Mummified Oligocene fruits of Schima (Theaceae) and their systematic and biogeographic implications

The genus Schima includes about 20 species and is distributed only in southern China and adjacent areas of Asia. The previous molecular phylogenetic analysis suggested Schima is in the tribe Gordoniae, along with Gordonia and Franklinia. However, because few fossils have been reported, the biogeographic origin of Schima is still poorly known. In this paper mummified fossil fruits of Schima are described from the upper Oligocene Yongning Formation of the Nanning Basin, Guangxi, South China. In gross morphology, the new fossil species, Schima kwangsiensis, is similar to the extant S. superba by its pentacarpellate, loculicidally dehiscent capsules, 5 imbricate sepals, pedicels with bracteoles and marginally winged seeds. Due to its excellent preservation, the new species may provide sufficient details for understanding the early evolutionary and phytogeographic history of the genus. Morphological clustering analysis shows that the new fossil species is closely related to two extant species (S. wallichii and S. superba) in the genus, implying that they may belong to an ancient taxon that occurs earlier than the others. More importantly, this discovery represents the earliest record of this genus in Asia and it explicitly moves the fossil record back to the late Oligocene in this region.


Systematics
Class   Etymology. The epithet "kwangsiensis" means the fossil is collected in Kwangsi (Guangxi) Province, China.   (Fig. 5C). Comparison. The new fossil species is characterized by its 5-loculed loculicidally dehiscent capsules, 5 imbricate sepals and pedicels with bracteoles. These features are consistent with the subfamily Theoideae of Theaceae and readily distinguish the fossils from fruits of other angiosperm families 18 . Modern Theoideae are divided into two main groups according to seed characters: one is composed of taxa bearing wingless seeds and includes Camellia, Tutcheria, Pyrenaria, Parapyrenaria, Apterosperma and Franklinia; the other encompasses taxa with winged seeds such as Gordonia, Laplacea, Schima, Hartia and Stewartia 6 . Furthermore, all the genera with winged seeds can be further divided into two groups based on seeds having either apical or marginal wings. Those genera with marginally winged seeds mainly include Schima, Stewartia and Hartia, but the columella in fruit of Stewartia and Hartia is incomplete or only extends ca. 1/2 the length of the locule, easily distinguishing these genera. As for Franklinia, its immature seeds sometimes have diminished wings, but the dehiscence mode of the capsules in Franklinia is unique that they have both loculicidal dehiscence and septicidal dehiscence, and the capsule valves rarely adheres to the central part of the columella 8,9 . Therefore, compared with other related genera, these fossil fruits belong to Schima because they have marginally winged seeds and persistent columellae extending most of the length of the locules. Initially, the genus Schima was established in 1823, based on the type species S. noronhae Reinwardt ex Blume from Indonesia; the species has long pedicels, two caducous bracteoles, five suborbicular, ca.5 mm sepals, globose 5-loculed capsules and marginally winged seeds 19 . Since then, additional Schima species have been reported from China and adjacent areas. At present, it is generally acknowledged that there are a total of 20 species belonging to Schima, and 13 of them are distributed in China 3 . Compared with extant species of Schima, the new fossil species is distinguished by its longer pedicels, smaller sepals and larger globose fruits. Among extant Schima plants in China, the sepals are normally larger than 5 mm in diameter, and there are only two species (S. argentea and S. superba) with sepals measuring 2 mm in diam.; but they both have shorter pedicels (1-2 cm). Moreover, the size of the fruits is different. In fruit gross morphology S. superba can be viewed as the closest relative to the new fossil species.
To date, three fossil fruits of Schima have been reported, among which two species, S. macrocalycalis Mai and S. lignitica (Menzel) Mai, are from Germany 14 ; another one, namely S. nanlinensis Li, Awasthi, Yang and Li., from China 15 . The fruit of S. lignitica is ovoid to oblong which can easily be distinguished from our fossils. The difference between the new species and S. macrocalycalis is that the latter has larger and suborbicular sepals. S.  Table 1.

