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Cycas chenii (Cycadaceae), a new species from China, and its phylogenetic position


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Cycas chenii X. Gong & W. Zhou sp. nov., a new species of Cycas L., is described and illustrated here. The morphological and karyomorpholoical comparisons are made between C. chenii and the closely related taxa for defining its taxonomical status as a new species. Moreover, the phylogenetic position of C. chenii within 16 Cycas species is determined using DNA sequences of two plastid regions, nuclear ribosomal ITS and two nuclear regions. Cycas chenii is readily distinguished from the related C. guizhouensis by an acaulescent stem. Phylogenetic evidence indicates that C. chenii is a distinct group related to C. guizhouensis in the Section Stangerioides. The distribution and conservation status of C. chenii are also discussed.
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Research Article
doi: 10.1111/jse.12153
Cycas chenii (Cycadaceae), a new species from China, and
its phylogenetic position
Wei Zhou
, Meng-Meng Guan
, and Xun Gong
Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201,
University of Chinese Academy of Sciences, Beijing 100049, China
*Author for correspondence. E-mail: Tel./Fax: 86-871-65223625.
Received 11 June 2014; Accepted 12 February 2015; Article first published online xx Month 2015
Abstract Cycas chenii X. Gong & W. Zhou sp. nov., a new species of Cycas L., is described and illustrated here. The
morphological and karyomorphological comparisons are made between C. chenii and the closely related taxa for
dening its taxonomical status as a new species. Moreover, the phylogenetic position of C. chenii within 16 Cycas
species is determined using DNA sequences of two plastid regions, nuclear ribosomal internal transcribed spacers,
and two nuclear regions. Cycas chenii is readily distinguished from the related C. guizhouensis K. M. Lan & R. F. Zou by
an acaulescent stem. Phylogenetic evidence indicates that C. chenii is a distinct group related to C. guizhouensis in
the Section Stangerioides. The distribution and conservation status of C. chenii are also discussed.
Key words: China, Cycas chenii, new species, phylogenetic position.
The genus Cycas consists of approximately 100 species, chiey
Indo-Chinese (about 40 species) and Australian (27 species).
The genus also occurs in the Malaysian region, Japan and
India, extending to Micronesia and Polynesia, Madagascar,
and East Africa (Lindstrom & Hill, 2007). In China, there are 22
Cycas species distinguished, based on wide investigations
(Hill, 2008). The Red River drainage area (China and Vietnam)
is recognized as a secondary diversication center of Cycas,
where more than 20 species occur. The majority of these
species are endemic to this area. During eld investigations in
the Red River drainage areas in southeastern Yunnan, China, a
species belonging to the genus Cycas was observed that did
not conform to morphological features of any known species
in Cycas. The small plants were easily distinguished from
known species by the absence of an obvious stem and the
presence of only a few leaves on the crown. However, these
plants showed similarities to C. guizhouensis K. M. Lan & R. F.
Zou, C. simplicipinna (T. Smitinand) K. D. Hill, and C. tanqingii
D. Y. Wang in certain aspects of its morphology. In this paper,
we present a comprehensive study based on morphology,
karyomorphology, and molecular phylogenetics for determin-
ing the taxonomic status and the phylogenetic relationships
of these plants.
Material and Methods
Plant materials
Specimens of Cycas chenii were collected during our eld
investigations in 2012, and some living individuals were
introduced and cultivated in Kunming Botanical Garden,
Kunming Institute of Botany (Kunming, China). DNA material
of eight individuals were sampled from four populations
(Dutian and Qingshuihe in Shuangbai county, Menglong and
Lianhua in Honghe county, Figs. 13), comprising two
individuals from each population. For the study of the
phylogenetic position of C.chenii,16Cycas species were
sampled (one to four individuals were sampled for each
species), being representatives of most sections of Cycas
based on morphological characters. Vouchers from all taxa
sequenced in this study are listed in Table 1 and the specimen
of C. chenii was deposited in the herbarium of Kunming
Institute of Botany, CAS.
Karyomorphological studies
For the observation of somatic chromosomes, we obtained
growing root tips from a living seedling of C.chenii. The root
tips were pretreated in 0.1% colchicine solution at 812 °C for
4 h then xed in acetic alcohol (3:1, absolute ethanol : glacial
acetic acid) at 1525°C for 1224 h. They were macerated in 1:1
mixture of 1 mol/L hydrochloric acid and 45% acetic acid at 60°C
for 8 min and then stained and squashed in 1% aceto-orcein
solution. Karyotype formulas were derived on measurements
of metaphase chromosomes from photomicrographs. The
nomenclature used to describe the karyotype followed Levan
et al. (1964).
DNA extraction, polymerase chain reaction amplification,
and sequencing
Total genomic DNA was extracted from silica gel dried leaves
following a modied CTAB protocol (Doyle, 1991). After
preliminary screening of some chloroplast fragments and
SE Journal of Systematics
and Evolution
XXX 2015 | Volume 9999 | Issue 9999 | 110 © 2015 Institute of Botany, Chinese Academy of Sciences
nuclear genes, we chose to generate DNA sequences of the
ve regions to reconstruct the phylogenetic relationship of C.
chenii within the genus Cycas. These regions included two
chloroplast internal transcribed spacer regions psbAtrnH
(Shaw et al., 2005) and trnLtrnF (Taberlet et al., 1991), the
nuclear internal transcribed spacer region (ITS4ITS5, White
et al., 1990), and two nuclear genes, the phytochrome spacer
region PHYP, and the RNA polymerase II largest subunit spacer
region RPB1. Details of polymerase chain reaction (PCR)
primers are given in Table 2.
