$¢PhytoKeys

PhytoKeys 234: 145-165 (2023)
DOI: 10.3897/phytokeys.234.108841

Research Article

Karyotype and genome size variation in Delphinium subg.
Anthriscifolium (Ranunculaceae)

Xiao-Yu Luo’?®, Tang-Jie Nie'2©, Heng Liu’?®, Xue-Fei Ding'2®, Ying Huang'2®, Chun-Ce Guo!®,

Wen-Gen Zhang!7&

1 Forestry College, Jiangxi Agricultural University, Nanchang 330045, China

2 Jiangxi Provincial Key Laboratory for Bamboo Germplasm Resources and Utilization, Nanchang 330045, China

3 Co-Innovation Center for Sustainable Forestry in Southern China, College of Biology and the Environment, Nanjing Forestry University, Nanjing 210037, China
Corresponding author: Wen-Gen Zhang (wgzhang@jxau.edu.cn)

OPEN Qaceess

Academic editor: Marco Pellegrini
Received: 30 June 2023

Accepted: 22 September 2023
Published: 18 October 2023

Citation: Luo X-Y, Nie T-J, Liu H,

Ding X-F, Huang Y, Guo C-C, Zhang
W-G (2023) Karyotype and genome
size variation in Delphinium subg.
Anthriscifolium (Ranunculaceae).
PhytoKeys 234: 145-165. https://doi.
org/10.3897/phytokeys.234.108841

Copyright: © Xiao-Yu Luo et al.

This is an open access article distributed under
terms of the Creative Commons Attribution
License (Attribution 4.0 International -

CC BY 4.0).

Abstract

Five taxa of Delphinium subg. Anthriscifolium have been karyologically studied through
chromosome counting, chromosomal measurement, and karyotype symmetry. Each tax-
on that we investigated has a basic chromosome number of x = 8, D. anthriscifolium var. sa-
vatieri, D. anthriscifolium var. majus, D. ecalcaratum, and D. callichromum were diploid with
2n = 16, while D. anthriscifolium var. anthriscifolium was tetraploid with 2n = 32. Monoploid
chromosome sets of the investigated diploid taxa contained 1 metacentric chromosome,
3 submetacentric chromosomes, and 4 subtelocentric chromosomes. Higher interchro-
mosomal asymmetry (CV,,) was present in D. ecalcaratum and D. callichromum than in
other taxa. The highest levels of intrachromosomal asymmetry (M,,) and heterogeneity
in centromere position (CV,,) were found in D. anthriscifolium var. majus. Diploid and tet-
raploid genome sizes varied by 3.02-3.92 pg and 6.04-6.60 pg, respectively. Karyotype
and genome size of D. anthriscifolium var. savatieri, D. anthriscifolium var. majus, D. calli-
chromum, and D. ecalcaratum were reported for the first time. Finally, based on cytological
and morphological data, the classification of Delphinium anthriscifolium was revised.

Key words: Columbines, Consolida, genome size, karyotype, ploidy, Ranunculales

Introduction

Delphinium L., ca. 385 species and 232 species in China (Ilarslan et al. 1997; Wang
2019; Hadidchi et al. 2020), is a species-abundant genus of tribe Delphinieae in
the buttercup family (Ranunculaceae) with great economic importance in terms
of both horticultural and pharmaceutical value (Ghimire et al. 2015; Wang 2019;
Wang et al. 2020). It is usually characterised by the following key traits: (1) In the
zygomorphic flower, there are 5 petaloid sepals, with the upper one spurred; (2) a
pair of dorsal petals are sessile, free, and spurred in the upper sepal, while a cou-
ple of lateral petals (i.e., staminodes) are spurless, each with a slender claw and
an expanded limb; (3) follicles 3 (Tamura 1993; Wang and Warnock 2001; Wang
2019). Except for a few species found in tropical Africa’s montane regions, the
genus is widely distributed in northern temperate regions (Milne-Redhead and
Turrill 1952; Chartier et al. 2016; Aleem et al. 2020; Kashin et al. 2021).

145

Xiao-Yu Luo et al.: Karyotype and genome size variation in Delphinium subg. Anthriscifolium

To date, the classification of subgenus or groups in Delphinium is still con-
troversial. For example, Wang (2019, 2020) divided Delphinium into subge-
nus Delphinastrum (DC.) Peterm. comprising sections Aconitoides W.T.Wang,
Elaopsis Huth, Delphinastrum DC. and Oligophyllon Dimitrova, and subgenus
Delphinium with section Anthriscifolium. However, molecular phylogenetic
studies indicated at least four monophyletic subgenera [i.e., D. subg. Consolida
(DC.) Huth, subg. Delphinium, subg. Delphinastrum, and subg. Anthriscifolium
(W.T.Wang) Wei Wang] should be accepted (Jabbour and Renner 2011, 2012;
Wang et al. 2013; Xiang et al. 2017; DuPasquier et al. 2021). Interestingly, the
taxon, including D. anthriscifolium Hance, is a monoclade, either a subgenus of
Delphinium (Xiang et al. 2017) or an independent group included in Delphinium
(Jabbour and Renner 2012; Wang et al. 2013).

As a recently erected subgenus, Delphinium subg. Anthriscifolium, includ-
ing ca. 3 species [i.e., D. anthriscifolium Hance, D. ecalcaratum S.Y.Wang &
K.F.Zhou, and D. callichromum Q.L.Gan & X.W.Li], is endemic to East Asia and
mainly distributed in the south of Zhongtiao Mountain and Qinling Mountain in
China (Ding et al. 1981; Gan and Li 2017; Wang 2019). Moreover, there are three
varieties of D. anthriscifolium |i.e., D. anthriscifolium var. anthriscifolium, D. an-
thriscifolium var. majus Pamp., and D. anthriscifolium var. savatieri (Franch)
Munz], among which there are obvious differences in flower size, colour, and
shape, which cause disagreements in the taxonomic circumscription of this
species and associated varieties.

Genome size refers to the amount of DNA contained in the gametes of a
species, which is broadly constant within an organism and is primarily indi-
cated by C-value (Pellicer et al. 2018; Twyman and Wisden 2018; Kocjan et
al. 2022). C-value estimation is not only crucial for genomic sequencing and
analysis (Gregory 2005) but also significant for the identification of species
and taxonomic positions (Bourge et al. 2018; Sliwinska 2018). Furthermore,
as an important character of genetic material, karyotype, including chromo-
some number, morphology, length, band type, and centromere position (de
Resende 2017; Ning et al. 2018; Vimala et al. 2021; Mahmoudi and Mirzagha-
deri 2023), was extensively used in the systematic and evolutionary study of
plants (Baltisberger and Hoérandl 2016; Peruzzi et al. 2017; Wang et al. 2020).
So far, there are few reports on the genome size and karyotype of Delphinium
subg. Anthriscifolium.

