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Journal of Systematics JSE and Evolution doi: 10.1111/jse.12190 Review

The Gnetales: Recent insights on their morphology, reproductive , chromosome numbers, biogeography, and divergence times

Stefanie M. Ickert-Bond1* and Susanne S. Renner2

1University of Alaska Museum of the North and Department of Biology and Wildlife, University of Alaska Fairbanks, 907 Yukon Dr., PO Box 756960, Fairbanks, Alaska 99775-6960, USA 2Institute of Systematic and Mycology, University of Munich (LMU), Menzinger Str. 67, 80638 Munich, Germany *Author for correspondence. E-mail: [email protected]. Tel./Fax: 1-907-474-6277/1-907-474-5469. Received 23 November 2015; Accepted 15 December 2015; Article first published online 12 January 2016

Abstract , , and constitute the Gnetales of still unclear phylogenetic relationships within . Here we review progress over the past 10 years in our understanding of their species diversity, morphology, reproductive biology, chromosome numbers, and genome sizes, highlighting the unevenness in the sampling of species even for traits that can be studied in preserved material, such as morphology. We include distribution maps and original illustrations of key features, and specify which species groups or geographic areas are undersampled. Key words: biogeography, chromosome numbers, fertilization, morphology, phylogenetics, pollen, , polyploidy.

Mais les Gnetophytes se presentent au botaniste, depuis In terms of their morphology and even basic ecology, longtemps, comme un ensemble d’un inter et^ exceptionnel et the Gnetales remain enigmatic, with surprising discoveries comme un enigme particulierement irritante. continuing to be made (e.g., Wetschnig, 1997; Mundry Pierre Martens, 1971 &Stutzel,€ 2004; Friedman, 2015; Ickert-Bond et al., 2015; Les Gnetophytes Rydin & Bolinder, 2015). Here we review progress over the past 10 years in our understanding of the species The Gnetales are a of three genera that is morpho- diversity, morphology, reproductive biology, chromosome logically and genetically so disparate from the remaining numbers, and genome sizes of the Gnetales, highlighting seed (, , angiosperms, and Coniferales) the unevenness in the sampling of species even for traits that its precise placement has remained unclear (Mathews, that can be studied in preserved material, such as pollen 2009; reviewed in Mathews et al., 2010; Fig. 1). The order morphology. Gnetales Luersson (or the subclass Pax) is characterized by compound cones with unisexual repro- ductive units borne in the axils of , with the The Gnetales: Three Disparate Mono- surrounded by 1-2 envelopes and the integument extending into a micropylar tube carrying the pollination generic Families droplets (Kubitzki, 1990). This combination of traits is Gnetum L. (Markgraf, 1930) and Ephedra L. (Cutler, 1939 for extremely rare in fossil forms (Krassilov, 2009). Tran- only) were monographed in the last century; scriptome data for 92 streptophytes, analyzed along with Welwitschia contains but a single species, endemic to the 11 complete plant genomes, support a position of Gnetales Desert (Leuenberger, 2001; Figs. 2, 3). Ephedra is sister either as sister to Coniferales, represented by seven to the other two genera and comprises about 54 species genera, or as sister to one of their families, the , distributed evenly between the deserts of the Old and New represented by Cedrus and Pinus (Wickett et al., 2014). A World (Stapf, 1889; Ickert-Bond, 2003; Figs. 2, 3; Table 1). placement of the Gnetales near to or inside Coniferales Gnetum has ten species in , two to four in would be consistent with previously published analyses tropical West (Biye et al., 2014; Figs. 2, 3; Table 1), and ca. of concatenated gene alignments that aimed to reduce 25 in tropical (Markgraf, 1930; Price, 1996; Won & Renner, long-branch attraction artifacts by implementing various 2006; Hou et al., 2015). Multi-locus analyses of nuclear and among-site rate heterogeneity models (e.g., Bowe et al., DNA sequences have shed light on species relation- 2000; Chaw et al., 2000; Burleigh & Mathews 2007a, 2007b; ships within Ephedra and Gnetum (Ickert-Bond & Wojciechow- Lee et al., 2011; Wu et al., 2011; Zhong et al., 2011; Ruhfel ski, 2004; Won & Renner, 2005a, 2005b, 2006; Ickert-Bond et al., 2014). et al., 2009; Rydin & Korall, 2009; Rydin et al., 2010; Loera et al.,

January 2016 | Volume 54 | Issue 1 | 1–16 © 2015 Institute of Botany, Chinese Academy of Sciences 2 Ickert-Bond & Renner

