Zhengyia shennongensis: A new bulbiliferous genus and species of the nettle family (Urticaceae) from central China exhibiting parallel evolution of the bulbil trait

Abstract

Zhengyia shennongensis is described here as a new genus and species of the nettle family (Urticaceae) from Hubei province, central China. The phylogenetic position of Z. shennongensis is determined using DNA sequences of nuclear ribosomal ITS and three plastid regions (rbcL, psbA-trnH, trnL-F). Zhengyia shennongensis is readily distinguished from the related genera Urtica, Hesperocnide, and Laportea in the tribe Urticeae by its seed (oblong-globose or subglobose and not compressed achenes, surface densely covered with nipple-shaped protuberances) and stipule morphology (large leaf-like stipules with auriculate and amplexicaulous base and united with stem). Phylogenetic evidence indicates that Zhengyia is a distinct group related to Urtica (including Hesperocnide) species and Laportea cuspidata in tribe Urticeae. The bulbiliferous
species of the tribe (L. bulbifera, L. cuspidata, Z. shennongensis) do not form a clade. This result indicates that the bulbil trait evolved in parallel within Urticeae. Our findings highlight the importance of shady and moist habitats in promoting species diversification and the parallel evolution of morphological traits that are likely to be adaptive.

Full text

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INTRODUCTION
Urticeae (= Urerareae Wedd.) is a moderately sized tribe
of the nettle family (Urticaceae) with eleven genera and ap-
proximately 220 species. Its members are often found in humid
habitats in forests or at forest margins and occur in both the
Old and New Worlds (Friis, 1993; Hadiah & al., 2008; Cohn
& Hadiah, 2009). The tribe is characterized by stinging hairs
and pistillate flowers with four tepals, of which frequently one
pair is larger than the other, and without staminodes (Friis,
1989, 1993). Because of the obvious morphological synapo-
morphies of Urticeae, it is not difficult to recognize a plant as
being a member of this tribe. Moreover, phylogenetic analysis
of Urticaceae using plastid DNA sequence data has shown
that Urticeae (including Poik ilospermum Zipp.) form a well-
supported clade (Hadiah & al., 2008).
The Shennongjia National Nature Reserve (SNNR) is lo-
cated in the Northwest of Hubei province, central China. Its
unique geographical location and complex topology make it
one of the most biodiverse areas in China (Ying, 2001; Xie,
2003). The Shennongjia Mountains are characterized by high
mountains and deep valleys, a dense network of streams, and
lush vegetat ion. The region is an impor tant hot-spot for south-
central Chinese biodiversity and contains many endemic plants
(Myers & al., 2000). During our recent in-depth floristic ex-
plorations of the SNNR, an unusual taxon caught our attention.
The plant was easily identified as belonging to Urticeae based
on the presence of stinging hairs, stipules, and pistillate f low-
ers with four tepals and without staminodes. In its paniculate
inf lorescences with many long branches and its four-lobed
perianth with larger dorsal than ventral lobes in its female
flowers, it superficially resembles Urtica L., a genus of about
30 species with a wide distribution in the northern temperate
region (Chen & al., 2003). However, based on its alternate leaf
arrangement, the presence of one to three woody bulbils in
sterile axils, intrapetiolar stipules in the leaf axils, and oblique
achenes with short stipes, we initially assigned the new taxon to
Laportea Gaudich., a genus with 30 species confined to tropical
and warm-temperate E Asia and eastern North America (Friis,
1993). Upon closer examination, however, it was clear that the
set of morphological characters did not match Urtica, Laportea
or any other genus of Urticeae (Table 1). The plant is described
below as a new genus with only one species, Zhengyia shen-
nongensis T. Deng, D.G. Zhang & H. Sun.
Bulbils are specialized propagules, allowing vegetative
reproduction and dispersal, and many herbaceous plants can
produce them (Wang & al., 2004; Walck & al., 2010). Pres-
ence or absence of bulbils has been recognized as a significant
Zhengyia shennongensis: A new bulbiliferous genus and species of
the nettle family (Urticaceae) from central China exhibiting parallel
evolution of the bulbil trait
Tao Den g,1,2,5 Changkyun Kim,1,5 Dai-Gui Zhang,3 Jian-Wen Zhang,1 Zhi-Ming Li,4 Ze-Long Nie1 & Hang Sun1
1 Key Laboratory of Biodiversity and Biogeography, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201,
Yunnan , P.R. China
2 University of Chinese Academy of Sciences, Beijing 100039, P.R. China
3 Key Laboratory of Plant Resources Conservation and Utilization, Jishou University, College of Hunan Province, Jishou 416000,
Hunan, P.R. China
4 Life Science School, Yunnan Nor mal Universit y, Kunming 650031, Yunnan, P.R. China
5 These authors contributed equally to the work.
Author for correspondence: Hang Sun, hsun@mail.kib.ac.cn
Abstract
Zhengyia shennongensis is described here as a new genus and species of the nettle family ( Urticaceae) from Hubei
province, central China. The phylogenetic position of Z. shennongensis is determined using DNA sequences of nuclear ribo-
somal ITS and three plastid regions (rbcL, psbA-trnH, trnL-F). Zhengyia shennongensis is readily distinguished f rom the
related genera Urtica, Hesperocnide, and Laportea in the tribe Urticeae by its seed (oblong-globose or subglobose and not
compressed achenes, surface densely covered with nipple-shaped protuberances) and stipule morphology (large leaf-like
stipules with auriculate and amplexicaulous base and united with stem). Phylogenetic evidence indicates that Zhengyia is a
distinct group related to Urtica (including Hesperocnide) species and Laportea cuspidata in t ribe Urticeae. The bulbiliferous
species of the tr ibe (L. bulbifera, L. cuspidata, Z. shennongensis) do not form a clade. This result indicates that the bulbil trait
evolved in parallel within Urticeae. Our findings highlight the importance of shady and moist habitats in promoting species
diversification and the parallel evolution of morphological traits that are likely to be adaptive.
