A fossil-calibrated relaxed clock for Ephedra indicates an Oligocene age for the divergence of Asian and New World clades and Miocene dispersal into South America



Abstract  Ephedra comprises approximately 50 species, which are roughly equally distributed between the Old and New World deserts, but not in the intervening regions (amphitropical range). Great heterogeneity in the substitution rates of Gnetales (Ephedra, Gnetum, and Welwitschia) has made it difficult to infer the ages of the major divergence events in Ephedra, such as the timing of the Beringian disjunction in the genus and the entry into South America. Here, we use data from as many Gnetales species and genes as available from GenBank and from a recent study to investigate the timing of the major divergence events. Because of the tradeoff between the amount of missing data and taxon/gene sampling, we reduced the initial matrix of 265 accessions and 12 loci to 95 accessions and 10 loci, and further to 42 species (and 7736 aligned nucleotides) to achieve stationary distributions in the Bayesian molecular clock runs. Results from a relaxed clock with an uncorrelated rates model and fossil-based calibration reveal that New World species are monophyletic and diverged from their mostly Asian sister clade some 30 mya, fitting with many other Beringian disjunctions. The split between the single North American and the single South American clade occurred approximately 25 mya, well before the closure of the Panamanian Isthmus. Overall, the biogeographic history of Ephedra appears dominated by long-distance dispersal, but finer-scale studies are needed to test this hypothesis.

Efforts to date the evolutionary divergences of the five extant seed plant lineages (Gingko L., cycads, gymnosperms, Gnetales, and angiosperms) with molecular clocks have been hampered by the still unresolved relationships between them (for a summary, see Mathews et al., 2009, in press). Another problem is the marked difference in the rate of molecular evolution among seed plant lineages. The Gnetales in particular have unusually high or low substitution rates (depending on genus) in all datasets examined so far (Sanderson et al., 2000; Magallón & Sanderson, 2002, 2005; Mathews, 2009). For example, the rbcL substitution rate in Ephedra L. is approximately 10-fold slower than that in its sister clade Gnetum (Renner & Grimm, 2008). Such heterogeneity among lineages, which is not accommodated by molecular substitution models, presents a challenge for molecular clock dating, whether strict or relaxed. In response to this challenge, local and relaxed clock methods have been proposed that permit different parts of a tree to have different rates (Rambaut & Bromham, 1998; Thorne et al., 1998; Yoder & Yang, 2000; Kishino et al., 2001; Rannala & Yang, 2007). Of these relaxed clock approaches, several rely on a Bayesian framework and assume that substitution rates are autocorrelated between branches, meaning that rate changes occur gradually between ancestors and descendants as a clade diversifies. Other Bayesian clock models assume that branch-specific rates are drawn from a single underlying distribution, such as a log normal, gamma, or exponential distribution, the parameters of which are estimated from the data (Drummond et al., 2006; Rannala & Yang, 2007). Studies that have tested the performance of the different approaches have found that relaxed clock models with uncorrelated rates can outperform other approaches (Ho et al., 2005; Drummond et al., 2006; Lepage et al., 2007; but see Ho, 2009).

The Gnetales comprise Ephedra L., Gnetum L., and Welwitschia Hook.f. and are one of the five major groups of extant seed plants. Studies over the past 17 years have been unable to securely resolve the phylogenetic relationships of Gnetales with the four other seed plant lineages (Mathews et al., 2009, in press). Most recently, plastid sequence data have placed the Gnetales as sister to all non-Pinaceae conifers or cupressophytes (“gnecup” clade; Nickrent et al., 2000; Doyle, 2006; Chumley et al., 2008; Braukmann et al., 2009; Rydin & Korall, 2009).

Resolving species-level relationships within Ephedra has been equally problematic. However, in contrast with the many well-supported, yet often mutually incompatible hypotheses on seed plant phylogeny, relationships in Ephedra have been largely unresolved owing to few informative characters in investigated gene regions and substantial plasticity in gross morphological traits (Ickert-Bond & Wojciechowski, 2004; Rydin et al., 2004; Huang et al., 2005). A recent study, with denser species sampling, provides support for several subgeneric clades (Rydin & Korall, 2009), but the deepest divergences in the genus are still ambiguous.

