A Whole Plant Herbaceous Angiosperm from the Middle Jurassic of China

In contrast to woody habit with secondary growth, truthful herbaceous habit lacking secondary growth is restricted to angiosperms among seed plants. Although angiosperms might have occurred as early as in the Triassic and herbaceous habit theoretically may have been well adopted by pioneer angiosperms, pre‐Cretaceous herbs are missing hitherto, leaving the origin of herbs and evolution of herbaceous angiosperms mysterious. Here we report Juraherba bodae gen. et sp. nov, a whole plant herbaceous angiosperm, from the Middle Jurassic (>164 Ma) at Daohugou Village, Inner Mongolia, China, a fossil Lagerstätten that is worldwide famous for various fossil finds. The angiospermous affinity of Juraherba is ensured by its enclosed ovules/seeds. The plant is small but complete, with physically connected hairy root, stem, leaves, and fructifications. The Middle Jurassic age recommends Juraherba as the earliest record of herbaceous seed plants, demanding a refresh look at the evolutionary history of angiosperms.

information sources for palaeobotanical studies because reconstructions based on separately preserved parts may be risky and is frequently inflicted by hard-to-detect artifacts and errors (Rothwell et al., 2009;Tekleva and Krassilov, 2009). Here we report an herbaceous angiosperm, Juraherba bodae gen. et sp. nov., with all parts physically connected from the Jiulongshan Formation (Jurassic, >164 Ma) of Inner Mongolia, China (Chen et al., 2004;Ji et al., 2005;Zhou et al., 2007;Pott et al., 2012). The herbaceous plant habit and enclosed ovules/seeds pin down the angiospermous affinity for Jurahbera. This discovery will re-shape the current thinking on the origin of herbaceous habit in seed plants and its role in the evolution of angiosperms.

Samples and Methods
The fossil is preserved as a compression specimen with coalified residues embedded in tuffaceous siltstone, and thus resulting in an impression, too. The specimen was photographed using a Nikon D300 digital camera. Details of the fossil were observed and photographed using a Leica MZ-16A stereomicroscope with a DFC290 digital camera. Afterward replicas of nitro cellulose (Zhu, 1983) were made on the specimen, and the replicas were cleaned with HCl and HF, coated with gold, and observed using a Leo 1530 VP SEM (scanning electron microscope) at the Nanjing Institute of Geology and Palaeontology (NIGPAS), Nanjing, China. The surface of the whole specimen was scrutinized usin g SEM and Leica fluorescent microscope to search for any trace of in situ pollen, but in vain, although many pollen grains were found in the adjacent sediment. One of the fructifications was dissected to expose the internal details (includ ing seed) inside. Fluorescent observation and micrography were performed using a Leica DM5000B using green fluorescent light. All photographs were saved in TIFF format and organized together for publication using Photoshop 7.0.
Generic diagnosis: Her baceous plant , small, including physically connected roots, stem, leaves, and fructifications. Root minute, borne on the bottom of the   Specific diagnosis: as of the genu s. Description: The fossil is pr eser ved as an impr ession/ compression embedded in grey tuffaceous siltstone, with some coalified residue preserved in the lower portion and fructifications (Fig. 2a). The fossil includes physically connected roots, a stem, leaves, fructifications, and is associated with an insect fossil (Fig. 2a) and pollen clumps (Fig. 5n). It is 38 mm long and 12 mm wide, including at least twelve leaves and four fructifications (Fig. 2a). The lower port ion is delimited from the above by a constriction, oval-shaped, 0.79 mm high and 1.16 mm wide, with scales and hairy roots (Figs. 2c, 3d-3e, 3g-3h). The scales have integral smooth surfaces and bear vertically inserted hairy roots up to 121 μm long and 33 μm wide (Figs. 3e-3h). The stem bears helically arranged leaves, with irregularly wrinkled surface (Figs. 2a,2b,2e,3i). The leaves are simple, linear, up to 40 mm long and only 1.3 mm wide, with a midvein, entire-margined, with acute tips, usually eclipsing the stem, and older leaves tend to abscise at their bases (Figs. 2a,2b,3i,3j,5n). The midvein is 0.3 mm wide and the lateral zone is 0.42 mm wide in the middle portion of the leaves, both   Labandeira et al. (2007)) are seen on the leaves (Figs. 2f, 2g, 5o). Four fructifications are inserted basally, arranged nearly at the same horizontal level (Figs. 2a,3d). The fructifications are fusiform, 2.2-4.1 mm long and 1.4-2.2 mm wide, borne on long pedicels, and surrounded by foliar parts (Figs. 2a, 2d, 5a-c). Different foliar parts distinguish each other by their surface texture and may have stomata (Figs. 4c, 5b). The pedicels are 14-15.5 mm long, longitudinally ridged, with sparsely helically arranged scaly leaves (Figs. 4b, 5a, 5e, 5i). The fructification terminus has an irregu lar margin, suggestive of an abscised distal part (Fig. 5h). Each fructification has several longitudinal rid ges and irregu lar wrinkles on its surface (Figs. 2d, 5a -5c, 5f, 5g, 5i). Inside one of the fructifications an ovule/seed 339 μm long is seen anchored to an internal structure and embedded in the fructification tissue (Figs. 5c, 5d). Another ovule appears in the same fructification (Fig.   5m). An oval body (possible seed) is seen in another fructification after removing the covering tissue (Figs. 5i-5l).
Etymology: bodae, dedicated to Boda (the Chinese abbreviation of Bohai University), the affiliation of the first author.
Holotype: PB21415, deposited in the Nanjing Institute of Geology and Palaeontology, Chinese Academy of Sciences, Nanjing, China.
The irregularly wrinkled surfaces of the stem and fructifications form a strong contrast against the relatively smooth leaf surface, implying the volume of these parts has been reduced during the fossilization and these parts used to be more or less fleshy in live form.