Keywords
bulbils; central China; new genus and species; parallel evolution; Urticaceae; Urticeae; Zhengyia shennongensis
Received: 14 May 2012; revision received: 1 Oct. 2012; accepted: 12 Nov. 2012.

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morphological trait in species delimitation in Urticeae (Chen
& al., 2003). The character is also useful to establish infra-
generic taxa in genera of Urticeae (e.g., Laportea; Chen & al.,
2003). To date, in Urticeae only two species of Laportea
(L. bulbifera Weed ., L. cuspidata Friis) have been reported
to be bulbiliferous; they both grow in shady, moist conditions
(Chen & al., 2003).
In the present study, we determine the phylogenetic
position of the new taxon based on morphological data, espe-
cially surface features of the achene examined using scanning
electron microscopy (SEM), and cytological and molecular data
(the three plastid regions rbcL, psbA-trnH intergenic spacer
[IGS], and trnL-F IGS; and nuclear ribosomal ITS [nrITS]).
Based on the inferred phylogeny, we also provide a discussion
of the evolution of the bulbil trait in Urticeae.
MATERIALS AND METHODS
Plant material. —
Samples of Z. shennongensis were col-
lected for morphological comparison from the only known
populatio n—Wusha nhu, Hub ei Provi nce—duri ng our field in-
vest ig at ions in 2011 (Fig. 1). Leave s and mat u re seed s were also
collected for SEM and cytological and molecular phylogenetic
studies. For comparison with the seed morphology of Z. shennon-
gensis, eight species of the closely related U. mairei H. Lév, U. di-
oica L., U. fissa E. Pritz., U. urens L., L. bulbifera Wed d., L. cus-
pidata Friis, L. canadensis Gaudich., and Girardinia diversifolia
(Link) Friis were examined.
In order to determine phylogenetic relationships in Urticeae
using molecular markers, we sampled 16 taxa (21 accessions)
in addition to Z. shennongensis, including two species of Den-
drocnide Miq. (two ac cessions), thre e subspecie s of Girardinia
diversifolia (three accessions), one species of Hesperocnide
Torr. (one accession), three species of Laportea (five acce s-
sions), one species of Poikilospermum (one accession), and
six species of Urtica (nine accessions). We also included three
species, Pilea plataniflora C.H. Wright of Elatostemateae
Gaudich., Boehmeria spicata Thunb. of Boehmerieae Gaudich.,
and Fatoua villosa Nakai of Moraceae as outgroups, based on
a previous phylogenetic analysis using rbcL and trnL DNA
sequence data (Hadiah & al., 2008). Voucher information and
GenBank accession numbers for all specimens used in this
study are listed in Appendix 1.
Seed observation. —
The mature seeds of 90 individuals
of the species listed above and our new taxon were mounted
on aluminum stubs with double-sided adhesive tape, sputter-
coated with gold to a maximum thickness of 20 μm, and ex-
amined using a KYKY-1000B scanning electron microscope
(SEM; Science Instrument Company, Beijing, China) with a
voltage of 30 kV. Microphotographs focused primarily on the
center of the seeds. Seed morphology was also examined under
the dissecting microscope (OLYMPUS BX53).
Cytological studies. —
Root-tip meristems were obtained
by germinating seeds on wet filter paper in Petri dishes at ap-
proximately 20°C. Root tips less than 1.5 cm long were cut and
treated with 0.002 M 8-hydroxyquinoline at room temperature
for 3–5 h before being fixed in Carnoy (glacial acetic acid:
absolute ethanol = 1 : 3), macerated in a 1 : 1 mixture of 45%
acetic acid and 1 M HCl for 2.5 min, and stained and squashed
in Carbol Fuchsine. Karyotypes of mitotic chromosomes at
metaphase were determined from at least f ive well-spread
Fig. 1.
Distribution of Zhengyia
shennongensis T. Deng, D.G.
Zhang & H. Su n. The circle indi-
cates the t ype locality of Z. shen-
nongensis.

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metaphases of three different roots. The designation of the
centromere position as median (m), submedian (sm) and sub-
terminal (st) followed Levan & al. (1964).
DNA extraction, PCR amplification, and sequencing. —
Total genomic DNA was isolated from silica gel-dried leaf
material using the Universal Genomic DNA Extraction Kit
(Takara, Dalian, China). Primer sets and protocols for PCR
followed specifications in previous publications: rbcL (prim-
ers Z1 and 1204R; Zurawski & al., 1981; from G. Zurawski
[DNAX Research Institute, Palo Alto, California, U.S.A.]),
psbA-trnH IGS (psbA_F and trn H _R; Hami lton, 1999), trnL-F
IGS (e and f; Taberlet & al., 1991), and nrITS (ITS1 and ITS4;
White & al., 1990; Kim & al., 2010). Amplif ied DNA samples
were analyzed by electrophoresis on 1.4% agarose gel, run in
a 0.5× TBE buffer and detected by ethidium bromide staining.
The PCR products were then purified using a QiaQuick gel
extraction kit (Qiagen, Inc., Valencia, California, USA) and
directly sequenced in both directions using the amplification
primers on an ABI 3730 automated sequencer (Applied Bio-
systems, Forster City, California, U.S.A.).
Phylogenetic analyses. —
DNA Baser v.3 (http://www
.dnabaser.com) was used to evaluate the chromatograms for
base confirmation and to edit contiguous sequences. Multiple-
sequence alignment was performed with MAFFT v.6 (Katoh
& al., 2009; available at http://www.genome.jp/tools/mafft)
using the default alignment parameters. Gaps were coded as
missing data. All datasets have been submitted to TreeBASE
(http://www.treebase.org/; study accession number, S12631).
The phylogenetic reconstruction of the sequences was
performed by maximum parsimony (MP) in PAUP* v.4.0b10
(Swofford, 2002). All characters were weighted equally and
unordered. Each dataset was analyzed separately and then a
simultaneous analysis was performed including all four re-
gions. Before combining the datasets, the incongruence length
difference (ILD) test was conducted to assess data congruency
(Farris & al., 1995) using PAUP* and 10,000 heuristic search
replications including only parsimony-informative characters.
