Introduction
Acer Linn. of the Aceraceae family is one of the largest
genera of deciduous forests in the northern hemisphere with over 130 species
(Xu 1998; Xu et al. 2013). This
genus is furnished with comprehensive fossil record and is often used as a
model plant to study the origin and evolution of woody plants (Yang
and Li 2010). Many of its taxa are also ideal materials for studying the
intermittent distribution of plants in East Asia-North America (Chang
et al. 1991). The Acer plants carry unique co-source
characteristics such as opposite leaves and samara, which are easy to
distinguish from the adjacent genera. However, other morphological
characteristics, such as leaf shape, fruit shape and inflorescence are highly
variable among species. Thus, classification and phylogenetic study of this
genus is comparatively complex (Pojárkova 1933; Tian et al. 2002).
In 1885, Pax established the first system of Acer, in which the genus was classified into 14 sections, mainly on
the basis of the relative position of stamens to discs (Pax 1885). Since then,
many researchers have successively studied the system of Acer in the
fields of morphology (Koidzumi 1911; Pojárkova 1933; Rehder 1936;
Ogata 1967; Fang 1981; De Jong 1994; Xu et
al. 2013), relic fossils (Wolfe and Tanai 1987),
palynology (Erdtmen 1952; Tian et al. 2001), isozyme (Liu et
al. 2001; Wang et al. 2007),
molecular systematics (Suh et al.
2000; Tian et al. 2002; Grimm et al. 2006) and branch taxonomy (Fang
1981; Tian and Li 2004). By now, the system of Acer has been
fundamentally defined. However, there are still disputes over classification of
some sections, which pose complications in species identification of Acer.
In addition, the infrageneric phylogenetic relationships in this genus are also
controversial. Although some evidence including gross morphology, seed
proteins, chemical composition, geographic distributions, fossils and molecular
information are available, the conclusions are not in consensus (Tian et al.
2002).
DNA barcode technology is a
molecular biological technique and is based on the principle of sequencing
method (Deef 2019; Afzal
et al. 2020). It has been
widely used in the field of biodiversity assessment, species identification,
phylogenetic analysis and ecological studies (Lin et al. 2017; Shinwari et al. 2018; Mitchell et al. 2020). For animals, the
mitochondrial cytochrome oxidase I (COI) gene has been considered as the
standard DNA barcode (CBOL Plant Working Group 2009). However, the choice of DNA barcoding in
plants is more complicated compared to animals owing to their uniparentally
inherited, nonrecombining and structurally stable genome (Kress et al.
2005). In recent years, many gene sequences such as matK, rbcL, psbA-trnH,
rpl16, atpF-atpH,
ycf1 and ITS, have been successively
used in distinguishing different taxonomic groups in plants, but no universal
DNA barcoding has been found yet (Kress et al. 2005; Yao et al. 2010; Dong et al. 2015; He et al. 2019; Prasad et al. 2020; Wu et al.
2020).
ITS2, a non-coding region of the
ribosomal DNA ITS, has been proven as a potential universal DNA barcode to
authenticate species (Feng et al. 2016; Mbareche et al. 2020; Shi et al. 2020). For herbs, the identification success rate
of ITS2 was up to 92.7% (Chen et al.
2010). It also performed well in species-level discrimination of Physalis L., Panax L. and Paris L. (Feng et
al. 2016; Sun et al. 2016). Compared with ITS sequence, ITS2 holds
advantages such as shorter sequence length and higher amplification efficiency
and is therefore considered as a suitable candidate sequence for
standard barcoding for plants (Liu et al.
2012; Dong et al. 2015; Zhao et al. 2015; Timpano
et al. 2020). Furthermore, ITS2
has been proven to be applicable in plant phylogenetic studies (Li et al. 2014; Feng et al. 2016;
Sun et al. 2016). In this study, ITS2
region was used to barcode Acer and to reconstruct the phylogenetic
relationships of Acer species.
Materials and Methods
Experimental material and sampling
A total of 60 samples from 50 Acer species were collected for this
study (Table 1). In addition, 277 published Acer
ITS2 sequences from 94 Acer species were downloaded from GenBank (Table
2). There were totally 105 species which represent the 23 sections of Acer of Xu’s system (Xu 1996; Xu et al. 2013). All samples were confirmed
using the botanical information from Chinese Virtual Herbarium
(http://www.cvh.org.cn/). Vouchers and digital images were deposited in the
Herbarium of Ningbo Key Laboratory of Landscape Plant Development, Ningbo City
College of Vocational Technology.
