Primotrapa gen. nov., an extinct transitional genus bridging the evolutionary gap between Lythraceae and Trapoideae, from the early Miocene of North China

Background Although Trapa is a well-defined genus of distinctive freshwater plants with accumulations of extensive morphological and embryological autapomorphies, its phylogenetic relationships have long been unclear. Formerly placed in the monotypic family Trapaceae, Trapa is now recognized as sister to Sonneratia within Lythraceae s.l., although both genera lack morphological synapomorphies. Thus, a split between the two taxa must have occurred in deep evolutionary time, which raises the possibility of finding transitional forms in the fossil record. Results Here we describe a new genus and species, Primotrapa weichangensis Y. Li et C.-S. Li (Lythraceae s.l.: Trapoideae), based on three-dimensionally preserved floral cups, fruits, and seeds from the early Miocene of Weichang County, Hebei Province, China. Primotrapa is characterized by a shallow, saucer-shaped floral cup, four distally barbellate sepals, four intersepal appendages alternating with the sepals at the rim of cup, a superior to basally inferior ovary, a fusiform or ovoid, one-seeded fruit with a ribbed surface, and a long persistent peduncle. Two fossil species of Hemitrapa are proposed as new combinations of Primotrapa, namely P. alpina (T. Su et Z.-K. Zhou) Y. Li et C.-S. Li comb. nov. and P. pomelii (Boulay) Y. Li et C.-S. Li comb. nov. Our phylogenetic analysis based on fifteen flower and fruit characters supports the placement of Primotrapa, Hemitrapa and Trapa in a monophyletic clade, which comprise subfamily Trapoideae. The phylogenetic analysis places Primotrapa at the base of Trapoideae. Conclusions In view of its superior ovary, which is a plesiomorphic character of Lythraceae s.l., the newly recognized genus Primotrapa and its three species likely represent transitional forms that bridge the evolutionary gap between the basal taxa of Lythraceae s.l., i.e. Lythrum, and the highly derived taxon Trapa.


Backgound
Trapa L., the water caltrop, is a genus of annual freshwater macrophytes, which grow in sluggish rivers, lakes, swamps, and ponds and are native to the tropical to warm temperate regions of Eurasia and Africa [1,2]. Trapa has accumulated extensive autapomorphic features such as a unique embryo, leaf margins with characteristic double-mucronate tooth apices, distinctively horned fruits, distally barbellate sepals (horns) (Fig. 1bf), and generally prolate-spheroidal crested pollen [3,4]. This suite of striking characters led to the assignment of all living species in the genus to the monotypic family Trapaceae [1,5,6]. However, the relationship of the Trapaceae to other families has remained unclear. Trapa was once thought to be related to Ludwigia L. (Onagraceae) [7] or Trapella Oliv. (Pedaliaceae) [8], but any morphological similarities have been dismissed as homoplasy (non-homologous similarity) [6,9]. A close relationship between Trapa and Lythraceae sensu stricto (s.str.) was also proposed, based on fossil evidence [9].
Molecular data unquestionably place Trapa and the other three distinct genera, namely Duabanga Buch.-Ham., Punica L., and Sonneratia L.f. in the family Lythraceae sensu lato (s.l.) (Fig. 1g) [10,[12][13][14]. From a morphological standpoint, Lythraceae s.l. lack morphological synapomorphies to define them, especially in regard to ovary position. It is a superior ovary in Lythraceae s.str., a superior to basally inferior ovary in Sonneratia, a semi-inferior ovary in Duabanga, and a nearly completely inferior ovary in Punica and Trapa [11]. Based on molecular phylogenetics, Trapa and Sonneratia are sister genera [10,[12][13][14], as unlikely as it may seem morphologically (Fig. 1a, b) [11]. Moreover, in regard to habit and habitat, Sonneratia is a tree growing in the brackish waters of mangrove and coastal environments, while annual Trapa is an aquatic plant floating in quiet bodies of freshwater. Hence, a link between Trapa and Sonneratia must exist in their common evolutionary history. Until now, the absence of this link in fossil record has greatly hampered our understanding of the evolutionary position of Trapa within Lythraceae s.l. and has led to a conflict between morphological and molecular systematics [10,11].
