RESEARCH ARTICLE

Parallel evolution of arborescent carrots (Daucus) in Macaronesia

Kamil E. Frankiewicz1,7 , Alexei Oskolski2,3, Łukasz Banasiak1, Francisco Fernandes4, Jean-Pierre Reduron5, Jorge-Alfredo Reyes-Betancort6, Liliana Szczeparska1, Mohammed Alsarraf1, Jakub Baczyński1, and Krzysztof Spalik1

Manuscript received 16 September 2019; revision accepted 2 January PREMISE: Despite intensive research, the pathways and driving forces behind the evolution 2020. of derived woodiness on oceanic islands remain obscure. The genus Daucus comprises 1 Department of Molecular Phylogenetics and Evolution, Institute mostly herbs (therophytes, hemicryptophytes) with few rosette treelets (chamaephytes) of Botany, Faculty of Biology, University of Warsaw, Biological and endemic to various Macaronesian archipelagos, suggesting their independent evolution. Chemical Research Centre, Żwirki i Wigury 101, 02-089 Warsaw, Poland To elucidate the evolutionary pathways to derived woodiness, we examined phylogenetic 2 Department of Botany and Plant Biotechnology, University of relationships and the habit and secondary xylem evolution in Daucus and related taxa. Johannesburg, PO Box 524, Auckland Park 2006, Johannesburg, METHODS: Sixty taxa were surveyed for molecular markers, life history, and habit traits. South Africa Twenty-one species were considered for wood anatomical characters. A dated phylogeny 3 Botanical Museum, Komarov Botanical Institute, Prof. Popov 2, 197376 St. Petersburg, Russia was estimated using Bayesian methods. The evolution of selected traits was reconstructed 4 Instituto das Florestas e Conservação da Natureza, Quinta Vila using parsimony and maximum likelihood. Passos, R. Alferes Veiga Pestana 15, 9054-505 Funchal, Madeira, RESULTS: Daucus dispersed independently to the Canary Islands (and subsequently to Portugal Madeira), Cape Verde, and the Azores in the late Miocene and Pleistocene. Life span, 5 Via Apia, 10 rue de l'Arsenal, 68100 Mulhouse, France reproductive strategy, and life form were highly homoplastic; the ancestor of Daucus was 6 Jardín de Aclimatación de la Orotava (ICIA), C/Retama no. 2, 38400 probably a monocarpic, biennial hemicryptophyte. Rosette treelets evolved independently Puerto de la Cruz, S/C de Tenerife, Spain in the Canarian-Madeiran lineage and in Cape Verde, the latter within the last 0.13 7 Author for correspondence (e-mail: [email protected]) Myr. Treelets and hemicryptophytes did not differ in wood anatomy. Pervasive axial Citation: Frankiewicz, K. E., A. Oskolski, Ł. Banasiak, F. Fernandes, J.-P. Reduron, J.-A. Reyes-Betancort, L. Szczeparska, et al. 2020. parenchyma in wood occurred more often in polycarpic rather than monocarpic species. Parallel evolution of arborescent carrots (Daucus) in Macaronesia. CONCLUSIONS: Life span and life form in Daucus are evolutionarily labile and may change American Journal of Botany 107(3): 394–412. independently of wood anatomy, which is related to plant reproductive strategy rather doi:10.1002/ajb2.1444 than to life form. Insular woodiness may evolve rapidly (as demonstrated in D. bischoffii), and in Daucus, it does not seem to be an adaptation to lower the risk of xylem embolism.

KEY WORDS Apiaceae; Daucinae; habit evolution; insular woodiness; Melanoselinum; molecular dating; Monizia; secondary woodiness; Tornabenea; wood anatomy.

Woodiness was most likely ancestral within angiosperms, and the shift herbaceous species favoring taller individuals; Carlquist (1974) postu- from trees and shrubs to herbs has long been considered a major trend lated that moderate climates with low seasonality and/or the absence in the evolution of flowering plants (e.g., Sinnott and Bailey, 1915); of large herbivores may be responsible for the shift; and recently, Lens however, a large body of evidence suggests that the opposite is also pos- et al. (2013b) and Dória et al. (2018) considered drought adaptation sible and that many woody taxa in various angiosperm lineages origi- to be the trigger because derived woody stems have higher embolism nated from herbaceous ancestors (reviewed, e.g., by Dulin and Kirchoff, resistance than herbaceous relatives. Each of those hypotheses has its 2010). Phenomenon of such evolutionary reversal, known as second- limitations; moreover, they are not mutually exclusive. To the contrary, ary or (phylogenetically) derived woodiness, is often associated with a single causal scenario explaining all cases of derived woodiness is insular woodiness, i.e., the tendency of herbaceous plants to evolve into unlikely (Kidner et al., 2015; Carlquist, 2017). Therefore, to explain rosette trees, shrubs, and other arborescent life forms after dispersal to the evolution of insular woodiness more synthetic studies combining islands. Both phenomena, and especially insular woodiness, have been anatomical and ecological approaches with robust phylogenetic back- extensively discussed since the 19th century, and numerous hypotheses ground are needed. have been proposed to explain them (e.g., Darwin, 1859; Wallace, 1878; Attempts to discover anatomical features that distinguish de- Carlquist, 1974). Darwin suggested increased competition between rived woody plants from ancestral woody ones led to the theory

American Journal of Botany 107(3): 394–412, 2020; http://www.wileyonlinelibrary.com/journal/AJB © 2020 The Authors. American Journal of Botany published by Wiley Periodicals, Inc. on behalf of Botanical Society of America. This is an open access article under the terms of the Creative​ Commo​ns Attri​bution License, which permits use, 394 • distribution and reproduction in any medium, provided the original work is properly cited. March 2020, Volume 107 • Frankiewicz et al.—Evolution of arborescence in insular carrots • 395