DNA Extraction, PCR amplification and sequencing
In order
to extract DNA, 2 mg of dried leaves were milled
with liquid nitrogen. This crushed blend was used to extract genomic DNA as per manufacturer’s recommendation (Lifefeng Co., Shanghai,
China). Using a NanoDrop 2000 spectrophotometer
(Thermo Fisher Scientific Inc., USA), the
quality and quantity of the extracted DNA was determined. All samples were
diluted to 100ng. μL-1
for later use. PCR amplification was performed using AG 22331 sequence
amplification apparatus (Eppendorf Ltd., Hamburg, Germany). Forward primer was
ITS4: 5’-TCCTCCGCTTATTGATATG-3’ and reverse primer was ITS5: 5’-GGAAGTAAAAGTCGTAACAAGG-3’. A total of 50 μL PCR reaction mix was
prepared containing 1 μL
genome DNA, 1 μL
forward and reverse primer (concentration is 10 μmol·L-1), 25 μL 2 × Taq PCR Mix (BioTeke Co., Beijing, China) and 22 μL ddH2O. The PCR
amplification conditions were applied as follow: pre-degeneration for 5 min at
94°C, degeneration for 30 s at 94°C, annealing for 30 s at 55°C, renaturation
for 1 min at 72°C and extend for 7 min at 72°C for a total of 35 cycles. The
PCR products were visualized in 1% agarose gel electrophoresis purified and
recovered by DNA Recovery Kit (Axygen, Hangzhou,
China). These PCR products sequenced using PCR primers in both directions by the Shanghai Sunny Biotechnology Co., LTD. Newly acquired sequences were submitted to GenBank (Table
1).
Data analysis
The raw sequences were edited using
CodonCode Aligner 5.1 software (CodonCode
Co., USA) to remove low-quality fragments, and the sequences less than 150 bp
were deprecated. The 5.8 s and 28 s region of all sequences was removed
according to the Hidden Markov model (HMM) to retain the complete ITS2 region
(Keller et al. 2009). Clustal
X2.1 software was used for multi-sequence comparison of sequences, and BioEdit V5.0.6 software was used to calculate the length
and GC contents (Hu et al. 2011). The K2P (kimura-2 parameter)
genetic distance between sequences was obtained by MEGA 6.0 software (Tamura et al. 2013). DNA barcoding gaps were plotted according to intra-
and inter-specific variations of the ITS2 sequences and Wilcoxon signed-rank
tests were performed (Slabbinck et al. 2008; Lee et al.
2016). TaxonDNA 1.0 software was used to evaluate the
identification efficiency of ITS2 region (Slabbinck et al. 2008). In addition, BLASTA1 method
was also applied to assess the discriminatory capability of ITS2 sequence (Gao et
al. 2010).
Phylogenetic analysis of Acer was performed using Bayesian inference (BI) method on MRBayes 3.1 (Huelsenbeck
and Ronquist 2001), and the best-fit model (GTR+G) was selected by the Akaike information criterion
(AIC) in MrModeltest
2.3 (Nylander
2004). Posterior probabilities (PP) for individual clades were computed with MrBayes. Dipteronia dyeriana was
selected as outgroup for its close relation to Acer species. Furthermore, the Neighbor-Net (NN) splits
phylogenetic network of Acer was
constructed using the SplitsTree 4.13.1 software
based on the uncorrected p-distance (Hu
et al. 2011).
Results
ITS2 sequence properties
Table
1: Voucher information of the Acer plants samples in this
study
|
Section |
Species |
Voucher No. |
Locality information |
GenBank/Accession No. |
|
Spicata |
A. ukurunduense
Trautv. et Meyer |
LN06 |
Kuandian, Dandong, Liaoning |
KY649425 |
|
Palmata |
A. palmatum Thunb. |
FH01 |
Mt. Siming, Ningbo, Zhejiang |
KU902463 |
|
|
A. linganense
Fang et P. L.