Most parsimonious trees were searched with a heuristic al-
gorithm using tree bisection-reconnection branch swapping,
MULPARS, and the alternative character state. Strict con-
sensus trees were constructed from the most parsimonious
trees. Bootstrap analyses (BP; 1000 pseudoreplicates) were
conducted to examine the relative level of support for individual
clades (Felsenstein, 1985). The consistency index (CI; Kluge
& Farris, 1969) and retention index (RI; Farris, 1989) were
calculated to measure the amount of homoplasy in the dataset.
Phylogenetic analyses of the nrITS and combined data sets
were also conducted using Bayesian Markov chain Monte Carlo
(MCMC) infere nce (BI; Yang & Ranna la , 1997 ) using MrBayes
v.3.12 (Ronqu ist & Huelsenbe ck, 2003). Modelte st v.3.1 (Posa da
& Crandall 1998) was used to determine the optimal model
of DNA evolution for the data based on the Akaike informa-
tion criterion (AIC; Akaike, 1974). Four chains of the MCMC
inference were run simultaneously, with sampling every 100
generations over a total of one million generations. The first
2500 trees (25%) of the sample trees from each run were dis-
carded as determined by Tracer v.1.5 (Rambaut & Drummond,
2007). A Bayesian consensus tree was constructed from the
remaining trees, yielding the posterior probability (PP) values
for each clade.
The single most parsimonious topology obtained from the
analysis of the combined molecular data (nrITS and three plas-
tid DNA regions) was employed to reconstruct the evolution of
the bulbil character. Character reconstruction was carried out
under the assumption of unordered and unweighted character
states with the Ancestral State Reconstruction Package in Mes-
qu it e v.2.75 (Ma ddiso n & Ma ddi son, 2011) using unambigu ou s
optimization.
RES U LT S
Morphological characters. —
Morphological characters
of Z. shennongensis and related genera, including Dendro-
cnide, Girardinia, Hesperocnide, Laportea, Poikilospermum,
and Urtica, are list ed in Table 1. The se ed char act eri st ics of our
new taxon, including shape and surface sculpturing, were found
to be unique when compared to the other genera. The achene
shape of Z. shennongensis was oblong-globose or subglobose
and extremely asymmetrical, and no infraspecific variation
was found (Fig. 2A). The seed surface of Z. shennongensis is
densely covered with nipple-shaped protuberances but smooth
and/or verrucose in the other genera (Fig. 2).
Chromosome counts and karyomorphology. —
The ch r o -
mosome number in mitotic metaphase cells was found to be
2n = 24, and the karyotype formula is 2n = 6m + 16sm + 2st
(Fig. 3).
Phylogenetic analyses. —
The characteristics and sta-
tistics for nrITS, the three plastid regions, and the combined
data sets for the MP analyses are presented in Table 2. Bayesian
analyses of all datasets resulted in the same tree topologies
as the corresponding MP analyses (data not shown). All MP
trees were generally congruent with respect to well-supported
clades, but there was an incongruence between the plastid and
nrITS analyses concerning the position of Z. shennongensis.
The combined plastid analysis resolved both Z. shennongensis
samples as well-supported sister to Urtica and Hesperocnide
species (BP = 92%, PP = 1.00), whereas in the nrITS tree the
species was sister to a clade including Urtica, Hesperocnide,
and Laportea species but with low statistical support (BP =
50%, PP = 0.64; data not shown).
ILD tests failed to identify significant conf lict among the
th ree partition s of the plasti d dat aset (rbcL, psbA-trnH, trnL-F;
P = 0.065) and between the nrI TS and the plas tid data sets (P =
0.052). When all molecular datasets were combined, the single
MP tree found was better resolved than any tree from sepa-
rate analyses. Phylogenetic analysis of the combined dataset
resulted in a single most parsimonious tree (tree length = 2302,
CI = 0.597, RI = 0.756). In the MP tree, tribe Urticeae formed
a monophyletic group (BP = 100%, PP = 1.00; Fig. 4). The two
individuals of Z. shennongensis were sister to Urtica (includ-
ing Hesperocnide) species, wit h high statistical support (BP =
92%, PP = 1.00). Laportea cuspidata was sister to the clade
comprising Zhengyia and Urtica + Hesperocnide sp ecies (BP =

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86% , PP = 1.00). The bulbilifer ous spe cies (L. bulbifera, L. cus-
pidata, and Z. shennongensis) were not closely related to each
other (Fig. 4).
DISCUSSION
Systematic position of Zhengyia shennongensis. —
Ac-
cording to the classification of Urticaceae by Friis (1989, 1993),
our new taxon Z. shennongensis is a member of tribe Urticeae.
In tribe Urticeae, the basic chromosome number most often
is x = 12 and 13, and less often x = 10, 11, and 19 (e.g. Wood-
land & al., 1976, 1982; Friis, 1993). The chromosome number
of Z. shennongensis was found to be 2n = 24 (x = 12) in this
study (Fig. 3). Thus, cytological evidence supports that our
new species should be included in Urticeae. Moreover, our
molecular phylogenetic results clearly confirmed that Z. shen-
nongensis is part of Urticeae (Fig. 4).
In general, genera of Urticeae have been recognized
primarily on the basis of stipule and fruit shape (Friis, 1993;
Chen & al., 2003). Zhengyia shennongensis has a distinctive
oblong-globose or subglobose achene with dense nipple-shaped
Table 1.
Morphological comparison of Zhengyia with other genera in Urticeae.
