How Old Are Sunflowers? a Molecular Clock Analysis of Key Divergences in the Origin and Diversification of Helianthus (Asteraceae)

Home , Asteroideae, Helianthus, Helianthus anomalus, Helianthus argophyllus, Helianthus deserticola, Helianthus niveus

HOW OLD ARE SUNFLOWERS? A MOLECULAR CLOCK ANALYSIS OF KEY DIVERGENCES IN THE ORIGIN AND DIVERSIFICATION OF HELIANTHUS (ASTERACEAE)

Chase M. Mason1,*

*Department of Biology, University of Central Florida, Orlando, Florida 32816, USA

Editor: Susana Magallón

Premise of research. For many questions in evolutionary ecology, it is quite valuable to have an estimate of the span of geologic time over which a group of species has arisen and diversified. This study uses a molecular clock approach to generate a first estimate of key divergences in the evolutionary history of the genus Helianthus, which to date has lacked such an estimate. Methodology. Divergence time analysis was performed with well-resolved phylogenies of Asteraceae and Helianthus in a stepping-stone manner using the RelTime maximum likelihood method with a variety of available macrofossil, fossil pollen, and molecular clock calibrations derived from the literature. Pivotal results. Mean estimates from nine individual calibration scenarios time the divergence of Helian- thus from the sister genus Phoebanthus at between 2.47 and 5.41 Ma. Composite calibration incorporating all literature estimates time this divergence at 3.63 Ma, with a 95% confidence interval spanning 0–8.26 Ma. Subsequent diversification within Helianthus is therefore confined to the last few million years. Conclusions. These findings place the origin and diversification of Helianthus into the temporal context of the Pliocene expansion of open habitats across North America and the subsequent vegetation fluctuations of the Pleistocene glacial-interglacial cycles.

Keywords: dating, divergence time, Helianthus, molecular clock, sunﬂower.

Online enhancement: appendix ﬁgures.

Introduction For many questions related to understanding the evolutionary history of Helianthus, it would be very useful to have an es- The genus Helianthus is an emerging model system in evolu- timate of the approximate age of the genus and the geologic tionary ecology. This group of species includes broad diversifi- periods during which this group has diversified. However, this cation in morphology, physiology, and life history across strong is an herbaceous genus (i.e., poorly fossilizing relative to woody environmental gradients (Heiser et al. 1969; Mason and Dono- plants) and lacks diagnostically unique pollen morphology (i.e., van 2015), allowing for the study of many aspects of trait evo- not useful for dating from sediment deposits). As such, no fos- lution under adaptation to diverse habitats. The genus also sils have been discovered for Helianthus or any close relatives contains a variety of hybridization and polyploidization events other than archaeological evidence of sunflower domestication that provide excellent arenas for the study of such processes in North America within the last five millennia (e.g., Crites (Rieseberg 2006; Kane et al. 2009; Bock et al. 2014). The 1993). This makes placing the diversification of Helianthus into amount of genetic resources for this group is steadily increasing, a geologic context difficult and limits the questions we can ad- with a larger number of sets of genetic markers in existence (Kane dress with phylogenetic comparative approaches. et al. 2013), several well-resolved phylogenies addressing both This study uses a molecular clock approach to generate a first the diploid backbone of the genus and various polyploid clades set of estimates for key dates in the evolutionary history of (Bock et al. 2014; Stephens et al. 2015), an association mapping Helianthus. To accomplish this, I exploit the existence of over- panel and associated linkage maps (Mandel et al. 2013), and the lap between two well-resolved phylogenies, one of diploid He- recent publication of a full reference genome for cultivated lianthus and one of a cross section of species spanning major Helianthus annuus (Badouin et al. 2017). Asteraceae tribes. By using a variety of divergence age estimates from the literature for major clades of Asteraceae and applying these to calibrate an Asteraceae phylogeny containing multiple species of Helianthus, estimates of divergence applicable to cal- 1 E-mail: [email protected]. ibration of the diploid Helianthus phylogeny are generated. This stepping-stone approach allows for the estimation of di- Manuscript received May 2017; revised manuscript received August 2017; electronically published January 24, 2018. vergence ages in a genus otherwise without useful calibrations.