Here, we aim to: (1) determine the chromosome number, karyotype, and ge-
nome size of the above five taxa (i.e., D. anthriscifolium var. anthriscifolium,
D. anthriscifolium var. majus, D. anthriscifolium var. savatieri, D. ecalcaratum,
and D. callichromum)); (2) evaluate the reliability of flow cytometry in genome
size determination to infer ploidy levels in D. subg. Anthriscifolium; and (3) pro-
vide cytological evidence for the taxonomic revision of D. anthriscifolium.

Materials and methods
Sampling

Materials of Delphinium subg. Anthriscifolium, including D. ecalcaratum, D. cal-
lichromum, D. anthriscifolium and its varieties (Fig. 1), were collected by field
investigations in China during 2017-2021 (see Table 1 in detail), of which rep-

PhytoKeys 234: 145-165 (2023), DOI: 10.3897/phytokeys.234.108841 146

Xiao-Yu Luo et al.: Karyotype and genome size variation in Delphinium subg. Anthriscifolium

gi SS a

D. anthriscifolium D. anthriscifolium
var. anthriscifolium var. Savatieri

A
Se

D. anthriscifolium var. majus

“iP

D. ecalcaratum D. callichromum

a ZZ
g
aa

Figure 1. Five taxa of Delphinium subg. Anthriscifolium A D. anthriscifolium var. anthriscifolium B D. anthriscifolium var.
savatieri C D. anthriscifolium var. majus D D. ecalcaratum E D. callichromum F flower front view of the above five taxa.
Scale bars: 5mm.

resentatives were transplanted to the garden of Jiangxi Agricultural University.
All vouchers were deposited in the herbarium of the College of Forestry, Jiangxi
Agricultural University, China (JXAU).

Flow cytometry (FCM) analysis

Twenty-two populations of Delphinium subg. Anthriscifolium were gathered
with silica gel-dried leaves for the assessment of genome size by using
flow cytometry (FCM; Table 1). In a petri dish containing pre-chilled MG?
dissociation solution, ca. 1 cm? of leaf material was quickly chopped using a
sharp blade. After 10 min on ice, the samples were filtered through a 40 um
filter into a tube with pre-chilled Pl (50 g/mL) and RNAase solution (50 ug/
mL), which were then placed on ice and kept from light for 0.5 to 1 hour. Using
BD FACSCalibur Flow Cytometer (USA), three replicates of each population
of D. subg. Anthriscifolium were estimated with the internal standard

PhytoKeys 234: 145-165 (2023), DOI: 10.3897/phytokeys.234.108841 147

Xiao-Yu Luo et al.: Karyotype and genome size variation in Delphinium subg. Anthriscifolium

Table 1. Chromosome number, ploidy, and genome size of Delphinium subg. Anthriscifolium in the study.

Pop
1

22

Taxa

D. anthriscifolium
var. anthriscifolium

D. anthriscifolium
var. anthriscifolium

D. anthriscifolium
var. anthriscifolium

D. anthriscifolium
var. anthriscifolium

D. anthriscifolium
var. anthriscifolium

D. anthriscifolium
var. anthriscifolium

D. anthriscifolium
var. anthriscifolium

D. anthriscifolium
var. anthriscifolium

D. anthriscifolium
var. savatieri

D. anthriscifolium
var. savatieri

D. anthriscifolium
var. savatieri

D. anthriscifolium
var. savatieri

D. anthriscifolium
var. Savatieri

D. anthriscifolium
var. savatieri

D. anthriscifolium
var. savatieri

D. anthriscifolium
var. Majus

D. anthriscifolium
var. Majus

D. anthriscifolium
var. majus

D. ecalcaratum
D. ecalcaratum

D. callichromum

D. callichromum

Voucher information

Bamboo Culture Park, Yifeng County, Jiangxi, China,
28°24'31"N, 114°50'3"E, 24 Apr 2018, Liu 1824
Huacheng Temple, Yichun City, Jiangxi, China, 27°48'40"N,
114°22'44"E, 17 Apr 2019, Zhang 1917
Guling Town, Lushan City, Jiangxi, China, 29°34'28'N,
115°59'19"E, 17 Apr 2019, Zhang 1904
Miaofeng Mountain, Fuzhou City, Fujian, China, 26°4'53'N,
119°14'59"E, 2 May 2017, Luo 1705
Jiaogiao Town, Nanchang City, Jiangxi, China, 28°46'6'N,
115°50'22"E, 16 Apr 2018, Liu 1876
Fujia County, Fuzhou City, Jiangxi, China, 27°45'40"N,
116°26'17"E, 17 Apr 2019, Nie 1917
Shangli County, Pingxiang City, Jiangxi, China, 27°50'37'N,
113°49'15"E, 17 Apr 2019, Zhang 1918
Guangxi Botanical Institute, Guangxi, China, 25°4'58'N,
110°18'45"E, 26 Mar 2020, Zhang 2026
Hanfeng, Liuyang County, Shaanxi, China, 33°20'26'"N,
105°59'43"E, 11 Apr 2020, Gao 2071

Baisha River, Zhuxi County, Hubei, China, 32°5'27'N,
109°55'25"E, 18 Apr 2019, Zhang 1818

Sun Yat-sen Mausoleum, Nanjing City, Jiangsu, China,
32°5'23'"N, 118°52'28"E, 19 Apr 2019, Nie 1919
Baohua Mountain, Gourong City, Jiangsu, China, 32°8'8"N,
119°5'40"E, 19 Apr 2019, Nie 1920
Nanjing Zhongshan Botanical Garden, Jiangsu, China,
32°3'38'N, 118°50'16"E, 19 Apr 2019, Nie 1921
Zhongtiao Mountain, Yuncheng City, Shanxi, China,
32°46'44'"N, 107°34'30"E, 21 May 2019, Ren 1921
Jiaogiao Town, Nanchang City, Jiangxi, China, 28°46'6'N,
115°50'22"E, 15 May 2021, Luo 2115
Hefeng County, Enshi City, Hubei, China, 30°3'57"N,
110°8'45"E, 18 Apr 2019, Zhang 1919
Songbai Town, Shennongjia, Hubei, China, 31°45'11"N,
110°40'5"E, 18 Apr 2019, Zhang 1925
Jiaogiao Town, Nanchang City, Jiangxi, China, 28°46'6'N,
115°50'22"E, 15 May 2021, Luo 2116
Jiaogiao Town, Nanchang City, Jiangxi, China, 28°46'6'N,
115°50'22"E, 15 May 2021, Luo 2117

