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

Stefanie Ickert-Bond

Ephedra, Gnetum, and Welwitschia constitute the gymnosperm order Gnetales of still unclear phylogenetic relationships within seed plants. 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 pollen morphology. We include distribution maps and original illustrations of key features, and specify which species groups or geographic areas are undersampled.

line a libr jou rna l/js e JSE Journal of Systematics and Evolution Volume 54 Number 1 January 2016 Journal of Systematics and Evolution Number 1 January 2016 Pages 1–92 RESEARCH ARTICLES 29 Hong Qian and Jian Zhang Are phylogenies derived from family-level supertrees robust for studies on macroecological patterns along environmental gradients? 37 Li-E Yang, Hang Sun, Friedrich Ehrendorfer, and Ze-Long Nie Molecular phylogeny of Chinese Rubia (Rubiaceae: Rubieae) inferred from nuclear and plastid DNA sequences 48 Yuan Xu, Chi-Ming Hu, and Gang Hao Pollen morphology of Androsace (Primulaceae) and its systematic implications 65 Daniel Vitales, Alfredo García-Fernández, Teresa Garnatje, Jaume Pellicer, and Joan Vallès Phylogeographic insights of the lowland species Cheirolophus sempervirens in the southwestern Iberian Peninsula 75 Manoj M. Lekhak, Siddharthan Surveswaran, and Shrirang R. Yadav Generic identity of Camptorrhiza indica (Colchicaceae) based on cytogenetics and molecular phylogenetics 83 Wei Gong, Ying Liu, Jing Chen, Yu Hong, and Hang-Hui Kong DNA barcodes identify Chinese medicinal plants and detect geographical patterns of Sinosenecio (Asteraceae) JSE Volume 54 REVIEWS 1 Stefanie M. Ickert-Bond and Susanne S. Renner The Gnetales: Recent insights on their morphology, reproductive biology, chromosome numbers, biogeography, and divergence times 17 Weronika B. Żukowska and Witold Wachowiak Utility of closely related taxa for genetic studies of adaptive variation and speciation: Current state and perspectives in plants with focus on forest tree species Journal of Systematics and Evolution w on iley / om ry.c Cover illustration: Morphological diversity in the Gnetales. Top left: Ephedra distachya subsp. helvetica showing twisted micropylar tube and red fleshy bracts of female cone in Embrun, Hautes Alpes, France (photo by Elke Zippel). Top right: Welwitschia mirabilis pollination droplets at micropylar tubes in female cone, cultivated (photo by Stefan Little). Bottom left: Gnetum gnemon bisexual cone with sterile ovules with pollination droplet above staminate reproductive units, cultivated (photo by Günther Gerlach). Middle right: Welwitschia mirabilis details of sticky pollen masses and sterile ovules in bisexual cone, cultivated (photo by Günther Gerlach). Bottom right: Weltwischia mirabilis, male (at left) and female cones (at right), cultivated (photo by Stefan Little). See Ickert-Bond et al., pp. 1–16 in this issue. I SSN 1 6 7 4 - 4 9 1 8 CN 11-5779/Q JSE_54(1)_Cover4_1.indd 1 Volume 54 Number 1 January 2016 29/12/15 3:00 PM JSE Journal of Systematics and Evolution doi: 10.1111/jse.12190 Review The Gnetales: Recent insights on their morphology, reproductive biology, chromosome numbers, biogeography, and divergence times Stefanie M. Ickert-Bond1* and Susanne S. Renner2 1 University 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 2 Institute of Systematic Botany and Mycology, University of Munich (LMU), Menzinger Str. 67, 80638 Munich, Germany *Author for correspondence. E-mail: smickertbond@alaska.edu. 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 Ephedra, Gnetum, and Welwitschia constitute the gymnosperm order Gnetales of still unclear phylogenetic relationships within seed plants. 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 pollen 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, pollination, polyploidy. Mais les Gn etophytes se pr esentent au botaniste, depuis longtemps, comme un ensemble d’un int er^ et exceptionnel et nigme particuli comme un e erement irritante. Pierre Martens, 1971 Les Gnetophytes The Gnetales are a clade of three genera that is morphologically and genetically so disparate from the remaining seed plant (cycads, Ginkgo, angiosperms, and Coniferales) that its precise placement has remained unclear (Mathews, 2009; reviewed in Mathews et al., 2010; Fig. 1). The order Gnetales Luersson (or the subclass Gnetidae Pax) is characterized by compound cones with unisexual reproductive units borne in the axils of bracts, with the ovules surrounded by 1-2 envelopes and the integument extending into a micropylar tube carrying the pollination droplets (Kubitzki, 1990). This combination of traits is extremely rare in fossil forms (Krassilov, 2009). Transcriptome data for 92 streptophytes, analyzed along with 11 complete plant genomes, support a position of Gnetales either as sister to Coniferales, represented by seven genera, or as sister to one of their families, the Pinaceae, represented by Cedrus and Pinus (Wickett et al., 2014). A placement of the Gnetales near to or inside Coniferales would be consistent with previously published analyses of concatenated gene alignments that aimed to reduce long-branch attraction artifacts by implementing various among-site rate heterogeneity models (e.g., Bowe et al., 2000; Chaw et al., 2000; Burleigh & Mathews 2007a, 2007b; Lee et al., 2011; Wu et al., 2011; Zhong et al., 2011; Ruhfel et al., 2014). January 2016 | Volume 54 | Issue 1 | 1–16 In terms of their morphology and even basic ecology, the Gnetales remain enigmatic, with surprising discoveries continuing to be made (e.g., Wetschnig, 1997; Mundry & St€ utzel, 2004; Friedman, 2015; Ickert-Bond et al., 2015; Rydin & Bolinder, 2015). Here we review progress over the past 10 years in our understanding of the species diversity, morphology, reproductive biology, chromosome numbers, and genome sizes of the Gnetales, highlighting the unevenness in the sampling of species even for traits that can be studied in preserved material, such as pollen morphology. The Gnetales: Three Disparate Monogeneric Families Gnetum L. (Markgraf, 1930) and Ephedra L. (Cutler, 1939 for North America only) were monographed in the last century; Welwitschia contains but a single species, endemic to the Namib Desert (Leuenberger, 2001; Figs. 2, 3). Ephedra is sister to the other two genera and comprises about 54 species distributed evenly between the deserts of the Old and New World (Stapf, 1889; Ickert-Bond, 2003; Figs. 2, 3; Table 1). Gnetum has ten species in South America, two to four in tropical West Africa (Biye et al., 2014; Figs. 2, 3; Table 1), and ca. 25 in tropical Asia (Markgraf, 1930; Price, 1996; Won & Renner, 2006; Hou et al., 2015). Multi-locus analyses of nuclear and plastid DNA sequences have shed light on species relationships within Ephedra and Gnetum (Ickert-Bond & Wojciechowski, 2004; Won & Renner, 2005a, 2005b, 2006; Ickert-Bond et al., 2009; Rydin & Korall, 2009; Rydin et al., 2010; Loera et al., © 2015 Institute of Botany, Chinese Academy of Sciences 2 Ickert-Bond & Renner Fig. 1. Extreme rate heterogeneity within Gnetales and gymnosperms based on matK and rbcL gene sequences. A, Unrooted maximum likelihood tree obtained from 558 matK gene sequences, downloaded from GenBank in mid2008. Values at nodes indicate statistical support from 100 bootstrap replicates under the GTR þG model of substitution. B, Unrooted maximum likelihood tree obtained from 792 rbcL gene sequences, downloaded from GenBank in mid- 2008. Values at nodes indicate statistical support from 100 bootstrap replicates under the GTR þG model of substitution. 2012, 2015; Hou et al., 2015); a few deep nodes within Ephedra and Gnetum still remain statistically poorly supported. Ephedra (Ephedraceae) All Ephedra are perennial and dioecious, and most species are shrubs (Price, 1996; Ickert-Bond, 2003; Fig. 3A); a few are climbers up to 4 m (e.g., Ickert-Bond, 2003: Fig. 3.1 E–F; Freitag, 2010: Fig. G2-02) or small trees up to 2 m (E. equisetina in Freitag, 2010: Fig. G2-01A). The nodes bear narrow, lanceolate leaves arranged in decussate or whorled phyllotaxis (Figs. 4A–4D). The leaves are 2–15 (40) mm long when fully expanded, but become non-functional (except for E. foliata and E. altissima) when the vegetative shoot ceases €rken, 2014). The vegetative elongation (Ickert-Bond, 2003; Do apical portion of each blade is free while the basal portions are fused into a sheath, with the extent of fusion a speciescharacteristic trait (Figs. 4A–4D). J. Syst. Evol. 54 (1): 1–16, 2016 The species of Ephedra occur in Old World and New World deserts, semideserts, desert steppes or in seasonally dry habitats, such as mediterranean-type evergreen or deciduous woodlands and subtropical thorn scrub (Fig. 2; Ickert-Bond, 2003; Freitag, 2010: Fig. G2-01A). The genus ranges from depressions below sea level (Death Valley of California and Dead Sea area) to about 5000 m in the Andes of Ecuador (E. rupestris, Ickert-Bond, 2005) and to 5300 m in the Himalayas (E. gerardiana, Fu et al., 1999). The desert species tend to be clonal, forming phytogenic mounds by accumulating sand, particularly in dune habitats. Branching in Ephedra is often broom-like with nearly parallel and fastigiate to ascending (virgate) green stems (Fig. 3A). Wood anatomical features of Ephedra include the presence of vessels that increase conducting efﬁciency as compared to tracheid-only systems in non-gnetalean gymnosperms (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-tracheids with abundant starch 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/whorl enclosing one to three seeds, 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 bundles evolved several times (Rydin et al., 2010). Fleshy bracts characterize the fruiting cones of 38 species (Fig. 3B), membranous (Fig. 4E) and winged bracts those of six or seven other species (see section on Seed dispersal). The seed envelopes are smooth (Fig. 4H) or papillate or bear transverse ridges (Ickert-Bond & Rydin, 2011; Figs. 4F, 4G). The male cones (staminate strobili, Fig. 4I) consist of two lateral strobili with 2–3 sterile bracts at the base, followed by 2–8 (10) fertile bracts, within each of which two median bracts enclose the stalked antherophore (Cutler, 1939; Hufford, 1996; Ickert-Bond, 2003; Mundry & St€ utzel, 2004). Each antherophore consists of two fused microsporophylls and bears 2–8 stalked or sessile synangia, which result from the fusion of two (rarely three) microsporangia (Hufford, 1996; Ickert-Bond, 2003; Mundry & St€ utzel 2004). Mundry and St€ utzel (2004) interpret the male