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Material and Methods 2012), thought to represent the minimum age of divergence of the Mutisioideae from the rest of the Asteraceae (represented Phylogenies by node B in fig. 1a), and another from fossil pollen dated to 16 Ma (Katinas et al. 2007), thought to represent the minimum Two recent phylogenies were used for divergence time analy- age of emergence of tribes within the Asteroideae (represented ses: a phylogeny of a cross section of representative Asteraceae by node D in fig. 1a). The remaining three studies used molecu- and Calyceraceae spanning disparate tribes (Mandel et al. 2014) lar clock approaches, with various fossil calibrations not directly and a phylogeny of the diploid backbone of Helianthus (Stephens transferable to the Asteraceae phylogeny used here (Kim et al. fi et al. 2015). The Asteraceae phylogeny ( g. 1a;Mandeletal. 2005; Bergh and Linder 2009; Barreda et al. 2015). One such 2014) was originally created using data on 763 nuclear genes study (Barreda et al. 2015) used both the fossils of Barreda et al. in 16 taxa using a target enrichment approach on conserved (2012) and newly described fossil pollen to date the divergence orthologous sequences, with topology and branch lengths esti- of Barnadesieae from the rest of the Asteraceae (represented by mated using maximum likelihood on a concatenated data set node A in fig. 1a) to between 53 and 76 Ma on the basis of five with GARLI (ver. 2.0; Zwickl 2006; Mandel et al. 2014). The different placement scenarios, all with similar results (one addi- fi fi tree le and concatenated alignment le were obtained from tional scenario places this date much older, but the accuracy of the Dryad Digital Repository (http://datadryad.org/resource the fossil placement in that scenario was contested, so this sce- /doi:10.5061/dryad.gr93t) for use in subsequent analyses. nario was not included in the analyses presented here; Barreda fi The Helianthus phylogeny ( g. 1b; Stephens et al. 2015) was et al. 2016; Panero 2016; Panero and Crozier 2016). Another of originally created using data on 170 nuclear genes on 38 taxa these studies (Bergh and Linder 2009) used Asteraceae fossil generated through target enrichment, with topology estimated pollen linked to the divergence of Heliantheae from Tageteae fi through two methods: rst, by maximum likelihood on a con- along with geological calibrations based on island emergence catenated data set in RAxML (ver. 04262012; Stamatakis 2006), within the genus Stoebe to derive an estimate of the divergence and second, by maximum pseudolikelihood estimation of the among the tribes of the Asteroideae (represented by node D in species tree (MP-EST; ver. 1.4; Liu et al. 2010), a multispecies fig. 1a). One additional study (Kim et al. 2005) avoided using coalescent approach that estimates a consensus species tree to- any internal Asteraceae fossils, given the relatively sparse fossil pology from multiple gene trees and is robust to the effects of record and controversy surrounding the identity and placement gene tree discordance from incomplete lineage sorting (Liu et al. of fossils at the time (something which remains today). They 2010). Because coalescent methods do not produce normal used a noncontroversial outgroup Cornus fossil to calibrate a fi branch lengths (e.g., coalescent units with xed terminal tip molecular clock analysis of an Asteraceae chloroplast gene in- lengths), branch lengths in substitution units were subsequently version data set, deriving