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Comparative Flower and Inflorescence Organogenesis among Genera of Betulaceae: Implications for Phylogenetic Relationships.


Betulaceae are one of six or seven families in Fagales (Manos & Steele, 1997; Qiu et al., 1998; Li et al, 2004; APG III, 2009; APG IV, 2016) and important economic trees and shrubs for wood, nuts, and horticulture. In addition, most species of Alnus show the capacity of fixing nitrogen due to their symbiotic association with actinomycetes (Bousquet et al., 1992; Maggia & Bousquet, 1994; Soltis et al., 1995; Werner et al, 2014; Li et al., 2015). The family includes six genera of deciduous, monoecious, and anemophilous woody plants with alternate and simple leaves, much reduced unisexual flowers with sessile or extremely short pedicels, and nuts. The inflorescences are compact thyrses (rarely racemes) with 3-flowered dichasia (rarely single flowers) as lateral branches (for terms, see Endress, 2010). The thyrses are unisexual, the male ones often pendulous as catkins, the female ones various in detail (the axis of the first order compact or elongate, the axes of the second order compact) (see also Li & Skvortsov, 1999).

The plants of the family are distributed primarily in temperate and subtropical or tropical montane regions of the Northern Hemisphere with a slight extension to Central and South America. There are 120 to 200 species in Betulaceae according to different authors (Winkler, 1904; Li & Cheng, 1979; Furlow, 1990; Chen, 1994b; Govaerts & Frodin, 1998; Li & Skvortsov, 1999). At the generic level, Corylus (ca. 14 spp.) and Ostrya (ca. 10 spp.) are disjunctly distributed among Europe, Asia, and North America, while Ostryopsis (3 spp.) is endemic to China. Alnus (ca. 26 spp.) has been divided into sect. Clethropsis, sect. Alnus, sect. Cremastogyne, and sect. Alnobetula. Betula (ca. 51 spp.) consists of sect. Betulaster and sect. Betula. Carpinus (31 spp.) includes sect. Distegocarpus and sect. Carpinus (see Table 1; Winkler, 1904; Jury, 1978; Chen, 1994a, b).

Both morphological and molecular studies have generally recognized two major lineages in Betulaceae either as tribes Betuleae (Alnus and Betula) and Coryleae (Corylus, Ostryopsis, Carpinus, and Ostrya) (Jussieu, 1789; Prantl, 1894; Winkler, 1904; Melchior, 1964; Li & Skvortsov, 1999), or as subfamilies Betuloideae and Coryloideae (Spach, 1841; Regel, 1861, 1868; Koehne, 1893; Rendle, 1925; Rehder, 1940; Hutchinson, 1967, 1973; Dahlgren, 1975, 1980, 1983; Jury, 1978; Takhtajan, 1980; Thome, 1973, 1983; Furlow, 1990; Bousquet et al., 1992; Chen et al., 1999; Forest et al., 2005; Grimm & Renner, 2013). Nevertheless, there are different viewpoints on the morphological evolution and phylogenetic relationships of genera in Betulaceae. Abbe (1935, 1974) studied floral vasculatures of all genera of Betulaceae and concluded that the ancestral inflorescences of Betulaceae were compound spikes consisting of cymes each with three flowers and the most primitive flowers are bisexual and trimerous with tricarpellary ovaries. According to him, Betuleae (Alnus, Betula) are earlier lineage than both Coryleae (Corylus, Ostryopsis) and Carpineae (Carpinus, Ostrya). In Betuleae or Betuloideae, some authors considered Betula as possessing more primitive features than Alnus (Spach, 1841; Regel, 1868; Bentham & Hooker, 1883; Prantl, 1894; Winkler, 1904), while others suggested that Betula might have gained more derived features than Alnus (Hall, 1952; Takhtajan, 1969, 1980; Furlow, 1979, 1983, 1990; Kikuzawa, 1982). Within the Coryloideae lineage, Winkler (1904) thought that Ostryopsis, Ostrya, Carpinus, and Corylus diverged successively. Bentham and Hooker (1883) thought that divergence order was Carpinus, Ostryopsis, Ostrya, and Corylus. Hall (1952) and Kikuzawa (1982) proposed that the earliest diverged genus was Corylus and youngest Carpinus based on wood anatomy and ecological characters. Hjehnqvist (1948) and Hardin and Bell (1986) proposed that Carpinus possessed more primitive characteristics than Corylus, based on the morphology of inflorescences and flowers and the trichome morphology of leaf epidermis, respectively. Through analyzing multiple vegetative and reproductive morphological characters, Furlow (1983) suggested that the divergence order was Carpinus, Ostrya, Ostryopsis, and Corylus. Crane (1989) and Chen (1994a,b) conducted cladistics analyses of morphological characters, and both proposed the phylogenetic sequence as Corylus, Ostryopsis, Carpinus, and Ostrya.

