / 99 Paraprenanthes oligolepis LAC 020 1 / 100 100 Paraprenanthes oligolepis LAC 022 100 1 / 100 Paraprenanthes triflora LAC 025 0.97 / 70 100 Paraprenanthes triflora LAC 266 63.5 Notoseris scandens LAC 147 1 / 100 Notoseris yakoensis LAC 054 99.9 Notoseris scandens LAC 052 0.98 / 83 1 / 100 Cephalorrhynchus kirpicznikovii LAC 235 1 80.1 1 / 100 99 Cephalorrhynchus microcephalus LAC 236 1 / 96 1 / 80 100 95.8 63.6 Cephalorrhynchus hispidus LAC 011 1 / 94 71.6 Cephalorrhynchus hispidus LAC 240 1 / 92 1 / 100 Cephalorrhynchus hispidus LAC 214 98.1 98.7 Cephalorrhynchus subplumosus LAC 226 Cephalorrhynchus cypricus LAC 215 E Mycelis muralisLAC 274 1 / 100 1 / 81 100 Cicerbita alpina LAC 133 1 / 69 Cicerbita pancicii LAC 273 0.99 / 73 90.8 Cicerbita pancicii LAC 272 1 / 100 Cicerbita petiolata LAC 271 B 99.5 Cicerbita petiolata LAC 228 1 / 100 Prenanthes purpurea LAC 013 0.92 / 74 100 Prenanthes purpurea LAC 242 53.1 1 / 100 Nabalus trifoliolatus LAC 288 1 / 100 99.6 Nabalus tatarinowii LAC 289 1 / 100 100 Soroseris erysimoides LAC 004 98.3 1 / 100 Lagoseriopsis popovii LAC 290 98.4 D 1 / 100 Faberia sinensis LAC 006 1 / 100 99.9 1 / 85 Faberia faberi LAC 010 100 Faberia nanchuanensis LAC 005 0.7 / 75 1 / 100 Youngia cineripappa LAC 292 0.61 / 57 99.6 Crepidiastrum tenuifolium LAC 295

0.53 / 52 1 / 64 Ixeris chinensis subsp. versicolor LAC 291 63.5 Lactuca triquetra LAC 277 0.75 / 75 0.93 / 73 Lactuca triquetra LAC 276 64 C 1 / 100 Lactuca triquetra LAC 278 Crepidinae 100 1 / 99 Lactuca triquetra LAC 285 1 / 96 A 95.9 Lactuca triquetra LAC 286 69.1 Prenanthes abietina LAC 294 // 1 / 100 // Crepis multicaulis LAC 003 100 // Crepis sancta LAC 293 Hypochaeridinae 1 / 99 // Hypochaeris radicata LAC 283 1 / 100 99.9 Hypochaeris maculata LAC 287 100 Hyoseridinae Leontodon tuberosus LAC 002 1 / 100 Launaea sarmentosa LAC 001 89.3 Reichardia dichotoma LAC 181 // Scorzonera hispanica LAC 296

Fig. 2A–C. Majority consensus phylogram of the Lactucinae from the Bayesian analysis (support values: Bayesian posterior probability / maxi- mum likelhood bootstrap, second line: maximum parsimony jackknife) based on the plastid DNA dataset. Designations and branch colours of lineages as in Fig. 1, illustrating incongruences at lineage level compared to the nrITS tree; species names in red indicate incongruences at species level; A, Prenanthes abietina; B, Prenanthes purpurea; C, Lactuca triquetra; D, Faberia; E, Cicerbita; F, Kovalevskiella; G, Notoseris; H, Paraprenanthes; I, Lactuca; J, Melanoseris.

684 Version of Record TAXON 66 (3) • June 2017: 675–703 Kilian & al. • Diversification of the Lactucinae (Compositae)

Melanoseris clade J (branches blue to blue-green; PP = 1, BS = comprises clades I4 to I9, representing the remainder of the 97, JK = 88.2) of the nrITS phylogeny are merged in the plastid Lactuca lineage. In the plastid DNA phylogeny, in contrast, DNA phylogeny, where the Lactuca lineage is deeply nested in the well-​supported clade I is polytomous and includes eight the Melanoseris clade, forming the clade I/J. strongly supported subclades. (4) The principal internal topology of the Lactuca clade I (5) The principal internal topology of the Melanoseris in the nrITS phylogeny, which received good statistical sup- clade J differs between both reconstructions. In the nrITS port in the BI and ML trees, is not revealed in the plastid phylogeny clade J1, comprising tropical African species hith- DNA phylogeny. In the ITS phylogeny, clade I1 with L. aurea erto assigned to Lactuca, is sister to a clade with two sub- (Vis. & Pančić) Stebbins and relatives is sister to a large clades, J2 comprising chiefly SW to Central Asian species, clade with two subclades. The first comprises clade I2, the and J3 comprising chiefly Sino-Himalayan species. In the L. indica L. clade, and I3, the core of Lactuca. The second plastid DNA phylogeny in contrast, the fairly well-supported Lactuca oyukludaghensis LAC 191 1 / 98 I1 95 Lactuca aurea LAC 193 Lactuca variabilis LAC 265 B 0.99 / 92 0.77 / 98 92.7 62.4 Melanoseris cyanea LAC 089 0.59 / 85 Melanoseris cyanea LAC 084 84.3 Melanoseris cyanea LAC 080 0.68 / 75 87.5 Melanoseris aff. cyanea LAC 257 1 / 100 1/ 90 Melanoseris brunoniana LAC 255 98.5 87.5 Melanoseris decipiens LAC 256 1 / 96 Melanoseris violifolia LAC 076 98.7 Melanoseris violifolia LAC 077 Melanoseris macrantha LAC 075 1 / 96 Melanoseris atropurpurea LAC 099 95.3 Melanoseris tenuis LAC 106 1 / 100 Melanoseris tenuis LAC 107 99.8 1 / 100 Melanoseris graciliflora LAC 112 0.91 / 81 99.9 81.9 1 / 95 Melanoseris atropurpurea LAC 101 63.4 Melanoseris likiangensis LAC 098 1 / 100 1 / 98 Melanoseris lessertiana LAC 071 98.4 100 Melanoseris qinghaica LAC 078 0.84 / 79 1 / 100 Melanoseris violifolia LAC 254 83.1 Melanoseris macrorhiza LAC 074 1 / 100 1 / 100 1 / 89 Melanoseris macrorhiza LAC 073 99.7 83.4 Melanoseris macrorhiza LAC 253 J3 J2b 1 / 100 Cicerbita thianschanica LAC 263 100 Cicerbita thianschanica LAC 264 1 / 98 Melanoseris souliei LAC 069 1 / 100 99.7 Melanoseris souliei LAC 070 0.67 / 59 98.2 51.5 Melanoseris souliei LAC 251 0.99 / 94 Lactuca praecox T3 LAC 204 79 Lactuca calophylla LAC 200 1 / 95 94.3 Lactuca praecox T2 LAC 203 Lactuca praecox T1 LAC 202 1 / 86 1 / 100 I/J 84.9 Lactuca setosa LAC 201 0.99 / 83 99.9 62.6 Lactuca ugandensis LAC 206 Lactuca schulzeana LAC 205 J1 Lactuca paradoxa LAC 198 1 / 97 70.7 Cephalorrhynchus kossinskyi LAC 217 1 / 100 Cephalorrhynchus kossinskyi LAC 243 99.9 Cephalorrhynchus kossinskyi LAC 220 J2a 1 / 100 Cephalorrhynchus kossinskyi LAC 212 100 Steptorhamphus crambifolius LAC 245 1 / 100 Steptorhamphus crassicaulis LAC 224 99.8 Steptorhamphus crassicaulis LAC 223 3.1 Steptorhamphus persicus LAC 284 J2d 1 / 100 Lactuca hazaranensis LAC 262 1 / 100 1 / 100 100 Lactuca rosularis LAC 209 99.9 100 Steptorhamphus pumilus LAC 261 1 / 97 86.3 Chaetoseris roborowskii LAC 216 1 / 100 Chaetoseris roborowskii yellow LAC 222 95.2 Chaetoseris roborowskii LAC 018 1 / 98 98.1 Chaetoseris roborowskii LAC 019 1 / 99 98.3 Stenoseris auriculiformis LAC 016 1 / 99 Stenoseris auriculiformis LAC 017 98.3 Lactuca mira LAC 267 0.97 / 66 Kovalevskiella zerawschanica LAC 239 63.6 Kovalevskiella zerawschanica LAC 238 F Kovalevskiella rosea LAC 232 1 / 100 Kovalevskiella rosea LAC 233 100 Kovalevskiella kovalevskiana LAC 241 1 / 93 86.3 Kovalevskiella zerawschanica LAC 269 1 / 99 Kovalevskiella zerawschanica LAC 270 96 Cicerbita azurea LAC 014 0.98 / 75 74.1 Cicerbita azurea LAC 015 Kovalevskiella kovalevskiana LAC 207 3 Lactuca alaica LAC 268 1 / 100 Notoseris henryi LAC 056 Version of Record 685 Kilian & al. • Diversification of the Lactucinae (Compositae) TAXON 66 (3) • June 2017: 675–703

Melanoseris subclade J2 (bright blue; PP = 1, BS = 75, JK = showed strong convergence. The independent relaxed molecular 72.8) of the nrITS phylogeny is heavily fragmented. Moreover, clock dating of the nrITS and plastid DNA datasets in general the terminal SW Asian clades J2e and J2f, the Sino-Himalayan produced age estimates with well-overlapping HDP intervals Melanoseris bracteata (C.B.Clarke) N.Kilian clade J2c and the for comparable clades. The results are summarised in Figs. 3 SW to Central Asian clade J2g (all bright blue) are nested in and 4 and Table 2. The median crown node age estimates, which the Lactuca clade I (yellowish orange to red). represent the minimum age estimates for the most recent com- Divergence time estimation. — Each of the four individ- mon ancestor (MRCA), obtained for the core Lactucinae in the ual chains per dataset of our BEAST analyses had an effective sense of Wang & al. (2013) are 12.2 (ITS tree) and 12.7 (plastid sampling size (ESS) of a multiple of 200 for all parameters and DNA tree) my, the median stem node age estimates are 13.4 and

0.99 / 75 64.6 1 / 96 Cephalorrhynchus brassicifolius LAC 225 1 / 100 99.2 Cephalorrhynchus brassicifolius LAC 227 C 100 1 / 100 Cephalorrhynchus brassicifolius LAC 218 1 / 100 J2g 98.8 Cephalorrhynchus polycladus LAC 213 1 / 98 100 Cephalorrhynchus polycladus LAC 282 92.4 Cephalorrhynchus polycladus LAC 219 0.51 / 87 Cephalorrhynchus takhtadzhianii LAC 234 1 / 100 Melanoseris bracteata LAC 244 I4 J2c 1 / 98 1 / 93 1 / 100 100 Melanoseris bracteata LAC 221 97.7 89.6 100 Melanoseris bracteata LAC 072 1 / 100 Cicerbita macrophylla LAC 190 1 / 100 99.9 Cicerbita macrophylla LAC 189 99.6 Cicerbita racemosa LAC 187 Cicerbita racemosa LAC 188 1 / 100 1 / 100 100 95.4 Lactuca sativa DQ383816 0.98 / 96 Lactuca serriola LAC 163 63 Lactuca serriola LAC 131 I3d 1 / 100 Lactuca serriola LAC 247 100 Lactuca georgica LAC 164 Lactuca virosa LAC 246 1 / 100 1 / 95 Lactuca saligna LAC 281 84.8 100 1 / 99 Lactuca tetrantha LAC 167 99.4 Lactuca viminea LAC 135 I3b 1 / 100 Lactuca alpestris LAC 168 100 Lactuca orientalis LAC 126

1 / 100 Lactuca acanthifolia LAC 162 99.8 Lactuca inermis LAC 119

I8 1 / 100 Lactuca inermis LAC 177 Lactuca inermis LAC 176 1 / 100 100 Lactuca tenerrima LAC 171 100 I5 1 / 90 Cephalorrhynchus picridiformis LAC 185 1 / 100 0.99 / 99 89.5 93.5 Lactuca floridana LAC 154 99.8 1 / 100 I7 Lactuca ludoviciana LAC 153 100 Lactuca graminifolia LAC 155 Cicerbita plumieri LAC 186 1 / 100 Lactuca tatarica LAC 248 I3a 1 / 100 100 1 / 76 Lactuca tatarica LAC 166 I 1 / 100 100 74.7 I3c Lactuca sibirica LAC 128 1 / 98 99.6 Lactuca quercina LAC 165 98 0.97 / 95 65.3 Cicerbita bourgaei LAC 260 J2e Cicerbita prenanthoides LAC 230 1 / 99 97.3 Cicerbita prenanthoides LAC 229 0.52 / 54 1 / 100 Lactuca indica LAC 170 I2 99.5 Lactuca indica LAC 121 1 / 95 1 / 100 Lactuca formosana LAC 122 94.3 100 Lactuca raddeana LAC 124 Cephalorrhynchus soongoricus LAC 210 J2f 1 / 100 Cephalorrhynchus soongoricus LAC 208 100 Cephalorrhynchus soongoricus LAC 211 0.63 / 95 63.1 Lactuca perennis LAC 334 1 / 100 Lactuca glauciifolia LAC 184 I6 100 1 / 100 Lactuca undulata LAC 115 1 / 100 100 Lactuca undulata LAC 114 100 1 / 100 Lactuca intricata LAC 183 100 Lactuca intricata LAC 182 1 / 100 Lactuca tuberosa LAC 180 I9 1 / 100 99.9 Lactuca tuberosa LAC 179 1 / 99 100 Lactuca tuberosa LAC 118 99.1 1 / 100 Lactuca dissecta LAC 116 0.99 / 76 100 Lactuca dolichophylla LAC 117 1 / 77 63.1 56.8 Lactuca glareosa LAC 196 1 / 98 Lactuca glareosa LAC 195 98.1 Cicerbita deltoidea LAC 194 0.97 / 69 Lactuca variabilis LAC 192 100 Lactuca oyukludaghensis LAC 191 1 / 98 I1 95 Lactuca aurea LAC 193 Lactuca variabilis LAC 265 0.99 / 92 0.77 / 98 92.7 62.4 Melanoseris cyanea LAC 089 686 Version of Record TAXON 66 (3) • June 2017: 675–703 Kilian & al. • Diversification of the Lactucinae (Compositae)

16.1 my respectively. Stem node age estimates show stronger considered in a molecular phylogenetic study, proved to be differences where incongruent sister-​group relationships are essential for the phylogenetic reconstruction. revealed in the two gene trees (compare Figs. 3 and 4, Table 2), The core Lactucinae as circumscribed by Wang & al. while crown node ages show stronger differences for clades (2013) are confirmed as a monophyletic lineage. In addition, within which incongruent topologies are revealed. evidence is provided here for the first time, not only from the The mean number of substitutions per nucleotide site per plastid DNA (Fig. 2) tree but also from the nrITS tree (Fig. 1) million years (subst./site/my) obtained is 5.47 × 10−3 for the nrITS and further by the Bayesian test of topological hypotheses, region and 1.51 × 10−3 for the five concatenated non-coding that Prenanthes purpurea is an early-diverging lineage of the plastid DNA markers (the petD region, psbA-trnH, 5′trnL(uaa)-​ Lactucinae. The circumscription of the Lactucinae with respect trnF, rpl32-trnL(uag) and 5′rps16-trnQ(uug)). to other lineages previously assigned to this subtribe is consid- Biogeography and ancestral area estimation. — The re- ered in more detail below. sults of the separate ancestral area estimations based on the Our new hypothesis for the relationships of the major nrITS and the plastid DNA trees using Bayesian binary analysis lineages of the core Lactucinae is inferred from congruent are shown in Figs. 3 and 4. Both estimations revealed similar re- topologies of the nrITS and plastid DNA tree, and for deeper sults for the core Lactucinae: the probabilities for the combined nodes from the plastid DNA tree only since these were not SW Asian-European area as the most likely ancestral area add to resolved with statistical support in the nrITS tree. Accordingly, 0.8/0.9 (first PP value nrITS, second PP value plastid DNA), but the Kovalevskiella lineage, the Notoseris and Paraprenanthes the results are not conclusive with respect to the narrower areas lineages, and finally the Cicerbita lineage are consecutive sis- (among them: SW Asia: PP 0.43/0.47, SW Asian-S-European ter groups to the Lactuca and Melanoseris lineages. The first region: PP 0.13/0.27, SW Asian-N + Central + E European re- diverging branch of the Lactucinae is Prenanthes purpurea, gion: PP 0.19/0.1). Similarly, for the Lactucinae in their wider which is confirmed as the only member of Prenanthes. While sense including Prenanthes purpurea, the probabilities for the it is sister to core Lactucinae in the plastid DNA phylogeny, it combined SW Asian-European region add to 0.93/0.99 (among is sister to the core Lactucinae plus the Faberia and Lactuca them: SW Asia: PP 0.32/0.27, the regions extended towards S triquetra lineages in the nrITS phylogeny. Europe: 0.13/0.18 or N + Central + E Europe: PP 0.21/0.19). In the Our separate divergence dating analyses of the nrITS and case of the Cicerbita clade as the first one diverging from the the plastid DNA matrices mostly revealed overlapping confi- core Lactucinae, the probabilities for the combined SW Asian– dence intervals for the node age estimates of comparable clades. European region add to 0.95/0.91 (among them: European– Our age estimates for the split between the Scorzonerinae and SW Asian region: PP 0.37/0.2, S European–SW Asian region: the two major core clades of the Cichorieae, which is the root of PP 0.2/0.34, SW Asian–N + Central + E European region: PP our trees (nrITS 24.9, 95% HPD 22–30.1 my / plastid DNA 24.4, 0.13/0.14, SW Asia: PP 0.07/0.23). Since the nodes of the core 95% HPD 22–28.9 my) are similar to those by Tremetsberger Lactucinae backbone in the dated nrITS trees otherwise are & al. (2012; 25.3, 95% HPD ca. 23.1– 30.2 my). In contrast, the unresolved or have no statistical support (see Figs. 1 and 3), crown node (thus the MRCA) of the core Lactucinae, which are the probabilities for area ranges to represent the ancestral state equivalent to the Lactucinae sensu Tremetsberger & al. (2012), of these nodes are meaningless and have been omitted (Fig. 3). has a significantly higher median minimum age, although with In contrast, the hypothesis of the relationships within the core still overlapping confidence intervals, in our analyses (nrITS Lactucinae as revealed in the topology of the plastid DNA trees 12.2, 95% HPD 9.4–14.7 my / plastid DNA 12.7, 95% HPD 9.9– (Figs. 2, 4) is statistically usually well supported. Clade 2 in 14.7 my) compared to Tremetsberger & al. (2012; 7.6, 95% HPD the core Lactucinae, which is sister to the Cicerbita clade, most 3.9–12.1 my). On the other hand, the most recent divergence likely has a SW Asian ancestral area (PP = 0.83). It bifurcates time estimates by Fernández-Mazuecos & al. (2016: fig. 2) into the E Asian Notoseris-Paraprenanthes clade and the clade based on a combined nrITS-matK dataset of the Cichorieae and with the remainder of the core Lactucinae, which again most by using the same calibrations as Tremetsberger & al. (2012) are likely has a SW Asian ancestral area (PP = 0.98). All basal very similar to ours for comparable nodes: they found a median nodes of this latter clade, including the Kovalevskiella, Lactuca crown node age for the core Lactucinae + Faberia of around and Melanoseris lineages, equally have a SW Asian ancestral 13 my (nrITS 13.4 my in our study) and a median age for the area with high PP values. The results for the more terminal split between Prenanthes purpurea and the Hypochaeridinae of clades in both reconstructions are considered in the discussion. around 18 my (nrITS 19 my, plastid DNA 18 my in our study). The mean substitution rate of 5.47 × 10−3 subst./site/my inferred in our analysis for the nrITS region is correspond- DISCUSSION ingly distinctly lower than that obtained by Tremetsberger & al. (2012) for their divergence time estimation across the entire Phylogenetic reconstruction, divergence times and bioge- Cichorieae (8.7 × 10−3 to 1.07 × 10−2 subst./site/my, the lower ography of the Lactucinae. — The present study provides the value in the unconstrained, the higher in the constrained re- first phylogenetic reconstruction of the widespread Lactucinae construction). The values by Tremetsberger & al. (2012) are on a global scale and considering all their species groups. The somewhat above the highest rates reported by Kay & al. (2006) inclusion of many representatives of the large SW to Central in their survey of ITS substitution rates across angiosperms, Asian portion of the subtribe, which had previously never been ranging from 0.38 × 10−3 to 8.34 × 10−3 subst./site/my. Kay &