Character Zhengyia Dendrocnide Girardinia Hespero cnide Laportea I
d
Laportea II
e
Poikilo-
spermum Urtica
Habit
a,b
robust herb shrub robust herb herb herb herb shrub or
woody
climber
herb
Bulbils
a,b
present absent absent absent present absent or
present in
L. bulbifera
absent absent
Leaf arrangement
a,b
alternate alternate alternate opposite alternate alternate alternate opposite
Stipules
a,b
intrapetiolar,
auriculate-
amplexicau-
lous base
united with
the stem,
persistent
intrapeti-
olar, subulate
or linear,
deciduous
intrapeti-
olar, subulate
or linear,
deciduous
lateral, subu-
late or linear,
persistent
intrapeti-
olar, subulate
or linear,
deciduous
intrapeti-
olar, subulate
or linear,
deciduous
intrapeti-
olar, subulate
or linear,
deciduous
lateral, subu-
late or linear,
persistent
Perianth
a
deeply
4-lobed, one
pair larger
4-lobed,
lateral ones
slightly larger
ovoid-
tubular, (2–)
3-toothed
almost tubu-
lar, minutely
2-toothed at
the apex
4-lobed,
strongly un-
equal, lateral
larger
4-lobed, one
minute or
absent
clavate-
tubular
deeply
4-lobed, one
pair larger
Inflorescences pairs solitary solitary or
pairs
pairs solitary solitary solitary pairs
Stigmas
a,b
short clavate linear or
ligulate
subulate,
acute, minute
capitate-
penicillate
linear, papil-
lose on one
side
linear, papil-
lose on one
side
capitate or
ligulate
capitate-
penicillate
Achene symmetry
a,b
asymmetric asymmetric asymmetric symmetric asymmetric asymmetric asymmetric symmetric
Achene shape
a
oblong-
globose or
subglobose,
not com-
pressed
elliposidal
to ovoid,
compressed
broadly
ovoid,
compressed
ovate,
compressed
ovoid to
semicircular,
compressed
ovoid to
semicircular,
compressed
oblong, ellip-
soid or ovoid,
compressed
ovoid,
compressed
Achene surface
a,c
with dense
nipple-shaped
protuberances
verrucose verrucose unknown smooth smooth or
with stripes
verrucose smooth or
verrucose
with sunken
dots
a
Friis (1993) and Chen & al. (2003).
d
Laportea I includes L. cuspidata.
b
Based on herbarium collections and field observation.
e
Laportea II comprises two species (L. bulbifera, L. interrupta).
c
Based on SEM.

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protuberances on the surface (Fig. 2A). The species also differs
from other genera of Urticeae by having large leaf-like stipules
with an auriculate-amplexicaulous base united with the stem
(Figs. 4, 5E). These differences are reflected in the MP tree
using the combined dataset where the new genus occupies a
distinct position in Urticeae with maximum support (BP =
100%, PP = 1.00; Fig. 4). Therefore, the recognition of Zhengyia
at the rank of genus is warranted based on morphological and
molecular evidence.
Our phylogenetic analyses showed that H. tenella Tor r.
was nested in the Urtica clade. This close relationship is
morphologically supported by opposite leaves, lateral stipules,
and straight achenes (Table 1). Zhengyia shennongensis is clos-
est relative of the Urtica + Hesperocnide clade with high sup-
port (BP = 92%, PP = 1.00; Fig. 4). Several morphological
characters (e.g., herbs with persistent stipules, deeply 4-lobed
perianth, inflorescences with many long branches) support
these relationships. Within this clade, Z. shennongensis can
be easily distinguished from Urtica and Hesperocnide species
by its alternate leaf arrangement (vs. opposite), large stipules
inserted in the axil of leaves (vs. 2 or 4 rather small and nar-
row, lateral stipules), and extremely oblique achenes (vs. erect;
Table 1; Fig. 4).
Our molecular data do not support the monophyly of
Laportea. Laportea cuspidata (Laportea I) is a sister to the
Zhengyia + Urtica (including Hesperocnide) lineage but not to
the other species of Laportea (Laportea II; Fig. 4). Zhengyia
shennongensis with one to three woody bulbils in sterile leaf
axils (Fig. 5F) resembles L. cuspidata, and these two species
share other morphological characters such as alternate leaves
and oblique achenes. However, the stigma of Z. shennongen-
sis is short and clavate, while that of L. cuspidata is linear
(Table 1). In addition, the achene surface of Z. shennongensis
differs from that of L. cuspidata in having markedly nipple-
shaped protuberances (Fig. 2A, G).
Of the other genera in tribe Urticeae, Z. shennongensis is
similar to Girardinia in that both are robust herbs with long
stinging hairs (> 5 mm). However, Z. shennongensis is distin-
guished from Girardinia by three morphological characters:
the presence of bulbils in leaf axils, branched inflorescences,
and the ornamentation of the achene surface (Table 1; Fig. 4).
Zhengyia shennongensis can be easily distinguished from
Dendrocnide and Poikilospermum by habit (herbs vs. shrub
or trees). Moreover, our molecular evidence shows that Z. shen-
nongensis is not closely related to Girardinia, Dendrocnide,
and Poikilospermum (Fig. 4).
Parallel evolution of bulbils. —
Many herbaceous plants
form bulbils (Okagami, 1979). Bulbils serve as a means of
clonal reproduction with the ability to colonize and seques-
ter resources quickly after initial introduction, particularly
in isolated populations (Callaghan & al., 1997; Abrahamson,
1980). Although bulbils are a valuable reproductive propert y,
they are found in only three species (L. bulbifera, L. cus-
pidata, Z. shennongensis) of Urticeae. In the combined MP
tree, the three bulbiliferous species did not group together
but were placed in three different clades, each with maximal
support except L. bulbifera (BP = 57%, PP = 0.79; Fig. 4). Two
Fig. 2.
Comparison of achene morphology and surface sculpting in
tribe Urticeae.
A,
Zhengyia shennongensis (Deng, Zhang & Sun 3431;
KUN);
B,
Urtica mairei (Pen g 16 07; KUN);
C,
U. dioica (Ca i 55102;
KUN);
D,
U. fissa (Liu 16628; KUN);
E,
U. urens (Qingzhang Exped.
318; KUN);
F,
Laportea bulbifera (Zhangdian Exped. 2575; KUN);
G,
L. cuspidata (Qingzhang Exped. 13367; KUN );
H,
L. canadensis
(Ko ya m a 74 01; KUN);
I,
Girardinia diversifolia (Nie & Deng 4248;
KUN).
a,
Dissecting microscope;
b,
SEM, low magnification;
c,
SEM,
ultrastructure of seed surface.