The divergence times of genera of Betulaceae were inferred in several studies based on rich and reliable fossils (e.g.. Crane, 1981, 1989; Crane & Stockey, 1987; Manchester & Crane, 1987; Crane et al., 1990; Sun & Stockey, 1992; Manchester & Chen, 1996, 1998; Pigg et al., 2003; Manchester et al., 2004) and molecular data (Bousquet et al, 1992; Forest et al., 2005; Grimm & Renner, 2013). However, the results varied with different datasets and dating methods. Using nuclear ribosomal DNA sequences (5S spacer and ITS), Forest et al. (2005) inferred the divergence times of Betulaceae. Based on maximum likelihood method, their median values of the molecular estimates are 119.0 million years old (Ma), 109.3 Ma, and 70.2 Ma for the crown groups of Betulaceae, Betuloideae, and Coryloideae, respectively. Grimm and Renner (2013) used both nuclear and plastid DNA sequences and estimated that the stem lineage of Betulaceae dated back to the late Cretaceous and the two subfamilies to the Paleocene; the most recent common ancestors of each of extant genera dated back to the mid- to late Miocene.

Along with the progress in the molecular phylogenetics of Betulaceae, only a few studies have been conducted on the micro-morphology of flowers and inflorescences in this family since Abbe (1935, 1938, 1974). Different from Abbe's work on the flower and inflorescence structures of Betulaceae based on a comparative observation of the vascular systems of reproductive organs, several organogenesis studies using scanning electron microscopy (SEM) provided micromorphological data on primordial occurrences of reproductive organs for some genera, which were very useful in clarifying the number, arrangement, and homology of each part of flower, cyme, and inflorescence in Betulaceae (Chen et al., 2001; Zhu & Lu, 2008; Lin et al., 2010; Zhu et al., 2014a, b, c). However, the comparative data in all genera are needed based on SEM observations. The importance of detailed and comprehensive morphological studies has been demonstrated in many other taxa (Endress & Stumpf, 1991; Endress & Friis, 2006; Endress & Matthews, 2006). The objectives of this study were to review floral ontogenetic patterns of Betulaceae based on SEM observations on inflorescence and flower development of all genera and sections, explore new structural characters, and evaluate their phylogenetic implications in the context of recent development in molecular phylogenetics of Betulaceae. Our results suggest that molecular and morphological data provide reciprocal support and evaluation for their contributions to our understanding of the evolutionary history of Betulaceae.

SEM Investigations

The staminate and pistillate reproductive buds and inflorescences of 21 species were collected from plants in natural areas or botanical gardens every 2-3 days from February to October. The vouchers were deposited in the Herbarium of the College of Life Sciences, Tonghua Normal University, China (Table 1). Pistillate flowers were artificially pollinated and collected twice daily to observe the process of pollen germination and growth.

The fresh materials were fixed in FAA (50% ethanol: acetic acid: formaldehyde = 89:6:5) for about 24 h and were transferred to 70% ethanol for storage. The floral buds and inflorescences were dissected in 95% ethanol, dehydrated through a graded series of ethanol: isoamyl acetate for 15-30 min (100:0, 75:25, 50:50, 25:75, 0:100%), critical-point dried in carbon dioxide with a Hitachi HCP-2, coated with gold palladium using a Hitachi E-1010 at 15 mA, and observed and photographed with a Hitachi S-3000 N SEM at 10.0 kV. Differences in both pistillate and staminate cymes and flowers were documented and compared across all genera of Betulaceae.

Development of Pistillate Cymes and Flowers

In all six genera of Betulaceae, the pistillate inflorescences consist of many cymes. The cymes are initiated acropetally and spirally on the inflorescence axis. Every cyme primordium differentiates a primary bract subtending a group of meristem cells (Fig. 1a1, b1, c1, d1, e1, f1), which generate two laminar in Alims (Fig. 1a5) and Betula (Fig. 1b5) or leaf-like secondary bracts in the other four genera (e.g., Fig. 1d5, e5), and two (Fig. 1a3-4, c3-4, d4, e4, f4) or three (Fig. 1b3-4; Betula) pistillate flowers. In Aims the cyme also includes two tertiary bracts (Fig. 1a3 -4).

The tepals of the pistillate flower are initiated from a common circular primordium at the base of the pistil in Cotylus, Ostryopsis, Carpinus, and Ostiya (Fig. 1c5, d5, e5, f5). These tepals with irregular shape and unstable number of lobes are adnate to the pistil up to the top to form an inferior ovary (Chen et al" 2001: Zhu et al., 2014a, b, c). There are no tepals in Alrtus (Fig. 1a4-5), and in Betula two tepals are initiated but stop developing early on, resulting in the lack of tepals in mature flowers of Betula (Fig. 1b5). In most genera there are two carpels in each pistillate flower (e.g., 1c5, 1f5), but in Betula, Carpinus monbeigiana, and Ostrya japonica three carpels are observed occasionally. Each bicarpellary pistil possesses two columnar stigmas. The two bicarpellate pistils change orientations from parallel to each other to oblique gradually during their development; this change is evident in Corylus (Fig. 1c3- 5) and Ostryopsis (Fig. 1d3-5), but less clear in Ostrya (Fig. 10-5). There are three flowers in a cyme of Betula, two of which are close to the primary bract while the third one is located between the first two (Fig. 1b3--5). The pollen tube enters the stigma either through papillae produced from the epidermal cells in Alnus (Fig. 4a), Corylus (Fig. 4c), Ostryopsis (Fig. 4f), Carpinus (Fig. 4g), and Ostrya (Fig. 4h), or through non-papillate epidermal cells and intercellular spaces in Alnus (Fig. 4b), Betula (Fig. 4e), and Corylus (Fig. 4d). The pollen tube of Corylus mandshurica is bead-like at the top (Fig. 4d), however that of Betula ermanii twines around the papillae, and then enters the stigma from the lateral side of the papillae (Fig. 4e).