The PCR was carried out using a PTC-200 thermal cycler (MJ
Research, Bruno, Canada) with a reaction volume of 40 mL,
containing 4.0 mL template DNA (2050 ng/mL), 4.0 mL10
PCR buffer, 2.4 mL MgCl
(25 mmol/L), 2.0 mL dNTP (10 mmol/
L), 2.0 mL DMSO, 0.7 mL each primer (10 mmol/L), 0.7 mLTaq
(5 U/mL; TaKaRa, Kyoto, Japan), and 24.6 mL double-distilled
water. The reaction of the nuclear genome was carried out
with an initial denaturation at 94 °C for 4 min, followed by 30
cycles of denaturation at 94 °C for 45 s, annealing at 53 °C for
1 min, extension 1 min at 72 °C, and a nal extension at 72 °C for
10 min. The reaction of the chloroplast genome was carried
out under an initial denaturation at 80 °C for 5 min, followed by
30 cycles of denaturation at 80 °C for 45 s, annealing at 48 °C
for 1 min, extension at 65 °C for 1 min, and a nal extension at
65 °C for 10 min. The puried PCR products were sequenced
with the same primers used for PCR amplications in an ABI
3770 automated sequencer at Shanghai Sangon Biological
Engineering Technology and Services (Shanghai, China). DNA
sequences were edited by SeqMan (DNAStar, Madison, WI,
USA) and aligned with ClustalX1.81 (Thompson et al., 1997). All
DNA sequences produced in this study were deposited in
GenBank (accession numbers are listed in Table 1).
Phylogenetic analyses
Maximum parsimony and Bayesian inference of phylogeny
were used for determining the phylogenetic position of the
newly collected plants. Maximum parsimony analyses (MP)
were carried out using Paup* v.4.0b10 (Swofford, 2002). All
characters were weighted equally and unordered, gaps were
treated as missing data, and the branch-swapping algorithm
was set as tree bisectionreconnection. Robustness of the
obtained phylogeny was calculated by bootstrap analysis with
1000 replicates (Felsenstein, 1985). Consistency index (Kluge
& Farris, 1969) and retention index (Farris, 1989) were
calculated to estimate the level of homoplasy. Strict
consensus trees were calculated if more than one most
parsimonious tree was recovered. The incongruence length
difference test as implemented in Paup was calculated to
assess data congruency (Farris et al., 1994) by Paup* before
Fig. 1. Cycas chenii X. Gong & W. Zhou sp. nov. A, Whole plant and habitat. B, Female cone. C, Male cone. D, Megasporophylls and
seed. E, Seedlings and female plant.
2 Zhou et al.
J. Syst. Evol. 9999 (9999): 110, 2015
Fig. 2. Holotype of Cycas chenii X. Gong & W. Zhou sp. nov. with details. A, Habit. B, Pinna. C, Microsporophyll. D,
Megasporophylls with seeds. E, Female cone. Drawn by G.-S. Yin.
Fig. 3. Distribution of Cycas chenii X. Gong & W. Zhou sp. nov in China. The circle indicates the type locality of C. chenii. DT, Dutian,
Shuangbai; LH, Lianhua, Honghe; ML, Menglong, Honghe; QS, Qingshuihe, Shuangbai. Original map downloaded from http://
A new Cycas species from China 3 J. Syst. Evol. 9999 (9999): 110, 2015
Table 1 List of taxa sampled and sequenced in this study, with distribution, located sections based on morphology, and voucher and GenBank accession numbers
Taxon Voucher Geographic
PHYP RPB1 trnLtrnF psbAtrnH ITS4ITS5 Located in
Cycas aculeata K. D. Hill &
H. T. Nguyen
T.-H. Nguyen & J. Liu,
CK752 (HN)
Vietnam KP117123 KP117177 KP117204 KP117150 KP117099 Stangerioides
Cycas diannanensis Z. T. Guan &
G. D. Tao
X. Gong, PG200044 China KP117124 KP117178 KP117205 KP117151 KP117100 Stangerioides
Cycas dolichophylla K. D. Hill,
T. H. Nguyen & K. L. Phan
T.-H. Nguyen & Y.-M. Shui,
CK182 (KUN, HN)
China and Vietnam KP117125 KP117179 KP117206 KP117152 KP117101 Stangerioides
Cycas guizhouensis K. M. Lan &
R. F. Zou
X. Gong, GX002 China KP117126 KP117180 KP117207 KP117153 KP117102 Stangerioides
Cycas media R. Br. S.-Z. Zhang, SZ461A Australia KP117127 KP117181 KP117208 KP117154 KP117103 Cycas
Cycas multipinnata C. J. Chen &
S. Y. Yang
X. Gong, HHJP008 China and Vietnam KP117128 KP117182 KP117209 KP117155 KP117104 Stangerioides
Cycas pectinata Buch-Ham. J. Liu et al., GXC01 India, Nepal, Bhutan, Burma,
China, and Indochina
KP117129 KP117183 KP117210 KP117156 KP117105 Indosinenses
Cycas panzhihuaensis L. Zhou &
S. Y. Yang
X. Gong, KBG20141 China KP117130 KP117184 KP117211 KP117157 KP117106 Panzhihuanses
Cycas parvulus S. L. Yang X. Gong, PG20010 China KP117131 KP117185 KP117212 KP117158 KP117107 Stangerioides
Cycas revoluta Thunb. X. Gong, KBG20142 China and Japan KP117132 KP117186 KP117213 KP117159 KP117108 Asiorientales
Cycas siamensis Miq. T.-S. Yi, Yi13531 Thailand and Vietnam KP117133 KP117187 KP117214 KP117160 KP117109 Indosinenses
Cycas silvestris K. D. Hill S.-Z. Zhang, SZ141A Australia KP117134 KP117188 KP117215 KP117161 KP117110 Cycas
Cycas simplicipinna (Smitinand)
K. D. Hill
T.-H. Nguyen et al., CK759 Thailand, Burma, Laos,
and Vietnam
KP117135 KP117189 KP117216 KP117162 KP117111 Stangerioides
Cycas szechuanensis W. C. Cheng
J. Liu, FL1401 China KP117136 KP117190 KP117217 KP117163 KP117112 Stangerioides
Cycas tanqingii D. Y. Wang X. Gong, HHJP001 China KP117137 KP117191 KP117218 KP117164 KP117113 Stangerioides
Cycas tropophylla K. D. Hill &
S. L. Yang
S.-Z. Zhang, SZ11134A Vietnam KP117138 KP117192 KP117219 KP117165 KP117114 Stangerioides
Cycas chenii DT5 W. Zhou & X. Gong,
China KP117139 KP117193 KP117220 KP117166 KP117115 Stangerioides
Cycas chenii DT9 W. Zhou & X. Gong,
China KP117140 KP117194 KP117221 KP117167 KP117116 Stangerioides
Cycas chenii LH9 W. Zhou & X. Gong,
China KP117141 KP117195 KP117222 KP117168 KP117117 Stangerioides
Cycas chenii LH18 W. Zhou & X. Gong,
China KP117142 KP117196 KP117223 KP117169 KP117118 Stangerioides
Cycas chenii ML3 W. Zhou & X. Gong,
China KP117143 KP117197 KP117224 KP117170 KP117119 Stangerioides
Cycas chenii ML7 W. Zhou & X. Gong, China KP117144 KP117198 KP117225 KP117171 KP117120 Stangerioides