Lingshan Mountain, Xinyang City, Henan, China, 31°54'46'N,

114°13'19"E, 19 Apr 2019, Luo 1919

Baisha River, Zhuxi County, Hubei, China, 32°5'27'N,
109°55'25"E, 18 Apr 2019, Luo 1978

Jiaogiao Town, Nanchang City, Jiangxi, China, 28°46'6'N,
115°50'22"E, 15 May 2021, Luo 2118

2n
32

32

32

32

32

32

32

32

16

16

16

16

16

16

16

16

16

16

16

16

16

16

Ploidy

4x

4x

4x

4x

4x*

Ax*

4x

4x

2X

2x*

2X

2x*

2x*

2x

2X

2x

2x*

2x

2X

2x*

2x

2x*

2C (pg)
6.26

6.20

3.40
3.43
3.36
3.32
3.31
3.92
3.80
a Bs)
3.02
3.03
3710

3.10

1Cx (pg)

T57

* Chromosome number and ploidy were validated by experimental analysis in the study, while others were inferred according to the
genome size by flow cytometry. Pop = population.

(Solanum lycopersicum L., 900 M bp; The Tomato Genome Consortium 2012).
According to Tian et al. (2011), the 2C-value of each sample was calculated
as the fluorescence intensity ratio. To remove the effect of genome size
resulting from recent polyploidisation, monoploid genome size value (1Cx;
Greilhuber et al. 2005) was used and calculated through the 2C-value.

PhytoKeys 234: 145-165 (2023), DOI: 10.3897/phytokeys.234.108841

148

Xiao-Yu Luo et al.: Karyotype and genome size variation in Delphinium subg. Anthriscifolium

Karyotype analysis

Somatic chromosomes were studied from the root tip cells of young seedlings.
About 1-2 cm long roots were first pretreated in a 0.1% colchicine solution at
15 °C for 2-3 hours, then fixed in Carnoy | (absolute ethyl alcohol and glacial ace-
tic acid in the proportions 3:1) for 30 minutes. After cleaning in purified water,
they were hydrolysed in a mixture of 1 M HCI and 45% acetic acid (1:1) at 60 °C
for 3-5 min and then stained with improved phenol magenta for 2 h. Five mitotic
cells per species were examined and photographed using an Axio Imager A.1
microscope (Carl Zeiss, Germany) with ZEN software at 1000x magnification.

Short arm length (s) and long arm length (I) were measured using Image J
(Collins 2007). Excel was used to determine additional chromosomal charac-
teristics such as arm ratio (r = I/s), centromeric indices (Cl), mean chromosome
length (CL), relative chromosome length (RL), and total haploid length (THL).
The coefficient of variation of chromosome length (CV,, ) [(S,, / X,,)* 100, where
S,,: standard deviation; X.,: mean chromosome length] (Lavania and Srivastava
1992; Paszko 2006), coefficient of variation of the centromeric index (CV) [(S,,
/ X,,) x 100, where S_,: standard deviation; X_,: mean centromeric index] (Paszko
2006), and mean centromeric asymmetry (M.,,) (A x 100; the calculation of A
is detailed in Watanabe et al. 1999) (Peruzzi and Eroglu 2013) were calculated.

To infer the formulas of karyotype, the arm ratio (r), as defined by Levan et al.
(1964), was used to categorise the chromosomes, and the homologous chro-
mosome was allocated based on the similarity in length and centromere posi-
tion using Photoshop CS6 software. The idiogram was constructed according
to the arm ratio and relative length of the chromosomes. In order to illustrate
karyotypic correlations between organisms, a bidimensional scatter plot was
also created, in which the parameters CV,, and M., are plotted on the x- and
y-axes, respectively, and dots indicate each sample.

Results
Genome size of Delphinium subg. Anthriscifolium

In the FCM analysis, all studied taxa and the internal standards exhibited clear
and sharp peaks (Fig. 2), and coefficients of variation were lower than 5%, sup-
porting the reliability of the flow cytometric assessments. Twenty-two popu-
lations of D. subg. Anthriscifolium, including five taxa, i.e., D. anthriscifolium
var. anthriscifolium, D. anthriscifolium var. savatieri, D. anthriscifolium var. ma-
jus, D. ecalcaratum, and D. callichromum, showed remarkable variation (3.02-
6.60 pg) in genome size (Table 1). Nearly twice as large as the others, D. an-
thriscifolium var. anthriscifolium had the greatest 2C-values (6.27 + 0.17 pg).
In contrast, D. ecalcaratum (3.03 pg) and D. callichromum (3.10 pg) had the
lowest values (Fig. 3A). The 1Cx values were highest in D. anthriscifolium var.
majus (1.91 + 0.04 pg), while lower in D. anthriscifolium var. anthriscifolium
(1.57 +0.04 pg), D. ecalcaratum (1.51 pg), and D. callichromum (1.55 pg) (Fig. 3B).
Additionally, the monoploid genome sizes of tetraploids (mean 1Cx = 1.57 pg)
are smaller than those of diploids (mean 1Cx = 1.69 pg). Thus, genome loss or
duplication events have occurred in the evolution of D. subg. Anthriscifolium.

PhytoKeys 234: 145-165 (2023), DOI: 10.3897/phytokeys.234.108841 149

Xiao-Yu Luo et al.: Karyotype and genome size variation in Delphinium subg. Anthriscifolium

120

BA 1 Peak Mean CV(%) B Peak Mean CV(%) C l Peak Mean CV(%)
| 1 1733 4.28 = l 1 19.92 3.55 2 1 1855 3.32
2 118.09 4.91 = 2 137-71 487 _ 2 6935 451
a]
at co)
| oO ©
S| = 5 re jay
eve = 58 : ES =
o | i iS) = is) ‘e
Ys = v = v0 s
‘ : 5 2 s 5 8 s 5
8 < 3 ° & . 2 Ss m
5 = nN S = v 2 =
= 3 = S S 3
a aa 4 | aa) os Q
f=) : f=) , fo)
10° 10! 102 103 104 10° 10! 102 108 104 109 10! 102 103 104
PI fluorescence (channels) PI fluorescence (channels) PI fluorescence (channels)
oS o
= =
'D 1 Peak Mean CV(%) 2 E 1 Peak Mean CV(%) F l Peak Mean CV(%)
| | 1 1836 3.29 a 1 17.19 3.79 re 1 1881 3.44
2) 2 7747 4283 2 57.84 432 a 2 64.71 4.96
| its)
| Ta =}
a
=e
=z 5 #§ oe :
= = = 3 Oo ~
6.) sl a ) = o “4
Vs 2 a a oe) A
| 5 > © S = 2 S =
= = = = © = 2
3 a S 5 3 S
8 S af ¢ S $ 2 S" s
< : S S i 2 :
4 Q w rex a fan)
on— - ————,——_—— ou - ——— on - ;
10° 10! 102 103 104 10° 10! 102 103 104 10° 10! 102 103 104
PI fluorescence (channels) PI fluorescence (channels) PI fluorescence (channels)