Divergence times from molecular clock analyses for the most recent common ancestors of living Gnetales genera range from 8–32 mya under a strict clock for Ephedra (Huang & Price, 2003) to 10–11 or 14 mya under a strict clock (Won & Renner, 2003, 2006) or 26–38 mya under a relaxed clock for Gnetum (Won & Renner, 2006). Estimates for the Gnetales crown group range from 120–131 mya (relaxed clock; Ickert-Bond & Wojciechowski, 2002) to 189 mya (relaxed clock; Schneider et al., 2004). Recent paleobotanical discoveries have further stirred up discussions about the age of Ephedra (Yang et al., 2005; Rydin et al., 2006; Friis et al., 2009), with some authors suggesting that Cretaceous fossil seeds resemble living species of Ephedra and may date the divergence of crown group Ephedra to ca. 125 mya (Yang et al., 2005; Rydin et al., 2006; Y. Yang, Institute of Botany, Beijing, pers. comm., 2008). However, to date, no ephedroid seed fossil has been unambiguously placed within crown group Ephedra, and these fossils are therefore of limited use as calibration points in molecular dating analyses. Conversely, coalified Ephedra seeds from the Drewry's Bluff locality of the Patuxent Formation in Virginia, USA, and from Buarcos and Torres Vedras localities in Portugal, which date to the late Barremian to early–middle Aptian age, have been assigned to stem group Ephedra based on two preserved features: (i) in situ Ephedra-type pollen, including discarded upcurled exines, which show that the pollen had germinated inside the ovules; and (ii) preserved papillae formed by the inner epidermis of the seed envelope. A combination of these features is unique to Ephedra (Rydin et al., 2006).

Here we apply relaxed molecular clock dating, using an uncorrelated rates model, to an Ephedra dataset that represents all the major clades found by Rydin and Korall (2009) with the goal of inferring the most probable age of the Ephedra crown group as well as the timing of key divergence events in the genus. The radiation of extant Ephedra is interesting because of its disjunct distribution in deserts north and south of the tropics, but not in the intervening regions (a classic amphitropical range; see Wen & Ickert-Bond, 2009). The genus comprises approximately 50 species, which are more-or-less equally distributed between the Old and New World deserts. In light of recent palaeobotanical evidence (above), availability of large molecular datasets for both Ephedra and Gnetum, as well as new approaches to dating that take into account topological uncertainty and rate heterogeneity among lineages, in the present paper we provide new age estimates for Ephedra and discuss their implications for the evolution of the genus.

1 Material and methods

1.1 Taxon sampling

To assemble the most useful phylogenetic dataset of Ephedra we included as many taxa and genes as possible from GenBank. We used PHYLOTA (Sanderson et al., 2008; http://loco.biosci.arizona.edu/pb/) to extract GenBank data. The browser returned 10 phylogenetic informative clusters. The genes comprising these clusters have been variously used for phylogenetic inference of the Gnetales and include the atpB gene (Rydin et al., 2002), the rbcL gene (Rydin et al., 2002, 2004; Rydin & Källersjö, 2002; Huang & Price, 2003; Won & Renner, 2003, 2006; Huang et al., 2005; Wang et al., 2005; Rydin & Korall, 2009), the matK gene (Won & Renner, 2003, 2006; Huang et al., 2005), the rps4 gene (Ickert-Bond & Wojciechowski, 2004; Rydin et al., 2004; Rydin & Korall, 2009), the psbA-trnH intergenic spacer (IGS) (Techen et al., 2006), the trnL gene, and the trnLtrnF IGS (Long et al., 2004), as well as nuclear ribosomal 18S (Rydin et al., 2002, 2004; Wang et al., 2005; Rydin & Korall, 2009), 26S (Rydin et al., 2002, 2004; Rydin & Korall, 2009), and internal transcribed spacer (ITS) 1 and ITS2 (Ickert-Bond & Wojciechowski, 2004; Rydin et al., 2004; Huang et al., 2005; Wang et al., 2005; Won & Renner 2005, 2006; Rydin & Korall, 2009). We excluded the cluster of the chlB gene (Boivin et al., 1996) from further consideration because it only contained four taxa. In addition to the clusters returned by PHYLOTA, we added the plastid rpl16 intron and the trnSUGAtrnfMCAU intron data from Rydin and Korall (2009). Because of the tradeoff between increasing gene and taxon sampling and limiting the amount of missing data, we reduced the initial matrix from 265 to 95 accessions. The reduced matrix included the most complete coverage for the genes used while maintaining sampling of the geographic and taxonomic diversity of Ephedra. This matrix included 53 accessions of Ephedra, 13 accessions of Gnetum, Welwitschia mirabilis, and 28 accessions of other gymnosperm taxa comprising the outgroup. Information regarding vouchers and GenBank accession numbers is given in Table 1.

