Bauhinia (Leguminosae) Fossils from the Paleogene of Southwestern China and Its Species Accumulation in Asia

: Extant Bauhinia (Leguminosae) is a genus of 300 species of trees, shrubs, and lianas, widely distributed in pantropical areas, but its diversiﬁcation history in southeastern Asia, one of its centers of highest diversity, remains unclear. We report new fossils of three Bauhinia species with cuticular preservation from the Paleogene of Puyang Basin, southwestern China. Our ﬁnding likely extends the emergence of Bauhinia in Asia to the late Eocene. Together with previously reported fossil records, we show that the diversiﬁcation of Bauhina in Asia and the phenomenon of a small region harboring multiple Bauhinia species in southwestern China could be traced back to the Paleogene.


Introduction
Bauhinia L. (Leguminosae) today comprises about 300 species of trees, shrubs, and lianas and is widely distributed in pantropical areas, with the largest diversity center being in the neotropics, and the second largest in southeastern Asia [1][2][3] (Figure 1). A typical leaf of this genus is simple and bilobed, rarely entire or two-foliolate, with pulvinus on both ends of the petiole [1]. Its fruit is flat, elliptic, oblong, or linear, woody or thinly valved. Species of Bauhinia are widely cultivated as ornamentals [1]. For example, the orchid tree (Bauhinia × blakeana Dunn) was chosen as the city flower of Hongkong. Several species of Bauhinia (e.g., B. purpurea L.) are used in local medicine and seeds of B. petersiana Bolle can be used as a coffee substitute [3].
This study reports new Bauhinia fossils from the late Eocene of southeastern China. First, we morphologically compared the macroscopic morphology and cuticle features of the fossils with those of the extant and fossil species in the genus. Then we discussed the implications of the fossils in the context of our current understanding of the evolutionary history of Bauhinia in Asia.

Geological Setting
The Puyang Basin (105.26° E, 23.48° N; 825 m asl) is a wedge-shaped strike-slip basin located in the southeastern Yunnan province, China [19][20][21] (Figure 2). The base of the basin is Cambrian limestone, with Cenozoic sediments unconformably lain above [20] ( Figure 3). The lower part of the Cenozoic basin fill is dominated by lignite beds representing swamp facies, and the upper part is mainly lacustrine grey to yellow mudstone [20]. Recently, a mammal fossil, belonging to Anthracotheriidae Leidy, similar to the late Eocene Bothriogenys hui from Yunnan and B. orientalis from Thailand [22,23], was recovered from the lignite (Figure 3). Pollen analysis also suggests a late Eocene age for the lignite bed (Yang et al. under review) and suggests that basin formation was roughly coeval with other regional basins such as Wenshan [13] and Lühe [24] which have been dated radiometrically. Our fossils are collected from the lacustrine mudstone above the lignite bed ( Figure 3) and are most likely also late Eocene in age. Fossils of Bauhinia have been documented in various forms of wood, leaf, and twig with attached fruit [9][10][11][12]. The earliest reliable fossils are leaves from the early Oligocene of China [11,13]. Later fossils of the genus are documented from the late Oligocene and Middle Miocene of China, the Oligocene of Mexico, and the early Miocene and middle Miocene-middle Pleistocene of India, the Pliocene of Uganda, and the Miocene of Ecuador [9,10,12,[14][15][16][17][18].
This study reports new Bauhinia fossils from the late Eocene of southeastern China. First, we morphologically compared the macroscopic morphology and cuticle features of the fossils with those of the extant and fossil species in the genus. Then we discussed the implications of the fossils in the context of our current understanding of the evolutionary history of Bauhinia in Asia.

