EMBRYOPSIDA Pirani & Prado

Gametophyte dominant, independent, multicellular, thalloid, with single-celled apical meristem, showing gravitropism; rhizoids +, unicellular; acquisition of phenylalanine lysase [PAL], phenylpropanoid metabolism [lignans +, flavonoids + (absorbtion of UV radiation)], xyloglucans +; plant [protoplasm dessication tolerant], ectohydrous [free water outside plant physiologically important]; cuticle +; cell wall also with (1->3),(1->4)-ß-D-MLGs [Mixed-Linkage Glucans]; chloroplasts per cell, lacking pyrenoids; glycolate metabolism in leaf peroxisomes [glyoxysomes]; centrioles in vegetative cells 0, metaphase spindle anastral, predictive preprophase band of microtubules, phragmoplast + [cell wall deposition spreading from around the spindle fibres], plasmodesmata +; antheridia and archegonia jacketed, stalked; spermatogenous cells monoplastidic; blepharoplast, bicentriole pair develops de novo in spermatogenous cell, associated with basal bodies of cilia [= flagellum], multilayered structure [4 layers: L1, L4, tubules; L2, L3, short vertical lamellae] + spline [tubules from L1 encircling spermatid], basal body 200-250 nm long, associated with amorphous electron-dense material, microtubules in basal end lacking symmetry, stellate array of filaments in transition zone extended, axonemal cap 0 [microtubules disorganized at apex of cilium]; male gametes [spermatozoids] with a left-handed coil, cilia 2, lateral; oogamy; sporophyte dependent on gametophyte, embryo initially surrounded by haploid gametophytic tissue, plane of first division horizontal [with respect to long axis of archegonium/embryo sac], suspensor/foot +, cell walls with nacreous thickenings; sporophyte multicellular, with at least transient apical cell [?level], sporangium +, single, dehiscence longitudinal; meiosis sporic, monoplastidic, microtubule organizing centre associated with plastid, cytokinesis simultaneous, preceding nuclear division, sporocytes 4-lobed, with a quadripolar microtubule system; spores in tetrads, sporopollenin in the spore wall, initially laid down in association with several trilamellar layers [white-line centred layers, i.e. walls multilamellate]; nuclear genome size <1.4 pg, LEAFY and KNOX1 and KNOX2 genes present, ethylene involved in cell elongation; chloroplast genome with close association between trnLUAA and trnFGAA genes.

Many of the bolded characters in the characterization above are apomorphies of subsets of streptophytes along the lineage leading to the embryophytes, not apomorphies of crown-group embryophytes per se.

All groups below are crown groups, nearly all are extant. Characters mentioned are those of the immediate common ancestor of the group, [] contains explanatory material, () features common in clade, exact status unclear.

STOMATOPHYTES

Abscisic acid, ?D-methionine +; sporangium - tapetum +, columella + [developing from endothecial cells], seta developing from basal meristem [between epibasal and hypobasal cells]; stomata +, anomocytic, cell lineage that produces them with symmetric divisions [perigenous]; underlying similarities in the development of conducting tissue and in rhizoids/root hairs; spores trilete; polar transport of auxins and class 1 KNOX genes expressed in the sporangium alone; shoot meristem patterning gene families expressed; MIKC, MI*K*C* and class 1 and 2 KNOX genes, post-transcriptional editing of chloroplast genes; gain of three group II mitochondrial introns.

[Anthocerophyta + Polysporangiophyta]: archegonia embedded/sunken in the gametophyte; sporophyte long-lived, chlorophyllous; sporophyte-gametophyte junction interdigitate, sporophyte cells showing rhizoid-like behaviour.

POLYSPORANGIOPHYTA†

Sporophyte branched, branching apical, dichotomous; sporangia several, each opening independently; spore walls not multilamellate [?here].

EXTANT TRACHEOPHYTA / VASCULAR PLANTS

Photosynthetic red light response; plant homoiohydrous [water content of protoplasm relatively stable]; control of leaf hydration passive; (condensed or nonhydrolyzable tannins/proanthocyanidins +); sporophyte soon independent, dominant, with basipetal polar auxin transport; lignins +; vascular tissue +, G- and S-type tracheids, sieve cells + [nucleus degenerating], tracheids +, in both protoxylem and metaxylem, plant endohydrous [physiologically important free water inside plant]; endodermis +; leaves spirally arranged, blades with mean venation density 1.8 mm/mm2 [to 5 mm/mm2]; sporangia adaxial on the sporophyll, derived from periclinal divisions of several epidermal cells, wall multilayered [eusporangium]; columella 0; tapetum glandular; gametophytes exosporic, green, photosynthetic; basal body 350-550 nm long, stellate array in transition region initially joining microtubule triplets; placenta with single layer of transfer cells in both sporophytic and gametophytic generations, root lateral with respect to the longitudinal axis of the embryo [plant homorhizic].

[MONILOPHYTA + LIGNOPHYTA]

Sporophyte branching ± indeterminate; root apex multicellular, root cap +, lateral roots +, endogenous; endomycorrhizal associations + [with Glomeromycota]; G-type tracheids +, with scalariform-bordered pits; leaves with apical/marginal growth, venation development basipetal, growth determinate; sporangia borne in pairs and grouped in terminal trusses, dehiscence longitudinal, a single slit; cells polyplastidic, microtubule organizing centres not associated with plastids, diffuse, perinuclear; blepharoplasts +, paired, with electron-dense material, centrioles on periphery, male gametes multiciliate; chloroplast long single copy ca 30kb inversion [from psbM to ycf2]; LITTLE ZIPPER proteins.