Table 1. Voucher information and GenBank accession numbers
 VoucherDistribution18S26S atpB rbcL rps4 matK ITS rpL16 trnS to trnfM psbA, psbA-trnH, trnH
E. alata Decne. C303 Anderberg 480 (S)MediterraneanAY755698AY755732 AY755805AY755851 AY755774FJ958074FJ958162 
E. altissima Desf. Bot. Dep. SU C7688 (S)North AfricaAY755697AY755731 AY755804AY755850 AY755773FJ958073FJ958161 
E. americana Humb. & Bonpl. ex Willd. Ickert-Bond 1105 (ASU)South America    AY591464 AY599143   
E. andina Poepp. ex C.A. Mey. Chase 10140 (K)South AmericaAY755670AY755707AY056538AY056573, AY755782*AY755821 AY755744FJ958045FJ958128 
E. antisyphilitica Berl. ex C.A. Mey. Huang20_1 (GA)US, Mexico   AY492031 AY492008AF429442   
E. aphylla Forssk. Anderberg 853 (S)MediterraneanAY755695AY755729 AY755802AY755848  FJ958071FJ958159 
E. aspera Engelm. ex S. Wats. Huang s.n. (GA)North America   AF489532 AY492010AF429443   
E. boelkei F.A. Roig Ickert-Bond 1252 (ASU)South America    AY591473 AY599175   
E. breana Phil. Ickert-Bond 1234 South America    AY591472 AY599177   
E. californica S. Watson Stedge O68_154 (O)North AmericaAY755676*AY755708AY056533AY056569AY755827 AY755750FJ958050FJ958135AY849358
E. chilensis C. Presl. Forbes 49_0542 (UC)North AmericaAY755691AY755725 AY755799AY755844 AY755767FJ958067FJ958155 
E. compacta Rose Puente 1901 (ASU)Mexico    AY591474 AY599157   
E. coryi Reed Ickert-Bond 953 (ASU)North America    AY591461 AY599153   
E. cutleri Peebles Ickert-Bond 1006 (ASU)North America    AY591456 AY599156   
E. distachya L. Rydin 69 (S)Asia–EuropeAY755686AY755719 AY755793AY755838 AY755761FJ958061FJ958149 
E. fasciculata A. Nelson Ickert-Bond 513 (ASU)North America    AY591457 AY599180  AY849360
E. fedtschenkoae Pauls. Ickert-Bond s.n. (ASU)Central Asia    AY591442 AY599158  AY849350
E. foliata Boiss. & C.A. Mey. Thulin 9975 (UPS)MediterraneanFJ957969FJ957988 FJ958030FJ958109 FJ958008FJ958085FJ958173 
E. foliata Boiss. & C.A. Mey. Thulin 10745 (UPS)MediterraneanFJ957971FJ957990 FJ958032FJ958111 FJ958010FJ958087FJ958175 
E. frustillata Miers Chase 10218 (K)South AmericaAY755674AY056490AY056528AY056564AY755825 AY755748FJ958048FJ958131 
E. funerea Coville & Morton Ickert-Bond 473 (ASU)North America    AY591454 AY599168   
E. gerardiana Wall. & Florin Chase 10141 (K)Central AsiaAY755671AY056486AY056524AY056560AY755822 AY755745FJ958046FJ958129 
E. gracilis Phil. Ickert-Bond 1201 (ASU)S. America    AY591465 AY599150   
E. intermedia Schrenk & C.A. Mey. Rydin 66 (S)Central–east AsiaAY755683AY755716 AY755790AY755835 AY755758FJ958058FJ958146 
E. laristanica Assadi Assadi & Sardabi 41781 (KAS)Iran    AY591437 AY599126   
E. laristanica Assadi Davis & Bokhari D56211B (E)IranFJ957980     FJ958020FJ958096FJ958182 