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reconstr uctions of bulbil evolution are equally parsimonious
in our phylogenetic tree (Fig. 4). Either bulbils evolved three
times independently in Urticeae or they evolved twice and
were lost once.
Bulbils have been recorded in many families and also in
different clades of single tribes (Givnish & al., 2000; Wang
& al., 200 4; Thomas & al., 2005; Kit aha ra & al., 2010) and may
have originated in response to strong selection in shady, moist
and pollinator-poor habitats (Wake & al., 2011), and indeed the
three bulbiliferous species of Urticeae grow mainly in shady
habitats along creeks, particularly on wet, dripping cliffs in
valley s. Th is tr ait probably re pl aces pr opagation and di spersal
by seeds or fruits. When compared with species without bul-
bils (e.g., Urtica, Girardinia), the bulbil iferous taxa appear to
have less seed set as judged from our field observations and
herbarium material, but statistical confirmation of this obser-
vation would require more detailed measurement. It might be
that the wind-pollinated bulbiliferous taxa of Urticeae have
evolved these propagules to cope with lack of seed set in the
windless conditions of their extremely shady, humid habitats.
Alternat ively, the bu lbils, which may be dispe rsed by grav it y,
water, animals, or birds (Thomas & al., 2005; Mizuki & Taka-
ha shi, 20 09), may be bet ter suited for sha dy ha bitats tha n seeds
because they are much larger than normal seeds of Urticaceae
and may store more nutrients needed for establishment.
TAXONOMIC TREATMENT
Zhengyia shennongensis T. Deng, D.G. Zhang & H. Sun, gen.
& sp. nov. – Holotype: China, cent ral China , Hubei prov-
ince, Shennongjia Forest District (SNNR), Yangri town,
Wushanhu, 31°32′37″ N, 110°50′35″ E, 450 m alt, 4 Sep
2011, T. Deng, D.G. Zhang & H. Sun 2295 (KU N; iso type s:
A, K, MO, PE). — Figures 5 and 6.
Description. – Perennial robust herbs with long stinging
hairs. Rhizomes stoloniferous, up to 2 m long. Stems erect,
1–3 m tall, terete, not longitudinally angular or sulcate, slightly
Table 2.
Tree statistics for the nrITS, rbcL, psbA-trnH, trnL-F, and combined datasets from maximum parsimony (MP) analysis.
Parameters nrITS rbcL psbA-trnH trnL-F
Combined
ptDNA nrITS + ptDNA
Number of sequences (ingroup/outgroup) 26 (23/3) 26 (23/3) 26 (23/3) 26 (23/3) 26 (23/3) 26 (23/3)
Aligned length [bp] 744 1013 337 426 1776 2520
Variable characters (%) 427 (57.4) 160 (15.8) 225 (66.8) 178 (41.8) 563 (31.7) 990 (39.3)
Parsimony informative characters (%) 307 (41.3) 87 (8.6) 131 (38.9) 113 (26.5) 331 (18.6) 638 (25.3)
Number of trees (MP) 186821
MP tree length 1169 260 537 303 1117 2302
Consistency index (CI)
a
0.577 0.591 0.623 0.727 0.632 0.597
Retention index (RI) 0.743 0.809 0.707 0.878 0.783 0.756
Model selected (AIC) GTR+I+G GTR+I+G GTR+G GTR+G GTR+I+G GTR+I+G
a
The consistency index is calculated excluding uninformative characters.
Fig. 3.
Mitotic metaphase of Zhengyia shennongensis T. Deng, D.G. Zhang & H. Sun.
A,
Micrograph of metaphase chromosomes;
B,
kar yoty pe
of mitotic metaphase chromosomes.

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95Version of Record (identical to print version).
woody at base, ca. 2 cm in diam. Sterile leaf axils often with
13 woody bulbils, fawn, globose or ovoid, 3–6 mm in diam,
often wit h adventi tious roo ts. Upper stem s and pet iole s densely
covered with stinging hairs and white pubescent. Stipules
greenish, leaf-like, herbaceous, persistent, solitary in leaf
axils, united with stem at base; stipule cordate or triangular-
ovate, 3–4 cm, margin subentire or minutely sparsely crenate,
base auriculate-amplexicaulous, apex long caudate-acuminate,
shallowly 2-cleft, basal veins 3. Leaves alternate; leaf blade
broadly ovate, 13–27 × 10 –26 cm, base shallowly cordate to
Fig. 4.
Single most par simon ious tree (tree le ng th = 2302, CI = 0.597, RI = 0.756) fro m the analysis of the combin ed nrITS and pt DNA sequen ces.
Numbe rs above branches indic ate boots tr ap suppor t (BP); numb er s below branches are Ba yes ian poste rior probabi litie s (PP); a dash (–) indic ates
that a node did not receive >80% BP in the MP analysis.
A,
achene shape;
B,
stipule position and shape (arrows indicate stipules);
C,
presence of
bulbils (arrows indicate bulbils);
D,
leaf arrangement.

96
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Fig. 5.
Images of living plants of Zhengyia shennongensis T. Deng, D.G. Zhang & H. Sun.
A,
Habitat;
B,
habit;
C,
population;
D,
inf lorescence;
E,
stipules;
F,
bulbils;
G,
root;
H,
inflorescence;
I,
staminate f lower;
J,
pedicel;
K,
fruit.

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Deng & al. • The new genus Zhengyia (Urticaceae)
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Fig. 6.
Holotype of Zhengyia shennongensis T. Deng, D.G. Zhang & H. Sun, gen. & sp. nov., with details.
A,
Habit;
B,
pistillate f lower (arrow
indicates stigma);
C,
staminate f lower;
D,
achene. — Drawn by X.-S. Zhang.

98
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98 Version of Record (identical to print version).
Abrahamson, W.G. 1980. Demography and vegetative reproduction.
Pp. 89–106 in: Solbr ig, O.T. (ed.), Demography and evolution in
plant populations. Oxford: Blackwell Scientific Publications.
Akaike, H. 1974. A new look at the statistical model identification.
