Fossil Fruits of Ceratophyllum from the Upper Eocene and Miocene of South China

Simple Summary Two fruit fossil species of Ceratophyllum L. are discovered from South China, namely C. cf. muricatum Chamisso from the upper Eocene of the Maoming Basin, Guangdong, and C. demersum L. from the Miocene of the Guiping Basin, Guangxi. Our findings provide evidence for the distribution of Ceratophyllum in South China in the late Eocene, and its wide expansion in subtropical China during the Miocene. Abstract Ceratophyllum L. is a cosmopolitan genus of perennial aquatic herbs that occur in quiet freshwaters. Fossils of this genus have been widely reported from the Northern Hemisphere, most of them occurring in the temperate zone. Here, we describe two species of fossil fruits discovered from subtropical areas of China. The fossil fruit discovered from the upper Eocene Huangniuling Formation of the Maoming Basin is designated as C. cf. muricatum Chamisso, and fruits discovered from the Miocene Erzitang Formation of the Guiping Basin are assigned to the extant species C. demersum L. The discovery of these two fossil species indicates that Ceratophyllum had spread to South China by the late Eocene and their distribution expanded in subtropical China during the Miocene.


Introduction
Ceratophyllaceae Gray is a family of submersed, hydrophilous, perennial, and herbaceous plants. It consists of only one cosmopolitan genus, Ceratophyllum L., and about six extant species [1]. Ceratophyllaceae is a sister group of the eudicots [2]. The infrageneric taxonomy of Ceratophyllum is largely based on fruit characters. It is generally divided into three sections, with two species in each section. Sect. Ceratophyllum has fruits with three to five long spines, sect. Muricatum has fruits with spiny and sometimes winged margins with stylar and basal spines and sect. Submersum has fruits with a spiny margin without stylar and basal spines [3,4].
In this paper, two species of Ceratophyllum fruit fossils are described from late Eocene and Miocene strata of South China. The new fossil occurrences provide important insights into the palaeophytogeography of this genus.

Materials and Methods
The fossil specimens investigated here were collected from two localities within South China (Figure 1). One specimen was collected from the upper part of the Huangniuling Formation (21°42′33.2″ N, 110°53′19.4″ E) of the Maoming Basin, Guangdong. This formation is composed mainly of gray, yellow to white sandstones, siltstones, and conglomerates [18]. The age of Huangniuling Formation is late Eocene according to palaeomagnetic data [19] and pollen assemblages [20]. Other fruit specimens were collected from the Erzitang Formation (23°23′09.67″ N, 110°09′55.21″ E) of the Guiping Basin, Guangxi. The Erzitang Formation is mainly composed of greyish yellow and red mudstone. The geological age of this formation is Miocene based on the mammal fossil Prolipotes yujiangensis Zhou, Zhou et Zhao [21,22].
Specimens were photographed and measured using a Sony Alpha 6400 Camera and a Nikon SMZ25 stereo microscope. The pericarp of the fossil fruit was examined using a JSM-6330F scanning electron microscope. The terminology for Ceratophyllum fruit description follows that used in the monograph of Les [3]. All fossils described here are deposited in the Museum of Biology, Sun Yat-sen University, Guangzhou.