The species of Ephedra occur in Old World and New World deserts, semideserts, desert steppes or in seasonally dry habitats, such as mediterranean-type or deciduous woodlands and subtropical thorn scrub (Fig. 2; Ickert-Bond, 2003; Freitag, 2010: Fig. G2-01A). The ranges from depressions below sea level (Death Valley of and Dead Sea area) to about 5000 m in the of (E. rupestris, Ickert-Bond, 2005) and to 5300 m in the (E. gerardiana, Fu et al., 1999). The desert species tend to be clonal, forming phytogenic mounds by accumulat- ing sand, particularly in dune habitats. Branching in Ephedra is often broom-like with nearly parallel and fastigiate to ascending (virgate) green stems (Fig. 3A). anatomical features of Ephedra include the presence of vessels that increase conducting efficiency as compared to -only systems in non-gnetalean (reviewed in Carlquist, 2012). The abundance of vessels and their diameter are greatest in the lianoid and scrambling species, while the alpine species have virtually no vessels (Carlquist, 1988; Motomura et al., 2007; Carlquist, 2012). Narrow vessels are characteristic for plants of very dry or desert habitats and probably provide conductive insurance by reducing embolisms (Carlquist, 2012). Nucleated fiber-trache- ids with abundant storage often form tangential bands in Ephedra and also appear an adaptation to extremely arid habitats. The female cones (ovulate strobili) of Ephedra consist of bracts in decussate or ternate (as a mode of verticillate) phyllotaxy, with the distal pair/ enclosing one to three , each surrounded by a seed envelope (Figs. 3B, 3C, 4E). An anatomical and histological study of pollination-stage female cones of 45 species inferred that a seed envelope with three vascular bundles is the ancestral state and that two Fig. 1. Extreme rate heterogeneity within Gnetales and bundles evolved several times (Rydin et al., 2010). Fleshy gymnosperms based on matK and rbcL gene sequences. bracts characterize the fruiting cones of 38 species (Fig. 3B), A, Unrooted maximum likelihood obtained from 558 membranous (Fig. 4E) and winged bracts those of six or seven matK gene sequences, downloaded from GenBank in mid- other species (see section on ). The seed 2008. Values at nodes indicate statistical support from 100 envelopes are smooth (Fig. 4H) or papillate or bear transverse bootstrap replicates under the GTR þG model of substitution. ridges (Ickert-Bond & Rydin, 2011; Figs. 4F, 4G). B, Unrooted maximum likelihood tree obtained from 792 rbcL The male cones (staminate strobili, Fig. 4I) consist of two gene sequences, downloaded from GenBank in mid- 2008. lateral strobili with 2–3 sterile bracts at the base, followed Values at nodes indicate statistical support from 100 by 2–8 (10) fertile bracts, within each of which two median bootstrap replicates under the GTR þG model of substitution. bracts enclose the stalked antherophore (Cutler, 1939; Hufford, 1996; Ickert-Bond, 2003; Mundry & Stutzel,€ 2012, 2015; Hou et al., 2015); a few deep nodes within Ephedra 2004). Each antherophore consists of two fused micro- and Gnetum still remain statistically poorly supported. and bears 2–8 stalked or sessile synangia, which result from the fusion of two (rarely three) Ephedra (Ephedraceae) (Hufford, 1996; Ickert-Bond, 2003; Mundry & Stutzel€ All Ephedra are perennial and dioecious, and most species are 2004). Mundry and Stutzel€ (2004) interpret the male (Price, 1996; Ickert-Bond, 2003; Fig. 3A); a few are cones of Ephedra as consisting of two units with four climbers up to 4 m (e.g., Ickert-Bond, 2003: Fig. 3.1 E–F; simple sporophylls, and propose homologies with parts in Freitag, 2010: Fig. G2-02) or small up to 2 m (E. equisetina the female cones of Welwitschia and Gnetum (see respective in Freitag, 2010: Fig. G2-01A). The nodes bear narrow, sections below). lanceolate arranged in decussate or whorled phyllo- The pollen of 45 species of Ephedra has been studied with taxis (Figs. 4A–4D). The leaves are 2–15 (40) mm long when light and scanning electron microscopy (Steeves & Barghoorn, fully expanded, but become non-functional (except for 1959; Zhang & Xi, 1983; Ickert-Bond, 2003; Ickert-Bond et al., E. foliata and E. altissima) when the vegetative ceases 2003; Doores et al., 2007; Bolinder et al., 2015a, 2015b; our vegetative elongation (Ickert-Bond, 2003; Dorken,€ 2014). The Table 1). Pollen is ellipsoidal, with characteristic ridges, apical portion of each blade is free while the basal portions and rather large (27–58 mm in average equatorial diameter; are fused into a sheath, with the extent of fusion a species- Figs. 4J, 4K). Based on a phylogenetic analysis of pollen traits, characteristic trait (Figs. 4A–4D). grains with unbranched valleys in the exine (pseudosulci of

J. Syst. Evol. 54 (1): 1–16, 2016 www.jse.ac.cn Biology and phylogeny of the Gnetales 3

Fig. 2. The distribution of the Gneales in the context of the World’s climates. A, Distribution plotted on WWW world ecoregions map (black line and at black arrowheads, Ephedra; yellow line, Gnetum; light blue line and at blue arrowhead, Welwitschia). B, Biomes in relation to mean annual temperature and mean annual precipitation based on worldclim climate layers (modified from Donoghue & Edwards, 2014). The distribution map of Old World Ephedra was kindly provided by H. Freitag, University of Kassel.

Bolinder et al., 2015b; Fig. 4J) represent the ancestral form, while grains with branched valleys are derived (Fig. 4K). Pollen ultrastructure in Ephedra appears to relate to pollination biology (Bolinder et al., 2015a; Bolinder et al., 2016). Using both wind- and insect- pollinated species of Ephedra, Bolinder and colleagues experimentally confirmed that the three- parted ectexine, composed of an undulating tectum, a granular infratectum and a narrow foot layer, influences grains’ settling velocity. Grains of the insect-pollinated E. foeminea have a thick tectum and a high density of granules in the infratectum, and they settle much faster than those of two wind-pollinated species (E. nevadensis and E. trifurca), which have a thin tectum and a spacious infratectum with a low density of granules and for which settling velocity measurements were available from earlier work (Niklas & Kerchner, 1986; Niklas et al., 1986; Niklas & Buchman, 1987). Four other species with pollen similar to E. nevadensis and E. trifurca also appear to be wind pollinated (Bolinder et al., 2015a). A revision of the New World species is currently in preparation by the first author, and the Asian species present the greatest taxonomic challenge. New species of Ephedra continue to be described, mostly from and , although they appear morphologically close to E. intermedia and E. saxatilis (Yang et al., 2003; Yang, 2005; Sharma & Uniyal, Fig. 3. Gross morphology of Gnetales. A, B Ephedra aphylla, 2009; Sharma et al., 2010; Sharma & Singh, 2015: Ephedra female plant with red fleshy seed envelope surrounding two rituensis Y. Yang, D.Z. Fu, G.H. Zhu, Ephedra dawuensis Y. Yang; seeds on ovulate cone, Wadi Musa, Egypt (Photo M. Hassan). Ephedra sumlingensis P. Sharma & P.L. Uniyal, Ephedra C, Ephedra minuta, female cone with pollination drop borne on kardangensis P. Sharma & P.L. Uniyal, Ephedra khurikensis P. micropylartube(Photo S. Little). D, ,femalewith Sharma & P.L. Uniyal, and Ephedra pangiensis Rita Singh & P. pollination drops, in greenhouse at Munich Botanical Garden. Sharma). The four Indian new species all come from the E, Gnetum cuspidatum, cluster of seeds formed on large trunk, Western Himalayas and, based on their descriptions, may Sulawesi (Photo J. Wen). F, Welwitschia mirabilis in , represent forms of E. intermedia. Their diagnoses rely heavily male, 1200 yearold specimen (Photo H. Freitag). G, Welwitschia on straight vs. coiled micropyles, a character of limited mirabilis,femalestrobili(PhotoS.Little).Scalebars:A¼ 50 cm; significance (Freitag & Maier-Stolte, 1993, 1994; Kakiuchi et al., B ¼ 5mm;C¼ 5mm;D,E¼ 5cm;F¼ 20 cm; G ¼ 15 cm. 2011). www.jse.ac.cn J. Syst. Evol. 54 (1): 1–16, 2016 4 Ickert-Bond & Renner