I. E. E. E. Trans. Automatic Control 19: 716 –723.
Callaghan, T.V., Jonasson, S. & Brooker, R.W. 1997. Arctic clonal
plants and global change. Pp. 381–403 in: De Kroon, H. & Van
Groenendael, J. (eds.), The ecology and evolution of clonal plants.
Leiden: Backhuys.
Chen, C.J., Lin, Q., Fr iis, I., Wilmot-Dear, C.M. & Monro, A.K.
2003. Urticaceae. Pp. 76–189 in: Wu, Z.Y., Raven, P.H. & Hong,
D.Y. (eds .), Flora of China, vol. 5. Bei ji ng: Scienc e Pr ess; St. Loui s:
Missour i Botanical Garden Press.
Cohn, B.J. & Hadiah, J.T. 2009. Nomenclature of tribes within the
Urticaceae. Kew Bull. 64: 349–352.
Farris, J.S. 1989. The retention index and the rescaled consistency
index. Cladistics 5: 417–419.
Farris, J.S., Källersjö, M., Kluge, A.G. & Bult, C. 19 9 5. Test ing
significance in incongruence. Cladistics 10: 315 –319.
Felsenstein, J. 1985. Confidence limits on phylogenies: An approach
using the bootstrap. Evolution 39: 783–791.
Friis, I. 198 9. The Urticaceae: A systematic review. Pp. 285–308 in:
Crane, P.R. & Blackmore, S. (eds.), Evolution, systematics, and fos-
sil history of the Hamamelidae, vol . 2. The System at ics Ass oc ia tion
special volume, no. 40B. Oxford: Clarendon Press.
Friis, I. 1993. Urticaceae. Pp. 612–630 in: Kubitzki, K., Rohwer, J.G.
& Bittrich, V. (eds.), The families and genera of vascular plants,
vol. 2, Flowering plants: Dicotyledons; Magnoliid, Hamamelid
and Caryophyllid families. Berlin: Springer.
Givnish, T.J., Evans, T.M., Zjhra, M.L., Patterson, T.B., Berr y,
P.E. & Sytsma, K. J. 2000. Molecular evolution, adaptive radia-
tion, and geographic diversification in the amphiatlantic family
Rapateaceae: Evidence from ndhF sequences and morphology.
Evolution 54: 1915–1937.
Hadiah, J.T., Conn, B.J. & Quinn C.J. 2008. Infra-familial phylogeny
of Urticaceae, using chloroplast sequence data. Austral. Syst. Bot.
21: 375–385.
Hamilton, M.B. 1999. Four pr imer pairs for the amplif ication of chlo-
roplast intergenic regions with intraspecific variation. Molec. Ecol.
8: 521–523.
IUCN 2001. IUCN Red List categories and criteria, version 3.1. IUCN
Species Survival Commission. Gland, Switzerland & Cambridge,
U.K.: IUCN.
cordate, margin dentate or lobed; lobes deltoid, denticulate,
slightly falcate; apex shortly acuminate; cystoliths minutely
punctiform; lateral basal veins reaching middle lobes, sec-
ondary veins 4– 6 on each side, reaching teeth or anastomos-
ing before margin, adaxial surface with sparse, stinging and
setulose hairs, abaxial surface densely setulose and sparsely
armed with stinging hairs on veins. Petiole 12–16 cm. In-
florescences unisexual, in axillary pairs; paniculate with
many long branches; male inf lorescences in proximal axils,
paniculate, erect, 15–25 cm; female inflorescence terminal
or in subterminal leaf axils, pendulous, 20–30 cm, peduncle
2–4 cm. Staminate flowers ca. 1.5 mm, shortly pedicellate or
subsessile; perianth lobes connate below middle, apex not cor-
niculate; stamens 4, filaments incurved, longer than perianth,
anthers peltate; pistillode terete, ca. 0.3 mm. Pistillate f low-
ers ca. 1.3 mm, subsessile; perianth lobes 4, connate at base,
strongly unequal, the 2 dorsal-ventral lobes larger, enclosing
the ovary, elliptic-ovate, setulose, as long as achene; lateral
lobes smaller, ovate-lanceolate, ca. 1/2 as long as dorsal lobe.
Ovary ca. 1.1 mm, shortly stipitate, asymmetrically ovoid;
stigma spirally winding, short clavate, ca. 0.4 mm. Achene yel-
lowish green, oblong-globose or subglobose, ca. 1.2–1.5 mm,
conspicuously oblique, with dense nipple-shaped protuber-
ances on surface, enclosed by persistent enlarged dorsal-ven-
tral perianth lobes; stipe ca. 0.1 mm.
Etymology. —
Zhengyia is named in honor of Prof.
Zhengyi Wu, a renowned Chinese botanist who has studied
Chinese plants for over 70 years. He deserves this homage in
recognition of his important contributions to the field of plant
taxonomy and floristics, to his deep involvement in training
new researchers and his tremendous contribution to our knowl-
edge of the flora of China.
Distribution and habitat. —
Despite extensive investiga-
tion s in ce ntr al Ch ina by the collect or s of th is ta xon, the species
has so far only been found in the area of Wushanhu Mountain
in the SNNR, in the southwest part of Hubei province, central
China (Fig. 1). The new species is probably calcicole. It prefers
shady and wet habitats with deep humus-rich soil. It grows in
small clusters in the valley and on limestone mountain slopes
mainly at 500 to 600 m. These ancient limestone mountains in
the region are deeply eroded and dissected by deep river val-
leys. The globose woody bulbils are probably associated with
a rain-splash dispersal mechanism: when bulbils are released
from parent plants, they are washed down the mountain slope
by rainwater and have the potential to spread more widely via
streams.
Conservation status. —
Endangered, based on the occur-
rence in an area smaller than 5000 km² and known at fewer
than five localities (IUCN, 2001).
Phenology. —
The peak f lowering period was observed
in September and fruiting specimens were found in October
and November.