4 Zhou et al.
J. Syst. Evol. 9999 (9999): 110, 2015
Table 1 Continued
Taxon Voucher Geographic
PHYP RPB1 trnLtrnF psbAtrnH ITS4ITS5 Located in
Cycas chenii QS2 W. Zhou & X. Gong,
China KP117145 KP117199 KP117226 KP117172 KP117121 Stangerioides
Cycas chenii QS11 W. Zhou & X. Gong,
China KP117146 KP117200 KP117227 KP117173 KP117122 Stangerioides
Cycas guizhouensis lw3 J. Liu et al., GZ011 China KP117147 KP117201 KP117228 KP117174 NA Stangerioides
Cycas guizhouensis lw5 J. Liu et al., GZ012 China KP117148 KP117202 KP117229 KP117175 NA Stangerioides
Cycas guizhouensis lw7 J. Liu et al., GZ010 China KP117149 KP117203 KP117230 KP117176 NA Stangerioides
Geographic distributions were referred from Hill et al. (2008). DT, Dutian, Shuangbai; HN, Herbarium of National Center for Natural Sciences and Technology, Vietnam; ITS, internal
transcribed spacer; KUN, Herbarium, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, China; LH, Lianhua, Honghe; ML, Menglong, Honghe; NA, missing data in
this study; QS, Qingshuihe, Shuangbai.
Table 2 DNA regions examined and primer information of two chloroplast internal spacers, internal transcribed spacer (ITS4ITS5), and two nuclear sequences used in this study
Region Primer sequences (50to 30) References
cpDNA, chloroplast DNA; F, forward; nDNA, nuclear DNA; R, reverse.
A new Cycas species from China 5 J. Syst. Evol. 9999 (9999): 110, 2015
Table 3 Morphological and karyotypic comparison of Cycas chenii with related Cycas species
Character C. chenii C. tanqingii C. simplicipinna C. guizhouensis
Habit Subtropical evergreen
forest, altitude
5001300 m
Scattered in
altitude <800 m
Scattered in tropical
rainforests at
lower altitude
Scattered or patched in shrubs and forests
along Nanpanjiang River Valley, altitude
400800 m, no more than 1100 m
Stem Acaulescent, 210
leaves in crown
Arborescent or
acaulescent, to 2 m
tall, 47 leaves in
Acaulescent, 25
leaves in crown
Arborescent or acaulescent, to 2 m tall
930 leaves in crown
Cataphylls Narrowly triangular Narrowly triangular Lanceolate Long deltoid
Megasporophylls 57 cm long, 25cm
wide, rhombic or
5.05.5 cm long,
5.06.5 cm wide
36 cm long, 35cm
subrhomboided or
410 cm long, 68 cm wide, ovate to
Soft spines
6969511 723
Obvious Unobvious Obvious Obvious
Ovules 242 2549
Flowering Apr.May Apr. Apr.May Apr.June
Seeds maturing Oct.Nov. Aug.Sep. Sep.Oct. Oct.Nov.
Karyotype 6m þ4sm þ12t 2m þ8sm þ2st þ10t 6m þ4sm þ12t 2m þ4sm þ4st þ12t
Authors Present authors Tian et al. (2002) Wang et al. (1996) Wu & Chen (1990)
6 Zhou et al.
J. Syst. Evol. 9999 (9999): 110, 2015
combining the datasets. The entire dataset was analyzed
using DnaSP 4.0 (Rozas et al., 2003).
Bayesian Markov chain Monte Carlo analyses (Yang &
Rannala, 1997) were carried out by MrBayes version 3.1
(Huelsenbeck & Ronquist, 2001). The best tting model of
sequences evolution was GTRþIþG for Akaike information
criterion and TPM2fuþI for Bayesian information criterion,
decided by JModelTest 2.1.5 (Posada, 2008; Dieg et al., 2012);
four simultaneous runs with four chains each were run for
combined data. Trees were sampled in every 1000 gener-
ations; the rst 2500 trees (25%) of the sample trees from each
run were discarded. The sampling data after Bayesian analysis
was examined and determined by Tracer version 1.6 (Rambaut
et al., 2014). Accomplishment of the Bayesian runs was
determined using the web-based program AWTY (Nylander
et al., 2008).
Morphological characters, chromosome counts, and
Morphological characters of Cycas chenii and its close relative
species, including C. guizhouensis,C. simplicipinna, and C.
tanqingii, are listed in Table 3. The new species is similar to C.
guizhouensis in morphology, but its stem is acaulescent, and
its megasporophylls are rhombic or ovate, 57 cm long by 2
5 cm wide, deeply pectinate with 1218 soft spines. Cycas
guizhouensis is arborescent, the megasporophylls are
subrotund to ovate-elliptic, 410 cm long by 68 cm wide,
deeply pectinate with 1446 soft spines. The new species is
also similar to C. simplicipinna, but differs in that leaves do not
turn black-brown when dry. The latter taxon is also distinct
from the new species by megasporophylls that are sub-
rhomboid or ovate, 36 cm long by 35 cm wide, simple
median pinnae is 1730 cm long by 0.91.3 cm wide, apical
spine distinct from lateral spines. The new species is also
closely related to C. tanqingii, but can easily be distinguished
by its shorter leaves and leaets.
