The Phylogenetic Significance of Fruit Structures in the Family Cornaceae of China and Related Taxa

The fruit morphological structures of the Cornaceae of China and related taxa were studied using the wax GMA semi-thin section method and other methods to identify characters useful in delimiting clades circumscribed in previous molecular phylogenetic studies. Maximum parsimony analyses of 27 fruit structural characters resulted in a generally poorly resolved strict consensus tree, yet one whose major clades matched those revealed previously. Cornaceae of China and related taxa are recognized in four significant clades with the following fruit structural features: (1) Helwingia, fruits lack trichome, the abdominal vascular bundles are close to the endocarp, and the endocarp sclereid is elongated; (2) Aucuba, single-cell lanceolate trichomes, pericarp without secretory structure; (3) Torricellia, polygon and elongated sclereids in the endocarp, pericarp without crystal and tannin; and (4) Cornus sensu lato, the trichome is T-shaped, the abdominal ventral bundle is absent, and the endocarp sclereid is nearly round. In Cornus sensu lato, this document supported that the cornelian cherries (CC, subg. Cornus) and the big-bracted dogwoods (BB, subg. Syncarpea) are sister groups. The dwarf dogwoods (DW, subg. Arctocrania) are sister to them, and the blue- or white-fruited dogwoods (BW, subg. Kraniopsis, subg. Yinquania, and subg. Mesomora) are the base of the Cornus sensu lato clade. The number of cell layers of endocarps and the types of crystals afford sound evidence for identifying their relationship. This study indicated that the fruit structures of Cornaceae might provide morphological and anatomical evidence for molecular phylogeny.


Introduction
The Cornaceae and related taxa are ecologically and horticulturally significant families, primarily trees or shrubs, but rarely herbs, which are found in the northern hemisphere's tropical, temperate, and circumpolar regions [1]. Harms (1898) and Hutchinson (1967) positioned Cornaceae in the order Apiales based on its umbel or compound umbel, 2-5 carpels, inferior ovary, and one anatropous ovule per locule [2,3]. However, some scholars shifted their classification from Apiales to Cornales based on the majority of ligneous plants, simple leaves opposite (rarely alternate), drupes (rarely berry), and cyclic enol ether terpene compounds [4,5]. Cornaceae is one of the most complex families of flowering plants due to the highly divergent and plastic characteristics of its members. Harms (1898) classified 15 genera (Alangium, Aucuba, Cornus, Helwingia, and Torricellia et al.) predicated on their wood, bract, involucre, and fruit sclereid characteristics [2]. Eyde (1988) moved Aucuba, Helwingia, and Torricellia from Cornaceae and retained Cornus, Camptotheca, Diplopanax, Davidia, Mastixia, and Nyssa based on the characteristics of their germination valves, the chromosome number of x = 11, and iridoid compounds [6]. Fang and Hu (1990) indicated that Cornaceae contains Cornus sensu lato (Bothrocaryum, Chamaepericlymenum, Cornus, Dendrobenthamia, Swida), Mastixia, Aucuba, Helwingia, and Torricellia, according to leaves vascular bundles on the endocarp surface are taxonomically significant characteristics [71]. Morozowska (2021) mentioned that the endocarp length and thickness, the number of vascular bundles, sclereid shape, and other characteristics were taxonomically essential to Cornus sensu lato [72]. Some researchers studied fossils of Cornaceae, but only in a few species [73][74][75][76][77]. However, the research on the fruit of Chinese Cornaceae is limited to just a few subgenera and especially lacks the pericarp structures' anatomical detail. This study aims to (1) describe more fully the fruit microstructure of Chinese Cornaceae and related taxa; (2) compare the differences in fruit morphology between the genus of Chinese Cornaceae and related taxa and evaluate the relationships among the inner groups of Cornus sensu lato; (3) to explore the taxonomical value and importance of fruit features; and (4) provide the fruit morphological evidence for Chinese Cornaceae and related taxa in molecular phylogenetic studies.

