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Morphological Re-Description and DNA Barcoding of Platycephalus indicus Collected from Beihai, China

Zhi Chen1 and Tianxiang Gao2,*

1The Institute of Evolution and Marine Biodiversity, Ocean University of China, Qingdao 266003, China

2Fisheries College, Zhejiang Ocean University, Zhoushan 316022, China

ABSTRACT

We collected Platycephalus indicus individuals from Beihai and accurately described the species. The diacritically meristic counts were listed as follows: first dorsal fin with a single small isolated spine anteriorly; gill rakers 2-3+4-6=6-9; pored lateral line scales 73-80 and caudal fin with a yellow marking on the middle when fresh. These conclusive characters were consistent with typical Platycephalus indicus individuals and could thoroughly separate them from other Platycephalus species. The fragment of cytochrome oxidase subunit I (COI) gene of mitochondrial DNA was also sequenced for the classification of specimens. The mean genetic distance within Platycephalus indicus was 0.8%, net genetic distance between Platycephalus indicus and other 13 species of the genus Platycephalus ranged from 10.3% to 26.1%. The phylogenetic analysis similarly supported the validity of Platycephalus indicus existed in the coastal waters of Beihai. Comments were made on some of the characters to more fully characterize the species and for phylogenetic studies.

Article Information

Received 22 January 2017

Revised 30 March 2017

Accepted 01 April 2017

Available online 22 August 2017

Authors’ Contribution

ZC and TG presented concept, analyzed the data and wrote the manuscript. TG supervised the study and arranged financial support.

Key words

Chinese taxonomy of flathead fishes, Isolated spine anteriorly, Yellow marking, Phylogenetic analysis.

DOI: http://dx.doi.org/10.17582/journal.pjz/2017.49.5.1647.1655

* Corresponding author: gaotianxiang0611@163.com

0030-9923/2017/0005-1647 $ 9.00/0

Introduction

Flathead fishes of the genus Platycephalus, family Platycephalidae, were widely distributed in the tropical and temperate areas of the Indo-West Pacific and eastern Mediterranean (Hureau, 1986; Shao and Chen, 1987; Imamura, 1996; Knapp, 1999; Qin et al., 2013). Before 2013, only one species of this genus, Platycephalus indicus (Linnaeus 1758), had been recorded and studied in China (Zhu et al., 1963; Chang et al., 1980; Chen, 1982; Chen and Zhao, 1986; Kong et al., 1994; Zhang et al., 1994; Jin, 2006; Tang, 2006; Qin et al., 2013). Qin et al. (2013) revealed that the common Platycephalus species widely distributed in the coastal waters of China was Platycephalus sp.1 in reality (Note: Platycephalus sp. 1 as well as Platycephalus sp. 2 was two valid but undescribed species. These two species were long-termly recognized and classified by Japanese ichthyologists with the Japanese names Yoshino-gochi and Ma-gochi (see Kamei and Ishiyama, 1968; Masuda et al., 1991, 1997; Osatomi et al., 2001; Nakabo, 2002; Yamada et al., 2007; Qin et al., 2013)). Further studies also indicate that all Chinese description of Platycephalus indicus was Platycephalus sp. 1 actually (Qin et al., 2013). Due to the undiscovered or rare existence of Platycephalus indicus in Chinese coastal waters, Qin et al. (2013) didn’t describe native Platycephalus indicus but just referred to foreign study of this species found locally. Thus his corrections created the description vacuum of Platycephalus indicus recorded in Chinese ichthyology.

Meeting this need, our purpose here was confirming the existence of true Platycephalus indicus and providing plentifully morphological characters of this species. Due to the complexities of morphological characters used in traditional taxonomy and taxonomic confusion as well as cryptic species has arisen in Platycephalus concerning the nomenclature (Imamura, 2006, 2008, 2012, 2013a, 2013b, 2015; Imamura et al., 2006; Imamura and Knapp, 2009). Employing solely morphological means to solve chaotically taxonomic problems was improper sometimes in traditional taxonomy. The mitochondrial cytochrome oxidase I gene (COI) varies noticeably between species and very little between the individuals of a given species (Gross, 2012). Therefore, a fragment of COI gene, as DNA barcoding (Hebert et al., 2003), has proven to be extremely effective at discriminating species (Domingues et al., 2013; Puckridge et al., 2013; Ming et al., 2015), discovering new-recorded and new species (Gao et al., 2011; Qin et al., 2013), uncovering cryptic species (Hajibabaei et al., 2007; Zemlak et al., 2009), identification of ichthyoplankton (Bian et al., 2008; He et al., 2011). In the present study, a mitochondrial DNA barcoding approach was also employed in order to better solve the taxonomic problems of Platycephalus indicus at genetic level. The results will contribute to Chinese Platycephalus species identification and be helpful to native fishery management, biodiversity conservation, and sustainable exploitation of this species.

