Consolida and Aconitella Are an Annual Clade of Delphinium (Ranunculaceae) That Diversified in the Mediterranean Basin and the Irano-Turanian Region

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TAXON 60 (4) • August 2011: 1029–1040 Jabbour & Renner • Consolida and Aconitella belong in Delphinium

Consolida and Aconitella are an annual clade of Delphinium (Ranunculaceae) that diversified in the Mediterranean basin and the Irano-Turanian region

Florian Jabbour & Susanne S. Renner

Systematic Botany and Mycology, Department of Biology, University of Munich, Menzinger Str. 67, 80638 Munich, Germany Author for correspondence: Florian Jabbour, [email protected]

Abstract The Mediterranean and Irano-Turanian biogeographic regions are biodiversity hotspots for mesophytic and xerophytic species, including many Delphinieae. This phylogenetically poorly understood tribe of Ranunculaceae consists of Consolida and Aconitella, with together ca. 52 species, and Delphinium and Aconitum, each with ca. 300 species. To infer the phylogeny of Consolida and Aconitella, we analyzed nuclear and chloroplast DNA sequences from 39 of their species and subspecies (44 taxa) plus a set of 30 exemplar species of Delphinium and Aconitum. We used a Bayesian relaxed clock model to estimate divergence times and a maximum likelihood approach to reconstruct ancestral areas. Aconitella forms a clade embedded in Consolida, and the latter is embedded in Delphinium. Consolida s.l. (including Aconitella) comprises two clades in the Irano- Turanian region and three in the Mediterranean basin. The latter clades’ inferred crown ages of 5.1, 4.4, and 2.8 Ma suggests that the repeated drying-up of the Mediterranean, concomitant with and following the Messinian salinity crisis (5.96–5.33 Ma), may have facilitated their westward expansion. While there is clear geographic structure towards the tips of the Consolida s.l. tree, a likely ancestral area could not be inferred. However, the initial diversification of Consolida s.l., which occurred ca. 17 Ma ago, falls in a period when the climate in the Anatolian region became more arid, which may have favoured the annual life cycle that characterizes all species in this clade. To achieve a classification of mutually monophyletic genera in Delphinieae may require transferring the species of Aconitella and Consolida into Delphinium.

Keywords Aconitella ; Bayesian relaxed clock; Consolida ; Delphinieae; Irano-Turanian region; Mediterranean region

Supplementary Material The alignment is available in the Supplementary Data section of the online version of this article (http://www.ingentaconnect.com/content/iapt/tax).

INTRODUCTION and Aconitella lies in the Mediterranean/Irano-Turanian region (Davis, 1965; Davis & Sorger, 1982; Munz, 1967a, b; Carlström, Plant clades that diversified in the Mediterranean biodi- 1984; Iranshahr, 1987, Misirdali & al., 1990; Chater, 1993), versity hotspots often have biogeographic links to the Irano- a few species occur east as far as Kazakhstan, Afghanistan, Turanian region and Central Asia (Quézel, 1985; Ribera and Pakistan (an example is Consolida schlagintweitii (Huth) & Blasco-Zumeta, 1998; Mansion & al., 2008, 2009; Lo Presti Munz; Munz, 1967b; Alam & Ali, 2010). & Oberprieler, 2009; Médail & Diadema, 2009; Blondel & al., Consolida and Aconitella are characterized by an an- 2010). The Irano-Turanian phytogeographic region encom- nual life cycle, probably as an adaptation to seasonal drought passes Anatolia, parts of Jordan, Syria, the Israeli-Palestinian (Constantinidis & al., 2001). Annuals survive unfavourable region, the Sinai Peninsula, upper Mesopotamia, a large part of periods as seeds and are statistically overrepresented in the the Armenian Highlands, southern and eastern Transcaucasia, Mediterranean biome (Raunkiaer, 1918). Delphinium also in- the Caspian shore of Iran, the Iranian Plateau (without the tropi- cludes a few annual species, all in subg. Delphinium. cal deserts), the westernmost Himalayas, and the arid territory of south-eastern European Russia to the Gobi desert (Takhtajan, 1986). This is an area with highly uneven botanical collecting Table 1. Generic classification of Consolida. and many diverse and poorly understood genera. Among the genera centred in this region and extending into the Mediter- Consolida included Consolida considered as in Delphinium a separate genus ranean are Consolida (DC.) S.F. Gray and Aconitella Spach. To- gether these genera comprise ca. 52 species, occurring from sea Candolle, 1824 (sect., 11 spp.) Gray, 1821 (monospec.: C. regalis) level to 2000 m altitude, often on dry stony slopes, in steppes, Boissier, 1867 (sect., 32 spp.) Soό, 1922 semi-deserts, or even deserts (Trifonova, 1990; our Fig. 1). Huth, 1895 (subg., 45 spp.) Davis, 1965 Their closest relatives are Delphinium L. and Aconitum L., Nevskii, 1937 (subg.) Munz, 1967a, b each with an estimated 300 species; morphological similarities Blanché & al., 1987 to Delphinium even led to the inclusion of Consolida in Del- phinium (Table 1). While the centre of diversity of Consolida Chater, 1993

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The mutual monophyly of Consolida and Aconitella and Materials and methods their relationship(s) with the remaining genera of Delphinieae, viz. Aconitum and Delphinium, have never been tested with Taxon sampling, DNA sequencing, cloning, alignment, adequate DNA sequence data. Studies so far have included and phylogenetic analyses. — The present study includes only C. ajacis (synonymous with C. ambigua) to represent 39 species and subspecies (44 taxa) of Consolida and Aco- Consolida (Ro & al., 1997) and one species each of Delphinium nitella, including the types of the generic names C. regalis and Aconitum (Ro & al., 1997; Wang & al., 2009). A study S.F. Gray (Delphinium consolida L.) and A. aconiti (L.) Soják that focused on the American larkspurs included 62 species (Delphinium aconiti L.). For C. ajacis, C. pubescens, and C. re- of Delphinium and two species of Aconitum (Koontz & al., galis we included two or three samples each to represent the 2004). Here we investigate the relationships and biogeography geographic ranges of these widespread species. Twenty-two of Consolida and Aconitella with a sampling of 75% of their sequences of Delphinium and eight of Aconitum were included, species plus representatives of the different subgenera of Del- with a focus on the short-lived species of Delphinium because phinium and Aconitum. Specific questions we are addressing of our interest in the evolution of an annual life cycle in Del- are: (1) Are Consolida and Aconitella mutually monophyletic phinieae. The subgeneric classification accepted for Delphin- and what is their relationship to Delphinium ? (2) When and ium is that of Malyutin (1987) who recognized two short-lived where did Consolida and Aconitella diversify? (3) What is the subgenera, subg. Staphisagria (J. Hill) Peterm. (three biennial relationship of the annual species of Delphinium to those in species, all sampled) and subg. Delphinium sect. Anthrisci- Consolida and Aconitella? folium Wang (three annual species, one sampled) and sect.