cones of Ephedra as consisting of two units with four simple sporophylls, and propose homologies with parts in the female cones of Welwitschia and Gnetum (see respective sections below). The pollen of 45 species of Ephedra has been studied with light and scanning electron microscopy (Steeves & Barghoorn, 1959; Zhang & Xi, 1983; Ickert-Bond, 2003; Ickert-Bond et al., 2003; Doores et al., 2007; Bolinder et al., 2015a, 2015b; our Table 1). Pollen is ellipsoidal, with characteristic ridges, and rather large (27–58 mm in average equatorial diameter; Figs. 4J, 4K). Based on a phylogenetic analysis of pollen traits, grains with unbranched valleys in the exine (pseudosulci of 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. Fig. 3. Gross morphology of Gnetales. A, B Ephedra aphylla, female plant with red ﬂeshy seed envelope surrounding two seeds on ovulate cone, Wadi Musa, Egypt (Photo M. Hassan). C, Ephedra minuta, female cone with pollination drop borne on micropylar tube (Photo S. Little). D, Gnetum gnemon, female with pollination drops, in greenhouse at Munich Botanical Garden. E, Gnetum cuspidatum, cluster of seeds formed on large trunk, Sulawesi (Photo J. Wen). F, Welwitschia mirabilis in Namibia, male, 1200 year old specimen (Photo H. Freitag). G, Welwitschia mirabilis, female strobili (Photo S. Little). Scale bars: A ¼ 50 cm; B ¼ 5 mm; C ¼ 5 mm; D, E ¼ 5 cm; F ¼ 20 cm; G ¼ 15 cm. www.jse.ac.cn 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 threeparted 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 India and China, although they appear morphologically close to E. intermedia and E. saxatilis (Yang et al., 2003; Yang, 2005; Sharma & Uniyal, 2009; Sharma et al., 2010; Sharma & Singh, 2015: Ephedra rituensis Y. Yang, D.Z. Fu, G.H. Zhu, Ephedra dawuensis Y. Yang; Ephedra sumlingensis P. Sharma & P.L. Uniyal, Ephedra kardangensis P. Sharma & P.L. Uniyal, Ephedra khurikensis P. Sharma & P.L. Uniyal, and Ephedra pangiensis Rita Singh & P. Sharma). The four Indian new species all come from the Western Himalayas and, based on their descriptions, may represent forms of E. intermedia. Their diagnoses rely heavily on straight vs. coiled micropyles, a character of limited signiﬁcance (Freitag & Maier-Stolte, 1993, 1994; Kakiuchi et al., 2011). 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 bract consistency Species (Distribution†) Bolinder et al., 2015§ 1. E. alata Decne. (SAH, AR) 2. E. altissima Desf. (MED) 3. E. americana Humb. & Bonpl. ex Willd. (SAm) 4. E. antisyphilitica Berlandier ex. C.A. Mey. (NAm) 5. E. aphylla Forssk. (MED, AR) 6. E. aspera Engelm. (NAm) 7. E. boelckei F.A. Roig (SAm) 8. E. breana Phil. (SAm) 9. E. californica S. Watson (NAm) 10. E. chilensis C. Presl. (SAm) 11. E. compacta Rose (NAm) 12. E. coryi E.L. Reed (NAm) 13. E. cutleri Peebles (NAm) 14. E. distachya L. (EU, AS) LM , SEM LM , SEM LM , SEM LM , SEM LM , SEM LM , SEM LM , SEM LM , SEM LM , SEM LM , SEM LM , SEM LM, SEM LM , SEM LM , SEM 15. E. equisetina Bunge (AS) 16. E. fasciculata A. Nelson (NAm) 17. E. fedtschenkoi Paulsen (AS) 18. E. foeminea Forssk. (MED, AR) 19. E. foliata Boiss. ex C.A. Mey. (MED, AR, AS) 20. E. fragilis Desf. (MED) LM , SEM LM , SEM LM , SEM LM , SEM El-Ghazaly et al., 1997 21. E. frustillata Miers (SAm) 22. E. funerea Coville & C.V. Morton (NAm) 23. E. gerardiana Wall. ex Stapf (AS) 24. E. glauca Regel (AS) 25. E. intermedia Schrenk & C.A. Mey. (AS) 26. E. laristanica Assadi (Iran) 27. E. likiangensis Florin (AS) 28. E. lomatolepis Schrenk (AS) 29. E. major Host (EU, AS) 30. E. milleri Freitag & M. Maier-Stolte (AR) 31. E. minuta Florin (AS) 32. E. monosperma J.G. Gmel. ex C.A. Mey. (AS) LM , SEM LM , SEM LM , SEM 33. E. multiflora Phil. ex. Stapf (SAm) 34. E. nevadensis S. Watson (NAm) 35. E. ochreata Miers (SAm) 36. E. pachyclada Boiss. (AR, AS) 37. E. pedunculata Engelm. ex S. Watson (NAm) 38. E. przewalskii Stapf (AS) 39. E. pseudodistachya Pachom. (AS) 40. E. regeliana Florin (AS) 41. E. rhytidosperma Pachom. (AS) 42. E. rupestris Benth. 43. E. sacrocarpa Aitch. & Hemsl. (AR, AS) 44. E. saxatilis (Stapf) Royle ex Florin (AS) 45. E. sinica Stapf (AS) ¼ synonym of E. dahurica 46. E. somalensis Freitag & Maier-St. (AR) 47. E. strobilacea Bunge (AS) 48. E. torreyana S. Watson (NAm) 49. E. transitoria Riedl (AS) 50. E. triandra Tul. (SAm) 51. E. trifurca Torr. ex S. Watson (NAm) LM , SEM LM , SEM LM , SEM LM , SEM LM , SEM TEM studies El-Ghazaly et al., 1997 Van Campo & Lugardon, 1973; Kurmann, 1992; El-Ghazaly et al., 1998 El-Ghazaly & Rowley, 1997 El-Ghazaly & Rowley, 1997; El-Ghazaly et al., 1998 LM , SEM LM , SEM LM , SEM LM , SEM LM , SEM LM , SEM Afzelius 1956, Erdtman 1957; Gullvåg, 1966 LM , SEM Ickert-Bond, 2003 LM , SEM LM , SEM LM , SEM LM , LM , LM , LM , LM , SEM SEM SEM SEM SEM El-Ghazaly et al., 1998 Ueno, 1960 Bract consistency‡ fl, pa fl fl fl fl me pa fl me/pa fl fl fl me fl fl me fl fl fl fl fl pa fl fl fl fl fl fl fl fl fl fl pa me fl fl fl pa fl fl fl fl fl fl fl fl fl pa fl fl pa Continued J. Syst. Evol. 54 (1): 1–16, 2016 www.jse.ac.cn Biology and phylogeny of the Gnetales 5 Table 1 Continued Species (Distribution†) €llner (SAm) 52. E. trifurcata Zo 53. E. tweediana Fisch. & C.A. Mey. (NAm) 54. E. viridis Coville (NAm) Bolinder et al., 2015§ LM , SEM LM , SEM LM , SEM