LIGNOPHYTA†

Sporophyte woody; lateral root origin from the pericycle; branching lateral, meristems axillary; cork cambium + [producing cork abaxially], vascular cambium bifacial [producing phloem abaxially and xylem adaxially].

SEED PLANTS†

Plants heterosporous; megasporangium surrounded by cupule [i.e. = unitegmic ovule, cupule = integument]; pollen lands on ovule; megaspore germination endosporic [female gametophyte initially retained on the plant].

EXTANT SEED PLANTS / SPERMATOPHYTA

Plant evergreen; nicotinic acid metabolised to trigonelline, (cyanogenesis via tyrosine pathway); primary cell walls rich in xyloglucans and/or glucomannans, 25-30% pectin [Type I walls]; lignins particularly with guaiacyl and p-hydroxyphenyl [G + H] units [sinapyl units uncommon, no Maüle reaction]; root stele with xylem and phloem originating on alternate radii, cork cambium deep seated; mitochondrial density in whole SAM 1.6-6.2[mean]/μm2 [interface-specific mitochondrial network]; stem with vascular cylinder around central pith [eustele], phloem abaxial [ectophloic], endodermis 0, xylem endarch [development centrifugal]; wood homoxylous, tracheids and rays alone, tracheid/tracheid pits circular, bordered; mature sieve tube/cell lacking functioning nucleus, sieve tube plastids with starch grains; phloem fibres +; cork cambium superficial; leaves with single trace from vascular sympodium [nodes 1:1]; stomatal pore with active opening in response to leaf hydration, control by abscisic acid, metabolic regulation of water use efficiency, etc.; axillary buds exogenous, (none; not associated with all leaves); prophylls two, lateral; leaves with petiole and lamina, development basipetal, blade simple; plant heterosporous, sporangia borne on sporophylls, sporophylls spiral; microsporophylls aggregated in indeterminate cones/strobili; grains monosulcate, aperture in ana- position [distal], primexine + [involved in exine pattern formation with deposition of sporopollenin from tapetum there], exine and intine homogeneous; megasporangium indehiscent; ovules with parietal tissue 2+ cells across, megaspore tetrad linear, functional megaspore single, chalazal, sporopollenin 0; gametophyte development initially endosporic, lacking chlorophyll, not photsynthesising, dependent on sporophyte, apical cell 0, rhizoids 0, development continuing outside the spore; male gametophyte with tube developing from distal end of grain, male gametes two, developing after pollination, with cell walls; female gametophyte initially syncytial, walls then surrounding individual nuclei; embryo cellular ab initio, endoscopic, plane of first cleavage of zygote transverse, suspensor +, short-minute, embryonic axis straight [shoot and root at opposite ends; plant allorhizic], cotyledons 2; plastid transmission maternal; ycf2 gene in inverted repeat, whole nuclear genome duplication [ζ - zeta - duplication], two copies of LEAFY gene, PHY gene duplications [three - [BP [A/N + C/O]] - copies], nrDNA with 5.8S and 5S rDNA in separate clusters; mitochondrial trans- nad2i542g2 and coxIIi3 introns present.

EXTANT GYMNOSPERMS / PINOPHYTA / ACROGYMNOSPERMAE

Biflavonoids +; cuticle wax tubules with nonacosan-10-ol; ferulic acid ester-linked to primary unlignified cell walls; phloem sieve area with small pores generally less than 0.8 µm across that have cytoplasm and E.R., joining to form a median cavity in the region of the middle lamella, Strasburger/albuminous cells associated with sieve tubes [the two not derived from the same immediate mother cell], phloem fibres +; sclereids +, ± tracheidal transfusion tissue +; stomatal poles raised above pore, no outer stomatal ledges or vestibule, epidermis lignified; buds perulate/with cataphylls; lamina development marginal; plants dioecious; microsporangium with exothecium; pollen tectate, infratectum alveolate [esp. saccate pollen], endexine lamellate at maturity, esp. intine with callose; ovule unitegmic, with pollen chamber formed by breakdown of nucellar cells, nucellus massive; ovules increasing considerably in size between pollination and fertilization, but aborting unless pollination occurs; ovule with pollination droplet; pollen grain lands on ovule, germinates in two or more days, tube with wall of pectose + cellulose microfibrils, branched, growing at up to 10(-20) µm/hour, haustorial, breaks down sporophytic cells; male gametophyte of two prothallial cells, a tube cell, and an antheridial cell, the latter producing a sterile cell and 2 gametes; male gametes released by breakdown of pollen grain wall, with >1000 cilia, basal body 800-900 nm long; fertilization 7 days to 12 months or more after pollination, to ca 2 mm from receptive surface to egg; female gametophyte initially with central vacuole and peripheral nuclei plus cytoplasm, cellularization/alveolarization by centripetal formation of anticlinal walls, the inner periclinal face open, with a single nucleus connected to adjacent nuclei by spindle fibres; seeds "large" [ca 8 mm3], but not much bigger than ovule, with morphological dormancy; testa mainly of coloured sarcoexotesta, scleromesotesta, and ± degenerating endotesta; first zygotic nuclear division with chromosomes of male and female gametes lining up on separate but parallel spindles, embryogenesis initially nuclear, embryo ± chlorophyllous; gametophyte persists in seed; plastid and mitochondrial transmission paternal; nuclear genome size 8-32(-76) pg [1 pg = 109 base pairs]; two copies of LEAFY gene [LEAFY, NEEDLY] and three of the PHY gene, [PHYP [PHYN + PHYO]], second intron in the mitochondrial rps3 gene [group II, rps3i2].