Fig. 1. Consolida and Aconitella plants and their natural habitats. A, Consolida persica (Iran, prov. Esfahan, 51°13′10″ E, 33°58′11″ N, altitude = 1360 m); B, Aconitella teheranica (Iran, prov. Mazandaran, Haraz road, 80 km NE Tehran); C, Garedji (Georgia), steppe region where Aconitella grows; D, Wardsia (Georgia), collecting site of Aconitella hohenackeri. Photos A and B: Shahin S. Zarre; C and D: Andreas Gröger.

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Delphinium (19 annual species, 10 sampled). The perennial lists all DNA sources, species names with authors, and Gen- species of Delphinium are represented by eight species from Bank accession numbers. The aligned trnL-trnF, trnK-matK, China and North America (subg. Delphinastrum (DC.) Peterm., trnS-trnG, and ITS matrices comprised 1338, 1560, 947 and subg. Oligophyllon Dimitrova). Aconitum is represented by 661 nucleotides respectively. There was no statistically sup- species from each of its three subgenera (classification of Ta- ported conflict (defined as > 80% bootstrap support) between mura, 1990: (1) the monospecific biennial subg. Gymnaconitum topologies obtained from the individual data partitions, and (Stapf) Rapaics is represented by Aconitum gymnandrum from the chloroplast and nuclear data were therefore combined, China; (2) the perennial subg. Lycoctonum (DC.) Peterm. (ca. yielding a matrix of 4506 characters and 76 taxa. All tree 50 species) by Aconitum septentrionale; and (3) the biennial searches relied on maximum likelihood (ML) as implemented subg. Aconitum (ca. 250 species) by six Asian species. Based in RAxML (Stamatakis, 2006; Stamatakis & al., 2008), using on the Ranunculaceae trees of Wang & al. (2009) we selected the GTR + G model (with four rate categories), with separate species of Actaea L. and Nigella L. to root our trees; the ap- partitions for the nuclear and chloroplast data. Statistical sup- propriateness of this choice was tested using Path-O-Gen v.1.3 port for nodes was assessed by bootstrap resampling of the data (Drummond & al., 2003), which can help infer the root of a under the same model (100 replicates). FindModel (http://hcv. tree as long as the molecular clock assumption fits the data lanl.gov/content/sequence/findmodel/findmodel.html; using reasonably well (below). the ModelTest script; Posada & Crandall, 1998) selected this DNA isolation and sequencing relied on commercial kits model or the slightly less parameter rich HKY85 model as the and the universal primers of Taberlet & al. (1991) for amplify- best fit for our combined data matrix. ing and sequencing the trnL intron and adjacent trnL-trnF Molecular clock analyses. — We used Path-O-Gen v.1.3 intergenic spacer. The trnK-matK spacer was sequenced using to test the clock-like behaviour of our concatenated dataset the 3914F and 1470R primers (Johnson & Soltis, 1994), in ad- (http://tree.bio.ed.ac.uk/software/pathogen/; Drummond & al., dition to two internal primers: trnK-rev (5′- GCT GAT AAG 2003). Divergence dating relied on Beast v.1.4.8 (Drummond GAA TCG ATT TAT TTA A -3′), and matK-for (5′- CTA TAT & al., 2006; Drummond & Rambaut, 2007), which employs a CCA CTT CTC TTT CAG GAA T -3′) designed for species Bayesian Markov chain Monte Carlo (MCMC) approach to co- belonging to the tribe Delphinieae. The trnS-trnG intergenic estimate topology, substitution rates, and node ages. All dating spacer was sequenced using the trnSgcu and trnGuuc primers runs relied on the GTR + G model (with six rate categories), (Hamilton, 1999). The 18S nuclear ribosomal internal tran- a Yule tree prior, with rate variation across branches uncor- scribed spacer 1 (ITS-1), 5.8S gene, and ITS-2, were ampli- related and log-normally distributed. The MCMC chains were fied with the external primers of Balthazar & al. (2000). The run for 30 million generations (burn-in 20%), with parameters rbcL gene was sequenced for a few exemplar species to extend sampled every 5000th step. Results from individual runs were the Delphinieae dataset for dating purposes, using the exter- combined, and effective sample sizes for all relevant estimated nal primers 1F (Fay & al., 1997) and 1460R (Olmstead & al., parameters and node ages were well above 200. Mixing of the 1992) and the internal primers 600F and 800R (Kocyan & al., chains was checked using Tracer v.1.5 (Rambaut & Drummond, 2007). Sequencing relied on Big Dye Terminator kits (Applied 2007). Final trees were edited in FigTree v.1.3.1 (http://tree.bio Biosystems, Foster City, California, U.S.A.) and an ABI 3100 .ed.as.uk/software/figtree/). Avant capillary sequencer (Applied Biosystems). Sequence To convert relative times into absolute times we used a assembly of forward strands and reverse strands was carried two-pronged approach. First, we constructed expanded rbcL out in MEGA v.4 (Tamura & al., 2007; Kumar & al., 2008), and trnL-trnF matrices (with sequences from GenBank added and sequences were BLAST-searched in GenBank and then to our own) so as to include Ranunculaceae nodes that have aligned by eye. been dated in other studies. For the rbcL clock, we accepted Our entire study is based on material from the herbaria M an age of 73 ± 3 Ma to constrain the crown group age of Ra- and MSB, one advantage being that vouchers are all located nunculaceae (Britton & al., 2007). For the trnL-trnF clock, (and available for future studies) at one place. Direct ampli- the split between Coptis and Xanthorhiza was set to 12 ± 3.75 fication via polymerase chain reaction (PCR) yielded single Ma and that between Ranunculus and Coptis + Xanthorhiza to bands and unambiguous base calls, except for the ITS region 45 ± 7 Ma, based on the work of Bell & al. (2010). The crown in a few species of Aconitum and Delphinium for which we group age of Consolida/Aconitella and the age for the node therefore cloned the ITS fragment. Bands were excised from equivalent to node A in Fig. 3, inferred with these two expanded agarose gel and purified using the QIAquick gel extraction kit matrices (rbcL, trnL-trnF), were then used as secondary cali- (Qiagen, Hilden, Germany). The fragment was ligated into bration points (using normal prior distributions) in a Delphin- the pGEM-T Easy plasmid (Promega, Mannheim, Germany) ieae-centered BEAST analysis of 76 taxa and 4506 characters. and transformed into competent subcloning efficiency DH5α The oldest Ranunculus, Glaucidium, Hydrastis, and Trautvet- Escherichia coli cells. Up to 15 positive clones per sample teria fossils are from the Oligocene (for Ranunculus: Dorofeev, were cultivated overnight. Modal consensus sequences were 1963, Łańcucka-Środoniowa, 1979; for the three latter genera: computed for each Aconitum and Delphinium species using the Li, 1952), but the dating and phylogenetic placements of these program g2cef following Göker & Grimm (2008). fossils are too imprecise to serve as calibration points. In all, 247 sequences (including the modal consensus se- To cross-validate results obtained with the secondary cali- quences) were newly generated for this study. The Appendix bration approach and plastid sequences, we also inferred ages