of paedomorphosis in wood evolution (Carlquist, 1962), which clade (Winter et al., 2008; Magee et al., 2009, 2010). Regardless of the has been extensively studied (Lens et al., 2005, 2007, 2009, 2012a; ancestral condition for subfamily Apioideae, the woody members Neupane et al., 2017) and reviewed in recent years (Carlquist, of Daucinae, as one of its crown groups, are definitely derived from 2009a, b, 2012, 2013; Dulin and Kirchoff, 2010; Lens et al., 2013a). herbaceous ancestors, and this character has been shown to have According to this theory, juvenile or “paedomorphic” traits are a substantial level of homoplasy in various clades of the subfamily expected to persist in the mature wood of derived woody plants. (Calviño et al., 2006; Oskolski and Van Wyk, 2008; Downie et al., However, Lens et al. (2013a) argued that at least some traits usu- 2010; Magee et al., 2010; Banasiak et al., 2016). ally interpreted as being indicators of protracted juvenilism result Recent phylogenetic and taxonomic studies of Daucinae have instead from some distinctive growth forms (rosette trees, stem shown that various woody species that had been previously scat- succulents, and slender-stem shrubs) irrespective of their derived tered among the genera Athamanta, Cryptotaenia, Melanoselinum, or ancestral woodiness. Also, several wood traits not necessarily in- Monizia, Rouya, and Tornabenea are nested within Daucus s.l. and terpreted as juvenile are known to correlate with plant habit (e.g., have been transferred to the latter (Banasiak et al., 2016). The for- Carlquist, 1984; Carlquist and Donald, 1996; Carlquist and DeVore, mer monotypic genera Melanoselinum and Monizia form a clade 1998; Olson and Carlquist, 2001; Carlquist and Grant, 2006; Arévalo (Daucus sect. Melanoselinum) closely related to Daucus s.s. (≡ sect. et al., 2017). Carlquist incorporated this view in his later works, Daucus, which includes the generitype, D. carota), whereas the stud- placing the emphasis of the explanation of protracted juvenilism ied representatives of Tornabenea, as well as Daucus rouyi (≡ Rouya not only with plant evolutionary history, but also with plant growth polygama) and Daucus della-cellae (≡ Athamanta della-cellae), are form (Carlquist, 2009b) and trait function (Carlquist, 2012, 2015a, b, nested within sect. Daucus. However, the phylogenetic positions of 2018). D. elegans from the Canary Islands and D. bischoffii (≡ T. bischoffii), Experimental data on the model plant Arabidopsis thaliana a rosette treelet from Cape Verde, remain obscure. The former was suggest that the gain of a prominent wood cylinder requires small resolved as either the sister to the two Madeiran Daucus species genetic changes (Chaffey et al., 2002; Ko et al., 2004; Melzer et al., (D. decipiens and D. edulis; Spalik and Downie, 2007; Banasiak et al., 2008; Lens et al., 2012b; Rowe and Paul-Victor, 2012; Davin et al., 2013) or included in sect. Daucus as the sister to the remaining spe- 2016); therefore, derived woodiness may evolve in a relatively short cies of sect. Daucus (Banasiak et al., 2016). The phylogenetic position time. However, even though derived woodiness is a widespread of D. bischoffii has never been investigated with molecular data. It is phenomenon found in many families (Park et al., 2001; Barber possible that D. bischoffii is nested within sect. Daucus, like other for- et al., 2002; Francisco-Ortega et al., 2002; Lee et al., 2005; Nürk et al., mer members of Tornabenea. However, as in the case of D. elegans, 2019), the ages of derived and, in particular, insular woody clades it is also possible that the species is related to the Madeiran endemics remain mostly unknown (Kim et al., 2008), obstructing our under- forming sect. Melanoselinum (Fig. 2), a scenario already considered standing of the possible causes and mechanisms leading to the evo- by Chevalier (1935). lution of derived woodiness. Resolving the phylogenetic positions of D. elegans and D. bi- Subtribe Daucinae (Apiaceae, Apioideae) is a promising group schoffii is crucial for understanding life-form evolution in sect. for testing the hypotheses explaining the relationships between habit Daucus and the evolution of wood traits in Daucinae. If D. elegans shifts, wood trait evolution, and insular diversification. The tribe com- and D. bischoffii are the closest relatives of the species from Madeira prises some 93 species distributed mostly around the Mediterranean, and Cape Verde, respectively, then the ancestor of sect. Daucus in Europe, western Asia, tropical Africa and in Macaronesia, of was most likely herbaceous, while the chamaephytic, or treelet, life which the cultivated carrot (Daucus carota; authorities are given form probably evolved independently in these two Macaronesian in Appendix S1) is the best-known representative (Banasiak et al., clades (Fig. 2A). However, if D. elegans is sister to the remaining 2013, 2016). Most species are herbaceous; however, there are a few species of sect. Daucus, then the Macaronesian treelets form a grade exceptions characterized by substantial height and deposition of a at the base of the group and the treelet life form is probably ances- significant amount of secondary xylem (Fig. 1). The most impressive tral for sect. Daucus (Fig. 2B). This scenario implies a loss of the examples are Daucus decipiens (≡ Melanoselinum decipiens) and its chamaephytic life form early in the evolution of sect. Daucus and sister species Daucus edulis (≡ Monizia edulis), both rosette trees its subsequent regain in the lineage of species formerly placed in from Madeira reaching 4 m in height (Lowe, 1923; Fernandes and Tornabenea. Carvalho, 2014). Stems with a prominent woody base of up to 0.5 m It should be kept in mind that woodiness and herbaceousness in height are also characteristic for the biennial Daucus elegans (≡ are highly ambiguous concepts that can refer either to habit or to Cryptotaenia elegans) from the Canary Islands. In addition, the for- stem anatomy. The term woodiness can be used to describe both mation of a slender woody stem up to 1 m tall has been reported plants having prominent perennial aboveground stems, as well as by Brochmann et al. (1997) for Daucus bischoffii (≡ Tornabenea bi- the ones producing a distinct wood cylinder that extends toward schoffii) and Daucus tenuissimus (≡ Tornabenea tenuissima) from upper stem parts (Kidner et al., 2015). Correspondingly, a plant Cape Verde. However, these two species are also considered to be can be recognized as herbaceous on the basis of either a life his- herbaceous (Martins, 1996). tory in which aboveground parts die off at the end of the growing Although shrubs and trees are uncommon in Apiaceae subfamily season or the absence or limited amount of secondary growth in Apioideae, some authors suggest that the woody habit—considered stems. These two approaches to the definition of woodiness and as shrubby or arborescent life form and, simultaneously, deposition herbaceousness are not necessarily correlated with each other; of secondary xylem—is plesiomorphic for the subfamily because its there are many examples of plants having a typical tree habit basal lineages are predominantly woody (Oskolski, 2001; Oskolski without secondary growth and of annual herbs with normal wood and Van Wyk, 2008; Stepanova and Oskolski, 2010; Long and cylinders (Dulin and Kirchoff, 2010; Schweingruber and Büntgen, Oskolski, 2018). However, formal ancestral state reconstruction in- 2013). The question whether wood evolution follows evolution- ferred herbaceousness for the most recent common ancestor of this ary change in life form remains unanswered in Daucinae. It might 396 • American Journal of Botany

FIGURE 1. Life form and life history variation among exemplar species under study. Monocarpic perennial Daucus decipiens (A) before and (B) after fruiting. Polycarpic perennial Daucus edulis with (C) prominent stem and (D) remains of previous-year umbels. (E) Monocarpic biennial Daucus elegans with (F) woody basal part of stem. (G) Polycarpic perennial Daucus rouyi; only upper parts of this sand dune plant are visible. March 2020, Volume 107 • Frankiewicz et al.—Evolution of arborescence in insular carrots • 397

AB

FIGURE 2. Phylogenetic hypotheses on the relationships among insular treelets in Daucus. In (A), Daucus elegans (marked in dotted line) is sister to a clade of Madeiran endemics (Daucus decipiens and Daucus edulis, i.e., Daucus sect. Melanoselinum). This scenario was reconstructed by Spalik and Downie (2007) and Banasiak et al. (2013). In (B), D. elegans is sister to the remaining species of Daucus sect. Daucus, scenario inferred by Banasiak et al. (2016). Clades comprising woody species are marked in yellow. Asterisks denote hypothetical alternative positions of Daucus bischoffii. turn out that both traits are highly correlated—hemicryptophytes MATERIALS AND METHODS being distinguishable from their chamaephytic/rosette treelet rel- atives based on wood anatomical traits—but it is equally likely Molecular analyses that both groups share similar xylem features. So far almost all anatomically studied Apioideae species have been shown to de- Based on a previous study (Banasiak et al., 2016), we selected 60 posit some amount of secondary xylem (e.g., Oskolski, 2001; species and subspecies representing all major lineages of sub- Oskolski and Van Wyk, 2008; Schweingruber and Landolt, 2010; tribe Daucinae and three species representing other subtribes of Stepanova and Oskolski, 2010; Long and Oskolski, 2018; our un- Scandiceae as an outgroup. Material obtained from herbaria or liv- published data), and for this reason, technically they all could be ing collections was used to isolate total DNA from ca. 20 mg of dried called woody. However, to identify the most dramatic shifts in leaves using DNeasy Plant Mini Kit (Qiagen, Venlo, Netherlands). the amount of wood deposition within Daucinae, we consider Four of the molecular markers used in this study—nuclear ribo- species with limited secondary growth, mostly therophytes and somal DNA internal transcribed spacer (nrDNA ITS) and plastid hemicryptophytes, as herbaceous, whereas as woody we describe rpoC1 and rps16 introns and rpoB-trnC intergenic spacer—have pre- only those with prominent secondary xylem, i.e., chamaephytes viously been used in phylogenetic analyses of the subtribe (Banasiak and rosette treelets. Simultaneously, we consider the evolution et al., 2016). Additionally, we analyzed sequence variation of nuclear of habit and wood anatomical traits separately using Raunkiaer ribosomal DNA external transcribed spacer (nrDNA ETS) and (1934) terms to characterize the life forms of species irrespective plastid rpl16 intron. Loci were amplified by PCR using previously of wood anatomical structure. developed protocols and primers (Logacheva et al., 2010; Banasiak Our study had two main objectives. First, to resolve the phy- et al., 2016). Sanger sequencing was performed by Genomed S.A. logenetic positions of D. elegans and D. bischoffii and to estimate (Warsaw, Mazovia, Poland), and obtained reads were assembled us- the timescale of the Daucinae radiation to reconstruct the evo- ing SeqMan Pro 13.0.2 (DNAStar, Madison, WI, USA). Newly ob- lution of life form in this group and particularly to estimate the tained sequences were deposited in GenBank (Appendix S1). timing of the origin(s) of insular treelets. Second, we investigate The sequences were aligned using the E-INS-i algorithm imple- wood anatomy of selected species (including all insular woody mented in MAFFT 7.271 (Katoh and Standley, 2013). Primer and ones) to determine whether there are any wood traits that dis- partial exon sequences flanking the noncoding regions were man- tinguish derived woody species of Daucus from their herbaceous ually trimmed in Mesquite 3.51 (Maddison and Maddison, 2018). relatives. Subsequently, the automated1 algorithm implemented in trimAl 1.2 398 • American Journal of Botany