Materials and Methods

Sampling

Specimens were collected from the coastal waters of Beihai (Fig. 1) from August 2012 to October 2016 (3 on August 17, 2012; 2 on November 08, 2014; 4 each on January 12 and October 23, 2016). All 13 individuals were identified based on morphological characteristics commonly-used by Imamura (2012, 2015). The body color and pigmentation were pictured in fresh fish and all measurements were made on preserved specimens. For genetic study, a piece of muscle tissue was obtained from each individual and preserved in 95% ethanol or directly extracted from frozen samples. All specimens examined were frozen and preserved at the Fishery Ecology Laboratory, Fisheries College, Ocean University of China in Qingdao.

Morphological study

Counts and measurements followed the standard methods given by Imamura (2012, 2015). The detailed guidance was listed as follows: gill rakers, defined as depressible bony elements and not including tooth plates, were counted on the right side. Other counts were routinely taken from the left side. Measurements of body lengths were done on a measuring board graduated in 1.0 mm intervals. All other measurements were taken using dial calipers and recorded to the nearest 0.1 mm. Fin rays were counted using a magnifier when specimens were too small. Small isolated anterior and posterior first dorsal-fin spines are given before and after the first dorsal-fin ray count (in Roman numerals), respectively. Orbital diameter was measured from the anteroventral to posteromedial portions, which was the greatest distance in many species of Platycephalus. Interorbital width was measured level with the eye center. When two small isolated anterior dorsal-fin spines were present, predorsal length was measured from the tip of the snout to the base of the second spine, since the latter was homologous with the single small isolated anterior spine present in many species of Platycephalus (see Imamura, 1996 for homology of such spines). Terminology of head spines follows Knapp et al. (2000) and Imamura (2015). The following abbreviations for counts and measurements were used: first dorsal-fin rays (D1), second dorsal-fin rays (D2), anal-fin rays (A), pectoral-fin rays (P1), branched caudal-fin rays (C), pored lateral line scales (LLS), oblique body scale rows slanting downward and backward above lateral line (OBS), gill rakers (GR), total length (TL), standard length (SL), head length (HL), predorsal length (PDL), length of first dorsal-fin base (LD1B), length of second dorsal-fin base (LD2B), length of anal-fin base (LAB), snout length (SNL), orbital diameter (OD), upper-jaw length (UJL), lower-jaw length (LJL), interorbital width (IW), postorbital length (POL), suborbital width (SW), pectoral-fin length (P1L), pelvic-fin length (P2L) and caudal-fin length (CL) (all words above were from Imamura, 2012, 2015). Some counts could not be determined because of the poor condition of this individual. In such instances the reported values were taken from the original description (for holotypes) or the specimen was discarded from analysis.

DNA extraction and sequencing

After morphometric measurements, all 13 specimens were selected for genetic studies. The classical phenol-chloroform technique was used for DNA extraction. Polymerase chain reaction (PCR) was subsequently conducted. The F and R sequences of the primers used for COI amplification were 5′-TCGACTAATCATAAAGATATCGGCAC-3′ and 5′-ACTTCAGGGTGACCGAAGAATCAGAA-3′ (Ivanova et al., 2007), respectively. PCR was carried out in a 25 μL reaction mix containing DNA template (1 μL, 50 ng/uL), forward primer (F, 1 μL, 10 uM/L), reverse primer (R, 1 μL, 10 uM/L), dNTPs (2 μL, 2.5 mM/L each), EasyTaq DNA Polymerase (0.15 μL, 5 U/μl) and 10× PCR buffer (2.5 μL, 25 uM/L). A Biometra thermal cycler (Göttingen, Germany) with the following given procedure: one initial denaturation (95°C, 5 min), thirty-five cycles consisting of denaturation (94°C, 50 s), annealing (54°C, 50 s) and extension (72°C, 48 s), and one final extension (72°C, 10 min), was employed to put PCR amplification into effect. PCR products were sent to Shanghai Majorbio Bio-Pharm Technology Co., Ltd. to get original COI sequences.