Materials and Methods
The external and internal structures of fruits were recorded from Chinese Cornaceae, representing 30 species and 4 genera following the Folar of China [7]. The two species of Araliaceae were also examined [78]. Sample names and voucher information are provided in Table 1. Morphological studies: For each sample, the shape of fruits was first investigated and photographed using an Olympus SZX7 stereomicroscope and an Olympus DP70 digital camera. The fruits were rehydrated, and the epidermis with trichomes was peeled and placed on a glass slide. A drop of 50% glycerol solution was added before mounting the sample with a cover slip. Then, the shape of sclereids and seeds was also observed. At least three mature fruits of each taxon were examined. Photographs were taken using an Olympus BX51 microscope and an Olympus DP70 digital camera.
Anatomical studies: Additional rehydrated fruits were placed in FAA (37% formalinglacial acetic acid and 70% alcohol = 5 mL:5 mL:90 mL) for a minimum of 24 h and then treated following the method of Feder and O'Brien (1968) for embedding in glycol methacrylate (GMA) [79]. A Leica Ultracut R microtome was used to prepare transverse sections about 3-4 µm in thickness. These sections were then stained using the periodic acid Schiff/toluidine blue method. At least three mature fruits of each taxon were examined. In the sections, fruit epidermal cells, mesocarp, endocarp, vascular bundles, secretory structures, and type of crystals were photographed by an Olympus BX51 microscope and an Olympus DP70 digital camera.
The pericarp of fruits was rehydrated and decoloured by sodium hypochlorite and then placed on a glass slide. One or two drops of 50% glycerol solution were added before mounting the sample with a cover slip. The pericarp cells separated from each other by beating the cover slip. These features were observed and photographed using an Olympus BX51 microscope and an Olympus DP70 digital camera and identified similar tannins in cells by ferrous salt [80]. Once more, at least three mature fruits of each taxon were examined.
Phylogenetic analysis: Coupled with our original observations of mature fruits and fruit sections, all-important micromorphological and anatomical characters used previously in systematic studies of Chinese Cornaceae and Araliaceae species were examined. Of these, 27 characters were potentially parsimony informative and included in the phylogenetic analysis ( Table 2). The data matrix of these characters for the same 32 taxa as examined above for fruit anatomy and micromorphology is presented in Table 3. Araliaceae was used to root the trees. Maximum parsimony (MP) analysis was carried out using PAUP* version 4.0a151 using 1000 random stepwise addition replicate searches and tree bisection and reconnection (TBR) branch swapping [81]. All character states were assumed unordered, and the options multrees, collapse branches, and accurate optimization were selected. Regardless of the number of states, scaling for equal character weighting did not affect the final tree topology. Bootstrap (BS) values were calculated from 1000 to replicate analyses, simple addition sequence of taxa, and TBR branch swapping. All character state changes inferred were mapped onto a single MP tree. Fruit length (mm) 0 = 2-3; 1 = 4-6; 2 = 7-9; 3 = 10-12; 4 = 13-25 7 Fruit type 0 = Berry drupe; 1 = Drupe; 2 = Aggregate drupe 8 Carpel number 0 = 2; 1 = 3; 2 = 4-6 9 Carpel shape 0 = Bilateral squashed; 1 = Dorsoventral squashed  Table 3. Data matrix of fruit characters used in the phylogenetic analysis of Chinese Cornaceae species and related taxa. Characters and states are described in Table 2. Characters 1-4 from Flora of China [7].

Results
The morphological and structural characteristics of the fruit were shown in Tables 2 and 3. The changes in the fruit structure were shown in Figures 1-4. The systematic relationships were shown in Figure 5.