Development of Staminate Cymes and Flowers

The staminate inflorescences consist of cymes that are spirally arranged. Every cyme primordium differentiates to form one primary bract and a group of meristem cells (Fig. 2al, bl, cl, dl, el, fl). There are two secondary bracts and two tertiary bracts in each cyme in Alnus (Fig. 2a2-3), while only two secondary bracts develop in Betula (Fig. 2b2-4) and Corylus (Fig. 2c2-4). However, in Ostryopsis, Carpinus, and Ostrya both secondary and tertiary bracts are absent (Fig. 2d2-4, e2-4, f2-4). Each cyme bears three staminate flowers with clear boundary between them in Alnus (Fig. 2a3-4) and Betula (Fig. 2b3-4). Each flower primordium produces four tepals and four globular stamens (Fig. 2a3-4) in Alnus, but four tepals and two stamens in Betula (Fig. 2a3-4). In the other four genera of Betulaceae, the flowers have no tepals. In Ostryopsis, Carpinus, and Ostrya, there are three flower areas recognizable at primordium stage but the boundary between the flowers is blurry as they mature (Fig. 2d2-3, e2-3, f2- 3). In Ostryopsis (Fig. 2d4) and Carpinus (Fig. 2e4), there are one to three stamens initiated in each of the lateral flower areas and four to six in the middle flower area. In Ostrya, there are three to four stamens initiated in each of the lateral flower areas and six in the middle flower area (Fig. 2f3-4). In Corylus (Fig. 2c2-4), each cyme has four stamens, but the flower areas cannot be recognized. The filaments of Corylus bifurcate completely from top to bottom (Fig. 2c5), whereas those of the other five genera divide from the top at different depths (Fig. 2a5, b5, d5, e5, f5).

Morphological Data Matrix and Phylogenetic Analysis

A morphological data matrix was constmcted including 11 characters from pistillate (Table 2) and staminate (Table 3) flowers and inflorescences based on the observations in this and previous studies (Chen et al., 2001; Zhu & Lu, 2008; Lin et al., 2010; Zhu et al" 2014a, b, c), and 14 characters in general morphology and anatomy from the literature (Hall, 1952; Hjelmqvist, 1957, 1960; Hardin & Bell, 1986; Crane, 1989; Chen, 1991; Chen & Zhang, 1991; Chen et al., 1999) (Appendix 1). Nothofagus (Nothofagaceae) was designated as the outgroup based on previous molecular systematic studies of Fagales (Manos & Steele, 1997; Qiu et al, 1998; Li et al., 2004; APGIV, 2016). In the morphological matrix (Appendix 2), most characters were coded as binary, while one character (leaf teeth) as multistate. Maximum Parsimony analysis was conducted using PAUP version 4.0b 10 (Swofford, 2002) with multistate characters treated as unordered, heuristic tree search with 100 replications of random addition, TBR branch swapping, and MULPARS in effect. Bootstrap analysis with 1000 replicates was conducted to assess relative support for clades with similar options as in the MP analysis. To evaluate the evolution of morphological traits, character states were mapped on the most parsimonious phylogenetic tree.

Maximum parsimony analyses generated one most parsimonious tree with 37 steps, CI = 0.730, and RI = 0.872 (Fig. 3). There are two major clades in the tree corresponding to Betuloideae (Alims and Betula', bootstrap support (BS) = 60%) and Coryloideae (Ostryopsis, Corylus, Carpinus, and Os try a: BS = 94%). The Betuloideae share features such as laminar secondary bracts and one to three flowers in the pistillate cyme, tepals in the staminate flower, tracheids in wood, pollen with arci and nutlets with wings. The Coryloideae clade is supported by seven synapomorphies including leaf-shaped secondary bracts and fewer than three flowers in the pistillate cyme, no tepals in the staminate flower, spiral thickenings in vessels, pollen without arci and nuts without wings. Within the Coryloideae clade, Corylus is sister to the clade containing the remaining genera with moderate support (BS = 81%). The Ostryopsis-Carpinus-Ostrya clade is supported by five nonhomoplasious characters, i.e., unisexual inflorescence, pollen tube entering the style only by papillae that originate from stylar epidermal cells (Fig. 4), no secondary bracts in the staminate cyme, pollen with operculum and endexine without thickening. Within the Ostryopsis-Carpinus-Ostrya clade, the Carpinus-Ostrya clade is strongly supported (88% BS) with three characters, i.e., vessels with simple perforations, wood with tyloses, and dispersal units of fruits with adnate persistent involucre (Hall, 1952; Kikuzawa, 1982).