The number of somatic chromosomes of C. chenii was
consistent with other Cycas species by showing a diploid
chromosome set of 2n¼22 and a karyotype formula of
2n¼2x¼6m þ4sm þ12t (Fig. 4; Table 3).
Phylogenetic analysis
Although the result of the incongruence length difference test
associated by all ve markers showed P¼0.01 in this study,
the 132 consensus trees obtained from the individual markers
did not display a robust signal of topological conicts
(bootstrap support values 80% and/or posterior probability
0.95). Sequence data from all markers were, therefore,
combined into a single dataset. All ve sequence markers
were aligned and generated a matrix of 3571 characters, of
which variable sites were 338 (total number of mutations
were 372), singleton variable sites were 216, and parsimony
informative sites were 122 when gaps were treated as missing
characters. Detailed information on each molecular marker is
listed in Table 4.
The Bayesian tree and MP bootstrap consensus tree
showed the same phylogenic results as the trees in Figs. 5A
and 5B, respectively, with only small differences at the
bootstrap and posterior condence value on each node
(Fig. 5). Both the MP and Bayesian consensus trees were
generally congruent with respect to well-supported clades,
Fig. 4. Mitotic metaphase of Cycas chenii.A, Micrograph of metaphase chromosomes. B, Karyotype of mitotic metaphase
Table 4 Tree statistics for chloroplast psbAtrnH,trnLtrnF, nuclear internal transcribed spacer (ITS4ITS5), PHYP,RPB1, and
combined datasets from maximum parsimony analysis using DnaSP 4.0
Chloroplast genes Nuclear genes Combined
Parameters psbAtrnH trnLtrnF ITS4ITS5 RPB1 PHYP cpDNA þnDNA
Number of sequences (ingroup/outgroup) 24 (23/1) 24 (23/1) 24 (23/1) 24 (23/1) 24 (23/1) 24 (23/1)
Aligned length (bp) 542 709 833 519 968 3571
Variable characters (%) 10 (1.8) 18 (2.4) 248 (29.8) 27 (5.2) 35 (3.62) 372 (10.4)
Parsimony informative characters (%) 7 (1.3) 6 (0.8) 83 (10) 11 (2.1) 15 (1.5) 122 (3.4)
Consistency index 0.92 1 0.7123 0.6111 0.75 0.6437
Retention index 0.08 0 0.2877 0.3889 0.25 0.3563
cpDNA, chloroplast DNA; nDNA, nuclear DNA.
A new Cycas species from China 7 J. Syst. Evol. 9999 (9999): 110, 2015
Fig. 5. A, Bayesian consensus tree from the analysis of the combined nuclear DNA and chloroplast DNA sequences. Numbers are
Bayesian posterior probabilities. B, Single most parsimonious tree (tree length ¼3571) from the analysis of the combined nuclear
DNA and chloroplast DNA sequences. Numbers indicate bootstrap values. DT, Dutian, Shuangbai; LH, Lianhua, Honghe; ML,
Menglong, Honghe; QS, Qingshuihe, Shuangbai.
8 Zhou et al.
J. Syst. Evol. 9999 (9999): 110, 2015
with all individuals of C. chenii located at the same clade.
This clade was nested in a polytomy sharing individuals of
C. guizhouensis. The two morphologically similar species
C. simplicipinna and C. tanqingii formed a separated clade.
The phylogram constructed by the MP method and Bayesian
inference both showed that populations of Cycas chenii nested
in the same clade. However, this clade was nested within a
polytomy also consisting of four individuals of C. guizhouensis.
The two other species considered as closely related,
C. simplicipinna and C. tanqingii, were found to be sufciently
separated in their genotype to allow unambiguous diagnoses
of species identity. These results are consistent with the
morphological characters described above. In contrast to the
available DNA data, C. chenii was distinct compared to all
three species mentioned. The lack of resolution concerning
C. guizhouensis may be caused by the lack of all ve regions for
all four specimens.
The karyotype formula of C. chenii is identical to that of
C. simplicipinna and C. taitungensis C. F. Shen, K. D. Hill, C. H.
Tsou & C. J. Chen, namely 2n¼2x¼6m þ4sm þ12t (Wang
et al., 1996). Furthermore, neither the karyotype formula of
C. guizhouensis nor C. tanqingii was the same as that of
C. chenii, with the former being 2n¼2x¼2m þ4sm þ4st þ
12t (Wu & Chen, 1990) and the latter 2n¼2x¼2m þ8sm þ2
st þ10t (Tian et al., 2002). Thus, C. chenii differed from
C. guizhouensis in all molecular phylogenetic, morphological,
and cytological attributes. Although all the reported species
within Cycas shared the same chromosome number of 2n¼22
and the same karyotypic component of m, sm, st, and T, as
well as most of them belonging to Stebbins3B type
(according to Stebbins, 1971), variations in karyotype could
be found throughout its distribution ranges or among
different sections of this genus (Hua & Chen, 1990; Tian
et al., 2002). Morphologically, C. chenii could be allocated to
Sect. Stangerioides with C. guizhouensis and C. tanqingii.
However, these three species share no known overlapping
geographic distribution area and occupy relatively discrepant
habitats, which may have derived different chromosomic
ssion and fusion patterns in karyotypical evolution and
formed their respective karyotypes (Moretti, 1990; Zheng
et al., 2002). However, as the karyotypical data of Cycas
seemed to be disordered and lacking detail, much work
should be carried out in the subsequent karyotypic research of
Cycas chenii has morphological similarities to C. simplicipinna
and C. tanqingii, but they were located in different subclades
on the phylogenetic tree. In terms of morphology, the
three Cycas species could easily be discriminated by their
megasporophylls, leaves, and seeds (see Table 3). The
karyotype of C. chenii was identical to C. simplicipinna and
C. taitungensis (Wang et al., 1996), which suggested that
different species might have the same karyotype. All evidence
from morphology, cytology, and molecular phylogeny validly
supported that C. chenii is an independent species from other
closely related species, although they appeared to be sister
lineages and shared distribution areas in southern Yunnan
with a similar habitat and subtropical climate.