COI analysis

All 13 individuals’ original sequences were successfully obtained and revised by DNASTAR software (DNASTAR Inc., Madison, WI, USA). One COI sequence

Table I.- GenBank accession numbers of related COI sequences downloaded from NCBI for phylogenetic tree study.

Species	GenBank accession number
Platycephalus aurimaculatus Knapp, 1987	JX488155, JX488255
Platycephalus bassensis Cuvier in Cuvier and Valenciennes, 1829	DQ107991
Platycephalus caeruleopunctatus McCulloch, 1922	DQ107994
Platycephalus conatus Waite and McCulloch, 1915	JX488183
Platycephalus endrachtensis Quoy and Gaimard, 1825	DQ108000
Platycephalus fuscus Cuvier in Cuvier and Valenciennes, 1829	DQ107974, DQ107989
Platycephalus grandispinis Cuvier in Cuvier and Valenciennes, 1829	DQ107959, DQ107960
Platycephalus indicus (Linnaeus, 1758)	JX972212
Platycephalus laevigatus Cuvier in Cuvier and Valenciennes, 1829	DQ107966, DQ107980
Platycephalus marmoratus Stead, 1908	DQ107950, DQ107964
Platycephalus orbitalis Imamura and Knapp, 2009	JX488284, JX488286, JX488150
Platycephalus richardsoni Castelnau, 1872	JX488214
Platycephalus westraliae (Whitley, 1938)	DQ107985, DQ107997
Platycephalus sp. 1	JX972210,JX972199
Coceilla crocodila Cuvier, 1829	JQ349911

of Coceilla crocodila and 23 sequences of Platycephalus were also downloaded from NCBI for phylogenetic tree study (Table I). These 37 COI sequences were then aligned using the above DNASTAR software. MEGA 5.0 (Tamura et al., 2011) was used to construct neighbor-joining (NJ) tree under the Kimura 2-parameter (K2P) model.

Results

Morphological characters

The generally morphological features were shown in Figure 2. Body was elongate and head strongly depressed. Rear edge of maxilla reached to about below middle of eye. Preopercular spines were 2, and the lower was longer than the upper. A trace of an accessory spine usually present on base of upper spine. Supraorbital ridge was pretty smooth. Spines and ridges on top and side of head weakly developed. Preorbital spine was lacking and a single preocular spine obscure in large adults. Suborbital ridge was smooth in adults, bearing a spine below rear of eye in juveniles. Teeth on vomer were in a single transverse band. Upper iris lappet was a simple, triangular lobe. Interopercular flap presented, finger-like in shape. Total gill rakers on first gill arch ranged from 6 to 9 (usually 8 or 9). In addition to 13 second dorsal-fin rays and 13 anal-fin rays, first dorsal-fin spines and pectoral-fin rays were I+VII+I and 18 to 19, respectively. Oblique body scale rows slanting downward and backward above lateral line ranged from 86 to 108. Lateral line scales were usually 73 to 80. Anterior-most scale usually had a one or two spine or ridge. Scale pores of lateral line had a single opening to the outside.

Besides above description, these specimens were also a species of Platycephalus with the following combination of characters: upper jaw without large caniniform teeth. Teeth absent on dorsal surface of anterolateral edge of upper jaw. Lip margins without papillae. First and second dorsal fin narrowly separated. Head and body covered with small brown flecks, whitish below, several indistinct dark bands crossing back in some; upper surface of eye without papillae; scales covering snout, a small area anteroventral to eye, interorbit, occipital region, nape, postorbital and opercular regions; suborbital region naked; first dorsal fin with a single small isolated spine anteriorly; body with two dark brown bands below second dorsal fin; first and second dorsal, pectoral and pelvic fins with small, dark brownish spots along rays; caudal fin with 2 or 3 horizontal dark bars, a prominent yellow blotch near middle of fin when fresh; head length 29.7–33.7% in SL; pectoral fin length 11.7-16.2% SL. Pelvic fin length 21.7-25.5% SL. Caudal fin usually slightly rounded or mostly straight posteriorly, length 15.0-19.7% SL. Postorbital length 52.2–59.6% HL; snout length 24.5-29.7% HL; interorbit narrower than orbital diameter in smaller specimens, becoming equal to or wider than orbital diameter with the development of specimens, width 22.7-28.0% HL; orbital diameter 12.6-18.4% HL.