(Capella-Gutiérrez et al., 2009) was used to remove ambiguously reference to plant habit or anatomy. We included all cases of pre- aligned positions. sumed insular woodiness (i.e., insular rosette treelets: D. decipiens, D. edulis, D. bischoffii, D. tenuissimus; and an insular chamaephyte Phylogeny estimation and node calibration with prominent woody base: D. elegans) and nearly all other spe- cies in the subtribe for which various degrees of woodiness were The BIC metric was used to choose the optimal partitioning reported (for example, Lowe, 1923; Brochmann et al., 1997; Table 1). scheme and nucleotide substitution models in PartitionFinder 2 Voucher specimens are listed in Appendix S1. Wood samples were (Lanfear et al., 2012). The substitution models were confined to taken from the basalmost part of the stem in the generative phase. those available in BEAST 1.10.0 (Drummond and Rambaut, 2007). Exceptionally, Daucus decipiens and D. edulis were sampled at both The congruence of phylogenetic signal between nuclear and plas- generative and vegetative phases from underground, basal, middle, tid molecular data sets was assessed using hierarchical likelihood and apical parts of the stem. Fresh material was immediately stored ratio test implemented in Concaterpillar 1.7.2. (Leigh et al., 2008). in 70% ethanol. Topology estimation was performed simultaneously with molecular Wood samples were soaked in boiling water until they sank. dating. Since Daucinae lack a reliable fossil record, we used four sec- They were then sectioned using a sledge microtome (SM2010R, ondary calibration points based on a Bayesian dating of subfamily Leica Biosystems, Wetzlar, Hesse, Germany) resulting in 20–60 μm Apioideae (Banasiak et al., 2013). All chosen nodes had a posterior thick transverse and longitudinal sections, which were subsequently probability of 1.0 in the analyses of Banasiak et al. (2013) and rep- stained with a 0.5% w/v aqueous solution of safranin and 0.5% w/v resented the most recent common ancestors of tribe Scandiceae aqueous solution of alcian blue. Sections were dehydrated in eth- (Fig. 3A), Daucus and Silphiodaucus (B), Daucus sect. Daucus and anol solutions of increasing concentration, and then mounted in sect. Melanoselinum (C), Daucus sect. Agrocharis and sect. Anisactis Euparal (Carl Roth, Karlsruhe, Baden-Württemberg, Germany). An (D). For each of these nodes, the age posterior distribution was eval- exceptionally fragile sample of D. della-cellae was fixed in 70% for- uated based on a sample of 10,800 phylogenetic trees from Banasiak malin–acetic acid–ethanol (2:1:7) solution, embedded in Technovit et al. (2013) using treeStat 1.10.0 (Rambaut and Drummond, 7100 resin (Kulzer, Hanau, Hesse, Germany), and sectioned on a 2018b). Because the posterior distributions did not considerably de- rotary microtome to obtain 7–10 μm thick transverse and longitu- viate from the normal distribution as assessed by normal quantile- dinal sections, which were stained with periodic acid–Schiff’s (PAS) quantile plots in R (R Core Team, 2008), the calibration points were reagent without dinitrophenylhydrazine (DNPH). Wood anatomi- defined as having normal distributions with mean and standard cal characters were examined using light microscopy and follow the deviation calculated based on the respective posterior samples. We International Association of Wood Anatomists’ list of microscopic used the random local clock model to accommodate differences features for hardwood identification whenever possible (IAWA in the rate of molecular evolution between lineages representing Committee, 1989). For traits that showed only limited variation, annual, biennial, and perennial taxa. Two independent Markov fewer character states were used. chains were run for 100,000,000 generations each and sampled ev- A length-on-age curve was prepared only for D. decipiens be- ery 10,000 generations. The initial 20% of trees from each run were cause the samples of other species had wood cylinders that were discarded as burn-in. Posterior distributions of parameter estimates too narrow. In D. decipiens, the wood was sliced into pieces 1 mm were inspected in Tracer 1.7.1 (Rambaut et al., 2018). Both runs thick from the pith to the bark. Lengths of vessel elements and fi- converged on the same stationary distribution and were combined bers were measured in each slice after maceration as described by for subsequent analyses using LogCombiner 1.10.0 (Rambaut and Franklin (1945). Drummond, 2018a). The resulting set of 16,000 trees was summa- rized using TreeAnnotator (Drummond and Rambaut, 2007) in Character evolution reconstruction a maximum clade credibility (MCC) tree. For diagnostic purposes, the posterior distributions of ages for nodes from the Banasiak et al. Reconstruction of the life history traits was conducted for all 60 (2013) serving as secondary calibration points were plotted against species or subspecies included in the phylogenetic inference, while the results of current study using the ggplot (Wickham, 2009) pack- the reconstruction of wood anatomical traits was done for 21 spe- age in R. Additionally, we compared median ages for all pairs of cies. All characters and their states are discussed in more detail in corresponding nodes between primary (Banasiak et al., 2013) and the Results section. Three discrete life history traits were assessed: secondary calibration (this study) fitting major axis with fixed zero (1) reproductive strategy (0, monocarpic; 1, polycarpic); (2) life intercept, i.e., optimizing only for slope parameter, using R package span (0, annual or wintering annual; 1, biennial or triennial; 2, smatr (Warton et al., 2012). perennial); and (3) Raunkiær’s life form (0, therophyte; 1, hemic- ryptophyte; 2, chamaephyte/rosette tree). Seven discrete wood Wood anatomical analyses anatomical traits were considered: (1) growth rings (0, absent; 1, only one ring; 2, distinct and recognizable); (2) wood porosity (0, Twenty-one species were considered for wood anatomical char- semi-ring-porous; 1, diffuse-porous); (3) ray formation (0, present acters: data for 10 species were gathered from the literature in early wood; 1, delayed or rays absent); (4) rays composed mostly (Schweingruber and Landolt, 2010), while 11 species were newly of upright and square cells (0, false; 1, true); (5) scalariform (and examined for this study. These newly examined species were rep- pseudoscalariform) intervessel pitting (0, uncommon; 1, common); resented by 15 specimens obtained from herbaria (E, FR, WA), (6) pervasive parenchyma (0, absent; 1, present); and (7) libriform living collections (Conservatoire Botanique de Mulhouse, France fibers (0, absent; 1, present). We define “pervasive parenchyma” as and Jardim Botânico da Madeira, Portugal), and wild populations abundant axial parenchyma replacing (at least partially) the im- (Tenerife, Anaga Mountains, Vueltas de Taganana). We tried to perforate tracheary elements in composition of the wood ground cover all species described as woody in the literature, regardless of tissue (Carlquist, 2001, p. 172). We avoided the distinction between March 2020, Volume 107 • Frankiewicz et al.—Evolution of arborescence in insular carrots • 399 400 • American Journal of Botany

FIGURE 3. Maximum clade credibility tree summarizing Bayesian MCMC analyses of Daucinae and outgroups with BEAST. Scatter plot shows cor- relation of median ages for corresponding nodes in primary (abscissa axis) and secondary (ordinate axis) calibrated tree. Solid and dashed violet lines represent major axis with 95% confidence interval compared to blue line expected under assumption of no systematic bias in age estimates. A, B, C, and D denote calibration points with respective plots of prior (blue curves) and posterior (violet histograms) age distributions. Pink histograms rep- resent posterior age distributions for the calibration points from previous study (Banasiak et al., 2013), which were used to calculate parameters for priors. Violet bars represent 95% highest posterior density intervals for age distributions. Red dots mark possible dispersals to and within Macaronesia. Branches in black were reconstructed with maximum parsimony as polycarpic, while those in blue as monocarpic. For clarity, only posterior probabil- ity values lower than 1.0 are shown. Taxa included in the anatomical study are marked in boldface; insular rosette treelets are marked with asterisks, and time scale is in millions of years ago.

