Comparative study of leaf architecture and cuticles of Nelumbo ...

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Botanical Journal of the Linnean Society, 2016, 180, 123–137. With 7 figures

Comparative study of leaf architecture and cuticles of Nelumbo changchangensis from the Eocene of Hainan Island, China, and the two extant species of Nelumbo (Nelumbonaceae) YA LI1,2, NILAMBER AWASTHI3, NATALYA NOSOVA4 and JIAN-XIN YAO1,2* 1

Institute of Geology, Chinese Academy of Geological Sciences, Beijing, 100037, China Key Laboratory of Stratigraphy and Paleontology, The Ministry of Land and Resources P.R.C, Beijing, 100037, China 3 Birbal Sahni Institute of Palaeobotany, Lucknow, 226007, India 4 Laboratory of Palaeobotany, Komarov Botanical Institute, Russian Academy of Sciences, St. Petersburg, 197376, Russia 2

Received 19 November 2014; revised 28 August 2015; accepted for publication 13 October 2015

Fossil leaves of Nelumbo changchangensis, collected from the Eocene of Hainan Island, China, were studied and compared with those of the extant species of Nelumbo, N. nucifera Gaertn. and N. lutea Willd. The fossil leaves have all the specialized features of extant Nelumbo in leaf architecture, except that the organization of the areolae looks much more irregular than that of extant Nelumbo. Comparisons of the cuticle and epicuticular ultrastructure indicate that: (1) N. changchangensis resembles N. nucifera in that anticlinal cell walls of the lower epidermis are straight along the major veins and near leaf bases and are shallowly undulate with U- to V-shaped undulations inside the areolae; (2) N. changchangensis differs from N. lutea in that anticlinal cell walls of the lower epidermis of the latter are deeply undulate with U-, V- to reversed Ω-shaped undulations inside the areolae; and (3) epicuticular wax crystals are more densely distributed on the leaves of N. changchangensis and N. nucifera than they are in N. lutea. These findings shed significant light on the cuticle differentiation of fossil and extant Nelumbo species. The morphometric comparisons indicate that almost all the synapomorphies of extant Nelumbo were already present by the Eocene. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 123–137.

ADDITIONAL KEYWORDS: areolae – epidermis – lotus – wax crystal.

INTRODUCTION Nelumbo Adans. (Nelumbonaceae), commonly known as the lotus plant, is an aquatic perennial herb that bears centrally peltate leaves, large showy flowers and numerous achenes embedded in the cavities of the strongly accrescent receptacles (Wiersema, 1997; Fu & Wiersema, 2001). Based on molecular data, Nelumbonaceae belong to an early diverging eudicot order Proteales, in which Nelumbonaceae are sister to Platanaceae plus Proteaceae (APG III, 2009). Nelumbo consists of two extant species: N. nucifera Gaertn. (the *Corresponding author. E-mail: [email protected]

sacred lotus) and N. lutea Willd. (the American lotus). They mainly differ in flower colour and geographical distribution. N. nucifera has pink or white tepals and is distributed in East, South and Southeast Asia and North Australia, whereas N. lutea has pale yellow tepals and occurs in Central and North America (Figs 1A, B, 2) (Wiersema, 1997; Fu & Wiersema, 2001). In addition, the central disc of the leaf is shallowly lobed in N. nucifera, but deeply lobed in N. lutea (Fig. 1C–F). It is reported that the sacred lotus has two ecological types: the hardy form that grows in temperate and subtropical regions, with a dormant period in autumn after the front part of rhizomes enlarges to form tubers, and the tropical form that

© 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 123–137

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Figure 1. Extant leaves and flowers of Nelumbo. A, flower of N. nucifera from Beijing Botanic Garden, China. B, flower of N. lutea (http://www.minnesotawildflowers.info), taken by Peter M. Dziuk at Lilydale Regional Park, Minnesota, USA. C, leaf of N. nucifera from Beijing Botanic Garden, China. D, central disc of the leaf in C. E, leaf of N. lutea (http://www.minnesotawildflowers.info), taken by P. M. Dziuk at Lilydale Regional Park, Minnesota, USA. F, central disc of the leaf in E.