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al. (2006), moreover, found no correlation between rates and Summarising our findings from the phylogenetic analy- phylogeny, the rate in Asteraceae, for example, spanning almost ses, molecular clock dating and ancestral area estimations, we the entire range. In contrast, they report a pattern of higher hypothesise the following scenario for the diversification of substitution rates in annuals compared to perennial herbs, and the Lactucinae in a geohistorical context. Like the other tribes for herbs compared to woody plants, which is in line with pre- of the Asteraceae, which are assumed to have evolved around vious findings from plastid DNA studies. 25–35 mya in the Oligocene (Funk & al., 2009), the origin The mean substitution rate of 1.51 × 10−3 subst./site/my we of the Cichorieae is estimated at around 26 (23.2–30.3) mya inferred for the non-coding plastid DNA loci is nearer to the (Tremetsberger & al., 2012). The Lactucinae, including upper end of published plastid DNA substitution rates compiled Prenanthes purpurea, originated most likely somewhere in for land plants by Villarreal & Renner (2014), which range from a SW Asian–European region (Figs. 3, 4) in the second half 1.0 × 10 − 4 to 1.9 × 10− 4 (woody plants) to 2.1 × 10−3 to 2.9 × 10−3 of the Early Miocene, around 18–19 mya (here and in the fol- (monocots and some herbaceous dicots). This is not surprising, lowing, unless otherwise stated, median age estimates from taking into account that the Lactucinae are primarily herba- the nrITS and plastid DNA analyses are given, see Table 2). ceous plants and that our rate refers to faster evolving non-cod- They started diversifying, similar to the other subtribes of ing loci, while the survey by Villarreal & Renner (2014) is the C2H2L clade, no later than around ca. 16–17 mya, at the mostly based on coding loci. beginning of the Middle Miocene. The SW Asian–European As stated above, the age estimates in our two reconstruc- region as its inferred area of origin corresponded to a more or tions showed overlapping confidence intervals for compa- less coherent landmass in the Miocene, the sub-Paratethyan rable, statistically well-supported and congruent clades. region, that was situated between the Tethys Sea (the precur- Significantly different age estimates were found in clades sor of the Mediterranean Sea) in the south and the Paratethys with hard topological incongruences in the nrITS and plastid Sea (from the present Black Sea eastwards extending to the DNA trees. An instructive example is the Cicerbita lineage. Aral Sea) in the north. This sub-Paratethyan region was iso- While the stem node age estimates are close to each other lated from Asia and /or western Europe for longer periods (e.g., (12.2 and 12.7 my), the split between its two clades is esti- Rögl, 1999; Popov & al., 2006). Diversification and expansion mated to have taken place around 8.7 mya according to the of the subtribe coincides with a series of global tectonic and analysis of the nrITS dataset but only around 5 mya according climatic changes during the Miocene, which led to essential to the plastid DNA analysis (median age estimates, Figs. 3, transformations of the earth’s surface. Increase in aridity and, 4 and Table 2). Similarly large differences were revealed for starting 14 mya, climatic cooling, caused an expansion of open the split between the Mycelis and Cephalorrhynchus sub- vegetation, especially of grasslands, at the expense of tropical clades (7.3 and 3.7 mya, respectively) and the MRCA of the broad-leaf forests. Extended orogenesis, uplifting in the region Cephalorrhynchus subclade (3.6 and 1.7 mya), while the age here concerned the Alpine-Himalayan belt and E Africa, cre- estimates for the MRCA of the Cicerbita subclade (4.3 and ated new habitats and migration corridors (Alpine-Himalaya 2.9 my) are closer to each other (Figs. 3, 4). Topological in- corridor). The closing of the oceanic gateway between the congruences exist within the Cicerbita and Cephalorrhynchus Mediterranean Sea and the Indian Ocean increased the conti- subclades, while the topology of the deeper nodes of the nentality in SW Asia and the Mediterranean region, but also Cicerbita clade is congruent. A possible explanation is that provided a new land bridge between Asia and Africa (Potts a reduction of sequence differences among the members of & Berensmeyer, 1992; Potter & Szatmari, 2009; Holbourn & either subclade by reticulation causes a compression of the al., 2013). time scale for the divergence of the entire lineage. Moreover, Diversification of the core Lactucinae, which also orig- as can be expected, strong differences in the age estimates inated in the SW Asian–European region, started at least are found in cases of different sister-​group relationships. 12.2–12.7 mya, following the Middle Miocene climate transi- Cephalorrhynchus picridiformis, nested in the Lactuca line- tion with its sharp decline in temperature (Potter & Szatmari, age in both reconstructions, provides an example. In the nrITS 2009). While the Cicerbita lineage diversified in the SW tree this species is sister to the L. perennis clade (Fig. 3) and Asian–European region around 5–8.7 mya, apparently with for its stem node a median age estimate of 6.9 my was found. a preference for tall forb communities, the Kovalevskiella- In the plastid DNA tree it is sister to the L. inermis clade, and Lactuca-Notoseris-Melanoseris-Paraprenanthes clade, with a stem node age estimate of only 3.3 my was found. an origin in SW Asia, started diversifying no later than 11.2

Fig. 3. Dated phylogram of the Lactucinae (maximum clade credibility tree, posterior probabilities ≥ 0.9 in italics and ages of nodes [my] as ◄ median node heights in blue, red arrow heads numbered 1 and 2 indicate the two calibrated nodes) based on the nrITS dataset and obtained with BEAST (Drummond & Rambaut, 2007) with probabilities of alternative ancestral ranges of nodes (in coloured pie charts) obtained with the Bayesian binary analysis implemented in RASP (Yu & al., 2015) shown for statistically supported basal nodes only. Insets illustrate (a) the colour codes of the possible ancestral ranges at the different nodes (black denotes other ancestral ranges) and (b) the coding of the distribution area units adapted from the TDWG level 2 units (Brummitt, 2001): A, N + Central + E Europe (TDWG units 10, 11 & 14); B, SW + SE Europe (TDWG units 12 & 13); C, N Africa + Macaronesia (TDWG units 20 & 21); D, Tropical Africa (TDWG units 22–26); E, S Africa (TDWG unit 27); F, Asia-Temperate W (TDWG units 32–35); G, Siberia + Russian Far East (TDWG units 30 & 31); H, Asia-Temperate E (TDWG units 36–38); I, Indian Subcontinent (TDWG unit 40); J, Asia-Tropical E (TDWG units 41–43); K, N America (TDWG unit 8); L, Central America + Caribbean (TDWG units 80 & 81).

688 Version of Record Lactuca graminifolia LAC 155 TAXON 0.2 66 (3) • June 2017: 675–703 Kilian & al. • Diversification1 0.4 Lactuca of the hirsuta Lactucinae HQ172901 (Compositae) 0.8 Lactuca canadensis HQ161956 1 2.3 Lactuca ludoviciana LAC 153 G 1 2.6 Lactuca floridana LAC 154 4.3 Lactuca biennis HQ161959 A Cicerbita plumieri LAC 186 1 Lactuca dolichophylla LAC 117 K B 7.3 0.96 2.6 5.1 Lactuca dissecta LAC 116 H 0.99 Lactuca tuberosa LAC 180 F 6.4 1 C 0.92 Lactuca inermis LAC 119 I 7.7 1.9 Lactuca tenerrima LAC 171 J 1 Lactuca glauciifolia LAC 184 1 1.2 D 1 3.2 Lactuca undulata LAC 115 8.2 Lactuca intricata LAC 183 6.9 2.5 Lactuca perennis LAC 334 Cephalorrhynchus picridiformis LAC 185 1 Cicerbita macrophylla LAC 190 1.3 Cicerbita racemosa LAC 187 1 Lactuca tetrantha LAC 167 0.7 Lactuca alpestris LAC 168 0.96 1 1.1 Lactuca acanthifolia LAC 162 9 0.4 0.96 1.4 Lactuca viminea LAC 127 5.2 Lactuca orientalis LAC 126 1 Lactuca sibirica LAC 128 2.7 Lactuca tatarica LAC 248 1 1 6.1 1.5 Lactuca virosa LAC 246 1 3 Lactuca georgica LAC 164 1 1 3.4 Lactuca serriola LAC 247 9.8 8.1 4.9 Lactuca saligna LAC 281 Lactuca quercina LAC 165 0.94 1 0.9 Lactuca raddeana LAC 124 1.9 Lactuca formosana LAC 122 Lactuca indica LAC 170 1 0.9 Lactuca glareosa LAC 196 2 Lactuca oyukludaghensis LAC 191 1 Cicerbita deltoidea LAC 194 2.4 Lactuca aurea LAC 193 * 1.5 Lactuca variabilis LAC 265 Melanoseris likiangensis LAC 098 0.2 Melanoseris monocephala LAC 231 A 0.99 0.4 0.99 0.7 Melanoseris atropurpurea LAC 101 1.3 Melanoseris macrantha LAC 075 0.96 2.1 Melanosris henryi LAC 280 AB 1 Melanoseris graciliflora LAC 112 3 1.4 Melanoseris tenuis LAC 106 1 Melanoseris cyanea LAC 089 0.93 1.8 Melanoseris qinghaica LAC 078 ABF 3.6 1 1.2 Melanoseris macrorhiza LAC 074 1 2.4 Melanoseris violifolia LAC 254 4.3 2.7 Melanoseris lessertiana LAC 071 AF 0.92 Prenanthes sumatrana LAC 279 6.9 1 Melanoseris decipiens LAC 256 2.4 Melanoseris brunoniana LAC 255 11.2 Melanoseris souliei LAC 069 B 1 1 1.8 Cephalorrhynchus brassicifolius LAC 218 0.93 2.8 Cephalorrhynchus polycladus LAC 219 4.6 Cephalorrhynchus takhtadzhianii LAC 234 BF 1 Cephalorrhynchus soongoricus LAC 210 8 5.5 1 1 0.1 Cicerbita bourgaei LAC 260 2.2 Cicerbita bourgaei LAC 150 F Cicerbita prenanthoides LAC 230 6.5 1 1.9 Steptorhamphus pumilus LAC 261 2.5 Lactuca rosularis LAC 209 FH 1 5 Lactuca hazaranensis LAC 262 7 5.8 Cicerbita tianschanica LAC 263 1 Melanoseris bracteata LAC 244 9 0.98 1 2.1 Steptorhamphus persicus LAC 284 4.4 Steptorhamphus crassicaulis LAC 223 Cephalorrhynchus kossinskyi LAC 217 0.39 Lactuca lasiorhiza LAC 199 0.44 3 Lactuca calophylla LAC 200 3.60.51 1 2.8 Lactuca praecox T1 LAC 202 4.2 Lactuca setosa LAC 201 1 1 Lactuca ugandensis LAC 206 7 1.8 Lactuca schulzeana LAC 205 1 Lactuca attenuata LAC 197 11.7 2.3 Lactuca paradoxa LAC 198 10.8 Paraprenanthes sororia LAC 037 0.2 Paraprenanthes diversifolia LAC 041 1 0.5 Paraprenanthes prenanthoides LAC 035 1 0.1 0.99 Paraprenanthes meridionalis LAC 036 3.9 Paraprenanthes yunnanensis LAC 044 1 1 Paraprenanthes melanantha LAC 046 5.9 1.2 1 Paraprenanthes wilsonii LAC 049 8.5 Paraprenanthes parishii LAC 028 1 Paraprenanthes oligolepis LAC 020 4.7 Paraprenanthes triflora LAC 266 1 Notoseris macilenta LAC 064 10.1 1 0.1 2 1 Notoseris henryi LAC 056 0.94 2.9 Notoseris khasiana LAC 066 1 1 3.2 Notoseris triflora LAC 059 12.2 4.2 Notoseris yakoensis LAC 054 Notoseris scandens LAC 147 1 1 0.2 Kovalevskiella kovalevskiana LAC 241 1 0.6 Kovalevskiella rosea LAC 232 1.7 Kovalevskiella zerawschanica LAC 238 4.3 Lactuca mira LAC 267 4.2 Cicerbita azurea LAC 015 1 Lactuca alaica LAC 268 5.2 1 Chaetoseris roborowskii LAC 216 2.8 Stenoseris auriculiformis LAC 017 1 1 Cephalorrhynchus kirpicznikovii LAC 235 13.4 0.4 0.95 1.7 Cephalorrhynchus microcephalus LAC 326 1 2.6 Cephalorrhynchus subplumosus LAC 226 3.6 Cephalorrhynchus cypricus LAC 215 7.3 Cephalorrhynchus hispidus LAC 214 1 Mycelis muralis LAC 274 8.7 1 1 1 2.7 Cicerbita petiolata LAC 271 4.3 Cicerbita pancicii LAC 272 17 Cicerbita alpina LAC 133 1 1 3.6 Faberia sinensis LAC 006 5.77 Faberia faberi LAC 010 Faberia nanchuanensis LAC 005 11.3 1 19 Lactuca triquetra LAC 278 0.6 Lactuca triquetra LAC 286 Prenanthes purpurea LAC 013 1 9.1 Hypochaeris maculata LAC 287 10.4 Hypochaeris radicata LAC 283 16.4 Leontodon tuberosus LAC 134 Prenanthes abietina LAC 294 1 1.3 Nabalus trifoliolata LAC 288 3.6 Nabalus altissima LAC 299 1 Nabalus sagittatus HQ161964 20.8 4.1 1 Soroseris glomerata LAC 297 1 0.3 3.1 Soroseris erysimoides LAC 004 5 Sonchella dentata LAC 300 Nabalus tatarinowii LAC 289 8.9 4.1 Dubyaea hispida LAC 298 1 10.2 Ixeris chinensis subsp. versicolor LAC 291 1 Lagoseriopsis popovii LAC 290 24.9 12.9 1 4 Crepis multicaulis LAC 003 11.3 Crepis sancta LAC 293 18.11 Youngia cineripappa LAC 292 9.3 Crepidiastrum tenuifolium LAC 295 1 Reichardia dichotoma LAC 181 11 Launaea sarmentosa LAC 001 Scorzonera hispanica LAC 296

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(8.5–13.7) mya (estimate from plastid DNA analysis) and mi- E Asian lineage of the subtribe and presumably also contributed grated eastwards. The Notoseris-Paraprenanthes lineages with the Lactucinae ancestor to the E Asian hybridogenous Faberia an origin in E Asia diverged first, around 11.2 (8.5–13.7) mya lineage, which arose around 9.1 (5.5–12.9) mya (estimate from (estimate from plastid DNA analysis). Its ancestors likely had plastid DNA analysis) and has the same habitat preference. The migrated along the Alpine-Himalayan orogenic belt, and diver- Kovalevskiella lineage originated in SW Asia and diversified sified from 7–8.5 mya onwards. Almost all members occupied from 2.8–5.2 mya onwards by migrating into Central Asia and moist montane habitats as tall forbs. They constitute the first further into E Asia, when adaptation, including to high montane

Table 2. Age estimates for relevant clades of the Lactucinae and relatives based on independent analyses of the nrITS and plastid DNA datasets. nrITS tree Plastid DNA tree Clade Node PP Median 95% HPD PP Median 95% HPD

Core Cichorieae clades Stem 24.9 22–30.1 24.4 22–28.9

C2H2L clade Crown [1] 20.8 15.1–26.1 [1] 20.2 14.6–25.1

Stem 18.1 12.4–23.5 18 13.2–22.6 Crepidinae 1 1 Crown 12.9 8.3–17.5 15.8 11–20.3

Stem 19 13.6–23.9 18 13.2–22.6 Lactucinae (with Prenanthes purpurea L.) [1] 1 Crown 17 12.1–21.5 16.1 11.7–20.3

Stem – – 9.1 5.5–12.9 Faberia Hemsl. lineage 1 1 Crown 5.8 2.7–9.1 2.5 0.5–5.1

Stem 13.4 9.8–16.7 16.1 11.7–20.3 Core Lactucinae [1] [1] Crown 12.2 9.4–14.7 12.7 9.9–14.7

Stem 12.2 9.4–14.7 12.7 9.9–14.7 Cicerbita Wallr. lineage 1 Crown 8.7 5.5–12 5 2.3–8 Kovalevskiella-Lactuca-Notoseris- Crown – – – 1 11.2 8.5–13.7 Melanoseris-Paraprenanthes clade

Paraprenanthes C.Shih lineage Crown 1 8.5 5.4–11.5 – – –

Notoseris C.Shih lineage Crown 1 4.3 2.1–6.6 – – –

Stem – – 11.2 8.5–13.7 Notoseris-Paraprenanthes lineage 1 Crown – – 7 4–10.3

Stem – – 9.9 7.3–12.4 Kovalevskiella Kamelin lineage 1 Crown 5.2 2.8–7.4 2.8 1.1–4.8

Stem – – 9.9 7.3–12.4 Lactuca-Melanoseris lineage – 0.9 Crown – – 9.4 6.9–11.8

Melanoseris Less. lineage Crown 1 9 6.5–11.6 – –

Stem – – 8 5.8–10.2 Lactuca L. lineage 1 1 Crown 9.8 7.1–12.3 7.4 5.3–9.4 Posterior probability (PP, bracketed numbers = topology constrained), the median and 95% highest posterior density (HPD) interval of the age [in my] for the stem nodes (as far as resolved with statistical support) and the crown nodes are given for the clades in the two resulting trees (Figs. 3, 4).