TABLE 1. Selected distribution and life span, reproductive strategy and habit traits of Daucinae species examined for anatomical characters. Slashes separate polymorphisms; asterisks indicate the dominant state. Shoot system Reproductive Species Insular woodiness Life span strategy Aboveground Underground Daucus bischoffii Yes (Cape Verde) Annual/perennial* Monocarpic Annual monocarpic stems Long unbranched with or without rosette rhizome Daucus carota No Annual/biennial*/perennial Monocarpic Annual monocarpic stem None or unbranched with basal rosette short rhizome Daucus decipiens Yes (Madeira) Biennial/perennial* Monocarpic Perennial monocarpic stem None with terminal rosette Daucus della-cellae No Perennial Polycarpic Annual monocarpic stems Short or long branched with basal rosette rhizome Daucus edulis Yes (Madeira) Perennial Polycarpic Perennial polycarpic stem None with terminal rosette Daucus elegans Yes (Canary Islands) Annual/biennial*/perennial Monocarpic Perennial monocarpic stem None without terminal rosette Daucus insularis Yes (Cape Verde) Annual/perennial Monocarpic Annual monocarpic stems Long unbranched without rosette rhizome Daucus rouyi Doubtful (Corsica, Perennial Polycarpic Perennial monocarpic stems Long branched N Africa) without rosette rhizome Daucus tenuissimus Yes (Cape Verde) Annual/perennial Monocarpic Annual monocarpic stems Short unbranched without rosette rhizome or none Laserpitium latifolium No Perennial Polycarpic Annual monocarpic stem Short unbranched with basal rosette rhizome Silphiodaucus prutenicus No Biennial Monocarpic Annual monocarpic stem Short unbranched with basal rosette rhizome scalariform and pseudoscalariform intervessel pitting, because the that annual plants could evolve directly into long-lived perennials only criterion proposed by Carlquist to distinguish these two types without intermediate state of biennial form. Also the opposite— is not practically applicable in the studied group (“pseudoscalari- shortening of a life span from perennial directly into annual— form pitting looks like … the product of lateral elongation of pits in seems biologically implausible and continuity of this trait has been an alternate pattern”; Carlquist, 2001, p. 76). demonstrated in natural populations (e.g., Hautekèete et al., 2002). As phylogenetic uncertainty was negligible, i.e., posterior prob- ability for all essential internal nodes was high, we mapped life his- tory and anatomical traits onto a MCC tree instead of taking into RESULTS account multiple trees from the Bayesian analysis (e.g., Lens et al., 2016; Arévalo et al., 2017). For maximum parsimony reconstruc- Phylogenetic relationships tion, we used the pace function from R package phangorn (Schliep, 2011) choosing unordered (Fitch) parsimony for all traits except In total, 35 new sequences were obtained for this study. The for life span considered ordered character (Wagner parsimony). ETS marker was particularly variable providing over 20% of all par- Maximum likelihood ancestral state estimates were calculated using simony informative sites, although its sequence was lacking for 77% the ace function in the R package ape (Paradis and Schliep, 2019) of the species (Appendix S2). Sequences of molecular markers were and the Akaike information criterion to select the best-fitted model partitioned into three groups: ITS, ETS, and chloroplast markers of evolution for each trait from three possible: equal rates (ER), sym- (rpoB-trnC intergenic spacer and rpoC1, rpl16, rps16 introns) with metrical (SYM), all rates different (ARD). Similarly to the approach SYM + G, HKY + I, and GTR + I + G nucleotide substitution mod- assumed in parsimony reconstruction, we applied a constraint of els, respectively. Although the relative likelihood ratio test returned ordered evolution before selecting best-fitted model for life span. P = 1.0 for the comparison of both nuclear markers—ITS and This trait is markedly continuous, and it seems biologically unlikely ETS—showing no evidence of incongruence in their phylogenetic March 2020, Volume 107 • Frankiewicz et al.—Evolution of arborescence in insular carrots • 401

signals, nuclear vs. plastid comparison rejected null hypothesis of years (Myr) with 95% highest posterior density interval (HPD) of congruence with P < 0.0001. However, excluding seven accessions 9.85–7.44 Myr ago (Ma). The divergence between the Canarian D. identified by Banasiak et al. (2016) as responsible for topological elegans and the Madeiran endemics took place 7.77 Ma (95% HPD differences at the generic level, mostly some species of Thapsia and 9.05–6.35), while the age of the ancestor of Madeiran D. decipiens Laserpitium, increased p-value to 0.127, indicating that the topolog- and D. edulis was estimated to be 3.54 Myr (95% HPD 5.32–1.73). ical conflict had a very limited scope. The Canary Islands were likely colonized first, because they are The topology of the maximum clade credibility tree was generally closer to Africa than Madeira is; from there, the common ancestor congruent with earlier studies (Spalik and Downie, 2007; Banasiak of D. decipiens and D. edulis dispersed to the latter. The divergence et al., 2013, 2016), i.e., the previously designated sections within between D. carota and the former genus Tornabenea occurred in Daucus—Daucus, Melanoselinum, Anisactis, and Agrocharis—were the Pleistocene, 0.57 Ma (95% HPD 1.01– 0.25), and coincided with retrieved. Posterior probabilities for most major clades were high the dispersal from the continent to Cape Verde. The age of the most (Fig. 3). The relationships among Ekimia, Thapsia, Laserpitium s.s., recent common ancestor of Cape Verde endemics was estimated to Siler, and Laserocarpum were somewhat different than in previous be 0.28 Myr (95% HPD 0.55–0.1), and D. bischoffii separated from analyses of this group; however, these taxa are uniform with respect its sister group 0.13 Ma (95% HPD 0.31–0.02). The ancestor of D. to their life form and life history. Hence, these differences did not carota subsp. azoricus dispersed to the Azores 0.05 Ma with 95% affect subsequent analyses of these traits.Daucus elegans was re- HPD of 0.25–0.0, i.e., including the period of human colonization solved as sister to the clade of the Madeiran endemics (D. decipiens of the archipelago. and D. edulis) with high support (posterior probability [PP] = 0.97). Previously placed in Tornabenea, the endemics of Cape Verde con- General observations on wood anatomy stituted a monophyletic group with Daucus bischoffii being sister to its former congeners: D. annuus and D. tenuissimus (PP = 1.0). Wood anatomy allows for delineation of two groups: (1) those These three species together with D. insularis formed a sister group depositing parenchymatous wood or both parenchymatous and to D. carota, while in previous studies, they were nested within the fibrous wood, (2) those depositing only fibrous wood. The first latter, albeit with a low support (Spalik and Downie, 2007; Banasiak group comprises D. della-cellae, D. edulis, Laserpitium latifolium, et al., 2016). It is noteworthy that the predominantly monocarpic and Silphiodaucus prutenicus from among the newly examined genera Daucus, Silphiodaucus, and Orlaya formed a crown clade, species and most species, for which the data were retrieved from hereafter, named the mostly monocarpic clade (MMC), while the the xylem database (Schweingruber and Landolt, 2010). These spe- basal grade included perennial polycarpic species, hereafter, named cies usually deposited wood cylinders composed of two distinct the perennial polycarpic grade (PPG). regions. Parenchymatous wood having abundant pervasive ax- ial parenchyma without libriform fibers was located in the inner Divergence time estimation and the age of insular lineages region of the secondary xylem cylinder, while fibrous wood with numerous libriform fibers was located in its outer region (Fig. 4). Prior and posterior age distributions of the four nodes selected as In most species, fibrous wood was predominant, whereas parenchy- calibration points differed to varying degree, although in all cases matous wood occurred only in the innermost part of the secondary there was considerable overlap between the previous analyses and xylem. In D. edulis, stem samples collected from vegetative shoots the current study (Fig. 3). The 95% confidence interval for the slope without floral buds had only parenchymatous wood, while samples parameter of the major axis was 0.91–1.14. Therefore, there is no from reproductive shoots had both parenchymatous and fibrous evidence for the systematic bias in secondary divergence time es- secondary xylem. Daucus della-cellae had exclusively parenchyma- timates as compared to primary calibration. The molecular mark- tous wood in the innermost region of the wood cylinder, whereas ers differed substantially in the mean rate of substitution, with ETS its outer wood had parenchymatous ground tissue with alternating having the fastest mean rate and plastid markers having the slow- bands of abundant and scanty fibers. Except for D. carota subsp. est rate (Table 2). Rate of molecular evolution also varied among gummifer, all species for which data were obtained from the xylem branches, with about 2-fold higher values for MMC than for PPG database (Schweingruber and Landolt, 2010) fell into this category, (Appendix S3). having either exclusively parenchymatous wood (Ferula communis, When estimating the age of insular endemics, we assumed that Laserpitium halleri, Siler montanum, Thapsia garganica, T. villosa) vicariance immediately followed dispersal—i.e., the time of dis- or xylem with parenchymatous and fibrous regions (Daucus pum- persal from the continent to Macaronesia is equivalent to the age ilus, Laserpitium gallicum, L. peucedanoides, Orlaya grandiflora). of the most recent common ancestor of the insular endemic clade The second group comprisesD. carota (including D. carota and its continental sister group. The stem node age of the lineage subsp. gummifer from the xylem database), D. decipiens, D. elegans, encompassing the three Canarian-Madeiran woody endemics D. insularis, D. rouyi, D. tenuissimus, and D. bischoffii. These species (D. elegans, D. decipiens, D. edulis) was estimated at 8.55 million deposited exclusively fibrous wood cylinders (Fig. 5). It is notewor- thy that both groups are polyphyletic and the insular treelets are in both: for example, Madeiran D. edulis produces both fibrous and TABLE 2. Clock rate statistics for ETS, ITS, and plastid markers used in phylogeny calibration. parenchymatous wood, while its sister species, D. decipiens, has only fibrous wood. Clock ratea ETS ITS Plastid markers Among the studied species, three paedomorphic traits were Mean 5.12×10–3 4.35×10–3 8.35×10–4 found: (1) delayed formation or absence of rays, (2) rays mostly SE 1.48×10–5 1.04×10–5 2.04×10–6 composed of upright and square cells, and (3) scalariform inter- –4 –4 –5 SD 5.53×10 3.46×10 6.43×10 vessel pitting. The first character was observed only in D. elegans: a Number of expected substitutions per site per million years. our sample was completely rayless, while in the specimen studied 402 • American Journal of Botany