Comparisons with Abbe's Observations

Abbe (1935) studied the flower and inflorescence structures of Betulaceae based on a comparative observation of the vascular systems of reproductive organs in 64 species, varieties, and hybrids of six genera. Extensive floral variations in each genus were carefully observed and discussed (Abbe, 1938). In our study, 28 species were used to compare the variations of inflorescence and floral organogenetic characteristics in all genera and sections of Betulaceae. Our observations on primordial occurrences of reproductive organs provide evidence for the number, arrangement, and homology of each part of flower, cyme, and inflorescence. We confirm Abbe's (1935, 1938) observations that there are one primary bract and one to three flowers in each staminate or pistillate cyme in all genera and sections of the family. However, our results differ from Abbe's observations in secondary and tertiary bracts (Table 2). For example, based on the number and branching pattern of vascular bundles, Abbe (1935) reported that each pistillate cyme had two secondary bracts and two or four tertiary bracts in all six genera except for Corylus, which lacked secondary bracts. We did not see the primordia of tertiary bracts in any of the genera except Aims, which has two laminar tertiary bracts in parallel with the primary bract on the abaxial side in both pistillate and staminate cymes. The secondary bracts in Aims and Betula are small, and scale-like in comparison with those in the other four genera. Abbe (1935) interpreted the involucre in Corylus as developed from two connate tertiary bracts, and that in Ostryopsis, Carpinus, and Ostiya as derived from the fusion of the secondary and tertiary bracts. In our observation, the involucre in Corylus, Ostryopsis, Carpinus, and Ostrya is initiated from a semicircular or circular primordium in one whorl and sometimes with several slight divisions (lobes) at the primordial stage that develops into different structures at maturation to enclose fruit, such as campanulate or tubular in Corylus and Ostryopsis, leaf- shaped in Carpinus, and saccate in Ostrya (Li & Skvortsov, 1999). We defined this structure as secondary bract here although fusion of secondary and tertiary bracts might have occurred during development due to the shorter tertiary axes noted by Abbe (1935). From the position and time of primordial formation, the secondary bract is comparable in the above four genera. In Ostryopsis and Carpinus, the margin of young bracts from a semi-circular primordium is entire, but becomes lobed usually with the abaxial side larger than the adaxial. The primordia of the secondary bracts in Corylus and Ostrya are almost circular surrounding the base of each flower, and finally developing into an involucre to partially (Corylus) or completely (Ostrya) enclose the fruit. Therefore the structure developed from the almost circular primordium in Corylus should be secondary bracts, rather than the first layered tepal as treated by Zhu et al. (2014a, b, c).

There are some differences between ours and Abbe's observations in the number of secondary or tertiary bracts in staminate cyme. Abbe (1935, 1938) reported two secondary bracts in Carpinus and Ostrya, but our study did not find any primordia of bracts except for a primary bract in their staminate cymes (Table 3). Abbe (1935, 1938) assumed that there were three flowers in each cyme in six genera of Betulaceae. In our study, we observed three-flower cymes in Alnus and Betula because each flower of the two genera is delimited by two to four tepals (Zhu & Lu, 2008; Lin et al, 2010). In the other genera, however, the flowers do not have tepals and the number of stamens varies, which results in the difficulty to delimit flowers. Nevertheless, at the primordial stage three flower areas can be seen in the cyme of Ostryopsis, Carpinus, and Ostrya, although the boundary between flowers is unclear at maturation. Albeit difficult to recognize flower areas in Corylus, the cyme seems to have one flower.

According to Abbe (1935, 1938), Alnus and Betula have 1-4 (6) tepals in staminate flowers and no tepals in pistillate flowers; while the other four genera have no tepals in staminate flowers and four tepals in pistillate flowers. Our observations show that in pistillate flowers, tepals are adnate to the top of the ovary in Corylus, Ostryopsis, Carpinus, and Ostrya, and develop from one circular primordium, making it difficult to determine the number of tepals, albeit with lobes at maturation (Table 3). In contrast, Abbe (1935, 1938) reported four tepals in Carpinus, Corylus, Ostryopsis, and Ostrya. In Betula, two primordia of tepals can be recognized in each flower, but do not further develop. In Alnus, we did not observe primordia of the tepals. For staminate flowers, our study is consistent with Abbe's observations that there are three flowers in one cyme, four tepals per flower with each tepal opposite one stamen in Alnus (Zhu & Lu, 2008), and two to four tepals per flower with two stamens in Betula (Lin et al, 2010).

The orientation of the two bicarpellate gynoecia in one cyme is diagonal in all genera at maturation (Table 2), which disagrees with Abbe (1935, 1938) who considered the gynoecia as transverse in Carpinus and Ostrya. Nevertheless, Chen et al. (2001) found that the two primordia of the bicarpellate gynoecia in a cyme are parallel to each other during early developmental stages in Carpinus and Ostryopsis. In this study, we also observe a parallel arrangement of young carpels in Alnus and Corylus (Figs. 3,13). Therefore, as suggested by Hjelmqvist (1948), the orientation of the gynoecia changes from parallel to diagonal during development.

Our observations of the floral development of betulaceous genera largely agree with Abbe (1935, 1938) with some exceptions in the absence of secondary and tertiary bracts. This may be due to the nature of the methods of investigations. Abbe (1935, 1938) used the data from the presence and absence of vasculature, while our study employed SEM to observe the developmental stages of floral organs. The vasculature at the potential site of a floral organ such as a secondary bract may be indicative of the early presence of the structure (Abbe, 1935, 1938). However, the floral organ may not develop into a visible primordium. Therefore, our study verifies and complements the results by Abbe (1935, 1938). The accurate description of the morphological traits lays the foundation for examining their phylogenetic implications.