Sequence analysis of the COI gene

Thirteen 654-bp-long sequences of COI gene fragments were obtained. After combined the downloaded COI sequences of Platycephalus, a total of 37 sequences were used for analysis. Table II reported the genetic distances between all species. The mean distance among species was 19.5%. Genetic distance between our 13 specimens and Platycephalus indicus downloaded from NCBI was only 0.8%. Net genetic distances between Platycephalus indicus and other 13 species of the genus Platycephalus ranged from 10.3% to 26.1%, which vastly exceeded the threshold of species delimitation.

Table II.- Comparative counts of Platycephalus indicus and P. sp. 1 from different records.

		Records in this study	Records in references
		Specimens	Qin, (2013)	Imamura, (2015)
		P. indicus (n = 13)	P. sp. 1 (n = 174)	P. indicus (n = 41)
	SL (mm)	148.0-546.7	122.0-482.8	40.2-379
Counts	D1	I+VII+I	II+VI-VII+0-I (usually II + VII+I)	I+VI-VIII+0-I
	D2	13	13-14 (usually 13)	13-14 (usually 13)
	A	13	13-14 (usually 13)	13
	P1	18-19	17- 19	18-20 (usually 19)
	C	11-12(usually 12)	11-14	9-12 (usually 12)
	LLS (spines)	73-80 (1-2)	83- 99 (1-2)	67-84 (1-3)
	OBS	86-108	103-121	83-112
	GR	6-9	11-17	7-10
As % SL:	HL	29.7-33.7	26.3-31.3	29.2-34.7
	PDL	28.7-34.2	27.8-41.0	29.9-38.1
	LD1B	16.2-22.3	14.0-22.1	14.8-21.8
	LD2B	30.7-34.2	30.2-38.0	31.8-36.2
	LAB	35.0-39.6	33.5-43.6	34.1-41.1
	SNL	7.4-8.8	7.6-9.8	7.6-9.5
	OD	3.2-6.7	3.1-9.9	3.9-8.5
	UJL	10.9-12.8	9.8-14.3	10.6-13.2
	LJL	14.9-17.6	13.5-19.9	14.8-18.4
	IW	2.7-5.2	2.6-6.1	2.5-5.5
	POL	16.8-19.4	15.9-22.3	16.5-19.3
	SW	2.6-3.3	2.3-3.7	2.5-3.1
	P1L	11.7-16.2	12.2-17.9	12.4-17.7
	P2L	21.7-25.5	19.6-26.7	21.2-25.6
	CL	15.0-19.7	14.1-20.8	14.7-19.9
As % HL:	SNL	24.5-29.7	23.8-31.3	25.3-28.4
	OD	12.6- 18 .4	10.9-17.2	13.0-23.6
	UJL	30.5-34.9	30.3-58.9	35.0-40.4
	LJL	48.9-52.9	46.8-57.1	48.6-54.3
	IW	22.7-28 .0	9.8-19.6	7.2-18.4
	POL	52.2-59.6	52.6-66.7	51.4-61.6
	SW	6.9-9.6	6.7-11.2	7.1-10.1

D1, first dorsal-fin rays; D2, second dorsal-fin rays; A, anal-fin rays; P1, pectoral-fin rays; C, branched caudal-fin rays; LLS, pored lateral line scales; OBS, oblique body scale rows slanting downward and backward above lateral line; GR, gill rakers; TL, total length; SL, standard length; HL, head length; PDL, pre-dorsal length; LD1B, length of first dorsal-fin base; LD2B, length of second dorsal-fin base; LAB, length of anal-fin base; SNL, snout length; OD, orbital diameter; UJL, upper-jaw length; LJL, lower-jaw length; IW, interorbital width; POL, postorbital length; SW, suborbital width; P1L, pectoral-fin length; P2L, pelvic-fin length; CL, caudal-fin length

Table III.- Net genetic distances (K2P) within (on the diagonal, bold font) and between (below the diagonal, normal font) species.