FIGURE 4. Wood anatomy of exemplar species with both parenchymatous and fibrous wood. (A–C) Daucus edulis. (D, E) Daucus della-cellae. (A) Cross section showing transition from parenchymatous to fibrous wood. (B) Radial section illustrating mostly upright and square ray cells in parenchyma- tous wood. (C) Radial section showing more numerous procumbent ray cells in fibrous wood. (D) Cross section with visible transition from paren- chymatous wood to wood of mixed constitution; the boundary is marked with red arrowheads. (E) Vessel elements with wide-aperture scalariform intervessel pitting. March 2020, Volume 107 • Frankiewicz et al.—Evolution of arborescence in insular carrots • 403

FIGURE 5. Wood anatomy of exemplar species with exclusively fibrous wood. (A, C, E, G) Daucus decipiens. (B, D, F) Daucus elegans. (H) Daucus tenuis- simus. (A) Cross section showing prominent rays. (B) Rayless wood seen in cross section. (C) Radial section with numerous procumbent ray cells. (D) Rayless wood seen in radial section, simple perforation plates and axial parenchyma are clearly visible. (E, F) Tangential sections. (G) Mostly alternate to transitional intervessel pitting seen in tangential section. (H) Low magnification of the cross section with growth rings or density fluctuations. 404 • American Journal of Botany

by Schweingruber and Landolt (2010) the formation of rays was very numerous in D. della-cellae (477 mm–²). Vessels are mostly ar- delayed. Rays mostly composed of upright and square cells usually ranged in clusters and radial to diagonal multiples (2–8 vessels, up occurred in species with a narrow (<1.5 mm) cylinder of secondary to 12 vessels in D. edulis). Vessel walls are thin in D. della-cellae xylem, while D. decipiens and D. edulis, in which the wood cylin- and S. prutenicus (1.1–3.3 μm and 1.3–4.7 μm thick, respectively) der was much wider (4–102 mm), had rays with more numerous and thicker (mostly 1.8–6.5 μm) in other species. Intervessel pit- procumbent cells. Scalariform intervessel pitting with wide pit ap- ting is almost exclusively scalariform (occasionally alternate) in D. ertures was found mostly in vessel element walls embedded in per- della-cellae, and mostly alternate, occasionally scalariform or tran- vasive parenchyma constituting background tissue. sitional to alternate, in other species. Opposite pitting occurs in S. prutenicus. Intervessel pits are minute to small (1.8–7.8 μm in ver- Wood anatomy of newly studied species with both tical size), rounded, and usually have narrow slit-like apertures (in parenchymatous and fibrous wood L. latifolium lens-like). Vessel-ray and vessel-axial parenchyma pits are distinctly bordered, similar in size and shape to intervessel pits. Growth rings are absent, i.e., wood is diffuse-porous (Fig. 4). Vessels Helical thickenings were not found, nor were vascular tracheids. are mostly angular, occasionally rounded in outline (rounded ves- Fibers are libriform, thin- to thick-walled (0.9–2.5 μm thick in sels are more common in D. edulis). Mean vessel element length D. edulis and between 1.1–4.1 μm thick in other species), nonsep- (not measured separately for parenchymatous and fibrous woods) tate, and with simple to minutely bordered pits located in radial ranges between 143 μm in D. della-cellae and 274 μm in D. edulis and tangential walls. Mean fiber length ranges from 207 μm (in D. (Appendix S4). Perforation plates are simple. della-cellae) to 415 μm (in S. prutenicus). Axial parenchyma is Rays are uniseriate and multiseriate, mostly 2–4 cells in width, scanty paratracheal (in L. latifolium also diffuse) in strands of 2–4 up to 6–7 cells wide in D. della-cellae and D. edulis, 9 cells wide in L. (D. della-cellae, S. prutenicus) to 3–5 cells (D. edulis, L. latifolium). latifolium and 14 cells wide in S. prutenicus. The shortest multiseri- ate rays (<900 μm) were found in D. della-cellae and the tallest ones Wood anatomy of newly studied species with exclusively (>4000 μm) in fibrous wood of D. edulis. They are composed of ex- fibrous wood clusively upright and square cells in D. della-cellae and mostly up- right cells with square and procumbent cells mixed throughout the Growth rings are generally indistinct, except for some samples rays in L. latifolium, S. prutenicus, and parenchymatous wood of D. of D. decipiens, D. bischoffii, and some sections of D. tenuissimus edulis. In fibrous wood of D. edulis, multiseriate rays consist mostly that are marked by differences in lignification of fiber walls be- of procumbent cells, with square and procumbent cells in 1–2 mar- tween early- and latewood detected by safranin staining (Fig. 5). ginal rows and solitary rows alternating with procumbent cells or Wood is diffuse-porous. Vessels are very narrow (<45 μm) in all as solitary sheath cells (Fig. 6). Scarce radial secretory canals occur species, except for D. insularis, where they are narrow (≤70 μm); only in D. edulis and S. prutenicus. Calcium oxalate crystals were they are few in D. rouyi and D. insularis (<50 mm–²), more nu- not found in ray cells. merous in D. decipiens and D. elegans (ca. 70 mm–²), and very numerous in D. bischoffii (159 mm–²), D. carota (176 mm–²) and Parenchymatous wood—Vessels vary from very narrow in D. D. tenuissimus (214 mm−2). Vessels are arranged mostly in small della-cellae (tangential diameter 5–19 μm), to narrow in D. edulis clusters and radial or diagonal multiples, which vary in number (25–82 μm) and L. latifolium and S. prutenicus (<55 μm). Vessels of vessels: 3–5 in D. elegans, up to 8 in D. carota, up to ca. 12 in are few in D. edulis (16 mm–²), more numerous in S. prutenicus D. decipiens, D. insularis, D. rouyi, D. bischoffii, and up to 23 (64 mm–²) and L. latifolium (124 mm–²), and very numerous in D. in D. tenuissimus. Solitary vessels are mostly angular (D. decip- della-cellae (206 mm–²), mostly arranged in small clusters and ra- iens, D. elegans, D. tenuissimus), angular to rounded (D. insu- dial to diagonal multiples (2–5 vessels) in D. edulis and in larger laris, D. bischoffii), or mostly rounded (D. rouyi). Vessel walls groupings in other species (up to 18 vessels in L. latifolium). are 2.3–6.8 μm thick in D. rouyi, and thinner (≤4.6 μm thick) in Vessel walls are thin in D. della-cellae (1.0–3.8 μm thick), thicker other species. Mean vessel element length ranges from 216 μm (>2.2 μm thick) in L. latifolium and S. prutenicus, and thickest (D. rouyi) to 481 μm (D. decipiens). Perforation plates are sim- in D. edulis (up to 9.4 μm). Intervessel pitting is exclusively sca- ple. Intervessel pitting is mostly opposite (occasionally alternate lariform in D. della-cellae, and mostly scalariform, occasionally and scalariform) in D. rouyi, alternate (occasionally transitional transitional to alternate, and alternate in other species. Opposite to scalariform) in D. bischoffii, alternate to scalariform in D. pitting occurs in S. prutenicus. Intervessel pits are minute to small tenuissimus, and mostly alternate (occasionally opposite and (1.8–6.8 μm in vertical size), mostly rounded (occasionally polyg- transitional to scalariform) in other species. Intervessel pits onal in D. edulis), and with wide lens-like apertures. Vessel-ray are minute to small (3.1–7.0 μm in vertical diameter), rounded, and vessel-axial parenchyma pits are distinctly bordered and usu- with narrow slit-like apertures. Vessel-ray and vessel-parenchyma ally similar in size and shape to intervessel pits (mostly opposite pitting is mostly similar to intervessel pitting in size and shape, in L. latifolium). Helical thickenings were not found, nor were distinctly bordered, and mostly alternate; in D. decipiens and vascular tracheids. Axial parenchyma is pervasive and scanty D. rouyi, it is sometimes scalariform. Helical thickenings were paratracheal in strands of 2–4 (D. della-cellae, S. prutenicus) or not found, nor were vascular tracheids. 3–5 cells (D. edulis, L. latifolium). Fibers are libriform, thin- to thick-walled, mostly between 1–3 μm thick, up to 2.3 μm thick in D. elegans, up to 3.7 μm thick in D. te- Fibrous wood—Vessels are very narrow in D. della-cellae (7–34 μm) nuissimus, and up to 4.3 μm thick in D. bischoffii, mostly nonseptate and L. latifolium (10–51 μm) and narrow in D. edulis (20–70 μm) (septate fibers occur in D. bischoffii) with simple to minutely bor- and S. prutenicus (8–61 μm), few in D. edulis (22 mm–²) and L. lat- dered pits in radial and tangential walls. Mean fiber length ranges ifolium (34 mm–²), more numerous in S. prutenicus (86 mm–²), and from 247 μm (D. rouyi) to 530 μm (D. decipiens). March 2020, Volume 107 • Frankiewicz et al.—Evolution of arborescence in insular carrots • 405