Both phylogeny and morphology supported that C. chenii
was located in section Stangerioides. Interestingly, C. aculeata
K. D. Hill & H. T. Nguyen and C. siamensis Miq. appeared to be
sister lineages, with both located in Section Indosinenses.
These results indicate some conict between morphological
characters and genotypic data because C. aculeata is most
similar to the non-sampled C. balansae Warb. This species is
currently assigned to section Stangerioides. A possible reason
for this phenomenon is long-branch attraction (LBA, Bergsten,
2005) or the result of convergent evolution for these most
recent divergent plants (Nagalingum et al., 2011).
Taxonomic treatment
Cycas chenii X. Gong & W. Zhou, sp. nov. Type: China. Yunnan:
Shuangbai county, Dutian, 24°31015.500N, 101°31055.800 E, 1100 m
alt., 2012, W. Zhou 201235 (holotype, KUN!).
(Figs. 1, 2).
Diagnosis: Species nova Cycas simplicipinna (Smitinand)
K. D. Hill et C. guizhouensis K. M. Lan & R. F. Zou afnis ab illo
foliis in sicco non nigro-brunneis, pinna apicali pinnis
lateralibus dissimili differ, ab hoc caule carente differ.
Description: Stem acaulescent, or subterranean; 28 leaves
in crown, leaves bright to deep green, highly glossy, 70
190 cm long, at (not keeled) in section (opposing pinnae
inserted at 160180° on rachis), with 2674 pinnae; with rusty
tomentum shedding as leaf expands, by rachis consistently
terminated by paired pinnae. Petiole 2080 cm long (2540%
of total leaf), glabrous spinescent for 90100% of length. Basal
pinnae are not gradually reducing to spines, the spines 0.2
0.3 cm long. Median pinnae simple, 1730 cm long, 0.91.3 cm
wide, inserted at 7080° to rachis, not decurrent, margins at
or undulate; apex acuminate, not spinescent; midrib raised
above, raised below. Cataphylls narrowly triangular, pilose,
36 cm long. Male cones yellowish-green, 1015 cm long,
68 cm wide, with rusty puberulous. Microsporophyll lamina
stiff, apical spine slender, closely appressed, 0.10.3 cm long.
Megasporophylls 1012 cm long, brown tomentose; ovules
24, glabrous; lamina rhombic or ovate, 57 cm long,
2.53.5 cm wide, deeply pectinate, with 1218 soft spines,
apical spine distinct from lateral spines. Seeds ovoid, 23cm
long, 1.52.6 cm wide; sarcotesta yellow.
Distribution and habitat: Cycas chenii is only known from
four populations that were found in Shuangbai and Honghe
counties of Yunnan Province, China. Based on the known
occurrences, we conclude that this species mainly occurs in
the upstream region of the Yuangjiang River in the central part
of Yunnan province. As a consequence of land clearing for
agriculture, these populations have to be considered
This species was found to grow on a range of substrates
from limestone to shale and schist, usually on steep slopes,
mainly in forests with the altitude ranging from 500 m to
1300 m. The vegetation type in the region is subtropical
evergreen broad-leaved forest. The dominant tree species of
the forest are Pistacia weinmannifolia J. Poisson ex Franch,
Phyllanthus emblica Linn, Dalbergia hupeana Hance, and Ficus
tinctoria subsp. gibbosa (Blume) Corner.
Conservation status: So far, there is no protected area
covering or adjacent to the known populations of C. chenii.
The total population size is estimated at less than 500. This
species will be assigned an IUCN Red List status of EN
A new Cycas species from China 9 J. Syst. Evol. 9999 (9999): 110, 2015
(endangered). Considering its living status, an urgent need of
in situ conservation should be carried out to protect the
existing populations of C. chenii. As a consequence of land
clearing for agriculture, the known populations must be
considered threatened.
Etymology: We named this species C. chenii after Professor
Jiarui Chen (Chia-Jui Chen), a botanist from the Institute of
Botany, Chinese Academy of Sciences, to honor his signicant
work on the genus Cycas in China.
The authors would like to thank Yu-Fa Zhou for help with
sample collection and Cheng-Cheng Tao for producing the
distribution map. We also own special thanks to Prof. H. Peng
for writing Latin descriptions of the new species. This research
was supported by the United Fund of the National Natural
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... For conifers, we referenced A Handbook of the World's Conifers (Farjon, 2017); for cycads, The genus Cycas (Cycadaceae) in China (Hill, 2008); and for gnetophytes and ginkgos, Species Catalogue of China, Vol.1: Plants (Jin and Yang, 2015). New records, such as Xanthocyparis vietnamensis (Meng et al., 2013) and Calocedrus rupestris (Nong et al., 2011), and new species, such as Cycas chenii (Zhou et al., 2015), were also included. Only taxa at the species level were considered for this purpose, excluding hybrids, cultivated species and uncertain species. ...
... The three unprotected species include two Cycas and one conifer species. For these species, no detailed distribution records were found for Cunninghamia konishii (Farjon, 2017), Cycas chenii was a newly described species from Honghe and Shuangbai counties in Yunnan province (Zhou et al., 2015), which is not protected in any nature reserve (Zheng et al., 2017), and Cycas shanyaensis lacked distribution reports except for the records in the original literature (Fu, 2006;Liu, 1998). Therefore, to fill the conservation gap of threatened gymnosperms in China, we should place more attention on these unprotected species, such as Cycas chenii. ...