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using ITS rates from the literature (Kay & al., 2006), namely Results a mean rate for herbaceous angiosperms (4.13 × 10–9 substitutions per site per year) or the Soldanella rate (Primulaceae, Main phylogenetic results. — Figure 2 shows the geo- 8.34 × 10–9 substitutions per site per year), to calibrate a strict graphic provenience of the 44 taxa of Consolida and Aconitella clock for the Delphinieae ITS sequences. The results from us- sampled for this study. The ML tree obtained from our 76-taxon ing the Soldanella ITS rate gave node ages that overlapped matrix (69 species and subspecies of Delphinieae) is shown in closely with those obtained from the 4506-nucleotide matrix Fig. 3. The tree is rooted on Actaea and Nigella, based on Wang and secondary calibrations. & al. (2009), and our experiments with the Path-O-Gen software Biogeographic analyses. — To reconstruct the biogeo- that indicated this as the most likely root placement. Aconitella graphic history of our focal group, we used ML as implemented and Consolida form a clade embedded in Delphinium (with a in Mesquite v.2.74 (Maddison & Maddison, 2009). The ML bootstrap support of 88%; node A, Fig. 3). The deepest diver- model incorporated information from genetic branch lengths gence in Consolida s.l. (including Aconitella) is between a clade and used the Markov k-state one-parameter model, which as- comprising C. lineolata, C. armeniaca, and C. olopetala, all sumes a single rate for all transitions between character states three endemic to Turkey, and the remaining species (both clades (Lewis, 2001). Analyses were carried out on the highest likeli- with 100% support). These remaining species fall in a polytomy hood tree with GTR + G branch lengths, and transition param- of five groups labelled I–V in Figs. 3–5. Three of the five groups eters were estimated based on the tip trait states (i.e., regions comprise species from the Mediterranean basin and the Near assigned to the species). Outgroup taxa and Aconitum species East, while one comprises species from Iran, Iraq, and Central were excluded from this analysis. Geographic regions were Asia and another species from Turkey to Afghanistan (all tra- coded as an unordered multi-state character using either five ditionally placed in Aconitella). Aconitella is clearly embedded or eight states (including three combined regions), namely: (1) within Consolida (node B, Fig. 3). Mediterranean (which included Northwest Africa and the north The closest relatives of Consolida s.l. may be the peren- of the Mediterranean basin); (2) Asia Minor (= Anatolia); (3) nial species of Delphinium from North America and China, Middle East and Central Asia; (4) 1 + 2; (5) 2 + 3; (6) 1 + 2 + 3; (7) but this grouping has a bootstrap support of only 64% (node China; and (8) North America. We also conducted an analysis C, Fig. 3). All annual species of Delphinium group together with six regions (the same as above, except the fourth, fifth, (in the Mediterranean/Chinese subg. Delphinum; Fig. 3). An and sixth region grouped into a new state, “equivocal”). Results unexpected discovery is that the perennial species D. balansae showed that the eight state coding was the most informative. apparently evolved from an annual ancestor (Fig. 3). The three An alternative approach to ancestral area reconstruction biennial species of Delphinium (D. pictum, D. requienii, and that requires a time-calibrated phylogeny and a transition D. staphisagria, forming subg. Staphisagria) form a strongly probability matrix specifying the rate of transition between supported clade (node D, Fig. 3: 100% bootstrap support), but geographic ranges as dispersal or extinction parameters is im- sequences from D. pictum were incomplete. This species is plemented in LAGRANGE (Ree & Smith, 2008). However, therefore not included in the final tree. LAGRANGE needs a fully bifurcated tree (Ree & Smith, 2008) The temporal and spatial unfolding of Consolida and Aco- and has the limitation that the number of biogeographic param- nitella. — The clock-like behaviour of the data assessed with eters to estimate from the data increases exponentially with the Path-O-Gen and the small variance of rate changes along the number of areas, decreasing the inferential power of the model tree (3.77 × 10–4) indicated that ancestor/descendant rates did (Ree & Sanmartín, 2009). not change drastically. Figure 4 shows the chronogram (i.e.,

Fig. 2. Geographic provenience of the material of Consolida and Aconitella sampled for this study. Numbers on the left/right of the slash refer to the number of sequenced Consolida/Aconitella herbarium samples.

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Fig. 3. Phylogenetic relationships in Delphin- 64 ieae with focus on Aconitella and Consolida 99 as inferred from the combined chloroplast and 76 nuclear data under maximum likelihood (ML) 100 90 V Aconitella optimization (76 taxa, 4506 aligned nucleotides); 52 numbers at nodes are ML bootstrap support 57 values from 100 replicates. The numbers I–V on the right mark clades discussed in the main 99 text. The three biennial species of Delphinium (D. pictum, D. requienii, D. staphisagria) form a 59 clade, but D. pictum sequences were incomplete 87 IV and therefore not included here. Where multiple accessions of the same species were sequenced, 86 we added voucher information. The colour coding refers to life history: blue = annuality; 53 green = annuality and/or bienniality, or pseudo- annuality; red = perenniality. 80 82 III 59 100

Consolida

69 B II 100 95 98

63 79

51 I 99

C 50 100 64

Delphinium subg. 97 Delphinastrum

100 Delphinium subg. A 88 Oligophyllon 93

68 52 80 Delphinium subg. 52 75 Delphinium 94 100 67 100

64 58 100 100 Aconitum 99 98 55

D 100 Delphinium subg. Staphisagria 100 Outgroup

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Fig. 4. Chronogram for Aconitella and Consolida obtained under a Bayesian relaxed clock with log-normally distributed rates (76 taxa, 4506 aligned nucleotides). Bars around node ages in- dicate the 95% highest posterior density (HPD) intervals for nodes with a posterior probability > 0.95. For further explanation see Fig. 3.

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Fig. 5. Ancestral area reconstruction for Aconitella and Consolida on a portion of the maximum likelihood tree (Fig. 3, see Material and Meth- ods). The arrow indicates the crown group of the Consolida s.l. clade. Numbers 1–3 refer to successive expansions towards the Mediterranean basin. These expansion events are numbered chronologically according to the dated tree (Fig. 4). A westward expansion can be inferred confidently for clade I, less confidently for clade II, and with poor confidence for clade III. Numbers I–V on the right refer to clades discussed in the main text. Within the Consolida s.l. clade, the precise geographic origin of samples is specified for all endemic species. Where multiple accessions of the same species were sequenced, we added voucher information. The colour coding of names refers to life history: blue = annuality, red = perenniality. Ir.-Tur. refers to a clade with a broad Irano-Turanian distribution; Med. to a clade with a broad Mediterranean distribution.