Table 1 List of currently recognized Ephedra species and whose pollen morphology is known for LM, SEM, and TEM as well as coding of ovulate consistency † § Species (Distribution ) Bolinder et al., 2015 TEM studies Bract ‡ consistency 1. E. alata Decne. (SAH, AR) LM, SEM fl, pa 2. E. altissima Desf. (MED) LM, SEM fl 3. E. americana Humb. & Bonpl. ex Willd. (SAm) LM, SEM El-Ghazaly et al., 1997 fl 4. E. antisyphilitica Berlandier ex. C.A. Mey. (NAm) LM, SEM fl 5. E. aphylla Forssk. (MED, AR) LM, SEM fl 6. E. aspera Engelm. (NAm) LM, SEM me 7. E. boelckei F.A. Roig (SAm) LM, SEM pa 8. E. breana Phil. (SAm) LM, SEM fl 9. E. californica S. Watson (NAm) LM, SEM me/pa 10. E. chilensis C. Presl. (SAm) LM, SEM fl 11. E. compacta Rose (NAm) LM, SEM fl 12. E. coryi E.L. Reed (NAm) LM, SEM fl 13. E. cutleri Peebles (NAm) LM, SEM me 14. E. distachya L. (EU, AS) LM, SEM Van Campo & Lugardon, 1973; fl Kurmann, 1992; El-Ghazaly et al., 1998 15. E. equisetina Bunge (AS) LM, SEM fl 16. E. fasciculata A. Nelson (NAm) LM, SEM me 17. E. fedtschenkoi Paulsen (AS) fl 18. E. foeminea Forssk. (MED, AR) LM, SEM fl 19. E. foliata Boiss. ex C.A. Mey. (MED, AR, AS) LM, SEM El-Ghazaly & Rowley, 1997 fl 20. E. fragilis Desf. (MED) El-Ghazaly et al., 1997 El-Ghazaly & Rowley, 1997; fl El-Ghazaly et al., 1998 21. E. frustillata Miers (SAm) LM, SEM fl 22. E. funerea Coville & C.V. Morton (NAm) LM, SEM pa 23. E. gerardiana Wall. ex Stapf (AS) LM, SEM fl 24. E. glauca Regel (AS) fl 25. E. intermedia Schrenk & C.A. Mey. (AS) LM, SEM fl 26. E. laristanica Assadi (Iran) fl 27. E. likiangensis Florin (AS) LM, SEM fl 28. E. lomatolepis Schrenk (AS) LM, SEM fl 29. E. major Host (EU, AS) fl 30. E. milleri Freitag & M. Maier-Stolte (AR) LM, SEM fl 31. E. minuta Florin (AS) LM, SEM fl 32. E. monosperma J.G. Gmel. ex C.A. Mey. (AS) LM, SEM Afzelius 1956, Erdtman 1957; fl Gullvåg, 1966 33. E. multiflora Phil. ex. Stapf (SAm) LM, SEM pa 34. E. nevadensis S. Watson (NAm) LM, SEM me 35. E. ochreata Miers (SAm) LM, SEM fl 36. E. pachyclada Boiss. (AR, AS) LM, SEM fl 37. E. pedunculata Engelm. ex S. Watson (NAm) LM, SEM fl 38. E. przewalskii Stapf (AS) pa 39. E. pseudodistachya Pachom. (AS) fl 40. E. regeliana Florin (AS) LM, SEM fl 41. E. rhytidosperma Pachom. (AS) fl 42. E. rupestris Benth. Ickert-Bond, 2003 El-Ghazaly et al., 1998 fl 43. E. sacrocarpa Aitch. & Hemsl. (AR, AS) LM, SEM fl 44. E. saxatilis (Stapf) Royle ex Florin (AS) LM, SEM fl 45. E. sinica Stapf (AS) ¼ synonym of E. dahurica LM, SEM Ueno, 1960 fl 46. E. somalensis Freitag & Maier-St. (AR) fl 47. E. strobilacea Bunge (AS) LM, SEM fl 48. E. torreyana S. Watson (NAm) LM, SEM pa 49. E. transitoria Riedl (AS) LM, SEM fl 50. E. triandra Tul. (SAm) LM, SEM fl 51. E. trifurca Torr. ex S. Watson (NAm) LM, SEM pa Continued

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Table 1 Continued † § Species (Distribution ) Bolinder et al., 2015 TEM studies Bract ‡ consistency 52. E. trifurcata Zollner€ (SAm) LM, SEM fl 53. E. tweediana Fisch. & C.A. Mey. (NAm) LM, SEM fl 54. E. viridis Coville (NAm) LM, SEM me † Distribution is presented in parentheses: AR, Arabian Penninsula; AS, Asia; EU, ; MED, Mediterranean; NAm, North ‡ § America; SAH, Sahara; Sam, South America. Bract consistency: fl, fleshy; me, membranous; pa, papery, winged. Taxa marked with an asterisk have been illustrated with the respective method.