While fossil remains of Trapa have been frequently reported from the Miocene swamp deposits of Europe and Asia [15][16][17][18][19][20], as well as from North America [21][22][23][24][25][26], unequivocal Trapa fossils first appear in the middle Miocene [2]. Hemitrapa Miki is an extinct genus of b Trapa natans L. from Xuanwu Lake, Nanjing, Jiangsu Province, China. c, d A leaf of T. natans L. and an enlargement (d) showing the characteristic double apices of the teeth on the leaf margin (red arrows). e A series of fruits of T. natans showing different developmental stages. f A sepal of T. natans with the cortex removed, showing dital, recurved barbs. g The position of the ovary in Lythraceae s.l. mapped on the Bayesian 50% majority-rule consensus of 4000 trees from the combined molecular data of four gene regions [10,11] fossil fruits initially described by Miki [17] from the late Miocene of Akazu and Hatagoya, Japan, and is thought to be closely related to Trapa [9]. Hemitrapa fruits are fusiform and contain a semi-inferior ovary. They also occur frequently in other mid-latitude localities in the Miocene of Eurasia and North America [27] and disappear after the Pliocene [2,27]. Based on comparative morphology, Miki hypothesized that living Trapa evolved from ancestral Lythrum L. through the extinct genus Hemitrapa [9]. However, Miki's hypothesis can no longer be considered tenable, as Trapa and Lythrum share only a family level relationship [2]. While other studies also consider Trapa to be a descendant of Hemitrapa [4,16], it is more likely that Trapa and Hemitrapa share a common ancestor [28]. It is worth pointing out that both Trapa and Hemitrapa are also morphologically very different from other taxa of Lythraceae s.l., including Trapa's molecular genetic sister Sonneratia.
In the present study, we describe a new, extinct member of Lythraceae s.l., Primotrapa weichangensis Y. Li et C.-S. Li gen. et sp. nov., on the basis of threedimensionally preserved floral cups, fruits and seeds from the early Miocene of Weichang County, Hebei Province, China (Fig. 2). A phylogenetic analysis shows that Primotrapa, Hemitrapa, and Trapa form a monophyletic clade, i.e. the subfamily Trapoideae. Phylogenetic analysis also reveals the transitional position of Primotrapa between Sonneratia and Trapa + Hemitrapa clade within Lythraceae s.l. The phytogeography of Primotrapa, its origin, and dispersal through geological time are also discussed. Generic diagnosis: Flowers perigynous or basally epigynous, bearing a short shallow, saucer-shaped floral cup, persistent on the fruit. Sepals 4, acicular, sparsely barbellate toward apex, inserted on floral axis in the same whorl, curved away from the floral axis, persistent on the fruit. Intersepal appendages 4, acicular, spiny, dichotomizing once, alternating with sepals at the rim of the cup, detaching from mature fruit. Ovary superior to basally inferior. Fruit fusiform, irregularly dehiscent, oneseeded, with an elongated apex, sparsely ribbed surface, and a long persistent peduncle.

Systematic paleontology
Etymology: The genus name refers to its primitive Trapa-like fruits.
Specific diagnosis: Floral cup perigynous with a superior ovary. Fruit fusiform or ovoid in shape, with an elongated and smooth (non-ribbed) apex. Barbs arise at a wide angle to the sepals. Fruit surface finely and sparsely ribbed.
Etymology: The species epithet refers to the fossil locality in Weichang County.
Repository: National Museum of the Plant History of China, Chinese National Herbarium, Institute of Botany, Chinese Academy of Sciences, Beijing, China.
Description: The floral cups (hypanthia) of these fossil flowers are very short, shallow, saucer-shaped, perigynous, and usually persistent on the fruits (Fig. 3a-c, g-n); however, some hypanthia are found detached from the immature fruits ( Fig. 3d-j). Sepals are four, acicular, curved outward at about their midpoint, 7-14 mm long and about 1 mm wide, and sparsely barbellate toward the apex (Figs. 3a-c, g-n, Fig. 4a-e), inserted on the floral axis in the same whorl and arising at a 30-40°a ngle. The barbs on the sepals are straight or recurved, forked once or unforked, about 1-2 mm long (Fig. 4e). The intersepal appendages are four, Y-shaped, spiny, dichotomized once, 3-5 mm long, up to 0.5 mm wide at their bases, nearly the same length as the apical forks ( Fig. 3a, g-j, Fig. 4f), and alternate with the sepals at the rim of the floral cup. The epidermis of the sepals is composed of elongated cells ( Fig. 4g-j). At the bottom of the floral cup is a single superior ovary ( Fig. 3k-n). The pedicel is very long (Fig. 3a-c).