Paratype. —
China. Hubei province, Shennongjia Forest
Dist r ict (SNNR), Yan gri town , Wugu Mountai n, 110°50′35″ E ,
31°32′37″ N, 450 m alt, 4 Sep 2011, T. Deng, D.G. Zhang
& H. Sun 2593 (KUN).
ACKNOWLEDGMENTS
This study was supported by grants-in-aid from the National
Natural Science Foundation of China (Grant no. NSFC, 31061160184),
Hundred Talents Program of the Chinese Academy of Sciences
(2011312D1102 2), Stra te gic Pri or ity Res ear ch Progra m of the Ch ine se
Acad em y of Scien ce s (XD B03030106), NSF C-Yu nna n Na tur al Scienc e
Foundation Unite Project (Grant no. U1136601), and the Research Pro-
gram from the Ministry of Science and Technology of China (Grant no.
2007FY110100) to H. Su n and the research program for Postdoctoral
Scholar, Ke y Lab or at ory of Biod iver sity an d Bioge ogr ap hy, Kunm i ng
Instit ute of Botany, CAS (Grant no. Y0205111L1) to C. Kim. The
authors wish to thank X.K. Fan for SEM operation and for preparing
the photos. We are also grateful to L.-E. Yang and G.-F. Chen for as-
sistance with the chromosome counts, and to X.-S. Zhang for the line
drawings. Finally, we would like to thank the anonymous reviewers
for their valuable comments and suggestions.
LITERATURE CITED

99
Deng & al. • The new genus Zhengyia (Urticaceae)
TAX ON
62 (1) • Februar y 2013: 89–99
99Version of Record (identical to print version).
Katoh, K., Asimenos, G. & Toh, H. 2009. Multiple alignment of DNA
sequences with MAFFT. Meth. Molec. Biol. 537: 39–64.
Kim, C., Shin, H., Chang, Y.-T. & Choi, H.-K. 2010. Speciation path-
way of Isoëtes (Isoëtaceae) in East Asia inferred from molecular
phylogenetic relationships. Amer. J. Bot. 97: 958–969.
Kitahara, M.V., Cairns, S.D., Stolarski, J., Blair, D. & Miller, D.J.
2010. A comprehensive phylogenetic analysis of the Scleractinia
(Cnidaria, Anthozoa) based on mitochondrial CO1 sequence data.
PLoS ONE 5: ell490, doi: 10.1371/journal.pone.0011490.
Kluge, A.G. & Farris, J.S. 1969. Quantitative phyletics and the evolu-
tion of anurans. Syst. Zool. 18: 1–32.
Levan, A., Fredya, K. & Sandberg, A. 1964. Nomenclat ure for cen-
tromeric position on chromosome. Hereditas 54: 201–220.
Maddison, W.P., Maddison, D.R . 2011. Mesquite: A modular system
for evolutionary analysis, version 2.75. http://mesquiteproject.org
(accessed 5 Apr. 2012).
Mizuki, I. & Takahashi, A. 2009. Secondary dispersal of Dioscorea
japonica (Dioscoreaceae) bulbils by rodents. J. Forest Res. 14:
95 –100.
Myers, N., Mit termeier, R.A., Mittermeier, C.G., Da Fonseca,
G.A.B. & Kent, J. 2000. Biodiversity hotspots for conservation
priorities. Nature 403: 853–858.
Okagami, N. 1979. Dormancy in bulbils of several herbaceous plants:
Effects of photoperiod, light, temperature, oxygen and gibberellic
acid. Bot. Mag. (Tokyo) 92: 39–58.
Posada, D. & Crandall, K. 1998. MODELTEST: Testing the model of
DNA substitution. Bioinformatics 14: 817–818.
Rambout, A. & Drummond, A.J. 2007. Tra ce r, ver si on 1.5. htt p: //t ree .
bio.ed.ac.uk/soft ware/tracer (accessed 3 Apr. 2012).
Ronquist, F. & Huelsenbeck, J.P. 2003. MrBaye s 3: Ba ye si an phy loge -
netic inference under mixed models. Bioinformatics 19: 1572–1574
Swofford, D.L. 2002. PAUP*: Phylogenetic analysis using parsimony
(*and other methods), version 4.0b10. Sunderland, Massachusetts:
Sinauer.
Taberlet, P., Gielly, L., Pautou, G. & Bouvet, J. 1991. Unive rsal pr im-
ers for amplification of th ree non-coding regions of chloroplast
DNA. Pl. Molec. Biol. 17: 1105–1109.
Appendix 1.
Voucher information and GenBank accession numbers for taxa used in this study. Information is presented i n the following order: Species
name, collection locality, abbreviation of OTU, collector and collection nu mber (herbarium acronym), Gen Bank accession number for nrITS, rbcL, psbA-
trnH, and trnL-F. (SNJ Exped. = Shennong jia Expedit ion).
INGROUP: Dendrocnide basirotunda (C.Y. Wu) Chew, Ch in a, Yun nan , dt 20 2, Deng 408 (K UN) , KC2 84962 , KC 28 498 8, KC28 50 40 , KC2 85014; Dendrocnide
urentissima (Gag ne p.) Chew, Ch in a, Yun nan , dt 20 3, Deng 40 9 ( KUN ), KC28 4963, KC2849 89, KC28 50 41, KC2 85 015; Girardinia diversifolia (Li nk) Frii s su bs p.