Molecular phylogenies of Ephedra by now have included 54 of the 56 species (Ickert-Bond & Wojciechowski, 2004; Rydin et al., 2004; Huang et al., 2005; Rydin & Korall, 2009; Rydin et al., 2010; Loera et al., 2015). In combination, they show that the six Mediterranean species form a grade at the base of the phylogeny, while the remaining 50 species from a well- supported clade. All 22 New World species form a clade that is nested within a paraphyletic Old World grade. Within the New World clade, the North American E. pedunculata is the earliest diverging species and sister to two , one with ten South American species and one with eleven North American ones (Rydin & Korall, 2009; Rydin et al., 2010; Loera et al., 2015). In the latter, the Mexican E. compacta is the earliest diverging species, albeit with low statistical support (Rydin & Korall, 2009; Loera et al., 2015). Phylogeographically, 107 populations of Ephedra were studied from the Qinghai-Tibetan Plateau (QTP) and the diversification was proposed to be linked to the rise of the QTP (Qin et al., 2013). A study that included 32 species of Ephedra and outgroups, calibrated with the welwitschioid fossil Cratonia (Rydin et al., 2003) from the Early of the Brazilian , which represents the Gnetales crown group, inferred a crown age of Ephedra of 30.39 (20.55–73.5) Ma and an Oligocene age for the divergence of Asian and New World clades (Ickert-Bond et al., 2009). An earlier study (Huang & Fig. 4. Morphology of Ephedra. A–D, diversity.E–H, Female Price, 2003) that used a strict molecular clock model and cone and seed morphology. I–K, Staminate cone and pollen rbcL sequences had placed the crown age at 8–32 Ma. diversity. A, Ephedra americana, with well developed free leaf tips and swollen dark, decussate leaf bases. B, , Gnetum (Gnetaceae) showing marked difference in development of the from Most of the c. 40 species of Gnetum are large woody climbers extremely large (left) to moderately developed (right), both (Fig. 3E); only G. gnemon (Fig. 3D) and G. costatum are free leavescomefromthe same plant.C, Ephedratorreyana,showing standing, and most species occur in mesic habitats (Fig. 2; unique whorls of four leaves at node, indicating possible Markgraf, 1930, 1951, 1972, 1977; Price, 1996). The decussate integration with E. aspera. D, , mature leaf, broad leaves of Gnetum with pinnate-reticulate venation showingtypicalsplittingofthelamina.E, , resemble the simple leaves of many dicots (Fig. 3D). pedunculate cone with two exserted seeds. F, Ephedra The vessels of Gnetum wood are much wider than those of torreyana, scanning electron micrograph of lance-ovoid seed Ephedra for both the lianoid and the tree species, but with elongated beak and transverse ridges on the seed surface. significantly smaller than those of lianoid angiosperms, G, , scanning electron micrograph showing probably because Gnetum species grow in understory, semi- details of transverse ridges. H, Ephedra aspera, scanning shaded habitats (Feild & Balun, 2008; Carlquist, 2012). Simple electron micrograph of ovoid seed. I, , perforation plates and torus-margo membranes in staminate cone showing numerous microspongiophores per tracheid-to-tracheid pits as well as in vessel-to-tracheid pits strobilus. J, , scanning electron micrograph of may provide insurance against air bubble formation and ancestral grain type with straight ridges and furrows. K, Ephedra embolisms (Carlquist, 2012), although given the mesic tropical coryi, scanning electron micrograph of derived pollen grain type habitats of Gnetum (Fig. 2) this functional role requires further with highly branched structure of the valleys and thickened study. Both axial parenchyma and nucleated fiber- ridge. Scale bars: A, B ¼ 3mm;C¼ 1.5 mm; D, H ¼ 2mm;E¼ 5 are common in Gnetum, potentially aiding in refilling collapsed mm; F ¼ 1mm;G¼ 200 mm; I ¼ 10 mm; J, K ¼ 20 mm. vessels (Carlquist, 2012). www.jse.ac.cn J. Syst. Evol. 54 (1): 1–16, 2016 6 Ickert-Bond & Renner

The ovulate cones of Gnetum have swollen collars and is the sampling of the Asian species. Pollen grains of Gnetum produce single large seeds (7 3 cm) that are surrounded by a are spherical, small (12-20 mm), inaperturate and micro- yellow or red fleshy or corky envelope (Markgraf, 1951; echinate, with a rather thin tectum, granular infratectum, Kubitzki, 1985; Figs. 3E, 5A, 5B). The staminate cones (Fig. 5C) and a lamellate endexine (Yao et al., 2004; Tekleva, 2015; usually are several cm long, with the microsporangium- Figs. 5D–5F). Gillespie and Nowicke (1994) recognized two bearing nodes separated by elongated internodes (Hufford, pollen types, grains with a uniformly thick tectum and conical 1996). The bracts at the nodes are highly synorganized, given blunt spines, characteristic of the Asian species (7 species them a collar-like appearance, and each collar bears numerous sampled), and grains with an irregular thickened tectum and staminate reproductive units. In some species, each node also spinules, found in the African G. africanum and the Neotropics bears a few ovulate ones (Fig. 5C), but such bisexual cones are (3 species sampled). The spinulose exine sculpture of Gnetum lacking in G. buchholzianum (Pearson, 1929) and G. cuspidatum is otherwise only known from the monospecific family (Kato et al., 1995). Mundry and Stutzel€ (2004) argued Sciadopityaceae (Tekleva & Krassilov, 2009). that Gnetum microsporophylls are simple, different from Two new species of Gnetum were recently described from the staminate cones of Ephedra and Welwitschia, which are East Africa, G. latispicum E.H. Biye and G. interruptum E.H. Biye the result of fusion of two lateral strobili. However, they did (Biye et al., 2014), and it is likely that further species await not examine Gnetum staminate cone development. discovery in tropical , given that most Gnetum The pollen of only 16 species of Gnetum has been studied are large canopy climbers or stragglers (Figs. 2E, 5A), a growth with light microscopy, scanning electron microscopy, and/or form that is difficult to collect and hence underrepresented in transmission electron microscopy (Gillespie & Nowicke, 1994; collections. Yao et al., 2004; Tekleva, 2015; our Table 2). Particularly sparse Molecular phylogenies of Gnetum have been based on plastid and nuclear sequences from most of its estimated 40 species (Won & Renner, 2003, 2005a, 2005b). Biye et al. (2014) first sequenced the African G. bucholzianum and their new African species, G. latispicum and G. interruptum, and Hou et al. (2015) added G. camporum, G. leptostachyum, and G. luofuense. The latter authors’ time-calibrated phylogeny includes 19 species and shows a crown age for Gnetum of 81 (64–98) Ma, while an earlier study had included 28 species and obtained a crown age of 44 (23–71) Ma (Won & Renner, 2006). The calibration closest to Gnetum in both studies is the above- mentioned Cratonia cotyledon fossil (Rydin et al., 2003), and the different ages are most likely due to the prior distribution assigned to this fossil in the different dating programs used in the 2006 and the 2015 study. Both studies inferred that the phylogeny of Gnetum is rooted between its South American clade and all remaining species. Phylogeographic studies are lacking for any of the ca. 40 species of Gnetum.

Welwitschia () When describing the genus Welwitschia, with the single species W. mirabilis, Hooker (1863) placed it near Ephedra and Gnetum and pointed out that all three in some traits resembled . Welwitschia is endemic in the Namib Desert of Namibia and and is one of the most bizarre species of seed plants (Figs. 2, 6A–6H). Its two strap-shaped leaves grow indefinitely from a basal intercalary , over the years becoming frayed at their ends. The leaves sit atop an unbranched short woody caudex (Figs. 3E, 6H), and the taproot can be several meters long (Cooper-Driver, 1994). The conductive system of Welwitschia shows typical desert Fig. 5. Morphology of Gnetum reproductive structures. adaptations, such as narrow vessels with simple perforation A, Gnetum cuspidatum, young ovulate cones from a lowland plates and tracheids, minimal torus-margo differentiation in rain in Sulawesi, (photo J. Wen). B, Gnetum vessel and tracheid pits, successive cambia, axial parenchyma, gnemon, ovulate cones producing copious pollination drop- and gelatinous walls in phloem fibers and sclereids (Carlquist, lets. C, Staminate cones with sterile ovules at arrowheads 2012). In situ experiments have also proven that Welwitschia (photo G. Gerlach). D, Gnetum gnemon, scanning electron mirabilis is able to take up CO2 at night and hence is a micrograph of pollen grain (courtesy of M. Kurmann). crassulacean acid metabolism (CAM) plant (Willert et al., E, F. Gnetum gnemon, transmission electron micrographs of 2005). exine stratification (courtesy of M. Kurmann). end, lamellate The female cones of Welwitschia are composed of 90–100 endexine; gr, infractectal granules; te, tectum. Scale bars: ovuliferous units (Endress, 1996; Figs. 6A, 6B, 6G). A bract A ¼ 1 cm, B ¼ 2 cm, C ¼ 5 mm, D ¼ 5 mm, E, F ¼ 1 mm. subtends each reproductive unit, and the integument is