FIGURE 6. Tangential sections of (A) parenchymatous and (B) fibrous wood in Daucus edulis, and (C) Daucus della-cellae.

Axial parenchyma is scanty paratracheal, in strands of 2–9 (D. hemicryptophyte (Figs. 3, 7, and 8; Appendix S6), and these plesio- decipiens), 3–6 (D. rouyi, D. tenuissimus, D. bischoffii), 8–10 (D. morphic character states were retained in the basal grade compris- carota) or 8–15 cells (D. elegans, D. insularis). ing Laserpitium, Ekimia, Thapsia, Siler, and Laser (PPG). A clade of Rays are absent in D. elegans, while in other species they are Orlaya, Silphiodaucus, and Daucus is predominantly monocarpic uniseriate and multiseriate with an average of 4 cells in width, up (MMC) with four reversals to polycarpy inferred for D. edulis, D. to 5 in D. carota, D. insularis, and D. tenuissimus, up to 9 in D. setifolius, D. rouyi, and a clade formed by D. della-cellae and D. mi- rouyi and D. bischoffii, and up to 15 in D. decipiens. In D. carota rabilis (Fig. 3; Appendix S6). and D. rouyi, multiseriate ray height exceeds the length of the en- Parsimony reconstructed the most recent common ancestor tire section (>2 mm and >4 mm, respectively). In the remaining of genus Daucus as a monocarpic biennial to triennial therophyte species, mean height ranges between 342 μm (D. decipiens) and or hemicryptophyte. However, because therophytes are annual by 689 μm (D. bischoffii). Rays are composed of procumbent cells, definition, one may exclude the combination of biennial/triennial sometimes with square and upright cells in 1–3 marginal rows life span and therophyte life form. The same character states were in D. decipiens, or exclusively of upright and square cells in D. inferred as ancestral for the most recent common ancestor of sect. carota, D. insularis, D. rouyi, D. tenuissimus, and D. bischoffii. Daucus and Melanoselinum with highly homoplastic subsequent Uniseriate rays are made of procumbent cells or also upright and evolution of six episodes of life span prolongation (from biennial square cells in D. decipiens and of square and upright cells in or triennial to perennial) and four independent cases of life span other species. Few radial secretory canals were found only in D. shortening (three of them from biennial or triennial to annual and bischoffii. The length-on-age curve for D. decipiens is ascending one from perennial to annual; Fig. 7). (Appendix S5). Parsimony revealed two independent shifts to the chamae- phyte/rosette tree life form, which occurred in the Canarian- Phylogenetic character mapping Madeiran lineage (D. elegans, D. decipiens, D. edulis) and in the species of Daucus from Cape Verde, formerly classified in The results of ancestral state reconstruction differed substantially Tornabenea (Fig. 7). between the two methods used. Parsimony resolved the most On the other hand, the maximum likelihood estimate for recent common ancestor of Daucinae as polycarpic perennial the most recent common ancestor of Daucinae was a monocarpic 406 • American Journal of Botany

FIGURE 7. Life form and life span evolution in Daucinae reconstructed using maximum parsimony (branch colors) and maximum likelihood esti- mates of ancestral states (pie charts represent relative likelihoods). Time scales are given in millions of years ago. Insular species are marked in bold- face; insular rosette treelets are marked with asterisks. The model all rates different (ARD) was chosen as the best fit for both traits, and the constraint of ordered evolution was applied for the life span character. March 2020, Volume 107 • Frankiewicz et al.—Evolution of arborescence in insular carrots • 407