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the maximum clade credibility tree with mean node ages from between Aconitella and species of Aconitum sect. Lycocto- the several thousand trees included in the post-burn-in sample) num, while Soják (1969) and Trifonova (1990) suggested that obtained for the Delphinieae dataset. Different from the ML Aconitella and Consolida might be sister groups, hypotheses topology (Fig. 3), the Bayesian tree obtained from the dating clearly rejected here. On the other hand, sect. Involutae sensu analysis (Fig. 4) shows clade II diverging below the remain- Kemularia-Nathadze (1939), Soják (1969), and Trifonova (1990) ing clades of the polytomy formed by the five main clades of (i.e., all Aconitella species, including A. barbata) is coherent Consolida (marked by an arrow in Fig. 4). Ages inferred using with clade V (Figs. 3–5), and Huth (1895) correctly grouped the 4506-nucleotide matrix and secondary calibration points the majority of the species in clade II (Fig. 3–5) together as a and those inferred with an ITS rate of 8.34 × 10–9 substitutions section (Consolida sect. Macrocarpae). Lastly, the species in per site per year (see Materials and Methods) applied just to our clade I (Figs. 3–5) all share a particular seed coat with discon- ITS sequences largely agreed. The split between Consolida s.l. nected scales, which in light of the molecular tree probably is and its Delphinium sister clade was dated to 21.7 ± 3 Ma (Fig. 4), a synapomorphy of these species (Constantinidis & al., 2001). and the crown group age of Consolida s.l. to 16.9 Ma (95% CI An unusual species that has been difficult to place based on 13.4–20.9 Ma). The diversification of major clades in Consolida morphology is Aconitella barbata. It has a Consolida-like peri- s.l. (clades I–V in Figs. 3–5) began around 8.8 (6.2–11.8) Ma. anth shape combined with an Aconitella-like petiole structure Figure 5 shows the reconstruction of ancestral areas on (Trifonova, 1990). Huth (1895) therefore placed A. barbata in the ML tree (Fig. 3), with the inset showing the relevant geo- a section of its own. Fitting with this classification, A. barbata graphic regions. The Consolida/Aconitella clade appears to is here placed as sister to all remaining Aconitella species. have undergone three expansions towards the Mediterranean While our sampling of Delphinium is biased towards subg. basin (labelled by boxed blue numbers in Fig. 5). Contrary to Delphinium, we made a point of including all biennial spe- this clear geographic structure in the tip clades, an ancestral cies, i.e., all species of subg. Staphisagria. Of the three spe- region of the entire group could not be inferred. Part of the cies, D. requienii is endemic to the Hyères Islands (southern problem is the statistically poorly supported sister-group rela- France), D. staphisagria is widespread due to its medicinal uses tionship between Consolida s.l. and the Chinese/North Ameri- (Orellana & al., 2009a), and D. pictum is found in Corsica, Sar- can Delphinium clade (represented by subg. Delphinastrum and dinia, and Majorca (D. pictum sequences were incomplete but subg. Oligophyllon in Figs. 3–5). If instead the Mediterranean did group with those of the other two species; Appendix). They subg. Delphinium were the sister group to Consolida s.l. an are similar in perianth shape and seed morphology (Blanché, ancestral region in the Mediterranean or Asia Minor would 1991) and exhibit ascending dysploidy, with D. staphisagria become likely. having 2n = 18 (Constantinidis & Kamari, 1995; Simon & al., 1995; Bosch & al., 2002), whereas the two other species have 2n = 16. Our phylogenetic results support the isolation of subg. DISCUSSION Staphisagria within Delphinium (Orellana & al., 2009b), but suggest that Delphinium and Aconitum may not be mutually Consolida and Aconitella may need to be included in Del- monophyletic (Fig. 3). Assessing this possibility, however, re- phinium. — Huth (1895) was the last worker to include Con- quires denser species sampling of these two large genera, each solida s.l. in Delphinium, an action that probably was easier with ca. 300 species. 115 years ago than it is now, with many of the 52 species of The spatio-temporal unfolding of Consolida and Aconi- Aconitella and Consolida having been treated in numerous tella. — Current understanding is that Delphinium differen- Floras and other botany texts. Modern workers therefore mostly tiated primarily in Central Asia (Tamura, 1993) and that the left Aconitella and Consolida as separate genera (Table 1), even Mediterranean region is a secondary centre of diversification when they inferred the evolution of Consolida from within (Orellana & al., 2009a). The Mediterranean sect. Delphinium Delphinium (Tamura, 1966; Malyutin, 1973). The phylogenetic has been suggested to stem from sect. Anthriscifolium (Wang, trees generated here now show unambiguously that Aconitella 1962; obviously using pre-cladistic thinking) during westward is part of Consolida (100% bootstrap support) and that both expansion from southwest China (Wu, 2001). This hypothesis are embedded in Delphinium (88%). A classification of mutu- needs further testing. Our results at least reveal that Consolida ally monophyletic genera might therefore require transferring diverged from Delphinium relatives in the Early to Middle Mio- Aconitella and Consolida into Delphinium. In terms of nomen- cene (Fig. 4), a period of increasing aridity, caused primarily clatural changes, this would involve only seven new names by decreases in sea level in the Mediterranean (Rögl, 1999; because for the remaining species, names in Delphinium are Peryt, 2006; de Leeuw & al., 2010) and desertification in Asia already available from Huth’s classification (1895). Alterna- (Guo & al., 2002). The retreat of the Paratethys Sea in western tively, one might split up Delphinium into several genera, a Central Asia during the Late Oligocene and Early Miocene step that would be premature with the current sampling of this (Ramsetin & al., 1997) and the uplift of the Tibetan-Himalayan genus (especially since the mutual monophyly of Aconitum and plateau, which intensified at 25–20 Ma, played a major role in Delphinium remains unclear; Fig. 3). the shift of the Central Asian climate from oceanic to continen- Few of the species relationships found in earlier stud- tal (Ramsetin & al., 1997), with accompanying increasing levels ies fit with the present molecular data. Thus, Spach (1839) of aridity. Open grass-dominated habitats expanded as a conse- and Kemularia-Nathadze (1939) inferred a close relationship quence of the increased aridity (Strömberg & al., 2007) probably