Fig. 4. Dated phylogram of the Lactucinae (maximum clade credibility tree, posterior probabilities ≥ 0.9 in italics and ages of nodes [my] as ◄ median node heights in blue, red arrow heads numbered 1 and 2 indicate the two calibrated nodes) based on the plastid DNA dataset and obtained with BEAST (Drummond & Rambaut, 2007) with probabilities of alternative ancestral ranges of nodes (in coloured pie charts) obtained with the Bayesian binary analysis implemented in RASP (Yu & al., 2015). Insets illustrate (a) the colour codes of the possible ancestral ranges at the different nodes (black denotes other ancestral ranges), and (b) the coding of the distribution area units adapted from the TDWG level 2 units (Brummitt, 2001), for details see caption of Fig. 3.

690 Version of Record 1 Lactuca dolichophylla LAC 117 TAXON 66 (3) • June 2017: 675–703 1Kilian & al. • Diversification1.4 of the Lactucinae (Compositae) 4.6 Lactuca dissecta LAC 116 Lactuca tuberosa LAC 180 Lactuca glareosa LAC 196 G 1 1.5 1.9 Lactuca oyukludaghensis LAC 191 A 6.4 1 3.3 Cicerbita deltoidea LAC 194 1 Lactuca aurea LAC 193 K B 1.5 Lactuca variabilis LAC 265 H 1 Cephalorrhynchus brassicifolius LAC 218 F 6.2 1 2.1 C 3 Cephalorrhynchus polycladus LAC 219 I Cephalorrhynchus takhtadzhianii LAC 234 J 1 3.7 1 Melanoseris bracteata LAC 244 D 4.1 Cicerbita macrophylla LAC 190 6.9 5.1 Cicerbita racemosa LAC 187 1 Lactuca ludoviciana LAC 153 1 1.1 Lactuca floridana LAC 154 0.99 1.8 4.4 Lactuca graminifolia LAC 155 1 Cicerbita plumieri LAC 186 5.4 1 Lactuca tenerrima LAC 171 1 1.5 3.3 Lactuca inermis LAC 119 Cephalorrhynchus picridiformis LAC 185 6.7 1 Lactuca sibirica LAC 128 1 2.3 3.9 Lactuca tatarica LAC 248 1 Lactuca quercina LAC 165 7.2 5.1 1 Cicerbita prenanthoides LAC 230 0.8 Cicerbita bourgaei LAC 260 Lactuca alpestris LAC 168 1 0.8 1 Lactuca viminea LAC 135 1 2 Lactuca tetrantha LAC 167 Lactuca acanthifolia LAC 162 2.2 1 Lactuca orientalis LAC 126 4.9 Lactuca serriola LAC 247 1 1 2 7.4 2.4 Lactuca saligna LAC 281 * 1 Lactuca georgica LAC 164 1.2 6.5 Lactuca virosa LAC 246 Lactuca formosana LAC 122 A 1 0.8 Lactuca raddeana LAC 124 1 1.6 4.8 Lactuca indica LAC 170 Cephalorrhynchus soongoricus LAC 210 AB Lactuca perennis LAC 334 1 2.5 1 2.8 Lactuca glauciifolia LAC 184 4.4 Lactuca undulata LAC 115 ABF Lactuca intricata LAC 183 1 Melanoseris tenuis LAC 106 1 0.2 Melanoseris graciliflora LAC 112 0.97 1 AF 8 1 Melanoseris likiangensis LAC 098 0.3 3.2 Melanoseris atropurpurea LAC 101 1 Melanoseris lessertiana LAC 071 1 B 1 Melanoseris qinghaica LAC 078 3.6 1 Melanoseris decipiens LAC 256 1 0.8 Melanoseris brunoniana LAC 255 1 1 1.3 BF 4.9 Melanoseris cyanea LAC 089 2.3 Melanoseris macrantha LAC 075 1 Melanoseris macrorhiza LAC 074 8.5 1 5.7 1.5 D 1 Melanoseris violifolia LAC 254 Cicerbita tianschanica LAC 263 6.4 Melanoseris souliei LAC 069 F Lactuca calophylla LAC 200 7.5 1 1.2 Lactuca praecox T1 LAC 202 1 1.6 3 Lactuca setosa LAC 201 1 1 Lactuca ugandensis LAC 206 FH 1 0.91 6 Lactuca schulzeana LAC 205 9.4 Lactuca paradoxa LAC 198 Steptorhamphus persicus LAC 284 FHI 1 0.2 1 0.8 Steptorhamphus crassicaulis LAC 223 2.6 Steptorhamphus crambifolius LAC 245 Caphalorrhynchus kossinskyi LAC 217 FI 1 Lactuca hazaranensis LAC 262 1 1 0.9 4.8 Lactuca rosularis LAC 209 9.9 Steptorhamphus pumilus LAC 261 G Kovalevskiella kovalevskiana LAC 241 1 0.1 Kovalevskiella zerawschanica LAC 238 0.3 2 Kovalevskiella rosea LAC 232 GH 0.99 Cicerbita azurea LAC 015 1 1 2.8 Lactuca alaica LAC 268 1 Stenoseris auriculiformis LAC 017 GHJ 1 1 Chaetoseris roborowskii LAC 216 1.6 Lactuca mira LAC 267 Paraprenanthes wilsonii LAC 049 GJ 1 0.2 11.2 1 0.4 Paraprenanthes prenanthoides LAC 035 1 0.6 Paraprenanthes meridionalis LAC 036 1.5 Paraprenanthes diversifolia LAC 041 H 2.4 Paraprenanthes yunnanensis LAC 044 0.94 Paraprenanthes sororia LAC 037 1.7 1 Paraprenanthes melanantha LAC 046 HI 2.8 1 Notoseris henryi LAC 056 1 0.1 1 1 Notoseris macilenta LAC 064 4 0.3 2 1.2 Notoseris triflora LAC 059 HIJ Notoseris khasiana LAC 066 6.3 1 Paraprenanthes parishii LAC 028 12.7 1 1 Notoseris yakoensis LAC 054 7 0.5 HJ Notoseris scandens LAC 147 1 Paraprenanthes triflora LAC 266 3.2 Paraprenanthes oligolepis LAC 020 1 I Cephalorrhynchus microcephalus LAC 236 0.98 0.2 1.1 Cephalorrhynchus kirpicznikovii LAC 235 1 1 Cephalorrhynchus hispidus LAC 240 16.1 1.70.99 Cephalorrhynchus subplumosus LAC 226 K 1 1 0.3 3.7 Cephalorrhynchus hispidus LAC 214 0.6 Cephalorrhynchus cypricus LAC 215 1 5 Mycelis muralis LAC 274 1 Cicerbita pancicii LAC 272 0.99 1.3 2.9 Cicerbita alpina LAC 133 Cicerbita petiolata LAC 271 Prenanthes purpurea LAC 013 1 Nabalus tatarinowii LAC 289 1 3.5 1 5.2 Nabalus trifoliolata LAC 288 18 7.4 Soroseris erysimoides LAC 004 1 Lagoseriopsis popovii LAC 290 9.1 1 Faberia faberi LAC 010 1 0.4 1 2.5 Faberia sinensis LAC 006 12.2 Faberia nanchuanensis LAC 005 1 Crepidiastrum tenuifolium LAC 295 7 13.5 11.4 Youngia cineripappa LAC 292 19.2 Ixeris chinensis subsp. versicolor LAC 291 1 14.5 0.8 Lactuca triquetra LAC 278 1 Lactuca triquetra LAC 286 15.8 Prenanthes abietina LAC 294 1 1 Crepis multicaulis LAC 003 20.2 7 Crepis sancta LAC 293 1 Hypochaeris radicata LAC 283 1 1 8.1 12.1 Hypochaeris maculata LAC 287 24.4 Leontodon tuberosus LAC 002 1 Launaea sarmentosa LAC 001 10.9 Reichardia dichotoma LAC 181 Scorzonera hispanica LAC 296

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habitats, took place. The Melanoseris and the Lactuca lineages congener of P. purpurea, and, moreover, there is no evidence also originated in SW Asia, split from their common ancestor for its classification in the Lactucinae, nor unequivocal sup- around 9.9 (7.3–12.4) mya (estimate from plastid DNA analysis) port for its classification in the Crepidinae. Consequently, the with crown nodes estimated to be 7.4–9.8 mya (Lactuca) and systematic position of this orphan lineage is pending further 9–9.4 mya (Melanoseris). One clade of Melanoseris took a new studies (A. Sennikov & al., in prep.). route that had become available through the final closure of the (2) The peculiar Lactuca triquetra, originally also de- former Tethys Sea by the drift of the African-Arabian against scribed as a species of Prenanthes, is endemic to Cyprus and the Asian plate in the Middle (Hamon & al., 2013) or early Late Lebanon (Kilian & Hand, 2004). In the nrITS phylogeny it ap- Miocene (Rögl, 1999) at 11–14 mya, and after Late Miocene pears in a polytomy with the core Lactucinae and Faberia, while orogeny had created a suitable corridor. They migrated into it is nested in the Crepidinae clade in the plastid DNA phylogeny tropical E Africa, subsequently spreading and diversifying in like Faberia, but in an even earlier diverging position. Ecology, all tropical Africa, most of them becoming adapted to savan- taxonomy and threat status of this species are subject of a sepa- nah habitats. A second clade, which diverged at around the rate forthcoming paper (N. Kilian & al., in prep.). same time as the first, migrated eastwards, started diversifying (3) Our placement of the allotetraploid (2n = 34 chromo- 6.4–6.9 mya in the Sino-Himalayan region with a preference somes) Chinese Faberia in a polytomy with the core Lactucinae for moist montane habitats, then further migrated into SE Asia and Lactuca triquetra in the nrITS phylogeny, while in the and, finally, reached Sumatra and Java. In contrast, the Lactuca Crepidinae clade in the plastid DNA phylogeny (see also Wang lineage diversified mainly in the SW Asian–S European region. & al., 2013), is in accord with and explained by further strong Its diversification seems to have been more affected by drier evidence from karyology (Liu & al., 2012; Liu & Ren, 2013; Liu climatic conditions than any other lineage. Only a minority of & Yang, 2014) and molecular phylogenetics (Liu & al., 2013; species of the Lactuca lineage are tall forbs of moist montane Wang & al., 2014) for the origin of this lineage by reticulate habitats. Migrations beyond the SW Asian–S European region evolution. The maternal ancestor (with 2n = 16 chromosomes) are represented by four clades: the L. indica clade, which split was apparently contributed by the Crepidinae, while the pater- off 6.5–8.1 mya and diversified in E Asia rather recently, the nal ancestor (with 2n = 18 chromosomes) by the Lactucinae. In L. tatarica (L.) C.A.Mey. clade, which split off 3.9–5.2 mya, view of the early split of the Notoseris-Paraprenanthes lineage migrated into Central and NE Asia and finally into North around 11.2 (8.5–13.7) mya (age estimate from plastid DNA America, the North American L. graminifolia Michx. clade, analysis) and their assumed migration into E Asia along the which diverged 4.3–4.4 mya from a European ancestor, and fi- Alpine-Himalaya-corridor, it is most likely that the parental nally the L. inermis Forssk. clade, which diverged 1.5–1.9 mya ancestor is from this lineage. and spread into tropical Africa. In contrast to Faberia, the monospecific Prenanthes abi- The historical biogeography of the Lactucinae as a line- etina and Lactuca triquetra lineages appear systematically age of sub-Paratethyan origin remarkably contrasts with the isolated in both the nuclear and plastid DNA trees. They are Hyoseridinae of the same major Cichorieae clade (C2H2L diploids with the plesiomorphic chromosome number of 2n = 18 main clade). The present-day distribution of the Hyoseridinae (here and in the following, chromosome number records refer in SW Asia and the Mediterranean region exemplifies an essen- to Watanabe, 2016) and present in SW Asia, which is indicated tially sub-Tethyan pattern, suggesting a corresponding origin. in our biogeographical analysis as (part of) the most likely This is most pronouncedly visible in Launaea (Kilian, 1997: ancestral area of the subtribe. They seem to have branched off fig. 1), which is completely absent from SE Europe and from at rather early stages of the rapid diversification of the C2H2L SW Asia northwest of 35°N, 45°E, and thus from Turkey and clade into its present subtribal lineages. Our age estimates in the Caucasus. The present-day distribution of the Lactucinae fact indicate that the P. abietina lineage split off in the Middle in SW Asia and the Mediterranean region in contrast is still Miocene (around 14.5 [plastid DNA] and 16.4 mya [nrITS]), in dominated by a sub-Paratethyan pattern, with few range expan- the same period where the C2H2L main clade of the Cichorieae sions at the eastern and western edges into the area to the south diversified (see Figs. 3 and 4 and Tremetsberger & al., 2012). of the Mediterranean Sea, the Cyrenaican endemic Lactuca L. triquetra and Faberia seem to be younger, the L. triquetra haimanniana E.A.Durand & Barratte being an odd exception. lineage having diverged between 11.3 (nrITS) and 13.5 (plastid Proto-Lactucinae and orphan lineages. — After confirma- DNA) mya, the Faberia lineage between 9.1 (plastid DNA) and tion of Prenanthes purpurea as a member of the Lactucinae in 11.3 (nrITS) mya. Both, thus, likely have evolved only after this study, three taxa that have been assigned to the Lactuca al- subtribes Lactucinae and Crepidinae had come into existence. liance (in the sense of Wang & al., 2013) for morphological rea- Future investigations will need to focus on the early diversifi- sons, still do not consistently appear nested in the Lactucinae cation of the C2H2L main clade by including all the orphan lin- clade in our phylogenetic reconstructions: eages associated with that clade (such as, e.g., Blanca (1) The Caucasian Prenanthes abietina appears in a ba- & C.Díaz, see Blanca & Díaz, 1985 and Fernández-Mazuecos sal polytomy with the core-Lactucinae-Faberia-Lactuca tri- & al., 2016). quetra-Prenanthes purpurea clade 1 and the Hypochaeridinae Prenanthoid Crepidinae: Nabalus. — Nabalus Cass., ac- clade in the nrITS phylogeny. In contrast, it is nested in the commodating Eastern Asian–North American species oth- Crepidinae clade in the plastid DNA phylogeny (with good erwise placed in Prenanthes, was treated as a of the statistical support in BA and ML). Hence, P. abietina is no Lactucinae by Bremer (1994) and Lack (2006). Kilian & al.