Chiu |
NJ04 |
Mt. Zhongshan, Nanjing, Jiangsu |
KX494348 |
|
|
A. japonicum Thunb. |
SH10 |
Chengdu, Sichuan |
KX494352 |
|
|
A. pseudosieboldianum
Komarov |
LN04 |
Kuandian, Dandong, Liaoning |
KX494353 |
|
|
A. flabellatum
Rehd. |
HZ07 |
Hangzhou,
Zhejiang |
KU902482 |
|
|
A. elegantulum
Fang & Chiu |
GL01 |
Yanshan, Guilin, Guangxi |
KU902460 |
|
|
A. elegantulum Fang & Chiu |
KM01 |
Kunming, Yunnan |
KU902461 |
|
|
A. elegantulum Fang & Chiu |
HZ04 |
Mt. Tianmu, Hangzhou, Zhejiang |
KU902487 |
|
|
A. elegantulum Fang & Chiu |
NB01 |
Mt. Siming, Ningbo, Zhejiang |
KU902488 |
|
|
A. sinense
Pax |
HZ06 |
Hangzhou,
Zhejiang |
KU902493 |
|
|
A. pubinerve Rehd. |
SH12 |
Chenshan, Shanghai |
KX494354 |
|
|
A. kweilinense
Fang & Fang |
SC01 |
Chengdu, Sichuan |
KU902496 |
|
|
A. wilsonii Rehd. |
SH04 |
Chenshan, Shanghai |
KU902481 |
|
|
A. oliverianum
Pax |
WH06 |
Wuhan, Hubei |
KU902485 |
|
|
A. fabri
Hance |
WH04 |
Wuhan, Hubei |
KU902466 |
|
|
A. fabri Hance |
GL03 |
Yanshan, Guilin, Guangxi |
KU902465 |
|
|
A.laevigatum Wall. |
WH03 |
Wuhan, Hubei |
KU902462 |
|
Platanoidea |
A. miaotaiense
Tsoong |
SH02 |
Chenshan, Shanghai |
KU902468 |
|
|
A.yangjuechi Fang & Chiu |
HZ05 |
Taoyuanling, Hangzhou, Zhejiang |
KU902489 |
|
|
A.yangjuechi Fang & Chiu |
WH07 |
Shennongjia Forestry District, Hubei |
KU902490 |
|
|
A. campestre
L. |
HZ15 |
Taoyuanling, Hangzhou, Zhejiang |
KY649427 |
|
|
A. acutum
Fang |
HZ02 |
Mt. Tianmu, Hangzhou, Zhejiang |
KU902475 |
|
|
A. acutum Fang |
SH03 |
Chenshan, Shanghai |
KU902473 |
|
|
A. acutum Fang |
BJ01 |
Fragrance Hill,
Beijing |
KU902474 |
|
|
A. truncatum Bunge |
WH08 |
Wuhan, Hubei |
KU902494 |
|
|
A. mono Maxim. |
HZ10 |
Mt. Tianmu, Hangzhou, Zhejiang |
KX494362 |
|
|
A. cappadocicum
Gled. var. sinicum Rehd. |
KM06 |
Kunming, Yunnan |
KU902486 |
|
|
A. longipes Franch.
ex Rehd. var. weixiense Fang |
KM05 |
Kunming, Yunnan |
KU902484 |
|
|
A. amplum subsp. tientaiense Chen |
HZ08 |
Mt. Tiantai, Taizhou, Zhejiang |
KY649428 |
|
Ginnala |
A. tataricum
subsp. ginnala
Maxim. |
SH06 |
Chenshan, Shanghai |
KU902495 |
|
Oblonga |
A. buergerianum
Miq. |
BJ02 |
Fragrance Hill,
Beijing |
KU902477 |
|
|
A. buergerianum Miq. |
FH02 |
Xikou, Fenghua, Zhejiang |
KU902478 |
|
|
A. buergerianum Miq. |
LS02 |
Mt. Lushan, Jiujiang, Jiangxi |
KU902479 |
|
|
A. paxii
Franch. |
KM02 |
Kunming, Yunnan |
KU902464 |
|
|
A. cinnamomifolium
Hayata |
KM08 |
Kunming, Yunnan |
KU902492 |
|
|
A. oblongum
Wall. ex DC. |
WH02 |
Yaowan, Wuhan, Hubei |
KU902459 |
|
|
A. wangchii Fang subsp. tsinyunense
Fang |
CQ01 |
MT. Jinyun, Chongqing |
KU902498 |
|
|
A. cordatum
Pax |
SH09 |
Chenshan, Shanghai |
KY649430 |
|
Macrantha |
A. davidii
subsp. grosseri Pax |
KM10 |
Anning District, Kunming,Yunnan |
KX494355 |
|
|
A. hookeri
Miq. |
HZ01 |
Hangzhou,
Zhejiang |
KU902472 |
|
|
A. davidii Franch. |
KM03 |
Kunming, Yunnan |
KU902471 |
|
|
A. capillipes
Maxim. |
SH07 |
Xuhui, Shanghai |
KU902502 |
|
|
A. pectinatum Wall. ex Nichols. |
KM15 |
Kunming, Yunnan |
KX494356 |
|
|
A. tegmentosum
Maxim. |
LN01 |
Kuandian, Dandong, Liaoning |
KU902470 |
|
|
A.komarovii Pojark. |
SX01 |
Xian, Shanxi |
KY649429 |
|
|
A. caudatifolium
Hayata |
HZ07 |
Taoyuanling, Hangzhou, Zhejiang |
KU902500 |
|
Lithocarpa |
A. sinopurpurascens
Cheng |
HZ03 |
Taoyuanling, Hangzhou, Zhejiang |
KU902483 |
|
|
A. tsinglingense Fang & Hsieh |
HN05 |
MT. Funiu, Luanchuan, Henan |
KU902469 |
|
|
A. sterculiaceum subsp. franchetii (Pax)
Murray |
WH01 |
Wuhan, Hubei |
KU902458 |
|
|
A. kungshanense
Fang & Chang |
KM11 |
Kunming, Yunnan |
KX494357 |
|
Pentaphylla |
A. pentaphyllum Diels |
KM12 |
Kunming, Yunnan |