FIGURE 8. Evolution of reproductive strategy, the presence of pervasive parenchyma, and the occurrence of scalariform intervessel pitting recon- structed using maximum parsimony (branch colors) and maximum likelihood estimates of ancestral states (pie charts represent relative likelihoods). The model equal rates/symmetrical (ER/SYM) was chosen as the best fit for evolution of reproductive strategy; the all rates different (ARD) model was chosen for all remaining traits. annual therophyte or biennial/triennial chamaephyte, while pattern, but has been uncovered for many Macaronesian endemic the most recent common ancestor of genus Daucus was inferred as clades (e.g., Crambe, Convolvulus, Sonchus; Francisco-Ortega et al., a monocarpic annual therophyte (Figs. 3 and 7). 2002; Carine et al., 2004; Lee et al., 2005). A second colonization of In Daucus, delayed formation of rays is autapomorphic for D. Macaronesia—from the continent to Cape Verde—led to the radi- elegans, while the presence of mostly upright and square ray cells is ation of the former genus Tornabenea. A third and a very recent synapomorphic for the Madeiran clade (not shown). Regardless of dispersal gave rise to D. carota subsp. azoricus. Given its young age the ancestral state estimation method, the most recent common an- with 95% HPD encompassing modern times and the results of ge- cestor of Daucus was reconstructed as lacking growth rings, diffuse nomic studies that do not support D. carota subsp. azoricus as a porous species with libriform fibers. (This is further supported by separate taxon (Arbizu et al., 2016), it is probable that this taxon presence of such fibers in all studied Daucus species, although not colonized the Azores with humans. necessarily throughout the whole life span; Appendix S6.) Presence of pervasive parenchyma and scalariform intervessel Incongruence between parsimony and ML reconstructions pitting was reconstructed with MP as plesiomorphic for Daucus, while ML method did not exclude any scenario, i.e., relative like- The most important difference between MP and ML methods is that lihoods of alternative character states were comparable. Pervasive the latter accommodate for branch lengths, while parsimony ne- parenchyma co-occurred with scalariform intervessel pitting except glects this information. Hence, ML infers changes as more probable for Daucus carota subsp. gummifer, and both traits were more com- on long branches rather than on short ones. Because branch lengths mon in polycarpic species than in monocarpic ones (Fig. 8). of a phylogenetic tree may be scaled to time (chronograms) or to the expected number of nucleotide substitution (phylogram), there comes a question of which one to use, or whether to abandon this DISCUSSION information altogether (Pagel, 1999). Unlike the case for the mo- lecular data, there is no good evidence that morphological traits, Dispersal of Daucus to Macaronesia which often are subject to natural selection, evolve stochastically as is assumed by Markov models employed by ML (Tuffley and Steel, Including the ETS marker in the phylogenetic analyses helped re- 1997; Cunningham et al., 1998; Goloboff et al., 2019). Moreover, se- solve the positions of two chamaephytic species, D. elegans and lection of chronogram or phylogram for ML reconstruction may D. bischoffii, that are crucial for understanding life-form evolution provide very distinct results, as is actually the case in the present and dispersal patterns in Daucinae. The first one was sister to the study, where ML reconstruction with chronogram inferred ances- Madeiran endemics, whereas the latter was placed together with tral states different from the ones resolved by MP, while the estima- other species formerly recognized in Tornabenea. The results of our tion using a phylogram (not shown) was in general congruent with study (Fig. 3) corroborate the biogeographic scenario inferred by MP. On the other hand, parsimony tends to be misleading when Spalik and Downie (2007), which postulated three independent dis- rates of evolution are high and when the probabilities of gains and persals of Daucus to Macaronesia that gave rise to (1) the Canarian- losses are not equal (Cunningham et al., 1998). For these reasons, Madeiran clade encompassing D. elegans, D. decipiens, and D. the results of ancestral state reconstruction should be interpreted edulis; (2) the Cape Verde clade including species formerly recog- with caution. nized in Tornabenea; and (3) the Azorean D. carota subsp. azoricus. Because the Canary Islands are closer to Africa than Madeira, the Habit evolution and the lability of wood ground tissue former archipelago was probably colonized first, and from there, the common ancestor of D. edulis and D. decipiens dispersed to Parsimony-reconstructed ancestral combination of traits for Madeira. Such a dispersal pattern is contrary to the dominant wind the most recent common ancestor of genus Daucus (as well as for 408 • American Journal of Botany

the ancestor of sections Daucus and Melanoselinum) was biennial to of wood traits among related species with unlike habits is also triennial, monocarpic therophyte or hemicryptophyte. Since thero- found in Bupleurum, another genus of umbellifers (Stepanova and phytes are annuals, it is reasonable to assume that the hemicrypto- Oskolski, 2010). phytic habit is plesiomorphic for the genus and its typical section (i.e., Daucus sect. Daucus). Life span is an ecologically labile trait: in Causes of insular woodiness in Daucus certain conditions, the usually biennial carrot (Daucus carota) may flower and die in its first year or it may postpone blooming until The shifts to chamaephytic life form in the clade comprising the third year (Lacey, 1988). The combination of character states re- Canarian D. elegans and Madeiran D. decipiens and D. edulis and in constructed for Daucus is intermediate between annual therophytes the former genus Tornabenea from Cape Verde represent cases of and perennial hemicryptophytes or chamaephytes/rosette treelets, insular woodiness. To explain such habit shifts, Lens et al. (2013b) thereby enabling subsequent evolution in both directions and for hypothesized that derived woodiness evolved as a result of selec- this reason is congruent with the observed pattern of life span and tion for greater air embolism resistance in the vascular system. This life form in the genus. hypothesis has recently been supported by ecophysiological data The chamaephyte/rosette treelet life form arose at least twice: in on shrubby species of Argyranthemum from the dry lowlands of the most recent common ancestor of Daucus elegans and Daucus the Canary Islands (Dória et al., 2018). This explanation, however, sect. Melanoselinum (D. decipiens and D. edulis) and in the former is not plausible for the chamaephytic Daucinae, as none of the in- genus Tornabenea within sect. Daucus. Such repetitive evolution has sular endemic species of this tribe is confined to dry coastal regions been documented multiple times on islands, including at least 38 of the Macaronesian islands. Instead, these species occur in more cases on the Canary Islands alone (e.g., Böhle et al., 1996; Kim et al., humid and mild habitats, usually at higher elevations (Martins, 2008; Lens et al., 2013b; Nürk et al., 2019). Aside from Daucinae, 1996; Brochmann et al., 1997; Press and Dias, 1998; Fernandes and other cases of insular woody apioids include Nirarathamnos asar- Carvalho, 2014; GBIF.org, 2018). The evolution of derived wood- ifolius Balf. f. from Socotra and Angelica lignescens Reduron & iness in Daucus may also be explained by other factors of insu- Danton from the Azores (Press and Dias, 1998; Oskolski, 2001). lar environment promoting longer life span, such as competition Two sister species comprising Daucus sect. Melanoselinum rep- for pollinators in a pollinator-poor setting and release from large resent strikingly different stem anatomies (solely fibrous wood in D. herbivore pressure (reviewed by Carlquist, 1972; Jorgensen and decipiens vs. parenchymatous and fibrous wood in D. edulis; Figs. 4 Olesen, 2001; Dulin and Kirchoff, 2010; Whittaker et al., 2017; see and 5) and life histories (monocarpic vs. polycarpic; Fig. 8). Hence, also Darwin, 1859; Wallace, 1878) or the effect of moderate climate it cannot be precluded that chamaephytic/rosette treelet habit (Carlquist, 1974). Assuming that biennial/triennial (MP recon- evolved in this clade twice or even three times (when the closely struction) or annual (ML reconstruction) habit was plesiomorphic related D. elegans is also taken into consideration). This scenario for the whole genus, the prolonged life span hypothesis may account is corroborated by the case of very rapid evolution of the chamae- for the treelet life form of Madeiran and Cape Verde perennials. In phyte with fibrous wood in stem, D. bischoffii, which originated case of D. elegans from the Canary Islands where prominent dry ca. 130,000 years ago—suggesting that habit shifts within Daucus season occurs (Mies, 1995; Cropper, 2013), embolism resistance is can occur rapidly—and by the ML reconstruction of ancestral life also a plausible explanation for evolution of woodiness, especially form. The stem node age of Daucus sect. Melanoselinum is 8.5 Myr that this species has not evolved markedly longer life span. (95% HPD 9.85–7.44), which would have allowed many evolution- ary changes to have taken place. As demonstrated for Arabidopsis Reproductive strategy and wood anatomical traits thaliana, the formation of secondary xylem cylinder may occur through mutations in only two genes regulating the timing of flow- Reproductive strategy, pervasive parenchyma, and scalariform in- ering (Lens et al., 2012b), which points to the lability of this trait. tervessel pitting seem to covary when reconstructed on a phyloge- However, only through genomic studies can we possibly answer the netic tree (Fig. 8). This relationship was asserted in previous studies question of whether D. decipiens and D. edulis evolved the chamae- (e.g., Sibout et al., 2008; Ragni et al., 2011). We additionally calcu- phytic habit independently or inherited it from their most recent lated Pagel’s correlations (1994; as implemented in Mesquite) be- common ancestor. tween each pair of these traits, i.e., reproductive strategy, presence Among the chamaephytic species, D. decipiens, D. elegans, D. of pervasive parenchyma, and presence of scalariform intervessel bischoffii, and D. tenuissimus have fibrous wood, whereas D. edu- pitting, which were statistically significant (P < 0.01). However, lis has abundant pervasive parenchyma in the inner region of the Maddison and FitzJohn (2015) demonstrated that current tests for wood cylinder (Figs. 4 and 5). Libriform fibers are present also in phylogenetic correlation between categorical characters—including secondary xylem of all newly studied species, but the amount var- Pagel’s (1994) test—do not eliminate pseudoreplication because ies considerably from an entire cylinder of fibrous wood to narrow they are susceptible to an effect of a single coevolutionary event in- bands or clusters of fibers embedded in pervasive parenchyma. herited by multiple descendants. Our data show, therefore, that the shifts to chamaephytes are not Daniel (1916) and Radkevich (1928) suggested that shorten- necessarily associated with an increase in fibrous wood deposition. ing of the internodes and formation of a leaf rosette during the Generally, all wood anatomical traits of insular woody species of vegetative phase of shoot growth might be responsible for devel- Daucus can be found also in other species of this genus, e.g., their opment of parenchymatous zones in the secondary xylem, while ground tissue may be composed either of fibers alone or of perva- the transition to flowering triggers a shift to the formation of sive parenchyma and fibers together. In other plant groups, the evo- wood. Sibout et al. (2008) showed that flowering might be respon- lutionary shift between herbaceous and woody life forms is also not sible for xylem expansion in hypocotyl and root of Arabidopsis. strictly linked to the evolution of specific wood traits (Stepanova Ragni et al. (2011) broadened this view by presenting evidence et al., 2007; Schweingruber and Büntgen, 2013) and similar diversity that, in plants with leaf rosettes, the appearance of fibers never March 2020, Volume 107 • Frankiewicz et al.—Evolution of arborescence in insular carrots • 409