Figure 2. Flow cytometric histograms of Delphinium subg. Anthriscifolium was analysed simultaneously with the internal
standard Solanum lycopersicum. In each histogram, the peaks are marked as follows: 1, nuclei of the internal standard
at the G, phase; 2, nuclei of the sample at the G, phase. The mean channel number (PI fluorescence) and coefficient of
variation value (CV, %) of each peak are also given; 3, nuclei of the internal standard at the G, phase.

A B
, 2.0 a
b
t Cc
1.5 .
ns: —
bf) bf)
ra¥ a.
——— ——* 1.0
'S) mo
NI OU
—
0.5
0.0
Dan Dsa Dma Dec Dea Dan Dsa Dma Dec Dea

Figure 3. Comparison of the 2C and 1Cx mean values among Delphinium subg. Anthriscifolium. The columns marked
with different index letters are significantly different at P < 0.05; those marked with the same index letters are not signifi-
cantly different at P < 0.05 (one-way ANOVA followed by Tukey's test). Error bars represent standard deviation.

Karyotypes of Delphinium subg. Anthriscifolium

Eight representative populations of D. subg. Anthriscifolium, including the above
five taxa, were karyologically studied. Karyomorphometric data, microphotographs
of metaphase plates, and idiograms are presented here (Tables 1-3; Figs 4-6).

PhytoKeys 234: 145-165 (2023), DOI: 10.3897/phytokeys.234.108841 150

Xiao-Yu Luo et al.: Karyotype and genome size variation in Delphinium subg. Anthriscifolium

Table 2. Karyomorphological parameters of Delphinium subg. Anthriscifolium in the study.

Taxa Pop Chromosome pair

D. anthriscifolium var. 5 |
anthriscifolium T

D. anthriscifolium var. 13 |
savatieri i

12 |

Vi
VII
VIII

PhytoKeys 234: 145-165 (2023), DOI: 10.3897/phytokeys.234.108841

CL (um)
7.42 + 0.72
6.35 + 0.48
5.85 + 0.12
4.28 + 0.25
3.25 + 0.50
2.76 + 0.08
2.54 + 0.02
2.41 + 0.02
2.34 + 0.03
2.19 + 0.01
2.06 + 0.07
1.95 + 0.02
1.76 + 0.08
1.61 + 0.09
1.40 + 0.01
1.31 + 0.06
5.60 + 0.05
4.14+0.13
4.62 + 0.16
3.43 + 0.52
2.75 + 0.20
2.24 + 0.23
1.81 + 0.08
1.90 + 0.04
1.78 + 0.29
1.88 + 0.04
1.76 £0.25
1.56 + 0.01
1.76 + 0.50
1.32 + 0.20
1.59 + 0.20
1.68 + 0.22
7.65 + 0.53
5.14+0.43
2.87 +0.11
2.73 + 0.03
2.67 + 0.03
2.15 + 0.26
1.53 + 0.08
1.39 + 0.00
8.15 + 1.58
6.14 + 0.20
3.13 + 0.16
2.62 + 0.08
2.46 + 0.09
2.37 + 0.02
1.82 + 0.00
1.55 +0.24

r
1.28 + 0.06
1.59:+0.05
2.10 + 0.42
2.45 + 0.55
2.79 + 0.26
2.06 + 0.42
3.34 + 0.21
3.15 + 0.00
3.38 $0.27
3.26:%:0:13
2.88 + 0.19
2.73 + 0.76
3.02 + 0.06
3.08 + 0.29
1.82 + 0.14
1.88 40.13
1.22 + 0.09
1.1440.13
1.90 + 0.02
2,1220,03
1,90:2 O17
2:30 20.79
3.09 + 0.04
3.01 + 0.00
2.03 + 0.43
1.73 + 0.01
3.254 0.01
3.08 + 0.07
3.23 + 0.24
3.10 + 0.02
2.91 + 0.06
2.60 + 0.12
1.05 + 0.04
2712031
4.23 + 0.91
3.36 + 0.03
3.60 + 0.49
3.53 + 0.14
2.90 + 0.01
2.90 + 0.13
1.24+0.17
2.04 + 0.06
3.80 + 0.01
4.47+1.41
3.752 0:02
3.05 + 0.02
2.82 + 0.18
2.89 + 0.14

Cl
0.44
0.39
0.33
0.29
0.26
0.33
0:23
0.24
0.23
0.23
0.26
0.20
0.25
0.25
0.30
0.35
0.45
0.47
0.35
0.32
0.35
0.31
0.33
0.37
0.24
0.25
0.24
0.24
0.26
0.28
0.24
0.24
0.49
0.27
0.19
0.23
0.22
0.22
0.26
0.26
0.45
0.33
0.21
0.19
0.21
0.25
0.26
0.26

RL (%)
14.47
12.38
11.40
8.35
6.34
5.38
4.95
4.71
4.57
4.27
4.01
3.79
3.42
3.14
2.72
2.56
13.90
10.27
11.45
8.50
6.81
5.55
4.49
4.70
4.42
4.66
4.37
3.86
4.37
3.28
3.94
4.16
28.15
18.91
10.54
10.05
9.84
7.90
5.64
5.12
27.24
20.52
10.45
8.75
8.23
7.93
6.09
5.20

Type

151

Xiao-Yu Luo et al.: Karyotype and genome size variation in Delphinium subg. Anthriscifolium

Taxa Pop
D. anthriscifolium var. 10
savatieri
D. anthriscifolium var. 17
majus
D. ecalcaratum 20
D. callichromum 22