[GNETALES + PINALES]: tree branched; wood pycnoxylic; tracheid side wall pits with torus:margo construction, bordered; phloem fibres 0; axillary buds at at least some of the nodes; microsporangiophore/filament simple with terminal microsporangia; microsporangia abaxial, dehiscing by the action of the hypodermis [endothecium]; plastid ndh genes lost/pseudogenized.

PINALES Gorozh.

Resin ducts/cells in phloem in vascular tissue [and elsewhere]; lignins lacking syringaldehyde [Mäule reaction negative]; cork cambium ± deep seated; bordered pits on tracheids round, opposite; compression wood +; nodes 1:1; leaves with single vein; plants monoecious; pollen exine thick [³2 µm thick]; ovulate strobilus compound, ovuliferous scales flattened, ± united with bract scales; ovules lacking pollen chamber; pollen tube unbranched, growing towards the ovule, wall with arabinogalactan proteins, gametes non-motile, lacking walls, released from the distal end of the tube, siphonogamy; seed coat dry, not vascularized; proembryo with 2 to 4 nuclear divisions, with upper tier or tiers of cells from which secondary suspensor develops, elongated primary suspensor cells and basal embryonal cells [or some variant]; germination phanerocotylar, epigeal, (seedlings green in the dark); plastid and mitochondrial transmission paternal, one duplication in the PHYP gene line, one copy of chloroplast inverted repeat missing. - 7 families, 68 genera, 545 species.

Age. Davies et al. (2011: 95% credibility intervals) suggested an age for this clade of (259-)219(-174) m.y.; Magallón et al. (2013) suggested that it was about 312 m.y.o., while (259-)219(-174) m.y. is the age suggested by Clarke et al. (2011); the age of a [Gnetales + Pinales] clade is estimated at 181-140.1 m.y. (Naumann et al. 2013).

GNETALES Blume  Main Tree.

Lignin with syringyl units common [G + S lignin, positive Maüle reaction]; stem apex with tunica/corpus construction; roots diarch; bark with sclereids; gelatinous fibres [g-fibres] with innermost layer of secondary cell wall rich in cellulose and poor in lignin; protoxylem tracheids with large circular bordered pits, vessels + [from circular bordered pits], also in metaxylem, both fibre tracheids and tracheids +; phloem fibres 0; nodes 1:2, vascular traces leaving stele one internode below exit; two primary veins in leaves (and cone bracts); resin canals 0, mucilage cells +; stomata paracytic [mesogenous]; leaves opposite, joined at the base, overall growth ± diffuse/marginal/from basal plate, axillary buds serial, collateral; strobili compound [micro- and megasporangium-bearing structures closely associated, one not fertile], megasporangia apical, ovules with pollination droplets, bracts opposite; microsporangiate cones with sterile ovules, microsporangia in synangia, surrounded by a tubular "bract", dehiscing apically by the action of the epidermis [exothecium]; pollen not saccate, surface striate, tectate and with granular layer; ovulate cone scale 0, ovules terminal, surrounded by a vascularized connate structure ["outer integument"/seed envelope], papillae on the inner surface around the micropyle; ovule erect, integument with much-elongated beak, ca 2 cell layers across, not vascularized, micropylar tube with inner epidermis lignified, nucellar cap well developed; pollen reaches nucellus in less than 7 days, both sperm nuclei fuse with gametophytic cells ["double fertilization"]; first zygotic nuclear division with one spindle, tiered proembryo 0, free nuclear stage in which each nucleus forms an embryo, secondary suspensor developing from upper embryonal tier, no primary suspensor; germination epigeal, (seedlings green in the dark), cotyledons with connate bases; plastid transmission maternal; plastid ndh genes and rps16 gene lost, loss of PHY0 gene, mitochondrial coxII.i3 intron 0; nuclear genome C value 1.4-3.5 pg. - 3 families, 3 genera, 96 species.

Age. Estimates of the age of crown-group Gnetales are (202-)155(-104) m.y. with eudicot calibration (Smith et al. 2010: see also Table S3), slightly older without. Magallón et al. (2013) suggested an age of 140-120 m.y., Won and Renner (2006) ages of (196-)159(-132) m.y., and Ickert-Bond et al. (2010: 95% h.p.d.) ages of (192.3-)166.6(-90.6) m.y..

Ephedra and Welwitschia had diverged by 110 m.y. ago or more, the welwitschioid seedling, Cratonia, from Brazil, being of this vintage (Rydin et al. 2003), while pollen and seeds attributed to a welwitschioid plant are known from the Lower Cretaceous both in Portugal and eastern North America (Friis et al. 2014). Crane (1996) summarized the fossil history of Gnetales (see Won & Renner 2006; Rydin & Friis 2010 for additional references). For a probable Gnetalean fossil from the Permian, some 250-270 m.y.a., see Wang (2004). Both Ephedra and Welwitschia have polyplicate pollen that has a fossil record of ³250 m.y., being common from the Late Triassic onwards. Dilcher et al. (2005) noted that Gnetalean-like (striate/ribbed) pollen was common in both N. and S. Hemispheres; in the former, records are from the Upper Triassic onwards, in the latter, especially in the early Cretaceous from the northern half of South America. The pollen found by Wang (2004) associated with his fossil, Palaeognetaleana auspicia, is of this general kind. However, that fossil was radiospermic and had two complete integuments, a possible third integument being represented by scales, and the arrangement of parts in the cone was spiral, so what it represents is unclear.