estimates for multiple divergences, in- fi estimated on the xed MP-EST topology using RAxML (Ste- cluding between Barnadesieae and the rest of Asteraceae, be- fi fi phens et al. 2015). Tree les and aligned FASTA les were ob- tween Cichorieae and the Asteroideae, and between Heliantheae tained from the Dryad Digital Repository (http://datadryad.org and Senecioneae (represented by nodes A, C, and D, respectively, /resource/doi:10.5061/dryad.4n28n) for use in subsequent anal- in fig. 1a). While these latter three studies represent secondary yses. Because both the concatenation and MP-EST trees con- calibrations (the primary fossil evidence they employ cannot fi tained multiple individuals per taxa, both the tree les and FASTA be directly transferred to the Asteraceae tree employed here), data sets were pruned to contain only the single individual per spe- the additional information provided by them is of significant cies or subspecies with the longest concatenated sequence length value. Both Bergh and Linder (2009) and Barreda et al. (2015) (i.e., most complete sequence). derive estimates from potentially valuable Asteraceae fossil evidence that we would otherwise not be able to include, and Kim et al. (2005) represents a set of estimates that are not subject to Fossil and Molecular Clock Calibrations the identity and placement controversies that plague many inter- To obtain divergence time estimates for nodes in the Astera- nal Asteraceae fossils. ceae phylogeny, a literature search was performed for examples Additionally, three other fossil pollen records within Aste- of Asteraceae macrofossils or fossil pollen as well as previous raceae were identified but not included in analyses because of molecular clock analyses that incorporate members of the As- large phylogenetic distances between the divergence represented teraceae. This yielded five relevant studies that provided age by the fossil and the closest divergence present in the Asteraceae estimates applicable to four nodes in the Asteraceae phylogeny phylogeny used here (fig. 1a). These all involved divergences (fig. 1a; table 1). These nodes include the split between the Bar- within Cichorieae, including a divergence between the basal Scor- nadesieae and the rest of the Asteraceae (fig. 1a, node A), the zonerinae and the rest of the Cichorieae (Cichorium intybus– split between the Mutisioideae (here represented by the Muti- type pollen; Hochuli 1978; Tremetsberger et al. 2013), a di- sieae) and the rest of the Asteraceae excluding Barnadesieae vergence within the Scorzonerinae (Scorzonera hispanica–type (fig. 1a, node B), the split between the Cichorieae and the Aster- pollen; Blackmore et al. 1986; Tremetsberger et al. 2013) and oideae (fig. 1a, node C), and the most recent common ancestor a divergence among genera within the Hyoseridinae (Sonchus of the tribes within the Asteroideae (fig. 1a, node D), here oleraceus–type pollen; Blackmore et al. 1986; Tremetsberger represented by the tribes Gnaphalieae, Senecioneae, Eupato- et al. 2013). None of the three are sound choices for calibration rieae, and Heliantheae. of nodes within the Asteraceae phylogeny used here (fig. 1a) be- Two of the five relevant studies provided estimates of diver- cause this phylogeny contains only two Cichorieae species. The gence directly from fossils, one from both a macrofossil and ac- two possible nodes these fossils could be applied to (node C or companying fossil pollen dated to 47.39 Ma (Barreda et al. the Lactuca-Taraxacum divergence) are either much older or