J. Syst. Evol. 54 (1): 1–16, 2016 www.jse.ac.cn Biology and phylogeny of the Gnetales 7

Table 2 List of currently recognized Gnetum species indicating whose pollen morphology has been studied with light microscopy (LM), scanning electron microscopy (SEM), or transmission electron microscopy (TEM). Gillespie & Nowicki Yao et al. Tekleva Others (1994) (2004) (2015) for TEM 1. G. acutum Mkgf. (AS) 2. G. africanum Welw. (AF) SEM SEM, TEM Oryol et al., 1986 3. G. arboreum Foxw. (AS) 4. G. bosavicum Mkgf. (AS) 5. G. buchholzianum Engl. (AF) 6. G. cleistostachyum C.Y. Cheng (AS) SEM, TEM 7. G. camporum (Mkgf.) Stevenson & Zanoni (SAm) 8. G. contractum Mkgf. (AS) 9. G. costatum K. Schum. (AS) SEM 10. G. cuspidatum Blume (AS) SEM 11. G. diminutum Mkgf. (AS) 12. G. globosum Mkgf. (AS) 13. G. gracilipes Cheng (AS) 14. G. hainanense C.Y. Cheng ex L.K. Fu, Y.F. Yu & SEM M.G. Gilbert (AS) 15. G. gnemon L. 16. G. gnemonoides Brongn. (AS) TEM 17. G. indicum Merr. (AS) SEM, TEM 18. G. interruptum E.H. Biye (AF) 19. G. klossii Merrill ex Mkgf. (AS) 20. G. latifolium Blume (AS) SEM, TEM var. funiculare (AS) 21. G. latispicum E.H. Biye (AF) 22. G. leptostachyum Blume (AS) SEM SEM, TEM 23. G. leyboldii Tul. (SAm) 24. G. loerzingii Mkgf. (AS) 25. G. luofuense C.Y. Cheng (AS) SEM 26. G. macrostachyum Hook. f. (AS) LM SEM, TEM 27. G. microcarpum Blume (AS) 28. G. montanum Markgr. (AS) SEM, TEM 29. G. neglectum Blume (AS) LM 30. G. nodiflorum Brongn. (SAm) LM 31. G. oxycarpum Ridl. (AS) 32. G. paniculatum Spruce ex Benth. (SAm) 33. G. parvifolium (Warb) Cheng (AS) SEM 34. G. raya Mkgf. (AS) 35. G. ridleyi Gamble (AS) 36. G. schwackeanum Taub. ex A. Schwenk (SAm) SEM 37. G. ula Brongn. (AS) Gullvåg, 1966 38. G. urens (Aubl.) Blume (SAm) SEM 39. G. venosum Spruce ex Benth. (SAm) Welwitschia mirabilis SEM TEM Ueno, 1960; Gullvåg, 1966; Hesse, 1984; Kedves, 1987 Distribution given in parentheses: AF, Africa; AS, Asia; Sam, South America. Taxa marked with an asterisk have been illustrated with the respective method. extended into a micropylar tube surpassing the bract a centrally located sterile (Mundry & Stutzel,€ 2004; (Carafa et al., 1992; Endress, 1996). Pollination drops are Fig. 6D). Based on morphological differences in the male presented at the tip of the micropylar tube (Fig. 6B). The strobili and allopatric distribution, Leuenberger (2001) recog- staminate strobili (Fig. 6C) consist of two axillary reproductive nized two subspecies within W. mirabilis: subsp. namibiana units that are fused into a tubular sporangiophore bearing Leuenberger from Namibia and the typical subsp. mirabilis six synangia (each composed of three microsporangia) and from Angola. www.jse.ac.cn J. Syst. Evol. 54 (1): 1–16, 2016 8 Ickert-Bond & Renner

Fig. 6. Morphology of Welwitschia mirabilis reproductive structures. A, Female cones (Photo S. Little). B, Detail of micropylar extensions (mi) and pollination droplets (Photo S. Little). C, Staminate cones (Photo S. Little). D, Detail of staminate cone with yellow sticky masses of pollen grains (po) and central sterile ovule (sov) surrounded by staminate reproductive units, note synangia composed of three fused sporangia (at arrows, Photo G. Gerlach). E, Scanning electron micrograph of Welwitschia mirabilis ellipsoidal, polyplicate pollen grain (photo M. Kurmann). F, Transmission electron micrograph of Welwitschia mirabilis exine stratification (photo M. Kurmann). G, Cultivated specimens in raised bed at the Berlin Botanical Garden greenhouse (photo E. Zippel). H, Welwitschia mirabilis in Namibia, female (photo H. Freitag). end, endexine; gr, infractal granules; mi, micropylar tube; po, pollen grains; sov, sterile ovule; sy, synangium; te, tectum. Scale bars: A ¼ 5 cm; B ¼ 1.5 cm; C ¼ 1 cm; D ¼ 3 mm; E ¼ 10 mm; F ¼ 5 mm.