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China is one of the diversity centers of gymnosperms. Nearly one-fifth (195 species) of gymnosperms are located in China, but 69 species are threatened. To date, the conservation status of gymnosperms, especially threatened gymnosperms in China, remains largely unknown, which seriously restricts the comprehensive protection of gymnosperms. Understanding the distribution pattern of species richness and exploring the relationships between species richness and environmental factors are key steps for their protection. In this study, we first constructed a database for the 69 threatened species of gymnosperms with 13 270 distribution records. It is found that 31 of the grid cells (50 km × 50 km) cover all threatened gymnosperm species in China, and the grid cells of threatened gymnosperms are mainly distributed in the southern area of the Yellow River, with a distribution center in the Western Sichuan Plateau. Then, we evaluated the conservation status of threatened gymnosperms, and the results indicate that 9 (13%) threatened gymnosperms are distributed outside of nature reserves. Therefore, there are still conservation gaps in the protection of threatened gymnosperms in China. We should give more attention to unprotected threatened gymnosperms and conduct taxonomic studies on the species without detailed distribution records. Finally, conservation priority areas and priority conservation levels of threatened gymnosperms in China were proposed. The Western Sichuan Plateau is the most important conservation priority area of threatened gymnosperms. This study will shed light on plant protection and forest management in China.
... Furthermore, the dioecious habit and rarity of reproductive organs in the wild has also caused ambiguity when circumscribing species (e.g. (Averyanov et al., 2014;Zhou et al., 2015)). The number of species of Cycas has increased from less than 20 in the early 20th century (Pilger, Hill and sect. ...
... A previous phylogenetic study had proposed that it was close to sect. Indosinenses, although the authors explained it by long-branch attraction (Zhou et al., 2015). Given that our results are equivocal, the classification of this species remains unclear. ...
The gymnosperm genus Cycas is the sole member of Cycadaceae, and is the largest genus of extant cycads. There are about 115 accepted Cycas species mainly distributed in the paleotropics. Based on morphology, the genus has been divided into six sections and eight subsections, but this taxonomy has not yet been tested in a molecular phylogenetic framework. Although the monophyly of Cycas is broadly accepted, the intrageneric relationships inferred from previous molecular phylogenetic analyses are unclear due to insufficient sampling or uninformative DNA sequence data. In this study, we reconstructed a phylogeny of Cycas using four chloroplast intergenic spacers and seven low-copy nuclear genes and sampling 90% of extant Cycas species. The maximum likelihood and Bayesian inference phylogenies suggest: (1) matrices of either concatenated cpDNA markers or of concatenated nDNA lack sufficient informative sites to resolve the phylogeny alone, however, the phylogeny from the combined cpDNA-nDNA dataset suggests the genus can be roughly divided into 13 clades and six sections that are in agreement with the current classification of the genus; (2) although with partial support, a clade combining sections Panzhihuaenses + Asiorientales is resolved as the earliest diverging branch; (3) section Stangerioides is not monophyletic because the species resolve as a grade; (4) section Indosinenses is not monophyletic as it includes Cycas macrocarpa and C. pranburiensis from section Cycas; (5) section Cycas is the most derived group and its subgroups correspond with geography.
... Y. Deng are mainly distributed along the Nanpan River; C. panzhihuaensis L. Zhou & S. Y. Yang is mainly distributed in the Jinsha River basin. Among these river drainages, the Red River basin across China and Vietnam is recognized as one of the diversity centers for Cycas, harboring nearly 20 species including Cycas dolichophylla, C. hongheensis S. Y. Yang & S. L. Yang ex D. Y. Wang, and C. multipinnata (Hill 2008;Zhou et al. 2015;Liu 2016;Xi et al. 2022). Since the Tertiary period, the Red River Fault Zone has undergone multiple significant sinistral and dextral strike-slip movements (Zhang 2009), potentially influencing the spatiotemporal diversification and population differentiation of Cycas in the Red River basin (Liu et al. 2015;Zheng et al. 2016). ...
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The intricate river systems forming various drainages play a crucial role as natural barriers, impeding the gene flow of extensively distributed taxa. However, there have been limited studies focused on examining the impact of river barriers on the phylogeography of plant lineages with restricted distributions. Cycas chenii X. Gong & W. Zhou, an endemic plant species found alongside rivers in the Yunnan region of China, possesses considerable research significance. By utilizing double digest restriction site associated DNA sequencing, we investigated the genetic diversity, genetic structure, and population historical dynamics for C. chenii to explore the influence of rivers on this species. We found C. chenii displayed greater genetic diversity in comparison to other Cycas species, with the HB population, a sole population situated in southwest Red River Region, exhibiting the highest genetic diversity at the population level. The AMOVA results revealed that the predominant genetic variations existed within populations (84.46%). Mantel test revealed a significant positive correlation between genetic differentiation and geographic distance in C. chenii. Both genetic structure and phylogenetic analyses strongly corroborate the categorization of the ten populations of C. chenii into three distinct clusters, aligning with their respective rivers and geographic distributions. Furthermore, the populations of C. chenii experienced three bottlenecks within the last five million years, which could be correlated with geological events. Based on these findings, specific conservation strategies were proposed for the three evolutionary significant units (ESUs) of C. chenii, each of which encounters distinct conservation challenges.
... However, molecular phylogenetic analyses indicated that the extant cycads underwent a synchronous global re-diversification and are not much more than 12 million years old (Nagalingum et al., 2011). In recent years, researchers have studied cycad phylogeny Wei et al., 2015), conservation (Feng et al., 2014;Feng et al., 2016;Feng et al., 2017;Gregory and Lopez-Gallego, 2018;Tang et al., 2018a;Zheng et al., 2017), biogeography (Gutiérrez-Ortega et al., 2017;Meerow et al., 2018;Cabrera-Toledo et al., 2019), pollination biology (Tang et al., 2018b;Santiago-Jimnez et al., 2019), and physiology (Vovides and Galicia, 2016). Cycads comprise two families (Cycadaceae and Zamiaceae), with 10 genera and 355 accepted species (Calonje et al., 2019). ...