Seeds of Ephedraceae are similar to those of Erdmanithecales (Rydin et al. 2006); for more detail see stem-group angiosperms and gymnosperm relationships.

Note: (....) denotes a feature common in the clade, exact status uncertain, [....] includes explanatory material. Possible apomorphies are in bold. However, the actual level at which many of these features, particularly the more cryptic ones, should be assigned is unclear. This is partly because many characters show considerable homoplasy, in addition, basic information for all too many is very incomplete, frequently coming from taxa well embedded in the clade of interest and so making the position of any putative apomorphy uncertain. Then there are the not-so-trivial issues of how character states are delimited and ancestral states are reconstructed (see above).

Evolution. Divergence & Distribution. Gnetales s.l., i.e., stem-group Gnetales and including the fossil groups above, show considerably more variation than perhaps might have been expected given the small size of the clade. In the Cretaceous in particular the diversity of Gnetales and the possibly related Bennettitales and Erdtmanithecale (all have an elongated micropyle, etc. - see Friis et al. 2011: chapter 5 and the Cycadales page), and several genera of Gnetalean affinity have been described from the Brazilian Crato formation, some 115-112 m.y.o. (Löwe et al. 2013 and references). Polyplicate (ephedroid) pollen was notably common 125-85 m.y.a. in middle latitudes (northern Gondwana), angiosperms and Gnetales perhaps growing togther then (Crane & Lidgard 1980; see also Friis et al. 2014).

Pollination Biology & Seed Dispersal. Ovules of all three extant genera - in Ephedra, including E. foemina, sister to the rest of the genus - secrete notably sucrose-rich pollination droplets (Ziegler et al. 1959: any further information?) and are visited by diptera, lepidoptera (moths), and other pollinators (see Kato & Inoue 1994; Labandeira 2005; Bolinder et al. 2015; Rydin & Bolinder 2015). For details of the time from pollination to fertilization, short for a gymnosperm, see Williams (2008 and references).

Genes & Genomes. The nuclear genome is small, C values being 1.4-3.5 picograms (Leitch et al. 2001, 2005). All three genera also have very small chloroplast genomes, Welwitschia rather less so than the others, and it has been suggested that this is because they grow in resource-poor environments, but genome size in Pinus, for example, may not be much bigger (see also C.-S. Wu et al. 2007, 2009 and references: other seed plants growing in similar environments?). Up to 18 genes have been lost from the chloroplast (McCoy et al. 2008; C.-S. Wu et al. 2009; Jansen & Ruhlman 2012 and references). Gnetales have a high substitution rates in their chloroplast genomes, dN/dS being lower than in other gymnosperms (B. Wang et al. 2015). Variation in the nad1 intron 2 needs clarification; it is absent in Welwitschia, present in Gnetum, and what is going on in Ephedra is not entirely clear (Gugerli et al. 2001).

Morphology, Anatomy, etc. Although vessels in Gnetum, for example, are commonly described as being derived from circular pits, this has been questioned (e.g. Rodin 1969; Muhammad & Sattler 1982). Although Rodin (1969) suggested that Gnetales lack pits with a margo-torus construction, they are clearly shown for Ephedra, but not Gnetum, by Eicke (1957). For gelatinous fibres (g-fibres), see Montes et al. (2012); in Ephedra, at least, their presence had nothing to do with bending and they are not associated with wood tissues, so they are not reaction wood (c.f. angiosperms; c.f. Tomlinson et al. 2014). There are nodal girdles of tissue very like transfusion tissue, at least in Ephedra (Beck et al. 1982). For the numbers of veins entering the leaves, see Rydin and Friis (2010). Boyce and Knoll (2002), Nardmann and Werr (2013), and others discuss leaf development; the scale leaves of Ephedra are reductions/

Interpretations of the parts of both the microsporangium- and megasporangium-bearing structures differ substantially (e.g. Gifford & Foster 1989; Hufford 1997a; Mundry & Stützel 2004). In microsporangiate plants of all three extant genera both stamens and non-functional ovules (although pollination droplets may still be produced) are closely associated, although this perhaps least marked in Ephedra (see also Flores-Rentería et al. 2011), and the microsporangiate cones can be interpreted as being compound (Mundry & Stützel 2004), rather like the megasporangiate cones of Pinales. The plants themselves are functionally dioecious. Gnetum ula is reported as having two sperm cells (Singh 1978). Plastid transmission appears to be maternal, at least in Ephedra distachya (Moussel 1978). The megaspore membrane is thin, but is definitely present (Doyle 2006).

For the morphology of Gnetales in the context of that of fossil gymnosperms, see e.g. Doyle and Donoghue (1986a, b) and especially Doyle (2006, 2008b, and references), for mycorrhizae, see Jacobson et al. (1993), and for pollen, see Osborn (2000: comparison with gymnospermous "anthophytes"), Yao et al. (2004: pollen of Gnetales compared with that of Nymphaea colorata), Rydin and Friis (2005: pollen germination) and Tekleva and Krassilov (2009: pollen morphology, inc. fossils). Martens (1971) provides an extensive treatment of the whole group (see also Gifford & Foster 1989), Friedman (1992), Carmichael and Friedman (1996) and Friedman and Carmichael (1997, and references) discuss double fertilization and Friedman (2015) that and much more, Carlquist (1997, 2012b) describes wood anatomy, Takaso (1985 and references) integument morphology, Endress (1997) details of megasporangiate structures, and Hufford (1997a) microsporangium arrangement.

