Karyomorphology and Chromosome Evolution in Nyssaceae (Cornales)

Abstract

Nyssaceae, consisting of 36 species in five genera (Camptotheca, Davidia, Diplopanax, Mastixia and Nyssa), are poorly known concerning their karyomorphology. Here we present karyomorphological features of seven species in all five genera and discuss chromosome evolution in the family. With a few dubious chromosome counts, information available shows that the chromosome number is consistent within a genus, i.e., 2n=42 in Campthotheca, Davidia and Diplopanax, 2n=44 in Nyssa, and 2n=26 in Mastixia. Their distribution on a phylogenetic tree suggests that 2n=42 is plesiomorphic with 2n=44 and 2n=26 apomorphic. Karyotype analyses show that 2n=42 of Camptotheca and Davidia comprise six sets of seven chromosomes, suggesting that Nyssaceae are of hexaploid origin with the basic number x=7.

Nyssaceae, one of seven families of the Cornales, is a small family of trees and shrubs, consisting of 36 species in five genera Camptotheca Decne. (two spp.), Davidia Baill. (one sp.), Diplopanax Hand.-Mazz. (two spp.), Mastixia Blume (20 spp.) and Nyssa L. (11 spp.) (Stevens 2001 onwards: http/www.mobot.org/MOBOT/research/APweb/, Mabberley 2017). While Nyssa is distributed disjunctively in the middle latitudes of China and the southern areas of North America, the four other genera are distributed in southern areas of Asia including subtropical China, Vietnam, and Indomalay (Wen and Stuessy 1993, Mabberley 2017). Until recently, the familial circumscription of Nyssaceae had been controversial. About 200 years ago Nyssaceae were regarded as consisting only of Nyssa (Dumortier 1829). Thereafter, Nyssa along with Camptotheca and Davidia had been placed in Cornaceae (Harms 1898). Handel-Mazzetti (1933) recognized a new genus Diplopanax in Araliaceae. Melchior (1964) placed Camptotheca and Nyssa in Nyssaceae, Davidia in Davidiaceae, Mastixia in Cornaceae, and Hutchinson (1967) and Cronquist (1981) recognized Nyssaceae as comprising Camptotheca, Davidia, and Nyssa, but placed Mastixia in Cornaceae. Takhtajan (1997, 2009) placed Camptotheca and Nyssa in Nyssaceae, Davidia in Davidiaceae, and Diplopanax and Mastixia in Mastixiaceae. Thorne (2001) and Thorne and Reveal (2007) assigned all the five genera to Nyssaceae. Molecular phylogenetic analyses have consistently suggested or supported close relationships among the five genera (Xiang et al. 2002, Fan and Xiang 2003, Xiang et al. 2011, Chen et al. 2016, Fu et al. 2017). Currently, APG IV (Angiosperm Phylogeny Group 2016) accepts Nyssaceae as comprising the five genera Camptotheca, Davidia, Diplopanax, Mastixaia, and Nyssa.

Thus, Nyssaceae now require a comparative study of morphological characters including karyomorphology throughout the family. As for chromosome numbers, Nyssa has n=22 from N. javanica (Blume) Wangerin (Mehra 1972, Mehra and Bawa 1969), and N. sylvatica Marshall (Goldblatt 1978) and 2n=44 from N. sinensis Oliv. (Huang et al. 1985, 1989) and N. sylvatica (Dermen 1932, Eyde 1966). In contrast, diverse chromosome numbers have been reported from Camptotheca, Davidia, and Mastixia. Camptotheca has 2n=42 (Raven 1975) and 2n=44 (Goldblatt 1978, Wu 1984, Li and Hsu 1986, He et al. 2004); Davidia has n=21 (Goldblatt 1978), 2n=40 (Yang and Zhu 1983) and 2n=42 (Li and Shang 1989, He et al. 2004); Mastixia has n=13 (Goldblatt 1978) and 2n=22 (Goldblatt 1978). Karyotypes were reported only from Camptotheca and Davidia (He et al. 2004). No chromosome information is available for two species of Diplopanax.

In this study, we report the karyomorphology of seven species of five genera, including that of Diplopanax, and discuss morphological features and evolution of the chromosomes in Nyssaceae.

Materials and methods

Seven species of five genera of Nyssaceae are listed in Table 1 along with their collection data, chromosome numbers, and karyotype formulae. Somatic chromosomes were examined using meristematic cells of young leaves collected from wild-growing and cultivated plants. The methods for observations follow those described elsewhere (Oginuma et al. 1992). Classification of chromosome morphology is based on the position of centromeres (Levan et al. 1964).