Vi
VII
Vill

Chromosome pair

CL (um)
10.43 +0.11
6.77 + 0.49
3.60 + 0.16
3.30 + 0.03
2.89 +0.14
2.45 + 0.10
2.18 + 0.10
1.84 40.15
11.08 + 0.48
7.10 + 0.04
4.40 + 0.06
3.75 + 0.31
3.26 + 0.15
3.07 + 0.00
2.67 + 0.05
1.86 + 0.31
8.17 + 0.10
5.39 + 0.03
2.61 + 0.02
2.51 +0.04
2.27 + 0.02
1.95 + 0.07
1.70 + 0.00
1.50 + 0.09
9.47 + 1.40
6.48 + 0.12
3.36 + 0.17
3.14 + 0.10
2.56 + 0.45
2.07 + 0.01
1.96 + 0.09
1.66 + 0.09

r
1330.03
2.95 + 0.01
4.31 +1.43
3.20 £032
4.60 + 0.23
3,23°2:0,02
2.55 + 0.40
2.64 40.18
1.05+0.05
2.97 + 0.01
3.43 + 0.10
3.43 + 0.34
4.75 + 1.40
4.03 + 0.84
2.86 + 0.01
2.84 + 0.06
1.06+0.05
2.66 + 0.26
3.14 + 0.04
3.12 + 0.10
S220 0.09
3.05.4 O72
2.65 + 0.31
2,43 £0:09
1.20+0.14
2.50 + 0.62
3.25-2:0.33
3.45 + 0.46
3.23 + 0.07
3 poe O20
2:01 £0.27
1.77 + 0.01

Cl
0.47
0.25
0.20
0.24
0.18
0.24
0.28
0.28
0.49
0.25
0.23
0.23
0.18
0.20
0.26
0.26
0.49
0.27
0.24
0.24
0.24
0.22
0.28
0.27
0.46
0.29
0.24
0.23
0.24
0.21
0.33
0.36

RL (%)
30.34
19.70
10.49
9°59
8.40
7.14
6.36
9.34
28.96
18.58
TL51
9.81
8.53
8.03
6.97
4.87
30.99
20.45
9:92
9.53
8.62
7.38
6.43
9.70
29.21
19.99
10.36
70
7.90
6.38
6.05
pe 15

Type
msat

sm

Cl = centromeric index; CL = chromosome length, mean value + standard deviation; m = metacentric chromosome; Pop = population, numbers
shown in Table 1; r = arm ratio, mean value + standard deviation; RL = relative chromosome length; sat = chromosome showing secondary
constriction; sm = submetacentric chromosome; st = subtelocentric chromosome.

Table 3. Karyotype parameters of Delphinium subg. Anthriscifolium in the study.

Taxa Pop Ploidy § 2n Karyotype formula THL CV. Mon CV,,,
D. anthriscifolium var. anthriscifolium 5 Ax 32 2n = 4m + 16sm + 12st 51.30 60.13 40.32 22.07
6 Ax 32 2n = 4m + 16sm + 12st 40.33 50.93 37.18 27.08
D. anthriscifolium var. savatieri 1S 2x 16 2n = 2m + 6sm + 8st 24.18 62.81 46.58 34.01
12 2x 16 2n = 2m + 6sm + 8st 29.91 65.20 47.18 30.83
10 2x 16 2n = 2m**+ 6sm + 8st 34.37 68.27 46.05 33.27
D. anthriscifolium var. majus leg 2x 16 2n = 2m + 6sm + 8st 38.24 63.10 47.59 35,/9
D. ecalcaratum 20 2x 16 2n = 2m + 6sm + 8st 26,39 68.87 44.65 29.84
D. callichromum 22 2X 16 2n = 2m+6sm + 8st 32.42 69.63 40.11 29.09

CV,, = Coefficient of Variation of Centromeric Index; CV,, = Coefficient of Variation of Chromosome Length; m = metacentric chromo-
some; M,, = Mean Centromeric Asymmetry; Pop = population, numbers shown in Table 1; sat = satellite chromosome; sm = submeta-
centric chromosome; st = subtelocentric chromosome; THL = total haploid length, um.

PhytoKeys 234: 145-165 (2023), DOI: 10.3897/phytokeys.234.108841 152

Xiao-Yu Luo et al.: Karyotype and genome size variation in Delphinium subg. Anthriscifolium

thriscifolium var. anthriscifolium (6), 2n = 32 C D. anthriscifolium var. savatieri (13), 2n = 16 D D. anthriscifolium var. savatieri
(12), 2n = 16 ED. anthriscifolium var. savatieri (10), 2n = 16 F D. anthriscifolium var. majus (17), 2n = 16 G D. ecalcaratum (20),
2n = 16 HD. callichromum (22), 2n = 16. Numbers in brackets represented populations shown in Table 1. Scale bars: 10 um.

1. Delphinium anthriscifolium var. anthriscifolium

In two populations (Pop 5 and Pop 6) of D. anthriscifolium var. anthriscifolium
from Jiangxi, China, the somatic and basic chromosome numbers were 2n = 32
and x = 8, respectively (Table 1; Fig. 4A, B). Two pairs of chromosomes (i.e., III)
are metacentric, eight pairs (i.e., III-VI, XI-XIl, and XV-XVI) are submetacentric,
and six pairs (i.e., VII-X and XIII-XIV) are subtelocentric (Tables 2, 3; Figs 5A, B,
6A, B). Thus, the karyotype formula is 2n = 4x = 32 = 4m + 16sm + 12st.

2. Delphinium anthriscifolium var. savatieri

In three populations (i.e., Pop 10 from Hubei, Pop 12 and Pop 13 from Jiangsu),
the somatic and basic chromosome numbers are 2n = 16 and x = 8, respec-
tively (Table 1; Fig. 4C-E). Pop 12 and Pop 13 have more similar karyotypes:
one pair of chromosomes (i.e., |) is metacentric, three pairs (i.e., Il, VII, and VIII)
are submetacentric, and four pairs (i.e., II-VI) are subtelocentric (Tables 2 and
3; Figs 5C, D, 6C, D). The karyotype formula is 2n = 2x = 16 = 2m + 6sm + 8st.
However, Pop 10 differed from Pop 12 and Pop 13 in that it has a secondary
constriction on the first pair of chromosomes (Figs 5E, 6E), so its karyotype
formula is 2n = 2x = 16 = 2ms* + 6sm + 8st.