TEM studies Bract consistency‡ fl fl me † Distribution is presented in parentheses: AR, Arabian Penninsula; AS, Asia; EU, Europe; 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. Fig. 4. Morphology of Ephedra. A–D, Leaf diversity. E–H, Female cone and seed morphology. I–K, Staminate cone and pollen diversity. A, Ephedra americana, with well developed free leaf tips and swollen dark, decussate leaf bases. B, Ephedra aspera, showing marked difference in development of the lamina from extremely large (left) to moderately developed (right), both leaves come from the same plant. C, Ephedra torreyana, showing unique whorls of four leaves at node, indicating possible integration with E. aspera. D, Ephedra trifurca, mature leaf, showing typical splitting of the lamina. E, Ephedra nevadensis, pedunculate cone with two exserted seeds. F, Ephedra torreyana, scanning electron micrograph of lance-ovoid seed with elongated beak and transverse ridges on the seed surface. G, Ephedra torreyana, scanning electron micrograph showing details of transverse ridges. H, Ephedra aspera, scanning electron micrograph of ovoid seed. I, Ephedra funerea, staminate cone showing numerous microspongiophores per strobilus. J, Ephedra californica, scanning electron micrograph of ancestral grain type with straight ridges and furrows. K, Ephedra coryi, scanning electron micrograph of derived pollen grain type with highly branched structure of the valleys and thickened ridge. Scale bars: A, B ¼ 3 mm; C ¼ 1.5 mm; D, H ¼ 2 mm; E ¼ 5 mm; F ¼ 1 mm; G ¼ 200 mm; I ¼ 10 mm; J, K ¼ 20 mm. www.jse.ac.cn 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 wellsupported 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 clades, 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 molecular clock study that included 32 species of Ephedra and outgroups, calibrated with the welwitschioid fossil seedling Cratonia cotyledon (Rydin et al., 2003) from the Early Cretaceous Crato Formation of the Brazilian Araripe basin, 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 & Price, 2003) that used a strict molecular clock model and rbcL sequences had placed the crown age at 8–32 Ma. Gnetum (Gnetaceae) Most of the c. 40 species of Gnetum are large woody climbers (Fig. 3E); only G. gnemon (Fig. 3D) and G. costatum are free standing, and most species occur in mesic habitats (Fig. 2; Markgraf, 1930, 1951, 1972, 1977; Price, 1996). The decussate broad leaves of Gnetum with pinnate-reticulate venation resemble the simple leaves of many dicots (Fig. 3D). The vessels of Gnetum wood are much wider than those of Ephedra for both the lianoid and the tree species, but signiﬁcantly smaller than those of lianoid angiosperms, probably because Gnetum species grow in understory, semishaded habitats (Feild & Balun, 2008; Carlquist, 2012). Simple perforation plates and torus-margo pit membranes in tracheid-to-tracheid pits as well as in vessel-to-tracheid pits may provide insurance against air bubble formation and embolisms (Carlquist, 2012), although given the mesic tropical habitats of Gnetum (Fig. 2) this functional role requires further study. Both axial parenchyma and nucleated ﬁber-tracheids are common in Gnetum, potentially aiding in reﬁlling collapsed vessels (Carlquist, 2012). J. Syst. Evol. 54 (1): 1–16, 2016 6 Ickert-Bond & Renner The ovulate cones of Gnetum have swollen collars and produce single large seeds (7 3 cm) that are surrounded by a yellow or red ﬂeshy or corky envelope (Markgraf, 1951; Kubitzki, 1985; Figs. 3E, 5A, 5B). The staminate cones (Fig. 5C) usually are several cm long, with the microsporangiumbearing nodes separated by elongated internodes (Hufford, 1996). The bracts at the nodes are highly synorganized, given them a collar-like appearance, and each collar bears numerous staminate reproductive units. In some species, each node also bears a few ovulate ones (Fig. 5C), but such bisexual cones are lacking in G. buchholzianum (Pearson, 1929) and G. cuspidatum (Kato et al., 1995). Mundry and St€ utzel (2004) argued that Gnetum microsporophylls are simple, different from the staminate cones of Ephedra and Welwitschia, which are the result of fusion of two lateral strobili. However, they did not examine Gnetum staminate cone development. The pollen of only 16 species of Gnetum has been studied with light microscopy, scanning electron microscopy, and/or transmission electron microscopy (Gillespie & Nowicke, 1994; Yao et al., 2004; Tekleva, 2015; our Table 2). Particularly sparse Fig. 5. Morphology of Gnetum reproductive structures. A, Gnetum cuspidatum, young ovulate cones from a lowland rain forest in Sulawesi, Indonesia (photo J. Wen). B, Gnetum gnemon, ovulate cones producing copious pollination droplets. C, Staminate cones with sterile ovules at arrowheads (photo G. Gerlach). D, Gnetum gnemon, scanning electron micrograph of pollen grain (courtesy of M. Kurmann). E, F. Gnetum gnemon, transmission electron micrographs of exine stratiﬁcation (courtesy of M. Kurmann). end, lamellate endexine; gr, infractectal granules; te, tectum. Scale bars: A ¼ 1 cm, B ¼ 2 cm, C ¼ 5 mm, D ¼ 5 mm, E, F ¼ 1 mm. J. Syst. Evol. 54 (1): 1–16, 2016 is the sampling of the Asian species. Pollen grains of Gnetum are spherical, small (12-20 mm), inaperturate and microechinate, with