KX494358 |
|
Trifoliata |
A. griseum (Franch.)
Pax |
NJ02 |
Mt. Zhongshan, Nanjing, Jiangsu |
KX494359 |
|
|
A. nikoense
(Franch.) Pax |
LS01 |
Mt. Lushan, Jiujiang, Jiangxi |
KU902467 |
|
|
A. triflorum
Komarov |
LN02 |
Kuandian, Dandong, Liaoning |
KU902476 |
|
|
A. mandshuricum Maxim. |
LN03 |
Kuandian, Dandong, Liaoning |
KX494360 |
|
Arguta |
A. barbinerve Maxim. |
LN08 |
Kuandian, Dandong, Liaoning |
KY649432 |
|
Rubra |
A. saccharinum
L. |
SX02 |
Xian, Shanxi |
KY649431 |
|
Cissifolia |
A. henryi
Pax |
SH08 |
Chenshan, Shanghai |
KX494361 |
|
Negundo |
A. negundo L. |
SH01 |
Chenshan, Shanghai |
KU902456 |
|
|
A. negundo L. |
LN05 |
Kuandian, Dandong, Liaoning |
KY649424 |
The ITS2 sequences from 60 Acer samples were obtained under this
study, and a total of 337 sequences (277 sequences downloaded from GenBank)
were used for analysis. It was noticed that the ITS2 sequence length ranges
from 208 to 254 bp with an average length of 234 bp. The GC contents were
different among species, with the lowest value of 57.63%, the highest value of
68.60% and the average value of 62.02%. Post-alignment analysis identified that
the sequence length was 296 bp, containing 107 conserved sites, 181 variable
sites and 158 reduced information sites. Thus, the ITS2 fragments of Acer species
displayed considerable variation in the length and GC
content.
Table
2: GenBank accession numbers
of Acer plants samples
and Dipteronia sinensis
(Outgroup) in this study
|
Section |
Species |
GenBank/Accession No. |
|
Parviflora |
A.nipponicum Hara |
AF020380, DQ366140, DQ366141, DQ366143 |
|
Distyla |
A.distylum Sieb. & Zucc. |
AF241485, AF401155, DQ238354, DQ238355 |
|
Spicata |
A. caudatum
Wall. |
AY605432, AY605433 |
|
|
A. ukurunduense
Trautv. et Meyer |
AY605434, AY605435 |
|
|
A. spicatum
Lam. |
U89911, AF241503, AF401122 |
|
Palmata |
A. palmatum Thunb. |
AB683975, JF980312, AB690435 |
|
|
A. linganense Fang et P. L. Chiu |
KX494348 |
|
|
A. japonicum Thunb. |
U57776, AF241489 |
|
|
A. pseudosieboldianum Komarov |
DQ238405, DQ238406 |
|
|
A. shirasawanum Koidzumi |
AY605428, DQ238409, DQ238409, DQ238410, DQ238411 |
|
|
A. circinatum Pursh |
AY605412, AY605413, HM352653 |
|
|
A. flabellatum Rehd. |
AY605417, DQ238394 |
|
|
A. sinense Pax |
HM352663 |
|
|
A. pubinerve Rehd. |
KP093224, AF401125 |
|
|
A. wilsonii Rehd. |
HM352665 |
|
|
A. oliverianum Pax |
AY605422, AY605423, AY605424 |
|
|
A. tutcheri Duth. |
KP093225 |
|
|
A. miaoshanicum Fang |
AF401124 |
|
|
A. erianthum Sch. |
EU720501, DQ238391, DQ238392, DQ238393 |
|
|
A. tonkinense Lec. |
HM352664 |
|
|
A. fabri Hance |
KP096075, KP093223, JF975777 |
|
|
A. crassum Hu &
Cheng |
AF401135 |
|
Glabra |
A. glabrum
Torrey |
DQ238338, AF056017, AF241488, AF401139, DQ238337, DQ238340 |
|
Platanoidea |
A. campestre L. |
LK022464, LK022604, LK022459, AF401158 |
|
|
A. miyabei
Maxim. |
AY605451, AY605452 |
|
|
A. truncatum Bunge |
AY605459, LK022669 |
|
|
A. mono Maxim. |
U57775, JF980310, AF241491 |
|
|
A. cappadocicum var. divergens (Pax) Murray |
LK022629, LK022630, LK022631, LK022632 |
|
|
A. cappadocicum Gled. |
AJ634579, DQ238439, DQ238440, DQ238444, LK022625, LK022626 |
|
|
A. platanoides L. |
AF401136, EF494236, LK022679, LK022672, U57773, DQ238461 |
|
Ginnala |
A. tataricum subsp.
ginnala Maxim. |
AF241487, AF401147 |
|
|
A. tataricum subsp.
semenovii (Regel & Herder) Murray |
AY605365, AY605366 |
|
|
A. tataricum L. |
AF401146, AM265511, JF975781, AM265512 |
|
|
A. tataricum subsp.
aidzuense Franchet |
AM113519, AM113520, AM113521 |
|
Acer |
A. caesium Wall. ex Brandis |
AY605293, AY605294, DQ366115, DQ366116, DQ366117 |
|
|
A.caesium Wall.
