Consolida and Aconitella are an annual clade of Delphinium

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Jabbour & Renner • Consolida and Aconitella belong in DelphiniumTAXON 60 (4) • August 2011: 1029–1040
INTRODUCTION
Plant clades that diversified in the Mediterranean biodiversity hotspots often have biogeographic links to the Irano- Turanian region and Central Asia (Quézel, 1985; Ribera & Blasco-Zumeta, 1998; Mansion & al., 2008, 2009; Lo Presti & Oberprieler, 2009; Médail & Diadema, 2009; Blondel & al., 2010). The Irano-Turanian phytogeographic region encom- passes Anatolia, parts of Jordan, Syria, the Israeli-Palestinian region, the Sinai Peninsula, upper Mesopotamia, a large part of the Armenian Highlands, southern and eastern Transcaucasia, the Caspian shore of Iran, the Iranian Plateau (without the tropi- 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 and many diverse and poorly understood genera. Among the genera centred in this region and extending into the Mediter- ranean are Consolida (DC.) S.F. Gray and Aconitella Spach. To- gether these genera comprise ca. 52 species, occurring from sea level to 2000 m altitude, often on dry stony slopes, in steppes, semi-deserts, or even deserts (Trifonova, 1990; our Fig. 1). Their closest relatives are Delphinium L. and Aconitum L., each with an estimated 300 species; morphological similarities to Delphinium even led to the inclusion of Consolida in Del- phinium (Table 1). While the centre of diversity of Consolida
and Aconitella lies in the Mediterranean/Irano-Turanian region (Davis, 1965; Davis & Sorger, 1982; Munz, 1967a, b; Carlström, 1984; Iranshahr, 1987, Misirdali & al., 1990; Chater, 1993), a few species occur east as far as Kazakhstan, Afghanistan, and Pakistan (an example is Consolida schlagintweitii (Huth) Munz; Munz, 1967b; Alam & Ali, 2010).
Consolida and Aconitella are characterized by an annual life cycle, probably as an adaptation to seasonal drought (Constantinidis & al., 2001). Annuals survive unfavourable periods as seeds and are statistically overrepresented in the Mediterranean biome (Raunkiaer, 1918). Delphinium also includes a few annual species, all in subg. 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).
Table 1. Generic classification of Consolida. Consolida included in Delphinium
Consolida considered as a separate genus
Candolle, 1824 (sect., 11 spp.) Gray, 1821 (monospec.: C. regalis) Boissier, 1867 (sect., 32 spp.) So, 1922 Huth, 1895 (subg., 45 spp.) Davis, 1965 Nevskii, 1937 (subg.) Munz, 1967a, b
Blanché & al., 1987 Chater, 1993
TAXON 60 (4) • August 2011: 1029–1040Jabbour & Renner • Consolida and Aconitella belong in Delphinium
The mutual monophyly of Consolida and Aconitella and their relationship(s) with the remaining genera of Delphinieae, viz. Aconitum and Delphinium, have never been tested with adequate DNA sequence data. Studies so far have included only C. ajacis (synonymous with C. ambigua) to represent Consolida (Ro & al., 1997) and one species each of Delphinium and Aconitum (Ro & al., 1997; Wang & al., 2009). A study that focused on the American larkspurs included 62 species of Delphinium and two species of Aconitum (Koontz & al., 2004). Here we investigate the relationships and biogeography of Consolida and Aconitella with a sampling of 75% of their species plus representatives of the different subgenera of Del- phinium and Aconitum. Specific questions we are addressing are: (1) Are Consolida and Aconitella mutually monophyletic and what is their relationship to Delphinium ? (2) When and where did Consolida and Aconitella diversify? (3) What is the relationship of the annual species of Delphinium to those in Consolida and Aconitella?