Geological Setting
The Puyang Basin (105.26 • E, 23.48 • N; 825 m asl) is a wedge-shaped strike-slip basin located in the southeastern Yunnan province, China [19][20][21] (Figure 2). The base of the basin is Cambrian limestone, with Cenozoic sediments unconformably lain above [20] (Figure 3). The lower part of the Cenozoic basin fill is dominated by lignite beds representing swamp facies, and the upper part is mainly lacustrine grey to yellow mudstone [20]. Recently, a mammal fossil, belonging to Anthracotheriidae Leidy, similar to the late Eocene Bothriogenys hui from Yunnan and B. orientalis from Thailand [22,23], was recovered from the lignite ( Figure 3). Pollen analysis also suggests a late Eocene age for the lignite bed (Yang et al. under review) and suggests that basin formation was roughly coeval with other regional basins such as Wenshan [13] and Lühe [24] which have been dated radiometrically. Our fossils are collected from the lacustrine mudstone above the lignite bed ( Figure 3) and are most likely also late Eocene in age.

Macroscopic Feature Observations and the Modern Distribution of Bauhinia
Fifteen leaf fossils and one fruit compression were recovered and photographed using a digital camera (Nikon D750, Kanagawa, Japan). Fine-scale details of the fossils were further examined under a stereo microscope (Leica S8APO, Wetzlar, Germany), and images were taken. The raw data for the extant occurrence of Bauhinia was download from Gbif [25], and first cleaned using an R program and then checked manually [26]. Finally, the cleaned data were imported into Arcgis 10.0 to prepare the distributional heat map.

Macroscopic Feature Observations and the Modern Distribution of Bauhinia
Fifteen leaf fossils and one fruit compression were recovered and photographed using a digital camera (Nikon D750, Kanagawa, Japan). Fine-scale details of the fossils were further examined under a stereo microscope (Leica S8APO, Wetzlar, Germany), and images were taken. The raw data for the extant occurrence of Bauhinia was download from Gbif [25], and first cleaned using an R program and then checked manually [26]. Finally, the cleaned data were imported into Arcgis 10.0 to prepare the distributional heat map.

Macroscopic Feature Observations and the Modern Distribution of Bauhinia
Fifteen leaf fossils and one fruit compression were recovered and photographed using a digital camera (Nikon D750, Kanagawa, Japan). Fine-scale details of the fossils were further examined under a stereo microscope (Leica S8APO, Wetzlar, Germany), and images were taken. The raw data for the extant occurrence of Bauhinia was download from Gbif [25], and first cleaned using an R program and then checked manually [26]. Finally, the cleaned data were imported into Arcgis 10.0 to prepare the distributional heat map.

Cuticle Preparation for Fossil and Extant Materials
Fossil leaf fragments were treated with HCl and HF to remove calcareous and siliceous materials, and then macerated using 3% NaClO solution for 30 min to one hour until they became translucent [27][28][29]. For extant materials, fragments from mature leaves were macerated using a 1:1 solution of CH 3 COOH and 30% H 2 O 2 at 80 • C for about one hour [30,31]. After the mesophyll tissue was removed, the adaxial and abaxial cuticles for both fossil and extant materials were stained for about 30 min using Safranin O, mounted in glycerine on glass slides, and then photographed using a light microscope (Leica DM 750 with a Leica DFC 295 camera). All cuticular slides are stored at Kunming Institute of Botany, Chinese Academy of Sciences. Description: Leaf is entire and bilobed, 28-34 mm long and 18-26 mm wide, ovate to elliptical in outline ( Figure 4A-D). The basal portion is cordate, slightly asymmetrical ( Figure 4A,B). The widest part is in the lower third of the leaf, and the lamina gradually tapers toward the apex ( Figure 4A,B). The apex is bifid to form two acute lobes at an angle of 31 • -49 • ( Figure 4A,B). The primary vein framework is palmate with nine basal veins ( Figure 4A Figure 6A,B). The basal portion is cordate and weakly asymmetrical ( Figure 6B). The widest part is in the middle of the leaf ( Figure 6A,B). Primary vein framework is palmate with seven basal veins ( Figure 6B).