3. Delphinium anthriscifolium var. majus

In Pop 17, the somatic and basic chromosome numbers are 2n = 16 and
x = 8, respectively (Table 1; Fig. 4F). Its chromosome set includes one pair of

PhytoKeys 234: 145-165 (2023), DOI: 10.3897/phytokeys.234.108841 153

Xiao-Yu Luo et al.: Karyotype and genome size variation in Delphinium subg. Anthriscifolium

1
Raa Chas Saad AédaGa
B m sm sm St
Sena Séaa &aas 680932
sm St St sm

C
AA Ah An aalt
St St sm sm
D
tT Lv.) éa £816
Sst St sm sm
E
| aa aA 6816
St Sst sm sm
F
6&8 aan 6d 846801.
St Sst sm sm
G oa ts de 8916
St St sm S

H tf Tt an | et “é YY ee 1 T
sm Sst st st

m st sm sm
Figure 5. Karyotypes of Delphinium subg. Anthriscifolium A D. anthriscifolium var. anthriscifolium (5), 2n = 32 B D. anth-
riscifolium var. anthriscifolium (6), 2n = 32 C D. anthriscifolium var. savatieri (13), 2n = 16 D D. anthriscifolium var. savatieri
(12), 2n = 16 ED. anthriscifolium var. savatieri (10), 2n = 16 F D. anthriscifolium var. majus (17), 2n = 16 G D. ecalcaratum
(20), 2n = 16H D. callichromum (22), 2n = 16. Numbers in brackets represented populations shown in Table 1. m = meta-
centric chromosome; sat = satellite chromosome; sm = submetacentric chromosome; st = subtelocentric chromosome.

PhytoKeys 234: 145-165 (2023), DOI: 10.3897/phytokeys.234.108841 154

Xiao-Yu Luo et al.: Karyotype and genome size variation in Delphinium subg. Anthriscifolium

A B
3.0
2.0
= 20 =
p=! &S 10
pa) ~~
a ; a
2 He a sub Uuususlsu
o o
a 1.0 i = 1 ; [ | i | | | | f : f
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DO 20 oO
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3.0
3,0
$.0 4.0 -
1 2 3 4 5 6 7 8 9 10 ll 12 13 14 15 16 1 2 3 4 5 6 7 8 9 10 il 12 13 14 15 16
Chromosome number Chromosome number
5.0 6.0
Cc D
4.0
o 3.0 |} 3 4.0
o of
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= : Sp 2.0
o o
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Chromosome number Chromosome number
6.0 8.0
E = F
4.0 6.0
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Chromosome number Chromosome number
5.0 7, 6.0
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S20} o
5 BN 20
5 1.0 | Ss
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oo —_ |
3 10 | 3
aw S20
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5.0 6.0
1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8
Chromosome number Chromosome number

Figure 6. Haploid idiograms of Delphinium subg. Anthriscifolium A D. anthriscifolium var. anthriscifolium (5) B D. anth-
riscifolium var. anthriscifolium (6) C D. anthriscifolium var. savatieri (13) D D. anthriscifolium var. savatieri (12) E D. anth-
riscifolium var. savatieri (10) F D. anthriscifolium var. majus (17) G D. ecalcaratum (20) H D. callichromum (22). Numbers
in brackets represented populations shown in Table 1.

metacentric chromosomes (i.e., I), three submetacentric (i.e., II, VII, and VIII),
and four subtelocentric chromosomes (i.e., III-VI; Tables 2, 3; Figs 5F, 6F).
Hence, the karyotype formula is 2n = 2x = 16 = 2m + 6sm + 8st.

4. Delphinium ecalcaratum

In Pop 20 from Xinyang City of Henan, China, the somatic and basic chromo-
some numbers are 2n = 16 and x = 8, respectively (Table 1; Fig. 4G). One pair of

PhytoKeys 234: 145-165 (2023), DOI: 10.3897/phytokeys.234.108841 155

Xiao-Yu Luo et al.: Karyotype and genome size variation in Delphinium subg. Anthriscifolium

CVor

metacentric chromosomes (i.e., |), three pairs of submetacentric chromosomes
(i.e., Il, VII, and VIII), and four subtelocentric chromosomes (i.e., II-VI) make up
the chromosome set of D. ecalcaratum (Tables 2, 3; Figs 5G, 6G). Therefore, the
karyotype formula is 2n = 2x = 16 = 2m + 6sm + 8st.

5. Delphinium callichromum

In Pop 22 collected from the type locality of Zhuxi County, Hubei, China, the
somatic and basic chromosome numbers are 2n = 16 and x = 8, respectively
(Table 1; Fig. 4H). Its chromosome set includes one pair of metacentric chro-
mosomes (i.e., |), three submetacentric (i.e., Il, Vil, and VIII), and four subtelo-
centric chromosomes (i.e., III-VI) (Tables 2, 3; Figs 5H, 6H). Accordingly, the
karyotype formula is 2n = 2x = 16 = 2m + 6sm + 8st.

Karyotype asymmetry analysis

In all five taxa of Delphinium subg. Anthriscifolium, the total haploid length
(THL) of D. ecalcaratum was probably the shortest (26.35), while that of D. an-
thriscifolium var. majus was the longest (up to 38.24). The highest level of in-
terchromosomal asymmetry, estimated via CV, was found in D. callichromum
(69.63). In contrast, the lowest level of CV,, was found in D. anthriscifolium
var. anthriscifolium (its mean value was 55.53). The highest values of both the
heterogeneity in centromere position (CV,,) and intrachromosomal asymmetry
(M.,,) were found in D. anthriscifolium var. majus (47.59 and 35.79, respectively;
Table 3). As seen in the scatter diagram (Fig. 7) drawn based on the parameter
CV, vs M.,, compared to D. anthriscifolium var. anthriscifolium and D. callichro-
mum, D. anthriscifolium var. savatieri, D. anthriscifolium var. majus, and D. ecal-
caratum gathered together, indicating that they might be more closely related.

D. ecalcaratum

D. callichromum® — @@ PD. ant. var. savatieri

D. ant. var. majus

D. ant. var. anthriscifolium

0

10 20 30 Mca 40 50 60

Figure 7. Scatter diagram of Delphinium subg. Anthriscifolium based on karyotype parameters CV,, vs. M,,. CV,, = Coef-
ficient of Variation of Chromosome Length; M,, = Mean Centromeric Asymmetry.