692 Version of Record TAXON 66 (3) • June 2017: 675–703 Kilian & al. • Diversification of the Lactucinae (Compositae)

(2009a) transferred Nabalus to the Crepidinae based on molec- lineage consists of two sister clades. One includes the wide- ular phylogenetic results, which confirmed the conclusion by spread European C. alpina L. (Stachurska-Swakoń & al., 2012), Stebbins (1940) that Nabalus is much closer to Dubyaea DC. providing the type of the name Cicerbita, the Balkan endemic and Soroseris Stebbins than to Prenanthes purpurea with re- C. pancicii (Vis.) Beauverd and the Caucasian-Turkish C. pet- spect to achene vascularisation. Its separation from Prenanthes iolata (K.Koch) Gagnidze. The other has two subclades, one and placement in the Crepidinae was corroborated by Zhang represented by Mycelis muralis (L.) Wallr., providing the type & al. (2011a) for Chinese, and by Schilling & al. (2015) for of the name Mycelis Cass. and being widespread in SW Asia North American Nabalus species. Zhang & al. (2011a) also and Europe (Clabby & Osborne, 1999), the other represented corroborated the relationship with Soroseris and Dubyaea by a few closely related and partly doubtfully distinct, chiefly and, moreover, provided a first indication that Nabalus is not SW Asian species of Cephalorrhynchus Boiss., among them the monophyletic, since the Chinese Nabalus species group in a SE European–SW Asian C. hispidus (DC.) Boiss., of which the different subclade of the Syncalathium clade than the North taxonomic synonym C. glandulosus Boiss. provides the type American N. altissimus (L.) Hook. Our nrITS tree (Fig. 1) of the name Cephalorrhynchus. shows that the eastern to southeastern North American N. tri- The members of this lineage are perennial herbs or tall forbs foliolatus Cass., which provides the type of the generic name of predominantly moist montane to subalpine habitats. As far (Flann & al., 2010), and N. altissimus, are sister to the clade as chromosome numbers are known, they all have 2n = 18. The that unites Soroseris and Sonchella Sennikov. The northwest- lineage has its origin in the SW Asian–European region accord- ern North American N. sagittatus (A.Gray) Rydb. is sister to ing to our biogeographic analysis (Figs. 3, 4) and we hypothesise these two clades. The Chinese N. tatarinowii (Maxim.) Nakai its ancestral area to range from SE Europe across Turkey to the again is sister to Dubyaea hispida DC., and these two spe- Caucasus. Phylogeographic and population genetic analyses, cies in turn are sister to all aforementioned clades within this respectively, of Cicerbita alpina (Michl & al., 2010; Stachurska- group (Fig. 1). Thus, apparently two different lineages of the Swakoń & al., 2012) and Mycelis muralis (Chauvet & al., 2004), Dubyaea-Nabalus-Soroseris-Syncalathium main clade of the although focussing on their glacial history, do not contradict Crepidinae (see Zhang & al., 2011a) have migrated into North this assumption. The age estimates for the split between the America, one diversified into the eastern to southeastern true two main clades of the Cicerbita lineage (8.7 and 5 mya) and for Nabalus (see Schilling & al., 2015), the other is represented by the split between the Mycelis and Cephalorrhynchus subclades the northwestern species pair N. sagittatus and N. alatus Hook. (3.3 and 3.7 mya, respectively) significantly differ between the The systematics of the main clade, however, is still unresolved, nrITS and the plastid DNA analyses, which is likely due to the so its generic reclassification would be premature. hard topological incongruences within the Cephalorrhynchus Prenanthes lineage. — The assumption by Kilian & and the Cicerbita subclades (see above). Gemeinholzer (2007), Kilian & al. (2009a) and Wang & al. Notoseris and Paraprenanthes lineages. — The phylogeny (2013) that Prenanthes purpurea forms an isolated lineage of of these lineages, centred in SW China, which are the sister its own, is confirmed in the present study. It has successively group to the clade comprising Kovalevskiella, Lactuca and been shown in the past that other species previously included Melanoseris according to the plastid DNA tree, has been dis- in Prenanthes are entirely unrelated (for a summary see Kilian cussed in detail in our previous paper (Wang & al., 2013). The & Gemeinholzer, 2007, and see also Wang & al., 2013). Now species of these lineages are usually tall forbs of the moist mon- we can confirm the exclusion of the last potential candidates tane zone, two are scandent perennial herbs overgrowing forest from that lineage: for the American Prenanthes species and the edges. As far as is currently known, their chromosome number Caucasian P. abietina see above; for the scandent Prenanthes is 2n = 18. The two scandent Notoseris species represent an species of Sumatra and Java and the lineage that for morpholog- early-diverging lineage according to the plastid DNA tree (Fig. ical reasons includes the tropical African P. subpeltata Stebbins 2, 4). The stem node age is around 6.3 my (plastid DNA), but see below under Melanoseris lineage. Since P. purpurea pro- this is significantly lower in the nrITS analysis (around 4.2 my), vides the type of the generic name Prenanthes, this genus, corresponding to the topological differences. It can be assumed contrary to traditional views, is consequently monospecific. that evolution of the scandent habit has been triggered by for- The emergence of the Prenanthes lineage some 16–17 mya ests covering the moist montane habitats of these hygrophilous precedes the diversification of the bulk of the subtribe by a few species, pushing them to natural forest edges. Increased climate million years. Prenanthes purpurea is a rhizomatous perennial cooling during and beyond the Late Miocene may then have herb native to S, W and Central Europa and northwestern SW opened further habitats suitable for tall herbs. Asia, which grows at edges and in openings of and mixed Kovalevskiella lineage. — This lineage was first recog- forests of the colline and montane zone. Its habitat preferences nised and provisionally referred to as “Cicerbita II lineage” thus show similarity to that of the early-diverging lineages of by Wang & al. (2013). Our study now provides the first com- the core Lactucinae. prehensive phylogeny of this chiefly Central Asian clade. It Cicerbita lineage. — The Cicerbita lineage received strong receives very strong support in both the nuclear and plastid statistical support and consistently is sister to the remainder of DNA phylogenies (Figs. 1, 2) and has diversified from 5.2 to the core Lactucinae in the nuclear and plastid DNA phylogenies 2.8 mya onwards (median age estimates from the nrITS and (Figs. 1, 2), corroborating the results by Wang & al. (2013) that plastid DNA analyses, Table 2) after migration from SW to were based on a much more restricted sampling. The Cicerbita Central Asia. The lineage includes Kovalevskiella (Kamelin,

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1993), a genus based on Cicerbita subg. Poicilachena Kirp., (median age estimates from the nrITS and plastid DNA anal- the name of which is typified by C. zeravschanica Kovalevsk. yses). The topology of clade J1 is fully in line with the recent (≡ Kovalevskiella zeravschanica (Kovalevsk.) Kamelin). findings by Wei & al. (2016, 2017) based on plastid DNA. Kamelin (1993) recognised six species in Kovalevskiella, (2) Clade J2 is largely restricted to SW and Central Asian distributed in Central Asia and extending to SW Asia and mountain ranges. Only Melanoseris bracteata (C.B.Clarke) the Himalayas, but the genus is biphyletic as our analysis N.Kilian is distributed in the Himalayas. The clade started shows. Only K. zeravschanica, K. rosea (Popov & Vved.) diversifying around 7 mya (nrITS tree) and consists of a Kamelin and K. kovalevskiana (Kirp.) Kamelin form a sub- smaller clade J2a and a larger polytomy. The smaller clade J2a clade of this lineage, whereas the three Western Himalayan unites species of Steptorhamphus Bunge (including S. cram- species K. decipiens (C.B.Clarke) Kamelin, K. aitchisoniana bifolius Bunge, which provides the type of that generic name; (Beauverd) Kamelin and K. rapunculoides (DC.) Kamelin are only represented in the plastid DNA dataset, see Fig. 2) with nested in the Melanoseris lineage. The true Kovalevskiella spe- Cephalorrhynchus kossinskyi (Krasch.) Kirp. as their sister. cies are mesic or hygrophilous, medium-sized perennial herbs The larger polytomous clade, which has no statistical support, of open woodlands, deciduous forests and meadows endemic includes the terminal clades J2b to J2g. Clade J2b comprises to the Pamiro-Alai and western Tian Shan mountain ranges only the Central Asian Cicerbita thianschanica (Regel & and have diversified only very recently (< 0.5 mya, see Figs. Schmalh.) Beauverd, J2c comprises the Himalayan M. bracte- 3, 4). They are sister to the acaulescent, scree inhabiting high ata and J2d includes the Lactuca rosularis Boiss. group (Kilian montane Lactuca mira Pavlov, which is a narrow endemic of & al., 2012), which is confined to the Iranian-Afghan highlands. the Western Tian Shan, in the nrITS tree (Fig. 1). The second J2e includes the closely related Caucasian-Turkish Cicerbita subclade in the nrITS tree includes Chaetoseris roborowskii bourgaei Boiss. and C. prenanthoides (M.Bieb.) Beauverd. It (Maxim.) C.Shih and Stenoseris auriculiformis C.Shih, two is, without support, sister to the well-supported clade includ- montane to high montane perennial herbs from N to NW China, ing the terminal clades J2f and J2g with SW to Central Asian and the little known Pamiro-Alai endemic Lactuca alaica species assigned to Cephalorrhynchus (Kirpicznikov, 1964; Kovalevsk., a tiny perennial, blue-flowered herb of alpine Tuisl, 1968). Chromosome numbers have been established for stone runs, where it grows among boulders. The third subclade several species; accordingly, subclades J2a, J2c and J2e have in the nrITS tree only includes the rather widespread Central 2n = 16, the only member of J2g counted, Cephalorrhynchus Asian Cicerbita azurea (Ledeb.) Beauverd, a perennial herb polycladus (Boiss.) Kirp., has 2n = 18 chromosomes. of montane deciduous and Pinus sibirica forests. The rela- (3) The Sino-Himalayan–SE Asian clade J3 mainly in- tionships among the three subclades are unresolved and their cludes the Sino-Himalayan members, which were treated in topologies differ in both reconstructions (Figs. 1, 2). The only some detail in our previous paper (Wang & al., 2013), where chromosome number known from this lineage is 2n = 16 for we hypothesised that extensive hybridisation and introgression K. zeravschanica. takes place among them. The clade started diversifying around Melanoseris lineage. — This lineage has been consid- 6.4–7 mya (median age estimates from the nrITS and plastid ered in our previous paper (Wang & al., 2013) with its Sino- DNA analyses). Four terminal clades or species groups can be Himalayan members only. Our global sampling now shows recognised in the polytomous main clade of J3. Statistical sup- that the Melanoseris lineage spans a distribution range from port for this topology is, however, weak: the Melanoseris gra- tropical Africa across SW and Central Asia to E and SE Asia. ciliflora (DC.) N.Kilian group, the M. atropurpurea (Franch.) Regarding species numbers, it is of a size similar to the Lactuca N.Kilian & Ze H.Wang group, the M. macrorhiza (Royle) lineage. The lineage started diversifying around 9 mya (Figs. N.Kilian group and the M. cyanea (D. Don) Edgew. group. 3, 4; Table 2) and is formed of three statistically well-supported The newly sampled, very rare and neglected M. monocephala major clades of very distinct geographical distribution. (C.C.Chang) Ze H.Wang (Wang & al., 2015) and M. henryi (1) Clade J1 is sister to the other two and includes exclu- (Dunn) N.Kilian (Zhang & al., 2016) group with the M. atro- sively species from tropical Africa. Its topology is essentially purpurea clade. A small group of little-known, mostly scan- the same in the nrITS and the plastid DNA trees. The clade dent species from high mountain forests of the Indonesian originated with high probability from SW Asia and split off islands Sumatra and Java is represented for the first time, around 7.5–9 mya (median age estimates from the nrITS and by Prenanthes sumatrana Tjitr. This species groups with the plastid DNA analyses). With the odd exception of Prenanthes Himalayan M. macrorhiza clade and its lineage seems to have subpeltata, the members of the African clade have so far unan- diverged around 2.7 mya (estimated median age, nrITS, see imously been assigned to Lactuca. The scandent species of Fig. 3). This also indicates that the scandent habit has evolved Afromontane forests (Stebbins, 1937; Jeffrey, 1966), to which three times independently in the Lactucinae: twice, in East also P. subpeltata belongs, are represented in our sampling by Tropical Africa and Melanesia, in the Melanoseris lineage, and L. attenuata Stebbins and L. paradoxa A.Rich. Chromosome a third time in subtropical southern China in the Notoseris line- numbers are known for two species with 2n = 16 and 32, re- age. The Western Himalayan M. decipiens (Hook.f. & Thomson spectively. The African scandent species form a sister clade to ex C.B.Clarke) N.Kilian & Ze H.Wang and M. brunoniana the rest of the tropical African species, which predominantly (Wall. ex DC.) N.Kilian & Ze H.Wang, form a well-supported inhabit savannahs. Of the latter, the only chromosome number sister clade to the polytomous main clade of J3. The high-alpine known is 2n = 18. The two clades diverged around 6–7 mya acaulescent M. souliei (Franch.) N.Kilian, alternatively treated

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as member of a genus of its own, Parasyncalathium souliei DNA analysis. The species composition of this clade is identical (Franch.) J.W.Zhang & al. (Zhang & al., 2011b), is sister, with in the nrITS and plastid DNA trees. Its species have 2n = 16 full support, to all other members of clade J3. This taxon is chromosomes, share many morphological features but have here shown to be deeply nested in the Melanoseris lineage, thus far never been considered to belong to one taxonomic unit. both in the nrITS and the plastid DNA phylogeny (Figs. 1, 2). Instead they were treated as species of Prenanthes, Cicerbita, Chromosome counts known of members of clade J3 all revealed Cephalorrhynchus, Mycelis, Lactuca and Lactucopsis. 2n = 16. The long branches of the M. souliei clade in both trees (2) The Lactuca indica clade I2 of E Asian distribution likely reflect the shift from predominantly montane habitats diverged around 6.5–8.1 mya and includes four closely related of the Asian members of the Melanoseris lineage to a high species (Shih & Kilian, 2011; Wang & al., 2013). Its radiation, alpine environment with a major reorganisation in habit and in contrast, took place only around 1.6–1.9 mya (Figs. 3, 4). All synflorescence architecture, which has happened, in contrast species have 2n = 18 chromosomes. It is sister to the following to the Crepidinae (e.g., Babcock & Stebbins, 1937; Stebbins, clade in the nrITS tree but forms a sister group with the mono- 1940; Enke & al., 2008; Zhang & al., 2011b), only exceptionally specific Cephalorrhynchus soongoricus (Regel) Kovalevsk. in the Lactucinae (see also Kovalevskiella). clade of the Melanoseris lineage in the plastid DNA tree. The incongruences in the topologies of the nrITS tree (3) The Lactuca sativa-quercina-viminea-tatarica clade and the plastid DNA tree with respect to the composition of I3 of predominantly SE European–Mediterranean–SW Asian the three Melanoseris clades mainly affect the SW to Central distribution and the L. indica clade I2 form the second main Asian clade J2 (compare Figs. 1 and 2). The general topology clade of the Lactuca lineage in the nrITS tree. All species have of the Melanoseris and Lactuca clades in the plastid DNA tree 2n = 18 chromosomes and its MRCA is dated to around 6.1 mya (Fig. 2) is very robust with respect to sampling, although not (Fig. 3). This clade is one of the few clades not present in the all of the deeper sister-​group relationships have very strong plastid DNA tree. There, its members are divided between two statistical support. The basic structure can already be seen in clades: one with the MRCA dated to 3.9 mya is formed of the the plastid DNA tree by Wang & al. (2013: fig. 2). Addition of L. tatarica subclade I3a with the L. quercina subclade I3c as African Melanoseris members always resulted in the central sister; the other, with the MRCA dated to 4.9 mya, is composed trichotomy seen in Fig. 2 of the L. sativa subclade I3d with the L. viminea subclade I3b Lactuca lineage. — The circumscription of Lactuca has as sister (Fig. 4). The analyses by Wang & al. (2013) revealed ranged from extremely broad to extremely narrow concepts, the same principal topological differences, and the ML tree of as has been reviewed by, e.g., Ferákova (1977), Koopman & al. the entire chloroplast genome by Wei & al. (2016) also corrob- (1998) and Wang & al. (2013). The uncertainty of its delimita- orates the sister-​group relationship between the L. sativa and tion reflects the difficulties to employ diagnostic morphological L. viminea subclades, while the sampling of both did not include characters for the classification of the Lactuca alliance, as has L. quercina. The clustering of the L. sativa and L. viminea sub- been briefly elucidated for several Lactuca segregates by Wang clades in both the nrITS and plastid DNA trees indicates that & al. (2013). All morphological characters used as diagnostic the L. viminea subclade s closer to the primary and secondary for generic classification in the Lactuca alliance are homoplas- lettuce gene pools (Harlan & Wet, 1971) represented by the wild tic as will be shown in a subsequent publication on character members of the L. sativa subclade than is any other of the sub- state evolution (N. Kilian & al., in prep.). In the present paper, clades. However, crossability of L. viminea subclade members we refer, as in the case of the other major clades, purposely to (sometimes generically segregated as Scariola F.W.Schmidt) the Lactuca lineage, because we consider it premature to equate with those of the L. sativa subclade has never been tested in these lineages with generic concepts. the extensive crossing experiments within Lactuca (Thompson The Lactuca lineage is composed of nine well-supported & al., 1941; Thompson, 1943; Lindqvist, 1960; Vries, 1990; terminal clades in the nrITS tree. With few exceptions, these Koopman & al., 2001). In the ML tree based on entire chloro- are also present in the plastid DNA tree, where, however, rela- plast genomes by Wei & al. (2016) the L. tatarica clade appears tionships differ or are not resolved. The three main clades in in a sister-group​ relationship with the L. indica clade I2, instead the nrITS tree are the L. aurea clade I1, which is sister to the of with the L. sativa-quercina-​ viminea​ clade as in the nrITS tree. remainder of the Lactuca lineage, the core Lactuca clade com- Crossing experiments in fact corroborate a closer relationship prising clades I2 and I3, and its sister clade, comprising clades of L. tatarica to L. indica than to L. sativa: while L. tatarica I4 to I9. A recently published ML analysis by Wei & al. (2016), produced fertile hybrids with the only other member of its own using the entire chloroplast genome of 19 samples of the Lactuca subclade, L. sibirica­ (Koopman & al., 2001), and sterile hybrids lineage plus some species of the African Melanoseris lineage with the L. indica clade (Thompson & al., 1941), crosses be- and outgroup taxa, could also resolve deeper nodes with full tween L. tatarica and L. sativa subclade members completely support. Accordingly, plastid DNA supports the nrITS results failed (Thompson & al., 1941). Moreover, the L. tatarica sub- of a core Lactuca clade comprising clades I2 and I3 and with a clade appears in a sister-​group relationship with the Cicerbita sister clade comprising the member of the nrITS clades I4 to I9. bourgaei subclade J2e of the Melanoseris lineage in our plastid (1) The Lactuca aurea clade I1 is of NE Mediterranean DNA tree. This sister-​group relationship is remarkable when distribution. It is the first lineage that diverged, already around considering that crosses between L. tatarica (2n = 18) and 9.8 mya (nrITS), whereas its radiation appears to be rather re- Cicerbita bourgaei (2n =16) produced vivid but sterile hybrids cent, dated to 2.4 mya in the nrITS and to 3.3 mya in the plastid (Thompson, 1943 under Lactuca marschallii Stebbins).