This content downloaded from 128.192.114.019 on February 01, 2018 21:01:48 PM All use subject to University of Chicago Press Terms and Conditions (http://www.journals.uchicago.edu/t-and-c). Fig. 1 Two phylogenetic trees used for RelTime analyses: a recent maximum likelihood tree of 16 select Asteraceae and Calyceraceae created with 763 nuclear genes using targeted enrichment on conserved orthologous sequences (Mandel et al. 2014) and the most recent phylogeny of diploid Helianthus created with 170 nuclear genes obtained through targeted enrichment (Stephens et al. 2015). Letters reﬂect the location of nodes with calibrations from fossils and previous molecular clock analyses (see table 1), and numbers reﬂect nodes shared between the two phylogenies and used to calibrate the Helianthus tree for RelTime analyses (see table 2).

Table 1 Details of the Nine Scenarios Used to Calibrate RelTime Analyses on the Asteraceae Tree Calibration Node Age range (Ma) Type Source A1 A 36–42 Molecular clock Kim et al. 2005 A2 A 53–76 Molecular clock Barreda et al. 2015 B (broad) B 47.39–76 Macrofossil 1 pollen Barreda et al. 2012 B (narrow) B 47.39–57.39 Macrofossil 1 pollen Barreda et al. 2012 CC32–38 Molecular clock Kim et al. 2005 D1 (broad) D 16–76 Fossil pollen Katinas et al. 2007 D1 (narrow) D 16–26 Fossil pollen Katinas et al. 2007 D2 D 26–39 Molecular clock Kim et al. 2005 D3 D 29.6–56.6 Molecular clock Bergh and Linder 2009 Note. Node letters refer to four divergences in the Asteraceae tree (Mandel et al. 2014) depicted in figure 1: A, the most recent common ancestor (MRCA) of Fulcaldea stuessyi and Helianthus annuus, representing the divergence between the Barnadesieae and the rest of the Asteraceae; B, the MRCA of Gerbera hybrida and H. annuus, representing the divergence between the Mutisioideae (represented by the Mutiseae) and the rest of the Asteraceae excluding Barnadesieae; C, the MRCA of Lactuca sativa and H. annuus, representing the divergence between the Cichorieae and the Asteroideae; and D, the MRCA of Senecio vulgaris and H. annuus, representing the common ancestor of the Asteroideae. For calibration scenarios derived from previous molecular clock analyses (A1, A2, C, D2, D3), age ranges reported in the original studies were used. For calibrations derived from fossils (B, D), two age ranges were used reflecting broad and narrow estimates: one with minimum age set by the fossil and a maximum age equivalent to the oldest age of divergence between Barnadesieae and the rest of Asteraceae among our scenarios (76 Ma), and the other with minimum age set by the fossil and maximum age set to 10 Ma earlier. much younger than the three divergences represented by each maximally conservative estimate allowing for potentially long fossil, so calibration with these fossils was not attempted. Care- spans between the emergence of a group and the fossilization ful calibration with these distant fossils could perhaps be at- of the member of that group that was found, accounting for poor tempted in a Bayesian analysis with appropriate priors, but given fossilization and/or lengthy periods where a group is not wide- the size of the data sets used here, a maximum likelihood ap- spread or numerous enough to appear in the fossil record; and proach (RelTime; Tamura et al. 2012) was favored in order (2) a less conservative estimate for comparison, one more typical to keep computation time reasonable, especially given the sen- of the 95% confidence ranges obtained from molecular clock sitivity analysis performed. This approach has been shown to analyses in Asteraceae. Additionally, given the controversial na- give highly similar results to Bayesian approaches with large data ture of the identity and placement of some Asteraceae fossils sets, with orders of magnitude faster computation time (Mello (Panero et al. 2014; Panero and Crozier 2016), a sensitivity anal- et al. 2017). The use of this maximum likelihood approach pre- ysis was performed by conducting analyses with each of the nine cluded the inclusion of the Cichorieae fossil records, given their different calibration scenarios individually and comparing diver- phylogenetic distance from available nodes, because constrain- gence estimates alongside a composite analysis combining cali- ing those nodes with minimum ages far from the divergences brations. In this way, the suite of results obtained here is not they represent would likely introduce substantial bias and un- strongly dependent on the validity of any one calibration. certainty into analyses when prior probability distributions are not modeled. The sensitivity analysis performed here itself dem- Divergence Time Analysis onstrates how changing minimum or maximum ages for a given node can substantially alter divergence time estimates. Given the Because the goal of this study was to estimate the timing of number of other calibrations available without phylogenetic dis- the origin of Helianthus and clades within the genus, and no fos- tance issues, the Cichorieae fossils were not included. sil calibrations exist for Helianthus, a stepping-stone approach For the four focal nodes in the Asteraceae phylogeny, there was used. Primary analysis was conducted on the Asteraceae were a total of seven divergence age estimates (one to three per phylogeny with the above-described literature-derived calibra- node; table 1), with sometimes conflicting estimates from differ- tions, and dates derived for nodes within Helianthus were then ent sources. Because there is much uncertainty inherent in as- transferred to the Helianthus phylogeny to calibrate that sec- signing ages to nodes, a conservative approach was employed. ondary analysis. Note that the age of Helianthus divergence First, when estimates were reported as ranges in the literature from its sister genus can be inferred directly from the primary (i.e., 95% confidence intervals [CIs] from molecular clock anal- analysis with the Asteraceae phylogeny, and the secondary analyses), these ranges were used in subsequent analyses as mini- ysis is only needed to infer the divergence of particular clades mum and maximum ages. Second, when estimates were reported within Helianthus. as point estimates from fossil dating analyses, two age ranges All divergence time analyses were performed using RelTime were used, reflecting broad and narrow estimates: the broad esti- (Tamura et al. 2012) to estimate local clock rates and diver- mate, with a minimum age set by the fossil and a maximum age gence times with the maximum likelihood analytical method equivalent to the oldest age of divergence between Barnadesieae in MEGA7 (Kumar et al. 2016). For each analysis, a general and the rest of Asteraceae among our scenarios (76 Ma), and the time reversible (GTR) nucleotide substitution model was used, narrow estimate, with a minimum age set by the fossil and max- with a discrete Gamma distribution (1G) with eight categories imum age set to 10 Ma earlier. This approach allows for (1) a used to model evolutionary rate differences among sites. The