E. likiangensis Florin Forbes 94_0389 (UC)ChinaAY755690AY755724 AY755798AY755843 AY755766FJ958066FJ958154AY849357
E. lomatolepis Schrenk & C.A. Mey. Baitulin (UPS)Central–east AsiaFJ957967FJ957986 FJ958028FJ958108 FJ958006FJ958083FJ958171 
E. major Host Uggla (S)Mediterranean–central AsiaFJ957976FJ957994 FJ958035FJ958117 FJ958016FJ958092FJ958178 
E. milleri Freitag & Maier-StolteE 7667OmanFJ957983FJ958002  FJ958121 FJ958024FJ958100FJ958186 
E. minuta Florin Rydin 63 (S)ChinaAY755681AY755714 AY755788AY755833 AY755756   
E. monosperma JG. Gmel. ex C.A. Mey. Hurka & Neuffer12182 (KAS)Central–east Asia    AY591443 AY599139   
E. monosperma Chase 10142 (K)Central–east AsiaAY755672AY056525,AY056525AY056561AY755823 AY755746   
 JG. Gmel. ex C.A. Mey.   AY056487        
E. multiflora Phil. ex Stapf Ickert-Bond 1211 (ASU)South America    AY591471 AY599173   
E. nevadensis S. Watson Forbes 66_1033 (UC)North AmericaAY755688AY755722 AY755796AY755841 AY755764FJ958064FJ958152 
E. ochreata Miers B380819 (B)South America    AY591463 AY599176   
E. pachyclada Boiss. Danin S-2455 (S)West AsiaAY755703AY755738 AY755810AY755857 AY755779FJ958080FJ958168AY849362
E. pedunculata Engelm. ex S. Watson Ickert-Bond 920 (ASU)Mexico, Texas    AY591460 AY599144   
E. regeliana Florin Wundisch 956 (KAS)Central–east Asia    AY591449 AY599160   
E. rhytidosperma Pachom. Wang 518 ChinaDQ028781  DQ028779 DQ028780DQ028782   
E. rupestris Benth. Forbes 87.1368 (UC)South AmericaAY755689AY755723 AY755797AY755842 AY755765FJ958065FJ958153 
E. sarcocarpa Aitch. & Hemsl. Allen & Esfandri 2703 (S)Central AsiaFJ957977FJ957995  FJ958118FJ958093FJ958017FJ958093FJ958179 
E. saxatilis Florin S. Hedin C-218 Central AsiaFJ957981     FJ958022FJ958098FJ958184AY849364
E. sinica Stapf J. Schonenberger s.n. (S)East AsiaAY755675AY056491AY056529AY056565AY755826 AY755749FJ958049FJ958134 
E. somalensis Freitag & Maier-Stolte Thulin10925A (UPS)Horn of AfricaFJ957966     FJ958004FJ958081FJ958169 
E. strobilacea Bunge Aellen & Esfandri 2703 (S)Central AsiaFJ957978     FJ958018FJ958094FJ958180 
E. strobilacea Bunge Rechinger 27161 (US)Central AsiaAY599162   AY591448 AY599162   
E. torreyana S. Watson 04_487 (S)North AmericaAY755684AY755717 AY755791AY755836 AY755759FJ958059FJ958147 
E. transitoria Riedl Collenette 9095B (?)West Asia FJ957999    FJ957999FJ958097FJ958183 
E. triandra Tul. Ickert-Bond 1227 (ASU)South America    AY591468 AY599165   
E. trifurca Torr. MO04630447 (MO)North AmericaAY755687AY755720 AY755794AY755839 AY755762FJ958062FJ958150 
E. tweediana Fisch. ex C.A. Mey. Forbes 66.0742 (UC)South AmericaAY755692AY755726 AY755800AY755845 AY755768FJ958068FJ958156 
E. viridis Coville Huang37_1 (GA)North America   AY492050 AY492028AF429436   
Gnetum africanum Welw.Tropical AfricaU43012  AY296527      