Results
Fruit. Species: cf. Bauhinia sp. (morphotype 4). Specimens: FN0465 ( Figure 6C-E). Description: Fruit is flat, elliptic to oblong, 42 mm long and 17 mm wide ( Figure 6C). The left flank of the proximal end is nearly straight, and the right flank is convex ( Figure 6D). The distal end is acuminate ( Figure 6E). The stigmatic remain is short and persistent ( Figure 6E). There is a constriction in the middle of the fruit ( Figure 6C). The suture lines are prominent, about 0.5 mm wide ( Figure 6D,E). The seed chambers are elliptic, 4.7-10.4 mm long and 3.6-4.6 mm wide ( Figure 6C,E). The angle between the long axis of the seed chambers and those of fruit is 94-100 • ( Figure 6C).   Figure 6A,B). The basal portion is cordate and weakly asymmetrical ( Figure 6B). The widest part is in the middle of the leaf ( Figure 6A,B). Primary vein framework is palmate with seven basal veins ( Figure 6B).  Description: Fruit is flat, elliptic to oblong, 42 mm long and 17 mm wide ( Figure 6C). The left flank of the proximal end is nearly straight, and the right flank is convex ( Figure  6D). The distal end is acuminate ( Figure 6E). The stigmatic remain is short and persistent ( Figure 6E). There is a constriction in the middle of the fruit ( Figure 6C). The suture lines are prominent, about 0.5 mm wide ( Figure 6D,E). The seed chambers are elliptic, 4.7-10.4 mm long and 3.6-4.6 mm wide ( Figure 6C,E). The angle between the long axis of the seed chambers and those of fruit is 94-100° ( Figure 6C).

Morphological Comparison
The fossil leaves are characterized by simple and bilobed leaves. As far as we know, such kinds of leaves are seen in several families including Ginkgoaceae Engl., Lauraceae Juss., Passifloraceae Juss. ex Roussel, Proteaceae Juss., and Leguminosae Juss. (Figure 7). However, venation of the Ginkgoaceae leaves is dichotomous, and so different from our fossils where the venation is reticulate. The leaves of Passifloraceae have a small middle lobe or a broad angle (larger than 90°) between the lobes, distinguishing them from our fossils, which are strictly bilobed and diverge at an angle less than 90°. The palmate venation of Dilobia Thours. in the Proteaceae (see cleared leaf in Pole and Bowman (1996) [32]) and some species in Lauraceae such as Sassafras albidum (Nutt.) Nees is suprabasal but that of our fossils is basal. In Leguminosae, Pueraria DC., Desmodium Desv., Christia Moench, and Bauhinia L. have bilobed leaves, but leaves of Pueraria are prominently deflective and asymmetric, differing from our fossils, which are nearly symmetric. Secondary veins of Desmodium leaves are parallel whereas those of our fossils extend towards the leaf apices. Leaves of Christia have pinnate venation, distinguishing them from our fossils which possess palmate venation. Overall, our fossil leaves are a close match with Bauhinia.