Phylogeny. For general discussion on the relationships of Gnetales, see also above. As mentioned there, a position of Gnetum, etc., in or near Pinales is becoming increasingly credible. Although Rydin et al. (2002) strongly questioned the possibility of any paraphyly of Pinales, a [Gnetales + Pinales] clade - the Gnetifer hypothesis - has frequently been recovered (e.g. Samigullin et al. 1999: not all analyses; Antonov et al. 2000; Sanderson et al. 2000; Chaw et al. 2000; Gugerli et al. 2001: rather strong support; de la Torre et al. 2006: much hidden support, but not from the chloroplast partition; Wu et al. 2007; Rydin & Korall 2009: Bayesian analysis; Ran et al. 2010: the mitochondrial rps3 gene; C.-S. Wu et al. 2013: some analyses; Magallón et al. 2013; Rothfels et al. 2015b), and it is the preferred topology in Englund et al. (2011) and Groth et al. (2011).

Gnetales may even be placed within Pinales, in particular being sister to Pinaceae; this is the Gnepine hypothesis (e. g. Chaw et al. 2000; Bowe et al. 2000; Gugerli et al. 2001; Hajibabaei 2003; Burleigh & Mathews 2004, 2007c: supermatrix analyses; Hajibabaei et al. 2006: genes from all three compartments, but sampling?; Qiu et al. 2007; Graham & Iles 2009; Finet et al. 2010: quite strong support; Soltis et al. 2011). This topology was also found by Zhong et al. (2011, see also 2010; also C.-S. Wu et al. 2011b) when the most variable sites in concatenated alignments were removed, so reducing the LBA/heterotachy problem and by the concatenation-based transcriptome analyses of Wickett et al (2014).

Gnetales - on a long branch - were found to be sister to Cupressaceae, the Gnecup hypothesis, in an analysis of an amino acid matrix derived from chloroplast genomes (Zhong et al. 2010; see also Ruhfel et al. 2014); both quickly-evolving proteins and also proteins in which there appeared to be much parallel evolution in Cryptomeria and the branch leading to all Gnetales were removed. If they were not removed, a clade [Cryptomeria + Gnetales] was obtained (Zhong et al. 2010; see also Moore et al. 2011; C.-S. Wu et al. 2013). Similarly, an analysis of variation in 83 plastid genes strongly suggested a grouping [Pinaceae [Gnetales + other Pinales]], although other relationships could not be entirely rejected (Chumley et al. 2008; see also Ruhfel et al. 2014). Finally, Raubeson et al. (2006) found that Welwitschia grouped with Podocarpus, but this may be due to rate heterogeneity.

Xi et al. (2013b), using much nuclear and plastid data, although they included only ten gymnosperms, found a poorly to moderately supported [Gnetum + Pinaceae] clade in analyses of nuclear genes only. In analyses of chloroplast data a relationship with Cupressaceae was preferred (see also Davis et al. 2014a for the influence of different genomes); in both cases the alternative topology was rejected with a p-value of 0.001. This suggested to Xi et al. (2013b) that the two genomes of Gnetum had different histories. See also X.-Q. Wang and Ran (2014) for discussion; they noted that analyses of different classes of genes resulted in different topologies,.

Thus despite a number of unresolved issues, a position somewhere around Pinales seems most likely for Gnetales. There are some specific points of genomic similarity between Gnetum, etc., and some or all Pinales. Some Pinaceae have lost a number of the chloroplast genes that are missing in Gnetales (Wu et al. 2009). All eleven NADH dehydrogenase genes in the chloroplast of Pinus thunbergii are absent - or are present, but as pseudogenes (Wakasugi et al. 1994); other work suggests that these genes are absent in all Gnetales and Pinales alone (Braukmann et al. 2009, also 2010; Martín & Sabater 2010; Wicke et al. 2011). The rps16 gene in Gnetales and Pinaceae is commonly lost (Wu et al. 2007, 2009). All Pinales sampled have but a single copy of the chloroplast inverted repeat (Strauss et al. 1988; Tsudzuki et al. 1992); nearly all other seed plants have two copies (Raubeson & Jansen 1992; Lackey & Raubeson 2008), and this may be marked by micromorphological changes in the genome. Interestingly, one end of the inverted repeat of Welwitschia has expanded (Welwitschia is derived within Gnetales) with duplication of trnI-CAU and partial duplication of pscbA gene region at the end of the Large Single Copy region, and these match those of the remnant inverted repeat known from Pinus and other Pinaceae, but not other members of Pinales (Margheim et al. 2006; McCoy et al. 2006, 2008: details of relationship depend on methods of analysis; see also Braukmann et al. 2009; Hirao et al. 2009).