Table 1. Studied taxa, their collection data, and chromosome number of Nyssaceae.

Species	Collection	Chromosome number and karyotype formula1)
Camptotheca acuminata Decne.	Cultivated, Kyoto Univ., Japan	2n=42=24m (2SC)+12sm+6st
Davidia involucrata Baill.	Cultivated, Kyoto Univ., Kamigamo Expr. Forest Station, Japan	2n=42=20m (2SC)+16sm+6st
Diplopanax stachyanthus Hand.-Mazz.	Chaina, Yunnan, Wenshen, Mao 04026 (KUN)	2n=42=30m (2SC)+12sm (2SC)
Mastixia kaniensis Melch.	Papua New Guinea, Morobe Prov. Finschhafen	2n=26=16m (2SC)+6sm (2SC)+4st
M. trichotoma Blume	Cultivated, Bogor Bot. Gard., Indonesia	2n=26=16m (2SC)+6sm (2SC)+4st
Nyssa sinensis Oliv.	Cultivated, Hiroshima Bot. Gard., Japan	2n=44=20m+12sm (2SC)+12st
N. sylvatica Marshall	Cultivated, Kyoto Univ., Kamigamo Expr. Forest Station, Japan, Oginuma 02001 (KYO)	2n=44=20m+12sm (2SC)+12st

¹⁾ Abbreviations; m, chromosomes with a centromere at median position; sm, chromosomes with a centromere at submedian position; st, chromosomes with a centromere at subterminal position; SC, chromosomes with a secondary constriction.

Results

Somatic chromosome numbers and karyotype formulae for seven species of five genera are summarized in Tables 1 and 2 along with previously reported chromosome counts. Somatic chromosomes at metaphase of the five species in Camptotheca, Davidia, and Nyssa are small, ranging from 0.5 to 1.7 µm long, while those of three species of Diplopanax and Mastixia are long, ranging from 0.9 to 3.8 µm long. Features of somatic chromosomes in individual genera and species are presented below.

Table 2. Chromosome data of Nyssaceae.

Genus/species	Total No. of species1)	No. of examinedspecies	n	2n	Reference	Basic number
Camptotheca	2	1				x=21
C. acuminata Decne.				42	Raven (1975)
				44	Wu (1984)
				44	Li and Hsu (1986)
				42	He et al. (2004)
				42	Present study
Davidia	1	1				x=21
D. involucrata Baill.				c.40	Dermen (1932)
				40	Eyde (1966)
			21		Goldblatt (1978)
				40	Yang and Zhu (1983)
				42	Li and Shang (1989)
				42	He et al. (2004)
				42	Present study
Diplopanax	2	1				x=21
D. stachyanthus Hand.- Mazz.				42	Present study
Mastixia	20	3				x=13
M. arborea (Wight) C.B. Clarke			13		Goldblatt (1978)
M. kaniensis Melch.				26	Present study
M. trichotoma Blume				22	Goldblatt (1978)
				26	Present study
Nyssa	11	3				x=22
N. javanica (Bl.) Wangerin			22		Mehra (1972)
			22		Mehra and Bawa (1969)
N. sinensis Oliv.				44	Huang et al. (1985, 1989)
				44	Present study
N. sylvatica Marshall				44	Dermen (1932)
				44	Eyde (1966)
			22		Goldblatt (1978)
				44	Present study

¹⁾ The number of species of each genus is based on Mabberley (2017).

Chromosomes at mitotic metaphase are 2n=42 in Camptotheca acuminata. The chromosome number 2n=42 agreed with that reported by Raven (1975) and He et al. (2004), but disagreed with 2n=44 reported by Wu (1984) and Li and Hsu (1986). The longest pair is about 1.5 µm long, and the shortest about 0.5 µm long. Among the 42 chromosomes, 24 have median centromeres (m), 12 have submedian centromeres (sm), and six have subterminal centromeres (st). A secondary constriction (SC) was observed at the proximal regions on the short arms in a pair of chromosomes with median centromeres (Fig. 1a, b). The karyotype formula is 2n=42=24m (2^SC)+12sm+6st.

Fig. 1. Somatic chromosomes at metaphase in three species of three genera in Nyssaceae. (a) and (b): Camptotheca acuminata (2n=42). (c) and (d): Davidia involucrata (2n=42). (e) and (f): Diplopanax stachyanthus (2n=42). (b), (d) and (f) are respective drawings of (a), (c) and (e). Arrows indicate sm-chromosomes. Open arrows indicate st-chromosomes. Arrowheads indicate m-chromosomes with SC. Open arrowheads indicate sm-chromosomes with SC. Scale bar=5 µm.