	A	B	C	D	E	F	G	H	I	J	K	L	M	N
A	0.002
B	0.2065	-
C	0.267	0.2701	-
D	0.2023	0.1932	0.3269	0.01
E	0.2348	0.2233	0.3132	0.22	0.03
F	0.2287	0.2259	0.3245	0.21	0.06	-
G	0.2266	0.2027	0.3257	0.29	0.04	0.06	-
H	0.1201	0.2254	0.2895	0.22	0.22	0.28	0.25	0.05
I	0.1028	0.2097	0.2744	0.23	0.26	0.24	0.28	0.16	0.08
J	0.1909	0.181	0.2966	0.09	0.29	0.21	0.21	0.21	0.21	0.00
K	0.2148	0.2171	0.2902	0.19	0.21	0.24	0.24	0.25	0.24	0.15	0.03
L	0.1816	0.1895	0.2815	0.16	0.19	0.21	0.17	0.22	0.19	0.15	0.15	0.00
M	0.1167	0.1986	0.2881	0.17	0.24	0.29	0.25	0.14	0.16	0.18	0.21	0.20	0.00
N	0.2067	0.1804	0.3003	0.19	0.27	0.25	0.24	0.21	0.25	0.17	0.14	0.16	0.20	-
O	0.1823	0.2155	0.3277	0.18	0.29	0.22	0.28	0.11	0.24	0.14	0.16	0.17	0.18	0.18

A, P. westraliae; B, P. endrachtensis; C, Coceilla crocodila; D, P. orbitalis; E, P. aurimaculatus; F, P. conatus; G, P. richardsoni; H, P. sp. 1; I, P. indicus; J, P. marmoratus; K, P. grandispinis; L, P. laevigatus; M, P. fuscus; N, P. bassensis; O, P. caeruleopunctatus.

A neighbor-joining phylogenetic tree was constructed using MEGA 5.0 (Fig. 3). Coceilla crocodila was chosen as the out-group to root the tree. All COI sequences of specimens in present study clustered in the same group, and 7 haplotypes were defined. All haplotype sequences were submitted to GenBank with the following accession numbers: KY463436-KY463442. The haplotype 2 (Hap-2), haplotype 4 (Hap-4) and haplotype 5 (Hap-5) were severally shared by 2 specimen and haplotype 7 (Hap-7) by 4 specimen. Remaining haplotypes were unique and each of them was shared by one specimen. At the same time, a large genetic distance (19.0%) between specimens and Platycephalus sp.1 indicated that they couldn’t be the same species.

Discussion

The morphological characters of specimens used in this study were photographed, counted and compared with previously representative records on Table I. These individuals were characterized by: first dorsal fin with a single small isolated spine anteriorly; gill rakers 2-3+4-6=6-9; pored lateral line scales 73-80 and caudal fin with a yellow marking on the middle when fresh. These phenotypic traits were consistent with the descriptions of typical Platycephalus indicus described by Imamura (2015).

Platycephalus sp. 1 distributed in China had previously been misidentified as Platycephalus indicus (Zhu et al., 1963; Chang et al., 1980; Chen, 1982; Chen and Zhao, 1986; Kong et al., 1994; Zhang et al., 1994; Tang, 2006; Jin, 2006), both species being characterized by usually 13 second dorsal- and anal-fin rays, interorbit and occipital region scaled, large caniniform teeth absent on the upper jaw, a finger-like interopercular flap and so on (Imamura, 2015; Qin et al., 2013). Besides, by reviewing all known references including our present results, it could be obviously found that Platycephalus indicus and Platycephalus sp. 1 were very similar in meristic values. Almost all morphological characteristics were overlapped. Such overlaps appeared to be very common among Platycephalus that were either closely related or lived in similar habitats (Imamura, 2015). These might be the reason why all Platycephalus species from the coastal waters of China permanently and routinely misidentified as Platycephalus indicus by native ichthyologists.

Although Platycephalus indicus was similar with Platycephalus sp. 1 and long-termly lacked correct descriptions, there was still some obvious difference that could separate Platycephalus indicus from other Platycephalus species. Contrast to the concentrated distribution in Australia, only four species (Platycephalus indicus; Platycephalus cultellatus; Platycephalus sp.1 and Platycephalus sp. 2) of whole Platycephalus were recorded in Northwest Pacific (Imamura 2006, 2015; Nakabo, 2002; Qin et al., 2013). Among above four species, Platycephalus indicus was the only one that had the first dorsal fin with a single small isolated spine anteriorly and the caudal fin with a yellow marking on the middle when fresh (Fig. 2). The other three species uniformly usually had a caudal fin without yellow color and two small isolated spines in front of first dorsal fin. As for commonly-seen and previously misidentified Platycephalus sp.1, the range of gill rakers and pored lateral line scales could also distinguish Platycephalus indicus from it (Table I).