Systematic implication.
Theaceae is a large and complex family in angiosperms that includes approximately 19 genera and 600 species 3,6 . The classification of Theaceae has been disputed since the family was established. Traditionally, it is made up of two subfamilies Theoideae and Ternstroemioideae 5, 20-22 . However, a series of molecular studies suggested that the two subfamilies should be regarded as separate families 10,[23][24][25] . In APG III system of 2009, Ternstroemioideae was removed from the Theaceae to form the Ternstroemiaceae together with Pentaphylax (Pentaphylacaceae) 26 . Nevertheless, evolutionary trends within the Theaceae s.s. ( = Theoideae) remain controversial, which was shown by quite different classification systems 6,27 . Specifically, the evolutionary significance of pedicel length has been subject to intense debate during the past few decades 6-8 . Keng (1980) even suggested that species with a long pedicel are more primitive than those with a short pedicel 28 , but the hypothesis is controversial due to the lack of definitive cladistic and fossil evidence. Hence, the mummified fossil fruits with intact pedicels described here could shed light on the early evolution of the genus Schima, and even of the family Theaceae.
In order to gain a clearer taxonomic position of Schima kwangsiensis within the genus, a clustering analysis based on eight fruit characters (Tables 2, 3) for two fossil species and 13 extant species of the genus in China was performed using a modification of Ward's method in R package 29 . The result shows that the species within Schima, including both the fossil and extant species, may be divided into four groups. As shown in the dendrogram (Fig. 6), clade A consists of only two species S. brevipedicellata and S. multibracteata, and is distinguished from the others by having short pedicels and large sepals. Clade B, represented by S. sinensis, S. sericans, S. noronhae, S. remotiserrata, S. villosa, S. crenata, and S. khasiana, mainly bears long pedicels and large sepals. Clade C, including the new fossil S. kwangsiensis and two extant species S. superba, S. wallichii, is characterized by having long pedicels and small sepals (there is one exception). The last clade, comprising one fossil species S. nanlinensis, and two extant species, S. argentea and S. parviflora, has small sepals and short pedicels. Furthermore, among 13 extant species, eight taxa have long pedicels and five taxa have short pedicels. It is obvious that the two morphological characters, pedicel and sepal size, are tightly correlated with each other in the genus Schima (Fig. 6). In general, the cluster analysis shows that the new fossil species is closely similar in pedicel and calyx size to two extant species (S. wallichii and S. superba) in the genus, implying that they may belong to an ancient taxon that predated the others.
Paleogeography and Paleoecology. The origin and biogeographic history of Schima is little known because of the paucity of the fossil records. Up to now, only five definitive fossil occurrences have been reported in the world. Three fossil species are recognized on the basis of fruits, including Schima macrocalycalis from the middle Eocene of Germany, Schima lignitica from the late Miocene of Germany 14 , and Schima nanlinensis from the Miocene of Yunnan, China 15 . One wood fossil, Schima protowallichii occurs in the Miocene of Japan 30 , and a leaf fossil with well preserved cuticle was described as Schima mataschensis, is from the late Miocene of Styria, Austria 31 . In addition, one seed fossil, Schima euryoidesonce was reported from the late Eocene of Germany. However, its identity is doubtful as it has horseshoe-shaped embryos, a feature not found in Schima 32 .
The Schima fruits reported here are the earliest fossils of this genus in Asia, and imply that this genus probably first appeared in Asia, its modern distribution area, by the late Oligocene. Moreover, the new fossil record is   important because of the excellent preservation. Extant Schima has a small and lightweight seed with a marginal wing and the special structure of seeds aids long distance dispersal by wind 6 . The seeds of Schima fossils are morphologically similar to those of the extant genus implying that little change has occurred in the shape of seeds' wing during the past 23 million years. In addition to the fossils considered above, four species of Schimoxylon-a wood genus resembling Schima were reported, among which Schimoxylon dachelense was from the Upper Cretaceous of Egypt 33 , Schimoxylon g ordonioides was from the Tertiary deposits of Borneo of uncertain age 34 , Schimoxylon altingioides was from the Eocene of Germany 35 , and Schimoxylon benderi was from the late middle Eocene of Myanmar 36 (Fig. 7). According to these fossil records, Schima possibly appeared by Late Cretaceous and achieved a widespread distribution from low-latitudes to middle latitudes during the Tertiary and was more widespread in the Northern Hemisphere than today. It suggests the ancient species of Schima probably originated in Northern Africa or Western Europe and further dispersed to the regions of Asia. However, the genus apparently experienced subsequent extinction both in Europe and in North Africa later and is now confined to subtropical and tropical zones within South China and Southeast Asia.   Extant Schima superba, which has similar fruits, is a tree up to ca. 30 m tall and occurs in evergreen broadleaved forests with a wide distribution in the subtropical and tropical montane areas at altitudes of 100-2500 m 37 . It is likely that Schima kwangsiensis may have been tall trees and grown in a similar environment. Additionally, preliminary investigations suggest that the mummified flora reported here mainly comprises Fagaceae, Theaceae, Bambusoideae, Anacardiaceae, Annonaceae, and Nyssaceae representing flowering plants, and Polyporaceae of the higher fungi 16 . These fossil taxa provide a glimpse of ancient ecological environments in Guangxi, indicating there has been a typical montane evergreen broad-leaved forest in southern China since at least the late Oligocene. The dominant species were mainly Schima kwangsiensis and some groups of Fagaceae in the tree layer, which grew under a warm moist forest environment.

Methods
The fossil fruits were exceptionally well-preserved, and include intact pedicels and seeds. The specimens were thoroughly washed in water and dried in air. They were observed and photographed using a stereoscopic microscope (Zeiss Stereo Discovery V20). The seed micromorphology was investigated using a scanning electronic microscope (SEM Quanta 400 F). Next, the resulting images were processed with Adobe Photoshop CS5 (Adobe Inc., San Jose, California, USA). The extant fruits of Schima superba for comparison were collected from Heishiding Nature Reserve, Guangdong Province. To prevent the potential fracturing on drying, all the megafossil specimens used herein are preserved in a mixed solution of 50% alcohol and 100% glycerol with the volume ratio of 10:1 and deposited at the Museum of Biology of Sun Yat-sen University (SYS), Guangzhou, China. The terminology used to describe the fruit and seed follows Keng 8 , and Min & Bartholomew 3 .
Clustering analysis was performed using R (http://www.r-project.org/). The Excel file containing the binary data was imported to R package and the 0/1 matrix was used to calculate Euclidean distance. The resultant distance matrix was employed to construct dendrograms using hierarchical cluster analysis with Ward's algorithm to infer genetic relationships 38 .