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(4) The clade I4 of Cicerbita racemosa (Willd.) Beauverd the Lactucinae into tropical Africa. This has taken place only (Lactuca racemosa Willd.) otherwise includes only Lactuca around 1.5–1.9 mya (median ages from nrITS and plastid DNA macrophylla (Willd.) A.Gray (Cicerbita macrophylla (Willd.) analyses of the split from L. tenerrima), and thus much later Wallr.) and has a chiefly SW Asian distribution. Both species than the migration of the Melanoseris lineage (Figs. 3, 4), and have 2n = 16 chromosomes. The clade diverged already around may have started in NW Africa. 8.2 mya (nrITS analysis, Fig. 3) and is sister to a polytomous (9) The Lactuca tuberosa clade I9 comprises the E main clade of the remaining terminal clades I5 to I9. Divergence Mediterranean–SW Asian L. tuberosa Jacq., the SW to Central of these two species occurred rather recently around 1.3 mya Asian L. dissecta D.Don and the SW Asian to Sino-Himalayan (nrITS analysis, Fig. 3). This clade is not present in the plastid L. dolichophylla Kitam. The species composition of this clade DNA tree (Figs. 2, 4) where the two species instead are consec- is identical in the nrITS and plastid DNA trees. This clade of utive sisters to a clade uniting the two Melanoseris subclades mesic perennial herbs with a chromosome number of 2n = 16 J2g and J2c. is of SW Asian origin from around 6.4 mya and has diversified (5) The Cephalorrhynchus picridiformis clade I5 is since 4.6–5.1 mya (median ages from nrITS and plastid DNA monospecific and restricted to Afghanistan and western analyses) while extending eastwards into the Himalayas. Pakistan. Cephalorrhynchus picridiformis (Boiss.) Tuisl is a Perspectives: Phylogeny and classification. — We have scoparious subshrub with 2n = 16 chromosomes. Being part of refrained in this study from drawing taxonomic conclusions the polytomous fourth main clade of the Lactuca lineage in the for the generic reclassification of the subtribe, since no satis- nrITS tree, the species appears as sister to the L. inermis clade fying solution is visible yet. We all agree with the statement in the plastid DNA tree, which is in good accordance with by one of us (Sennikov in Lazkov & al., 2014: 93), reflect- morphology. Divergence of the species is dated significantly ing the consequences of the taxonomic conclusions drawn by different, with 6.9 my in the nrITS and 3.3 my in the plastid Wang & al. (2013) from their phylogenetic reconstruction of DNA analysis, corresponding to the incongruent sister-​group the subtribe and applied by Shih & Kilian (2011). He noted relationships revealed, which suggest a hybrid nature of this that due to convergent character evolution the morphologi- species. cal differences between the genera defined according to the (6) The Lactuca perennis clade I6, to which also L. glaucii- molecular phylogenetic lineages have become rather incon- folia Boiss., L. intricata Boiss. and L. undulata Ledeb. belong, spicuous and impractical to use, since species in one genus is centred in the E Mediterranean and in SW Asia but extends may share more similarities with species in another genus than widely into Europe and Central Asia. All species have 2n = 18 with other species of the same genus. Pragmatic needs such chromosomes. The species composition of this clade is identical as inventorying and making biodiversity accessible by means in the nrITS and plastid DNA trees. It diverged around 6.9–7.4 of checklists and Floras sometimes demand the application of mya and its diversification started around 3.2–4.4 mya (median premature classifications. It remains the task of systematics, ages from nrITS and plastid DNA analyses). however, to translate reconstructed phylogenies into natural but (7) The Cicerbita plumieri–Lactuca graminifolia clade I7 nevertheless practicable classifications. Generic classification links the allopolyploid North American endemic Lactuca spe- consequently should recognise monophyletic as well as diag- cies with 2n = 34 chromosomes to the European Lactuca plum- nosable entities, and be based on knowledge about character ieri (L.) Gren. & Godr. (≡ Cicerbita plumieri (L.) Kirschl.) with evolution in a group of organism. This we will be achieving in 2n = 16 chromosomes, a relationship equally revealed in the a subsequent contribution. plastid DNA tree. Lactuca plumieri, a montane tall forb, is dis- tributed from the Cantabrian mountains and Pyrenees over the French Central Massif to the S Vosges, S Black Forest and the ACKNOWLEDGEMENTS Alps (Wegmüller, 1994), with an isolated, disjunct occurrence in the Bulgarian Rila Mts. Notably, the species composition of We thank the curators of the herbaria B, BEO, BEOU, BR, CAS, this clade is identical in the nrITS and plastid DNA trees. The E, GAT, GFW, H, IRAN, KUN, L, LE, MW, PRC, US, W and WAG clade diverged around 5.4–7.3 mya (median age estimates from as well as Hossein Akhani (Tehran, Iran), Robert von Blittersdorff the nrITS and plastid DNA analyses). The North American (Frankfurt a. M., Germany), Ralf Hand, Gerald Parolly and Eckhard L. graminifolia subclade diverged around 4.3–4.4 mya (median von Raab-Straube (Berlin, Germany), Magda Bou Dagher Kharrat age estimates from the nrITS and plastid DNA analyses) from (Beirut, Lebanon), Georg and Sabine Miehe (Marburg, Germany), its European ancestors. More in-depth studies, focussing on the Marjan Niketić and Gordana Tomović (Belgrade, Serbia), Michael position of the Azorean L. watsoniana Trel. and the origin of Ristow (Potsdam, Germany) and Alexander Sukhorukov (Moscow, the allopolyploid North America subclade are under way (E.F. Russia) for providing material for our analyses. Funding of molecular Dias & al., in prep.; K. Jones & al., in prep.). lab work by the Verein der Freunde des BGBM Berlin and the use of (8) The Lactuca inermis clade I8 includes, apart from the computing resources of the Soroban high-performance computing tropical African–SW Asian perennial herb L. inermis, also the system at the Scientific Computing Service of the Freie Universität W Mediterranean–NW African L. tenerrima Pourr., both hav- Berlin are gratefully acknowledged. Special thanks are due to Jana ing 2n = 16 chromosomes and a yellowish pappus. The species Bansemer, Ilona Danßmann, Helga Fleischer-Notter and Julia Pfitzner composition of this clade is identical in the nrITS and plastid (all Berlin) for excellent technical assistance in the molecular lab DNA trees. Lactuca inermis constitutes a second migration of and to Michael Rodewald (Berlin) for graphical assistance. The first

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author is grateful to his colleagues Katy Jones and Michael Grünstäudl Duchêne, S., Lanfear, R. & Ho, S.Y.W. 2014. The impact of calibra- (Berlin) for valuable discussions, to Katy Jones also for linguistic tion and clock-model choice on molecular estimates of divergence times. Molec. Phylogen. Evol. 78: 277–289. improvement of the text. Finally we like to thank the Editors and two https://doi.org/10.1016/j.ympev.2014.05.032 anonymous reviewers for their valuable comments, which considerably Edgar, R.C. 2004. MUSCLE: Multiple sequence alignment with high helped to improve this contribution. accuracy and high throughput. Nucl. Acid Res. 32: 1792–1797. https://doi.org/10.1093/nar/gkh340 Enke, N., Kilian, N., Nemomissa, S. & Gemeinholzer, B. 2008. LITERATURE CITED Afro-alpine Dianthoseris actually a congener of Crepis s.str. (Compositae, Cichorieae, Crepidinae). Bot. Jahrb. Syst. 127: 389– 405. https://doi.org/10.1127/0006-8152/2008/0127-0389 Babcock, E.B & Stebbins, G.L. 1937. 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Wiens, J.J. & Morrill, M.C. 2011. Missing data in phylogenetic analy- Stebbinsia, and Syncalathium (Asteraceae, Cichorieae), endemic sis: Reconciling results from simulations and empirical data. Syst. to the Tibetan Plateau, SW China. Taxon 60: 15–26. Biol. 60: 719–731. https://doi.org/10.1093/sysbio/syr025 Zhang, J.-W., Boufford, D.E. & Sun, H. 2011[b]. Parasyncalathium Yu, Y., Harris, A.J., Blair, C. & He, X.J. 2015. RASP (Reconstruct J.W. Zhang, Boufford & H. Sun (Asteraceae, Cichorieae): A new Ancestral State in Phylogenies): A tool for historical biogeography. genus endemic to the Himalaya-Hengduan Mountains. Taxon 60: Molec. Phylogen. Evol. 87: 46–49. 1678–1684. https://doi.org/10.1016/j.ympev.2015.03.008 Zhang, J.-W., Kilian, N., Deng, T., Souliya, O. & Sun, H. 2016. Zhang, J.-W., Nie, Z.L., Wen, J. & Sun, H. 2011[a]. Molecular phy- Melanoseris henryi (Asteraceae-Cichorieae) revisited: A new logeny and biogeography of three closely related genera, Soroseris, record of genus and species from the flora of Lao PDR with its systematic position. J. Jap. Bot. 91 (Spec. Issue): 107–114.

Appendix 1. Taxon sampling, voucher data and INSDC (International Nucleotide Sequence Database Collaboration, including GenBank/EMBL/DDBJ) accession numbers. Taxon name as used in the phylograms (Figs. 1–4) in bold in alphabetical order within the outgroup, the orphan lineages and Proto-Lactucinae, and the core Lactucinae lineages recognised; unique sample identifier as used in the phylograms and, in square brackets where applicable, unit ID of preserved DNA sample in the GGBN data portal (Droege & al., 2014); abbreviated voucher data (country, locality, collecting date, collectors and collecting number, herbarium code) are given for all samples of newly generated sequences; INSDC accession numbers in the following sequence: nrITS, petD, psbA-trnH, 5′trnL(uaa)-trnF, rpl32- trnL(uag) and 5′rps16-trnQ(uug), with missing sequences indicated by a dash (–), unless only the nrITS, in the first position, is present, and newly generated sequences indicated by an asterisk (*). Outgroup: Crepis multicaulis Ledeb.: LAC-003: KF485539 / ​​KF485665 / ​KF485794 / ​KF486050 / ​KF485922 / ​KF486178. Crepis sancta (L.) Bornm.: LAC-293 [DB0052]: Armenia, Armavir Province, vicinity of Echmiadzin town, 40.15°N, 44.33°E, 11.06.2002, C. Oberprieler 10032 (B), *LT722020 / *LT722528 / ​ ​ *LT722059 / ​*LT722182 / ​*LT722297 / ​*LT722412. Crepidiastrum tenuifolium (Willd.) Sennikov: LAC-295 [DB26539]: Russland, W Sibirien, Altai, ca. 4 km W Cherga, 51°34′N, 85°30′E, 550 m, 26.07.1997, M. Burkart (herb. M. Ristow), *LT721999 / ​*LT722527 / ​*LT722058 / ​*LT722181 / ​*LT722296 / ​*LT722411. Dubyaea hispida DC.: LAC-298 [DB26570]: China, Tibet, Yadong, Tuojia, 4109 m, 27°22′52.5″N, 88°53′23.4″E, 23.09.2009, Nie & al. Nie0954 (KUN), *LT721947. Hypochaeris maculata L.: LAC-287 [DB5185]: Italy, Piemont, Cuneo, oberhalb Morinesio, 44.52°N, 07.13°E, 12.07.2009, M. Ristow & al. 592/09 (B), *LT721997 / ​*LT722529 / ​*LT722060 / ​*LT722183 / ​*LT722298 / ​*LT722413. Hypochaeris radicata L.: LAC-283 [DB7983]: cult. BG Berlin, 19.08.2010, M. Cubr 47380 (B 100346414) *LT721977 / ​*LT722530 / ​*LT722061 / ​*LT722184 / ​*LT722299 / ​*LT722414. Ixeris chinensis subsp. versicolor (Fisch. ex Link) Kitam.: LAC-291 [DB26501]: China, Sichuan, Derong, Ding Qu, 2600–2900 m, 28°46′54″N, 99°18′27″E, 21.07.2004, D.E. Boufford & al. 31022 (KUN), *LT722018 / ​ *LT722531 / ​*LT722062 / ​*LT722185 / ​*LT722300 / ​*LT722415. Lagoseriospsis popovii (Krasch.) Kirp.: LAC-290 [DB26499]: Tadschikistan, N Olimabad, 600 m, 1982–1986, Botschantzev & al. (LE), *LT721934 / *LT722532 / ​ *LT722063 / ​ *LT722186 / ​ *LT722301 / ​ *LT722416.​ Launaea sarmentosa (Willd.) Kuntze: LAC- 001: KF485537 / ​KF485663 / ​KF485792 / ​KF486048 / ​KF485920 / ​KF486176. Leontodon tuberosus L.: LAC-134: AF528487; LAC-002 [DB4947]: – / ​KF485664 / ​ KF485793 / ​KF486049 / ​KF485921 / ​KF486177. Nabalus altissimus (L.) Hook.: LAC-299 [DB26571]: Canada, New Brunswick, Woodstock 24.08.1956, H.J. Scoggan 13555 (W 1961-11317), *LT722050. Nabalus saggitatus (A.Gray) Rydb.: HQ161964. Nabalus tatarinowii (Maxim.) Nakai: LAC-289 [DB26486]: cult. BG Berlin, acc. no. 235-01-01-10 from China, Beijing, Mentougou Qu, Donglingshan, 1200 m, 27.7.2001, leg. E. v. Raab-Straube & M. Steinhof 9, 30.07.2003, M. Cubr 40927 (B), *LT722031 / *LT722533 / ​ *LT722064 / ​ *LT722187 / ​ *LT722302 / ​ *LT722417.​ Nabalus trifoliolatus Cass.: LAC-288 [DB0230]: Canada, Gander District, Gander river, Joe Batt’s brook, 6.–8.7.1960, E. Rouleau 5611 (US), *LT722014 / *LT722534 / ​ *LT722065 / ​ *LT722188 / ​ *LT722303 / ​ *LT722418.​ Reichardia dichotoma (DC.) Freyn: LAC-181 [DB0045]: Armenia, Vayotsdzor Province, ca. 6 km SSE of Yeghegnadzor, 39.7°N, 45.35°E, 26.06.2002, C. Oberprieler 10157 (B), *LT722054 / ​*LT722535 / ​*LT722066 / ​*LT722189 / ​*LT722304 / ​*LT722419. Scorzonera hispanica L.: LAC-296 [DB8866]: cult. in BG Berlin, 30.06.2009, M. Cubr 46433 (B 10 0348435), *LT721963 / ​*LT722536 / ​*LT722067 / ​*LT722190 / ​*LT722305 / ​*LT722420. Sonchella dentata (Ledeb.) Sennikov: LAC-300 [DB26565]: Russland, Sibirien, Tuvniskaja ASSR, 04.08.1972, V. Chanminčun & al. (B), *LT721925. Soroseris erysimoides (Hand.-Mazz.) C.Shih: LAC-004: KF485540 / KF485666 / ​ KF485795 / ​ KF486051 / ​ KF485923 / ​ KF486179.​ Soroseris glomerata (Decne.) Stebbins: LAC-297 [DB26572]: China, Qinghai, Bayan Har pass, 4700 m, 12.08.1996, T.N. Ho 1692 (CAS 939054), *LT721919. Youngia cineripappa (Babc.) Babc. & Stebbins: LAC-292 [DB26536]: China, Chongqing, Nanchuan, Jinfoshan, 29°02′21.5″N, 107°12′30.3″E, 1270–1800 m, 04.07.2009, J.W. Zhang & W.D. Zhu ZZ09041 (KUN), *LT722046 / *LT722537 / ​ ​*LT722068 / ​ *LT722191 / ​*LT722306 / ​*LT722421. Orphan lineages and Proto-Lactucinae: Prenanthes abietina (Boiss. & Balansa) Kirp.: LAC-294 [DB26502]: Caucasus, 07.08.1977, Dolukhanov s.n. (LE), *LT721995 / ​*LT722538 / ​*LT722069 / ​*LT722192 / ​*LT722307 / ​*LT722422. Lactuca triquetra (Labill.) Boiss.: LAC-276 [DB26519]: cult. BG Berlin, acc. no. 033-08-99-10 from Cyprus, Gerasa, valley above road to Kalo Chorio, 470 m, 12.10.1998, leg. Buttler 32460, 17.08.2001, M. Cubr 38916 (B), *LT722047 / ​ *LT722539 / *LT722070 / ​ *LT722193 / ​ *LT722308 / ​ *LT722423;​ LAC-277 [DB8130]: Cyprus, Akrounta, rivulet, 200 m, 34°46′42.45″N, 33°05′14.04″E, 23.09.2010, R. Hand 5731 (B), *LT721973 / *LT722540 / ​ *LT722071 / ​ *LT722194 / ​ *LT722309 / ​ *LT722424;​ LAC-278 [DB8132]: Cyprus, ca. 2 km SSW Dierona, brook valley, ca. 610 m, 34°48′29.97″N, 33°05′3.44″E, 23.09.2010, R. Hand 5730 (B), *LT721970 / ​*LT722541 / ​*LT722072 / ​*LT722195 / ​*LT722310 / ​*LT722425; LAC-285 [DB8972], Lebanon, Hamat, 34°18′09.86″N, 35°40′51.58″E, 145 m, 24.05.2014, M. Bou dagher-Kharrat s.n. (B 10 0517440), *LT721961 / *LT722542 / ​ *LT722073 / ​ ​ *LT722196 / ​*LT722311 / ​*LT722426; LAC-286 [DB8971]: Lebanon, Baabda, 33°50′07.22″N, 35°32′49.96″E, 304 m, 15.07.2014, M. Bou dagher-Kharrat s.n. (B 10 0517439), *LT721962 / ​*LT722543 / ​*LT722074 / ​*LT722197 / ​*LT722312 / ​*LT722427. Faberia nanchuanensis C.Shih: LAC-005: KF485541 / ​KF485667 / ​ KF485796 / ​KF486052 / ​KF485924 / ​KF486180. Faberia sinensis Hemsley: LAC-006 [DB3264]: KF485542 / ​KF485668 / ​KF485797 / ​KF486053 / ​KF485925 / ​ KF486181. Faberia faberi (Hemsley) N.Kilian: LAC-010: KF485546 / ​KF485672 / ​KF485801 / ​KF486057 / ​KF485929 / ​KF486185. Prenanthes L. lineage: Prenanthes purpurea L.: LAC-013: KF485548 / ​KF485675 / ​KF485804 / ​KF486059 / ​KF485931 / ​KF486187; LAC-140: HQ161931; LAC- 141: cult. in GAT from BG Zürich, wild source from Switzerland, Kanton St. Gallen, Alt St. Johann, Churfirsten, 1407 m, 2003, B. Gemeinholzer 209 (GAT), *AJ633343; LAC-142: cult. in GAT from BG Dijon, 2003, B. Gemeinholzer 57 (GAT), *AJ633342; LAC-242 [DB0399]: Germany, Bavaria, 1 km NE Sommerau, 49°09′N, 13°07′E, 680 m, 05.08.1995, R. Eisenblätter & E. Willing 2009D (B 10 0209196), – / ​*LT722544 / ​*LT722075 / ​*LT722198 / ​*LT722313 / ​*LT722428. Cicerbita Wallr. lineage: Cicerbita alpina (L.) Wallr.: LAC-012: AJ633340; LAC-133 [DB7934]: Norway, Hordaland, Hardangarvidda, 15.08.2008, T. Dürbye 4350 (B), *LT721978 / ​KF485674 / ​KF485803 / ​KF486058 / ​KF485930 / ​KF486186; LAC-137: France, Auvergne, west of Monte Dore, along the road, 45°57′N, 02°80′ E, ca. 900 m, 08.07.2002, F. Blattner & S. Jakob BJ02-067 (GAT), *AJ633324. Cicerbita petiolata (K.Koch) Gagnidze: LAC-136: cult. in GAT from BG Frankfurt a. M., 2003, B. Gemeinholzer 233 (GAT), *AJ633326; LAC-271 [DB26521]: Turkey, A7 Trabzon, Doğu Karadeniz Dağlari, above Maçka, slope below the Sumela Monastiri, 1050–1150 m, 29.08.1999, G. Parolly & al. 7426 (B), *LT722026 / ​*LT722545 / ​*LT722076 / ​*LT722199 / ​*LT722314 / ​*LT722429; LAC-228 [DB26542]: Russia, Krasnodar Region, Khosta, 20.09.1973, Mordak 1105 (LE), – / ​– / ​*LT722077 / ​*LT722200 / ​*LT722315 / ​*LT722430. Mycelis mu- ralis (L.) Dumort.: LAC-138: cult. in GAT from BG Udine, 2003, B. Gemeinholzer 101 (GAT), *AJ633339; LAC-139: cult. in GAT from BG Antwerpen, acc. no. 259-15-06-6, 2003, B. Gemeinholzer 43 (GAT), *AJ633338; LAC-274 [DB2446]: Germany, Berlin, Schöneberg, Winterfeldtstraße 62, 52.497°N, 13.35°E, 07.06.2008, M. Ristow 605/08 (B), *LT722055 / ​*LT722546 / ​*LT722078 / ​*LT722201 / ​*LT722316 / ​*LT722431. Cicerbita pancicii (Vis.) Beauverd: LAC-272 [DB26540]: Serbia, between Nova Varoš town and Mt Zlatibor, 02.07.2006, M. Niketić & G. Tomović (BEO, BEOU), *LT722015 / *LT722547 / ​ ​*LT722079 / ​ *LT722202 / ​*LT722317 / *LT722432;​ LAC-273 [DB26541]: F.Y.R. Macedonia, Mt Jablanca, between village Gornja Belica and Malo Sedlo peak, 1400–1600 m, 15.06.2006, M. Niketić & G. Tomović s.n. (BEO, BEOU), *LT721948 / *LT722548 / ​ *LT722080 / ​ *LT722203 / ​ *LT722318 / ​ *LT722433.​ Cephalorrhynchus hispidus