The pollen of Welwitschia is ellipsoidal, large (51 mm; as to the of intermediate populations (Jacobson & compared to Ephedra and Gnetum), monosulcate, and Lester, 2003). polyplicate with psilate plicae (Tekleva, 2015; Fig. 6E). The exine is composed of a lamellate endexine and a three-parted Polyploidy ectexine composed of a granular infratectum, a narrow foot Three ancient whole-genome duplications have been inferred layer and a solid tectum (Kurmann, 1992; Tekleva, 2015; in gymnosperms based on sequencing, one in Fig. 6F). the ancestor of cupressophyte conifers, one in Pinaceae, and a ratios in wild populations in Namibia were obtained third in Welwitschia (Li et al., 2015). While polyploidy is rare in by Henschel & Seely (2000), who found them to be male- gymnosperms (Murray, 2013), it is common in Sequioa and biased in upper Messum Wash (males:females ¼ 253:195), at Cupressus, and—especially strikingly—the Gnetales (Ickert- Welwitschia Wash (125:80), and near Brandberg (368:311). Bond et al., 2015). Ephedra is chromosomally highly variable, Several other populations showed no sex bias, and female- with counts ranging from 2n ¼ 14–56 and 69% of the 52 species biased ratios were not found at all. A phylogeographic study being polyploid (Ickert-Bond et al., 2015). Ephedra is also the of five populations from Namibia, using random amplified gymnosperm with the greatest variation in 1C-values, which polymorphic DNA markers, inferred little gene flow between range from 8.09–38.34 pg and include the largest genome of populations separated by as little as 18 km (Jacobson & any gymnosperm, , with 2n ¼ 8x ¼ 56 Lester, 2003), implying limited transport of the pollen and and a 1C value of 38.34 pg (Ickert-Bond et al., 2015). The seeds (see the section on Dispersal below). The disjunction frequency of polyploidy in all clades of Ephedra may point to a of 440 km between populations north and south of the particular role of chromosomal speciation in arid regions. The Grant escarpment in Namibia probably is the result of genome of Welwitschia mirabilis, with 2n ¼ 42 (Khoshoo & desertification during the Tertiary and , leading Ahuja, 1962, 1963) and a 1C-value of 7.20 pg (Leitch et al., 2001)

J. Syst. Evol. 54 (1): 1–16, 2016 www.jse.ac.cn Biology and phylogeny of the Gnetales 9 also appears of polyploid origin (Khoshoo & Ahuja, 1963; Murray, 2013), which is supported by the above-mentioned transcriptomic study (Li et al., 2015). The Gnetales also include the smallest genome size so far reported in gymnosperms, namely in the Indian Gnetum ula, with 2n ¼ 22 and a 1C-value of 2.25 pg (Leitch & Leitch, 2012). Two of the three published chromosome counts in Gnetum appear to reflect tetraploidy (Gnetum montanum,2n ¼ 44; Hizume et al., 1993; Gnetum ula,2n ¼ 22; Ohri & Khoshoo, 1986; Gnetum gnemon,2n ¼ 44, Ickert-Bond et al., 2015).