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favouring the diversification of the Consolida s.l. clade, with its Acknowledgements annual life strategy and open, dry-habitat-adapted grow forms (Fig. 1). Delphinieae flowers are bee-pollinated (Bosch, 1997; We thank C. Blanché for constructive discussions, E.M. Friis Bosch & al., 1997) and the seeds wind-dispersed, both traits well for information on fossils, and A. Gröger and S. Zarre for providing suited to dry and open areas. The morphological bases of floral pictures of Consolida and Aconitella. Guido W. Grimm provided much innovations in Delphinieae are the focus of ongoing research in appreciated advice about the g2cef software. F.J.’s research was sup- our laboratory (see Jabbour & al., 2009). ported by DFG grant RE 603/12-1. Ancestral area reconstruction was relatively straightfor- ward for the densely sampled and geographically restricted tip clades (Fig. 5). Land bridges are known to have permitted biotic Literature cited expansions across the Mediterranean and Paratethys during the Oligocene and Miocene (Rögl & Steininger, 1983; Oosterbroek Alam, J. & Ali, S.I. 2010. Contribution to the Red List of the plants of & Arntzen, 1992). Each sea level regression event (associated Pakistan. Pakistan J. Bot. 42: 2967–2971. with biotic expansion) was followed by a new marine transgres- Bell, C.D., Soltis, D.E. & Soltis, P.S. 2010. The age and diversification of the angiosperms re-revisited. Amer. J. Bot. 97: 1296–1303. sion that restored connections between the Mediterranean and Balthazar, M. von, Endress, P.K. & Qiu, Y. 2000. Phylogenetic rela- the Paratethys and resulted in East-West vicariance of trans- tionships in Buxaceae based on nuclear internal transcribed spac- Mediterranean lineages (Oosterbroek & Arntzen, 1992). As a ers and plastid ndhF sequences. Int. J. Pl. Sci. 161: 785–792. result, Mediterranean clades often present disjunct distributions Blanché, C. 1991. Revisiό biosistemàtica del gènere Delphinium L. a between the western and eastern Mediterranean, or on a larger la Península Ibèrica i a les Illes Balears. Arxius de la Secciό de scale, between the western Mediterranean and Central Asia, Ciències 98. Barcelona: Institut d’Estudis Catalans. Blanché, C., Molero, J. & Simon, J. 1987. Estudis taxonomics en the so-called Kiermack disjunctions (examples in Oosterbroek Delphinium L. i Consolida S.F. Gray a la Peninsula Iberica i les & Arntzen, 1992; Ribera & Blasco-Zumeta, 1998). Based on illes Balears: Fitodermologia. Butl. Inst. Catalana Hist. Nat., Secc. the inferred clade ages (Fig. 4), the western expansions of the Bot. 54: 55–64. Consolida s.l. clade must have occurred in this setting, with the Blondel, J., Aronson, J., Bodiou, J.-Y. & Boeuf, G. 2010. The Mediter- drying up of the Mediterranean Sea around 5.96–5.33 Ma (Hsü ranean region: Biological diversity through time and space, 2nd ed. Oxford: Oxford University Press. & al., 1973; Krijgsman & al., 1999; Fauquette & al., 2006; Popov Boissier, E. 1867. Flora Orientalis, vol. 1. Basel: H. Georg. & al., 2006) probably permitting Consolida to disperse as far as Bosch, M. 1999. Biologia de la reproducciό de la tribu Delphinieae a Spain and Morocco (crown group ages of clade I: 2.8 Ma, clade la Mediterrània occidental. Arxius de les Seccions de Ciències, III: 4.4 Ma, and the group of Mediterranean species in clade II: 120. Barcelona: Institut d’Estudis Catalans. 5.11 Ma; Figs. 4, 5). Subsequent diversification may be linked to Bosch, M., Simon, J. & Blanché, C. 2002. Reports 1305–1311. In: the renewed interruption of gene flow caused by the re-flooding Kamari, G., Blanché, C. & Garbari, F. (eds.), Mediterranean chromosome number reports. Fl. Medit. 12: 470–475. of the Mediterranean basin (see Salvo & al., 2010). Of the three Bosch, M., Simon, J., Blanché, C. & Molero, J. 1997. Pollination clades, clade II (Fig. 5) has achieved the widest distribution: It ecology in tribe Delphinieae (Ranunculaceae) in W Mediterra- includes C. orientalis, found in Northwest Africa, the Iberian nean area: Floral visitors and pollinator behaviour. Lagascalia Peninsula, the Balkans, Caucasia, Syria, Iran, and Central Asia, 19: 545–562. a range covering the entire distribution area of Consolida s.l. Bosch, M., Simon, J., Molero, J. & Blanché, C. 2001. Breeding sys- Some Consolida species, however, occur in cultivated fields, tems in tribe Delphinieae (Ranunculaceae) in the western Mediter- ranean area. Flora 196: 101–113. and their ranges may be partly anthropogenic. Britton, T., Anderson, C.L., Jacquet, D., Lundqvist, S. & Bremer, The evolution of the annual life cycle in Delphinieae. — K. 2007. Estimating divergence times in large phylogenetic trees. In the most comprehensive phylogeny of Ranunculaceae to Syst. Biol. 56: 741–752. date (Wang & al., 2009), the closest relative of Delphinieae is Candolle, A.P. de. 1824. Prodromus systematis naturalis regni vege- Nigella, which consists of annuals. Within Delphinieae, Aco- tabilis, vol. 1. Paris: Treuttel & Würtz. Carlström, A. 1984. New species of Alyssum, Consolida, Origanum nitum comprises annual and biennial species, but also peren- and Umbilicus from the SE Aegean Sea. Willdenowia 14: 15–26. nial ones, such as A. septentrionale sampled here. Delphinium Chater, A.O. 1993. Consolida (DC.) S.F. Gray. Pp. 260–262 in: Tutin, likewise comprises annuals, biennials, and perennials (Orellana T.G., Burges, N.A., Chater, A.O., Edmondson, J.R., Heywood, & al., 2009b). The annual strategy of the Mediterranean Del- V.H., Moore, D.M., Valentine, D.H., Walters, S.M. & Webb, D.A. phinieae probably is advantageous in environments with un- (eds.), Flora Europaea, 2nd ed., vol. 1. Cambridge: Cambridge even and seasonal rainfall (Bosch & al., 1997, 2001; fitting with University Press. Constantinidis, T. & Kamari, G. 1995. Reports 401–414. In: Kamari, Raunkiaer’s (1918) finding that annuals are especially common G., Felber, F. & Garbari, F. (eds) Mediterranean chromosome num- in the Mediterranean). Based on our current tree (Fig. 3), the ber reports. Fl. Medit. 5: 265–268. annual strategy in Consolida s.l. evolved independently of that Constantinidis, T., Psaras, G.K. & Kamari, G. 2001. Seed morphol- in Delphinium subg. Delphinium; however, the crucial node ogy in relation to infrageneric classification of Consolida (DC.) has only 64% bootstrap support. If subg. Delphinium were the Gray (Ranunculaceae). Flora 196: 81–100. sister group to Consolida s.l., then the annual strategy would Davis, P.H. 1965. Consolida (DC.) S.F. Gray. Pp. 119–134 in: Davis, P.H. (ed.), Flora of Turkey and the East Aegean islands, vol. 1. parsimoniously have evolved only once, in their common an- Edinburgh: Edinburgh University Press. cestor, perhaps in Southwest Asia during times of increasing Davis, P.H. & Sorger, F. 1982. Two new Anatolian species of Con- aridity (see previous section). solida. Notes Roy. Bot. Gard. Edinburgh 40: 89–91.