G. costatum K. Sch. Chase 10219 (K)AsiaAY755661AY056497 AY056576AY755812  FJ958102FJ958132 
G. cuspidatum BlumeAsia   AY296530AY591430     
G. gnemon L. Swenson et al. s.n. (S)AsiaAY755660AF036488 L12680AY755811  FJ958101FJ958122 
G. gnemonoides Brongn.Asia   AY296539AY591429     
G. indicum Merr. E00130257 (E)AsiaAY755663AY056495 AY056574AY755814  FJ958104FJ958139 
G. leyboldii Tul.South AmericaL24045  U72820AY591432     
G. montanum Markgr. E00130261 (E)Asia–AustraliaAY755664AY056496 AY056575AY755815  FJ958105FJ958140 
G. nodiflorum Brongn.AsiaU42415  AY296564      
G. parvifolium (Warb.) W.C. Cheng Rydin s.n. (S)AsiaAY755662AY755704 AY056577AY755813  FJ958103FJ958133 
G. schwackeanum Taub. ex SchenckAsia   AY296567      
G. ula Brongn.Asia   AY296568AF313610     
G. urens BlumeSouth AmericaU42417  AY296569      
Welwitschia mirabilis Hook. f Stedje 67–1177 (O)NamibiaAF207059AY056484 AJ235814AY188246  FJ958106FJ958137 
Araucaria Juss. AF051792U90690 U96467AY188260     
Calocedrus Kurz D85293U90707 L12569AY188281     
Cephalotaxus Siebold & Zucc. ex Endl. D38241U90697 AF227461AY188264     
Chamaecyparis Spach  AY056506 L12570AY188283     
Cupressus L. AF051797  L12571AY188282     
Juniperus L. D38243AY056504 L12573AY188279     
Metasequoia Miki L00970AY056512 AJ235805AY188268     
Phyllocladus Rich. ex Mirb. D38244  AB027315AY188258     
Podocarpus Labill. AF051796U90685 AF307931AY188252     
Sciadopitys Siebold & Zucc. D85292U90698 L25753AY188262     
Sequoia Endl. AY686598U90701 L25755AY188266     
Sequoiadendron J. Buchholz    AY056580AY188267     
Taxodium Rich. EF053176U90702 AF127427AY188270     
Taxus L. D16445AY056513 AJ235811X84145     
Thuja L.  AY056503 L12578AY188276     
Thujopsis Siebold & Zucc.  AY056505 L12577AY188277     
Bowenia Hook.  AY056480 L12671      
Ceratozamia Brongn.  AY056482 AY056558      
Cycas L. D85297U90674 L12674EU016841     
Cycas revoluta Thunb. AB029356U90673 AY056556AF313609     
Dioon sp. Lindl.  AY056483 AF531203      
Encephalartos Lehm.  AY056479 L12676      
Zamia furfuracea L. f. AB029357U90677 AF202959      
Zamia pumila L. M20017AY056481 AY056557AY188209     
Ginkgo biloba L. D16448U90672 AJ235804AF313611     
Abies Mill.  AY056508 AB029646AY188224     
Cedrus Duham. AB026936AY056507 X63662AY188222     
Larix Mill. D85294AY056502 X63663      
Picea asperata Mast. L07059AY056509 AY056578AY188226     
Picea breweriana S. Watson  AY056510 AY056579      
Pinus cembra L.  U90681 AB019795      
Pinus L. AF051798U90680 AB019819AF313612     
Pinus mugo Turra  AY056500 AB063372      
Pinus peuce Griseb.  AY056499 AB019803      
Pinus strobus L.  AY056501 AB019798      
Pinus wallichiana A.B. Jacks. X75080  X58131AY188212     
Pseudotsuga Carrière AB026941AY056498 X52937AY188223     
Tsuga Carrière AB026942AY056511 AY056581AY188220     