occurred before flowering, unlike in related species without ro- For these reasons, Daucus makes an interesting model for further settes. The authors studied selected species of Asteraceae and testing of hypotheses regarding evolution of insular woodiness. Brassicaceae, but leaf rosettes are a common feature of Daucinae We observed that monocarpic species tend to have only fibrous (Table 1), making a similar scenario plausible also in this group. wood, while polycarpic species deposit parenchymatous and fibrous The mechanism of interdependence between flowering and fibers xylem, and that scalariform intervessel pitting is predominantly deposition remains obscure, but it is clear that this connection present in parenchymatous wood. Although it is possible that a exists (Chaffey et al., 2002; Lens et al., 2012b). The formation of common mechanism, similar to that advocated in previous studies, fibrous wood may be induced by gibberellic acid, which plays an is responsible for development of these three traits, the limited num- important role both in the developmental switch to flowering ber of sampled species preclude making any definitive statements. (Conti, 2017) and in stimulating secondary xylem differentiation and expansion of cambial derivatives, including fiber elongation (Moritz et al., 2000; Israelsson et al., 2005; Mauriat and Moritz, ACKNOWLEDGMENTS 2009; Ragni et al., 2011; Strabala and MacMillan, 2013; Ye and Zhong, 2015; Ragni and Greb, 2018). If so, the shift from paren- We thank Stefan Dressler of Senckenberg Herbarium and Wolfram chymatous to fibrous wood could be explained by the switch from Lobin for help with obtaining Tornabenea wood samples, Paulina vegetative to reproductive growth in the plant life history. Trzeciak for help with sectioning Daucus della-cellae, Cabildo de Among species for which wood anatomical data were available in Tenerife for the collection permits of Daucus elegans, John H. Chau the study, parenchymatous wood was often concomitant with poly- for linguistic corrections and useful comments, S. Carlquist for his carpic reproductive strategy, while monocarpic species deposited review and critique of the earlier version of our manuscript, Mark solely fibrous wood. This pattern could be explained by longer veg- P. Simmons (Associate Editor), Frederic Lens, and one anonymous etative growth in the former group, followed by floral shift leading reviewer for their inquisitive questions which greatly enhanced to deposition of libriform fibers. In the monocarps, flowering shift our study. This work was supported by National Science Centre, occurs early in plant development leading to the deposition of fibrous Poland (grant no. 2015/19/B/NZ8/00163 to K.S.), the Russian wood from the beginning of secondary growth. Such a shift may also Foundation for Basic Research (grant no. 16-04-00725a to A.O.), have mechanical explanation because fibrous wood provides more the Komarov Botanical Institute (institutional research project support for orthotropic shoots with long internodes and terminating no. AAAA-A18-118030690081-1 to A.O.), the National Research in heavy inflorescences (Ko et al., 2004; Sibout et al., 2008). Foundation of South Africa (incentive grant 109531 to A.O.), and Simultaneously, the co-presence of pervasive parenchyma the University of Johannesburg (to A.O. and K.F.). and scalariform intervessel pitting has been observed in other groups, e.g., Phacelia (Boraginaceae; Carlquist and Eckhart, 1984), Potentilla (Rosaceae; Stepanova et al., 2007), Bupleurum AUTHOR CONTRIBUTIONS (Apiaceae; Stepanova and Oskolski, 2010), and also in succulents including Crassulaceae (Carlquist, 2001, 2009b) and Cactaceae K.F. prepared and analyzed wood samples, gathered the ecologi- (Gibson, 1973). The shift to wide scalariform intervessel pit aper- cal and morphological data, performed phylogenetic analyses, and tures might be the result of relaxation of mechanical constraints wrote the manuscript. A.O. supervised wood anatomical analyses in stems supported primarily by turgor of parenchyma cells, and wrote the manuscript. Ł.B. supervised the phylogenetic analy- which may be an adaptation to expansion and contraction of the ses and reviewed the manuscript. F.F., J.-P.R., JARB collected sam- stem with fluctuating turgor (Carlquist, 2009b; Lens et al., 2012a; ples in the field and provided information about their ecology. L.S., Kedrov, 2013). M.A., and J.B. obtained new DNA sequences. K.S. designed and su- pervised the study, and edited the manuscript. The authors have no conflicts of interest to declare. CONCLUSIONS

The most likely ancestral habit of Daucus was biennial to tri- DATA AVAILABILITY ennial monocarpic hemicryptophyte, and these traits were re- tained in the most recent common ancestor of sect. Daucus and Matrix with aligned molecular sequences before and after trim- Melanoselinum. This habit makes a convenient starting point for ming, maximum clade credibility tree, and data matrices with habit both shortening and extending of the life span and is congruent and anatomical data are available online in TreeBase (http://purl.org/ with observed highly homoplastic evolution of habit. At least two phylo/​treeb​ase/phylo​ws/study/​TB2:S2490​2?x-access-code=c8c88​ independent shifts to chamaephytes/rosette treelets occurred in a0b3b​02f32​f309a​4bb82​21d05​68&forma​t=html). the genus and represent cases of insular woodiness in Canarian- Madeiran lineage of D. elegans, D. decipiens, and D. edulis, and, a very rapid one, in former Tornabenea of Cape Verde. Wood anat- SUPPORTING INFORMATION omy of insular, “woody” species show the same set of traits as ob- served among their continental, “herbaceous” relatives suggesting Additional Supporting Information may be found online in the that in Daucus, life-form evolution is not constrained by any fea- supporting information tab for this article. tures of their wood structure. At the same time, sister species of ro- APPENDIX S1. Accession table. sette treelets—D. decipiens and D. edulis—have strikingly different stem anatomies. This dissimilarity opens a possibility of treelet life APPENDIX S2. Characteristics of the data set used in phylogenetic form evolving multiple times in the Canarian-Madeiran lineage. analysis table. 410 • American Journal of Botany

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