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Appendix. Species names and authorities, geographical provenience, and herbarium vouchers for the material included in this study. GenBank accession numbers are given for the five markers sequenced, trnL-trnF, trnK-matK, trnS-trnG, ITS and rbcL (new sequences indicated by an asterisk); an n-dash denotes a missing marker. Outgroups: Actaea racemosa L., UMBI-SG-200903, GQ409514, –, –, –, –; Actaea racemosa L., UMBI-SG-200902, –, –, –, GQ409510, –; Nigella damascena L., Luo & al., (unpub.), AY150260, –, –, –, –; Nigella damascena L., BG Mainz 041773, –, –, –, EU699446, –; Aconitella Spach: A. aconiti (L.) Soják, Turkey: Konya, 2.5 km E Zaferiye köyii, road to Ilgĭn (1060 m), Buttler 20006 (M), **JF331679, **JF331746, *JF331810, *JF331874, –; A. anthoroidea (Boissier) Schröd., Iran: Kurdistan, 6 km SE Bijar, road to Hamadan, Rechinger 42468 (M), *JF331680, *JF331747, *JF331811, *JF331875, *JF331666; A. barbata (Bge) Schröd., Afghanistan: Baghlan prov., Pul-i-Kumri, Podlech 11320 (M), *JF331681, *JF331748, *JF331812, *JF331876, –; A. hohenackeri (Boiss.) Soják, Turkey: Erzurum, Dutlu Daĝi, orman Evi valley (1850 m), Nydegger 44486 (MSB), *JF331682, *JF331749, *JF331813, *JF331877, –; A. saccata (Huth) Soják, Kurdistan: Mardin prov., Rischemil, Sintenis 1186 (M), *JF331683, *JF331750, *JF331814, –, –; A. scleroclada (Boiss.) Soják var. rigida (Freyn & Sint.), Turkey: Erzincan, 10 km N Kemah, Refahiye (1250 m), Nydegger 43806 (MSB), *JF331684, *JF331751, *JF331815, –, –; A. stenocarpa (Davis & Hossain) Soják, Turkey: Konya, 1.9 km W Topraklĭ köyü (1030 m), Buttler 20012 (M), *JF331685, *JF331752, *JF331816, *JF331878, –; A. thirkeana (Boiss.) Soják, Turkey: Eskişehir, 500 m SE Asagi Söğlitönü köyü (800 m), Buttler & Erben 17987 (M), *JF331686, *JF331753, *JF331817, *JF331879, –; Aconitum L.: A. baicalense (Regel) Turczaninow ex Rapaics, Russia: Transbaikal (mountainous region), Graf Zedtwitz 1936 (M), *JF331723, *JF331788, *JF331852, –, –; A. baicalense (Regel) Turczaninow ex Rapaics, Russia: Siberia, Tuwa, TNS9027196, –, –, –, AB004941–AB004945, –; A. ciliare DC., China: Jilin prov., Cang Bai Shan, along road to Antu, 3 km N of Baihe railway station, Herrmann 238 (M), *JF331724, *JF331789, *JF331853, –, –; A. ciliare DC., Japan: Kumamoto, Kita 951120, –, –, –, AB004952–AB004954, –; A. delphinifolium subsp. delphinifolium DC., Alaska: Kenai peninsula, Turnagain pass, Lang s.n. (coll. date 28. 7. 1996) (M), *JF331725, *JF331790, *JF331854, –, –; A. delphinifolium DC., Wells 1777, –, –, –, AF258681, –; A. ferox Nallich ex Peringe, Nepal: Khumbu, near Tutkosital (3600–4000 m), Pcelt 29 (M), *JF331726, *JF331791, *JF331855, –, –; A. ferox Wall. ex Seringe, Nepal: Mt. Shiwapuri, Minaki & al. 9100909 (TI), –, –, –, AB004961–AB004962, –; A. gymnandrum Maxim., China: Xizang, E Tibet, upper Salween basin, Riwoqe, Dengqen, Da Qu, 5 km W of Songtung (3650 m; 95°52′ E, 31°17′ N), Dickoré 9111 (MSB), *JF331727, *JF331792, *JF331856, –, *JF331677; A. gymnandrum Maxim., Luo & al. (2002), –, –, –, AY150238, –; A. gymnandrum Maxim., China: Qinghai-Tibetan Plateau, Chen 2004013_1, –, –, –, FJ418136, –; A. gymnandrum Maxim., China: Qinghai-Tibetan Plateau, Liu Jianquan 1610 (HNWP), –, –, –, FJ418137, –; A. gymnandrum Maxim., China: Qinghai- Tibetan Plateau, Liu Jianquan A073 (HNWP), –, –, –, FJ418138, –; A. gymnandrum Maxim., China: Qinghai-Tibetan Plateau, Liu Jianquan A027 (HNWP), –, –, –, FJ418139, –; A. gymnandrum Maxim., China: Qinghai-Tibetan Plateau, Liu Jianquan A027 (HNWP), –, –, –, FJ418140, –; A. gymnandrum Maxim., China: Qinghai- Tibetan Plateau, Liu Jianquan A040 (HNWP), –, –, –, FJ418141, –; A. gymnandrum Maxim., China: Qinghai-Tibetan Plateau, Liu Jianquan 2713 (HNWP), –, –, –, FJ418142, –; A. gymnandrum Maxim., China: Qinghai-Tibetan Plateau, Liu Jianquan 20020728 (HNWP), –, –, –, FJ418143, –; A. gymnandrum Maxim., China: Qinghai-Tibetan Plateau, Liu Jianquan 20020728 (HNWP), –, –, –, FJ418144, –; A. gymnandrum Maxim., China: Qinghai-Tibetan Plateau, Liu Jianquan A087