MaTeRIals aND MeThODs
Taxon sampling, DNA sequencing, cloning, alignment, and phylogenetic analyses. — The present study includes 39 species and subspecies (44 taxa) of Consolida and Aco- nitella, including the types of the generic names C. regalis S.F. Gray (Delphinium consolida L.) and A. aconiti (L.) Soják (Delphinium aconiti L.). For C. ajacis, C. pubescens, and C. regalis we included two or three samples each to represent the geographic ranges of these widespread species. Twenty-two sequences of Delphinium and eight of Aconitum were included, with a focus on the short-lived species of Delphinium because of our interest in the evolution of an annual life cycle in Del- phinieae. The subgeneric classification accepted for Delphin- ium is that of Malyutin (1987) who recognized two short-lived subgenera, subg. Staphisagria (J. Hill) Peterm. (three biennial species, all sampled) and subg. Delphinium sect. Anthrisci- 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 species of Delphinium are represented by eight species from China and North America (subg. Delphinastrum (DC.) Peterm., subg. Oligophyllon Dimitrova). Aconitum is represented by species from each of its three subgenera (classification of Ta- mura, 1990: (1) the monospecific biennial subg. Gymnaconitum (Stapf) Rapaics is represented by Aconitum gymnandrum from China; (2) the perennial subg. Lycoctonum (DC.) Peterm. (ca. 50 species) by Aconitum septentrionale; and (3) the biennial subg. Aconitum (ca. 250 species) by six Asian species. Based on the Ranunculaceae trees of Wang & al. (2009) we selected species of Actaea L. and Nigella L. to root our trees; the ap- propriateness of this choice was tested using Path-O-Gen v.1.3 (Drummond & al., 2003), which can help infer the root of a tree as long as the molecular clock assumption fits the data reasonably well (below).
DNA isolation and sequencing relied on commercial kits and the universal primers of Taberlet & al. (1991) for amplify- ing and sequencing the trnL intron and adjacent trnL-trnF intergenic spacer. The trnK-matK spacer was sequenced using the 3914F and 1470R primers (Johnson & Soltis, 1994), in ad- dition to two internal primers: trnK-rev (5′- GCT GAT AAG GAA TCG ATT TAT TTA A -3′), and matK-for (5′- CTA TAT CCA CTT CTC TTT CAG GAA T -3′) designed for species belonging to the tribe Delphinieae. The trnS-trnG intergenic spacer was sequenced using the trnSgcu and trnGuuc primers (Hamilton, 1999). The 18S nuclear ribosomal internal transcribed spacer 1 (ITS-1), 5.8S gene, and ITS-2, were ampli- fied with the external primers of Balthazar & al. (2000). The rbcL gene was sequenced for a few exemplar species to extend the Delphinieae dataset for dating purposes, using the external primers 1F (Fay & al., 1997) and 1460R (Olmstead & al., 1992) and the internal primers 600F and 800R (Kocyan & al., 2007). Sequencing relied on Big Dye Terminator kits (Applied Biosystems, Foster City, California, U.S.A.) and an ABI 3100 Avant capillary sequencer (Applied Biosystems). Sequence assembly of forward strands and reverse strands was carried out in MEGA v.4 (Tamura & al., 2007; Kumar & al., 2008), and sequences were BLAST-searched in GenBank and then aligned by eye.
Our entire study is based on material from the herbaria M and MSB, one advantage being that vouchers are all located (and available for future studies) at one place. Direct amplification via polymerase chain reaction (PCR) yielded single bands and unambiguous base calls, except for the ITS region in a few species of Aconitum and Delphinium for which we therefore cloned the ITS fragment. Bands were excised from agarose gel and purified using the QIAquick gel extraction kit (Qiagen, Hilden, Germany). The fragment was ligated into the pGEM-T Easy plasmid (Promega, Mannheim, Germany) and transformed into competent subcloning efficiency DH5α Escherichia coli cells. Up to 15 positive clones per sample were cultivated overnight. Modal consensus sequences were computed for each Aconitum and Delphinium species using the program g2cef following Göker & Grimm (2008).
In all, 247 sequences (including the modal consensus sequences) were newly generated for this study. The Appendix
lists all DNA sources, species names with authors, and Gen- Bank accession numbers. The aligned trnL-trnF, trnK-matK, trnS-trnG, and ITS matrices comprised 1338, 1560, 947 and 661 nucleotides respectively. There was no statistically supported conflict (defined as > 80% bootstrap support) between topologies obtained from the individual data partitions, and the chloroplast and nuclear data were therefore combined, yielding a matrix of 4506 characters and 76 taxa. All tree searches relied on maximum likelihood (ML) as implemented in RAxML (Stamatakis, 2006; Stamatakis & al., 2008), using the GTR + G model (with four rate categories), with separate partitions for the nuclear and chloroplast data. Statistical support for nodes was assessed by bootstrap resampling of the data under the same model (100 replicates). FindModel (http://hcv. lanl.gov/content/sequence/findmodel/findmodel.html; using the ModelTest script; Posada & Crandall, 1998) selected this model or the slightly less parameter rich HKY85 model as the best fit for our combined data matrix.