Phylogenetic Implications of Morphological Characters

The phylogenetic relationships of six genera in Betulaceae have been extensively studied using morphological and molecular evidence (Behnke, 1973; Brunner & Fairbrothers, 1979; Kuprianova, 1983; Li, 2008). There are two main clades in the family: Betuloideae (Aims and Betula) and Coryloideae (Corylus, Ostryopsis, Carpinus, and Ostrya) or Betuleae and Coryleae (Crane, 1989; Bousquet et al., 1992; Savard et ah, 1993; Chen, 1994a, b; Chen et ah, 1999; Laroche & Bousquet, 1999; Forest et ah, 2005; Grimm & Renner, 2013). The major dispute in the systematics of Betulaceae lies in the systematic position of Ostryopsis. Several early studies considered it as sister to Corylus (Abbe, 1938; Kato et ah, 1999). A phylogenetic study based on nuclear ribosomal DNA ITS sequences showed that Ostryopsis was sister to Corylus-Carpinus-Ostrya clade (Yoo & Wen, 2007), whereas other molecular phylogenetic studies have well resolved this genus as sister to Carpinus and Ostrya (e.g., Chen et ah, 1999; Ma et ah, 2015). Our phylogenetic analysis based on morphological traits (mostly from floral organogenesis) also supports the sister relationship of Ostryopsis with Carpinus-Ostrya.

Abbe (1935, 1938, 1974) discussed an old hypothesis of cyme to explain the structure and variations of flowers and inflorescences and to understand the morphological evolution and phytogeny in Betulaceae. He considered the cyme of Betulaceae as a branching system (see also Endress, 2010). In the axil of the primary bract develops the secondary axis, bearing two sets of secondary bracts and terminating in a secondary flower. In the axils of each of the secondary bracts are the tertiary axes, each bearing a single set of tertiaiy bracts and terminating in tertiary flowers (Figs. 1 and 2 in Abbe, 1938). Each cyme in ancestral state should consist of up to eight flowers and three secondary bracts, six tertiary bracts, and eight quaternary bracts. He also hypothesized that ancestral flowers were bisexual and trimerous, each with six tepals and six stamens in two whorls, and a tricarpellate gynoecium. Our mapping well documented organogenetic characters on the phytogeny with Nothofagus as the outgroup (Fig. 3) shows that in both pistillate and staminate cymes and flowers Betulaceae have evolved in the direction of reduction in the number of flowers and bracts, although the staminate flowers show an increase in the number of stamens per flower. The ancestral character states include more flowers and bracts in each cyme, and flowers with tepals and tricarpellate gynoecia (Fig. 3).

Alnus and Betula show more plesiomorphic characters, such as more bracts and flowers in female cyme, tepals in male flower, and tracheids in wood (Hall, 1952; Zhu & Lu, 2008; Lin et al., 2010). In contrast, the four genera of Coryloideae clade with 94% BS are supported by synapomorphies such as female cyme with fewer bracts and flowers, staminate flowers much reduced to have no tepals, and spiral thickenings in vessels (Hall, 1952; Zhu et al., 2014a, c). The evolutionary patterns of bracts, flowers, and floral parts in Betulaceae reflect the biological principle that structures determine functions. For example, the delayed development of tepals in Coryloideae is associated with the loss of their mechanical protective function, the replacement of the protective function by the subtending primary bracts, the irregular shape and unstable number, and their gaining of secretory protective function (Endress, 2008). Morphological analysis also indicates the intermediate position of Corylus between Betuloideae and Coryloideae by having some characters in this genus, such as male cyme with secondary bracts, pollen tube entering the style by stigmatic epidermal cells or intercellular spaces (Fig. 4), pollen endexine thickening, and operculum of pollen absent (Chen & Zhang, 1991; Zhu et al., 2014a, b). In Coryloideae, Ostryopsis, Carpinus, and Ostiya are crown lineages in Betulaceae with many derived characters, such as more winter scales of bud for cold habitat, pollen tube entering the stigma only via papillae (Fig. 4), and dispersal units of fruits with adnate persistent involucre that are highly adaptive for wind pollination (Kikuzawa, 1982; Hardin & Bell, 1986; Chen & Zhang, 1991; Zhu et al., 2014b). Carpinus and Ostiya have also evolved vessels with simple perforations and wood with tyloses (Hall, 1952).


Our ontogenetic study of inflorescence and flowers in Betulaceae supported most observations by Abbe (1935, 1938), and provided new structural characters for phylogenetic analysis of the family using non-molecular data. Our analyses produced a well- supported phylogeny of Betulaceae with similar intergeneric relationships as suggested by recent molecular phylogenetic studies. Betulaceae may have evolved in the direction of floral reduction and wind pollination with various entries of pollen tubes into the stigma.

Acknowledgments We thank Dr, Limin Lu for her morphological analyses and Professor Peter K. Endress for his critical review, in particular suggestions on the usage of flower and inflorescence terms that help improve the manuscript. This study was supported by the National Natural Science Foundation of China (NNSF 31170180 and NNSF 31270268), Field work was partially supported by CAS International Research & Education Development Program (Grant No. SAJC201315), and MOST Science and Technology Basic Work (2013FY112100).