Discussion
Our results indicate that most fruit features can be used as taxonomic evidence to distinguish the genera and subgenera. For example, the difference in the carpel, trichome, the number of cell layers of the mesocarp and endocarp, the ratio of mesocarp thickness to endocarp thickness, the number of vascular bundles, abdominal vascular bundle, secretory structure, sclereid of the mesocarp and endocarp, and cell inclusion. Structural details of these (and other non-fruit) characters and their phylogenetic significance are provided below with reference to the clades identified in the MP analysis ( Figure 5) and recent molecular systematic studies of Cornaceae of China and related taxa [35][36][37][38][39][40][41][42][43][44][45].
Helwingiaceae (removed Helwingia from the Cornaceae): Chao (1954) studied the wood anatomy and pollen morphology of nine genera of Cornaceae and concluded that Helwingia is located between Cornaceae and Araliaceae [82]. Smith (1975) suggested that Helwingia should be promoted to family status because of the distribution of iridoid glycosides, procyanidins, and other compounds [25]. Eyde (1988) recommended that Helwingia should be elevated to the family Helwingiaceae based on the flower characteristics (e.g., dioecious, each round of 3-5 tepals, male stamens alternate with tepals) [6]. Wang

Discussion
Our results indicate that most fruit features can be used as taxonomic evidence to distinguish the genera and subgenera. For example, the difference in the carpel, trichome, the number of cell layers of the mesocarp and endocarp, the ratio of mesocarp thickness to endocarp thickness, the number of vascular bundles, abdominal vascular bundle, secretory structure, sclereid of the mesocarp and endocarp, and cell inclusion. Structural details of these (and other non-fruit) characters and their phylogenetic significance are provided below with reference to the clades identified in the MP analysis ( Figure 5) and recent molecular systematic studies of Cornaceae of China and related taxa [35][36][37][38][39][40][41][42][43][44][45].
Helwingiaceae (removed Helwingia from the Cornaceae): Chao (1954) studied the wood anatomy and pollen morphology of nine genera of Cornaceae and concluded that Helwingia is located between Cornaceae and Araliaceae [82]. Smith (1975) suggested that Helwingia should be promoted to family status because of the distribution of iridoid glycosides, procyanidins, and other compounds [25]. Eyde (1988)