grows all year round and does not develop tubers (Zhang & Wang, 2006; Huang, 2009; Yang et al., 2013). The fossil record indicates that Nelumbo had a nearly cosmopolitan distribution from the Cretaceous to the Quaternary (Snigirevskaya, 1964; Li et al., 2014a; Li et al., 2014b; Fig. 2). Snigirevskaya (1964) made a critical re-evaluation of the fossil record of Nelumbo and excluded some of them from the genus, e.g. N. nymphaeoides Ettingshausen and N. buchii Ettingshausen. She also doubted the affinities of some fossil species with Nelumbo, e.g. N. caspariana Heer, N. ettingshauseni Sieber and N. bactriana Ovcz. Seventeen fossil Nelumbo species are reported from the Cretaceous of Europe, Asia, North America, South America and Africa, including N. lusitanica Saporta, N. choffati Saporta, N. weymouthi Brown, N. kempii Hollick, N. intermedia Knowlton, N. primaeva Berry,

N. laramiensis Hollick, N. tenuifolia (Lesquereux) Knowlton, N. lakesiana (Lesquereux) Knowlton, N. crossii Knowlton, N. dawsoni Hollick, N. puertae Gandolfo & Cúneo, N. provinciale Saporta, N. arctica Heer, N. amurensis Baikovskaya, N. orientalis Matsuo and N. schweinfurthi Couyat & Fritel (Heer, 1882; Saporta, 1890, 1894; Hollick, 1894, 1904; Knowlton, 1900, 1930; Berry, 1903; Couyat & Fritel, 1910; Brown, 1933; Teixeira, 1945; Matsuo, 1954; Baikovskaya, 1956; Gandolfo & Cúneo, 2005), but many, if not all, of these species lack one or more synapomorphies of the crown group of Nelumbo. Thirteen fossil species of Nelumbo are reported from the Cenozoic of Europe, Asia and North America, including N. protolutea Berry, N. aureavallis Hickey, N. changchangensis X.Y.He & J.H.Jin, N. nagalensis Bhattacharyya, N. nipponica Endo, N. weylandi


COMPARATIVE STUDY OF THE LEAVES OF NELUMBO

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Figure 2. Geographical map showing the present distribution of Nelumbo and past distribution of fossils attributed to the genus (modified from Snigirevskaya, 1964).

Dotzler, N. protospeciosa Saporta, N. endoana Tanai, N. hungarica Tuzson, N. pacifica A.Pojark., N. lacunosa Macginitie, N. megalopolitana Weyland & Pflug and N. minima Reid & Reid (Saporta, 1891; Tuzson, 1914; Reid & Reid, 1915; Berry, 1917; Endo, 1934; Pojarkova, 1935; Dotzler, 1938; McGinitie, 1941; Tanai, 1961; Weyland & Pelug, 1961; Hickey, 1977; Bhattacharyya, 1983; He, Shen & Jin, 2010). Among them, N. lacunosa, N. megalopolitana and N. minima were established on the basis of fossil receptacles and the others were mainly based on fossil leaves. However, in many cases, these Cenozoic Nelumbo species still need critical evaluation to confirm if they are truly separate species, because many of the descriptions and illustrations lack enough information to allow detailed comparisons (Gandolfo & Cúneo, 2005) and some species are only based on single specimens, e.g. N. weylandi and N. nagalensis. These fossil and the modern species are generally indistinguishable by their leaf sizes and the number of primary veins (Snigirevskaya, 1964), which are often used in specific diagnoses. To investigate the usefulness of leaf cuticle characters to distinguish

fossil and extant Nelumbo species, we carried out detailed comparative studies between one fossil species, N. changchangensis from the Eocene of Hainan Island, China, and the two extant Nelumbo species. Fossil leaves of Nelumbo from Hainan were first reported by Guo (1979), who identified them as N. protospeciosa. Later, He et al. (2010) comprehensively described co-occurring fossil leaves, rhizomes, tubers, receptacles and fruits of Nelumbo, and considered them as a new species, N. changchangensis. Li et al. (2014a) supplementarily described the reproductive organs of this species. Wang et al. (2014b) reported fossil chloroplasts with internal ultrastructure in a leaf of N. changchangensis. In this paper, we describe in detail the leaf architecture and cuticle for this species.