This content downloaded from 128.192.114.019 on February 01, 2018 21:01:48 PM All use subject to University of Chicago Press Terms and Conditions (http://www.journals.uchicago.edu/t-and-c). MASON—MOLECULAR CLOCK DATING OF HELIANTHUS 000 rate variation model allowed some sites to be invariant (1I), rest of the Asteraceae (node A) ranging from as early as 84.29 Ma with 32.1% of sites invariant for the Asteraceae data set (Man- (the D1 broad calibration; fig. A4) to as late as 38.48 Ma (the del et al. 2014) and 41.2% of sites invariant in the Helianthus D1 narrow calibration; fig. A4). The composite calibration, how- data set (Stephens et al. 2015). All sites with less than 50% cov- ever, places the mean divergence between these two groups at erage (e.g., missing data, alignment gaps) were eliminated, for a 56.52 Ma, with a 95% CI spanning 47.39–76.00 Ma (fig. A5). total of 74,257 sites used for analysis in the Asteraceae data set For the divergence between Phoebanthus and Helianthus (our and 74,960 sites used for analysis in the Helianthus data set. focal divergence), the nine individual calibration scenarios yielded First, divergence time analysis was performed on the Aster- mean estimates ranging from 2.47 to 5.41 Ma (again from the aceae phylogeny independently for each of the nine individual narrow and broad D1 calibrations; table 2; figs. 2, A4). Esti- calibration scenarios derived from the literature (table 1) as well mates derived from the other calibration scenarios cluster within as in a composite calibration using the minimum and maximum this range, with a mean of 3.92 Ma and a pooled 95% CI span- ages for each node across literature sources. Ninety-five percent ning 0.37–12.83 Ma (table 2; fig. 2). The composite calibration CIs were estimated for each node in the Asteraceae phylogeny in places the mean divergence estimate very closely nearby at 3.63 Ma, each analysis. All calibrated timetrees generated from these with a 95% CI spanning from 0 to 8.26 Ma (table 2; figs. 2, A5). analyses are presented in figures A1–A5 (figs. A1–A11 are avail- For all nodes within Helianthus, mean estimates derived from able online). the nine individual calibration scenarios all fall within 4.5 Ma Second, divergence time analysis was performed on both the before present, while estimates from the composite calibration concatenation and MP-EST phylogenies of Helianthus using all span between 1.54 and 3.01 Ma (table 2). For divergences age estimates for the five nodes overlapping between the Aster- within Helianthus, CIs for individual calibration scenarios of- aceae and Helianthus trees (fig. 1; table 2). Three sets of age ten include the present day (0 Ma), but a substantial number ranges were used for RelTime analysis, reflecting (1) the range do not, and the pooled CI across the nine individual calibra- of minimum and maximum age estimates for each node ob- tion scenarios includes only 0 Ma for the divergence between tained on the Asteraceae tree under the nine scenarios used, the sister species Helianthus annuus and Helianthus argophyllus (2) the pooled 95% CI of the mean of the age estimates obtained (table 2). for each node, and (3) the 95% CI around node ages obtained under the composite calibration (table 2). All calibrated time- Helianthus Analysis trees generated from these analyses are presented in figures A6– A11. Divergence time analysis on the two Helianthus trees yielded very similar results for a given calibration, despite slight differ- fi – Results ences in topology ( gs. A6 A11). The pooled CI calibration generated the oldest estimates, with the basal Helianthus porteri diverging from other Helianthus 5.19 Ma and only one diver- Asteraceae Analysis gence less than 1 Ma before present (between subspecies of He- Divergence time analysis of the Asteraeae tree yielded esti- lianthus debilis; fig. A9). The range of estimates calibration gen- mates for the mean divergence between the Barnadesieae and the erated the youngest estimates, with H. porteri diverging from