ex Brandis subsp. giraldii Murray |
AY605296, DQ366121, AY605295, AF406969 |
|
|
A. pseudoplatanus
L. |
DQ366132, AY605338, AY605340, AY605346, DQ366131, DQ366133 |
|
|
A. heldreichii Orphanides
ex Boissier |
AY605301, AY605302, AM238280, AY605303, AY605304 |
|
|
A. trautvetteri
Medvedev |
AY605351, AF401126, AY605355, AM238285 |
|
|
A. velutinum Boissier |
AM238291, AM238294, AY605358, DQ366132, DQ366137 |
|
Saccharodendron |
A. saccharum
L. |
EU720502, AF401152 |
|
|
A. saccharum
ssp. skutchii (Rehd.) Murray |
FJ906753, FJ906754, FJ906755 |
|
|
A. saccharum
ssp. floridanum (Chap.) Desma. |
DQ366138, DQ366139 |
|
Pubescentia |
A. pilosum Maxim. |
DQ238344, DQ238345, DQ238346 |
|
Oblonga |
A. buergerianum Miq. |
AF401133, U89908, AY605466 |
|
|
A. buergerianum ssp. formosanum
Hance |
FN651690, FN651694, FN651695 |
|
|
A. paxii Franch. |
AF401132 |
|
|
A. cinnamomifolium Hayata |
DQ238468, DQ238470 |
|
|
A. oblongum Wall. ex DC. |
AF241494 |
|
|
A. albopurpurascens
Hayata |
DQ238471, FN651702, FN651712, |
|
|
A. poliophyllum Fang |
AF401134 |
|
|
A. cordatum Pax |
HM352654 |
|
Goniocarpa |
A. monspessulanum L. |
AY605321, AF401127, AM238361, DQ366128 |
|
|
A. hyrcanum Fisch.
& Mey. |
DQ366129, DQ366130, AY605305, AY605306 |
|
|
A. obtusifolium Sibthorp & Smith |
AM238327, AM238331, AM238332 |
|
|
A. opalus Mill. |
AF401128, AY605328, AM238302, AY605331, AY605332 |
|
|
AY605352, AY605353, DQ366123 |
|
|
Macrantha |
A. davidii subsp. grosseri Pax |
HM008383, HM008394, HM008397, AY605396 |
|
|
A. davidii Franch. |
AF401144, HM008393 |
|
|
A. capillipes
Maxim. |
DQ238368, DQ238371 |
|
|
A. laxiflorum Pax |
HM008386 |
|
|
A. crataegifolium Siebold
& Zucc. |
AY605391, DQ238376, DQ238378, DQ238379 |
|
|
A. micranthum Siebold
& Zucc. |
HM008404, HM008407, AF020369 |
|
|
A. rufinerve Siebold
& Zucc. |
AY605399, AY605400, DQ238372, DQ238373, DQ238374 |
|
|
A. komarovii
Pojark. |
HM008405 |
|
|
A. maximowiczii
Pax |
HM008400, HM008401, HM008402 |
|
|
A. pectinatum Wall. ex Nichols. |
KX494356, JF975779 |
|
|
A. tegmentosum
Maxim. |
DQ366113, AF241505 |
|
|
A. caudatifolium Hayata |
DQ238380 |
|
|
A. pensylvanicum
L. |
AY605398, AF020370, AF241497 |
|
|
A. wardii Smith |
DQ366146, DQ238413, DQ238415, DQ238416, DQ238418 |
|
Lithocarpa |
A. sterculiaceum
subsp. franchetii (Pax)