Fruits are small, 7.0-16.0 mm long, 3.0-4.5 mm wide, fusiform or ovoid with an elongated apex, obtuse base, sparsely ribbed surface ( Fig. 5a-g). Pericarps are thinner and less woody than Trapa. The ribs are probably the remains of a fleshy exocarp, and tightly adnate to the endocarp, acicular, arising longitudinally from the base of the fruit, and extending beyond the broadest part of the fruit, but not reaching the apex of the fruit (Fig. 5d). Sepals are persistent, mostly not barbellate ( Fig. 5a-g). Intersepal appendages are absent in mature fruits ( Fig.  5a-g), probably due to abscission. Peduncles are slender, curved and broadening towards the floral cup, up to 4 cm long, 1-2 mm wide ( Fig. 5a-g). Fruits are likely oneseeded ( Fig. 5f) and irregularly dehiscent (Fig. 5b, c, f). Seeds are obovoid or elliptoid, 5.4-7.5 mm long and 4.9-6.5 mm wide ( Fig. 5h-j). The surface of the testa  [29], and the stratigraphic column is modified from [30] consists of irregularly shaped, pentagonal to hexagonal cells (Fig. 5k, [32][33][34][35][36]. Remarks: This species represents the oldest fossil species of the subfamily Trapoideae and is morphologically much different from the majority of the species of Hemitrapa. It is transferred here from Hemitrapa to Primotrapa, because its sepals are inserted near the base of the fruit [32][33][34][35][36] as they are in Primotrapa, rather than inserted about mid-length along the fruit as is characteristic for Hemitrapa [9,17,27]. The main difference between P. pomelii and P. weichangensis is that the fruit surface of P. pomelii is more ribbed [32,34,36] than P. weichangensis ( Fig. 5a-g), and the fruit apex gradually narrows distally into a conical structure in P. pomelii [32,34,36], instead of the narrowly elongated apex seen in P. weichangensis ( Fig. 5a-g). However, some specimens of P. pomelii from the late Oligocene of Rott and the early Miocene of Otterwisch, Germany [33,35,36] show definite similiarities to P. weichangensis, including some immature fruits with a shallow, saucer-shaped floral cup, as observed in our specimens (Fig. 3e-j). However, the floral cups of the German fossils are very incomplete and lack complete sepals and intersepals, hampering more detailed comparison. Furthermore, the barbs on their sepals are different, arising at a narrow angle to the sepal, as in Trapa (Fig. 1f), whereas in our species the barbs arise at a wide angle to the sepal (Fig. 4e). Remarks: The fruits of Primotrapa alpina are elongate or fusiform in shape and are not borne on persistent peduncles, differentiating them from P. pomelii and P. weichangensis. However, the absence of a peduncle in P. alpina may be due to taphonomic bias or an abscission of a mature fruit from the peduncle at maturity [37]. Most specimens of P. alpina possess a basal, inferior ovary, whereas our specimens of P. weichangensis each bear a superior ovary. Moreover, the apex of the fruit is finely ribbed and conical in P. alpina, while it is not ribbed and narrowly elongated in P. weichangensis.

Carpological comparisons with related fossil and extant aquatic taxa
Primotrapa is superficially similar to three Cretaceous genera, i.e. Beipiaoa Dilcher, Sun et Zheng, Palaeotrapa Golovneva and Prototrapa Vassiljev, each of which consists of three species [38][39][40]. Beipiaoa is characterized by having apically three-or four-horned fruits, which were reported from the Early Cretaceous Yixian Formation of Beipiao, Liaoning, China [39,41] (Fig. 6a-c). Palaeotrapa is represented by two-horned fruits found in association with Quereuxia leaves in the Maastrichtian of the Koryak Upland, Russia [38] (Fig. 6d-f). Prototrapa was established for very small fruits (only 1-3 mm long) with two long horns from the Aptian-Albian of southeastern Australia [40] (Fig. 6g-i). Beipiaoa, Palaeotrapa, and Prototrapa all have horned Trapa-like fruits with a poorly developed fruit head and a distinctive fruit body [27], but they lack barbellate sepals, intersepal appendages, and finely ribbed fruit surface, which differentiate all these three genera from Primotrapa. The fruit of Trapa is also quite dissimilar to these three genera [2,27,34].
Instead, Primotrapa more closely resembles the nowextinct, Cenozoic genus Hemitrapa, as they both have fusiform fruits with a persistent floral cup, four distally barbellate sepals and four intersepal appendages a Muddy siltstone with three P. weichangensis fruits exposed on the surface. PEPB70631. b-g Laterally compressed fruits, each showing a persistent floral cup with 1-4 remaining sepals, ribbed fruit surface, persistent peduncle, and probably one seed in the fruit (f). PEPB70632-70637. h-j Laterally compressed seeds of P. weichangensis. PEPB70681-70683. k-l SEM of the seed surface, showing irregularly shaped, pentagonal to hexagonal cells arranged alternately at the rim of the floral cup, a less woody pericarp, a ribbed surface (Primotrapa) or a striated protrusion (the exserted portion) of the fruit (Hemitrapa), and a long, persistent peduncle. However, they differ in that Primotrapa has a very small, shallow, saucer-shaped floral cup, a superior to basally inferior ovary, and Y-shaped intersepal appendages, while Hemitrapa possesses a bowl-shaped floral cup, a semi-inferior ovary, and spine-like intersepal appendages (Fig. 7).