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Historical geology, climatic oscillations, and seed dispersal capabilities are thought to influence the population dynamics and genetics of plants, especially for distribution-restricted and threatened species. Investigating the genetic resources within and among taxa is a prerequisite for conservation management. The Cycas taiwaniana complex consists of six endangered species that are endemic to South China. In this study, we investigated the relationship between phylogeographic history and the genetic structure of the C. taiwaniana complex. To estimate the phylogeographic history of the complex, we assessed the genetic structure and divergence time, and performed phylogenetic and demographic historical analyses. Two chloroplast DNA intergenic regions (cpDNA), two single-copy nuclear genes (SCNGs), and six microsatellite loci (SSR) were sequenced for 18 populations. The SCNG data indicated a high genetic diversity within populations, a low genetic diversity among populations, and significant genetic differentiation among populations. Significant phylogeographical structure was detected. Structure and phylogenetic analyses both revealed that the 18 populations of the C. taiwaniana complex have two main lineages, which were estimated to diverge in the Middle Pleistocene. We propose that Cycas fairylakea was incorporated into Cycas szechuanensis and that the other populations, which are mainly located on Hainan Island, merged into one lineage. Bayesian skyline plot analyses revealed that the C. taiwaniana complex experienced a recent decline, suggesting that the complex probably experienced a bottleneck event. We infer that the genetic structure of the C. taiwaniana complex has been affected by Pleistocene climate shifts, sea-level oscillations, and human activities. In addition to providing new insights into the evolutionary legacy of the genus, the genetic characterizations will be useful for the conservation of Cycas species.
... This genus was recently classified into six sections (Hill, 2008;Lindstrom et al., 2008), which is inconsistent with the previous infrageneric classification by De Laubenfels and Adema (1998), which divided Cycas into four subgenera containing 30 species. In addition, a few new species have been documented in recent years, and several species complexes were also proposed ( Zhou et al., 2015;Singh, 2017), all of which further complicated the classifications within the genus. A comprehensive infrageneric classification incorporating both molecular phylogeny and morphological characters is therefore needed; however, the genetic diversity and classification of Cycas species remain unclear due to the lack of informative markers ( Liu et al., 2018). ...
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Premise: Cycas is an important gymnosperm genus, and the most diverse of all cycad genera. The C. taiwaniana complex of species are morphologically similar and difficult to distinguish due to a lack of genomic resources. Methods: We characterized the transcriptomes of two closely related and endangered Cycas species endemic to Hainan, China: C. hainanensis and C. changjiangensis. Three single-copy nuclear genes in the C. taiwaniana complex were sequenced based on these transcriptomes, enabling us to evaluate the species boundaries using the multispecies coalescent method implemented in the Bayesian Phylogenetics and Phylogeography program. Results: We obtained 68,184 and 81,561 unigenes for C. changjiangensis and C. hainanensis, respectively. We identified six positively selected genes that are mainly involved in stimulus responses, suggesting that environmental adaptation may have played an important role in the relatively recent divergence of these species. The similar KS distribution peaks at 1.0 observed for the paralogs in the two species indicate a common whole-genome duplication event. Our species delimitation analysis indicated that the C. taiwaniana complex consists of three distinct species, which correspond to the previously reported morphological differences. Discussion: Our study provides valuable genetic resources for Cycas species and guidance for the taxonomic treatment of the C. taiwaniana complex, as well as new insights into evolution of species within Cycas.
... They have survived through periods of dramatic tectonic activity, climate fluctuation and environmental variations, making this plant group of great significance for research (Zheng et al., 2017). Cycas chenii X. Gong & W. Zhou was described and illustrated to honor Professor Jiarui Chen (Chia-Jui Chen), a botanist from the Institute of Botany, Chinese Academy of Sciences, for his significant work on the genus Cycas in China (Zhou et al., 2015). Research by Feng et al. (2016) involving species of the Cycas segmentifida D.Y. Wang & C.Y. Deng complex significantly altered our understanding of this group. ...
The development of new taxonomical theories and approaches, particularly molecular phylogenetics, has led to the expansion of traditional morphology-based taxonomy into the concept of “integrative taxonomy.” Taxonomic knowledge has assumed greater significance in recent years, particularly because of growing concerns over the looming biodiversity crisis. Since its establishment in 1938, the Kunming Institute of Botany (KIB), which is located in Yunnan province in Southwest China, has focused attention on the taxonomy and conservation of the flora of China. For the forthcoming 80th anniversary of KIB, we review the achievements of researchers at KIB and their associates with respect to the taxonomy of land plants, fungi, and lichen. Major taxonomic advances are summarized for families of Calymperaceae, Cryphaeaceae, Lembophyllaceae, Neckeraceae, Polytrichaceae and Pottiaceae of mosses, Pteridaceae and Polypodiaceae of ferns, Taxaceae and Cycadaceae of gymnosperms, Asteraceae, Begoniaceae, Ericaceae, Euphorbiaceae, Gesneriaceae, Lamiaceae, Orchidaceae, Orobanchaceae, Poaceae, Theaceae and Urticaceae of angiosperms, Agaricaceae, Amanitaceae, Boletaceae, Cantharellaceae, Physalacriaceae Russulaceae, Suillaceae and Tuberaceae of fungi, and Ophioparmaceae and Parmeliaceae of lichens. Regarding the future development of taxonomy at KIB, we recommend that taxonomists continue to explore the biodiversity of China, integrate new theories and technologies with traditional taxonomic approaches, and engage in creative monographic work, with support from institutions, funding agencies, and the public.
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Taxonomic data is essential to advance the discovery and description of biodiversity, as well as the study of evolutionary processes. Emerging large-scale datasets and new methods of analysis have provided different approaches to describe biodiversity. Here, we present a review of the taxonomic history in Cycadales including an analysis of historical taxonomic concepts and approaches used for species delimitation. We examine the trends in the publication of new species following taxonomic works in books, journals and horticultural catalogues, monographic projects and floras where species treatments were published. In addition, we review the studies concerning species delimitations using the literature available in scientific journals appearing in the database ISI Web of Knowledge. The approaches used were discussed throughout all research focused on empirical and theoretical considerations in each study. We review the current state of the studies on causal processes that have given rise to the currently recognized diversity. The trend shows that taxonomic work on discovery and description of species has been intensive in the last 40 years culminating in 38.8% of binomials published. As a result, we consider the relevance of the monographs and floras for identification of species for other biological disciplines and the content of these contributions is compared and discussed. A total of six criteria (diagnosability, phenetic, phylogenetic, genotypic cluster, niche specialization and coalescent) were detected from the following three approaches to species delimitation within Cycadales: traditional, integrative taxonomy, and monophyletic. In all cases, the results from these species delimitations not only provided a taxonomic treatment or proposed a new species, but also supposedly clarified the other species involved as a result of the new taxonomic concept of the new species described. Most investigations of species delimitation used the traditional approach or a phenetic criteria. Finally, we discuss evolutionary studies on causal processes involved in cycad diversity. This is considered in the context of species delimitation as hypothesis testing for a successful evaluation of variation in both genetic and morphological understanding.