Murray |
DQ366145 |
|
|
A. kungshanense
Fang & Chang |
AF401143 |
|
|
A. diabolicum
Blime |
AF241484, AY605382, AY605383, AF020366 |
|
Marcophylla |
A. macrophyllum
Pursh |
AY605387, AY605388, DQ238347, DQ238350, AF401156 |
|
Pentaphylla |
A. pentaphyllum Diels |
DQ238477, DQ238478, AF241498, AF401137 |
|
Trifoliata |
A. griseum (Franch.) Pax |
DQ238480, DQ238481, AF401131, AY605469 |
|
|
A. nikoense (Franch.) Pax |
DQ238483, DQ238487, AJ698721, AJ698722 |
|
|
A. triflorum Komarov |
AF241506, AJ698128 |
|
|
A. mandshuricum Maxim. |
DQ238473, DQ238474, DQ238476, AF401129 |
|
Hyptiocarpa |
A. decandrum (Merr.)
Murray |
AF401149 |
|
|
A. laurinum Hasskarl |
DQ366114, AM113541, AM113542, AM113543 |
|
Arguta |
A. stachyophyllum Hiern |
AY605373, AY605374, AY605375, AY605376 |
|
|
A. stachyophyllum
subsp. betulifolium Maximowicz |
AY605373, AY605374 |
|
|
A. acuminatum Wall. |
AY605370, AY605371, AY605372 |
|
|
A. argutum
Maxim. |
AF401153, AF241480 |
|
|
A. barbinerve Maxim. |
AJ634569, AJ634571, AJ634573 |
|
Rubra |
A. pycnanthum
Koch. |
AM113528, AM113529 |
|
|
A. rubrum
L. |
AY605461, AF401150, AF020385 |
|
|
A. saccharinum
L. |
AF401151, AY605462, AY605463, AM113531 |
|
Indivisa |
A. carpinifolium Siebold
& Zucc. |
AF401148, AY605377, AY605379, AY605380 |
|
Cissifolia |
A. henryi Pax |
AY605404, AY605405, AF401141, AJ634574 |
|
|
A. cissifolium (Sieb.
& Zucc.) Koch. |
AY605401, AY605402, AF241483, AF401140 |
|
Negundo |
A. negundo L. |
AF401142, U89909, DQ238362, DQ238356 |
|
Outgroups |
D. sinensis Oliv. |
AY605290, EU720445, AF401121 |
Genetic variation within and between Acer species
The genetic variation of the Acer species samples were evaluated by MEGA 6.0 and six parameters (average
inter-specific distance, theta prime, the minimum inter-specific distance,
average intra-specific distance, theta and coalescent depth) were used to characterize inter- and intra-specific variation. Table 3 exhibited the calculated results of six
parameters that the divergence of congeneric was relatively higher than that of conspecific. The average inter-specific genetic distance (0.0766
± 0.0299) was 15 times of the average intra-specific genetic distance (0.0045 ± 0.0096), and the
minimum inter-specific genetic distance (0.0728 ± 0.0299) was significantly
higher than the maximum average intra-specific genetic distance (0.0073 ±
0.0129).
Table
3: Interspecific
and Intraspecific variation of the ITS2 sequence in 337 samples of 105 Acer
species
|
Measurement |
K2P value |
|
Average interspecific distance |
0.0777±0.0293 |
|
Theta prime |
0.0766±0.0299 |
|
The minimum interspecific
distance |
0.0728±0.0299 |
|
Average intraspecific distance |
0.0048±0.0108 |
|
Theta |
0.0045±0.0096 |
|
Coalescent depth |
0.0073±0.0129 |
Table
4: Authentication efficiency for ITS2 by using different
methods
|
Parameter |
Correct
identification |
Ambiguous
identification |
incorrect
identification |
No
match |
|
All species barcodes |
216 (64.09%) |
100 (29.67%) |
15 (4.45%) |
6 (1.78%) |
|
Best match |
218 (64.68%) |
88 (26.11%) |
31 (9.19%) |
0 |
|
Best close match |
216 (64.09%) |
88 (26.11%) |
27 (8.01%) |
6 (1.78%) |
|
BLASTA1 |
222 (65.87%) |
86 (25.52%) |
29 (8.61%) |
0 |
Barcoding gap test
The genetic distances of ITS2
sequences were calculated by TaxonDNA 1.0 software,
and the barcoding gap of genetic variation distribution within and between Acer species was plotted (Fig. 1). There was an obvious
barcoding gap in ITS2. These results highlight that ITS2 gene can potentially
be applied to identify and differentiate species. Meanwhile, Wilcoxon test was
used to further analyze the inter-specific and intra-specific divergence of ITS2 sequences. The analysis showed that the
inter-specific divergence of ITS2 sequences was significantly (P<0.001) greater than the
intra-specific variation.
Authentication ability of ITS2 region
TaxonDNA 1.0 software was used to evaluate the identification
efficiency of ITS2 region, and three criteria (Best Match: BM; Best Close
Match: BCM; and All Species Barcodes: ASB) were selected to analyze the
authentication ability of ITS2 sequences (Table 4). The results showed that
ITS2 region had relatively higher species identification success rates
(>64%) and low misidentification rates (<10%) based on the BM, BCM and
ASB analysis. For BLASTA1 analysis, similar data were obtained (Table 4). In
addition, TaxonDNA 1.0 software was also applied to
estimate the discriminatory capability of ITS2 region to sister species. Nearly
two-thirds (64.76%) of the ITS2 sequences had considerable inter-specific
heterogeneity that were larger than intra-specific variation (Fig. 2), which
revealed that the ITS2 sequences had obvious inter-specific boundaries for most
species of Acer.
Phylogenetic analysis
%201582-1590_files/image002.png)
Fig.
1: Relative distribution of inter-specific distance between Acer
species and intra-specific variation in the ITS2 region using K2P genetic
distances
%201582-1590_files/image004.png)
Fig.
2: The heterogeneity and separation for individual taxa of
ITS2 based on 105 Acer species by TaxonGap
The left side gives the list of Acer species used in this study. The right side represents the within species
heterogeneity (showed as light gray horizontal bar) and between-species separation (presented as dark gray horizontal bar)
%201582-1590_files/image006.png)
Fig.
3: Bayesian
phylogenetic tree based on ITS2 sequences for Acer
species
Posterior
probabilities (PP) ≥50 are shown above/down the branch
%201582-1590_files/image008.png)
Fig.