Molecular clock analyses. — We used Path-O-Gen v.1.3 to test the clock-like behaviour of our concatenated dataset (http://tree.bio.ed.ac.uk/software/pathogen/; Drummond & al., 2003). Divergence dating relied on Beast v.1.4.8 (Drummond & al., 2006; Drummond & Rambaut, 2007), which employs a Bayesian Markov chain Monte Carlo (MCMC) approach to co- estimate topology, substitution rates, and node ages. All dating runs relied on the GTR + G model (with six rate categories), a Yule tree prior, with rate variation across branches uncor- related and log-normally distributed. The MCMC chains were run for 30 million generations (burn-in 20%), with parameters sampled every 5000th step. Results from individual runs were combined, and effective sample sizes for all relevant estimated parameters and node ages were well above 200. Mixing of the chains was checked using Tracer v.1.5 (Rambaut & Drummond, 2007). Final trees were edited in FigTree v.1.3.1 (http://tree.bio .ed.as.uk/software/figtree/).
To convert relative times into absolute times we used a two-pronged approach. First, we constructed expanded rbcL and trnL-trnF matrices (with sequences from GenBank added to our own) so as to include Ranunculaceae nodes that have been dated in other studies. For the rbcL clock, we accepted an age of 73 ± 3 Ma to constrain the crown group age of Ra- nunculaceae (Britton & al., 2007). For the trnL-trnF clock, the split between Coptis and Xanthorhiza was set to 12 ± 3.75 Ma and that between Ranunculus and Coptis + Xanthorhiza to 45 ± 7 Ma, based on the work of Bell & al. (2010). The crown group age of Consolida/Aconitella and the age for the node equivalent to node A in Fig. 3, inferred with these two expanded matrices (rbcL, trnL-trnF), were then used as secondary calibration points (using normal prior distributions) in a Delphin- ieae-centered BEAST analysis of 76 taxa and 4506 characters. The oldest Ranunculus, Glaucidium, Hydrastis, and Trautvet- teria fossils are from the Oligocene (for Ranunculus: Dorofeev, 1963, acucka-rodoniowa, 1979; for the three latter genera: Li, 1952), but the dating and phylogenetic placements of these fossils are too imprecise to serve as calibration points.
To cross-validate results obtained with the secondary calibration approach and plastid sequences, we also inferred ages
using ITS rates from the literature (Kay & al., 2006), namely a mean rate for herbaceous angiosperms (4.13 × 10–9 substitutions per site per year) or the Soldanella rate (Primulaceae, 8.34 × 10–9 substitutions per site per year), to calibrate a strict clock for the Delphinieae ITS sequences. The results from using the Soldanella ITS rate gave node ages that overlapped closely with those obtained from the 4506-nucleotide matrix and secondary calibrations.
Biogeographic analyses. — To reconstruct the biogeographic history of our focal group, we used ML as implemented in Mesquite v.2.74 (Maddison & Maddison, 2009). The ML model incorporated information from genetic branch lengths and used the Markov k-state one-parameter model, which as- sumes a single rate for all transitions between character states (Lewis, 2001). Analyses were carried out on the highest likelihood tree with GTR + G branch lengths, and transition parameters were estimated based on the tip trait states (i.e., regions assigned to the species). Outgroup taxa and Aconitum species were excluded from this analysis. Geographic regions were coded as an unordered multi-state character using either five or eight states (including three combined regions), namely: (1) Mediterranean (which included Northwest Africa and the north of the Mediterranean basin); (2) Asia Minor (= Anatolia); (3) Middle East and Central Asia; (4) 1 + 2; (5) 2 + 3; (6) 1 + 2 + 3; (7) China; and (8) North America. We also conducted an analysis with six regions (the same as above, except the fourth, fifth, and sixth region grouped into a new state, “equivocal”). Results showed that the eight state coding was the most informative.
An alternative approach to ancestral area reconstruction that requires a time-calibrated phylogeny and a transition probability matrix specifying the rate of transition between geographic ranges as dispersal or extinction parameters is implemented in LAGRANGE (Ree & Smith, 2008). However, LAGRANGE needs a fully bifurcated tree (Ree & Smith, 2008) and has the limitation that the number of biogeographic parameters to estimate from the data increases exponentially with the number of areas, decreasing the inferential power of the model (Ree & Sanmartín, 2009).