The chromosomes at mitotic metaphase are 2n=42 in Davidia involucrata. The chromosome number 2n=42 corresponded to that reported by Goldblatt (1978), Li and Shang (1989), and He et al. (2004), but disagreed with 2n=40 reported by Dermen (1932) and Yang and Zhu (1983). The longest pair is about 1.5 µm long, and the shortest about 0.7 µm long. Among 42 chromosomes, 20 have centromeres at median positions, 16 have centromeres at submedian, and six have subterminal positions. The SC was observed at the proximal regions on the short arms of a pair of m-chromosomes (Fig. 1c, d). The karyotype formula is 2n=42=20m (2^SC)+16sm+6st.

Karyomorophological feature of Diplopanax stachyanthus was reported for the first time. Chromosomes at mitotic metaphase are 2n=42. The longest pair is about 1.8 µm long, and the shortest about 0.9 µm long. Among 42 chromosomes, 30 chromosomes have centromeres at median positions, and 12 have centromeres at submedian positions. The SC was observed both at the interstitial region of the long arms in a pair of large m-chromosomes and at the distal region of the short arms in a pair of sm-chromosomes (Fig. 1e, f). The karyotype formula is 2n=42=30m (2^SC)+12sm (2^SC).

Two species of Mastixia kaniensis and M. trichopoda were studied. Chromosomes at mitotic metaphase of the two species are 2n=26. The chromosome number 2n=26 of M. kaniensis is a new count for the species, and 2n=26 in M. trichopoda disagreed with 2n=22 previously reported by Goldblatt (1978). In M. kaniensis the longest pair was about 3.0 µm long, and the shortest about 1.5 µm long (Fig. 2a, b), while in M. trichopoda the longest is about 3.8 µm long, and the shortest about 1.5 µm long (Fig. 2c, d). Chromosome features at metaphase are similar in the two species examined. Among 26 chromosomes, 16 chromosomes have centromeres at median positions, six chromosomes have centromeres at submedian positions, and four chromosomes have centromeres at subterminal positions. The SC was observed at the proximal regions of short arms not only in two pairs of large m-chromosomes but also in a pair of medium-sized sm-chromosomes. The karyotype formulae of two species are same as 2n=26=16m (2^SC)+6sm (2^SC)+4st.

Fig. 2. Somatic chromosomes at metaphase in four species of two genera in Nyssaceae. (a) and (b): Mastixa kaniensis (2n=26). (c) and (d): M. trichotoma (2n=26). (e) and (f): Nyssa sinensis (2n=44). (g) and (h): N. sylvatica (2n=44). (b), (d), (f) and (h) are respective drawings of (a), (c), (e) and (g). Arrows indicate sm-chromosomes. Open arrows indicate st-chromosomes. Arrowheads indicate m-chromosomes with SC. Open arrowheads indicate sm-chromosomes with SC. Scale bar=5 µm.

Nyssa sinensis and N. sylvatica were studied. The chromosome number 2n=44 corresponded to that reported by Huang et al. (1985, 1989) for N. sinensis, and that reported by Dermen (1932), Eyde (1966), and Goldblatt (1978) for N. sylvatica. In N. sinensis the longest pair is about 1.5 µm long, and the shortest about 0.7 µm long (Fig. 2e, f), while in N. sylvatica the longest is about 1.7 µm long, and the shortest 0.6 µm long (Fig. 2g, h). Chromosome features are similar in the two species examined. Among 44 chromosomes, 20 have centromeres at the median position, 12 have centromeres at the submedian position, and 12 have centromeres at the subterminal position. The SC was observed in the distal regions on the short arms in a pair of sm-chromosomes. The karyotype formulae of the two species are uniformly 2n=44=20m+12sm (2^SC)+12st.

Discussion

Chromosome numbers of individual genera

Taken together with the results of the present study, chromosome information has become available for nine species in all the five genera of Nyssaceae (Table 2). Chromosome count 2n=42 we showed for C. acuminata agreed with that reported by Raven (1975) and He et al. (2004), but it disagreed with 2n=44 reported by Wu (1984) and Li and Hsu (1986). Likewise, the chromosome count 2n=42 we showed for D. involucrata agreed with that reported by Li and Shang (1989) and He et al. (2004), but it disagreed with 2n=40 reported by Eyde (1966) and Yang and Zhu (1983). The chromosome count 2n=26 we showed for M. kaniensis and M. trichotoma agreed with its haploid number (n=13) reported by Goldblatt (1978) for M. arborea, but it disagreed with 2n=22 reported by Goldblatt (1978) for M. trichotoma. Because of the lack of any concrete evidence for them like micrographs showing chromosomes, the previous chromosome counts of 2n=40 for both Camptotheca (Wu 1984, Li and Hsu 1986) and Davidia (Dermen 1932, Eyde 1966, Yang and Zhu 1983), as well as of 2n=22 of M. trichotoma (Goldblatt 1978) need reconfirmation. When such dubious chromosome counts were excluded, the chromosome number is consistent within every genus, and it is 2n=42 in Camptotheca, Davidia, and Diplopanax, 2n=44 in Nyssa, and 2n=26 in Mastixia (Table 2).