There are also some morphological similarities between Pinales and Gnetales, and within the former, perhaps particularly with Pinaceae. The binucleate sperm cells, basic proembryo structure, development of polyembryony, etc., of Ephedra agree with Pinales in general and perhaps Pinaceae in particular. Some Pinus species have mesogenous stomata in which the subsidiary cells are produced from the same initial that gives rise to the guard cells (Gifford & Foster 1989; see also Mundry & Stützel 2004), as in Gnetales. Strobili with both micro- and megasporangia are common as abnormalities in Pinales (Chamberlain 1935; Rudall et al. 2011a) and occur normally in Gnetum. However, wherever Gnetales are placed, they will have numerous apomorphies. Thus although nearly all Pinales have megasporangiate strobili with spirally-arranged ovuliferous scales or modifications of them, Gnetales have decussating bracts (Magallón & Sanderson 2002); loss of the ovuliferous scale, etc., might also be apomorphies (Finet et al. 2010).

However, given the uncertainty in our knowledge of the relationships between the major seed-plant clades, direct links are provided to Cycadales, Ginkgoales, flowering plants, and Pinales here.

Within Gnetales relationships are [Ephedra [Gnetum + Welwitschia]] (e.g. Price 1996).

Classification. If the evidence continues to point to a [Pinaceae + Gnetales] clade, Gnetales will disappear.


Ephedraceae Welwitschiaceae Gnetaceae Gnetales

Includes - Ephedraceae, Gnetaceae, Welwitschiaceae

Synonymy: Ephedrales Dumortier, Tumboales Wettstein, Welwitschiales Reveal - Ephedridae Reveal, Gnetidae Pax, Welwitschiidae Reveal - Ephedropsida Reveal, Gnetopsida Thomé, Welwitschiopsida B. Boivin - Gnetophytina Reveal - Gnetophyta Bessey

EPHEDRACEAE Dumortier Back to Pinales

Xeromorphic small trees and shrubs (climbers); cyclopropyl amino acids +; nodes 1:2; leaves much reduced (not); (microsporangiate cones lacking sterile ovules), microsporangiophores with 2-8 synangia, each with 2(-4) sporangia, dehiscence porose; pollen inaperturate, smooth, pseudosulcate, pseudosulci unbranched (with short branches at right angles); micropyle blocked by mucilaginous secretion; pollen exine shed during microgametophyte germination [microgametophyte naked]; pollen germinates in 1-2 hours, reaches nucellus in 10-16 hours, one gamete fuses with nucleus of ventral canal cell; archegonia exposed at base of deep pollen chamber formed by breakdown of overlying nucellar tissue, archegonial neck very long; (bracts below ovule become fleshy); seed with papillae on the inner side of the outer covering; n = 7, polyploids [to octaploids] frequent; 1C genome size 8.1-38.3 pg; loss of two more group II mitochondrial introns.

Ephedraceae

1/65. North (warm) temperate, W. South America; drier habitats (map: see Frankenberg & Klaus 1980; Caveney et al. 2001). [Photos - Ripe seed, Megasporangia, Habit, Microsporangia, Dwarf plant.]

Age. Ickert-Bond et al. (2009; see also Rydin & Ickert-Bond 2010; Rydin et al. 2010) estimate that divergence with the genus occurred quite recently, only (73.5-)30.4(-20.55) m.y.a. (see also Huang & Price 2003: 32-8 m.y.), but c.f. the fossil record below.

Fossils apparently assignable to Ephedraceae are known from the lower Cretaceous in China (Zhou et al. 2003). Rothwell and Stockey (2009) report a fossil from the Lower Cretaceous that has purportedly ancestral characters for Ephedra - two ovules together, and absence of a tubular micropyle and of a structure surrounding the ovule (seed envelope above), but this is unlikely to be assignable to crown group Gnetales (other Ephedra from this period may have three ovules - Yang & Wang 2013). The distinctive pollen of Ephedra has been found inside fossil seeds that are morphologically also Ephedra in late Aptian to Early Albian (early Cretaceous) deposits from Portugal, suggesting that diversification in the genus occurred some 127-110 m.y.a. (Rydin et al. 2004). Indeed, Early Cretaceous fossils of Ephedra have a "modern" morphology, E. paleorhytidosperma having distinctive seeds very like those of the extant E. rhytidosperma (Yang et al. 2005). The dating situation is further confused because derived pollen of Ephedra, i.e. with laterally-branched pseudosulci, is known from the Cretaceous-Raritan (Bolinder et al. 2015b), i.e. from around 95 m.y. ago.

Evolution. Divergence & Distribution. The history of Ephedra has a number of surprises. Polyplicate (ephedroid) pollen was notably common 125-85 m.y.a. in northern Gondwana (Crane & Lidgard 1980; see also Friis et al. 2014), and Ephedra (or ephedroid) seeds with a variety of morphologies are known from both South America and East Asia, and include fleshy fruits from 125-120 m.y. (Y. Yang & Wang 2013). The genus went into a severe decline at the end of the Cretaceous, the crown group age being Oligocene or younger. Extant taxa show little genetic divergence and most relationships have little support (Rydin et al. 2010). Ephedra moved from the Old to the New World in the Oligocene (41.5-)29.6(-8.8) m.y.a. and to South America in the Miocene (Ickert-Bond et al. 2009). A shift from entomophily to anemophily may perhaps be connected with Caenozoic diversification in the clade (Bolinder et al. 2012). There has been parallel evolution in micromorphological details of the seed envelope (Ickert-Bond & Rydin 2011).