Interpreting structures
The fructifications surrounded by foliar parts without any trace of spores in Juraherba are clearly different from sori or sporangia of ferns, in which sori and sporangia are closely related to pinnae except in Salviniales (Haupt, 1953;Smith et al., 2006). Fructification pedicels with Feb. 2016 ACTA GEOLOGICA SINICA (English Edition) http://www.geojournals.cn/dzxben/ch/index.aspx http://mc.manuscriptcentral.com/ags scaly leaves are distinct from smooth setae or pseudopodia of bryophytes (Gradstein et al., 2001), therefore the fructifications of Juraherba cannot be interpreted as capsules of bryophytes. These differences are sufficient to distingu ish Juraherba from bryophytes and most ferns.
The general morphology of Juraherba might appear similar to a short shoot of Czekanowskiales (includ ing Phoenicopsis, Czekanowskia, and Tianshia), which are frequently seen in the Mesozoic (Zhou and Zhang, 1998;Sun et al., 2009). However, the single vein and acute leaf tip of Juraherba are distinct from the multiple veins and rounded leaf tips of Phoenicopsis and Tianshia (Zhou and Zhang, 1998). The linear, non-branching leaves of Juraherba are distinct from the filiform, branched leaf of Czekanowskia (Sun et al., 2009). Furthermore the distinction between the terminal fusiform fructifications surrounded by foliar parts in Juraherba and lateral naked bivalvate units in Leptostrobus (Czekanowskiales) eliminates any relationship between them. If belonged to Czekanowskiales, then the base of Juraherba should be truncated and broken with rough surface and no hairs. However, the base of Juraherba has an integral surface (Fig. 3f) and hairy roots (Figs. 3e, 3 g, 3h). All these convince us that Juraherba is a whole plant rather than a short shoot of any plants.

Herbaceous habit
The whole plant of Juraherba is only 38 mm tall, implying an herbaceous habit. This conclusion is supported by comparison with other b ig herbaceous and small woody plants. On one hand, primary gro wth may produce organs bigger than those of Juraherba. For example, the early land plant Rhynia may produce an axis up to 3 mm in diameter (Edwards, 2003), in contrast to only 1.16 mm wide lower portion of Ju raherba. This contrast suggests that the size of Juraherba is with in the scope of primary growth. On the other hand, plants with litt le secondary growth have axes much thicker than Juraherba. The lower portion of Juraherba is much smaller than that of the so-called "herbaceous" conifer (Aethophyllum stipulare, Rothwell et al., 2000, their Plate I, Fig. 3,), the fertile one of the latter is more than 30 cm tall, suggesting that smaller Juraherba has no secondary growth. Similarly, shrubby Ephedra (Gnetales) is much bigger than Juraherba and does have an active cambium (Martens, 1971). Therefore actually there is no truthful herbaceous gymnosperm without secondary growth. These comparisons indicate that mature Juraherba (as implied by its fructifications) is truthfully herbaceous. Thus at least Juraherba can be distingu ished from all known gymnosperms by its herbaceous habit (Bierhorst, 1971;Biswas and Johri, 1997).
The tiny size and herbaceous habit suggests that Juraherba has a short life cycle, an ecological strategy that is paramount for the success of angiosperms (Stebbins, 1981) and adopted by some basal angiosperms such as the Hydatellaceae (Gandolfo et al., 1998;Saarela et al., 2007). This is in line with the ecophysiological analyses of extant and fossil plants, which conclude that herbaceous habit may well have been adopted by early angiosperms (Stebbins, 1981;Taylor and Hickey, 1990, 1992, 1996Carlquist, 1996;Royer et al., 2010). This conclusion has been favored by recent studies on Early Cretaceous plants (Jud, 2015;Friis et al., 2015). Juraherba unequivocally confirms that early angiosperms have already adopted this strategy at least back to the Middle Jurassic (Figs. 7a-7c), although their success did not come until much later. The discovery of Juraherba adds first-hand material for studying the origin and evolution of herbs, favoring the Paleoherb Theory that was advanced more than decades ago.