Morphological Comparison
The fossil leaves are characterized by simple and bilobed leaves. As far as we know, such kinds of leaves are seen in several families including Ginkgoaceae Engl., Lauraceae Juss., Passifloraceae Juss. ex Roussel, Proteaceae Juss., and Leguminosae Juss. (Figure 7). However, venation of the Ginkgoaceae leaves is dichotomous, and so different from our fossils where the venation is reticulate. The leaves of Passifloraceae have a small middle lobe or a broad angle (larger than 90 • ) between the lobes, distinguishing them from our fossils, which are strictly bilobed and diverge at an angle less than 90 • . The palmate venation of Dilobia Thours. in the Proteaceae (see cleared leaf in Pole and Bowman (1996) [32]) and some species in Lauraceae such as Sassafras albidum (Nutt.) Nees is suprabasal but that of our fossils is basal. In Leguminosae, Pueraria DC., Desmodium Desv., Christia Moench, and Bauhinia L. have bilobed leaves, but leaves of Pueraria are prominently deflective and asymmetric, differing from our fossils, which are nearly symmetric. Secondary veins of Desmodium leaves are parallel whereas those of our fossils extend towards the leaf apices. Leaves of Christia have pinnate venation, distinguishing them from our fossils which possess palmate venation. Overall, our fossil leaves are a close match with Bauhinia.
Based on macroscopic and cuticular morphology, the fossil leaves can be divided into three morphotypes. Morphotype 1 has a cordate base, acute apex, single-layered stomatal rim, and sinuolate adaxial epidermal walls. Morphotype 2 has a straight base, round apex, double-layered stomatal rim, and straight arched epidermal walls, whereas morphotype 3 has a cordate base and a round apex. Although leaf and epidermal cell shape display intraspecific variability, the characteristics of the stomatal rim are considered stable at intraspecific level [33,34], so morphotypes 1 and 2 should represent different species. Whether morphotype 3 is another species will be discussed below. The three morphotypes are further compared with 46 extant species based on macroscopic morphology and cuticle features (see images and tables in Zou [35]), and then they are compared with fossil species.
Morphotype 1 is similar to B. acuminata L., B. comosa Craib, and B. esquirolii; Gagnep. in gross macroscopic morphology (Figures 4 and 8). However, the abaxial cuticle of morphotype 1 has paracytic and tetracytic stomatal complexes with sunken guard cells ( Figure 4J,K), and so is different from those of P. comosa and P. esquirolii that have paracytic stomatal complexes and the guard cells are not sunken ( Figure 8F,I). The abaxial cuticle of morphotype 1 is similar to that of B. acuminata in features including paracytic and tetracytic (some atypical) stomatal complexes with sunken guard cells and single-layered stomatal rim ( Figure 8C). A combination of macroscopic and cuticular features suggests morphotype 1 is possibly a close relative of B. acuminata. However, it is worth noting that stomata also appear on the adaxial epidermis of B. acuminata in small number ( Figure 8B), but we did not observe any from the adaxial epidermis of morphotype 1 ( Figure 4E,F). This may be because the region from which we successfully extracted epidermis lacks stomata while the rest of leaf has them, or that morphotype 1 is a hypostomatic leaf. When compared with the reported fossil species (Table 1), morphotype 1 is similar to B. wenshanensis H. H. Meng et Z. K. Zhou found from the early Oligocene of Yunan, China [11]. In consideration of its near identical age, we assign morphotype 1 to B. wenshanensis. However, cuticular features have not yet been reported for B. wenshanensis, so our description of the cuticle for morphotype 1 may be taken as a tentative description for B. wenshanensis, but this should be used with caution. Future studies may obtain cuticle for B. wenshanensis from its type locality. Based on macroscopic and cuticular morphology, the fossil leaves can be divided into three morphotypes. Morphotype 1 has a cordate base, acute apex, single-layered stomatal rim, and sinuolate adaxial epidermal walls. Morphotype 2 has a straight base, round apex, double-layered stomatal rim, and straight arched epidermal walls, whereas morphotype 3 has a cordate base and a round apex. Although leaf and epidermal cell shape display intraspecific variability, the characteristics of the stomatal rim are considered stable at intraspecific level [33,34], so morphotypes 1 and 2 should represent different species. Whether morphotype 3 is another species will be discussed below. The three morphotypes are further compared with 46 extant species based on macroscopic morphology and cuticle features (see images and tables in Zou [35]), and then they are compared with fossil species.
Morphotype 1 is similar to B. acuminata L., B. comosa Craib, and B. esquirolii Gagnep. in gross macroscopic morphology (Figures 4 and 8). However, the abaxial cuticle of morphotype 1 has paracytic and tetracytic stomatal complexes with sunken guard cells (Figure 4J,K), and so is different from those of P. comosa and P. esquirolii that have paracytic compared with the reported fossil species (Table 1), morphotype 1 is similar to B. wenshanensis H. H. Meng et Z. K. Zhou found from the early Oligocene of Yunan, China [11]. In consideration of its near identical age, we assign morphotype 1 to B. wenshanensis. However, cuticular features have not yet been reported for B. wenshanensis, so our description of the cuticle for morphotype 1 may be taken as a tentative description for B. wenshanensis, but this should be used with caution. Future studies may obtain cuticle for B. wenshanensis from its type locality.   Note. Carpenter et al. [41] reported bilobed leaves from the Cenozoic of Australia and assigned these fossils to cf. Cercideae/Detarieae. It is clear that the veins diverge from the midvein and extend into the apex of the lobes in these fossils, which distinguishes them from Bauhinia in which two of the basal veins extend into the lobe apices. Moreover, Biagolini et al. [42] documented a fragment of a leaf, lacking apex and cuticle, from the Paleogene of Brazil. Their fossil seems to have palmate venation, a kind of venation pattern that exists in many families such as Malvaceae, Euphorbiaceae and Lauraceae. This makes the assignment of the leaf to Bauhinia superficial.
Morphotype 2 is similar to B. purpurea L., B. viridescens Desv., B. tomentosa L., and B. racemosa Lam. in terms of macroscopic morphology (Figure 9). However, the adaxial and abaxial epidermis of B. purpurea do not have trichome bases ( Figure 9C,D), and so are different from those of morphotype 2 that possesses single-celled trichome bases ( Figure 5G,K). The adaxial epidermal walls of B. viridescens are sinuolate ( Figure 9G) whereas those of morphotype 2 are straight ( Figure 5D-F). The abaxial epidermis of B. tomentosa has single-celled glandular trichome bases ( Figure 9L), distinguishing it from morphotype 2 that has regular single-celled trichome bases ( Figure 5G,K). The trichome bases for adaxial and abaxial epidermises of B. racemose are multicellular ( Figure 9O,P) whereas those of morphotype 2 are unicellular ( Figure 5G,K). In addition, B. purpurea, B. viridescens, and B. tomentosa have paracytic stomatal complexes ( Figure 9D,H,L), and so are different from morphotype 2 with paracytic and tetracytic stomatal complexes ( Figure 5H-J). Moreover, these four extant Bauhinia species exhibit a single-layered stomatal rim (Figure 9C,D,G,H,K,L,O,P), whereas morphotype 2 has a double-layered stomatal rim ( Figure 5H-I). Overall, the four extant species above are different from morphotype 2 in their cuticular features. When compared to the fossil species, morphotype 2 is similar to Bauhcis moranii Calvillo-Canadell et Cevallos-Ferriz from the Oligocene of Mexico [14]. Due to no cuticular information in Bauhcis moranii and limited preservation of morphotype 2, we leave the nomenclature open for discussion.
Morphotypes 3 and 4, are similar to leaves and fruits of two species, i.e., Bauhinia touranensis Gagnep. and B. damiaoshanensis T. Chen ( Figure 10). The two morphotypes possibly represent the same species, but this species is distinguished from those represented by morphotypes 1 and 2 because the fruit (morphotype 4) is different from those of the close recent relatives of morphotypes 1 and 2. When compared to fossils, morphotype 3 is different from any previously reported species. Bauhinia larsenii D.X. Zhang et Y.F. Chen from the late Oligocene of Ningming Basin, southern China [12], is the only fruit fossil assigned to the genus so far. However, morphotype 4 is banded with an acuminate stigmatic remnant, distinguishing it from B. larsenii which is elliptical with an acute stigmatic remnant. We here treat the morphotypes 3 and 4 as undetermined species.
To conclude, our fossils represent at least three species. Morphotype 1 is assigned to B. wenshanensis. Morphotype 2 constitutes the second species (B. sp.), and morphotypes 3 and 4 are possibly the third one (B. sp.).