When compared with the fruits of extant aquatic plants, Primotrapa superficially resembles Ceratophyllum L. (Ceratophyllaceae) in having a similarly-sized, fusiform fruit with an elongated apex. However, Ceratophyllum differs in having two long basal spines and sometimes two more spines on the upper part of the fruit [43]. Primotrapa is similar to Nymphoides Ség. (Menyanthaceae) in having a perigynous floral cup deeply lobed to near its base, a superior ovary, and a long persistent peduncle. However, Nymphoides has (4or) 5-merous flowers without intersepal appendages, and few-seeded capsular fruits [44].
Primotrapa is similar to Trapa in a series of characters, such as a persistent floral cup, four distally barbellate sepals and four intersepal appendages arranged alternately at the rim of the cup, and a ribbed fruit surface [42] (Figs. 1, 7). The main differences between Primotrapa and Trapa are that the former has a very small, shallow, saucer-shaped floral cup with a superior to basally inferior ovary, four sepals in the same level, Yshaped intersepal appendages, and a long persistent peduncle, whereas the latter has a epigynous floral cup with a nearly completely inferior ovary, four sepals that initiate at the same level and later develop into 0 to 2 pairs of horns (sepals) at different levels, tubercle-like intersepal appendages (sometimes absent), and a sessile mature fruit with a distinctly contacted fruit neck (Figs. 1, 7). In summary, the fruit of Primotrapa is morphologically  [38]. g-i Prototrapa douglasii V. Vassiljev (g), Prototrapa praepomelii V. Vassiljev (h), and Prototrapa tenuirostrata V. Vassiljev (i) from the Early Cretaceous of southeastern Australia [40] most similar to those of the extinct genus Hemitrapa and the living genus Trapa.
Phylogeny and evolution of Primotrapa within Lythraceae s.l.
Our morphological phylogenetic analysis places Primotrapa, Hemitrapa, and Trapa in the monophyletic subfamily Trapoideae (Fig. 7). It also suggests that Primotrapa is the basal taxon of the subfamily, sister to a Hemitrapa + Trapa clade (Fig. 7). Derivation of Sonneratia and Trapoideae from a common ancestor is strongly supported by molecular evidence [10,[12][13][14] (Figs. 1, 7). As in Primotrapa, Sonneratia also has a superior to basally inferior ovary, which suggests that this type of ovary is a plesiomorphic character for Sonneratia and the Trapoideae.
Lythrum, an early-branching genus of Lythraceae s.l. (Fig. 1g), has 6-merous flowers [11]; however, a fossil Lythrum with 4-merous flowers was described from the late Miocene of Akazu and Obata, Japan, which was considered to represent a form ancestral to Hemitrapa [9]. In having a superior ovary, which is a plesiomorphic character of Lythraceae s.l. [10], P. weichangensis is potentially basal to Primotrapa and may also represent an example with primitive morphology of basal Lythraceae s.l. Interestingly, our investigation of the ontogeny of the fruit in extant Trapa (Fig. 1f) reveals that it has a nearly semi-inferior ovary in its early developmental stages, which gradually becomes nearly completely inferior as fruit matures. Thus, the ontogenetic development of Trapa fruit appears to reflect the phylogeny of the Trapoideae.