MATERIALS AND METHODS The fossils described here were collected from the upper member of the Changchang Formation, Changchang Basin (19°37′56″N, 110°26′43″E; Fig. 3), in


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Figure 3. Map showing the position of the Changchang Basin on Hainan Island, China.

Hainan Island, China, during March 2009. On the basis of lithological properties, the Changchang Formation is subdivided into two members, the lower member of variegated lacustrine sediments and the upper member of dark lake-swamp coal-bearing series (Zhou & Chen, 1988; Lei et al., 1992). Based on the palynological assemblages, the lower member is dated as earliest early Eocene and the upper one as late early Eocene to early late Eocene (Lei et al., 1992). The Changchang Formation contains abundant plant fossils including leaves of Alseodaphne Nees, Amesoneuron Göppert, Livistona Brown, Nageia Gaertn., Sabalites Saporta and Salvinia Ség. (Li et al., 2009; Jin et al., 2010; Zhou et al., 2013; Wang, Xu & Jin, 2014a), wood material of Altingioxylon Kramer and Paraphyllanthoxylon Bailey (Feng, Yi & Jin, 2010; Oskolski, Kodrul & Jin, 2012) and fruits of Acer L., Craigia W.W.Sm. & W.E.Evans, Morinda L.

and Palaeocarya Saporta (Jin, 2009; Jin et al., 2009; Shi et al., 2012). Palynological studies indicate that the Changchang flora is composed of subtropical evergreen and deciduous elements and temperate floral components (Lei et al., 1992; Yao et al., 2009). The leaf compressions described here are represented by seven specimens (PEPB70512, 70514, 70517, 70518, 70520, 70521 and 70523). Leaves of N. nucifera (hardy form) were collected from Baiyangdian, Heibei Province. Leaves of N. nucifera (tropical form, introduced from Thailand) and N. lutea (introduced from the USA) were collected from the Chinese Lotus Research Center, Wuhan City, Hubei Province, China. Fossil cuticles were prepared by removing inorganic rock matrix (mainly CaCO3 and SiO2), for which the leaf compressions were treated with 10% HCl and followed by 5% HF. For removing extra organic debris adhered to the cuticles, the materials


COMPARATIVE STUDY OF THE LEAVES OF NELUMBO were treated with 30% HNO3 and then washed thoroughly in water. The cuticles were put in xylol solution for dehydration and finally mounted on glass slides using balsam. The extant cuticles were obtained with a mixture of ≥ 99% CH3COOH and 30% H2O2 at 60 °C. After staining in a 0.5% solution of safranin, the leaf cuticles were mounted on slides, embedded in glycerin and observed under a microscope. Photographs were obtained using a Canon PowerShot G9 camera, Nikon SMZ1000 stereomicroscope, Leica DM 2500 microscope and FEI Quanta-200 environmental scanning electron microscope. All the fossil and extant materials, including cuticle slides, were deposited at the Museum of Palaeobotany, Institute of Botany, Chinese Academy of Sciences, Beijing, China. The terminology followed in describing the leaf architecture and cuticle follows Dilcher (1974), Ellis et al. (2009) and Estrada-Ruiz et al. (2011).