4: Neighbor-net splits network for Acer species
computed with uncorrected p-distances based on ITS2 sequences
According to the taxonomic treatment of Acer in Xu’s system (Xu 1996; Xu et al.
2013), all the Acer
species used in this study belonged to 23 sections (Table 1, 2). By using BI method, a phylogenetic tree was constructed based on the ITS2 sequences, and all the Acer
species were clustered into five main groups (Fig. 3). Group I
contained 31 Acer species from eight sections, and was further classified into three subgroups, among which subgroup I-2 was
a monophyletic group formed by sect. Platanoidea. Subgroup I-1 comprised 18 species: three from sect. Lithocarpa, fourteen from sect. Macrantha, and one from sect. Marcophylla. Subgroup I-3 included those species from
sect. Parviflora, sect. Spicata, sect. Distyla and sect. Negundog. Group II involved 19 species from four sections,
was also been further categorized into three subgroups. Subgroup II-1 contained
two species (A. caesium and A.caesium
subsp. giraldii) belonging to sect. Acer.
Subgroup II-2 was formed by five
species from sect. Arguta. All species from sect. Goniocarp and sect. Saccharodendron and four species (A. pseudoplatanus, A. heldreichii, A. trautvetteri and A. velutinum) from sect. Acer were
clusted into subgroup II-3. Group III contained 22 species which clustered into three subgroups. Subgroup III-1 included all species from sect. Pubescentia, sect. Oblong, sect. Trifoliata and sect. Pentaphylla. Subgroup III-2 was composed of two species (A. decandrum
and A. laurinum) from sect. Hyptiocarpat. Subgroup III-3 contained the species from sect. Rubra and
sect. Ginnala. A total of 22 species were assigned group IV, which was further subdivided into two subgroups. Subgroup IV-1 included one species (A. carpinifolium)
from sect. Indivisa. Subgroup IV-2 contained 21 species, in addition to the species from sect. Palmata,
all species (A. henryi and A. cissifolium) from Sect. Cissifolia and the
species A. wardii from sect. Macrantha were clustered in this subgroup. A. Glabrum, a species from
Sect. Glabra was distant from other Acer species, and which constituted a separate group V.
In order to further clarify the phylogenetic
relationship of Acer, a NN splits graph was constructed. Resultant NN splits graph exhibited a similar phylogenetic relationship among Acer to bayesian analysis (Fig. 4).
Discussion
In previous studies, ITS2 has been
proven to have good species-identification capability and therefore been
suggested as a standard barcode to identify plant species (Chen et al.
2010; Yao et al. 2010; Feng et al. 2016; Sun et al. 2016). In our study, the ITS2 locus exhibited sufficient genetic variability among
congeneric Acer species and also displayed a relatively high
discrimination efficiency (>64% for BM, BCM, ASB and BLASTA1 analysis). For most Acer species, they could be successfully identified based
on their ITS2 locus. However,
the ITS2 locus was less effective
to discriminate morphologically similar species of Sect. Palmata. For instance, four species (A. elegantulum, A. sinense, A. pubinerve and A. kweilinense)
in sect. Palmata could not be
distinguished due to their
identical ITS2 sequences. Thus, some other DNA barcodes should be explored and
applied to identify these closely relative sect. Palmata species. Here, it should be pointed
out that the taxonomy of sect. Palmata
has always
been controversial and many species in this section such as A. olivaceum, A. changhuaense,
A. schneiderianum and A. anhweiense have been redefined in the Flora of China (Fang 1981; De Jong 1994; Xu et al. 2013). Our result
implied that there might be more species of this section to be revised.
Many studies have
confirmed that ITS2 could not only barcode plant species, but also provided a
superior phylogenetic marker for plant systematics and evolutionary research
(Chen et al. 2010; Liu et al. 2012; Zhao et al. 2015; Feng
et al. 2016). In this study, the ITS2 region exhibited sound
applicability in the identification of Acer, and it also provided a
taxonomic signature for Acer taxonomy. As shown in the dendrogram
generated from ITS2 data, the genus Acer was
revealed to be monophyletic (Fig. 3). However, monophyletic
groups could not be formed in some sections of Acer, such as Macrantha, Spicata, Acer and Oblonga (Fig. 3). As for sect. Macrantha, 14 species were sampled
representing three different series (Micrantha,
Tegmentosa and Crataegifolia)
of this section, and were categorized into two
clades. The species from ser. Tegmentosa and
ser. Crataegifolia formed two monophyletic
clades, and the species from ser. Micrantha
were grouped together with the species from sect. Marcophylla, it made Macrantha a possible paraphyletic
section. It should be noted that A. pectinatum in ser. Tegmentosa was nested within ser. Micrantha
species (Xu 1996) with relatively high support (PP=83), indicating A. pectinatum should be reassigned from ser. Tegmentosa
to ser. Micranthum. In addition,
as reported earlier (Grimm et al. 2006), A. wardii
from sect. Macrantha was included within group IV (IV-2) together with the species from sect. Palmata.