Pollination Biology & Seed Dispersal. Pollination in extant species is mostly by wind (Niklas 2015). However, Ephedra foemina is pollinated by insects and i.a. has pollen with a faster settling velocity than that of wind-pollinated taxa, while some fossil "ephedroid" pollen also has characteristics of insect pollination with a thick tectum and dense infratectal layer (Bolinder et al. 2015a; c.f. Hall & Walter 2011 in part). Pollination in E. foemina is probably by nocturnal moths, pollen being released around the time of the full moon, both the sterile ovules in male strobili and the ovules in female strobili producing pollination droplets (Rydin & Bolinder 2015).


Because the pollen exine of Ephedra is shed on germination (shed eximes can be seen in some fossils - Bolinder et al. 2015b), the male gametophyte is naked. Fertilization occurs 10-15 hours after pollination.

As the seeds ripen, the "outer integument" surrounding the ovule may become fleshy and brightly coloured, or it may dry and become a wing, or it may be faintly nondescript, the seeds then being dispersed by scatter-hoarding rodents (Hollander & Vander Wall 2009).

Genes & Genomes. There has been a great increase in the rate of synonymous substitutions in the mitochondrial genome, and chloroplast and nuclear sequences are also divergent compared with those of other seed plants (Mower et al. 2007 and references).

The nuclear genome is very variable in size, and can be huge (Ickert-Bond et al. 2014b). This is connected with extensive variability in chromosome numbers, unusual for gymnosperms, linked with polyploidy, but not with subsequent genome downsizing, unlike the common situation in angiosperms (Ickert-Bond et al. 2015).

Chemistry, Morphology, etc. Species of Ephedra are pharmacologically very active and contain a number of distinctive secondary metabolites (Caveney et al. 2001). (

Biswas and Johri (1997) mention the "deep origin of the periderm", a position that should be confirmed. Early Cretaceous fossils are described as having a dichasial branching pattern and linear leaves with two parallel veins (Y. Yang & Wang 2013). For leaf and nodal anatomy of extant species of Ephedra with well developed leaves, i.e. long and linear, see Dörken (2014) and Deshpande and Keswani (1963); the xylem of the two vascular bundles faces each other, and the leaf has been ventralised.

For some general information, see Rydin et al. (2004) and the Gymnosperm Database, and for nodal anatomy, see Marsden and Steeves (1955) and Singh and Maheshwari (1962).

Phylogeny. There is little strong phylogenetic structure along the backbone of a 7 plastid gene-2 compartment analysis of extant species of Ephedra, indeed, there is notably little molecular divergence within the genus (Rydin & Korall 2009; Rydin et al. 2010: see also above). The insect-pollinated Ephedra foeminea (see above) may be sister to the rest of the genus.

Clasification. For a classification of Ephedraceae, including fossil members, see Yang (2014).

[Gnetaceae + Welwitschiaceae]: cyclopropenoid fatty acids in seed oil, polysaccharide gums +; (successive cambia in roots); pits lacking margo-torus construction; nodes multilacunar, three [or more] primary veins proceeding to the leaves; branched sclereids +; cataphylls 0; leaves with second order venation; microsporangiate strobili with abortive apical ovules; male gametophyte with one ephemeral prothallial cell, sterile cell absent; micropyle blocked by tissue from expanded integument [by periclinal cell divisions, also radial cell expansion]; female gametophyte tetrasporic in development, chalazal portion densely cytoplasmic, nuclei scattered, wall formation not centripetal, cells enclosing groups of nuclei that later fuse, archegonia not obvious; ovules with additional pair of bracts; no archegonia per se; both male gametes fuse with egg nuclei; embryo cellular, some cells of embryonal mass elongate, (cleavage polyembryony +), embryo with lateral protrusion of the hypocotylar axis ["feeder"]; germination hypogeal.

Age. Ickert-Bond et al. (2010: 95% highest posterior density) suggest ages of (127-)111.3(-87.2) m.y. for divergence within this clade, Won and Renner (2006) ages of (175-)138(-112) m.y., and Magallón et al. (2013) an age of around 81.9 m.y.; on the other hand, the age in Magallón et al. (2015: note topology) was around 239 m.y. ago. See below for fossils placed in Welwitschiaceae.

Siphonospermum, a fossil from the Lower Cretaceous from Northeast China, may be assignable to this part of the tree (Rydin & Friis 2010).

Chemistry, Morphology, etc. For cyclopropenoid acids, similar to those in Malvales, see Aitzetmüller and Vosmann (1998). Rodin (1968) suggested that the reticulate venation of Gnetum, at least, was a modified dichotomizing system.

GNETACEAE Blume Back to Pinales

Gnetaceae

Plant trees or lianoid, ectomycorrhizae +; (Si02 accumulation - Gnetum gnemon]); (successive cambia in shoots - climbers); vessel elements with vestured pits; sieve tubes with companion cells [derived from different cells]; laticifers +; leaves petiolate, lamina hierarchical-reticulate venation [more than two orders], development dispersed, veins (4.4-)5.7(-7.4) mm/mm2; (plant monoecious), ovules and microsporangiophores at same node in staminate plant; microsporangiophore with (1-)2(-4) sporangia; pollen surface spinose; additional ovule envelope formed by connate bracts ["integument"]; pollen reaches nucellus in up to 7 days, both gametes fuse with nuclei in the syncytium; outer ovule envelope becomes fleshy; embryo with elongated suspensor tubes initially formed, nucleus at end divides forming a embryonal mass; n = 11; 2C genome size 4.5-8 pg, one copy of the LEAFY gene.