RESULTS DESCRIPTION

OF FOSSIL LEAVES OF

NELUMBO

CHANGCHANGENSIS

The foliage leaves are flat or funnel-form and centrally peltate, with a centrally positioned central disc (Fig. 4A–C). The foliage leaves are partially preserved with the margin part missing, 6.2–15.0 cm in diameter. The complete leaves are estimated to be 12–30 cm in diameter, on the basis of the distance from leaf base to first bifurcation of the primary vein being about onequarter of the diameter (Matsuo, 1954; Snigirevskaya, 1964). These leaves often fall into the mesophyll- to macrophyll-size classes, and were probably membranous in texture (Fig. 4A–D). Primary venation is actinodromous with 20–23 primary veins diverging radially from the centre of the lamina at acute angles (10–20°) in relation to other primary veins (Fig. 4A–C). They dichotomize at least once or twice near the margin (see fig. 3a in He et al., 2010). Secondary veins stemming from the unbranched primary vein (the midvein) are not observed in our specimens. Tertiary veins emerging from primary veins are transversely orientated, probably alternate or opposite percurrent (Fig. 4D). Quaternary and higher venations are also missing in our specimens. The areolae are well developed, isodiametric and 130–535 μm in diameter. They are predominantly six-sided and occasionally four-, five- or seven-sided, like those of extant Nelumbo, but the organization appears much more irregular than that of extant Nelumbo (Fig. 4E, F). Inside the areolae, freely ending ultimate veins are absent (Fig. 4E). The upper epidermis of the leaves is poorly preserved and difficult to obtain. Only possible papillae are present, but the epidermal cells and stomata cannot be clearly seen (Fig. 5A). The lower epidermis is

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well preserved. It consists of isodiametric to rectangular cells. The cells are four- to six-sided, 24–60 μm (average 36 μm) long and 14–36 μm (average 23 μm) wide (Fig. 5B, C). The anticlinal cell walls are straight along the major veins or near the leaf base (Fig. 5B) or shallowly undulate inside the areolae (Fig. 5C). The undulations are U- or V-shaped. Stomata were not observed. Scanning electron microscopy (SEM) of the leaf surface shows that it is densely covered with a layer of tubular wax crystals (Fig. 6A–C).

DESCRIPTION

OF EXTANT LEAVES OF

NELUMBO

The upper epidermis of N. nucifera (hardy type) consists of papillate cells and anomocytic (ranunculaceous) stomata and the epidermal cells are isodiametric and mostly six-sided (Fig. 5D). The lower epidermis is composed of isodiametric to rectangular, four- to six-sided cells, and the epidermal cells are 20–87 μm (average 31 μm) long and 15–43 μm (average 24 μm) wide (Fig. 5E, F). The anticlinal cell walls are straight along the major veins or near the leaf base (Fig. 5E), or shallowly undulate inside the areolae (Fig. 5F). The undulations are U- or V-shaped. Stomata are absent. Epidermal features of N. nucifera (tropical type) (Fig. 5G–I) are consistent with those of N. nucifera (hardy type). The upper epidermis of N. lutea (Fig. 5J) is similar to that of N. nucifera, whereas the lower epidermis is slightly different. In N. lutea, the anticlinal cell walls are straight along the major veins or near the leaf base (Fig. 5K), but deeply undulate inside the areolae (Fig. 5L). The undulations are U-, V- or reversed Ω-shaped. Two specimens from two leaves of each ecotype and species were observed by SEM. Investigations of the leaf surfaces indicate that the upper and lower epidermises of N. nucifera (hardy type) are covered with clusters of tubular wax crystals (Fig. 6D–F). The wax crystals on the upper epidermis (Fig. 6E) are smaller but denser than those of the lower epidermis (Fig. 6F). Nelumbo nucifera (tropical type) (Fig. 6G–I) is the same as N. nucifera (hardy type). Nelumbo lutea (Fig. 6J–L) also has similar structures on both the upper and the lower epidermis, but differs from N. nucifera in having sparsely covered crystals on the lower epidermis.

DISCUSSION COMPARISON OF NELUMBO CHANGCHANGENSIS EXTANT NELUMBO SPECIES

WITH

To understand the architecture of fossil leaves accurately, Upchurch, Crane & Drinnan (1994) described and summarized the leaf features of extant Nelumbo. Later, Estrada-Ruiz et al. (2011) described the foliar architecture of extant Nelumbo more completely. The


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Figure 4. Leaves of Nelumbo changchangensis (A–E) from the Eocene of Hainan, China, and N. nucifera (F). A, a peltate leaf with 23 actinodromous primary veins emerging radically from the centre of the leaf. PEPB70512. Scale bar = 3 cm. B, a folded membranous leaf. PEPB70521. Scale bar = 2 cm. C, a membranous leaf. PEPB70514. Scale bar = 3 cm. D, white rectangular area of C magnified showing tertiary veins (arrows) intercalated within primary veins. Scale bar = 6 mm. E, leaf fragment with mostly six-sided areolae. PEPB70512. Scale bar = 600 μm. F, leaf fragment with mostly six-sided areolae. Scale bar = 600 μm. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 123–137