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Appendix 1

Morphological characters including floral developmental data used in phylogenetic analysis (Hall, 1952; Hjelmqvist, 1957, 1960; Hardin & Bell, 1986; Crane, 1989; Chen et al., 1990; Chen, 1991; Chen & Zhang, 1991;Manos, 1997; Manos et al., 2001; Chen et al., 1999; Chen et al, 2001; Zhu & Lu, 2008; Lin et al" 2010; Zhu et al, 2014a, b, c; and this study)

1. Inflorescence--bisexual (0), unisexual (1)

2. Female cyme--secondary bract laminar (0), leaf-shaped (1)

3. Female cyme--tertiary bract present (0), absent (1)

4. Female cyme--1 to 3 flowers (0), 1 or 2 flowers (1)

5. Female flower--pollen tube entering the stigma by papillae, absent (0), present (1)

6. Female flower--pollen tube entering the stigma by stylar epidermal cells or intercellular spaces, present (0), absent (1)

7. Male cyme--secondary bracts 2 (0), none (1)

8. Male cyme--Tertiary bracts 2 (0), none (1)

9. Male flower--tepal present (0), absent (1)

10. Male flower--pistillode present (0), absent (1)

11. Male flower--thecae and filaments not or slightly divided longitudinally (0), thecae separated, filaments partly divided (1), thecae separated, filaments completely divided (2)

12. Wood--vessel perforations scalarifonn (0), simple (1)

13. Wood--vessel with spiral thickening absent (0), present (1)

14. Wood--tracheids present (0), absent (1)

15. Wood--rays heterocellular (0), homocellular (1)

16. Wood--tyloses absent (0), present (1)

17. Leaves--embedded glands present (0), absent (1)

18. Leaves--brachyparacytic stomatal apparatus absent (0), present (1)

19. Leaves--teeth absent (0), simple (1), compound (2)

20. Pollen--endexine thickening present (0), absent (1)

21. Pollen--operculum absent (0), present (1)

22. Pollen--arci present (0), absent (1)

23. Fruits--laterally winged nutlets present (0), absent (1)

24. Fruits--dispersal units with adnate persistent involucre absent (0), present (1)

25. Seeds--germination epigeal (0), hypogeal (1)
Appendix 2
Table 4 Data matrix for morphological characters used in this
analysis (including the outgroup Nothofagus-, "?" indicating
not applicable

Taxon \ character                 1   2   3   4   5   6   7   8

Nothofagus                        1   0   7   0   ?   7   ?   7
Alnus section Clcthropsis         0   0   0   0   i   0   0   0
Alnus section Cremastogync        0   0   0   0   i   0   0   0
Alnus section Alnobctula          0   0   0   0   i   0   0   0
Betula section Betulaster         0   0   1   0   0   0   0   1
Betula section Betula             0   0   1   0   0   0   0   1
Corylus                           0   I   1   1   1   0   0   1
Ostryopsis                        1   1   1   1   1   1   1   1
Carpinus section Carpinus         1   1   1   0   1   1   1   1
Carpinus section Distcgocarpus    1   1   1   0   1   1   1   1
Ostrya                            1   1   1   1   1   1   1   1

Taxon \ character                 9   10   11   12   13   14   15

Nothofagus                        0   1    0    1    7    0    0
Alnus section Clcthropsis         0   0    0    0    0    0    1
Alnus section Cremastogync        0   0    0    0    0    0    1
Alnus section Alnobctula          0   0    0    0    0    1    0
Betula section Betulaster         0   1    1    0    0    0    1
Betula section Betula             0   1    1    0    0    0    1
Corylus                           1   0    2    0    1    1    0
Ostryopsis                        1   0    1    1    1    1    0
Carpinus section Carpinus         1   1    1    1    1    1    1
Carpinus section Distcgocarpus    1   1    1    1    1    1    1
Ostrya                            1   0    1    1    1    1    1

Taxon \ character                 16   17   18  19  20  21  22

Nothofagus                        1    0    0   0   0   0   1
Alnus section Clcthropsis         0    0    0   0   0   0   0
Alnus section Cremastogync        0    0    0   0   0   0   0
Alnus section Alnobctula          0    0    0   0   0   0   0
Betula section Betulaster         0    0    0   1   0   0   0
Betula section Betula             0    0    0   1   0   0   0
Corylus                           0    1    0   2   0   0   1
Ostryopsis                        0    0    0   2   1   1   1
Carpinus section Carpinus         1    1    1   1   1   1   1
Carpinus section Distcgocarpus    1    1    1   1   1   1   1
Ostrya                            1    1    0   2   1   1   1

Taxon \ character                 23    24    25

Nothofagus                         0     0    0
Alnus section Clcthropsis          0     0    0
Alnus section Cremastogync         0     0    0
Alnus section Alnobctula           0     0    0
Betula section Betulaster          0     0    0
Betula section Betula              0     0    0
Corylus                            1     0    1
Ostryopsis                         1     0    0
Carpinus section Carpinus          1     1    0
Carpinus section Distcgocarpus     1     1    0
Ostrya                             1     1    0

Junyi Zhu (1,5) * Lifan Zhang (1) * Baoqing Ren (2) * Min Chen (3) * Ruiqi Li (3) * You Zhou (1) * Yu Liang (1) * Jianhua Li (1,4,5) * Zhiduan Chen (3,5)

(1) College of Life Science, Tonghua Normal University, Tonghua 134002, China

(2) Constructional and Preparation Office of Taiyuan Taishan Botanical Garden, Taiyuan 030025, China