Table 2 RelTime Estimates of Divergences for Helianthus Species in the Asteraceae Tree (Ma before Present) Node 3: Node 5: Node 1: Phoebanthus Node 2: H. verticillatus– Node 4: H. niveus H. argophyllus– Calibration tenuifolius–H. annuus H. porteri–H. annuus H. annuus tephrodes–H. annuus H. annuus A1 2.50 (.82–4.43) 2.08 (.54–3.84) 1.54 (.23–3.05) 1.30 (.09–2.70) 1.07 (.00–2.47) A2 4.14 (.36–9.23) 3.43 (.02–8.05) 2.55 (.00–6.48) 2.15 (.00–5.75) 1.76 (.00–5.36) B (broad) 4.56 (.09–11.08) 3.78 (.00–9.68) 2.82 (.00–7.81) 2.38 (.00–6.94) 1.94 (.00–6.49) B (narrow) 3.87 (1.39–6.80) 3.21 (.95–5.89) 2.39 (.46–4.68) 2.02 (.24–4.13) 1.65 (.00–3.77) C 3.45 (1.90–5.24) 2.86 (1.43–4.52) 2.13 (.91–3.54) 1.80 (.66–3.12) 1.47 (.33–2.80) D1 (broad) 5.41 (.00–28.44) 4.48 (.00–25.25) 3.34 (.00–20.93) 2.82 (.00–18.80) 2.30 (.00–18.26) D1 (narrow) 2.47 (.34–5.55) 2.05 (.14–4.83) 1.52 (.00–3.88) 1.29 (.00–3.44) 1.05 (.00–3.19) D2 3.82 (.93–7.77) 3.17 (.58–6.74) 2.36 (.19–5.38) 1.99 (.01–4.76) 1.63 (.00–4.37) D3 5.07 (.00–13.72) 4.20 (.00–12.00) 3.13 (.00–9.71) 2.64 (.00–8.64) 2.16 (.00–8.12) Range of estimates 2.47–5.41 2.05–4.48 1.52–3.34 1.29–2.82 1.05–2.30 Mean of estimates 1 pooled CI 3.92 (.37–12.83) 3.25 (.20–11.29) 2.42 (.08–8.92) 2.04 (.02–8.24) 1.67 (.00–7.85) Composite calibration 3.63 (.00–8.25) 3.01 (.00–7.25) 2.24 (.00–5.89) 1.89 (.00–5.25) 1.54 (.00–4.96) Note. RelTime mean estimates with 95% conﬁdence intervals (CIs) for Helianthus nodes in the Asteraceae tree (Mandel et al. 2014) are presented (Ma before present). Letters represent the nine individual calibration scenarios described in table 1. Three additional derived summaries were created and used for subsequent analysis with the Helianthus tree (Stephens et al. 2015): a simple range of the estimates derived with the nine individual calibration scenarios, the mean of the nine individual calibration scenario estimates with pooled 95% CI, and a composite calibration made using the minimum and maximum ages used for each node across sources (table 1; A p 36–76 Ma; B p 47.39–76 Ma; C p 32–38 Ma; D p 16–56.6 Ma).