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Appendix. Continued. (HNWP), –, –, –, FJ418145, –; A. gymnandrum Maxim., China: Qinghai-Tibetan Plateau, Liu Jianquan A093 (HNWP), –, –, –, FJ418146, –; A. gymnandrum Maxim., China: Qinghai-Tibetan Plateau, Liu Jianquan A093 (HNWP), –, –, –, FJ418147, –; A. gymnandrum Maxim., China: Qinghai-Tibetan Plateau, Liu Jianquan A090 (HNWP), –, –, –, FJ418148, –; A. pendulum Busch, China: Sichuan prov., Songpan Xian, S of the Jiuzhaigou Xian line and N of the city of Songpan on hwy 301 on E side of highway at Awong Gou (3575 m; 103°44′07″ E, 33°01′26″ N), Boufford & al.. 40471 (MSB), *JF331728, *JF331793, *JF331857, –, –; A. pendulum Busch, Luo & al. (2002), –, –, –, AY150235, –; A. pentheri Hayek, Serbia: road from Pec to Anrijevica, E slopes of the Cakor pass (1820 m), Podlech & Lippert 26275 (M), *JF331729, *JF331794, *JF331858, *JF331905–*JF331918, –; A. septentrionale Koelle, Norway: Oppland, Rindatal, camping Hammarseter, street 257, W Rindseter, Dietrich 5808/09 (M), *JF331730, *JF331795, *JF331859, –, *JF331678; A. septentrionale Koelle, Russia: Ural,–, A.Y. Yarosheko & N. Maslinnikova ZZT5, –, –, –, AF216552, –; A. septentrionale Koelle, Norway: Oppdal, 545, E. Zogg ZZT3, –, –, –, AF216555, –; Consolida (DC.) S.F. Gray: C. ajacis (L.) Schur, Germany: Bavaria, Munich, Romanplatz (wild), Merxmüller 8524 (M), *JF331687, *JF331754, *JF331818, *JF331880, *JF331667; C. ajacis (L.) Schur, France : Var, Hyères, Ceinturon, Ruppert D3663 (M), *JF331688, *JF331755, *JF331819, *JF331881, –; C. ajacis (L.), U.S.A. : Texas, Austin County, Bellville, in flower bed on E side of house at 951 S Holland, Franklin Susen 35 (M), *JF331689, *JF331756, *JF331820, *JF331882, *JF331668; C. armeniaca (Stapf ex Huth) Schröd., Turkey: 47 km N Erzincan, between Erzincan and Gümfişane (1580 m), Nydegger 16993 (MSB), *JF331690, *JF331757, *JF331821, *JF331883, *JF331672; C. aucheri (Boissier) Iranshahr, Afghanistan: Kandahar prov., Nurmohammadkhan, 24 km W Kandahar (975 m), Podlech & Jarmal 28879 (MSB), *JF331691, *JF331758, *JF331822, *JF331884, –; C. axilliflora (DC.) Schröd., Turkey: Anatolia, Hatay, Antioch, Iskenderun, W. Amik-Gölü (120 m), Brachfeld & Graben 42414 (MSB), *JF331692, *JF331759, *JF331823, *JF331885, *JF331669; C. brevicornis (Visiani) Soό, Italy: Southern Adriatic coast, Lesina, Batteni s.n. (M 2825), *JF331693, –, –, –, –; C. camptocarpa (Fisch. & Mey.) Nevski, Kazakhstan: Taldy-Kurgan prov., next to Balchasch lake, between Karatal and Aksu rivers, Rusanovitsch & Kramarenko 14029 (M), *JF331694, *JF331760, *JF331824, *JF331886, –; C. flava (DC.) Schröd., Iraq: Haswa desert, 20 km W Rutba, Ramadi Liwa, Rechinger 148 (M), *JF331695, *JF331761, *JF331825, *JF331887, –; C. glandulosa (Boiss. & Huet) Bornm., Turkey: Anatolia, Maraş, E Elbistan, from Göksun to Elbistan (1180 m), Nydegger 16741 (M), *JF331696, *JF331762, *JF331826, *JF331888, –; C. hellespontica (Boiss.) Chater, Turkey: Konya, Koraş, 38 km S Ayranci (1580 m), Nydegger 44206 (MSB), *JF331697, *JF331763, *JF331827, *JF331889, –; C. hispanica (Costa) Greuter & Burdet, Germany: Bavaria, natural reserve, W B17 between Kaufering and Hurlach, S Augsburg, Angerer s.n. (coll. date 25.7.1984) (M), *JF331698, *JF331764, *JF331828, *JF331890, –; C. incana (Clarke) Munz, Israel: Upper Galilee, Malkiya, Zacai s.n. (coll. date 25.06.1979 MSB), *JF331699, *JF331765, *JF331829, –, –; C. kabuliana (Akhtar) Iranshahr, Afghanistan: Kabul province, Sarobi (ca. 1000 m; 69°45′ E, 34°35′ N), Volk 2630 (M), *JF331700, *JF331766, *JF331830, *JF331891, –; C. kandaharica Iranshahr, Afghanistan: Farah province, 20 km SW Sherzad, road from Shindand to Farsi (1600 m), Podlech 22009 (MSB), *JF331701, *JF331767, *JF331831, *JF331892, –; C. leptocarpa Nevski, Afghanistan: Takhar prov., Naqel, road from Khanabad to Taluqan (620 m), Anders 5897 (MSB), *JF331702, *JF331768, *JF331832, –, –; C. lineolata Huber-Morath & Simon, Turkey: between Ermenek and Mut, 43 km SW Mut (940 m), Nydegger 13313 (MSB), *JF331703, –, –, *JF331893, –; C. mauritanica (Cosson) Munz, Morocco: Er-Rachidia prov., High Atlas, ca. 8 km W Tounfite, road to Arhballa (2100 m; 5°19′ W, 32°28′ N), Podlech 47566 (MSB), *JF331704, *JF331769, *JF331833, *JF331894, –; C. oliveriana (DC.) Schröd., Turkey: Elaziĝ prov., 34 km W Bingöl (1680 m), Nydegger 17239 (MSB), *JF331705, *JF331770, *JF331834, –, –; C. olopetala (Boissier) Hayek, Turkey: Erzincan, 8 km N Erzincan, Çayirli (1470 m), Nydegger 43795 (MSB), *JF331706, *JF331771, *JF331835, *JF331895, –; C. orientalis (Gay.) Schröd., Iran: Isfahan prov., Isfahan to Shahreza road (2150 m), Parishani 14382 (M), *JF331707, *JF331772, *JF331836, *JF331896, –; C. persica (Boiss.), Iran: Kurdistan, 107–109 km SW Zanjan, versus Bijar (1700 m), Rechinger 42445 (M), *JF331708, *JF331773, *JF331837, *JF331897, *JF331673; C. pubescens (DC.) Soό var. dissitiflora (Coss.) A. Dubuis, Algeria: Djelfa department, between El Mesrane and Hassi-Bahba, 40 km N Djelfa, against the dunes at the edge of Zahrez Chergui (900 m), Dubuis 11222 (M), *JF331709, *JF331774, *JF331838, *JF331898, –; C. pubescens (DC.) Soό, Spain: Zaragoza prov., SE of Sierra de Alcabierre, SW of Castejon de Monegros (500 m), Lambinon 79E568 (MSB), *JF331710, *JF331775, *JF331839, *JF331899, –; C. raveyi (Boiss.) Schröd., Turkey: Paphlagonia, Wilajet Kastambuli, Tossia, Sabadja, Sintenis 4293 (M), *JF331711, *JF331776, *JF331840, –, –; C. regalis S.F. Gray, Germany: Bavaria, Oberpflaz, Schwandorf, Münchshofen moutain (1.5 km NW Teublitz an der Naab), field margins (450–500 m), Förther 8339 (MSB), *JF331712, *JF331777, *JF331841, –, –; C. regalis