Phytogeographic history of Primotrapa
The oldest fossils of Primotrapa appear in the late Eocene diatomite of Kučlín, Czech Republic [34], then during the Oligocene, Primotrapa appears in Xizang, China [37], and Rott, Germany [33]. By the early Miocene, Primotrapa had spread to Otterwisch, Germany [33], Gergovie, France [32,33], and Weichang, China (this paper).  [42]; Hemitrapa is represented by the type species H. trapelloidea Miki on the left, modified from Miki [9], and by the Chinese species H. shanwangensis Wang on the right, modified from Wang [27]; Primotrapa weichangensis is represented by an immature fruit on the left and a mature fruit on the right; Sonneratia is represented by the extant species S. apetala Buch.-Ham, drawn from Fig. 1a; Lythrum is represented by the fossil species L. tetrasepalum Miki on the left and the extant species L. anceps (Koehne) Makino on the right, both modified from Miki [9] Overall, this pattern suggests that the genus likely originated during the Eocene of Eurasia, remaining restricted to the mid-latitudes of Europe and East Asia during the mid-Cenozoic (Fig. 8), then becoming extinct after the early Miocene. However, the last surviving member of subfamily Trapoideae, Trapa, has a broad current Old World distribution (Fig. 8). Similarly, its sister genus Sonneratia grows in Old World mangrove communities from East Africa to Indo-Malesia, Australia, New Guinea, and the West Pacific islands [11,45] (Fig. 8). Li et C.-S. Li comb. nov. The close morphological similarity of Primotrapa, Hemitrapa and Trapa suggests a close evolutionary relationship within subfamily Trapoideae rather than convergent evolution, with morphological cladistic analysis placing Primotrapa as sister to the remainder of subfamily Trapoideae. In view of its superior ovary, a plesiomorphic character of Lythraceae s.l., Primotrapa seems to be intermediate between basal Lythraceae s.l. taxa (e.g. Lythrum) and more highly derived genera such as Trapa.

Geological setting
The fossil plant material described here was all collected from an outcrop at the Gaoshanjian locality (42°07′33″ N, 117°50′28″ E; 1406 ± 16 m), near Guangfayong Town, Weichang County, Chengde City, Hebei Province, China (Fig. 2). Collecting of fossils does not need any permits in this area. The outcrop consists of nine layers of Fig. 8 Distribution of fossil Primotrapa, as well as extant Trapa and Sonneratia, on a world map with Miller cylindrical projection. The base map is from https://www.naturalearthdata.com/. Fossil sites were plotted on the map using MapInfo Professional 8.5 SCP. The present-day distribution range of living Trapa and Sonneratia were modified from Mai [35] and Mao and Foong [45] lacustrine deposits (Fig. 2). The sedimentary rock and its underlying basalts belong to the Hannuoba Formation, according to the 1: 200,000 geological map of Qipanshan section (K-50-16) [29]. Fossil specimens of Primotrapa were collected from the third (muddy siltstone) and ninth layers (shale) (Fig. 2).

Fossil preparation, photography and repository
The fossil remains of Primotrapa are charcoalified, and preserved as compressions in a matrix of muddy siltstone and lignite. They were exposed from the matrix by the technique of dégagement [52]. Selected specimens were treated with 10% HCl and 48% HF, and then rinsed with water and air dried. Specimens were imaged using a digital camera (Canon PowerShot G15), a stereomicroscope (ZEISS SteREO Discovery.V20), scanning electron microscope (LEO1530VP), and a Micro-CT (ZEISS Xra-dia520 Versa). In total, 40 floral cups, many detached sepals and intersepal appendages, 50 fruits, and 20 seeds were assigned the inventory numbers PEPB70591-70700 and deposited at the National Museum of the Plant History of China, Chinese National Herbarium, Institute of Botany, Chinese Academy of Sciences in Beijing. Ya Li and Cheng-Sen Li carried out the formal identification of the samples and provided details of the voucher specimens deposited.

Morphological phylogenetic analysis
To reconstruct the phylogenetic relationship of Primotrapa, three extant genera and two fossil genera of Lythraceae s.l. were chosen for cladistic analysis, employing a morphological matrix of 15 characters (Tables 1 and 2). Lythrum was chosen as the outgroup because it is a basal member of the superclade I of the family (Fig. 1g), according to molecular phylogenetic results of Lythraceae s.l. [10]. Since the relationships among the extant genera could not be resolved by morphological data alone [10], the backbone constraint tree approach was used to determine the phylogenetic position of Primotrapa. The constrained tree is modified from the Bayesian tree of the combined molecular data proposed by Graham et al. [10]. All characters were unordered and equally weighted, except one character, namely, the position of the ovary. This character is considered to be irreversible, because a superior ovary is plesiomorphic in Lythraceae s.l., and semi-inferior to  Note: a indicates characters and codes cited from Graham et al. [10] inferior ovaries are secondarily derived [10]. Maximum parsimony (MP) analysis was run in PAUP* 4.0a167 [53] using heuristic search algorithms with random addition (RA), 1000 replicates holding 1 tree at each step, tree bisection and reconnection (TBR) branch swapping and MULTREES settings, with the steepest descent option off. Bootstrap values were calculated from 10 replicates and RA sequences with 1000 replicates holding one tree.   Note: See Table 1 for codes. ? = missing data; a indicates character matrix cited from Graham et al. [10]