1.2 Sequence and phylogenetic analyses

Maximum likelihood (ML) searches were performed in RAxML 7.2.1 (Stamatakis, 2006), using the GTR+Γ model. Model parameters were estimated over the duration of runs and searches started from random parsimony trees. Statistical support was measured by ML bootstrapping in RAxML, with 100 replicates.

1.3 Estimation of divergence time

We used a Bayesian relaxed clock as implemented in BEAST 1.4.8 (Drummond et al., 2006; Drummond & Rambaut, 2007). To reduce topological uncertainty in parts of the tree, which prevented the Markov chain Monte Carlo (MCMC) chains from reaching a stationary state, we gradually reduced the 95-taxon matrix to 67, 54, 46, and 42 taxa (with varying taxon combinations), and we also took out partitions with more than 30% empty cells, which left a matrix of 7736 aligned nucleotides. After tuning the operators using the auto-optimization option in BEAST, analyses used a speciation model that followed a Yule tree prior, with rate variation across branches uncorrelated and lognormally distributed. The MCMC chains were run for between 40 and 60 million generations (burn-in 10%), with parameters sampled every 1000th step. Results from individual runs were combined as recommended, and effective sample sizes for all relevant estimated parameters and node ages were above 100. Because the oldest described ephedroid fossils place somewhere along the stem lineage of Ephedra (see above), we used a single constraint, namely a lognormal prior probability that the split between Gnetum and Welwitschia is at least 110 mya old (with a 95% confidence interval of 10 mya), based on the welwitschioid fossil seedling Cratonia cotyledon (Rydin et al., 2003) from the Early Cretaceous of Brazil. This fossil is slightly younger than the oldest Ephedra seeds (125 mya) and clearly belongs to crown group Gnetales, based on the presence of an embryo feeder and a unique venation pattern, shared by the fossil and Welwitschia.

2 Results

The ML tree obtained from the 10 locus–95 taxon dataset shows relationships within Ephedra (Fig. 1) that are similar to those recovered by Rydin and Korall (2009), although species sampling in the present study is smaller. Ephedra foeminea is sister to the rest of Ephedra. The next diverging clade is one of strictly Mediterranean taxa (E. altissima, E. aphylla, E. milleri, E. alata and E. fragilis), sister to the rest of Ephedra (“core Ephedra” sensu Rydin & Korall, 2009). Core Ephedra comprises several subclades of Mediterranean and Asian distribution (e.g. E. laristanica and E. foliata), but there is no statistical support for their precise composition and relationships. However, the New World clade of Ephedra is strongly supported (bootstrap (BS) 94%) and consists of North American and South American clades. The large substitution rate heterogeneity among Gnetales is evident from the phylogram (Fig. 1), particularly the long branches leading to the three genera compared with the significantly reduced branch lengths within Ephedra.

Figure 1.

Maximum likelihood (ML) phylogeny, inferred from 10 combined chloroplast and nuclear ribosomal DNA (nrDNA) loci for 95 taxa of gymnosperms; branch lengths computed using RAxML. The ML bootstrap support values above 80% are indicated above the branches. The biogeographic distribution of Ephedra taxa is shown on the right.

Relationships among the fewer species included in the molecular clock runs (Fig. 2) differ in part from those obtained from the ML analysis (Fig. 1) and have slightly better statistical support because the matrix includes many fewer missing nucleotides. Table 2 lists divergence times obtained for key nodes within Ephedra (labeled 3–9 in Fig. 2). Although deep divergences have originated in the Oligocene, most of the tip clades have diverged more recently in the Late Miocene or Pliocene (Fig. 2).

Figure 2.

Chronogram based on 42 accessions of Ephedra, Gnetum, and Welwitschia from combined chloroplast and nuclear ribosomal DNA (nrDNA) loci obtained under a model of uncorrelated rate change using one fossil-based constraint (see text for details). Node heights are median ages, with gray bars indicating the 95% highest posterior density intervals (see Table 2 for details). The maximum likelihood bootstrap support values are indicated below the branches. The distribution of Ephedra taxa is indicated on the right with node labels (black circles) pointing to particular nodes of biogeographic interest (see Table 2 and text for details). Pal., Paleocene; Oligoc., Oligocene; Mioc., Miocene; Pl., Pleistocene.

Table 2. Time estimates (in million years) and confidence intervals for significant nodes for the crown group Ephedra obtained from combined analysis of the 7736-nucleotide matrix under an uncorrelated rates molecular clock (see Material and Methods)
Node no.Clade nameFossil evidenceAge (million years)95% Highest posterior density intervals
  1. *Used as a constraint.

  2. n.a., not available.