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Figure 5. The cuticles of fossil and extant leaves of Nelumbo: N. changchangensis (A–C), N. nucifera (hardy form) (D–F), N. nucifera (tropical form) (G–I) and N. lutea (J–L). A, upper epidermis showing possible papillae. Pa, papilla. Scale bar = 50 μm. B, lower epidermis showing straight anticlinal cell walls. Scale bar = 100 μm. C, lower epidermis showing shallowly undulate anticlinal cell walls with the U- to V-shaped undulations. Scale bar = 100 μm. D, upper epidermis showing papillate cells and stomata. Scale bar = 50 μm. E, lower epidermis along the major veins or near leaf base showing straight cell walls. Scale bar = 100 μm. F, lower epidermis inside the areolae showing shallowly undulate cell walls with the U- to V-shaped undulations. Scale bar = 100 μm. G, upper epidermis. Scale bar = 50 μm. H, lower epidermis along the major veins or near leaf base. Scale bar = 100 μm. I, lower epidermis inside the areolae. Scale bar = 100 μm. J, upper epidermis showing papillate cells and stomata. Scale bar = 50 μm. K, lower epidermis along the major veins or near leaf base showing straight cell walls. Scale bar = 100 μm. L, lower epidermis inside the areolae showing shallowly undulate anticlinal cell walls with the U-, V- to reversed Ω-shaped undulations. Scale bar = 100 μm.


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Figure 6. Scanning electron micrographs of the leaf surfaces of Nelumbo changchangensis (A–C), N. nucifera (D–I) and N. lutea (J–L). A, the fossil leaf surface. Scale bar = 20 μm. B, magnification of A showing leaf surface densely covered with wax crystals. Scale bar = 5 μm. C, magnification of B showing details of the wax crystals. Scale bar = 3 μm. D, upper leaf surface of N. nucifera (hardy form) showing papillate cells and stomata. Scale bar = 20 μm. E, magnification of D showing leaf surface densely covered with small tubular wax crystals. Scale bar = 3 μm. F, lower leaf surface of N. nucifera (hardy form) densely covered with big tubular wax crystals. Scale bar = 3 μm. G, upper leaf surface of N. nucifera (tropical form) showing papillate cells and stomata. Scale bar = 20 μm. H, magnification of G. Scale bar = 3 μm. I, lower leaf surface of N. nucifera (tropical form). Scale bar = 3 μm. J, upper leaf surface of N. lutea showing papillate cells and stomata. Scale bar = 20 μm. K, magnification of J. Scale bar = 3 μm. L, lower leaf surface of N. lutea sparsely covered with tubular wax crystals. Scale bar = 3 μm. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 123–137