Pollination Biology of the Gnetales Bolinder et al. (2016) argue for a shift to wind pollination within crown-group Ephedra that could explain the puzzling geological history of the Ephedra lineage (see below Recent studies of the fossil record). Initially, in the , insect-pollinated stem relatives diversified, subsequently declined to near-extinction in the , and after a shift to wind pollination early in diversification of the crown group in the Tertiary the Ephedra lineage resurged (Bolinder et al., 2016). While wind pollination is the prevalent mode of pollination in extant Gnetales (Niklas & Buchmann, 1987; Kubitzki, 1990; Bolinder et al., 2015) and most gymnosperms (Takaso & Owens, 1996; Owens et al., 1998; Nepi et al., 2009), field observations and experimental studies have docu- mented insect visitation in all three genera of the Gnetales. Specifically, small moths and flies feed on the pollination droplets of Gnetum, (Kato & Inoue, 1994; Kato et al., 1995; Gong et al., 2015), flies and beetles on those of Welwitschia (Pearson, 1907; Wetschnig, 1997; Wetschnig & Depisch, 1999), and small wasps, flies (Bino et al., 1984a, 1984b; Bolinder et al., 2016), and ants of the subfamilies Formicidae and Myrmicinae on the droplets of Ephedra (Figs. 7A–7G; Bolinder et al., 2016). Fig. 7. Ant visitation on Ephedra trifurca in the Sonoran Just as insect visitation, bisexual cones also have been Desert, . A, Ovulate strobilus with pollination drop documented in all three genera and may represent the formation at the micropylar tube. B, Ant of Myrmecocystus cf. ancestral condition (Thompson, 1916; Endress, 1996; Haycraft mimicus Wheeler, 1908 feeding on pollination droplet of & Carmichael, 2001; Mundry & Stützel, 2004; Jorgensen€ & ovulate cone in E. trifurca. C, Myrmecocystus cf. mimicus Rydin, 2015). The organization of the bisexual cones differs foraging in staminate cones of E. trifurca. D, Myrmecocystus cf. among the three genera: in Welwitschia the male cones are mimicus covered in Ephedra pollen. E, Detail of Myrmecocystus made up of reproductive units that are structurally bisexual, cf. mimicus head with Ephedra pollen grains indicated by with microsporophylls and a sterile ovule, whereas in Ephedra arrowheads near the mandibles. F, G, Close-up of E showing foeminea (the only Ephedra species with bisexual cones) and details of characteristically polyplicate pollen grains of most species of Gnetum the male cones are made up of Ephedra and setae on Myrmecocystus cf. mimicus. Scale separate male reproductive units and sterile female repro- bars: A–C ¼ 10 mm; D ¼ 1 mm; E ¼ 200 mm; F, G ¼ 50 mm. ductive units. It appears that the presence of sterile ovules with pollination formation in the male cones is linked to pollination drops glittering in the full-moonlight help attract rewards, and insect pollination may be the and guide these nocturnal insects. Further fieldwork on the ancestral condition in the Gnetales (Jorgensen€ & Rydin, 2015; pollination of Ephedra, Gnetum, and Welwitschia is highly Rydin & Bolinder, 2015; Bolinder et al., 2016). desirable. The significance of insect pollination (and not just visitation) Ephedra pollination drops have relatively high in Ephedra is highlighted by the discovery that the exact timing concentrations (Porsch, 1910; Moussel, 1980; Bino et al., of pollination in E. foeminea in Greece and Croatia correlates 1984a,1984b, Meeuse et al., 1990; von Aderkas et al., 2015) and with the full moon of July (Rydin & Bolinder, 2015). During are produced by the nucellus (Rydin et al., 2010). The proteins peak full moon, all cones secreted pollination drops from the that are found in trace amounts in these droplets (as in many micropylar opening (Fig. 3C). When the moon was new, drop gymnosperm pollination drops) are the product of nucellar secretion was weak to non-existent, and even cones of the breakdown during pollen chamber formation (i.e., degra- appropriate developmental stage produced no drops. dome, von Aderkas et al., 2015), as well as export from the and moths feed on this species’ droplets and pollinate it in cytoplasm (i.e., secretome, von Aderkas et al., 2015). The the process (Rydin & Bolinder, 2015), and apparently, the micropyle for a typical species (E. distachya L.) is 1 mm in inner www.jse.ac.cn J. Syst. Evol. 54 (1): 1–16, 2016 10 Ickert-Bond & Renner minimum diameter and produces multiple pollination drops, striate or reticulate due to convex or depressed periclinal each following a previous pollination episode, but droplet walls (Ickert-Bond & Rydin, 2011; Figs. 4F–4H). Micromorphol- secretion ceases when the reaches the pollination ogy of the seed envelope is not useful for subclade chamber (Moussel, 1980). delineation, and parallel evolution of similar micro-morpho- Chemical analysis of pollination drops in 13 species logical patterns in unrelated groups is evident. Nevertheless, representing the main lineages of extant gymnosperms features of extant seed envelopes have been used to draw (Ginkgo, cycads, Coniferales, and Gnetales) reveals a correla- relationships between Early Cretaceous fossil seeds with tion between wind or insect pollination and total sugar affinity to Ephedra (Yang et al., 2005). In recent work, content, fructose concentration, proline and amino acid however, Yang has re-interpreted the same fossil seeds as concentration (Nepi et al., 2016 in review). Droplets of insect- extinct stem relatives of extant species (Yang et al., 2015). pollinated gymnosperms (Zamia furfuracea, Welwitschia Ephedra dispersal involves wind, birds, or terrestrial animals mirabilis, Gnetum gnemon, ) and those of (Ickert-Bond, 2003; Ickert-Bond & Wojciechowski, 2004; and Ephedra minuta, whose pollination mode is Hollander & Vander Wall, 2009; Hollander et al., 2010; Loera unclear, have higher levels of carbohydrates, lower levels of et al., 2015). Wind-dispersed seeds have dry, winged bracts of amino acid, and specific and amino acids profiles than the strobili; bird- and lizard-dispersed seeds are enclosed in gymnosperms shown to be wind pollinated in experimental fleshy, brightly colored bracts (e.g., Rodrıguez-Perez et al., studies. Most probably, insects shifted from fluid feeding 2012), and seeds dispersed by seed-caching are large on the ovular secretions of gymnosperms to feeding on and enclosed in dry, membranous bracts (Fig. 4E). Dispersal by angiosperm nectar as the latter became abundant and species wind and frugivores occurs in both Old and the New World rich (Nepi et al., 2016 in review). species, while dispersal has been documented only from New World species, but likely occurs in Old World deserts as well (Hollander et al., 2010; Loera et al., 2015; Fertilization in the Gnetales H. Freitag, University of Kassel, pers. comm. to SIB in 2015). Species dispersed by birds have higher phylogenetic niche Fertilization in the three lineages of the Gnetales is as different divergence for mean annual temperature (and to a lesser as their morphologies are. The process of in extent mean annual precipitation) and occupy a broader set of angiosperm, that is, the regular fusion of one nucleus temperature regimes than rodent-dispersed species (Loera with the egg nucleus and of the second sperm nucleus (from et al., 2015), which has been attributed to the higher dispersal the same pollen tube) with two nuclei of the polar cell to form ability of birds compared to scatter-hoarding rodents (Hol- the triploid (Nawaschin, 1898; Guignard, 1899), was lander & Vander Wall, 2009; Loera et al., 2012, 2015). The short- long considered a synapomorphy of flowering plants and it was distance movement of seeds from the mother plant due to thought that fertilization in Ephedra might involve similar rodents may be especially beneficial in arid conditions (Beck & paired fusion events. Over the past 20 years, however, the work Vander Wall, 2010; Vander Wall & Beck, 2012). of Friedman and colleagues has revealed that both basal Most species of Gnetum have yellow or red seed envelopes angiosperms and Gnetales have a range of fertilization modes. that are fed on by birds, rodents, or monkeys (e.g., Ridley, For example, in some Nymphaeales and Austrobaileyales the 1930; Markgraf, 1951; Kubitzki, 1985; Van Roosmalen, 1985; second sperm cell fuses with a single haploid polar nucleus so Forget et al., 2002). Catfish also feed on the when they that the endosperm is diploid rather than triploid. Here we fall into Amazonian rivers, and this may help upstream discuss findings only for the Gnetales. In Ephedra trifurca and E. dispersal (Kubitzki, 1985). Dispersal by water has been nevadensis, the second sperm nucleus regularly fuses with the inferred for species with a fibrous endotesta, giving their ventral canal nucleus within the egg cytoplasm (Friedman, seeds buoyancy (Kubitzki, 1985). 1990a, 1990b), while in Gnetum gnemon, free nuclei aggregate Welwitschia seeds have wings that develop from the bracts, around the pollen tube (Friedman & Carmichael, 1996). but due to their considerable weight they seldom become Welwitschia mirabilis lacks a second fertilization event (Fried- airborne (Fig. 6G, Bornman, 1978). In greenhouse experiments man, 2015). In addition, the three genera differ in their using wild-collected seeds from the Namib Desert, Whitaker development. The female gametophyte in et al. (2004) showed that removal of the bracts doubled Welwitschia shows tubular extensions (prothallial tubes) that success as compared to untreated seeds. grow through the nucellus to meet with downward growing pollen tubes (Friedman, 2015), and while Ephedra megagame- tophytes still contain archegonia and have a pollen chamber (similar to other gymnosperms), those of Gnetum and Recent Studies of the Fossil Record Welwitschia lack archegonia (Carmichael & Friedman, 1995; Fossils of the Ephedra lineage are known from the late Friedman & Carmichael, 1996; Friedman, 2015). The evolution of (Bolinder et al., 2016) with an increase of Ephedra- endosperm remains unclear (Linkies et al., 2010), and further like pollen during the Early Cretaceous (Crane & Lidgard, 1989) comparative cytogenetic studies of the fertilization events in and numerous and diverse Ephedra-like plants reported from Gnetales and other gymnosperms are needed. the (Krassilov, 1986; Yang et al., 2005; Rydin et al., 2006a; Wang & Zheng, 2010). During the Cretaceous diversity declined dramatically (Crane & Lidgard, 1989). Seeds and Seed Dispersal in the Gnetales Since the last comprehensive review of the Gnetales fossil In the 48 species of Ephedra whose seed morphology has record (Crane, 1996), a number of especially well-preserved been studied, the outer seed envelope is smooth, slightly fossils have come to light, most important among them the