Ceratophyllum cf. muricatum Chamisso
Family: Ceratophyllaceae Gray Genus: Ceratophyllum L.  Specimen: MMJ3-2907 Locality: Maoming Basin, Guangdong, South China Geological horizon and age: Huangniuling Formation, late Eocene Description: Achene axis symmetric, elliptic to suborbicular, 3.6 mm long, 3.2 mm wide (excluding the spines), with a length/width ratio of about 1.1 (Figure 2A,C). The surface of the fruit has granular ornamentation ( Figure 2B). The fruit body has five prominent spines: one stylar spine (incomplete, ca. 0.9 mm long), two lateral spines 0.9-1 mm long, and two basal spines 2.7-3.8 mm in length (Figure 2A,C). The impression of a marginal wing is clear, and slightly toothed at the apex ( Figure 2A).
Remarks: The characteristics of Ceratophyllum fruit include shape, size, spine, surface, and wings [3], while the most informative taxonomic characters are the fruit size and the spines [25]. The morphological comparison of our fossil to other Ceratophyllum fruit fossils is summarized ( Table 1).
The fossil fruit from Maoming is characterized by it being an axially symmetrical fruit with marginal wings and five spines: a stylar spine, two lateral spines and two basal spines. Accordingly, this fruit can be assigned to sect. Muricatum. This section includes two modern species, C. tanaiticum Sapjegin and C. muricatum Chamisso.
The fossil from Maoming in compared to Ceratophyllum tanaiticum has fewer lateral spines. C. tanaiticum is a Pontic-Caspian endemic relict plant [3]. Fruit of C. tanaiticum possess several short lateral and basal spines, with the length of spines about 0.35-3.15 mm and a minute stylar spine [4,26,27]. Meanwhile, our fossil has only two clear lateral spines and longer basal spines.
Instead, our fossil is more morphologically similar to Ceratophyllum muricatum. C. muricatum have at least two basal spines of achene margin, and fruit body length is less than 4.5 mm [27]. C. muricatum has three subspecies, among which, fruits of C. muricatum subsp. australe (Grisebach) Les and subsp. muricatum Cham. have more lateral spines compared with those of subsp. kossinskyi (Kuzen.) Les [4]. Our fossil fruit can be easily distinguished from previously reported fossil species assigned to sect. Muricatum based on the number of lateral spines: our fossil fruit has only two lateral spines, while those reported fossil species have at least five lateral spines [7,8,14]. Despite minor intraspecific variation, there is very small amount of evolutionary change in Ceratophyllum [28]. Therefore, we prefer to assign the fossil to Ceratophyllum cf. muricatum Chamisso.  (Figure 2A,C). The surface of the fruit has granular ornamentation ( Figure 2B). The fruit body has five prominent spines: one stylar spine (incomplete, ca. 0.9 mm long), two lateral spines of 0.9-1 mm long, and two basal spines of 2.7-3.8 mm in length (Figure 2A,C). The impression of a marginal wing is clear, and slightly toothed at the apex (Figure 2A).
Remarks: The characteristics of Ceratophyllum fruit include shape, size, spine, surface, and wings [3], while the most informative taxonomic characters are the fruit size and the spines [25]. The morphological comparison of our fossil to other Ceratophyllum fruit fossils is summarized ( Table 1).
The fossil fruit from Maoming is characterized by it being an axially symmetrical fruit with marginal wings and five spines: a stylar spine, two lateral spines and two basal spines. Accordingly, this fruit can be assigned to sect. Muricatum. This section includes two modern species, C. tanaiticum Sapjegin and C. muricatum Chamisso.
Fossil from Maoming in compare to Ceratophyllum tanaiticum has fewer lateral spines. C. tanaiticum is a Pontic-Caspian endemic relict plant [3]. Fruit of C. tanaiticum possess several short lateral and basal spines, with the length of spines about 0.35-3.15 mm and a minute stylar spine [4,26,27]. While our fossil has only two clear lateral spines and longer basal spines.
Instead, our fossil is more morphologically similar to Ceratophyllum muricatum. C. muricatum have at least two basal spines of achene margin, and fruit body length is less than 4.5 mm [27]. C. muricatum has three subspecies, among which, fruits of C. muricatum subsp. australe (Grisebach) Les and subsp. muricatum Cham. have more lateral spines compared with those of subsp. kossinskyi (Kuzen.) Les [4]. Our fossil fruit can be easily distinguished from previously reported fossil species assigned to sect. Muricatum based on the number of lateral spines: our fossil fruit has only 2 lateral spines, while those reported fossil species have at least 5 lateral spines [7,8,14]. Despite minor intraspecific variation, there is very small amount of evolutionary change in Ceratophyllum [28]. Therefore, we prefer to assign the fossil to Ceratophyllum cf. muricatum Chamisso.    Figure 3A,B), with a length/width ratio of 1.48-1.79. The surfaces of the fruit bodies are tuberculate, with striated grooves near the edge (Figure 3A,E,G,I,J). The achenes have an apical stylar spine and a pair of basal spines ( Figure 3A). The largest stylar spine is 2.79 mm in length and 0.33 mm in width ( Figure 3K). The length of stylar spines ranges from 0.22 to 2.79 mm, with a mean of 0.76 mm. The length of basal spines ranges from 0.15-1.35 mm, with a mean of 0.59 mm. Cotyledons are observed when the fruits are longitudinally split into two halves ( Figure 3D,F,H). Two symmetrical holes are present on the base, which represent the pedicel connection ( Figure 3C). There is a small projecting part at the base ( Figure 3G,L).
The outer surface of the fruit exocarp consists of irregular polygonal cells with lengths of 10-25 µm ( Figure 4A,B,E), with some of the cells having parallel partitions ( Figure 4C,D). The inner surface of the endocarp is composed mainly of rectangular cells, about 20 µm × 40 µm in size ( Figure 4F).
Remarks: Our fossils have three spines, two basal spines, and one stylar spine. The fruits have a rough surface and no marginal wings. These characteristics are consistent with the fruit features of sect. Ceratophyllum (fruits with three to five spines but no winged margins). Sect. Ceratophyllum includes two species: C. platyacanthum Chamisso and C. demersum L. The fruit of C. platyacanthum has five spines, which include two distinctive facial spines. Our three-spined fruit fossils can be distinguished from this species. In comparison, the fruit of C. demersum is 3.5-6 × 2-4 mm in size, with two basal spines and one stylar spine, lacks a marginal wing, and its facial spines are absent.
By carefully comparing the shape, size, and spines of our specimens and the fruit characters of the extant species C. demersum, we cannot discern any significant morphological differences between them. Additionally, the morphological data of our fossil materials is close to other fossils designated as C. demersum [11,17] (Table 1). So, these fossil fruits are assigned to C. demersum L.