ResUlTs
Main phylogenetic results. — Figure 2 shows the geographic provenience of the 44 taxa of Consolida and Aconitella sampled for this study. The ML tree obtained from our 76-taxon matrix (69 species and subspecies of Delphinieae) is shown in Fig. 3. The tree is rooted on Actaea and Nigella, based on Wang & al. (2009), and our experiments with the Path-O-Gen software that indicated this as the most likely root placement. Aconitella and Consolida form a clade embedded in Delphinium (with a bootstrap support of 88%; node A, Fig. 3). The deepest divergence in Consolida s.l. (including Aconitella) is between a clade comprising C. lineolata, C. armeniaca, and C. olopetala, all three endemic to Turkey, and the remaining species (both clades with 100% support). These remaining species fall in a polytomy of five groups labelled I–V in Figs. 3–5. Three of the five groups comprise species from the Mediterranean basin and the Near East, while one comprises species from Iran, Iraq, and Central Asia and another species from Turkey to Afghanistan (all tra- ditionally placed in Aconitella). Aconitella is clearly embedded within Consolida (node B, Fig. 3).
The closest relatives of Consolida s.l. may be the perennial species of Delphinium from North America and China, but this grouping has a bootstrap support of only 64% (node C, Fig. 3). All annual species of Delphinium group together (in the Mediterranean/Chinese subg. Delphinum; Fig. 3). An unexpected discovery is that the perennial species D. balansae apparently evolved from an annual ancestor (Fig. 3). The three biennial species of Delphinium (D. pictum, D. requienii, and D. staphisagria, forming subg. Staphisagria) form a strongly supported clade (node D, Fig. 3: 100% bootstrap support), but sequences from D. pictum were incomplete. This species is therefore not included in the final tree.
The temporal and spatial unfolding of Consolida and Aco- nitella. — The clock-like behaviour of the data assessed with Path-O-Gen and the small variance of rate changes along the tree (3.77 × 10–4) indicated that ancestor/descendant rates did 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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Delphinium subg. Delphinastrum
Delphinium subg. Oligophyllon
Fig. 3. Phylogenetic relationships in Delphin- ieae with focus on Aconitella and Consolida as inferred from the combined chloroplast and nuclear data under maximum likelihood (ML) optimization (76 taxa, 4506 aligned nucleotides); numbers at nodes are ML bootstrap support values from 100 replicates. The numbers I–V on the right mark clades discussed in the main text. The three biennial species of Delphinium (D. pictum, D. requienii, D. staphisagria) form a clade, but D. pictum sequences were incomplete and therefore not included here. Where multiple accessions of the same species were sequenced, we added voucher information. The colour coding refers to life history: blue = annuality; green = annuality and/or bienniality, or pseudo- annuality; red = perenniality.
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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 the several thousand trees included in the post-burn-in sample) obtained for the Delphinieae dataset. Different from the ML topology (Fig. 3), the Bayesian tree obtained from the dating analysis (Fig. 4) shows clade II diverging below the remaining clades of the polytomy formed by the five main clades of Consolida (marked by an arrow in Fig. 4). Ages inferred using the 4506-nucleotide matrix and secondary calibration points and those inferred with an ITS rate of 8.34 × 10–9 substitutions per site per year (see Materials and Methods) applied just to our ITS sequences largely agreed. The split between Consolida s.l. and its Delphinium sister clade was dated to 21.7 ± 3 Ma (Fig. 4), and the crown group age of Consolida s.l. to 16.9 Ma (95% CI 13.4–20.9 Ma). The diversification of major clades in Consolida s.l. (clades I–V in Figs. 3–5) began around 8.8 (6.2–11.8) Ma.
Figure 5 shows the reconstruction of ancestral areas on the ML tree (Fig. 3), with the inset showing the relevant geographic regions. The Consolida/Aconitella clade appears to have undergone three expansions towards the Mediterranean basin (labelled by boxed blue numbers in Fig. 5). Contrary to this clear geographic structure in the tip clades, an ancestral region of the entire group could not be inferred. Part of the problem is the statistically poorly supported sister-group relationship between Consolida s.l. and the Chinese/North Ameri- can Delphinium clade (represented by subg. Delphinastrum and subg. Oligophyllon in Figs. 3–5). If instead the Mediterranean subg. Delphinium were the sister group to Consolida s.l. an ancestral region in the Mediterranean or Asia Minor would become likely.