(3) State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing 10093, China

(4) Biology Department, Hope College, Holland, Ml 49423, USA

(5) Author for Correspondence; e-mail:;; Published online: 26 October 2017

Caption: Fig. 1 Development of pistillate inflorescences, cymes, and flowers in six genera of Betulaccae. al,bl". .fl Early stage showing primary bract (Bl) of cyme; a2,b2, ... f2 Primordia of secondary bracts and flowers; a3,b3, ... f3 Secondary bracts, tertiary bracts (only in A/nus, a3), and young flowers; a4,b4, ... f4 Developed secondary bracts, tertiary bracts (only in Alnus, a4), and flowers; a5,b5, ... f5 Initiation of carpels and tepals (except for Alnus, a5 no tepals); a6,b6, ... f6 Diagrams of pistillate cymes and flowers in six genera of Bctulaceae. B1, primary bract; B2, secondary bract; B3, tertiary bract; t, tepal. a 1-5 Alnus (a 1-4 A. mandshurica, a5 A. sieboldiana); bl--5 Betula (bl B. ermanii, b2-5 B. luminifera); c1-5 Corylus (c1,c3 C. yunnanensis, c2,c5 C. heterophylla, c4 C. mandshurica); dI--5 Ostryopsis (O. davidiana); el-5 Carpinus (e1-4 C. cordata, e5 C. turezaninowii); f1-5 Ostrya (O. japonica)

Caption: Fig. 2 Development of staminate inflorescences, cymes, and flowers in six genera of Betulaceae. al,bl, ... fl Early stage showing primary bract (Bl) of cyme; a2,b2, ... f2 Initiation of cyme showing secondary in Alnus (a2). Betula (b2), and Corylus (c2), as well as three flowers (a2,b2) and three flower tissue zones (d2,e2,f2); a3,b3, ... f3 Initiation of tepals in Alnus (a3) and Betula (b3), and stamens in the other four genera; a4,b4, ... f4 Developed tepals and stamens; a5,b5, ... f5 Mature stamens with filaments completely divided in Corylus (c5), and only partly divided in the other five genera; 31-36 Diagrams of staminate cymes and flowers in six genera of Betulaceae, Bl, primaiy bract; B2, secondary bract; B3, tertiary bract; t, tepal. A1-5 Altius (a1-3,a5 A. sieboldiana, a4 A. sibirica); b1-5 Betula (b1, b4 B. luminifera,b2, b3,b5 B. platyphylla)', c1--5 Corylus (c1-2,c5 C. yunnanensis, c3 C. heterophylla, c4 C. mandshurica); dl 5 Ostryopsis (O. davidiana); el-5 Carpinus (C. cordata);f1--5 Ostrya (O. japonica)

Caption: Fig. 3 Most parsimonious tree of Betulaceae based on morphological characters (see Appendix 2) with Nothofagus as outgroup. Multistate characters are treated as unordered. Length = 37 steps, CI = 0.730, RI = 0.872. Bootstrap values are shown above branches. Changes of morphological character states within Betulaceae are indicated with boxes on branches. Filled boxes represent non- homoplastic characters, whereas empty boxes indicate homoplastic changes. Numbers above and below boxes indicate character number and state respectively

Caption: Fig. 4 Stigma types and pollen tube growth process in Betulaceae. One type of stigma has epidermal papillae through which the pollen tube enters the stigma (a, c, f, g, h), and another type has no papillae so that pollen tube enters the stigma via non-papillate epidermal cells or intercellular spaces (b, d, e). The pollen tube of Corylus mandshurica is bead-like at the top (d), and that of Betula ennanii twines around the papillae, and then enters the stigma from the lateral sides of the papillae (e). a-b Alnus (a A. mandshurica, b A. sibirica); c-d Corylus (c C. heterophylla x avellana, d C. mandshurica): e Betula (B. ermanii); f Ostryopsis (O. davidiana); g Carpinus (C. cordata); h Ostrya (O. japonica)
Table 1 Number of species and geographical distribution of each
section among six genera of Bctulaceae, and species sampled for SEM
observation in our previous and this studies. Section division and
distribution pattern based on the following references: (Winkler,
1904; Li, 1976; Li and Cheng, 1979; Furlow, 1979, 1990; Chen,
1991, 1994a,b; Chen and Zhang, 1991; Chen et al., 1999; Li and
Skvortsov, 1999; Yoo and Wen, 2007. The species number based on
Chen, 1994a, b)

Genus        Section         No. of    Species

Alnus        Clethropsis     3
                                       Alnus nepalensis

             Alnus           17
                                       A. sibirica
                                       A. japonica

                                       A. sibirica var.
             Crcmastogyne    3
                                       A. cremastogyne
             Alnobctula      3

                                       A. sieboldiana
                                       A. mandshurica

Betula       Bctulastcr      7
                                       Betula luminifera

             Betula          44
                                       B. alnoides
                                       B. platyphylla
                                       B. dahurica

                                       B. schmidtii
                                       B. fruticosa

                                       B. ermanii

Corylus                      14

                                       Corylus heterophylla

                                       C. mandshurica

                                       C. yunnanensis

                                       C. maxima

                                       C. avellana

                                       C. chinensis

                                       C. heterophylla
                                       x avellana
Ostryopsis                   3
                                       O. davidiana

                                       O. nobilis

Carpinus     Distcgocarpus   4
                                       C. cordata

             Carpinus        27

                                       Europe, Asia,
                                       North America
                                       C. turczaninowii

                                       C. londoniana

                                       C. monbeigiana

Osttya       10                        Europe, Asia,
                                       North America
                                       O. japonica

Genus        Geographic              Collection locality

Alnus        Asia, North America
                                     Kunming Institute of Botany,
                                     the Chinese
                                     Academy of Sciences
             North Temperate
                                     Tonghua city, Jilin Province
                                     Huanrcn County, Liaoning
                                     Campus, Changchun University