a rather thin tectum, granular infratectum, and a lamellate endexine (Yao et al., 2004; Tekleva, 2015; Figs. 5D–5F). Gillespie and Nowicke (1994) recognized two pollen types, grains with a uniformly thick tectum and conical blunt spines, characteristic of the Asian species (7 species sampled), and grains with an irregular thickened tectum and spinules, found in the African G. africanum and the Neotropics (3 species sampled). The spinulose exine sculpture of Gnetum is otherwise only known from the monospeciﬁc family Sciadopityaceae (Tekleva & Krassilov, 2009). Two new species of Gnetum were recently described from East Africa, G. latispicum E.H. Biye and G. interruptum E.H. Biye (Biye et al., 2014), and it is likely that further species await discovery in tropical Southeast Asia, given that most Gnetum are large canopy climbers or stragglers (Figs. 2E, 5A), a growth form that is difﬁcult to collect and hence underrepresented in collections. 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 abovementioned 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 (Welwitschiaceae) 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 conifers. Welwitschia is endemic in the Namib Desert of Namibia and Angola 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 meristem, 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 adaptations, such as narrow vessels with simple perforation plates and tracheids, minimal torus-margo differentiation in vessel and tracheid pits, successive cambia, axial parenchyma, and gelatinous walls in phloem ﬁbers and sclereids (Carlquist, 2012). In situ experiments have also proven that Welwitschia mirabilis is able to take up CO2 at night and hence is a crassulacean acid metabolism (CAM) plant (Willert et al., 2005). The female cones of Welwitschia are composed of 90–100 ovuliferous units (Endress, 1996; Figs. 6A, 6B, 6G). A bract subtends each reproductive unit, and the integument is 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 (1994) 1. G. acutum Mkgf. (AS) 2. G. africanum Welw. (AF) 3. G. arboreum Foxw. (AS) 4. G. bosavicum Mkgf. (AS) 5. G. buchholzianum Engl. (AF) 6. G. cleistostachyum C.Y. Cheng (AS) 7. G. camporum (Mkgf.) Stevenson & Zanoni (SAm) 8. G. contractum Mkgf. (AS) 9. G. costatum K. Schum. (AS) 10. G. cuspidatum Blume (AS) 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 & M.G. Gilbert (AS) 15. G. gnemon L. 16. G. gnemonoides Brongn. (AS) 17. G. indicum Merr. (AS) 18. G. interruptum E.H. Biye (AF) 19. G. klossii Merrill ex Mkgf. (AS) 20. G. latifolium Blume (AS) var. funiculare (AS) 21. G. latispicum E.H. Biye (AF) 22. G. leptostachyum Blume (AS) 23. G. leyboldii Tul. (SAm) 24. G. loerzingii Mkgf. (AS) 25. G. luofuense C.Y. Cheng (AS) 26. G. macrostachyum Hook. f. (AS) 27. G. microcarpum Blume (AS) 28. G. montanum Markgr. (AS) 29. G. neglectum Blume (AS) 30. G. nodiflorum Brongn. (SAm) 31. G. oxycarpum Ridl. (AS) 32. G. paniculatum Spruce ex Benth. (SAm) 33. G. parvifolium (Warb) Cheng (AS) 34. G. raya Mkgf. (AS) 35. G. ridleyi Gamble (AS) 36. G. schwackeanum Taub. ex A. Schwenk (SAm) 37. G. ula Brongn. (AS) 38. G. urens (Aubl.) Blume (SAm) 39. G. venosum Spruce ex Benth. (SAm) Welwitschia mirabilis Yao et al. (2004) SEM Tekleva (2015) Others for TEM SEM , TEM Oryol et al., 1986 SEM , TEM SEM SEM SEM TEM SEM , TEM SEM , TEM SEM SEM , TEM SEM LM SEM , TEM SEM , TEM LM LM SEM SEM Gullvåg, 1966 SEM 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 (Carafa et al., 1992; Endress, 1996). Pollination drops are presented at the tip of the micropylar tube (Fig. 6B). The staminate strobili (Fig. 6C) consist of two axillary reproductive units that are fused into a tubular sporangiophore bearing six synangia (each composed of three microsporangia) and www.jse.ac.cn a centrally located sterile ovule (Mundry & St€ utzel, 2004; Fig. 6D). Based on morphological differences in the male strobili and allopatric distribution, Leuenberger (2001) recognized two subspecies within W. mirabilis: subsp. namibiana Leuenberger from Namibia and the typical subsp. mirabilis from Angola. 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 compared to Ephedra and Gnetum), monosulcate, and polyplicate with psilate plicae (Tekleva, 2015; Fig. 6E). The exine is composed of a lamellate endexine and a three-parted ectexine composed of a granular infratectum, a narrow foot layer and a solid tectum (Kurmann, 1992; Tekleva, 2015; Fig. 6F). Sex ratios in wild populations in Namibia were obtained by Henschel & Seely (2000), who found them to be malebiased in upper Messum Wash (males:females ¼ 253:195), at Welwitschia Wash (125:80), and near Brandberg (368:311). Several other populations showed no sex bias, and femalebiased ratios were not found at all. A phylogeographic study of ﬁve populations from Namibia, using random ampliﬁed polymorphic DNA markers, inferred little gene ﬂow between populations separated by as little as 18 km (Jacobson & Lester, 2003), implying limited transport of the pollen and seeds (see the section on Dispersal below). The disjunction of 440 km between populations north and south of the Grant escarpment in Namibia probably is the result of desertiﬁcation during the Tertiary and Quaternary, leading J. Syst. Evol. 54 (1): 1–16, 2016 to the extinction of intermediate