PhytoKeys 234: 145-165 (2023), DOI: 10.3897/phytokeys.234.108841 156

Xiao-Yu Luo et al.: Karyotype and genome size variation in Delphinium subg. Anthriscifolium

Discussion

In Ranunculaceae, taxonomic position and evolutionary history were generally
inferred by using chromosomal data (Tamura 1993; Yang 2001; Cires et al. 2010;
Soza et al. 2013; Orooji et al. 2022). So far, nearly 60 species of Delphinium have
been karyologically studied (Tjebbes 1927; Kolar et al. 2012; Gupta et al. 2018;
Bosch et al. 2019; see www.iaptglobal.org/chromosome-data). The basic num-
ber of haploid chromosomes in Delphinium was typically 8 (Legro 1961; Orellana
et al. 2007; Yuan and Yang 2008), with 9 (Blanché and Molero 1983; Bosch 1999;
Bosch et al. 2002) and 10 (Sarkar et al. 1982) occasionally occurring in some cir-
cumstances. The chromosome number of most Delphinium plants was 2n = 16,
while a few were 2n = 32, such as D. denudatum (Al-Kelidar and Richards 1981),
D. chrysotrichum (Yuan 2006), and D. spirocentrum (Yuan and Yang 2008). Here,
chromosome numbers of five taxa in D. subg. Anthriscifolium (i.e., D. anthriscifo-
lium var. anthriscifolium, D. anthriscifolium var. majus, D. anthriscifolium var. sa-
vatieri, D. ecalcaratum, and D. callichromum) are reported. All studied taxa have a
basic chromosome number of x = 8, D. anthriscifolium var. savatieri, D. anthrisci-
folium var. majus, D. ecalcaratum, and D. callichromum are diploid with 2n = 16,
while D. anthriscifolium var. anthriscifolium is tetraploid with 2n = 32.

Furthermore, the karyotypes of Delphinium taxa were very consistent, typical-
ly consisting of one pair of large metacentric, one pair of large submetacentric,
five pairs of medium-sized subtelocentric, and one pair of smaller submetacen-
tric (rarely subtelocentric) chromosomes (Lewis et al. 1951; Yang 2001; Yuan
and Yang 2008; Kolar et al. 2012). In the study, we found that the karyotype of
the diploid cytotype in D. subg. Anthriscifolium shared the traits listed below: (1)
the first pair (metacentric chromosomes) and the second pair (submetacentric
chromosomes) of chromosomes are significantly larger than the remaining six
pairs; (2) the proportion of subtelocentric chromosomes is relatively high; and
(3) intrachromosomal asymmetry and interchromosomal asymmetry are both
high. Two pairs of large metacentric, eight pairs of submetacentric, and six
pairs of subtelocentric chromosomes make up the tetraploid cytotype in D. an-
thriscifolium var. anthriscifolium. The karyotype formula of D. anthriscifolium
var. anthriscifolium is 2n = 4m + 16sm + 12st, consistent with the results of
Yuan and Yang (2008). The karyotype formulas of D. anthriscifolium var. sa-
vatieri, D. anthriscifolium var. majus, D. ecalcaratum, and D. callichromum are
2n = 2m + 6sm + 8st, consistent with the karyotype formulas of D. caeruleum,
D. maximowiczii, D. kamaoense var. glabrescens, D. nangchienense, and D. can-
delabrum var. monanthum (Yang 1996; Liu and Ho 1999).

On the genome size of Ranunculaceae, few related studies involving ten
genera (i.e., Ranunculus, Eranthis, Helleborus, Hepatica, Thalictrum, Delphinium,
Anemone, Ficaria, Adonis, and Trollius), showed that the 2C-value of diploid
taxa significantly ranged from 0.5 to 57.3 pg and from 14.8 to 89.2 pg for tet-
raploid taxa (Zonneveld 2001; Mabuchi et al. 2005; Cires et al. 2009; Cires et
al. 2010; Zonneveld 2010; Soza et al. 2013; Zonneveld 2015; Mitrenina et al.
2020; Mitrenina et al. 2021; Salvado et al. 2022; Seidl et al. 2022). According
to Salvado et al’s (2022) report on the genome size of Delphinium, the tetra-
ploid D. montanum had a 1C value of 10.32 pg. Here, the 2C-value of D. subg.
Anthriscifolium was 3.02—3.92 pg for diploids and 6.04—6.60 pg for tetraploids,
respectively. Chromosome counts were completed for selected taxa to confirm

PhytoKeys 234: 145-165 (2023), DOI: 10.3897/phytokeys.234.108841 157

Xiao-Yu Luo et al.: Karyotype and genome size variation in Delphinium subg. Anthriscifolium

ploidy and further calibrate the flow cytometry results. However, the above data
lacks comparability due to the difference in experimental conditions and refer-
ence genome species.

Interestingly, in the study, the monoploid genome sizes of tetraploids (mean
1Cx = 1.57 pg) are less than those of diploids (mean 1Cx = 1.69 pg; see Fig. 3B),
maybe showing a general tendency toward genome downsizing in the evolu-
tion of Delphinium subg. Anthriscifolium. Following polyploidisation, chromo-
some counts and genome size may change independently or dependently due
to sequence loss and gain, chromosomal elimination, or chromosome fusions
and fissions (Heslop-Harrison et al. 2023). Typically, the loss of repetitive DNA,
such as retroelements or retrotransposons, caused the decline in monoploid
genomes (Leitch and Bennett 2004; Bennetzen et al. 2005; Simonin and Roddy
2018). In addition, genome size data can be used to estimate ploidy in closely
related taxa when properly calibrated with known cytological standards (Shear-
er and Ranney 2013; Lattier et al. 2014; Hembree et al. 2019). Delphinium anth-
riscifolium var. anthriscifolium is tetraploid with a genome size of about 6.28 pg.
In comparison, the remaining diploid taxa have a genome size of approximately
3.38 pg, meaning that polyploidisation occurred in the D. subg. Anthriscifolium.

Taxonomic treatment

1. Delphinium anthriscifolium Hance. In J. Bot. 5: 207. 1868.

= D. calleryi Franch. in Bull. Mens. De la Soc. Linn. De Paris, 1: 329. 1882. =D. an-
thriscifolium var. calleryi (Franch.) Fin. & Gagnep. in Bull. Soc. Bot. Fr. 51: 471.
1904. syn. nov. Type: China: Aomen (Macao), 1841, Callery 6 (Holotype P!);
Aomen, 1844, Callery 57 (Isotypes P!).

= D. cavaleriense Lévi. et Vant. in Bull. Acad. Géog. Bot. 11: 49. 1902., syn.
nov. Type: China: Guizhou (Kweichow), “environs de Tou-chan, belles fleurs
bleues”, 2 June 1898, J. Cavalerie 2344 (Holotype E!; lsotypes K!).

= D. cerefolium Lévl. et Vant. in Bull. Acad. Géog. Bot. 11: 49. 1902., syn. nov.
Type: China: Guizhou (Kouy-Tcheou), Guiyang (Kouy-Yang), “mont du Col-
lege”, 2 June 1898, Chaffanjon s.n. (Holotype E!).