DIsCUssION
Consolida and Aconitella may need to be included in Del- phinium. — Huth (1895) was the last worker to include Con- solida s.l. in Delphinium, an action that probably was easier 115 years ago than it is now, with many of the 52 species of Aconitella and Consolida having been treated in numerous Floras and other botany texts. Modern workers therefore mostly left Aconitella and Consolida as separate genera (Table 1), even when they inferred the evolution of Consolida from within Delphinium (Tamura, 1966; Malyutin, 1973). The phylogenetic trees generated here now show unambiguously that Aconitella is part of Consolida (100% bootstrap support) and that both are embedded in Delphinium (88%). A classification of mutually monophyletic genera might therefore require transferring Aconitella and Consolida into Delphinium. In terms of nomen- clatural changes, this would involve only seven new names because for the remaining species, names in Delphinium are already available from Huth’s classification (1895). Alterna- tively, one might split up Delphinium into several genera, a step that would be premature with the current sampling of this genus (especially since the mutual monophyly of Aconitum and Delphinium remains unclear; Fig. 3).
Few of the species relationships found in earlier studies fit with the present molecular data. Thus, Spach (1839) and Kemularia-Nathadze (1939) inferred a close relationship
between Aconitella and species of Aconitum sect. Lycocto- num, while Soják (1969) and Trifonova (1990) suggested that Aconitella and Consolida might be sister groups, hypotheses clearly rejected here. On the other hand, sect. Involutae sensu Kemularia-Nathadze (1939), Soják (1969), and Trifonova (1990) (i.e., all Aconitella species, including A. barbata) is coherent with clade V (Figs. 3–5), and Huth (1895) correctly grouped the majority of the species in clade II (Fig. 3–5) together as a section (Consolida sect. Macrocarpae). Lastly, the species in clade I (Figs. 3–5) all share a particular seed coat with discon- nected scales, which in light of the molecular tree probably is a synapomorphy of these species (Constantinidis & al., 2001). An unusual species that has been difficult to place based on morphology is Aconitella barbata. It has a Consolida-like perianth shape combined with an Aconitella-like petiole structure (Trifonova, 1990). Huth (1895) therefore placed A. barbata in a section of its own. Fitting with this classification, A. barbata is here placed as sister to all remaining Aconitella species.
While our sampling of Delphinium is biased towards subg. Delphinium, we made a point of including all biennial species, i.e., all species of subg. Staphisagria. Of the three species, D. requienii is endemic to the Hyères Islands (southern France), D. staphisagria is widespread due to its medicinal uses (Orellana & al., 2009a), and D. pictum is found in Corsica, Sar- dinia, and Majorca (D. pictum sequences were incomplete but did group with those of the other two species; Appendix). They are similar in perianth shape and seed morphology (Blanché, 1991) and exhibit ascending dysploidy, with D. staphisagria 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. Staphisagria within Delphinium (Orellana & al., 2009b), but suggest that Delphinium and Aconitum may not be mutually monophyletic (Fig. 3). Assessing this possibility, however, requires denser species sampling of these two large genera, each with ca. 300 species.
The spatio-temporal unfolding of Consolida and Aconi- tella. — Current understanding is that Delphinium differen- tiated primarily in Central Asia (Tamura, 1993) and that the Mediterranean region is a secondary centre of diversification (Orellana & al., 2009a). The Mediterranean sect. Delphinium has been suggested to stem from sect. Anthriscifolium (Wang, 1962; obviously using pre-cladistic thinking) during westward expansion from southwest China (Wu, 2001). This hypothesis needs further testing. Our results at least reveal that Consolida diverged from Delphinium relatives in the Early to Middle Mio- cene (Fig. 4), a period of increasing aridity, caused primarily by decreases in sea level in the Mediterranean (Rögl, 1999; Peryt, 2006; de Leeuw & al., 2010) and desertification in Asia (Guo & al., 2002). The retreat of the Paratethys Sea in western Central Asia during the Late Oligocene and Early Miocene (Ramsetin & al., 1997) and the uplift of the Tibetan-Himalayan plateau, which intensified at 25–20 Ma, played a major role in the shift of the Central Asian climate from oceanic to continen- tal (Ramsetin & al., 1997), with accompanying increasing levels of aridity. Open grass-dominated habitats expanded as a conse- quence of the increased aridity (Strömberg & al., 2007) probably
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favouring the diversification of the Consolida s.l. clade, with its annual life strategy and open, dry-habitat-adapted grow forms (Fig. 1). Delphinieae flowers are bee-pollinated (Bosch, 1997; Bosch & al., 1997) and the seeds wind-dispersed, both traits well suited to dry and open areas. The morphological bases of floral innovations in Delphinieae are the focus of ongoing research in our laboratory (see Jabbour & al., 2009).