COMPARATIVE STUDY OF THE LEAVES OF NELUMBO main points are listed as follows. (1) The foliage leaves are usually large (mesophyll or macrophyll size), entire-margined and peltate, with an orbicular lamina and a centrally positioned petiole. (2) On the adaxial leaf surface, directly above the petiole, is the central disc that is bilaterally lobed with the median line of symmetry defined by the unbranched primary vein. (3) Primary venation is actinodromous, with 18–24 primary veins. One primary vein is straight and unbranched and runs directly to the leaf margin. The other primary veins dichotomize twice or three times near the margin and form an inner and outer set of intramarginal loops. (4) In some foliage leaves, the unbranched primary vein gives rise to one or two pairs of homologues of secondary veins that are thinner than the primary veins and thicker than the tertiary veins. (5) Primary veins are interconnected by numerous tertiary veins and their venation is a mix of alternate and opposite percurrent. (6) Quaternary venation is a mix of opposite and alternate percurrent. (7) Areoloation is isodiametric and predominantly hexagonal, with a mix of six- and fivesided areoles; freely ending veinlets are absent. The fossil species N. changchangensis presents the following characters: peltate orbicular leaves, 20–23 thick primary veins with radial organization, primary veins bifurcating at least once or twice, probably alternate or opposite percurrent tertiary venation and mostly hexagonal areolation. Thus, N. changchangensis exhibits the combined characters of extant Nelumbo in foliar architecture. The small difference observed is that the areolae in N. changchangensis have a tendency to be six-sided, like those of extant Nelumbo, but the organization appears much more irregular than that of extant Nelumbo (Fig. 4E, F). However, this irregular organization may also be due to the effect of fossilization. With respect to the cuticles and epicuticular ultrastructure, N. changchangensis generally resembles extant Nelumbo, showing similar features. The upper epidermis consists of papillate cells, whereas the lower epidermis is composed of epidermal cells with straight or undulate anticlinal walls, but has no stomata (Fig. 5). The leaf surface is covered with a layer of wax crystals (Fig. 6). On modern leaves of N. nucifera and N. lutea, stomata are abundant on the uppr epidermis, but they are less well developed on the lower epidermis, gradually degenerating and finally disappearing in the adult stage (Gupta, Paliwal & Ahuja, 1968). This indicates that the fossil leaves are probably adult. The wax crystals on the leaf surface of extant Nelumbo were reported by Barthlott et al. (1996). This material often provides effective water repellence, and protects the leaf from contaminating particles, including spores and conidia of pathogens (Barthlott & Neinhuis, 1997). Compared

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with the two extant species, N. changchangensis is more similar to N. nucifera and differs from N. lutea in the lower epidermis. The epidermal cells of the fossil species have shallowly undulate anticlinal walls with U- to V-shaped undulations inside the areolae (Fig. 5C, F, I), whereas N. lutea has deeply undulate anticlinal walls with U-, V- to reversed Ω-shaped undulations inside the areolae (Fig. 5L). The wax crystals are more densely distributed on fossil leaves (Fig. 6A–C) than on the leaves of N. lutea (Fig. 6L). Thus, it is quite evident that the Eocene species N. changchangensis already possessed almost all the synapomorphies of extant Nelumbo and is particularly similar to N. nucifera.

COMPARISON

NELUMBO-LIKE FOSSILS NELUMBO SPECIES

WITH

FOSSIL

AND

Some Cretaceous and Palaeogene Nelumbo-like leaves are placed within Nelumbites Berry, Paleonelumbo Knowlton, Nelumbago McIver & Basinger and Exnelumbites Estrada-Ruiz, Upchurch, Wolfe & Cevallos-Ferriz (Table 1). These extinct genera probably represent branches of the stem lineage of Nelumbonaceae. The leaves of Nelumbites have the petiole displaced toward the base of the lamina, no central disc, fewer primary veins, multiple pairs of secondary veins that derive from the midvein, and irregular reticulate tertiary and higher order venation (Berry, 1911; Upchurch et al., 1994; Estrada-Ruiz et al., 2011). Leaves of Paleonelumbo differ from those of Nelumbo in the pronounced marginal lobes entered by the ribs and the secondary branches in the upper portion of the ribs instead of equal forks (Knowlton, 1930), and leaves of Nelumbago differ in having orthogonal reticulate or more random tertiary veins and commonly four-sided areoles (McIver & Basinger, 1993). Leaves of Exnelumbites are more primitive than those of Nelumbo in having no central disc, fewer primary veins, less highly organized tertiary venation and predominantly non-hexagonal areolation (Estrada-Ruiz et al., 2011). The fossil record of Nelumbo appears to indicate that the genus had significantly higher species diversity during the geological past, especially during the Late Cretaceous, than it does today. Using the manual of leaf architecture (LAWG, 1999), Gandolfo & Cúneo (2005) described the leaves of N. puertae from the Late Cretaceous of Patagonia, Argentina. Estrada-Ruiz et al. (2011) pointed out that N. puertae falls outside the Nelumbo crown group because it lacks multiple advanced features currently restricted to Nelumbo (e.g. central disc, percurrent tertiary venation, hexagonal areolation). They further argued that this fossil species probably fits best in the extinct genus Nelumbago. Another two Cretaceous forms,