The Diversification of Bauhinia in Southeastern Asia
Southeastern Asia is one of the diversity centers of Bauhinia (Figure 1). A recent molecular phylogenetic study suggests that the diversification of Asian Bauhinia can be traced back to the Paleocene (~60 Ma) [11]. However, Asian Bauhinia fossils are only known from the early Oligocene so far (Table 1). This forms a gap between the fossil evidence and molecular dating. Our finding is most likely the earliest reliable fossil records of Bauhinia in Asia, extending the existence of the genus to the late Eocene. Bauhinia wenshanensis has been reported from the early Oligocene of Wenshan, and four species, i.e., B. larsenii D.X. Zhang et Y.F. Chen, B. ningmingensis Q. Wang, B. cheniae Q. Wang, and Bauhcis moranii have also been found from the late Oligocene of Ningming Basin, Guangxi, China [9,11,12]. This provides evidence that Bauhinia apparently diversified in Asia in the Oligocene. In the Miocene and later periods, B. An interesting phenomenon for the extant distributional pattern of Bauhinia in southwestern China is that a small area can harbor many species. For example, 10 species have been found living in the Laojun Mountain area while 14 species have been recorded from Dawei Mountain, southeastern Yunnan [43,44], close to the locality that yielded fossils in this study. Of primary interest is when this kind of pattern formed. The discovery of three Bauhinia species from the late Eocene Puyang Basin and four species in the late Oligocene Ningming Basin (Table 1) shows that the two basins once harbored multiple Bauhinia species. Therefore, the phenomenon of many Bauhinia species coexisting in a small area can now be traced back to at least the Paleogene. 9C,D,G,H,K,L,O,P), whereas morphotype 2 has a double-layered stomatal rim ( Figure  5H-I). Overall, the four extant species above are different from morphotype 2 in their cuticular features. When compared to the fossil species, morphotype 2 is similar to Bauhcis moranii Calvillo-Canadell et Cevallos-Ferriz from the Oligocene of Mexico [14]. Due to no cuticular information in Bauhcis moranii and limited preservation of morphotype 2, we leave the nomenclature open for discussion. Morphotypes 3 and 4, are similar to leaves and fruits of two species, i.e., Bauhinia touranensis Gagnep. and B. damiaoshanensis T. Chen ( Figure 10). The two morphotypes possibly represent the same species, but this species is distinguished from those represented by morphotypes 1 and 2 because the fruit (morphotype 4) is different from those et Y.F. Chen from the late Oligocene of Ningming Basin, southern China [12], is the only fruit fossil assigned to the genus so far. However, morphotype 4 is banded with an acuminate stigmatic remnant, distinguishing it from B. larsenii which is elliptical with an acute stigmatic remnant. We here treat the morphotypes 3 and 4 as undetermined species.
To conclude, our fossils represent at least three species. Morphotype 1 is assigned to B. wenshanensis. Morphotype 2 constitutes the second species (B. sp.), and morphotypes 3 and 4 are possibly the third one (B. sp.).