COI sequence was recognized as an effective and reliable method for species identification (Domingues et al., 2013; Hebert et al., 2003; Masuda and Ozawa, 2000; Qin et al., 2013). The validity of Platycephalus indicus has also been demonstrated from genetically reconstructed phylogenetic relationships of Platycephalus, showing that specimens from Beihai, with a yellow marking on the mid-caudal fin and Platycephalus indicus sequence from NCBI were included in a same monophyletic clades. As an indicator of speciation, Hebert et al. (2004) proposed the ‘10×rule’, whereby barcoded individuals are flagged as possible another species if they diverge by 10 times or more the average intraspecific variability of the group. A different approach was taken by Ward et al. (2009), who analyzed barcode data from about 1000 fish species and showed that at a level of 2% distance or greater, individuals were much more likely to be congeneric than conspecific. We identified that Platycephalus indicus and other Platycephalus species were distinguished by distance = 0.103~0.261. All other pairwise divergence among who species exceeded distance = 0.195, and the highest values were observed between Platycephalus indicus and Platycephalus aurimaculatus (distance = 0.261). The mean evolutionary distance within the species Platycephalus indicus was 0.8%, use of either the 10× or 2% rule suggests that the genetic distance between groups was significantly higher than the average genetic distance within the group, which indicated that the COI gene used as a barcode of Platycephalus indicus was effective at identifying Platycephalus species. Thus, these reality provided our studies a strongly morphological and genetic level support that the species Platycephalus indicus in the coastal waters of China was correctly identified and described.

It was also reported that Platycephalus indicus was widely distributed in the coastal waters of China (Zhu et al., 1963; Chang et al., 1980; Chen, 1982; Chen and Zhao, 1986; Kong et al., 1994; Zhang et al., 1994; Jin, 2006; Tang, 2006). We have also tried our best to collect more specimens from all Chinese seas. But it seemed that fishermen and researchers could hardly see the occurrence of Platycephalus indicus. Only 13 Platycephalus indicus was successfully collected in the past four years and all of them came from Beihai. The inundant distribution of Platycephalus sp. 1 and infrequent presence of Platycephalus indicus made a sharp contrast. A single South China Sea distribution of Platycephalus indicus suggested this species might prefer warm water and live in lower latitude areas. Further domestic and overseas specimen collection is also indispensable in order to define its clearly geographic limits.

Accurate identification of fish is essential and would assist in managing fisheries for long-term sustainability, and improve ecosystem research and conservation. Resolution of cases of this nature will require careful morphological analysis from expert taxonomists before any final recommendations can be made (Ward et al., 2005; Xiao et al., 2016). Mitochondrial sequence divergences are strongly linked to the process of speciation, DNA barcoding and morphological analysis should go hand-in-hand. The data presented here would aid more information and explicit species taxonomy and avoid numerous misidentification and erroneous distributional records within Platycephalus genus. We hope this study will not only promote the sustainable exploitation, biodiversity conservation and fisheries management of Platycephalus distributed in China but also contribute to species identification within this genus in the future.

Conclusion

The morphological characters and COI sequence analysis revealed that specimens collected from the coastal waters of Beihai were consistent with typical Platycephalus indicus individuals and could thoroughly separate them from other Platycephalus species. Our present study confirmed the existence of true Platycephalus indicus and providing plentifully morphological characters of this species. We hope this study will not only promote the sustainable exploitation, biodiversity conservation and fisheries management of Platycephalus but also contribute to species identification within this genus in the future.

Acknowledgements

Mr. Jianwei Zou contributed much to specimen collection. Thanks were also given to Yan Qin and Zhiyang Wang for paper writing. This work was supported by the National Natural Science Foundation of China (41576145) and the Special Fund for Agroscientific Research in the Public Interest (201303048, 201303050).

Statement of conflict of interest

Authors have declared no conflict of interest.

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