Estrada-Ruiz et al., 2011 Berry, 1911; Upchurch et al., 1994

Commonly four-sided Late Cretaceous to Palaeocene McIver & Basinger, 1993; Van Boskirk, 1998 No data Late Cretaceous to Palaeocene Knowlton, 1930; Brown, 1962 Polygonal Early Cretaceous

Bifurcating Orthogonal reticulate

Literature

Areoles Age

Primary venation Tertiary venation

Bifurcating Regular, opposite percurrent Predominantly six-sided Early Cretaceous to Present Snigirevskaya, 1964; Upchurch et al., 1994; Estrada-Ruiz et al., 2011

Not bifurcating Irregular, reticulate

Not bifurcating No data

Centrally peltate With broad glandular teeth Not bifurcating Alternate percurrent to reticulate Four- to six-sided Late Cretaceous Centrally peltate Entire Centrally peltate Obtusely toothed or lobed Centrally peltate Entire Position of petiole Leaf margin

Excentrically peltate Entire or crenate

Nelumbago Nelumbo Character

Nelumbites

Paleonelumbo

Exnelumbites

Y. LI ET AL.

Table 1. Comparisons of leaf features between Nelumbo and the stem group of Nelumbonaceae

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Nelumbo sp. from the Cenomanian Chulym flora of western Siberia (Goloneva & Nosova, 2012) and Brasipeltis gregaria Krassilov, Lewy, Nevo & Silantieva from the Turonian of the Negev, Israel (Krassilov et al., 2005), have numerous primary veins, like extant Nelumbo, and a structure that appears to represent the central disc, but their areolation is not preserved. Nine fossil species of Nelumbo from the Cenozoic, N. protolutea, N. changchangensis, N. nagalensis, N. nipponica, N. weylandi, N. protospeciosa, N. endoana, N. hungarica and N. pacifica, have been established mainly based on leaves (Fig. 7). However, only N. aureavallis can be distinguished from the other Cenozoic fossil species and the modern taxa by having more primary veins (about 35–45), whereas the others have c. 18–25. Zhilin & Snigirevskaya (1974) studied the fossil leaves of Nelumbo from the Miocene of Altyn-Chokusu, Kazakhstan, and found that they had large variation in leaf sizes and the numbers of primary veins. Snigirevskaya (1974) attributed all fossils of Nelumbo from the Palaeogene and Neogene of Russia to one species, N. protospeciosa.

MORPHOLOGICAL

STASIS IN

NELUMBO

Morphometric statistics and comparisons of N. changchangensis with modern taxa included the following characters: tuber and leaf sizes, number of primary veins, areola arrangement and size, shape and size of epidermal cells, receptacle shape and size, number of stamens and fruits, and fruit length/width (Table 2). It is evident that the Eocene fossil N. changchangensis not only bears all the specializations of extant Nelumbo, but also has quantifiable traits that fall within the ranges of extant Nelumbo. The close morphological similarity of N. changchangensis with extant Nelumbo species may be explained by morphological stasis, namely the lack of significant morphological change in an individual taxon for a long period of time (Williamson, 1987). Such examples exist in Metasequoia occidentalis Chaney, a Late Cretaceous species that is similar to modern Metasequoia glyptostroboides Hu & W.C.Cheng (LePage, Yang & Matsumoto, 2005), and Donlesia Dilcher & Wang which was described from the Early Cretaceous of Kansas, USA, and close to the living genus Ceratophyllum L. (Dilcher & Wang, 2009). This concept has also been applied frequently to explain the morphological similarities between eastern Asian–eastern North American disjunct taxa, e.g. Liquidambar L., Osmorhiza Raf. and Phryma L. (Hoey & Parks, 1991; Wen et al., 2002; Nie et al., 2006). Two mechanisms, evolutionary constraints and stabilizing selection, have been proposed for generating and maintaining morphological stasis (Williamson,