700 Version of Record TAXON 66 (3) • June 2017: 675–703 Kilian & al. • Diversification of the Lactucinae (Compositae)

Appendix 1. Continued. (DC.) Boiss.: LAC-011 [DB0050]: KF485547 / ​KF485673 / ​KF485802 / ​*LT722206 / ​*LT722321 / ​*LT722436; LAC-275 [DB0051], Armenia, 16 km to East from Ararat town, 39.80°N, 44.97°E, 23.06.2002, C. Oberprieler 10129 (B), *LT722022; LAC-214 [DB0049]: Armenia, Kotayk Province, ca. 3 km N Yeghvard, 40.38°N, 44.28°E, 29.08.2002, C. Oberprieler 10189 (B), *LT722035 / ​*LT722549 / ​*LT722081 / ​*LT722204 / ​*LT722319 / ​*LT722434; LAC-240 [DB26523]: Russia, Krasnodar region, Aanapa District, Sukko river valley in 5–7 km upstream the Sukko village, 07.06.1989, A. Dolmatova & al. 1717 (LE), *LT721939 / ​ *LT722550 / ​*LT722082 / ​*LT722205 / ​*LT722320 / ​*LT722435. Cephalorrhynchus cypricus Rech.f.: LAC-215 [DB26497]: Cyprus, Stavros tis Psokas, Cedar Valley, along Tripylos trail, ca. 1200 m, 13.05.1998, R. Hand 2456 (herb. Hand), *LT722019 / ​*LT722552 / ​*LT722083 / ​*LT722207 / ​*LT722322 / ​*LT722437. Cephalorrhynchus subplumosus Kovalevsk.: LAC-226 [DB26546]: Tajikistan, drainage basin of Varzob river, ascent from Kvak to Rundasht, 14.06.1970, R. Kamelin 107 (LE), *LT721971 / *LT722553 / ​ *LT722084 / ​ *LT722208 / ​ *LT722323 / ​ *LT722438.​ Cephalorrhynchus kirpicznikovii Grossh.: LAC-235 [DB26530]: Azerbaijan, Nakhichevan Republic, Karagut Range, near Alma-bulaq spring, ca. 1900 m, 11.06.1947, Grossheim & al. (LE), *LT722040 / *LT722554 / ​ *LT722085 / ​ ​ *LT722209 / ​*LT722324 / ​*LT722439. Cephalorrhynchus microcephalus (DC.) Schchian: LAC-236 [DB26563]: Azerbaijan, Nakhichevan Republic, Ordubad District, Nyus-Nyus village, 2000 m, 08.06.1980, Yu.L. Menitsky & al. 320 (LE), *LT721958 / ​*LT722555 / ​*LT722086 / ​*LT722210 / ​*LT722325 / ​*LT722440. Notoseris C.Shih lineage: Notoseris henryi (Dunn) C.Shih: LAC-056: KF485591 / ​KF485718 / ​KF485846 / ​KF486102 / ​KF485974 / ​KF486230; LAC-062: KF485597 / KF485724 / ​ KF485852 / ​ KF486108 / ​ KF485980 / ​ KF486236.​ Notoseris khasiana (C.B.Clarke) N.Kilian: LAC-066: KF485601 / KF485728 / ​ KF485856 / ​ ​ KF486112 / ​KF485984 / ​KF486240; LAC-068: KF485603 / ​KF485730 / ​KF485858 / ​KF486114 / ​KF485986 / ​KF486242. Notoseris macilenta (Vaniot & H.Lév.) N.Kilian: LAC-064: KF485599 / KF485726 / ​ KF485854 / ​ ​KF486110 / ​KF485982 / KF486238;​ LAC-065: KF485600 / KF485727 / ​ KF485855 / ​ ​KF486111 / ​KF485983 / ​ KF486239; LAC-145 [DB26566]: China, Jiangxi Prov., Wunging Xian, 650 m, 28.11.1996, C.M. Tan 9611138 (B), *LT722038; LAC-143: EU046560. Notoseris scandens (Hook.f.) N.Kilian: LAC-052: KF485587 / ​KF485714 / ​KF485842 / ​KF486098 / ​KF485970 / ​KF486226; LAC-147 [DB3266]: China, Yunnan, 1300 m, 21.11.1990, coll. ignot. 621 (KUN 0725673), *LT722002 / ​*LT722556 / ​*LT722087 / ​– / ​*LT722326 / ​*LT722441. Notoseris triflora (Hemsl.) C.Shih: LAC-059: KF485594 / KF485721 / ​ KF485849 / ​ KF486105 / ​ KF485977 / ​ KF486233;​ LAC-061: KF485596 / KF485723 / ​ KF485851 / ​ KF486107 / ​ KF485979 / ​ KF486235;​ Notoseris triflora (Hemsl.) C.Shih × khasiana (C.B.Clarke) N.Kilian: LAC-151: EU436698. Notoseris yakoensis (Jeffrey) N.Kilian: LAC-054: KF485589 / ​KF485716 / ​ KF485844 / ​KF486100 / ​KF485972 / ​KF486228. Paraprenanthes C.Shih lineage: Paraprenanthes diversifolia (Vaniot) N.Kilian: LAC-041: KF485576 / KF485703 / ​ KF485831 / ​ KF486087 / ​ KF485959 / ​ KF486215.​ Paraprenanthes melanantha (Franch.) Z.H.Wang: LAC-046: KF485581 / ​KF485708 / ​KF485836 / ​KF486092 / ​KF485964 / ​KF486220; LAC-047: KF485582 / ​ KF485709 / ​KF485837 / ​KF486093 / ​KF485965 / ​KF486221. Paraprenanthes oligolepis (C.C.Chang ex C.Shih) Z.H.Wang: LAC-020: KF485555 / KF485682 / ​ ​ KF485810 / KF486066 / ​ KF485938 / ​ KF486194;​ LAC-022: KF485557 / KF485684 / ​ KF485812 / ​ KF486068 / ​ KF485940 / ​ KF486196.​ Paraprenanthes parishii [= P. um- brosa sensu Wang & al., 2013]: LAC-028: KF485563 / ​KF485690 / ​KF485818 / ​KF486074 / ​KF485946 / ​KF486202; LAC-029: KF485564 / ​KF485691 / ​KF485819 / ​ KF486075 / ​KF485947 / ​KF486203. Paraprenanthes prenanthoides (Hemsl.) C.Shih: LAC-035: KF485570 / ​KF485697 / ​KF485825 / ​KF486081 / ​KF485953 / ​ KF486209. Paraprenanthes sororia (Miq.) C.Shih: LAC-037: KF485572 / ​KF485699 / ​KF485827 / ​KF486083 / ​KF485955 / ​KF486211; LAC-038: KF485573 / ​ KF485700 / KF485828 / ​ KF486084 / ​ KF485956 / ​ KF486212.​ Paraprenanthes triflora (Chang & C.Shih) Z.H.Wang & N.Kilian: LAC-025: KF485560 / KF485687 / ​ ​ KF485815 / ​KF486071 / ​KF485943 / ​KF486199; LAC-152: EU046559; LAC-266 [DB26490]: Nepal, Pokahara region, betweem Tikhedhunga and Ghorepani, 12.09.2008, A. Suchorukow N-151 (B), *LT722011 / ​*LT722557 / ​*LT722088 / ​– / ​– / ​–. Paraprenanthes wilsonii (C.C.Chang) Z.H.Wang: LAC-049: KF485584 / ​ KF485711 / ​KF485839 / ​KF486095 / ​KF485967 / ​KF486223; LAC-051: KF485586 / ​KF485713 / ​KF485841 / ​KF486097 / ​KF485969 / ​KF486225. Paraprenanthes yunnanensis (Franch.) C.Shih: LAC-044: KF485579 / ​KF485706 / ​KF485834 / ​KF486090 / ​KF485962 / ​KF486218; LAC-490 [DB0291]: China, Yunnan, Kuming, Xishan, 2100–2350 m, 1984, Sino-Amer Exped. 1542 (B), *LT722024; LAC-491 [DB26577]: China, Yunnan, Gaoligong Shan, 1650 m, Gaoligong Sahn Biodiv. Survey 27650 (CAS 1067483), *LT721946. Kovalevskiella Kamelin lineage: Chaetoseris roborowskii (Maxim.) C.Shih: LAC-018: KF485553 / ​KF485680 / ​KF485808 / ​KF486064 / ​KF485936 / ​KF486192; LAC-019: KF485554 / ​KF485681 / ​KF485809 / ​KF486065 / ​KF485937 / ​KF486193; LAC-222 [DB8367]: China, Sichuan, Liangshan Prefecture, Muli Xian, ca. 15 km NNW of Muli town on road no. 216 near Lizigou, 28°02′34″N, 101°11′29″E, 19.08.2012, N. Kilian & al. 10809 (B), *LT722009 / ​*LT722558 / ​*LT722089 / ​ *LT722211 / *LT722327 / ​ *LT722442;​ LAC-216 [DB26553]: China, Qinghai, 3550–3650 m, 24.08.1996, T.N. Ho & al. 2287 (CAS 939360), *LT721921 / *LT722559 / ​ ​ *LT722091 / ​*LT722212 / ​*LT722328 / ​*LT722443. Cicerbita azurea (Ledeb.) Beauverd: LAC-014: KF485549 / ​KF485676 / ​KF485805 / ​KF486060 / ​KF485932 / ​ KF486188; LAC-015: KF485550 / ​KF485677 / ​KF485806 / ​KF486061 / ​KF485933 / ​KF486189. Kovalevskiella kovalevskiana (Kirp.) Kamelin: LAC-207 [DB7121]: Kyrgyzstan, W Thien-Shan, Sary Chelek, 24.07.1998, H. Gottschling s.n. (GFW), *LT721982 / *LT722560 / ​ *LT722092 / ​ *LT722213 / ​ *LT722329 / ​ *LT722444;​ LAC- 241 [DB26529]: Kyrgyzstan, Alay Range, Gulcza river valley, 03.08.2004, G.A. Lazkov s.n. (LE), *LT721945 / ​*LT722561 / ​*LT722093 / ​*LT722214 / ​*LT722330 / ​ *LT722445. Kovalevskiella rosea (Popov & Vved.) Kamelin: LAC-232 [DB26548]: Uzbekistan, Kurama Range, W of Kamchik pass, 2500 m, 28.07.1973, R.V. Kamelin 398 (LE), *LT721989 / ​*LT722562 / ​*LT722094 / ​*LT722215 / ​*LT722331 / ​*LT722446; LAC-233 [DB26547]: Uzbekistan, Bolshoi Chimgan Range, upper course of Chimganka river, 2500 m, 21.07.1972, R.V. Kamelin 254 (LE), *LT721980 / *LT722563 / ​ *LT722095 / ​ *LT722216 / ​ *LT722332 / ​ *LT722447.​ Kovalevskiella zeravschanica (Kovalevsk.) Kamelin: LAC-238 [DB26549]: Tajikistan, Turkestan Range, Isfara river basin, Puli-Oftobruy, archa [Juniperus] forest, 27.06.1970, R.V. Kamelin 495 (LE), *LT721972 / ​*LT722564 / ​*LT722096 / ​*LT722217 / ​*LT722333 / ​*LT722448; LAC-269 [DB26555]: Uzbekistan, Samarkand Mts (Kyrtau), above Urgut village, 18.06.1982, Transcaucasian-Central Asian Expedition 1591 (LE), – / *LT722566 / ​ *LT722099 / ​ *LT722220 / ​ *LT722336 / ​ *LT722451;​ LAC-270 [DB26554]: Tajikistan, Western Hissar, Kashka-Darya-Aksu river basin, upper reaches of Gilyandar river, 13.06.1982, Transcaucasian-Central Asian Expedition 1186 (LE), *LT721924 / ​– / ​*LT722097 / ​*LT722218 / ​*LT722334 / ​*LT722449; LAC-239 [DB26552]: Tajikistan, N side of Karategin Range, 4–5 km SWW of Ramit village, glades in maple forest with Caragana, thick tall forb vegetation, 10.07.1962, T. Ivanova & T. Strizhova 1976 (LE), *LT722030 / ​*LT722565 / ​*LT722098 / ​ *LT722219 / ​*LT722335 / ​*LT722450. Lactuca alaica Kovalevsk.: LAC-268 [DB26527]: Kyrgyzstan, N side of Alay Range, Kök-Suu river valley, 39.867°N, 71.917°E, 3200 m, G.A. Lazkov s.n. (H), *LT722033 / ​*LT722567 / ​*LT722100 / ​*LT722221 / ​*LT722337 / ​*LT722452. Lactuca mira Pavlov: LAC-267 [DB26528]: Kyrgyzstan, Pskem Range, Kara-Korum river valley, 41.857°N, 70.68°E, 3500 m, 25.08.2006, G.A. Lazkov s.n. (H), *LT721920 / ​*LT722568 / ​*LT722101 / ​ *LT722222 / ​*LT722338 / ​*LT722453. Stenoseris auriculiformis C.Shih: LAC-016 [DB26551]: China, Qinghai, Huzhu, 06.07.1991, T.N. Ho 1730 (CAS 934678), KF485551 / ​KF485678 / *LT722090 / ​KF486062 / ​KF485934 / ​KF486190; LAC-017: KF485552 / KF485679 / ​ ​KF485807 / ​KF486063 / ​KF485935 / ​KF486191. Melanoseris Less. lineage: Cephalorrhynchus brassicifolius (Boiss.) Tuisl: LAC-218 [DB26507]: Iran, Golestan, Jahan Nama Protected Area, 36°39′30″N, 54°13′20″E, 06.07.1999, H. Akhani 13646 (IRAN), *LT721929 / *LT722569 / ​ *LT722102 / ​ *LT722223 / ​ *LT722339 / ​ *LT722454;​ LAC-225 [DB26535]: Turkmenistan, NW Kopetdagh, 3 km S of Uylakushluk village, 25.04.1987, J. Kurbanov s.n. (LE), *LT721931 / ​*LT722570 / ​*LT722103 / ​*LT722224 / ​*LT722340 / ​*LT722455; LAC-227 [DB26556]: Turkmenistan, NW Kopetdagh, 13.06.1988, V. Botschantzev & J. Kurbanov 11 (LE), *LT721943 / ​*LT722571 / ​*LT722104 / ​*LT722225 / ​ *LT722341 / ​*LT722456. Cephalorrhynchus kossinskyi (Krasch.) Kirp.: LAC-212 [DB26537]: Turkmenistan, Kopetdagh, Dusin, 26.05.1984, J. Kurbanov s.n. (LE), *LT722029 / ​*LT722572 / ​*LT722105 / ​*LT722226 / ​*LT722342 / ​*LT722457; LAC-217 [DB26531]: Iran, Khorasan, NE border of Loestan National Park, 10 km NW of Mirzabaylu towards Soolezerd, Yakhtikalan pass, 37°23′43″N, 56°12′01″E, 1730 m, 22.06.2003, H. Akhani 16837 (herb. Akhani), *LT721942 / ​ *LT722573 / *LT722106 / ​ *LT722227 / ​ *LT722343 / ​ *LT722458;​ LAC-220 [DB26511]: Iran, Razavi Khorasan, Tandooreh National Park, Dargaz County, between Tivan and Ortebolagh, [ca. 37°35′N, 58°40′E], 2215–2280 m, 12.06.2004, Memariani & Zangooei FMUH 35533 (B), *LT722003 / ​*LT722574 / ​*LT722107 / ​ *LT722228 / ​*LT722344 / ​*LT722459; LAC-243 [DB26493]: Iran, Khorasan, Kopet Dagh, 2300 m, 13.06.1975, Rechinger 53447 (B), *LT721952 / ​*LT722575 / ​ *LT722108 / ​*LT722229 / ​*LT722345 / ​*LT722460. Cephalorrhynchus polycladus (Boiss.) Kirp.: LAC-213 [DB26559]: Tajikistan, Kuhistoni Badakhshon Autonomous Province, N side of Sarez lake, Sary-Tugay, moraine on the ancient terrace of Murgab river, 3700 m, 24.08.1958 Yu. Gusev 5864 (LE), *LT721951 / ​ *LT722576 / ​*LT722109 / ​*LT722230 / ​*LT722346 / ​*LT722461; LAC-219 [DB26510]: Iran, Khorasan, Moghan, 2500 m, 03.06.2001, Joharchi FMUH 33677 (B), *LT721927 / ​*LT722577 / ​*LT722110 / ​*LT722231 / ​*LT722347 / ​*LT722462; LAC-282 [DB26509]: Kyrgyzstan, Batken region, Kadamjay Distr., Alay Range (N side), Sürmö-Tash Nature Reserve, near Langar, 17.07.2012, G.A. Lazkov s.n. (B), *LT721928 / ​*LT722578 / ​*LT722111 / ​*LT722232 / ​*LT722348 / ​*LT722463.