diversifolia, Chin a, Yun na n, dt115, Nie 4248 (KU N), KC284955, KC28 4981, KC285033, KC285007; Girardinia diversifolia subsp. suborbiculata (C.J. Ch en)
C.J. Chen & Friis, China, Hubei, dt094, SN J Exp ed . 20111107 004 (KU N), KC284950, KC284976, KC285028, KC285002; Girardinia diversifolia subsp. triloba
(C.J. Chen) C.J. Chen & Friis, China, Hubei, dt079, SNJ Exped. 20110906024 (KUN ), KC28 4945, KC28 49 71, KC28502 3, KC28 49 97; Hesperocnide tenella Tor r.,
U.S.A., California, sm336, B. Trusk 188 ( US) , KC2 84 967, KC2849 93 , KC2 85045 , KC2 85019; Laportea bulbifera (Siebold & Zu cc .) Wed d. , Ch ina , Hu be i, dt0 76,
SNJ Exped. 20110730020 (KUN ), KC284942, KC284968, KC285020, KC284994; China, Yunnan, dt109, Nie 3717 (KUN), KC284951, KC284977, KC285029,
KC28 500 3; Laportea cuspidata (Wedd.) Friis, China, Hubei, dt078, SNJ Exped. 20110714028 (KUN), KC284944, KC284970, KC285022, KC284996; China,
Hubei, dt083, SNJ Exped. 20110723090 (KUN), KC284946, KC284972, KC285024, KC284998; Laportea interrupta (L.) Chew, China, Yunnan, dt113, Nie
4263 (K U N) , KC2 84 954, KC 28 498 0, KC285032, KC2850 06; Poikilospermum suaveolens (Blume) Merr., China, Yunnan, dt205, De ng 411 (KU N), KC28496 4,
KC284990, KC285042, KC285016; Urtica dentata Hand.-Ma zz., Chi na, Hubei, dt230, SNJ Ex ped. 20110724077 (KUN), KC284966, KC284992, KC285044,
KC2 85 018; Urtica fissa E. Pr itz., China, Hubei, dt198, SN J E x ped . 201111120 01 (KUN), KC284960, KC284986, KC285038, KC285012; Urtica laetevirens
Maxim., China, Hunan, dt124, D.G. Zhang 134 (KU N), KC284956, KC2 84982, KC285034, KC2850 08; Urtica mairei H. Lév., Ch in a, Yunn an , dt110, Nie 4292
(KUN ), KC284952, KC284978, KC285030, KC285004; China , Sichuan, dt127, Liu & Yuan MY-121 (KUN ), KC284957, KC284983, KC285035, KC285009;
China , Hunan, dt199, Deng 406 (KU N) , KC28 4961, KC284987, KC285039, KC285013; Urtica thunbergiana Siebold & Zucc., China, Yunnan, dt112, Nie 4 275
(KUN ), KC284953, KC284979, KC285031, KC285005; Urtica triangularis Hand.-Mazz., China, Sichuan, dt133, Qingzhang Exped . 5801 (KUN), KC28 4958,
KC284984, KC285036, KC285010; China, Xizang, dt135, Qingzhang Exped. 12157 (KUN ), KC2849 59, KC28 498 5, KC285037, KC2 85011; Zhengyia shennon-
gensis T. Deng & al., Chi na , Hub ei, dt08 8, SNJ Ex ped . 20110904001 (KUN), KC2849 48, KC2849 74, KC285026, KC28500 0; Ch in a, Hu bei , dt0 91, SNJ Exped.
20111107 0 01 (KU N), KC28 4949, KC 28 4975, KC285 027, KC2850 01. OUTGROUP: Boehmeria spicata Thun b., Chin a, Hub ei , dt 07 7, SNJ Exped. 20110812020
(KU N), KC284943, KC2 84969, KC285021, KC 2849 95; Pilea plataniflora C.H. Wright, China, Hubei, dt228, SNJ Expe d. 20110714007 (KUN), KC284965,
KC284991, KC2850 43, KC285017; Fatoua villosa Nak ai, Chi na , Hube i, dt086 , SNJ Exped. 20110802047 (KUN), KC284947, KC284973, KC285025, KC284999.
Thomas, J.R., Gibson, D.J. & Middleton, B.A. 2005. Water dispersal
of vegetative bulbils of the invasive exotic Dioscorea oppositifo-
lia L. in southern Illinois. J. Torrey Bot. Soc. 132: 187–196.
Wake, D.B., Wake, M.H. & Specht, C.D. 2011. Homoplasy: From
de te cti ng pat ter n to dete rmi n in g pr oce ss and mechan ism of evolu-
tion. Science 331: 1032–1035.
Walck, J.L., Cofer, M.S. & Hidayati, S.N. 2010. Understanding the
ge rmi nat ion of bulbils from an ec ological per spe ct ive: A cas e st udy
on Chi nes e ya m (Dioscorea polystachya). Ann. Bot. (Oxdord) 106:
945 –955.
Wang, C.N., Moller, M. & Cronk, Q.C.B. 2004. Aspects of sexual
failure in the reproductive processes of a rare bulbiliferous plant,
Titanotrichum oldhamii (Ge sn er iac ea e), in subtr op ic al Asi a. Sexual
Pl. Reprod . 17: 23–31.
White, T.J., Bruns, T., Lee, S. & Taylor, J. 1990. Amplif icat ion and di-
rect sequencing of f ungal ribosomal RNA genes for phylogenetics.
Pp. 315–322 in: In n is , M. A. , Gelf an d, D.H ., Shi nsk y, J. J. & White,
T. J. (eds.), PCR protocols: A guide to methods and applications.
San Diego: Academic Press.
Woodland, D.W., Bassett, I. J. & Crompton, C.W. 1976. The an nual
species of stinging nettle (Hesperocnide and Urtica) in Nor th
America. Canad. J. Bot. 54: 374–383.
Woodland, D.W., Bassett, I. J., Crompton, C. & Forget, S. 1982.
Biosystematics of the perennial North-American taxa of Urtica.
1. Chromosome-number, hybridization, and palynology. Syst. Bot.
7: 269–281.
Xie, Z. 2003. Characteristics and conservation priority of threatened
plants in the Yangtze valley. Biodivers. & Conser vation 12: 65–7 2.
Yang, Z. & Rannala, B. 1997. Bayesian phylogenetic inference using
DNA sequences: A Markov Chain Monte Carlo method. Molec.
Biol. Evol. 14: 717–724.
Ying T.S. 2001. Species diversity and distribution pattern of seed plants
in China. Biodivers. Sci. 9: 393–398.
Zurawski, G., Perrot, B., Bottomley, W. & Paul, R.W. 19 81. The
structure of the gene for the large subunit of ribulose 1,5-bispho-
sphate carboxylase from spinach chloroplast DNA. Molec. Biol.
9: 3251–3270.