1/30. Tropical, rather disjunct (map: see Renner 2005b). [Photos - Collection]

Age. Crown-group Gnetaceae are (77-)44-26(-13) m.y.o., or using a strict clock, as little as 6 m.y. (Won and Renner 2006); (98-)81(-64) m.y. is the age suggested by C. Hou et al. (2015).

Evolution. Divergence & Distribution. For biogeographical relationships in the genus, basically a story of post-Eocene diversification and dispersal, see Renner (2005b) and Won and Renner (2006).

Pollination Biology & Seed Dispersal. Entomophily has been reported from Malesian species of Gnetum (Kato & Inoue 1994).

Bacterial/Fungal Associations. Brundrett (2008, seen viii.2012) summarizes information on the mycorrhizal status of members of Gnetales as a whole.

Genes & Genomes. Horizontal gene transfer of the mitochondrial nad1 intron 2 from flowering plants (an asterid) to an Asian clade of Gnetum seems to have occurred within the last 5 m.y. (Won & Renner 2003).

Chemistry, Morphology, etc. Not surprisingly, the wood of the lianoid taxa is distinctive, with serial cambia being formed. The reaction wood in Gnetum consists of gelatinous extra-xylary (reaction) fibres in the adaxial position (Tomlinson 2001b, 2003; see also Höster & Liese 1966); it is not typical tension wood. See Martens (1971) for the vascularization of the leaves; pairs of vascular bundles leave the central stele in close proximity.

There is vascular tissue in the two outer coverings of the ovule, but vascular bundles barely enter the base of the inner integument. The outer covering in definitely bilobed early in development, the lobes alternating with bracts, but the middle covering is only weakly bilobed (Takaso & Bouman 1986). Although some gametophyte development occurs after fertilization, the ovule increases appreciably in size between pollination and fertilization (Leslie & Boyce 2012).

For reproductive morphology and development, see Sanwal (1962), for mycorrhizae, see Onguene and Kuyper (2001), and for general information, see the Gymnosperm Database.

Phylogeny. Relationships within the genus are strongly correlated with geography, i.e. [South America [Africa + S.E. Asia-Malesia]] (Won & Renner 2006), somewhat elaborated as [South America [Africa [S. Asia - 2 arborescent spp. + The Rest]]], diversification being much older than in Ephedra (C. Hou et al. 2015).

Synonymy: Thoaceae Kuntze

WELWITSCHIACEAE Caruel Back to Pinales

Welwitschiaceae

Stem apex lacking tunica-corpus construction?; ?bark; fibre tracheids 0; successive cambia in root, derived from phelloderm; no typical vascular cambium in stem; leaf traces in cortex?; sclereids with crystals in wall; leaves amphistomatic; stem apex aborts, plant three pairs of leaves, second pair of leaves persisting for the life of the plant, leaf development from a basal cambium, venation parallel; ovules and microsporangiophores in intimate association; microsporangiophores 6, basally connate, with synangia of three sporangia, dehiscence radial; additional bracts free; only one sperm nucleus functional, fertilization in prothallial tube; whole female gametophyte densely cytoplasmic, nuclei scattered, wall formation not centripetal, cells enclosing groups of nuclei that later fuse, micropylar cells with separate nuclei, growing upwards through nucellus forming prothallial tubes; seed with seed envelope forming membranous wing; proembryo pushed back down gametophytic tube by elongating embryonal suspensor; seedling with collar; n = 24; 2C genome size ca 13.1 pg, 2nd intron in nad1 lost.

1/1: Welwitschia mirabilis. S.W. Africa, desert close to the Atlantic ocean. [Photos - Collection.]

Evolution. Divergence & Distribution. Cratonia cotyledon is a fossil seedling with distinctive cotyledon vasculature very like that of the leaves of Welwitschia, the secondary veins leaving from the primary veins fuse to form an inverted "Y" (Rydin et al. 2003). Cratonia was found in N.E. Brazil and is late Aptian or early Albian in age, perhaps 114-112 m.y. old; other fossils of welwitschiaceous or more generally gnetalean affinity have been found in the same area (Dilcher et al. 2005; Löwe et al. 2013).

Ecology & Physiology. Welwitschia mirabilis grows in the Namib desert close to the ocean; although there is little rain, fogs are frequent - but not where Welwitschia grows (von Willert 1985). Plants may be some hundreds of years old, the two persistent leaves growing at the base and fraying at the apex.

Pollination Biology & Seed Dispersal. Pollination appears to be by diptera (Wetschnig & Depisch 1999; Bolinder et al. 2015 and references).

Genes & Genomes. The chloroplast genome of Welwitschia mirabilis is the smallest plastid genome of all non-parasitic land plants that still have inverted repeats (McCoy et al. 2008).

Chemistry, Morphology, etc. Because of the abundant, branched sclereids in the plant, "One might as well try to cut sections of a thick Scotch plaid blanket as to try and cut a stem of Welwitschia without imbedding." (Chamberlain 1935: pp. 388-389).

Kaplan (1997, vol. 1:6) described the seedling as having a haustorial collet (collar). Serial axillary buds are added throughout the course of the long life of the plant, the youngest buds being in the centre of the axil - a branch-like organization? See Martens (1971) for the vascularisation of the bracts of the megasporangia and the complex organisation of the axis of the megasporangiate strobilus.

For female gametophyte development and fertilization, see Friedman (2014, esp. 2015: remarkable), for general information, see the Gymnosperm Database.

Synonymy: Tumboaceae Wettstein