populations (Jacobson & Lester, 2003). Polyploidy Three ancient whole-genome duplications have been inferred in gymnosperms based on transcriptome sequencing, one in the ancestor of cupressophyte conifers, one in Pinaceae, and a third in Welwitschia (Li et al., 2015). While polyploidy is rare in gymnosperms (Murray, 2013), it is common in Sequioa and Cupressus, and—especially strikingly—the Gnetales (IckertBond et al., 2015). Ephedra is chromosomally highly variable, with counts ranging from 2n ¼ 14–56 and 69% of the 52 species being polyploid (Ickert-Bond et al., 2015). Ephedra is also the gymnosperm with the greatest variation in 1C-values, which range from 8.09–38.34 pg and include the largest genome of any gymnosperm, Ephedra antisyphilitica, with 2n ¼ 8x ¼ 56 and a 1C value of 38.34 pg (Ickert-Bond et al., 2015). The frequency of polyploidy in all clades of Ephedra may point to a particular role of chromosomal speciation in arid regions. The genome of Welwitschia mirabilis, with 2n ¼ 42 (Khoshoo & Ahuja, 1962, 1963) and a 1C-value of 7.20 pg (Leitch et al., 2001) 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 Early Cretaceous, insect-pollinated stem relatives diversiﬁed, subsequently declined to near-extinction in the Late Cretaceous, 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 documented 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). Just as insect visitation, bisexual cones also have been documented in all three genera and may represent the ancestral condition (Thompson, 1916; Endress, 1996; Haycraft € rgensen & & Carmichael, 2001; Mundry & Stützel, 2004; Jo Rydin, 2015). The organization of the bisexual cones differs among the three genera: in Welwitschia the male cones are made up of reproductive units that are structurally bisexual, with microsporophylls and a sterile ovule, whereas in Ephedra foeminea (the only Ephedra species with bisexual cones) and most species of Gnetum the male cones are made up of separate male reproductive units and sterile female reproductive units. It appears that the presence of sterile ovules with pollination formation in the male cones is linked to pollinator nectar rewards, and insect pollination may be the ancestral condition in the Gnetales (J€ orgensen & Rydin, 2015; Rydin & Bolinder, 2015; Bolinder et al., 2016). The signiﬁcance of insect pollination (and not just visitation) in Ephedra is highlighted by the discovery that the exact timing of pollination in E. foeminea in Greece and Croatia correlates with the full moon of July (Rydin & Bolinder, 2015). During peak full moon, all cones secreted pollination drops from the micropylar opening (Fig. 3C). When the moon was new, drop secretion was weak to non-existent, and even cones of the appropriate developmental stage produced no drops. Flies and moths feed on this species’ droplets and pollinate it in the process (Rydin & Bolinder, 2015), and apparently, the www.jse.ac.cn Fig. 7. Ant visitation on Ephedra trifurca in the Sonoran Desert, Arizona. A, Ovulate strobilus with pollination drop formation at the micropylar tube. B, Ant of Myrmecocystus cf. mimicus Wheeler, 1908 feeding on pollination droplet of ovulate cone in E. trifurca. C, Myrmecocystus cf. mimicus foraging in staminate cones of E. trifurca. D, Myrmecocystus cf. mimicus covered in Ephedra pollen. E, Detail of Myrmecocystus cf. mimicus head with Ephedra pollen grains indicated by arrowheads near the mandibles. F, G, Close-up of E showing details of characteristically polyplicate pollen grains of Ephedra and setae on Myrmecocystus cf. mimicus. Scale bars: A–C ¼ 10 mm; D ¼ 1 mm; E ¼ 200 mm; F, G ¼ 50 mm. pollination drops glittering in the full-moonlight help attract and guide these nocturnal insects. Further ﬁeldwork on the pollination of Ephedra, Gnetum, and Welwitschia is highly desirable. Ephedra pollination drops have relatively high sugar concentrations (Porsch, 1910; Moussel, 1980; Bino et al., 1984a,1984b, Meeuse et al., 1990; von Aderkas et al., 2015) and are produced by the nucellus (Rydin et al., 2010). The proteins that are found in trace amounts in these droplets (as in many gymnosperm pollination drops) are the product of nucellar breakdown during pollen chamber formation (i.e., degradome, von Aderkas et al., 2015), as well as export from the cytoplasm (i.e., secretome, von Aderkas et al., 2015). The micropyle for a typical species (E. distachya L.) is 1 mm in inner J. Syst. Evol. 54 (1): 1–16, 2016 10 Ickert-Bond & Renner minimum diameter and produces multiple pollination drops, each following a previous pollination episode, but droplet secretion ceases when the pollen tube reaches the pollination chamber (Moussel, 1980). Chemical analysis of pollination drops in 13 species representing the main lineages of extant gymnosperms (Ginkgo, cycads, Coniferales, and Gnetales) reveals a correlation between wind or insect pollination and total sugar content, fructose concentration, proline and amino acid concentration (Nepi et al., 2016 in review). Droplets of insectpollinated gymnosperms (Zamia furfuracea, Welwitschia mirabilis, Gnetum gnemon, Ephedra fragilis) and those of Ginkgo biloba and Ephedra minuta, whose pollination mode is unclear, have higher levels of carbohydrates, lower levels of amino acid, and speciﬁc sugars and