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 expansions across the Mediterranean and Paratethys during the Oligocene and Miocene (Rögl & Steininger, 1983; Oosterbroek & Arntzen, 1992). Each sea level regression event (associated with biotic expansion) was followed by a new marine transgres- sion that restored connections between the Mediterranean and the Paratethys and resulted in East-West vicariance of trans- Mediterranean lineages (Oosterbroek & Arntzen, 1992). As a result, Mediterranean clades often present disjunct distributions between the western and eastern Mediterranean, or on a larger scale, between the western Mediterranean and Central Asia, the so-called Kiermack disjunctions (examples in Oosterbroek & Arntzen, 1992; Ribera & Blasco-Zumeta, 1998). Based on the inferred clade ages (Fig. 4), the western expansions of the Consolida s.l. clade must have occurred in this setting, with the drying up of the Mediterranean Sea around 5.96–5.33 Ma (Hsü & al., 1973; Krijgsman & al., 1999; Fauquette & al., 2006; Popov & al., 2006) probably permitting Consolida to disperse as far as Spain and Morocco (crown group ages of clade I: 2.8 Ma, clade III: 4.4 Ma, and the group of Mediterranean species in clade II: 5.11 Ma; Figs. 4, 5). Subsequent diversification may be linked to the renewed interruption of gene flow caused by the re-flooding of the Mediterranean basin (see Salvo & al., 2010). Of the three clades, clade II (Fig. 5) has achieved the widest distribution: It includes C. orientalis, found in Northwest Africa, the Iberian Peninsula, the Balkans, Caucasia, Syria, Iran, and Central Asia, a range covering the entire distribution area of Consolida s.l. Some Consolida species, however, occur in cultivated fields, and their ranges may be partly anthropogenic.
The evolution of the annual life cycle in Delphinieae. — In the most comprehensive phylogeny of Ranunculaceae to date (Wang & al., 2009), the closest relative of Delphinieae is Nigella, which consists of annuals. Within Delphinieae, Aco- nitum comprises annual and biennial species, but also perennial ones, such as A. septentrionale sampled here. Delphinium likewise comprises annuals, biennials, and perennials (Orellana & al., 2009b). The annual strategy of the Mediterranean Del- phinieae probably is advantageous in environments with uneven and seasonal rainfall (Bosch & al., 1997, 2001; fitting with Raunkiaer’s (1918) finding that annuals are especially common in the Mediterranean). Based on our current tree (Fig. 3), the annual strategy in Consolida s.l. evolved independently of that in Delphinium subg. Delphinium; however, the crucial node has only 64% bootstrap support. If subg. Delphinium were the sister group to Consolida s.l., then the annual strategy would parsimoniously have evolved only once, in their common ancestor, perhaps in Southwest Asia during times of increasing aridity (see previous section).
aCkNOwleDgeMeNTs
We thank C. Blanché for constructive discussions, E.M. Friis for information on fossils, and A. Gröger and S. Zarre for providing pictures of Consolida and Aconitella. Guido W. Grimm provided much appreciated advice about the g2cef software. F.J.’s research was supported by DFG grant RE 603/12-1.
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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 Ilgn (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 Dai, 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 Toprakl köyü (1030 m), Buttler 20012 (M), *JF331685, *JF331752, *JF331816, *JF331878, –; A. thirkeana (Boiss.) Soják, Turkey: Eskiehir, 500 m SE Asagi Sölitönü 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
(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 Gümfiane (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) So, 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, Mara, E Elbistan, from Göksun to Elbistan (1180 m), Nydegger 16741 (M), *JF331696, *JF331762, *JF331826, *JF331888, –; C. hellespontica (Boiss.) Chater, Turkey: Konya, Kora, 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: Elazi 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.) So 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.) So, 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) So, Montenegro: Dalmatia, W Budva, coastline (3 m), ernoch 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) So, Greece: Attica, Mt Parnes, Pinatzi s.n. (coll. date 1937; M), *JF331721, *JF331786, *JF331850, –, –; C. uechtritziana (Huth) So, 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 Ascatrn (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 Domusnvas 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: Nide, 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 (Bakirdai), Zarre 53 (MSB), *JF331745, *JF331809, *JF331873, *JF332030–*JF332031, –.
Appendix. Continued.

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Consolida and Aconitella are an annual clade of Delphinium