recommended that
Helwingia should be elevated to the family Helwingiaceae based on the flower characteristics (e.g., dioecious, each round of 3-5 tepals, male stamens alternate with tepals) [6]. Wang and Chen (1990) described that the pollen of Helwingia lacks a central cavity and a covering layer, compared to the pollen of Araliaceae studied by Shang and Callen (1988) [26,83]. They pointed out that the pollen morphology of Helwingia differs from that of Cornaceae and Araliaceae. Noshiro and Baas (1998) proposed that Helwingia should be excluded from Cornaceae based on the wood anatomy characters of Helwingia (e.g., apotracheal parenchyma absent or rare, fibres septate) [84]. Ao and Tobe (2015), based on the flower and embryology characters (loss of petals, poorly developed disc nectary, tenuinucellate ovules with a mature female gametophyte filled), suggested that Helwingia should be raised to family status [29]. Molecular phylogenetic studies supported this conclusion. Morgan and Soltis (1993) determined that Helwingia has a distant relationship with Cornaceae based on rbcL sequence analysis [85]. Xiang (1993), according to rbcL sequence data analysis, believed Helwingia does not belong to the Cornaceous clade [35]. Based on an analysis of the 18S rDNA sequence, Soltis and Soltis (1997) concluded that there is a distant relationship between Helwingia and other genera of Cornaceae [86]. Olmstead (2000) established a phylogenetic tree based on chloroplast ndhF gene sequence data analysis, which supported this conclusion [40]. Savolainen (2000) combined analysis of plastid atpB and rbcL gene sequences [39], and Li (2002) based on rbcL sequence analysis reached the same conclusion [37]. Our research supports previous molecular systematics studies. Helwingia has several morphological and anatomical characters of fruit that differentiate it from other species examined herein, including berry drupe (7-0), subglobose (5-4), 3-5 carpels (occasional six carpels) (8-2,3), absence of trichomes (10-0), abdominal vascular bundles close to the endocarp , an endocarp which is separate at the commissure of carpels (18-0), and an elongated sclereid . These distinguishing characteristics support that Helwingia was separated from other species of Cornaceae.
Aucubaceae (removed Aucuba from the Cornaceae): Wang and Chen (1990) described that Aucuba is not the same as other genera of Cornaceae based on dense drumstick pattern pollen [26]. Noshiro and Baas (1998) noted that the wood anatomy of Aucuba (rays up to and over six cells wide, typically in two sizes) differed from other genera of Cornaceae and concluded that Aucuba should be excluded from Cornaceae [84]. Molecular evidence confirmed that Aucuba has a distant relationship with Cornaceae. Analysis of rbcL sequence data by Xiang (1993) revealed that Aucuba is not closely related to the Cornaceae [35]. The rbcL sequences phylogenetic tree established by Morgan and Soltis (1993) proved that Aucuba is distantly related to Cornaceae [85]. Savolainen (2000) combined analysis of plastid atpB and rbcL gene sequences and considered that Aucuba and Garrya are sister branches [39]. Li (2002) determined that Aucuba has a distant relationship with Cornus sensu lato and Torricellia based on rbcL sequence analysis [37]. Ngondya (2013) analyzed cpDNA and ITS sequences and believed that the relationship between Aucuba and other Cornaceae genera is distant [38]. Chen (2021) proposed that Aucuba is a member of Garryaceae by analyzing plastome DNA sequence data [87]. Huang (2022) indicated that Aucuba is monophyletic, a sister branch of Garrya, and has a distant relationship with Cornaceae, according to the analysis of 68 plastid protein-coding gene data [88]. Our study supports previous studies and molecular systematics. Members of Aucuba have some morphological and anatomical features of fruit that differentiate them from other species studied herein, including berry drupe (7-0), oval (5-1), 10-12 mm (6-3), two carpels (8-0), single-cell lanceolate trichome (10-1), the ratio of mesocarp thickness to endocarp thickness is 4-7 (14-3), vascular bundles number per carpel is 6-9 (15-1), absence of sclereid in mesocarp (17-0), absence of secretory structure (24-0), 2-3 layers of cells in the endocarp , and nearly-round sclereid . These distinguishing features separate Aucuba from other species of Cornaceae. In determining Aucuba's relationship with Garryaceae, more research is required.
Torricelliaceae (removed Torricellia from the Cornaceae): Wang and Chen (1990) described that Torricellia has acrogenous racemose panicles, which distinguishes it from other Cornaceae groups. As a result, they substantiated its status as an independent family [26]. Noshiro and Baas (1998) pointed out that the wood of Torricellia has helical thickenings throughout the vessel elements, which is different from other genera of the Cornaceae, and believed that Torricellia should be excluded from this family [84]. Molecular phylogenetic studies provided proof for this conclusion. Savolainen (2000) indicated that Torricellia is not closely related to other genera of Cornaceae by using a combined analysis of plastid atpB and rbcL gene sequences [39]. Li (2002) analyzed the rbcL sequences and concluded that Torricellia and Cornaceae have a distant genetic relationship [37]. In this study, Torricellia displays some morphological and anatomical characteristics of the fruit that set it apart from other species, including drupe (7-1), oval (5-1), three carpels (8-1), single-cell lanceolate trichome (10-1), absence of sclereid in mesocarp (17-0), absence of secretory structure (24-0), sclereid polygon and elongated , and the absence of crystal and tannin (25-0, 26-0, 27-0). These distinguishing features separate Torricellia from other species of Cornaceae.

Conclusions
Previous molecular phylogeny studies identified four major branches of Chinese Cornaceae and related taxa studied herein, each of which is now supported by fruit anatomical and micromorphological features. These characteristics are valuable in providing readily observable features for diagnosing monophyletic groups. Characteristics of fruit structure can provide cogent morphological evidence to support the phylogenetic relationships inferred by molecular evidence. They also are useful for predicting the phylogenetic placement of species that have yet to be sampled in molecular studies. However, fruit characteristics on their own cannot wholly resolve species-level relationships, which require combination with additional micromorphological structures of flowers and trophic organs to improve the understanding of this complex group.