Morphologically, A. wardii
was similar to
sect. Macrantha, but some characters, such as
conspicuous bracts, reflexed sepals and amphistaminal
disk, were atypical features of Macrantha. Thus, de Jong’s treatment of placing this
species in a monotypic section was supported here (De Jong 1994). The species from sect. Spicata
were grouped into subgroup I-3 together with species from sect. Distyla and Parviflora, indicating a
close relationship among the sections, although the internal support was
relatively weak (PP=62). This was consistent with Momotani’s and de Jong’s treatments
by placing sect. Distyla and sect. Parviflora under sect.
Parviflora as another two series.
Actually, these three sections shared similar morphological features of
cotyledon, samara, endocarp and pollen (Ogata 1967). A. negundo was strongly supported (PP=96) as a sister species to A. distylum of sect. Distyla. In gross morphology, these two species are obviously different, such as the type of inflorescences, the number of
bud-scales and the arrangement of leaves. In Xu’s system (2013), A. negundo was combined with sect. Cissifolia for
their compound leaves. Our result indicated that A.
negundo should be treated as a separate section rather than a species under sect. Cissifolia. It was also supported by palynological evidence of Acer (Tian et al. 2001).
The species
from sect. Acer (except A. caesium and A.caesium subsp.
giraldii) were grouped into
subgroup II-3 together with the species from sect. Goniocarpa and sect. Saccharodendron
(Fig. 3), indicating the
phylogenetic relationships among the sections were
close. In fact, due to the gross morphological
similarities, these sections were reduced to the rank of series and put under sect. Acer by De Jong
(1994). It was also backed by Ogata’s study on the wood rays of Acer (Ogata 1967). Therefore, we supported de Jong’s treatment of merging the three
sections into sect. Acer (De Jong 1994).
However, our finding didn’t support
de Jong’s
division of this section into three series (ser. Acer, ser. Monspessulana and ser. Saccharodendron). A. caesium and its subspecies failed to be included into subgroup II-3 and
formed subgroup II-1. It is possibly caused by the long-time geographical
isolation, as the two species are endemic to Southwest China, while other sect.
Acer species are
distributed in Northern America and Southern Europe—Western Asia. Thus, our results tend to place A. caesium and its subspecies into a separate series. In addition,
as reported
earlier (Ogata 1967; Tian et al. 2001),
A. pseudoplatanus was distant from other de Jong’s ser. Acer species
and formed an independent clade in this study. Morphologically, A. pseudoplatanu
was obviously different from
other ser. Acer species, e.g. (i)
inflorescence long paniculate, (ii) filament hairy
and (iii) pollen exine sculpture arranged very
irregularly (Ogata 1967; Tian et al. 2001). Therefore, it is suggested
that A. pseudoplatanu should be treated as a monotypic series.
The close relationships among
sect. Oblonga, sect. Pentaphyllum and sect. Trifoliata
were supported in our study, which were also backed by De Jong (1994) and Tian et al. (2002). However, the systematic relationships
among the three sections were still
controversial. In De Jong
(1994) system, sect. Oblonga was treated as a series (i.e.
ser. Trifida) under sect. Pentaphyllum. It was different from other
treatments (Ogata 1967; Fang 1981; Xu et al. 2013), but was supported here, as sect. Pentaphyllum species formed a sister-clade to sect. Oblonga species (except A.
buergerianum). A. buergerianum was strangely placed as a sister species to A. pilosum in sect. Pubescentia (Fig. 3). Though the
two species shared many similar morphological traits, some important taxonomic
features were obviously different; for instance, the number of stamens and the type of leaf
margin. Additionly, A. mandshuricum from sect.
Trifoliata expressed a
closer relationship with sect. Oblonga, although there were obvious differences in leaf
shapes. Thus, it is necessary to use more
methods to address the relationships among the three sections.
Conclusion
The ITS2 sequence carried
comparatively high identification efficiency at the section level of Acer, and it also proposed reliable
identification efficiency for most of the species in genus Acer. In
addition, the phylogenetic tree constructed based on ITS2 sequences revealed
the phylogenetic relationship of Acer
and highlighted that the ITS2 sequences are potentially applicable in the
identification and phylogenetic investigation of Acer species.
Acknowledgement
This study was supported by the
Ningbo Scientific and Technological Innovation 2025 Major Projects (NO.
2019B10012), Key Scientific
Research Project of Ningbo City College of Vocational Technology (Grant No.
ZZX18126), and A Project Funded by the Priority Academic Program Development of
Jiangsu Higher Education Institutions. We are grateful to Nian
Wang, Yan Wei, Liwen Han, Yexin
Zhang, Xuexiao Zhang, Yue Chen and Gengguo Tang for their kind help for providing samples for
this study.
Author Contributions
Li Lin performed
the experiments, analyzed the data and wrote the manuscript; Zhiyong Zhu, Lejing Lin and Yuan
Zhou provided essential reagents and materials; Tao Fu and
Feng Liu provide technical assistance in molecular experiments and data
analysis; Wen Li and Yulong Ding gave suggestions to revise the manuscript.
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