Karyotype analyses and chromosome evolution

When the generic chromosome numbers are mapped on a phylogenetic tree of the Nyssaceae (Fig. 3), a parsimony analysis indicates that 2n=42 common to the three genera Camptotheca, Davidia, and Diplopanax is plesiomorphic, and 2n=26 of Mastixia and 2n=44 of Nyssa apomorphic. Although it is uncertain how they occurred, 2n=26 and 2n=44 were derived independently from 2n=42.

Fig. 3. Somatic chromosome numbers mapped on a phylogenetic tree of Nyssaceae. The tree is modified from Fu et al. (2017). Note that 2n=42 occurs in three (Camptotheca, Diplopanax, and Davidia) of the five genera in Nyssaceae.

Given that 2n=42 is a plesiomorphy in chromosome evolution in the family, the karyotypes of 2n=42 in Camptotheca, Davidia, and Diplopanax require deeper analyses. Like chromosome number, the karyotype of each genus is likely consistent, because in both Mastixia and Nyssa the two species examined had identical karyotype formulae (Table 1). As for karyotypes of Camptotheca and Davidia, we showed those of C. acuminata and D. involucrata as 2n=42=24m (2^SC)+12sm+6st and 2n=42=20m (2^SC)+16sm+6st, respectively. They resemble the karyotypes reported by He et al. (2004) as those of C. acuminata and D. involucrata but do not coincide with them in detail. According to He et al. (2004), the karyotype is 2n=42=22m (2^SC)+14sm+4st+2t in C. acuminata and 2n=42=20m (2^SC)+16sm+4st+2t in D. involucrata. The difference between the karyotypes reported by He et al. (2004) and those we reported seems to be owing to differences in the mitotic phase when the chromosomes were observed.

Noticeably the karyotypes of Camptotheca and Diplopanax show that the somatic chromosomes appear to consist of six sets of seven chromosomes. Each set comprises 4m+2sm+1st in Camptotheca and 5m+2sm in Diplopanax. Although it is uncertain which set represents a more ancestral constitution in Nyssaceae, 2n=42 is likely hexaploid in both Camptotheca and Diplopanax. This may be the case of 2n=42 of Davidia, because its somatic chromosomes contain six st-chromosomes. Thus, Nyssaceae are very likely of hexaploid origin with their original basic number x=7. After polyploidization of x=7 resulting in a hexaploidy in an ancestral Nyssaceae, evolutionary changes seem to have occurred from 2n=42 to 2n=26 as in Mastixia as well as to 2n=44 as in Nyssa within Nyssaceae (Fig. 3).

The chromosome number 2n=42 in Nyssaceae might be shared by other cornalean families. Molecular phylogenetic analyses suggest that Nyssaceae have close relationships with Hydrangeaceae, Hydrostachyaceae, and Loasaceae, but exact relationships within the order are still unclear (Xiang et al. 2011, Stevens 2001 onward: http/www.mobot.org/MOBOT/research/APweb/). We searched 2n=42 (or n=21) in the other six families [for the information on chromosome numbers see “Chromosome numbers of flowering plants” edited by Fedorov (1974) and “Index to plant chromosome numbers” edited by Ornduff (1967, 1968), Moore (1973, 1974, 1977), Goldblatt (1981, 1984, 1985, 1988), and Goldblatt and Johnson (1990, 1991, 1994, 1996, 1998, 2000, 2003, 2006, 2010)]. Consequently, n=21 (an archaic chromosome number in Nyssaceae) was found in all the eight species of Encnide which were examined by Thompson and Ernst (1967) and Baker and Parfitt (1986). Since Eucnide is a most basally divergent genus in Loasaceae, the shared chromosome number x=21 may suggest affinities between Loasaceae and Nyssaceae. For more critical comparison with Nyssaceae, karyotype analyses are required for Eucnide in the future.

Acknowledgments

We thank Mr. Zhijian Gu and Zong-Shu Yue of Kunming Institute of Botany, Chinese Academy of Sciences, the People’s Republic of China for supporting materials of Diplopanax.

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