This content downloaded from 128.192.114.019 on February 01, 2018 21:01:48 PM All use subject to University of Chicago Press Terms and Conditions (http://www.journals.uchicago.edu/t-and-c). Fig. 2 RelTime estimates of divergence between Helianthus and the sister genus Phoebanthus using nine different individual calibration scenarios (table 1) on the Asteraceae tree (fig. 1). Diamonds indicate the mean date of divergence, while gray bars reflect the 95% confidence interval (CI) around each estimate (see table 2). Dark gray bars reflect derived summaries, including the range of estimates, the mean of estimates with a pooled 95% CI, and a composite calibration made using the minimum and maximum ages used for each node across sources (see table 2).

This content downloaded from 128.192.114.019 on February 01, 2018 21:01:48 PM All use subject to University of Chicago Press Terms and Conditions (http://www.journals.uchicago.edu/t-and-c). MASON—MOLECULAR CLOCK DATING OF HELIANTHUS 000 other Helianthus only 2.82 Ma, although still with only three the range of estimates found here for Helianthus are likely to be divergences less than 1 Ma before present (between subspecies reliable. of H. debilis and Helianthus niveus and sister species Helian- Working from CIs, the majority of calibration scenarios place thus angustifolius and Helianthus floridanus; fig. A11). The the divergence between Helianthus and the sister genus Phoe- composite calibration generated intermediate estimates, al- banthus at sometime between the mid-Miocene and the Pleis- though all three calibrations estimate the major divergences tocene (fig. 2). Mean estimates across scenarios are spread among the three major clades of Helianthus (fig. 1) at between throughout the Pliocene, with the range of estimates extending means of approximately 2.33 and 4.55 Ma (figs. A9, A11). just a few thousand years into both the Miocene and the Pleis- However, CIs for both the composite calibration and the pooled tocene (fig. 2). Both the mean of estimates and the composite CI calibration were quite large (on the order of several million calibration place this divergence in the Zanclean age of the Pli- years for deeper nodes), making precise inferences of divergence ocene (fig. 2). Simulation work on divergence time analysis sug- times unreliable (figs. A6, A8). The range of estimates calibra- gests that deeper node calibrations tend to give more accurate tion generated much smaller CIs, most under 1.5 Ma (fig. A10), estimates and that using multiple calibrations give more accu- by virtue of the smaller ranges used for calibration (table 2). rate estimates than single calibrations (Duchêne et al. 2014), so estimates from the node A scenarios and the composite cali- Discussion bration should perhaps be given more subjective weight than some of the other individual calibration scenarios. Regardless, First and foremost, it is important to note the uncertainty in- most of the various calibrations agree rather well with one an- herent in the methods used in these analyses and molecular other in placing the Phoebanthus-Helianthus divergence. clock dating methods generally (Donoghue and Benton 2007; In recent years, paleoclimate and vegetation reconstructions Pulquério and Nichols 2007; Tamura et al. 2012; Duchêne et al. have become much more well developed and allow for useful 2014; Mello et al. 2017). The approach used here attempts to insights in comparison to the divergence estimates obtained here. be conservative and preserve uncertainty in the dating estimates Interestingly, historical temperature reconstructions during this derived, but it is important not to interpret these results as more period show that global average surface temperature declined than a first approximation of the timeline of the origin and ra- significantly between the late Miocene and mid-Pliocene (fig. 3; diation of Helianthus. Hansen et al. 2013). This cooling trend resulted in a concomi- Dates obtained from the Asteraceae analysis appear consis- tant drying trend across North America, and paleobiome vege- tent with geologic evidence relating to early Asteraceae history. tation reconstructions from the Tortonian (fig. 3; Forrest et al. Inference from the breakup of Gondwana and the uplift of the 2015) and Piacenzian (fig. 3; Salzmann et al. 2008) show a ma- Southern Andes suggests that Asteraceae diverged from the sis- jor reduction of closed forest habitat between the late Miocene ter genus Calyceraceae sometime between 50 and 90 Ma before and mid-Pliocene and an increase in open shrubland, grassland, present (Funk et al. 2005), so the estimates obtained here for the and desert habitats across the continent. This