                                     Ya'an City, Sichuan Province
             Eurasia to North
                                     Ya'an City, Sichuan Province
                                     The north slope of Changbai
Betula       Asia
                                     Kunming Institute of Botany,
                                     the Chinese Academy of
             North Temperate
                                     Jinxi County, Guangxi Province
                                     Tonghua, Jilin Province
                                     Tonghua County. Jilin Province

                                     Ji'an City, Jilin Province
                                     Hani River Wetland, Jilin
                                     The north slope of Changbai
Corylus      Europe, Asia,
             North America
                                     Tonghua City, Jinlin Province

                                     Tonghua City, Jinlin Province

                                     Kunming Institute of Botany,
                                     the Chinese Academy of Sciences
                                     Institute of Botany, the
                                     Chinese Academy of Sciences
                                     Institute of Botany, the
                                     Chinese Academy of Sciences
                                     Institute of Botany, the
                                     Chinese Academy of Sciences
                                     Thonghua City, Jilin Province

Ostryopsis   China
                                     Taiyuan City, Shanxi Province

                                     Kunming Institute of Botany,
                                     the Chinese Academy of Sciences
Carpinus     Asia
                                     Campus, Tonghua Normal

             Institute of Botany,    70
             the Chinese Academy
             of Sciences
             Kunming Institute of    2000
             Botany, the Chinese
             Academy of Sciences
             Kunming Institute of    2000
             Botany, the Chinese
             Academy of Sciences

             Kunming Institute of    2000
             Botany, the Chinese
             Academy of Sciences

Genus        Altitude   Collection    Reference

             2000       2012.3        This study

             560        2005.3        Zhu and Lu (2008)
             620        2012.3        This study

             260        2013.3        This study

                        2012.3        This study

                        2012.3        This study
             1820       2005.3        Zhu and Lu (2008)

             2000       2013.2        This study

             470        2008.3        Lin ct al. (2010)
             420        2013.3        This study
             460        2013.3        This study

             207        2013.3        This study
             556        2012.3        This study

             1810       2013.3        This study


             660        2013.3        Zhu et al.
                                      (2014a, b, c)
             660        2013.3        Zhu ct al.
                                      (2014a, b. c)
             2000       2012.2        Zhu ct al.
                                      (2014a, b. c)
             70         2013.3        This study

             70         2013.3        This study

             70         2013.3        This study

             620        2013.3        Zhang et al.
             810        2013.2        Chen ct al.
                                      (2001); this
             2000       2013.2        This study

             420        2012.3        Zhu et al.
                                      (2014a, b, c)

             2013.2     Chen et
                        al. (2001);
                        this study
             2013.2     This study

             2013.2     This study


             2013.2     This study

Table 2 A comparison of female flower and cyme among genera
in Betulaceae

         Alnus         Betula            Corylus

No. PB   1             i                 1
No. SB   2 (laminar)   2 (laminar)       2 (leaf-shaped)
No. TB   2             0                 0
No. FP   2(3)          3                 2
No. C    2 or 3        2 or 3            2
No. TF   0             2 (undeveloped)   1 (circular)
SP       1             0                 1
SE       0             0                 0

         Ostryopsis         Carpinus           Ostrya

No. PB   1                  1                  1
No. SB   2 (leaf-shaped)    2 (leaf-shaped)    2 (leaf-shaped)
No. TB   0                  0                  0
No. FP   2                  2(3)               2
No. C    2 or 3             2                  2
No. TF   1 (circular)       1 (circular)       1 (circular)
SP       1                  1                  1
SE       1                  1                  1

PB = primary bract; SB = secondary bract; TB = tcrtiaiy bract; FP =
female flower primordium; C = carpel; TF = tepal of female flower;
SP = pollen tube entering the stigma via papillate stigmatic cells,
absent (0), present (1); SE = pollen tube entering the stigma via
non-papillate stigmatic cells or intercellular spaces, present (0),
absent (1)

Table 3 A comparison of male flower and cyme among
genera in Betulaceae

              Alnus             Betula          Corylus

No. PB        1                 1               1
No. SB        2                 2               2
No. TB        2                 0               0
No. MP        3                 3               3 MA
No. SP        4 (each flower)   2 (each         2 on lateral sides, 2
                                flower)         in the center)
No. SP        12                6               4
(each cyme)
No. TM        4                 2-4             0
Filament      shallow split     shallow split   split to the base and

              Ostryopsis                Carpinus

No. PB        1                         1
No. SB        0                         0
No. TB        0                         0
No. MP        3 MA                      3 MA
No. SP        2-4 on lateral sides, 2   4-6 on lateral sides, 4-6
              in the center)            in the center)
No. SP        4-6                       8-12
(each cyme)
No. TM        0                         0
Filament      shallow split             shallow split


No. PB        1
No. SB        0
No. TB        0
No. MP        3 MA
No. SP        6-8 on lateral sides, 5-6
              in the center)
No. SP        11-14
(each cyme)
No. TM        0
Filament      shallow split

PB = primary bract; SB = secondary bract; TB = tertiary bract;
MA = male flower areas; SP = stamen primordium; TM = tepal of
male flower
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Author:Zhu, Junyi; Zhang, Lifan; Ren, Baoqing; Chen, Min; Li, Ruiqi; Zhou, You; Liang, Yu; Li, Jianhua; Che
Publication:The Botanical Review
Article Type:Report
Date:Mar 1, 2018
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