1Split: Welwitschia vs. Gnetum Cratonia cotyledon 110*111.3587.21, 127.01
2Divergence of Ephedra from Gnetum and Welwitschia Ephedra archaerhytidosperma 125166.6190.62, 192.34
3Divergence Mediterranean clade from core Ephedra  30.3920.55, 73.5
4Divergence of NW clade from the rest of core Ephedra  29.568.84, 41.53
5Divergence of China clade from rest of mixed Asia clade  27.6314.45, 49.36
6Divergence of Middle Eastern/Horn of Africa from African Mediterranean members  25.815.37, 55.53
7Divergence of North American clade from South American clade  24.788.84, 41.53
8Divergence of Asia 2 clade from combined Asia 1/Horn of Africa and Asia clade  20.6114.35, 49.36
9Divergence of Asia 1 clade from Horn of Africa/Asia clade  15.516.18, 32.5

3 Discussion

The results of the present study provide strong evidence for a recent radiation of extant Ephedra. Given the few clear morphological differences among species, it has been suggested that the lack of molecular divergence in Ephedra plastid genomes may be the result of hybridization and polyploidization, which appears to be rampant in the genus (Cutler, 1939; Ehrendorfer, 1976; Choudry, 1984; Wendt, 1993). Plants with montane distribution also frequently exhibit rapid diversification, likely because of small-scale habitat heterogeneity (Bell & Donoghue, 2005; Hughes & Eastwood, 2006).

Studies with a comprehensive species sampling of Ephedra (the Bayesian analysis in Rydin & Korall, 2009; the ML analysis in the present study) indicate a basal grade of Mediterranean species and thus a possible origin of the crown group of Ephedra in the Mediterranean region (northern Africa, southern Europe, the Near East). However, these basal divergences still have little statistical support owing to the limited signal in the loci so far included (Ickert-Bond & Wojciechowski, 2004; Ickert-Bond et al., 2009; Rydin & Korall, 2009; present study). Parsimony analysis (Rydin & Korall, 2009), as well as Bayesian analysis of the reduced dataset in the present study (Fig. 2), results in a Mediterranean clade sister to the remaining Ephedra. The respective divergence may have taken place some 30 mya (Fig. 2, node 3; Table 2). Within the Mediterranean clade, the Near Eastern E. foliata (Arabia and Somalia) and E. laristanica (Iran) split from the western Mediterranean species ca. 26 mya. Turning to the (mostly) Asian clade of Ephedra, the prevailing pattern appears to be westward dispersal (Fig. 3), with an estimated divergence of a strictly Chinese clade from the rest of the Asian/African clade at 28 mya (Fig. 2, node 5; Table 2). Dispersal into the Horn of Africa from the Asia 1 clade (Fig. 2, node 9; Table 2) may date back to 16 mya.

Figure 3.

Distribution of Ephedra (green shading) and hypothesized intercontinental (solid arrows) and intracontinental dispersal routes (dashed arrows).

New World species are monophyletic and estimated to have diverged from their mostly Asian sister clade some 30 mya. This timeframe is corroborated by many other Beringian plant disjunctions (for a review, see Wen & Ickert-Bond, 2009). In turn, the New World species split into a North American and a South American clade (Fig. 2), which appear to have diverged approximately 25 mya; that is, well before the closure of the Panamanian Isthmus (Fig. 2, node 7; Table 2). These results mirror other studies indicating that significant dispersal took place between Mesoamerica and South America before the closure of the Isthmus of Panama during the Oligocene or Miocene (e.g. mammals: Marshall & Sempere, 1993; Melastomeae: Renner & Meyer, 2001; Ruprechtia (Polygonaceae) and Nissolia (Leguminosae): Pennington et al., 2004; and Platymiscium (Leguminosae): Saslis-Lagoudakis et al., 2008).