Type material. Lecotype: CHINA: Guangdong (Kwantung), “necnon prope rupem
calcaream kai-kun-shek, secus eundem fluvium’, June 1867, Sampson, Hance
no. 10125 (Holotype K!; Isotypes BM! NY! P! JE! GH).

2. Delphinium savatieri Franch. In Bull. Mens. De la Soc. Linn. De Paris 1:
330. 1882.

= D. anthriscifolium var. savatieri (Franchet) Munz., J. Armold Arbor. 48: 261.
1967. Type: China: Zhejiang (Tche-kiang/Chekiang), “in siccis ad pedem
montium Shao-Shin, prope Ning-po”, May 1863, Lud. Savatier (Holotype P!;
Isotype P!).

= D. robertianum Lévl. et Vant. in Bull. Acad. Géog. Bot. 11: 49. 1902., syn. nov.
Type: China: Guizhou (Kouy-tcheou), Guiyang (Kouy-yang), 9 Dec 1897, no.
2025 (Holotype E!).

PhytoKeys 234: 145-165 (2023), DOI: 10.3897/phytokeys.234.108841 158

Xiao-Yu Luo et al.: Karyotype and genome size variation in Delphinium subg. Anthriscifolium

= D. minutum Lévl. et Vant. in Bull. Herb. Boiss. sér. 2, 6: 505. 1906., syn. nov.
Type: China: Guizhou, 2 Mar 1904, Jos. Esquirol no. 23 (Holotype E!).

= D. kweichowense W.T.Wang in Acta Bot. Sin., 10: 283. 1962., syn. nov. Type:
China: Guizhou, Huishui, 18 July 1930, Y. Jiang 8577 (PE!).

Note. Morphologically, D. savatieri differs from D. anthriscifolium in that the
staminode limb is ovate (vs. dolabriform), 2-lobed (vs. 2-parted), and its base
is broadly cuneate (vs. subtruncate). Cytologically, D. savatieri also differs from
D. anthriscifolium in that its karyotype formula is 2n = 2x = 16 = 2m + 6sm + 8st
(vs. 2n = 4x = 32 = 4m + 16sm + 12st).

3. Delphinium zanlanscianense W.G.Zhang & X.Y.Luo, nom. nov.
urn:lsid:ipni.org:names:77328947-1

= Delphinium anthriscifolium var. majus Pamp. in Nuovo Giorn. Bot. Ital., n.s.,
20: 288. 1915.

= D. anthriscifolium f. latilobulatum W.T.Wang in Acta Bot. Sin., 10: 279. 1962.,
syn. nov. Type: China: Hunan, Xue-Feng-Shan, 1954, Z. T. Li 2377 (Holotype
PE!; Isotype PE!).

Type material. Lecotype: CHINA: Hubei (Hu-peh), Zhanglang County (Zan-lan-
scian), 1913, P C. Silvestri no. 3917 (Holotype FI!).

Note. Morphologically, D. anthriscifolium var. majus differs from D. anthrisci-
folium var. anthriscifolium in that the flowers are 2.3-3.4 cm long (vs. 1.0-
1.8 cm), spur 1.7—2.2 cm (vs. 0.5-2.2 cm) and its base 3.0-4.0 mm (vs. 1.5-
4.0 mm) in diam., other sepals 1.2-1.6 cm (vs. 0.6-1.6 cm), staminode limb
broadly ovate (vs. dolabriform or ovate). Cytologically, D. anthriscifolium var.
majus differs from D. anthriscifolium var. anthriscifolium in that its karyotype
formula is 2n = 2x = 16 = 2m + 6sm + 8st (vs. 2n = 4x = 32 = 4m + 16sm + 12st).

When elevating D. anthriscifolium var. majus to the rank of species, the name
is already occupied by D. majus (W.T.Wang) W.T.Wang (Wang and Hsiao 1965),
making it necessary to propose a replacement name. Thus, we propose the
name ‘zanlanscianense’ based on the locality of its lectotype.

Conclusions

In the present study, comparative karyomorphological analyses and genome
size determinations of five taxa of Delphinium subg. Anthriscifolium have
been carried out. The chromosome numbers of D. savatieri, D. zanlanscian-
ense, D. callichromum, and D. ecalcaratum were determined for the first time.
Karyotypes of D. subg. Anthriscifolium were shown to have both common
and species-specific features related to chromosome number, size, and mor-
phology. All studied taxa have the basic chromosome numbers x = 8, dip-
loid, or polyploid cytotypes, and the monoploid genome size (C-value) deter-
mined by flow cytometry varies more than twice. Additionally, the monoploid
genome sizes of tetraploids (mean 1Cx = 1.57 pg) are smaller than those
of diploids (mean 1Cx = 1.69 pg). Thus, genome loss or duplication events
have occurred in the evolution of D. subg. Anthriscifolium. Finally, based on

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Xiao-Yu Luo et al.: Karyotype and genome size variation in Delphinium subg. Anthriscifolium

cytological and morphological evidence, D. anthriscifolium var. savatieri was
restored to species rank, and D. anthriscifolium var. majus was elevated and
renamed as D. zanlanscianense.

Acknowledgements

We are grateful to Yu-Cai Luo (South China Botanical Garden, Chinese Academy
of Sciences), Shao-Dong Wu (Lushan Botanical Garden, Chinese Academy of
Sciences), and Qiang Zhang (Guangxi Institute of Botany, Chinese Academy of
Sciences) for the work in field surveys and sampling. We thank the editor (Mar-
co Pellegrini), Dr. José Ignacio Marquez-Corro and two anonymous reviewers
for constructive comments and suggestions.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Funding

This research was funded by the National Natural Science Foundation of China (grants
31500189). The authors declare no conflict of interest.

Author contributions

Conceptualization, Data curation, Writing — original draft: XYL. Methodology, Visualiza-
tion: TIN. Data curation, Visualization: HL, YH, XFD. Conceptualization, Resources, Su-
pervision, Writing — review and editing: WGZ, CCG.

Author ORCIDs

Xiao-Yu Luo © https://orcid.org/0009-0005-81 53-7348
Tang-Jie Nie © https://orcid.org/0000-0003-2405-8904
Heng Liu © https://orcid.org/0009-0008-4900-4025
Xue-Fei Ding © https://orcid.org/0009-0008-2034-5459
Ying Huang ® https://orcid.org/0009-0004-7731-6916
Chun-Ce Guo ® hitps://orcid.org/0000-0003-3376-1116
Wen-Gen Zhang ® https://orcid.org/0000-0003-0946-8614

Data availability

All of the data that support the findings of this study are available in the main text.

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