Version of Record 701 Kilian & al. • Diversification of the Lactucinae (Compositae) TAXON 66 (3) • June 2017: 675–703

Appendix 1. Continued. Cephalorrhynchus soongoricus (Regel) Kovalevsk.: LAC-208 [DB7134]: Uzbekistan, Zeravshanski Krebet, Aman Kutan, 1500–1800 m, 21.05.1974, V. Vašák s.n. (B), *LT721926 / ​*LT722579 / ​*LT722112 / ​*LT722233 / ​*LT722349 / ​*LT722464; LAC-210 [DB26557]: Kazakhstan, Bala-Boguty Mts, SW slope in 2 km W of a spring, 1200–1300 m, 04.06.1985, V.Grubov & al. 27 (LE), *LT721933 / *LT722580 / ​ *LT722113 / ​ *LT722234 / ​ *LT722350 / ​ *LT722465;​ LAC-211 [DB26543]: Uzbekistan, Kuhitang Mts, right side of Tangi-Duval valley, 10 km upstream Khatak village, 14.05.1978, Nabiev & al. 123 (LE), *LT722043 / *LT722581 / ​ ​ *LT722114 / *LT722235 / ​ ​*LT722351 / ​*LT722466. Cephalorrhynchus takhtadzhianii (Sosn.) Kirp.: LAC-234 [DB26524]: Artashat District, between Garni and Zovashen villages, gypsaceous slopes, 1000–1200 m, 22.06.1970, A.L. Takhtajan & T.N. Popova s.n. (LE), *LT721996 / *LT722582 / ​ ​*LT722115 / ​*LT722236 / ​ *LT722352 / ​*LT722467. Cicerbita bourgaei (Boiss.) Beauverd: LAC-150: cult. in GAT from BG Meise, 2003, B. Gemeinholzer 287 (GAT), *AJ633329; LAC- 260 [DB26520]: Turkey, A8 Rize, Doğu Karadeniz Dağlari, near Sivrikaya, 40°40′45″N, 40°42′52″E, 1950 m, 28.08.1999, G. Parolly & al. 7418 (B), *LT722021 / ​ *LT722583 / *LT722116 / ​ *LT722237 / ​ *LT722353 / ​ *LT722468.​ Cicerbita prenanthoides (M.Bieb.) Beauverd: LAC-229 [DB26562]: Russia, Pyatigorsk, 29.07.1990, Yu.L. Menitsky & al. 18 (LE), *LT721956 / *LT722584 / ​ ​*LT722117 / ​*LT722238 / ​*LT722354 / ​*LT722469; LAC-230 [DB26561]: Russia, Pyatigorsk, Beshtau Mt, 26.08.1990, Yu.L. Menitsky 36 (LE), *LT721960 / ​*LT722585 / ​*LT722118 / ​*LT722239 / ​*LT722355 / ​*LT722470. Cicerbita thianschanica (Regel & Schmalh.) Beauverd: LAC-263 [DB26538]: Kyrgyzstan, N side of Teskey Ala-Too, Jylandy, experimental forestry, spruce forest, 29.07.1965, Konnov & al. 1210 (LE), *LT721954 / ​*LT722586 / ​*LT722119 / ​*LT722240 / ​*LT722356 / ​*LT722471; LAC-264 [DB26544]: Kyrgyzstan, S side of Küngöy Ala-Too, floodplain of Tüp river, Bosharyn village, 14.07.1983, R. Aydarova s.n. (LE), *LT721974 / ​*LT722587 / ​*LT722120 / ​*LT722241 / ​*LT722357 / ​*LT722472. Lactuca attenuata Stebbins: LAC-197 [DB7116]: Kongo, Kivu, Kalehe, Mt Kahuzi, 2200 m, 07.07.1971, J. Ntakiyimana 87 (BR), *LT721984. Lactuca calophylla C.Jeffrey: LAC-200 [DB8134]: Tanzania, Rukwa, Sumbawanga, 15.01.2011, R. v. Blittersdorff 34 (B), *LT721968 / ​*LT722588 / ​*LT722121 / ​*LT722242 / ​*LT722358 / ​ *LT722473. Lactuca hazaranensis Djavadi & N.Kilian: LAC-262 [DB26503]: Iran, Kerman, Rayen, 2850 m, 10.08.2010, Eskandari & Torabi s.n. (IRAN 55199), *LT721922 / ​*LT722589 / ​*LT722122 / ​*LT722243 / ​*LT722359 / ​*LT722474. Lactuca lasiorhiza (O.Hoffm.) C.Jeffrey: LAC-199 [DB1334]: Cameroun, savanna woodland on slopes of the Nganha Mts, near Ndigou, about 60 km E of Ngaoundéré, ca. 1500 m, 03.12.1964, W.J.J.O. de Wilde & al. 4531b (WAG), *LT722008. Lactuca paradoxa A.Rich.: LAC-198 [DB26568]: Ethiopia, Near Menagesha, ca. 30 km along Amba road W of Addis Ababa, 2100 m, 27.11.1965, W.J.J.O. de Wilde 8964 (B), *LT721957. Lactuca setosa C.Jeffrey: LAC-201 [DB8135]: Tanzania, Rukwa, Sumbawanga, 14.01.2011, R. v. Blittersdorff 32 (B), *LT721967 / *LT722590 / ​ *LT722123 / ​ *LT722244 / ​ *LT722360 / ​ *LT722475.​ Lactuca praecox R.E.Fr.: T1: LAC-202 [DB26504]: Tanzania, Rukwa, Sumbawanga, 21.01.2012, R. v. Blittersdorff 38 (B), *LT721975 / ​*LT722591 / ​*LT722124 / ​*LT722245 / ​*LT722361 / ​*LT722476; T2: LAC-203 [DB26505]: Tanzania, Rukwa, Sumbawanga, 18.10.2011, R. v. Blittersdorff 39 (B), *LT721935 / ​*LT722592 / ​*LT722125 / ​*LT722246 / ​*LT722362 / ​*LT722477; T3: LAC-204 [DB26506]: Tanzania, Rukwa, Sumbawanga, 12.01.2012, R. v. Blittersdorff 40 (B), *LT721941 / *LT722593 / ​ ​*LT722126 / ​*LT722247 / ​*LT722363 / ​*LT722478. Lactuca rosularis Boiss.: LAC-209 [DB26489]: Iran, prov. Khorasan, in montibus serpentinicus probe Robat-e-Safirid inter Mashhad et Torbat-eHeydariyeh, 1800–2000 m, 29.05.1977, K.H. Rechinger 55980 (B), *LT722000 / ​*LT722594 / ​*LT722127 / ​*LT722248 / ​*LT722364 / ​*LT722479. Lactuca schulzeana Büttner: LAC-205 [DB7119]: Kongo, Massif de Bangu, 01.02.1987, F. Billiet & B. Jadin 4013 (BR), *LT721983 / ​– / ​*LT722128 / ​– / ​*LT722551 / ​–. Lactuca ugandensis C.Jeffrey: LAC-206 [DB8133]: Tanzania, Rukwa, Sumbawanga, 02.04.2011, R. v. Blittersdorff 36 (B), *LT721969 / ​*LT722595 / ​*LT722129 / ​*LT722249 / ​ *LT722365 / ​*LT722480. Melanoseris atropurpurea (Franch.) N.Kilian & Z.H.Wang: LAC-099: KF485633 / KF485760 / ​ ​KF485888 / ​KF486144 / ​KF486016 / ​ KF486272; LAC-101: KF485635 / KF485762 / ​ KF485890 / ​ KF486146 / ​ KF486018 / ​ KF486274.​ Melanoseris bracteata (C.B.Clarke) N.Kilian: LAC-072: KF485607 / ​ KF485734 / KF485862 / ​ KF486118 / ​ KF485990 / ​ KF486246;​ LAC-221 [DB26534]: Bhutan, between Barshong and Dolam Kencho, Thimphu Chu, 27°41′N, 89°38′E, 3300 m, 05.10.1984, I.W.J. Sinclair & D.G. Long 5560 (E 000360977), *LT722006 / ​*LT722596 / ​*LT722130 / ​*LT722250 / ​*LT722366 / ​*LT722481; LAC-244 [DB26533]: Bhutan, near Gasa Dhong, 2750 m, 27°55′N, 89°46′E, 14.09.1984, I.W.J. Sinclair & D.G. Long 5017 (E 00081859), *LT722042 / ​*LT722597 / ​ *LT722131 / ​*LT722251 / ​*LT722367 / ​*LT722482. Melanoseris brunoniana (Wall. ex DC.) N.Kilian & Ze H.Wang: LAC-255 [DB26491]: cult. BG Berlin, acc. no. 261-39-83-10, from Pakistan, Swat, Bhan Tal, 2600 m, 20.9.1983, leg. H. Ern 7639, 23.07.1986, Schwertfeger 20570 (B), *LT722023 / – / ​ *LT722132 / ​ *LT722252 / ​ ​ *LT722368 / ​*LT722483. Melanoseris cyanea (D.Don) Edgew.: LAC-080: KF485614 / ​KF485741 / ​KF485869 / ​KF486125 / ​KF485997 / ​KF486253; LAC-084: KF485618 / KF485745 / ​ KF485873 / ​ KF486129 / ​ KF486001 / ​ KF486257;​ LAC-089: KF485623 / KF485750 / ​ KF485878 / ​ KF486134 / ​ KF486006 / ​ KF486262;​ LAC-257 [DB26567]: Nepal, Pokahara region, Ghorepani village, 2800 m, 12.09.2008, A. Sukhorukov N-152 (B), *LT721990; LAC-492 [DB26564]: Nepal, 5 km NE Jumla village, 29°17′N, 82°05′E, 3000 m, 02.10.2010, A. Suchorukow 336 (E00425055), *LT721986 / *LT722598 / ​ *LT722133 / ​ *LT722253 / ​ *LT722369 / ​ *LT722484.​ Melanoseris decipiens (Hook.f. & Thomson ex C.B.Clarke) N.Kilian & Ze H.Wang: LAC-256 [DB26492]: Pakistan, Nanga Parbat, 3100 m, 21.07.1994, M. Nüsser 1598 (B), *LT722028 / – / ​ *LT722134 / ​ – / ​ – / ​ –.​ Melanoseris graciliflora (DC.) N.Kilian: LAC-112: KF485646 / KF485773 / ​ KF485901 / ​ KF486157 / ​ KF486029 / ​ ​ KF486285; LAC-249 [DB26573]: China, Sichuan, Kangding Xian, downstream from Mugecho lake, 30°10′16″N, 101°52′08″E, 3300–3500 m, 28.08.1997, Boufford & al. 27661 (CAS 1053926), *LT721936; LAC-250 [DB8355]: China, Yunnan, Lijiang Shi, Ninglang Xian, ca. 36 km NW of Ninglang on dirt road towards Bai Yan Zi mountain, 27°33′07″N, 100°38′33″E, 27.08.2012, N. Kilian & al. 11072 (B), *LT721991 / – / ​ ​*LT722135 / ​– / ​– / ​–; LAC-259 [DB8359]: China, Yunnan, Lijiang Shi, Yulong Xian, S part of Yulong Xueshan, ca. 2.3 km NNE of field station near mountain path towards south peak, 27°01′22″N, 100°11′10″E, 29.08.2012, N. Kilian & al. 11264 (B), *LT721992 / ​– / ​*LT722136 / ​– / ​– / ​–. Melanoseris henryi (Dunn) N.Kilian: LAC-280: KU746878. Melanoseris lessertiana (DC.) Decne.: LAC-071: KF485606 / KF485733 / ​ KF485861 / ​ KF486117 / ​ KF485989 / ​ KF486245.​ Melanoseris likiangensis (Franch.) N.Kilian & Z.H.Wang: LAC- 098: KF485632 / ​KF485759 / ​KF485887 / ​KF486143 / ​KF486015 / ​KF486271; LAC-258 [DB8364]: China, Sichuan, Liangshan Prefecture, Muli Xian, ca. 15 km NNW of Muli town on road no. 216 near Lizigou, 28°02′34″N, 101°11′29″E, 23.08.2012, N. Kilian & al. 10808 (B), *LT722004 / – / ​ *LT722137 / ​ – / ​ – / ​ –.​ Melanoseris macrantha (C.B.Clarke) N.Kilian & J.W.Zhang: LAC-075: KF485610 / ​KF485737 / ​KF485865 / ​KF486121 / ​KF485993 / ​KF486249. Melanoseris macrorhiza (Royle) N.Kilian: LAC-073: KF485608 / ​KF485735 / ​KF485863 / ​KF486119 / ​KF485991 / ​KF486247; LAC-074: KF485609 / ​KF485736 / ​KF485864 / ​KF486120 / ​ KF485992 / ​KF486248; LAC-253 [DB7131]: Nepal, Mustang, S of Samar, 28°57′N, 83°48′E, 3700 m, 27.08.2001, S. & G. Miehe & K. Koch 01-091-04 (herb. Miehe), *LT722052. Melanoseris monocephala (C.C.Chang) Ze H.Wang: LAC-231 [DB26574]: China, NW Yunnan 22.08.2010, Y. Niu s.n. (KUN [lost]), *LT721950. Melanoseris qinghaica (S.W.Liu & T.N.Ho) N.Kilian & Z.H.Wang: LAC-078: KF485613 / KF485740 / ​ KF485868 / ​ KF486124 / ​ KF485996 / ​ KF486252.​ Melanoseris souliei (Franch.) N.Kilian: LAC-069: KF485604 / ​KF485731 / ​KF485859 / ​KF486115 / ​KF485987 / ​KF486243; LAC-070: KF485605 / ​KF485732 / ​ KF485860 / KF486116 / ​ KF485988 / ​ KF486244;​ LAC-251 [DB26500]: China, Sichuan, 4100–4300 m, 23.07.1998, Boufford & al. 29089 (CAS 1010664), *LT721923 / ​ – / ​*LT722138 / ​– / ​– / ​–; LAC-252 [DB8365]: China, Sichuan, Liangshan Prefecture, Muli Xian, mountains NE of Muli, ca. 12 km NNE of Muli town on country road no. 015 to Da Kuahuona massif, 28°01′42″N, 101°19′E, 3900 m, 25.08.2012, N. Kilian & al. 10941 (B), *LT721955. Melanoseris tenuis (C.Shih) N.Kilian: LAC-106: KF485640 / KF485767 / ​ KF485895 / ​ KF486151 / ​ KF486023 / ​ KF486279;​ LAC-107: KF485641 / KF485768 / ​ KF485896 / ​ KF486152 / ​ KF486024 / ​ KF486280;​ LAC-237 [DB26575]: China, Sichuan, Muli, Yanyashan, 3228 m, 27°41′11″N, 101°13′20″E, 08.10.2009, Y. Nie & al. Nie1159 (KUN), *LT721949. Melanoseris violifolia (Decne.) N.Kilian: LAC-076: KF485611 / ​KF485738 / ​KF485866 / ​KF486122 / ​KF485994 / ​KF486250; LAC-077: KF485612 / ​KF485739 / ​KF485867 / ​ KF486123 / ​KF485995 / ​KF486251; LAC-254 [DB8204]: Nepal, trecking route Rara lake to Jumla, from Bota village to Jumla, 29°25′EN, 82°10′E, ca. 2200 m, 01.10.2010, A. Sukhorukov 328 (MW), *LT721965 / ​*LT722599 / ​*LT722139 / ​*LT722254 / ​*LT722370 / ​*LT722485. Prenanthes sumatrana Tjitr.: LAC-279 [DB7136]: Indonesia, Sumatra, Atjeh, Gunung Leuser West top, ca. 25 km SW of Blang Kedjeren, 2900–3200 m, 11.04.1975, De Wilde & De Wilde-Duyfjes 16338 (L 348795), *LT721979. Steptorhamphus crambifolius Bunge: LAC-245 [DB26525]: Tajikistan, drainage basin of Varzob river, on slopes between villages Zudda and Gushara, 1800 m, 20.05.1965, A. Takhtajan s.n. (LE), – / ​*LT722602 / ​*LT722142 / ​*LT722257 / ​*LT722373 / ​*LT722488. Steptorhamphus crassicaulis (Trautv.) Kirp.: LAC-223 [DB26558]: Kazakhastan, east Balkhash area, Arakhly Mts, 28.05.1976, V.P. Botschantzev & V.V. Botschantzeva 1023 (LE), *LT721944 / ​*LT722600 / ​*LT722140 / ​*LT722255 / ​*LT722371 / ​*LT722486; LAC-224 [DB26545]: Kyrgyzstan, N side of Talas Ala-Too, foothills, left side of Shilbili-say river, 21.06.1969, R. Aydarova s.n. (LE), *LT722032 / ​*LT722601 / ​*LT722141 / ​*LT722256 / ​*LT722372 / ​*LT722487. Steptorhamphus persicus (Boiss.) O.Fedtsch. & B.Fedtsch.: LAC-284 [DB26512]: Pakistan, Quetta, inter Bostan (30°23′N, 67°00′E) at Saran Tangai, 1700–1800 m, 10.05.1965, K.H. Rechinger 29178 (B), *LT722041 / ​*LT722603 / ​*LT722143 / ​*LT722258 / ​*LT722374 / ​*LT722489. Steptorhamphus pumilus (Rech.f. & Tuisl) Tuisl: LAC-261