Dispersal in Ephedra may have been facilitated by the ovulate bracts, which, in some species of Ephedra, are bright red and fleshy and indicative of endozoochory (Stapf, 1889; Freitag & Maier-Stolte, 1994; Danin, 1996; Hódar et al., 1996). Bird dispersal has also been observed directly (Ridley, 1930; Hollander et al., 2009). In contrast, dry, wing-bracted strobili are adapted for anemochory (Stapf, 1889; Danin, 1996). The seeds of North American E. aspera, E. californica, E. funerea, E. nevadensis, and E. viridis are not fleshy and their ovulate bracts are not winged. Seeds of these “intermediate bracted” taxa often accumulate at the stem base, and seed-caching rodents have been observed as dispersers (Ickert-Bond, 2003; Ickert-Bond & Wojciechowski, 2004; Hollander & Vander Wall, 2009; Hollander et al., 2009). Wind-dispersed Ephedra typically inhabit marginal habitats, such as hyperarid deserts or dry salt lakes devoid of animal life (Danin, 1996), and, in general, dispersal biology in Ephedra appears to relate to habitat, rather than being phylogenetically conservative (Hollander et al., 2009).

Recent studies have found that reliable topologies may be obtained even in the face of large amounts of missing data (e.g. Wiens, 2003, 2006; McMahon & Sanderson, 2006; Smith et al., 2009). However, for molecular clock dating, missing data present a so-far insurmountable challenge. This is because estimation of divergence time depends on accurate estimates of branch lengths, which can only be obtained with large numbers of nucleotides (Sanderson, 1998). When the BEAST dating runs failed to reach stable distributions, we first reduced the number of empty cells by deleting data partitions that lacked sequences for more than 30% of the included species; next, we deleted species that lacked sequences for more than five loci. Even so, a combined run length of 108 million generations was needed for each parameter to converge on a stationary distribution.

A caveat with all molecular clock dating is that the absolute ages obtained depend on the calibration used. An earlier study that concentrated on Gnetum and only included three species of Ephedra, using a Bayesian relaxed clock and an auto-correlated model, explored the effects of three different constraints (Won & Renner, 2006). In one experiment, these authors used 125-mya-old Ephedra seeds to constrain the crown group age of Ephedra. This had the effect of roughly doubling within-Gnetum estimates compared with the ages obtained when these seeds were assigned to the Ephedra stem (Won & Renner, 2006, table 1). In the present study, we initially included representatives of all major lineages of gymnosperms so that we could constrain the Ephedra stem to a minimum of 125 mya. However, this introduced the problem of the uncertain placement of Gnetales within seed plants, a problem that Won & Renner (2006) circumvented by conducting dating runs under four different seed plant topologies.

With just the Gnetales included, as in Fig. 2, one cannot infer a Bayesian probability distribution around the split between Ephedra and Gnetum/Welwitschia. Instead, we decided to rely exclusively on the Cratonia cotyledon fossil from the Early Cretaceous of Brazil, the assignment of which is unambiguous because it clearly represents the Welwitschia stem group (Rydin et al., 2003). This calibration yielded an age of 167 mya (91–192 mya confidence interval) for the divergence between Ephedra and the other two genera. This age range is too large to be very useful, but fits the placement of Gnetales within conifers, perhaps as sister to the non-Pinaceae conifers (“gnecup” clade; Nickrent et al., 2000; Doyle, 2006; Chumley et al., 2008; Braukmann et al., 2009; Rydin & Korall, 2009), and also with the Ephedra pollen and seed fossil record.

The present biogeographic analysis (Figs. 2, 3) corroborates other molecular studies that have found New World clades of Oligocene age evolving out of Asian paraphyletic residuals, which is the classic pattern of Beringian disjunctions (for summaries, see Wen & Ickert-Bond, 2009). Our work also adds to a growing body of studies reporting long-distance dispersal between arid floras in North and South America (Moore et al., 2006). Finer-scale studies are now needed to test the broad-brush biogeographic scenario for Ephedra developed here.


Acknowledgements  This study was supported, in part, by the National Science Foundation (USA)-Emerging Frontiers, Assembling the Tree of Life, Collaborative Research: Gymnosperms on the Tree of Life: Resolving the Phylogeny of Seed Plants (Grant No. EF-0629657 to SMI-B) and by the Swedish Research Council (grants to CR). The authors thank Sinian CHEN and Jordan METZGAR for help assembling the matrices. Highschool student intern, Sinian CHEN, West Valley Highschool, Fairbanks, Alaska, USA and Graduate student, Jordan METZGAR, UA Museum of the North Herbarium, Fairbanks, Alaska, USA.