pattern largely subsequent divergence between the basal Barnadesieae clade continued until the present day, with further cooling and drying and the rest of the Asteraceae (e.g., composite calibration with through the Pleistocene (Hansen et al. 2013). Modern members a mean of 56.52 Ma and a 95% CI of 47.39–76.00 Ma) are con- of the genus Helianthus are largely heliophytes, with most spe- sistent with that evidence. This bodes well for the validity of the cies occupying high-light open-vegetation environments, such other estimates derived from the Asteraceae analysis. Addition- as deserts, grasslands, herbaceous wetlands, rock outcrops, and ally, another recent molecular clock analysis focusing on whole- pine savannas (Heiser et al. 1969; Mason and Donovan 2015). genome duplications across the family places the divergence be- An increase in the availability of open habitats occurring at the tween Barnadesieae and the rest of the Asteraceae at between same time as the origin and subsequent diversification of Helian- 52.2 and 72.1 Ma (Huang et al. 2016), consistent with the re- thus fits well with what we know of the ecology of the genus to- sults found here. That study also found generally consistent esti- day, and assuming reasonable accuracy in divergence estimates, mates at other nodes shared with the analyses performed here, the climatic shifts during the late Miocene and Pliocene may have including placing the divergence between the Cardueae and generated ecological opportunities for Helianthus to diversify Asteroideae at between 42.3 and 54.8 Ma (as compared with across the continent. between 32.0 and 56.5 Ma for the 95% CI of the results of the Within the genus, CIs from the analysis of the Helianthus tree composite calibration here). Other node estimates differ some- are large and highly overlapping for all but the range of esti- what, for instance, the divergence among Asteroideae tribes is mates calibration (figs. A6–A11). With such large CIs, mean di- dated to between 36.1 and 46.6 Ma (Huang et al. 2016), while vergence estimates are largely uninterpretable, other than to ob- here our composite calibration places that divergence at between serve that species diversification likely took place from the late 16 and 38 Ma on the basis of the constraints of calibrations at C Pliocene through the Pleistocene until the present day. Diversi- and D nodes (though estimates for two-thirds of individual cali- fication through the lengthy glacial-interglacial cycles of the brations have 95% CIs overlapping the range of estimates from Pleistocene is interesting, because these climate cycles are known Huang et al. 2016). Some of the differences between the results to have generated vegetation shifts that would have repeatedly found here and those of Huang et al. (2016) may arise from the mimicked the initial opening up of closed habitats between the far larger number of taxa included in that study as well as the late Miocene and mid-Pliocene (Axelrod 1985; Williams et al. large number of non-Asteraceae fossil calibrations (eight) that 2004; Anderson 2006; fig. 3). This phenomenon may perhaps they were able to include, given the larger taxonomic scope of have also contributed to the widespread reticulate evolution ob- their data set. Regardless, taken as a whole, the general concor- served in Helianthus (Rieseberg 1991; Timme et al. 2007) be- dance of results for these deeper nodes in Asteraceae suggest that cause repeated vegetation shifts could have conveniently brought

This content downloaded from 128.192.114.019 on February 01, 2018 21:01:48 PM All use subject to University of Chicago Press Terms and Conditions (http://www.journals.uchicago.edu/t-and-c). Fig. 3 Paleobiome and historical temperature reconstructions relative to Helianthus-Phoebanthus divergence. Top, paleobiome vegetation reconstructions of North America during the Late Miocene (adapted from Forrest et al. 2015) and the Middle Pliocene (adapted from Salzmann et al. 2008), two well-modeled periods that happen to overlap with the confidence intervals (CIs) of many of the Helianthus-Phoebanthus divergence estimates (fig. 2). Note in particular the increasing abundance of open habitats between the Tortonian and Piacenzian. Bottom, reconstruction of global average surface temperature (adapted from Hansen et al. 2013) overlaid with both the Tortonian (green) and Piacenzian (or- ange) and the three derived summary estimates for Helianthus-Phoebanthus divergence. Thick solid line reflects a 500,000-yr moving average fit over the more fine-scale temperature estimates contained by the gray area (adapted from Hansen et al. 2013).

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