amino acids proﬁles than gymnosperms shown to be wind pollinated in experimental studies. Most probably, insects shifted from ﬂuid feeding on the ovular secretions of gymnosperms to feeding on angiosperm nectar as the latter became abundant and species rich (Nepi et al., 2016 in review). Fertilization in the Gnetales Fertilization in the three lineages of the Gnetales is as different as their morphologies are. The process of double fertilization in angiosperm, that is, the regular fusion of one sperm nucleus with the egg nucleus and of the second sperm nucleus (from the same pollen tube) with two nuclei of the polar cell to form the triploid endosperm (Nawaschin, 1898; Guignard, 1899), was long considered a synapomorphy of ﬂowering plants and it was thought that fertilization in Ephedra might involve similar paired fusion events. Over the past 20 years, however, the work of Friedman and colleagues has revealed that both basal angiosperms and Gnetales have a range of fertilization modes. For example, in some Nymphaeales and Austrobaileyales the second sperm cell fuses with a single haploid polar nucleus so that the endosperm is diploid rather than triploid. Here we discuss ﬁndings only for the Gnetales. In Ephedra trifurca and E. nevadensis, the second sperm nucleus regularly fuses with the ventral canal nucleus within the egg cytoplasm (Friedman, 1990a, 1990b), while in Gnetum gnemon, free nuclei aggregate around the pollen tube (Friedman & Carmichael, 1996). Welwitschia mirabilis lacks a second fertilization event (Friedman, 2015). In addition, the three genera differ in their gametophyte development. The female gametophyte in Welwitschia shows tubular extensions (prothallial tubes) that grow through the nucellus to meet with downward growing pollen tubes (Friedman, 2015), and while Ephedra megagametophytes still contain archegonia and have a pollen chamber (similar to other gymnosperms), those of Gnetum and Welwitschia lack archegonia (Carmichael & Friedman, 1995; Friedman & Carmichael, 1996; Friedman, 2015). The evolution of endosperm remains unclear (Linkies et al., 2010), and further comparative cytogenetic studies of the fertilization events in Gnetales and other gymnosperms are needed. Seeds and Seed Dispersal in the Gnetales In the 48 species of Ephedra whose seed morphology has been studied, the outer seed envelope is smooth, slightly J. Syst. Evol. 54 (1): 1–16, 2016 striate or reticulate due to convex or depressed periclinal cell walls (Ickert-Bond & Rydin, 2011; Figs. 4F–4H). Micromorphology of the seed envelope is not useful for subclade delineation, and parallel evolution of similar micro-morphological patterns in unrelated groups is evident. Nevertheless, features of extant seed envelopes have been used to draw relationships between Early Cretaceous fossil seeds with afﬁnity to Ephedra (Yang et al., 2005). In recent work, however, Yang has re-interpreted the same fossil seeds as extinct stem relatives of extant species (Yang et al., 2015). Ephedra dispersal involves wind, birds, or terrestrial animals (Ickert-Bond, 2003; Ickert-Bond & Wojciechowski, 2004; Hollander & Vander Wall, 2009; Hollander et al., 2010; Loera et al., 2015). Wind-dispersed seeds have dry, winged bracts of the strobili; bird- and lizard-dispersed seeds are enclosed in rez et al., ﬂeshy, brightly colored bracts (e.g., Rodrıguez-Pe 2012), and seeds dispersed by seed-caching rodents are large and enclosed in dry, membranous bracts (Fig. 4E). Dispersal by wind and frugivores occurs in both Old and the New World species, while rodent 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; H. Freitag, University of Kassel, pers. comm. to SIB in 2015). Species dispersed by birds have higher phylogenetic niche divergence for mean annual temperature (and to a lesser extent mean annual precipitation) and occupy a broader set of temperature regimes than rodent-dispersed species (Loera et al., 2015), which has been attributed to the higher dispersal ability of birds compared to scatter-hoarding rodents (Hollander & Vander Wall, 2009; Loera et al., 2012, 2015). The shortdistance movement of seeds from the mother plant due to rodents may be especially beneﬁcial in arid conditions (Beck & Vander Wall, 2010; Vander Wall & Beck, 2012). Most species of Gnetum have yellow or red seed envelopes that are fed on by birds, rodents, or monkeys (e.g., Ridley, 1930; Markgraf, 1951; Kubitzki, 1985; Van Roosmalen, 1985; Forget et al., 2002). Catﬁsh also feed on the fruits when they fall into Amazonian rivers, and this may help upstream dispersal (Kubitzki, 1985). Dispersal by water has been inferred for species with a ﬁbrous endotesta, giving their seeds buoyancy (Kubitzki, 1985). Welwitschia seeds have wings that develop from the bracts, but due to their considerable weight they seldom become airborne (Fig. 6G, Bornman, 1978). In greenhouse experiments using wild-collected seeds from the Namib Desert, Whitaker et al. (2004) showed that removal of the bracts doubled germination success as compared to untreated seeds. Recent Studies of the Fossil Record Fossils of the Ephedra lineage are known from the late Mesozoic (Bolinder et al., 2016) with an increase of Ephedralike pollen during the Early Cretaceous (Crane & Lidgard, 1989) and numerous and diverse