regalis S.F. Gray, Italy: Aosta valley, hill W. Châtillon, Doppelbaur 2393 (M), *JF331713, *JF331778, *JF331842, –, *JF331671; C. regalis regalis S.F. Gray, Austria: Parndorf plain, ruderal plants between Parndorf and Neusiedl, Hertel 6560 (M), *JF331714, *JF331779, *JF331843, *JF331900, –; C. regalis regalis S.F. Gray, France: Basses-Alpes department, Vaucluse, Gubian, 4 km N Revest, road to Onglet, fallow (680 m), Podlech 50694 (MSB), *JF331715, *JF331780, *JF331844, –, –; C. regalis divaricata (Ledeb.) Schröd., Armenia: Vayotsdzor province, Yeghegnadzor district, ca. 10 km SW Yeghegnadzor, ca. 4 km SE Areni, valley N. of monastery Noravank, along river (1500 m; 45°13′ E, 39°41′ N), Fayvush 1600 (M), *JF331716, *JF331781, *JF331845, –, *JF331670; C. regalis S.F. Gray paniculata (Host) Soό, Montenegro: Dalmatia, W Budva, coastline (3 m), Černoch 32452 (MSB), *JF331717, *JF331782, *JF331846, *JF331901, –; C. rugulosa (Boiss.) Schröd., Afghanistan: Bamian prov., Ajar Tal (Kamard Tal), Roysang (1700 m), Anders 6295 (MSB), *JF331718, *JF331783, *JF331847, –, –; C. songorica (Kar. & Kir.), Kazakhstan: “Songoriae” dune near Lepsa river, Karelin & Kiriloff 1165 (MSB), *JF331719, *JF331784, *JF331848, *JF331902, –; C. stocksiana (Boiss.) Nevski, Afghanistan: Kabul prov., Sarobi (ca. 1000 m; 69°45′ E, 34°35′ N), Volk 1585 (M), *JF331720, *JF331785, *JF331849, *JF331903, –; C. tenuissima (Sibthorp & Smith) Soό, Greece: Attica, Mt Parnes, Pinatzi s.n. (coll. date 1937; M), *JF331721, *JF331786, *JF331850, –, –; C. uechtritziana (Huth) Soό, Greece: Epirus, Mt. Timphi, Arisiti village, Rechinger 21108 (M), *JF331722, *JF331787, *JF331851, *JF331904, –; Delphinium L.: D. anthriscifolium Hance, China: Shanxii prov., northern valley of western Taipaishan (ca. 1400 m; 107°48′ E, 34°04′ N), Podlech 55468 (MSB), *JF331731, *JF331796, *JF331860, *JF331919–*JF331930, *JF331674; D. bakeri Ewan, U.S.A.: California, Marin Co., Snow 1230 (RPBG 89.181), AF258652, –, –, AF258697, –; D. balansae Boiss. & Reuter, Morocco: Marrakech prov., High Atlas, near Oukaimeden (2700–3200 m; 7°51′ W, 31°11′ N), Sammet & Ilitz s.n. (coll. date June/July 1991) (MSB), *JF331732, *JF331797, *JF331861, *JF331931–*JF331944, –; D. balcanicum Pawl., Serbia: road ca. 30 km northern Kumanovo, near Bujanovac (400 m), Rössler 6570 (M), *JF331733, *JF331798, *JF331862, *JF331945–*JF331954, –; D. cardinale Hooker, U.S.A.: California, Santa Barbara Co., Santa Barbara Botanical Garden, Mort 1374 (no voucher), AF258648, –, –, AF258740, –; D. cossonianum Batt., Morocco: Meknes prov., 14 km northern the diversion between the road Fes-Sidi-Kacem (P3) and the road to Ouezzane (P28) (90 m; 5°29′ W, 34°14′ N), Podlech 46655 (MSB), *JF331734, *JF331799, *JF331863, *JF331955–*JF331964, *JF331675; D. delavayi Franch., China: Lijiang, Yulong Shan, McBeath & al. CLD0895 (UCBG 91.362), AF258659, –, –, AF258705, –; D. favargeri Blanche, Molero & Simon, Morocco: Middle Atlas, 3 km from Azrou along road to Midelt at junction of road to Ain-Leuh (1475 m; 5°11′54″ W, 33°25′38″ N), Jury & Ait Lafkih 19781 (M), *JF331735, *JF331800, *JF331864, *JF331965–*JF331976, –; D. gracile DC., Spain: N. Teruel prov., Azalla, road to Ascatrόn (300 m), Lambinon 79/E/534 (MSB), *JF331736, *JF331801, *JF331865, *JF331977–*JF331981, –; D. grandiflorum L., China: Rd from Songpan to Juizhaigou, Erskine & al. SICH205 (UCBG 89.0411), AF258630, –, –, AF258761, –; D. halteratum Sm., Italy: Abruzzo, L’Aquila prov., Barisciano, San Colombo, road embankment near the entrance of the National Park S Colombo (1000 m), Dunkel, MTB 3647.4 (M), *JF331737, *JF331802, *JF331866, *JF331982–*JF331987, –; D. hirschfeldianum Heldr. & Holzm., Greece: Mykonos island, sine coll. 1604 (coll. date 4.–22.07.1901) (M), *JF331738, *JF331803, *JF331867, *JF331988– *JF331995, –; D. macropetalum DC., Morocco: Er-Rachidia prov., High Atlas, Tizlit lake, N Imilchil (2250 m; 5°38′30″ W, 32°11′ 47″ N), *JF331739, *JF331804, *JF331868, *JF331996–*JF332000, –; D. parryi Gray, U.S.A.: California, Ventura Co., Hwy 33, Edwards s.n. (RPBG 88.240), AF258636, –, –, AF258694, –; D. patens Bentham, U.S.A.: California, Colusa Co., Bear Valley Rd., Richter 11 (WS 341959), AF258658, –, –, AF258734, –; D. peregrinum L., Macedonia: 4 km NE of the village of Openica, about half-way on the main road between Resen and Ohrid (880 m), Franzén & al. 870 (M), *JF331740, *JF331805, *JF331869, *JF332001– *JF332008, –; D. pictum Willd., Balearic Islands: Majorca, Puntas de Covas, top of sea cliffs (100 m), Rumsey 15012 (M), *JF331741, –, –, –, –; D. polycladon Eastwood, U.S.A.: California, Fresno Co., Line Creek, Bacigalupi 6508 (WS 256518), AF258642, –, –, AF258743, –; D. requienii DC., Sardinia: Cagliari prov., between San Giovanni Grotto in Domusnόvas and Sa Duchesse, ca. 1 km after the diversion after Perdu Carta (180 m), Erben s.n. (coll. date 6.6.1991) (M), *JF331742, *JF331806, *JF331870, *JF332009–*JF332021, *JF331676; D. staphisagria L., Greece: Crete, Nomos Lassithiou, ravine between Zákros and Kato Zákros (70 m), Vitek 02-205 (M), *JF331743, *JF331807, *JF331871, *JF332022–*JF332023, –; Delphinium trolliifolium Gray, U.S.A.: California, Bald Hills Road, RPBG (Regional Parks Botanic Garden) 94.73, AF258650, –, –, AF258686, –; D. venulosum Boiss., Turkey: Niğde, 8 km N Çamardi (1400 m), Nydegger 15452 (MSB), *JF331744, *JF331808, *JF331872, *JF332024–*JF332029, –; D. virgatum Poiret, Turkey: Kayseri, 55 km to Saimbeyli from Tasçi (Bakirdaği), Zarre 53 (MSB), *JF331745, *JF331809, *JF331873, *JF332030–*JF332031, –.

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