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Based on the IUCN Red List Categories and Criteria (Version 3.1), we assessed native gymnosperm species and infraspecific taxa found in China between March of 2010 and December of 2012. Results indicated that 37 species were critically endangered (CR), 35 species were endangered (EN), 76 species were vulnerable (VU), 87 species were of least concern (LC), and 16 species were data deficient (DD). Up to 59% of the 251 native species of gymnosperms found in China were classified as threatened. Threatening factors impacting gymnosperm species in China were ascribed into seven categories, among which habitat degradation, restricted distribution, and over exploitation were listed as the top three threats. According to results of red list assessments and conservation practices of gymnosperms in China, we propose that conservation of endangered gymnosperm species should have a targeted program. Otherwise, over-protection could result in additional threats to endangered species.
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In the quantitative phyletic approach to evolutionary taxonomy, quantitative methods are used for inferring evolutionary relationships. The methods are chosen both for their operationism and for their connection to evolutionary theory and the goals of evolutionary taxonomy. As an example of this approach, a detailed analysis of a set of anuran characters is presented and taxonomic conclusions based on those characters are drawn. The methods and conclusions of the quantitative phyletic analysis are compared and contrasted with the methods of previous workers in the field of anuran classification.
Chromosome numbers and karyotypes of species from four American Zamiaceae (Cycadales) are reported. Zamia shows interspecific and intraspecific chromosome variation, whereas Microcycas, Ceratozamia, and Dioon have constant karyotypes within each genus. In Zamia, all karyotypes have the same number of submetacentric and acrocentric chromosomes, but they differ in the number of metacentric and telocentric chromosomes. Centric fission of metacentric chromosomes is proposed to explain the karyotypic variation in this genus. Zamia shows karyological relationships with Microcycas and Ceratozamia, whereas Dioon appears very distinct from the other American cycad genera. Affinity among Zamia, Ceratozamia, and Microcycas karyotypes and distinctiveness of Dioon karyotypes are supported by comparative analysis of phenotypic characters in the four genera.
The recently-developed statistical method known as the "bootstrap" can be used to place confidence intervals on phylogenies. It involves resampling points from one's own data, with replacement, to create a series of bootstrap samples of the same size as the original data. Each of these is analyzed, and the variation among the resulting estimates taken to indicate the size of the error involved in making estimates from the original data. In the case of phylogenies, it is argued that the proper method of resampling is to keep all of the original species while sampling characters with replacement, under the assumption that the characters have been independently drawn by the systematist and have evolved independently. Majority-rule consensus trees can be used to construct a phylogeny showing all of the inferred monophyletic groups that occurred in a majority of the bootstrap samples. If a group shows up 95% of the time or more, the evidence for it is taken to be statistically significant. Existing computer programs can be used to analyze different bootstrap samples by using weights on the characters, the weight of a character being how many times it was drawn in bootstrap sampling. When all characters are perfectly compatible, as envisioned by Hennig, bootstrap sampling becomes unnecessary; the bootstrap method would show significant evidence for a group if it is defined by three or more characters.
The genus Cycas is reviewed within China. Twenty two species are enumerated, one with two subspecies, and the species are placed within an infrageneric classification previously outlined. C. panzhihuaensis is placed in the new Section Panzhihuaenses. Lectotypes are designated for Cycas revoluta, C. balansae and C. miquelii. Distribution of all taxa is mapped, conservation status discussed and a key to species provided.
This procedure has been used with success on a wide variety of plant groups and even some animals. The method is used to isolate total genomic DNA (nuclear, chloroplast, and mitochondrial). It is a rapid, inexpensive method that is suitabie for use in conjunction with other protocois, such as isolation of DNA enriched for cpDNA. it is also easy to scale down for use in population sampling, using 0.01g or less of fresh tissue. Other applications include isolation of DNA from herbarium specimens (Doyle & Dickson, 1987. Taxon 36:715–722), and isolation of RNA. A brief word on the history of the protocol is in order. This procedure was modified by us (Doyle and Doyle, 1987. Phytochemical Bulletin 19:11–15) for use with fresh plant tissue from a method of Saghai-Maroof et al. (1984, PNAS USA 81:8014–8019) who used lyophilized tissue. They in turn had developed their procedure from earlier protocols. We were recently asked to publish a slightly modified version of our procedure (Doyle and Doyle, 1990 Focus 12:13–15). We recently learned from Brian Taylor (Texas A&M University, USA) that he had published a virtually identical procedure for fresh tissue, also in Focus, in 1982 (Taylor & Powell, Focus 4:4–6) of which we (and apparently the editors of Focus!) were entirely unaware. It is indeed a useful procedure, thus independently confirmed.
— We studied sequence variation in 16S rDNA in 204 individuals from 37 populations of the land snail Candidula unifasciata (Poiret 1801) across the core species range in France, Switzerland, and Germany. Phylogeographic, nested clade, and coalescence analyses were used to elucidate the species evolutionary history. The study revealed the presence of two major evolutionary lineages that evolved in separate refuges in southeast France as result of previous fragmentation during the Pleistocene. Applying a recent extension of the nested clade analysis (Templeton 2001), we inferred that range expansions along river valleys in independent corridors to the north led eventually to a secondary contact zone of the major clades around the Geneva Basin. There is evidence supporting the idea that the formation of the secondary contact zone and the colonization of Germany might be postglacial events. The phylogeographic history inferred for C. unifasciata differs from general biogeographic patterns of postglacial colonization previously identified for other taxa, and it might represent a common model for species with restricted dispersal.