702 Version of Record TAXON 66 (3) • June 2017: 675–703 Kilian & al. • Diversification of the Lactucinae (Compositae)

Appendix 1. Continued. [DB26550]: Afghanistan, prov. Ghazni, in faucibus Say Khoshkak, ca. 33°53′N, 67°50′E, 06.07.1962, K.H. Rechinger 17862 (W), *LT722045 / ​*LT722604 / ​ *LT722144 / ​*LT722259 / ​*LT722375 / ​*LT722490. Lactuca L. lineage: Cephalorrhynchus picridiformis (Boiss.) Tuisl: LAC-185 [DB7135]: Afghanistan, W Sharestan, 2200 m, 02.07.1967, Rechinger 36813 (B), *LT721994 / *LT722624 / ​ *LT722164 / ​ *LT722279 / ​ *LT722395 / ​ *LT722510.​ Cicerbita deltoidea (M.Bieb.) Beauverd: LAC-194 [DB26526]: Russia, Northern Caucasus, Verkhnie Tuby, 31.07.1946, A.V. Gavrilevich s.n. (LE), *LT721937 / ​*LT722608 / ​*LT722148 / ​*LT722263 / ​*LT722379 / ​*LT722494. Cicerbita macro- phylla (Willd.) Wallr.: LAC-189 [DB26494]: cult. BG Berlin-Dahlem, acc. no. 195-02-74-80, 19.06.2003, M. Cubr 40734 (B), *LT722034 / *LT722621 / ​ *LT722161 / ​ ​ *LT722276 / *LT722392 / ​ *LT722507;​ LAC-190 [DB26560]: Azerbaijan, Murovdag Mts, Kanyaz Mt, 09.07.1970, Yu.L. Menitsky s.n. (LE), *LT721959 / *LT722622 / ​ ​ *LT722162 / ​*LT722277 / ​*LT722393 / ​*LT722508. Cicerbita plumieri (L.) Kirschl.: LAC-149: France, Auvergne, west of Clermont-Ferand, south of Orcival, tall forb community at forest edge, 1070 m, 45°57′N, 02°80′E, 08.07.2003, F. Blattner & S. Jakob s.n. (GAT), *AJ633325; LAC-186 [DB8373]: Schweiz, Valais, cult. BG Berlin-Dahlem, acc. no. 137-02-89-14, 04.07.1997, M. Cubr 35816 (B), *LT721964 / ​*LT722625 / ​*LT722165 / ​*LT722280 / ​*LT722396 / ​*LT722511. Cicerbita racemosa (Willd.) Beauverd: LAC-187 [DB26488]: Kaukasus, Elbrus, Maksan Tal, 2050 m, 1967, Quasdorf 216 (B), *LT722053 / ​*LT722627 / ​ *LT722167 / ​*LT722282 / ​*LT722398 / ​*LT722513; LAC-188 [DB292]: Turkey, Sarikanis, 2200 m, 1981, Raus 4424 (B), *LT722036 / ​*LT722628 / ​*LT722168 / ​ *LT722283 / – / ​ *LT722514.​ Lactuca acanthifolia (Willd.) Boiss.: LAC-162 [DB2444]: Greece, Chalki, an der Straße zwischen Pontamos und Chorio, 11.04.2008, M. Ristow 144-08 (B 10 0340953), *LT722007 / *LT722605 / ​ ​*LT722145 / ​*LT722260 / ​*LT722376 / ​*LT722491. Lactuca alpestris (Gand.) Rech.f.: LAC-168 [DB7123]: Greece, Kreta, Lefka Ori, Jul–Aug 1993, Bergmeier & Matthäs 3384 (B), *LT721981 / *LT722606 / ​ *LT722146 / ​ *LT722261 / ​ *LT722377 / ​ *LT722492.​ Lactuca aurea (Vis. & Pančić) Stebbins: LAC-193 [DB26495]: Greece, Rhodopi, 9 km along road N of the forest station of Zagradenia, area called Likolakka (Bulgarien name: Daliboska), 1800–1900 m, 09.08.1979, A. Strid & Papanicolaou 16497 (B), *LT722039 / ​*LT722607 / ​*LT722147 / ​*LT722262 / ​*LT722378 / ​ *LT722493. Lactuca biennis (Moench) Fernald: LAC-158: HQ161959. Lactuca canadensis L.: LAC-156: HQ161956. Lactuca dissecta D.Don: LAC-116: KF485649 / ​KF485777 / ​KF485905 / ​KF486161 / ​KF486033 / ​KF486289. Lactuca dolichophylla Kitam.: LAC-117: KF485650 / ​KF485778 / ​KF485906 / ​KF486162 / ​ KF486034 / ​KF486290. Lactuca floridana (L.) Gaertn.: LAC-154 [DB0238]: U.S.A., Florida, Sumter County, just W of Oxford, 28.08.1999, M.T. Strong 2116 (US), *LT722010 / ​*LT722609 / ​*LT722149 / ​*LT722264 / ​*LT722380 / ​*LT722495; LAC-157: HQ161957. Lactuca formosana Maxim.: LAC-122: KF485655 / ​ KF485783 / ​KF485911 / ​KF486167 / ​KF486039 / ​KF486295. Lactuca georgica Grossh.: LAC-164 [DB26532]: Iran, Golestan, Golestan National Park, 6 km E of Tangegol, Golzar, 37°22′N, 56°00′E, 800–900 m, 09.07.1994, H. Akhani 9624 (herb. Akhani), *LT722049 / ​*LT722610 / ​*LT722150 / ​*LT722265 / ​*LT722381 / ​ *LT722496. Lactuca glareosa Boiss.: LAC-195 [DB26517]: Turkey, C5 Nigde, Daglari above Meydan, 37°23′N, 34°34′E, 2900–3000 m, 01.08.1999, G. Parolly & al. 7000 (B), *LT722017 / ​*LT722611 / ​*LT722151 / ​*LT722266 / ​*LT722382 / ​*LT722497; LAC-196 [DB26518]: Turkey, C5 Nigde, Aladaglari, upper Yalak Deresi terminal moraine shortly above Yalak Bogazi gorge, 2700–2750 m, 09.08.1999, G. Parolly & al. 7106 (B), *LT721988 / *LT722612 / ​ *LT722152 / ​ *LT722267 / ​ ​ *LT722383 / ​*LT722498. Lactuca glauciifolia Boiss.: LAC-184 [DB3267]: Afghanistan, prov. Kabul, in faucibus inter Mollah Jakup et Dahan-e Siah Darreh, 4 km E Panjao, ca. 34°25′N, 67°10′E, ca. 2800 m, 23.07.1962, K.H. Rechinger 18647 (B), *LT721998 / *LT722613 / ​ *LT722153 / ​ *LT722268 / ​ *LT722384 / ​ *LT722499.​ Lactuca graminifolia Michx.: LAC-155 [DB0237]: U.S.A., Florida, Marion County, 1 mile W of Fort McCoy, 20.04.1963, D.B. Ward & S.G. Shetler 3443 (US), *LT722013 / *LT722614 / ​ *LT722154 / ​ *LT722269 / ​ *LT722385 / ​ *LT722500;​ LAC-160: HQ161958. Lactuca hirsuta Nutt.: LAC-159: HQ172901. Lactuca indica L.: LAC-121: KF485654 / ​KF485782 / ​KF485910 / ​KF486166 / ​KF486038 / ​KF486294; LAC-169 [DB26569]: South Africa, Natal, 1962, Strey 4199 (B), *LT722027; LAC-170 [DB8352]: China, Yunnan, Nujiang Prefecture, Lushui Xian, W side of Salween [= Nu Jiang] valley ca. 7 km N of bridge in Liuku town, 850 m, 25°54′41″N, 98°50′18″E, 02.09.2012, N. Kilian & al. 11381 (B), *LT721993 / ​*LT722615 / ​*LT722155 / ​*LT722270 / ​*LT722386 / ​*LT722501. Lactuca inermis Forssk.: LAC-119: KF485652 / KF485780 / ​ ​KF485908 / KF486164 / ​ KF486036 / ​ KF486292;​ LAC-176 [DB26484]: Tanzania, Usambara Mts, 1997, Schlage 252 (B), *LT721976 / ​*LT722616 / ​*LT722156 / ​*LT722271 / ​*LT722387 / ​*LT722502; LAC-177 [DB26514]: Tanzania, North, Marangu village, 1300–1500 m, 17.09.2003, J. Meister s.n. (B), *LT721938 / ​*LT722617 / ​*LT722157 / ​*LT722272 / ​*LT722388 / ​*LT722503. Lactuca intricata Boiss.: LAC-182 [DB26513]: Türkei, Antalya, Tahtali Dag, 2050 m, Strid & al. 23585 (B), *LT721930 / ​*LT722618 / ​*LT722158 / ​*LT722273 / ​*LT722389 / ​*LT722504; LAC-183 [DB26508]: Greece, Mt Timfi, 1981, Franzén & al. 668 (B), *LT722025 / *LT722619 / ​ *LT722159 / ​ *LT722274 / ​ *LT722390 / ​ *LT722505.​ Lactuca ludoviciana (Nutt.) Riddell: LAC-153 [DB0227]: U.S.A., Nebraska, Morrill County, 1 mile SE from Broadwater, 24.07.1992, S.B. Rolfsmeier 10789 (US), *LT722016 / ​*LT722620 / ​*LT722160 / ​*LT722275 / ​ *LT722391 / *LT722506.​ Lactuca orientalis (Boiss.) Boiss.: LAC-126: KF485659 / KF485787 / ​ KF485915 / ​ KF486171 / ​ KF486043 / ​ KF486299.​ Lactuca oyukluda- ghensis (Parolly) N.Kilian & Parolly: LAC-191 [DB26516]: Turkey, C4 Karaman, Oyuklu Dagi N of Ermenek, 1850 m, 22.07.1999, G. Parolly & al. 6869 (B), *LT721953 / ​*LT722623 / ​*LT722163 / ​*LT722278 / ​*LT722394 / ​*LT722509. Lactuca perennis L.: LAC-113: AJ633334; LAC-161: L48143 + L48144; LAC-334 [DB5188]: Italy, Piemont. Cuneo, 12.07.2009, M. Ristow & al. MiRi 578/09 (B), *LT721987 / ​KF485774 / ​KF485902 / ​KF486158 / ​KF486030 / ​KF486286. Lactuca quercina subsp. wilhelmsiana (DC.) Ferákova: LAC-165 [DB0048]: Armenia, Vayotsdzor Province, Yeghegnadzor Distr., ca. 12 km N Yeghegnadzor, around village Eghegis, 39.87°N, 45.37°E, 27.06.2002, C. Oberprieler 10168 (B), *LT722048 / *LT722626 / ​ ​*LT722166 / ​*LT722281 / ​*LT722397 / ​*LT722512. Lactuca raddeana Maxim.: LAC-124: KF485657 / KF485785 / ​ KF485913 / ​ KF486169 / ​ KF486041 / ​ KF486297.​ Lactuca saligna L.: LAC-148: cult. in GAT from BG Mainz, acc. no. 199637190, 2003, B. Gemeinholzer 182 (GAT), *AJ633336; LAC-281 [DB26498]: Greece, Ilias, Krestena, 25.09.2003, R. & E. Willing 117.851 (B 10 0142322), *LT721966 / *LT722629 / ​ *LT722169 / ​ *LT722284 / ​ *LT722399 / ​ *LT722515.​ Lactuca sativa L.: LAC-130: AJ633337; LAC-132: – / DQ383816 / ​ DQ383816 / ​ ​ DQ383816 / DQ383816 / ​ DQ383816.​ Lactuca serriola L. LAC-131: KF485662 / KF485791 / ​ KF485919 / ​ KF486175 / ​ KF486047 / ​ KF486303;​ LAC-144: Turkey, Vilayet Antalya, coast ca. 60 km E of Antalya, riverbed, 36°49′N, 31°20′E, 14.06.2003, J. Ochsmann 8206 (GAT), *AJ633332; LAC-163 [DB26522]: Zentralasien, Koped Dag, Kara-Kala, Chukur, 800–1100 m, 17.09.1976, V. Vašak s.n. (B), *LT721932 / *LT722630 / ​ *LT722170 / ​ *LT722285 / ​ *LT722400 / ​ *LT722516;​ LAC-247 [DB1417]: Germany, Brandenburg, Odergebiet, N-Rand von Mallnow, 52.44°N, 14.54°E, 20.09.2007, M. Ristow & B. Gemeinholzer 739/07 (B), – / ​*LT722631 / ​ *LT722171 / *LT722286 / ​ *LT722401 / ​ *LT722517.​ Lactuca sibirica (L.) Maxim.: LAC-128: KF485660 / KF485789 / ​ KF485917 / ​ KF486173 / ​ KF486045 / ​ KF486301.​ Lactuca tatarica (L.) C.A.Mey.: LAC-166 [DB26487]: Russia, Altay Rep., Kosh-Agachskiy Rayon, Chuyskaya Step, 13 km SE Kosh-Agach, 2000 m, 49°55′N, 88°50′E, 24.07.2002, E. v. Raab-Straube 020349 (B), *LT721985 / *LT722632 / ​ ​*LT722172 / ​*LT722287 / ​*LT722402 / ​*LT722518; LAC-248 [DB2191]: Germany, Brandenburg. Niederlausitz, Bahnhofsgelände Finsterwalde, 51.64°N, 13.715°E 22.06.2008, M. Ristow 718/08 (B), *LT722057 / ​*LT722633 / ​*LT722173 / ​ *LT722288 / ​*LT722403 / ​*LT722519. Lactuca tenerrima Pourr.: LAC-171 [DB26485]: cult. BG Berlin, acc. no. 249-45-90-10, from Morocco, Hoher Atlas, Piste nach Toufrine, 31°35′N, 6°57′W, 1600 m, leg. C. Oberprieler 3524, 10.07.1991, M. Cubr 28809 (B), *LT722001 / ​*LT722634 / ​*LT722174 / ​*LT722289 / ​ *LT722404 / ​*LT722520. Lactuca tetrantha B.L.Burtt & P.H.Davis: LAC-167 [DB26496]: Cyprus, Hand 3991 (B, herb. Hand), *LT722051 / ​*LT722635 / ​ *LT722175 / *LT722290 / ​ ​*LT722405 / *LT722521.​ Lactuca tuberosa Jacq.: LAC-118: KF485651 / KF485779 / ​ KF485907 / ​ KF486163 / ​ KF486035 / ​ KF486291;​ LAC- 178 [DB26576]: Greece, Rhodos, Attavarios, ca. 1.8 km S des Hauptgipfels, 36°11′36″N, 27°51′35″E, 850 m, 19.02.2012, M. Ristow & A. Schmiedgen 376/12 (B, herb. Ristow), *LT722005; LAC-179 [DB0046]: Armenia, Ararat Province, Ararat Distr., 8 km NE of Vedi village, between villages Urtsadzor and Azizkend, 39.95°N, 44.88°E, 20.06.2002, C. Oberprieler 10177 (B), *LT722012 / ​*LT722636 / ​*LT722176 / ​*LT722291 / ​*LT722406 / ​*LT722522; LAC-180 [DB0047]: Armenia, Vayotsdzor Province, Yeghegnadzor Distr., ca. 6 km SSE of Yeghegnadzor, vicinity of Agarakadzor village, Ajar gorge, 39.7°N, 45.35°E, 26.06.2002, C. Oberprieler 10155 (B), *LT721940 / *LT722637 / ​ *LT722177 / ​ *LT722292 / ​ *LT722407 / ​ *LT722523.​ Lactuca undulata Ledeb.: LAC-114: KF485647 / KF485775 / ​ ​ KF485903 / ​KF486159 / ​KF486031 / ​KF486287; LAC-115: KF485648 / ​KF485776 / ​KF485904 / ​KF486160 / ​KF486032 / ​KF486288. Lactuca variabilis Bornm.: LAC-265 [DB26515]: Turkey, B3/C3 İsparta, Barla Daği, lateral summit above Çamdaği, 38°04′95″N, 30°45′66″E, 1800–1050 m, 30.07.2000, Eren & G. Parolly 7708 (B), *LT722037 / ​*LT722639 / ​*LT722179 / ​*LT722294 / ​*LT722409 / ​*LT722525; LAC-192 [DB290]: Turkey A4, Çankırı, Küçük Ilgaz Dağ, 1900–2100 m, 03.08.1982, T. Raus 6893 (B), *LT722044 / ​*LT722638 / ​*LT722178 / ​*LT722293 / ​*LT722408 / ​*LT722524. Lactuca viminea (L.) J.Presl & C.Presl: LAC-127: AJ633333; LAC-135 [DB0126]: – / ​KF485788 / ​KF485916 / ​KF486172 / ​KF486044 / ​KF486300. Lactuca virosa L.: LAC-146: cult. in GAT from BG Mainz, acc. no. 199804701, 2003, B. Gemeinholzer 181 (GAT), *AJ633335; LAC-246 [DB7962]: cult. BG Berlin, from Berlin-Zehlendorf, Teltow-Kanal, 01.08.1985, leg. E. Royl & Wiechert, 20.08.2010, M. Cubr 47387 (B), *LT722056 / ​*LT722640 / ​*LT722180 / ​*LT722295 / ​*LT722410 / ​*LT722526.

Version of Record 703