Ephedra-like plants reported from the Aptian (Krassilov, 1986; Yang et al., 2005; Rydin et al., 2006a; Wang & Zheng, 2010). During the Cretaceous diversity declined dramatically (Crane & Lidgard, 1989). Since the last comprehensive review of the Gnetales fossil record (Crane, 1996), a number of especially well-preserved fossils have come to light, most important among them the www.jse.ac.cn Biology and phylogeny of the Gnetales seedling Cratonia cotyledon from the Early Cretaceous of Brazil, which represents the Gnetales crown group and resembles Welwitschia (Rydin et al., 2003; Dilcher et al., 2005; Friis et al., 2014a). Additional seed fossils with afﬁnities to stem lineage Gnetales have also been described (Guo et al., 2009; Friis et al., 2014b). The Early Cretaceous Yixian Formation of Northeast China has yielded seeds described as Ephedra archaeorhytidosperma (Yang et al., 2005), a reproductive shoots of Liaoxia (Rydin et al., 2004), Siphosospermum simplex (Rydin & Friis, 2010), a ﬂeshy cone described as Ephedra carnosa (Yang & Wang, 2013), a leafy shoot system with reproductive organs described as Chengia laxispicata (Yang et al., 2013), and a macrofossil with strap-shaped leaves, reduced female cones, and seeds described as Ephedra multinervia (Yang et al., 2015). Yang (2014) has arranged all fossils of the Ephedra lineage into a single classiﬁcation, in a study that is also useful in bringing together much relevant literature. Since the taxa Ephedra and Ephedraceae have the same composition in the extant ﬂora, one might restrict the lower-rank name (Ephedra) to the crown group and the higher-rank name (Ephedraceae) to the crown group plus its stem relatives, to reﬂect that the extinct taxa are more closely related to Ephedra than they are to Welwitschia and Gnetum (Cantino et al., 2007; Doyle & Endress, 2014). Phase-contrast X-ray microtomography links charcoaliﬁed seeds from the Early Cretaceous (144 to 100 Ma) with the Gnetales but also the extinct seed plant lineages Bennettitales and Erdmanithecales (Friis et al., 2007, 2009). These seeds are c. 0.5–1.8 mm long and have two layers surrounding the nucellus: an inner, thin, membranous integument, formed by thin-walled cells; and a robust, outer, sclerenchymatous envelope that completely encloses the integument except for the micropylar opening. The integument itself is extended into a long, narrow micropylar tube, which bore the pollination droplets. Outlook Historically, the Gnetales have been critical to the development of hypotheses of cone origin (Arber & Parkin, 1908), and with new genomic tools they may continue to play this role. Modern detailed developmental studies of reproductive structures are mostly lacking for the Gnetales, and few taxa have been included in detailed developmental studies historically (Thoday & Berridge, 1912; Thompson, 1912, 1916; Thoday, 1921; Pearson, 1929; Martens, 1971; Takaso, 1984, 1985; Takaso & Bouman, 1986). Gene expression studies have already provided many insights into ﬂoral organ identity among seed plants and indicate that determination of reproductive organ identity is similar across this clade (Winter et al., 1999; Wang et al., 2010; Mathews & Kramer, 2012). They also imply that the last common ancestor of seed plants may have possessed the developmental machinery for speciﬁc ﬂoral organ identity (Theißen & Becker, 2004; Wang et al., 2010). Evolutionary developmental studies might shed light on the evolution of the diverse cone types in the Gnetales and help shed light on homologies with other gymnosperm cones. With the short generation time of some of its species, Ephedra could also become a gymnosperm model to underpin understanding of development and gene regulation in seed www.jse.ac.cn 11 plant evolution. Lastly, studies of Gnetales would beneﬁt from more ﬁeldwork and experiments on cone and bract function, and the role of insects in pollination. Noteworthy is that pollenkitt is supposedly lacking in Gnetales (Hesse, 1980, 1984), yet the pollen of all three genera is sticky (Bolinder et al., 2015b; pers. obs.). Acknowledgements We thank G€ unther Gerlach (Munich Botanical Garden), Helmut Freitag (University of Kassel), Moshira Hassan (Free University of Berlin), Marie Kurmann (Sursee, Switzerland), Stefan Little Paris-Sud), Jun Wen (Smithsonian Institution) and (Universite Elke Zippel (Berlin Botanical Garden and Botanical Museum) for micrographs and photos; Catarina Rydin, James A. Doyle, and Helmut Freitag for their insightful critique of the manuscript; Philip S. Ward (University of California, Davis) for the ant identiﬁcations; Douglas Walker, University of California at Davis Science Laboratory and Greenhouse, for taking care of the Ephedra farm; and Angelo Razeto for taking care of the Ephedra collection at the Munich Botanical Garden. The ﬁrst author thanks the German Academic Exchange Service (DAAD) for a one-year fellowship supporting her sabbatical. References Afzelius BM. 1956. Electron-microscope investigations into exine stratiﬁcation. Grana Palynologica 1: 22–37. 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The Gnetales: Recent insights on their morphology, reproductive biology, chromosome numbers, biogeography, and divergence times.

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