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Figure 7. Line drawings of the leaves and areolae of the fossil Nelumbo species from the Cenozoic. A, N. weylandi from the Oligocene of Germany. Scale bar = 5 cm. B, N. endoana from the Miocene of Japan. Scale bar = 2 cm. C, N. hungarica from the Late Miocene of Hungary. Scale bar = 10 cm. D, N. nipponica from the Late Palaeogene of Japan. Scale bar = 10 cm. E, areolae of N. nipponica. Scale bar = 0.5 mm. F, N. nagalensis from the Eocene of India. Scale bar = 2 cm. G, N. changchangensis from the Eocene of Hainan, China. Scale bar = 2 cm. H, areolae of N. changchangensis. Scale bar = 0.5 mm. I, N. protospeciosa from the Miocene of France. Scale bar = 5 cm. J, areolae of N. protospeciosa. Scale bar = 0.5 mm. K, N. protolutea from the Eocene of Mississippi, USA. Scale bar = 5 cm. L, N. aureavallis from the Eocene of North Dakota, USA. Scale bar = 10 cm. M, areolae of N. aureavallis. Scale bar = 0.5 mm. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 123–137

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21–90 × 14–43 c. 200 Obconical No data 12–25 No data < 1.25 North American Hall & Penfound, 1944; Wiersema, 1997; this study 20–87 × 15–43 100–400 Obconical 3.8–4.5 × 7.5–9.6 1–40 c. 18 × 10 > 1.5 Asia and Australia WBGCAS, 1987; Wiersema, 1997; this study 24–60 × 14–36 200 Obconical 3.8–4.0 × 4.4–10.0 20–26 10.0–17.5 × 6.5–10.0 1.54 Hainan, China He et al., 2010; Li et al., 2014a; this study

1987). Both mechanisms may play roles in causing morphological stasis for N. changchangensis. Nelumbo species are aquatic plants with a sheltered and stable environment (Hutchinson, 1975). As a consequence, the ecological niche and selective pressures are very similar (Borsch & Barthlott, 1994).

CONCLUSIONS We investigated the leaf architecture, cuticle and epicuticular ultrastructure of N. changchangensis from the Eocene of Hainan Island, China, and compared it with the extant species of Nelumbo, N. nucifera and N. lutea. This reveals that: (1) N. changchangensis possesses all the characters of extant Nelumbo in leaf architecture, except that it has much more irregular organization of the areolae; (2) with respect to the cuticle and epicuticular ultrastructure, there is no morphological difference between the tropical and hardy forms of N. nucifera; and (3) N. changchangensis is similar to the Asian–North Australian species N. nucifera, but differs slightly from the North American species N. lutea. Based on morphometric analysis, our study also reveals that almost all the synapomorphies of extant Nelumbo were already present by the Eocene, which probably indicates morphological stasis for N. changchangensis.

ACKNOWLEDGMENTS We thank Feng Qin, Zlatko Kvacˇek, Jirˇí Kvacˇek and Vasilis Teodoridis who participated in Hainan palaeontological expeditions. We also appreciate the associate editor and three anonymous reviewers for their constructive and inspiring comments. This work was a contribution to the National Natural Science Foundation of China (Nos. 41210001, 41472030). This study was also supported by the Ministry of Science and Technology of the People’s Republic of China (No. 2015FY310100).

References

Fruit

Cell size (μm) Stamen number Shape Size (cm) Fruit number Size (mm) Length/width Locality

REFERENCES

Stamen Receptacle

Lower epidermis

2–30 × 1–10 20–25 7–85 × 7–68 Four- to seven-sided 129–661 Polygonal or shallowly undulate 4.5–13.0 × 2.2–5.1 20–23 12–30 Four- to seven-sided 130–535 Polygonal or shallowly undulate Size (cm) No. of primary veins Diameter (cm) Arrangement of areolae Size of areolae (μm) Anticlinal wall pattern Tuber Leaf

No data 20–25 Ca. 60 Four- to seven-sided 148–634 Polygonal or deeply undulate

N. nucifera N. changchangensis Character Organ

Table 2. Morphometric statistics and comparisons of Nelumbo changchangensis with extant N. nucifera and N. lutea

N. lutea

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