The Diversification of Bauhinia in Southeastern Asia
Southeastern Asia is one of the diversity centers of Bauhinia (Figure 1). A recent molecular phylogenetic study suggests that the diversification of Asian Bauhinia can be traced back to the Paleocene (~60 Ma) [11]. However, Asian Bauhinia fossils are only known from the early Oligocene so far (Table 1). This forms a gap between the fossil evidence and molecular dating. Our finding is most likely the earliest reliable fossil records of Bauhinia in Asia, extending the existence of the genus to the late Eocene. Bauhinia wenshanensis has been reported from the early Oligocene of Wenshan, and four species, i.e., B. larsenii D.X. Zhang et Y.F. Chen, B. ningmingensis Q. Wang, B. cheniae Q. Wang, and Bauhcis moranii have also been found from the late Oligocene of Ningming Basin, Guangxi, China [9,11,12]. This provides evidence that Bauhinia apparently diversified in Asia in the Oligocene. In the Miocene and later periods, B. krishnanunnii A. K. Mathur comes from the early It is worth noting that although the Neotropics today host the largest diversity of Bauhinia species, few early fossil records have been documented there (Table 1 and note  therein). Moreover, a recent molecular work points to a Neogene diversification of Neotropical Bauhinia species [11]. This scenario suggests that Asia is probably an ancient diversification center of Bauhinia, while the Neotropics is a more recent one, although this could result from under investigation of fossil records.