J. Syst. Evol. 54 (1): 1–16, 2016 www.jse.ac.cn Biology and phylogeny of the Gnetales 11 seedling Cratonia cotyledon from the Early Cretaceous of . Lastly, studies of Gnetales would benefit from , which represents the Gnetales crown group and more fieldwork and experiments on cone and bract function, resembles Welwitschia (Rydin et al., 2003; Dilcher et al., 2005; and the role of insects in pollination. Noteworthy is that Friis et al., 2014a). Additional seed fossils with affinities to pollenkitt is supposedly lacking in Gnetales (Hesse, 1980, stem lineage Gnetales have also been described (Guo et al., 1984), yet the pollen of all three genera is sticky (Bolinder 2009; Friis et al., 2014b). The Early Cretaceous Yixian et al., 2015b; pers. obs.). Formation of Northeast China has yielded seeds described as Ephedra archaeorhytidosperma (Yang et al., 2005), a Acknowledgements reproductive of Liaoxia (Rydin et al., 2004), Siphoso- spermum simplex (Rydin & Friis, 2010), a fleshy cone described We thank Gunther€ Gerlach (Munich Botanical Garden), Helmut as Ephedra carnosa (Yang & Wang, 2013), a leafy shoot system Freitag (University of Kassel), Moshira Hassan (Free University with reproductive organs described as Chengia laxispicata of Berlin), Marie Kurmann (Sursee, Switzerland), Stefan Little (Yang et al., 2013), and a macrofossil with strap-shaped leaves, (Universite Paris-Sud), Jun Wen (Smithsonian Institution) and reduced female cones, and seeds described as Ephedra Elke Zippel (Berlin Botanical Garden and Botanical Museum) multinervia (Yang et al., 2015). Yang (2014) has arranged all for micrographs and photos; Catarina Rydin, James A. Doyle, fossils of the Ephedra lineage into a single classification, in a and Helmut Freitag for their insightful critique of the study that is also useful in bringing together much relevant manuscript; Philip S. Ward (University of California, Davis) literature. Since the taxa Ephedra and Ephedraceae have the for the ant identifications; Douglas Walker, University of same composition in the extant flora, one might restrict the California at Davis Science Laboratory and Greenhouse, for lower-rank name (Ephedra) to the crown group and the taking care of the Ephedra farm; and Angelo Razeto for taking higher-rank name (Ephedraceae) to the crown group plus its care of the Ephedra collection at the Munich Botanical Garden. stem relatives, to reflect that the extinct taxa are more closely The first author thanks the German Academic Exchange related to Ephedra than they are to Welwitschia and Gnetum Service (DAAD) for a one-year fellowship supporting her (Cantino et al., 2007; Doyle & Endress, 2014). sabbatical. Phase-contrast X-ray microtomography links charcoalified seeds from the Early Cretaceous (144 to 100 Ma) with the References Gnetales but also the extinct seed plant lineages and Erdmanithecales (Friis et al., 2007, 2009). These seeds are Afzelius BM. 1956. Electron-microscope investigations into exine stratification. Grana Palynologica 1: 22–37. c. 0.5–1.8 mm long and have two layers surrounding the nucellus: an inner, thin, membranous integument, formed by Arber EAN, Parkin J. 1908. Studies on the evolution of the thin-walled cells; and a robust, outer, sclerenchymatous angiosperms. The relationship of the angiosperms to the Gnetales. Annals of Botany 22: 489–515. envelope that completely encloses the integument except – for the micropylar opening. The integument itself is extended Beck MJ, Vander Wall SB. 2010. Seed dispersal by scatter hoarding – into a long, narrow micropylar tube, which bore the rodents in arid environments. Journal of Ecology 98: 1300 1309. pollination droplets. Bino RJ, Dafni A, Meeuse ADJ. 1984a. in the dioecious gymnosperm Ephedra aphylla Forsk. (¼E. alte C.A. Mey.), with some notes on E. campylopoda C.A. Mey. I. Aspects of the entomophilous syndrome. Proceedings of the Koninklijke Neder- Outlook landse Akademie von Wetenschappen. Series C: Biological and Medical Sciences 87: 1–13. Historically, the Gnetales have been critical to the develop- Bino RJ, Devente N, Meeusse ADJ. 1984b. Entomophily in the ment of hypotheses of cone origin (Arber & Parkin, 1908), and dioecious gymnosperm Ephedra aphylla Forsk. (¼E. alte C.A. Mey.) with new genomic tools they may continue to play this role. with some notes on E. campylopoda C.A. Mey. II. Pollination Modern detailed developmental studies of reproductive droplets, nectaries, and nectarial secretion in Ephedra. Proceed- structures are mostly lacking for the Gnetales, and few taxa ings of the Koninklijke Nederlandse Akademie von Wetenschappen. have been included in detailed developmental studies Series C: Biological and Medical Sciences 87: 15–24. historically (Thoday & Berridge, 1912; Thompson, 1912, 1916; Biye EH, Balkwill K, Cron GV. 2014. A clarification of Gnetum L. Thoday, 1921; Pearson, 1929; Martens, 1971; Takaso, 1984, 1985; (Gnetaceae) in Africa and the description of two new species. Takaso & Bouman, 1986). Gene expression studies have Plant Systematics and Evolution 300: 263–272. fl already provided many insights into oral identity Bolinder K, Humphreys AM, Ehrlen J, Alexandersson R, Ickert-Bond among seed plants and indicate that determination of SM, Rydin C. 2016. From near extinction to diversification by reproductive organ identity is similar across this clade (Winter means of a shift in pollination mechanism in the gymnosperm et al., 1999; Wang et al., 2010; Mathews & Kramer, 2012). They relict Ephedra (Ephedraceae, Gnetales). Botanical Journal of the also imply that the last common ancestor of seed plants may Linnean Society, in press. have possessed the developmental machinery for specific Bolinder K, Niklas KJ, Rydin C. 2015a. Aerodynamics of pollen floral organ identity (Theißen & Becker, 2004; Wang et al., ultrastructure in Ephedra. American Journal of Botany 102: 1–14. 2010). Evolutionary developmental studies might shed light on Bolinder K, Norback€ Ivarsson L, Humphreys AM, Ickert-Bond SM, Han the evolution of the diverse cone types in the Gnetales and F, Hoorn C, Rydin C. 2015b. Pollen morphology of Ephedra help shed light on homologies with other gymnosperm cones. (Gnetales) and its evolutionary implications. Grana. doi:10.1080/ With the short generation time of some of its species, Ephedra 00173134.2015.1066424. could also become a gymnosperm model to underpin Bornman CH. 1978. Welwitschia: Paradox of a Parched Paradise. Cape understanding of development and gene regulation in seed Town: C Struik. www.jse.ac.cn J. Syst. Evol. 54 (1): 1–16, 2016 12 Ickert-Bond & Renner

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