Discussion
Species of the genus Ceratophyllum are known to be highly tolerant of a wide range of temperature and salinity values, leading to their broad distribution ( Figure 5). The aquatic environment in which representatives of this genus live provides relatively buffered conditions minimizing pressure on its evolution and distribution [30].
Fossils of Ceratophyllum have been widely reported from the Northern Hemisphere ( Figure 5). The earliest fossil C. lesii was found in the Late Cretaceous of Mexico [6] and is similar to the modern species C. demersum. An extinct genus Donlesia Dilcher et Wang with affinities to Ceratophyllaceae was reported from the Lower Cretaceous of Kansas, USA [31,32]. Based on these findings, it is considered that Ceratophyllaceae may have originated in North and Central America during the Cretaceous, in accordance with the divergence time of Ceratophyllaceae from the rest of the eudicots based on a time-calibrated global angiosperm phylogeny [5].
The earliest Ceratophyllum fossil of sect. Muricatum was discovered from the Paleocene in the Fort Union Formation in the United States, designated as C. furcatispinum [7]. Later, C. muricatum subsp. incertum was discovered from the lower and middle Eocene in North America [7], and C. aff. muricatum from the middle Eocene of China [8]. The two species share a great number of morphological similarities. The discovery of fossils of Ceratophyllum in China suggests a floristic exchange between Asia and North America during the Eocene [8]. Our discovery of C. cf. muricatum from the late Eocene of Maoming Basin provides further evidence supporting this supposition, which also suggests Ceratophyllum was distributed in the subtropical region at that time.
Ceratophyllum fossils have also been reported from Asia in the Oligocene, such as C. zaisanicum Avakov from the Zaysan Basin of Kazakhstan [33], C. submersum L., and C. tenuicarpum Dorof. from Siberia, Russia [10,11]. The distribution area of this genus expanded during the Miocene, and Ceratophyllum fossils have been widely reported in Asia and Europe [11,12,16,17], such as the fruit fossils C. miocenicum Dorof. and C. pannonicum Dorof. from the Orlovka and Lgov, Russia, respectively [11], and C. lusaticum Mai from Leipzig, Germany [12]. From the middle Miocene Shanwang Formation of eastern China, stems and fruits referred to Ceratophyllum have been reported [9,14]. The discovery of Miocene fruit fossils of C. demersum from the Erzitang Formation of the Guiping Basin, Guangxi, together with the discovery of the same species from late Miocene of Huaning

Discussion
Species of the genus Ceratophyllum are known to be highly tolerant of a wide range of temperature and salinity values, leading to their broad distribution ( Figure 5). The aquatic environment in which representatives of this genus live provides relatively buffered conditions minimizing pressure on its evolution and distribution [30]. County, Yunnan, Southwest China [17], suggest that C. demersum was widely distributed in subtropical China in the Miocene.

Conclusions
Two fruit fossil species of Ceratophyllum are reported from low latitude of Asia in this paper. By morphological comparison with modern and fossil species, fruit fossil found from the upper Eocene Huangniuling Formation of the Maoming Basin, Guangdong, is assigned to C. cf. muricatum Chamisso. The discovery of this fossil indicates that Ceratophyllum has been distributed in South China by the late Eocene. Fossils discovered from the Miocene Erzitang Formation of the Guiping Basin, Guangxi, are designated to the extant species C. demersum L. The emergence of these fossils, together with the discovery of Fossils of Ceratophyllum have been widely reported from the Northern Hemisphere ( Figure 5). The earliest fossil C. lesii was found from the Upper Cretaceous of Mexico [6] and is similar to the modern species C. demersum. An extinct genus Donlesia Dilcher et Wang with affinities to Ceratophyllaceae was reported from the Lower Cretaceous of Kansas,