Affinity
The oval bodies inside the fructification are of crucial importance for the affinity of Juraherba. The oval bodies in the fructification seen in Figs. 5d and 5l cannot be interpreted as either microspores or pollen grains because of their large size (over 300 μm long, falling well within the scope of megaspores (Bateman and Dimichele, 1994)), therefore they have to be interpreted as seeds/ovules/ megaspores rather than microspores/pollen. Besides seed plants, megaspores are seen in Lycophyta, Sphenophyta, and Salviniales (Scott, 1962;Smith et al., 2006;Taylor et al., 2009). The reproductive organs of Lycophyta and Sphenophyta are usually organized in cone-like structures (Scott, 1962;Taylor et al., 2009), and completely different from Juraherba. Salviniales can be easily distingu ished from Juraherba by their leaf morphology (Smith et al., 2006). Another possibility is insect egg laid inside the fructification. The shape of the oval body in Juraherba (Fig. 5d) is not symmetrical, as expected for an insect egg. Instead the oval body is slightly elongated at the bottom, anchored and embedded in the fructification tissue in Fig.  5d. This interpretation is further strengthened by another possible ovule embedded in the same fructification seen in Fig. 5m, in which a mass of dense organic material is anchored by a pedicel and its lack of obvious testa implies that it is either an ovule or a seed in its very early development. Finally, one more oval body that was originally covered inside the fructification became exposed after the covering tissue was removed . Its large size (over 200 μm long) comparable to the two above possible ovules (Figs. 5d, 5m) rather than microspores or pollen grains implies that it may be an ovule or a seed. Its Feb. 2016 Vol. 90 No. 1 25 ACTA GEOLOGICA SINICA (English Edition) http://www.geojournals.cn/dzxben/ch/index.aspx http://mc.manuscriptcentral.com/ags spatial position within the fructification (angiospermy) is rather sign ificant because it is a feature expected for angiosperms. The irregular scar at the fructification tip (Fig. 5h) suggests that there used to be a distal part (probably style) in Juraherba, in agreement with the occurrence of angiospermy/angio-ovuly in Juraherba. Abscised styles are frequently seen in angiosperms after pollination but never in gymnosperms (Goldschmidt and Leshem, 1971;Simons, 1973;Keighery, 2004;Liu et al., 2009), it is logical to state that Juraherba is a Middle Jurassic angiosperm. It is noteworthy that, among angiosperms, Juraherba resembles Hydatellaceae (one of the basal-most angiosperms) in general morphology and habit (Rudall et al., 2007;Sokoloff et al., 2013), although certain differences do exist between them, too. Lack of both herbaceous growth habit and angio-ovuly in gymnosperms also helps to reinforce the angiospermous affinity for Juraherba. Apparently, Juraherba is an interesting Jurassic herbaceous angiosperm that deserves further attention.

Aquatic habita t
The hairy roots of Juraherba are very tiny in size, simple in organization, only about one-cell wide, apparently very short and not well-developed (Figs. 2c, 3d-3h, 6). They are even shorter than the earliest known rooting system in the Early Devonian (Hao et al., 2009). These features imply that Juraherba most likely lives in an environment with litt le water stress where strong anchorage and mechanical support are unnecessary. The arrangement of all four fructifications nearly at the same horizontal level in Juraherba implies that these reproductive organs might be close to the water surface when pollinated. This assemblage of information suggests that Juraherba is most likely aquatic (Figs. 7a-7c).

Interaction with animals
The wrinkled fructification surface (Figs. 5c, 5f, 5g), in a strong contrast against the smooth leaf surface with little distortion (Fig. 3c) in the same fossil, su ggests that Juraherba's fructifications are fleshy. According to the study of early angiosperms (Eriksson et al., 2000), some animals may have involved in the dispersal of fleshy diaspores of angiosperms in the Early Cretaceous. Thus the occurrence of fleshy fructifications in the Jurassic is not too surprisin g. The M iddle Jurassic age of Juraherba suggests that the co-evolution between fleshy fructifications of angiosperms and animals can be dated back to the Middle Jurassic. Before this report, Na et al.

Conclusions
(1) Juraherba bodae gen. et sp. nov from the Middle Jurassic of Inner Mongolia, China is preserved as a whole plant, including roots, stem, leaves, and fructifications.
(2) The small size of Juraherba with fructifications enclosing ovules/seeds and lacking secondary growth indicates that it is a truthful herbaceous angiosperm.
(3) The Middle Jurassic age of Juraherba marks the earliest record of herbaceous seed plants and angiosperms.
(4) Analyses indicate that Juraherba lived in an aquatic habitat. The unexpected morphology of Juraherba undermines the mainstream thinking about angiosperm evolution.
(5) Fleshy fructifications and insect damage seen in Juraherba suggest that animal-angiosperm interaction may be much longer than previously assumed.