Morphological and Molecular Evidence Supports the Occurrence

Acta Oceanol. Sin., 2014, Vol. 33, No. 8, P. 44–54 DOI: 10.1007/s13131-014-0457-y http://www.hyxb.org.cn E-mail: [email protected]

Morphological and molecular evidence supports the occurrence of a single species of Zebrias zebrinus along the coastal waters of China WANG Zhongming1,2,4, KONG Xiaoyu1*, HUANG Liangmin1, WANG Shuying1,4, SHI Wei1, KANG Bin3 1 Key Laboratory of Marine Bio-resources Sustainable Utilization, Marine Biodiversity Collection of South China Sea, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China 2 Key Laboratory of Sustainable Utilization of Technology Research for Fishery Resource of Zhejiang Province, Marine Fisheries Research Institute of Zhejiang, Zhoushan 316100, China 3 Asian International Rivers Center, Yunnan University, Kunming 650091, China 4 University of Chinese Academy of Sciences, Beijing 100049, China

Received 11 November 2012; accepted 28 May 2013

©The Chinese Society of Oceanography and Springer-Verlag Berlin Heidelberg 2014

Abstract The so-called zebra sole includes a group of small flatfishes characterized by transverse band pairs on the ocular side and distributed throughout shallow waters along the coast of the Indo-West Pacific Ocean. Sev- eral species of the zebra sole have been recorded from the coastal waters of China. Morphological analysis of 1 107 specimens of the zebra sole from 15 successive localities along the China’s coast demonstrated that no significant variations among these localities were found on the basis of meristic counts and morphometric characters. Phylogenetic analysis based on COI gene sequences of 14 individuals and D-loop of 22 individuals from eight localities showed that they were indistinguishable among these localities. Therefore, both morphological and molecular evidence supported the occurrence of a single species of the zebra sole along the China’s coast. The available name for this species is Zebrias zebrinus (Temminck and Schlegel, 1846) in- stead of Z. zebra (Bloch, 1787). Zebrias fasciatus (Basilewsky, 1855) and Solea ommatura (Richardson, 1846) are considered here as two synonyms of Z. zebrinus. Key words: Zebrias, zebra sole, China’s coast, morphological and molecular evidence, species validity, synonyms Citation: Wang Zhongming, Kong Xiaoyu, Huang Liangmin, Wang Shuying, Shi Wei, Kang Bin. 2014. Morphological and molecular evidence supports the occurrence of a single species of Zebrias zebrinus along the coastal waters of China. Acta Oceanologica Si- nica, 33(8): 44–54, doi: 10.1007/s13131-014-0457-y

1 Introduction pairs of transverse dark bands on the ocular side and yellow The genus Zebrias belongs to the family Soleidae of the Pleu- marks on the caudal fin; interorbital space narrow and scaly; ronectiformes, and includes a group of species characterized by dorsal and anal fins branched and completely confluent with a combination of the following characters: eyes and color on the caudal fin (the last dorsal and anal rays as long as adjacent cau- right side, with black transverse bands more or less arranged in dal rays); and pectoral fin on the ocular side longer than the one pairs; pectoral fin of the blind side rudimentary or wanting, dor- on the blind side (Li and Wang, 1995). However, the taxonomy of sal and anal fins confluent with caudal fin; teeth minute, only the zebra sole is poorly understood. present on blind side or absent (Jordan and Starks, 1906; Cheng Bloch (1787) initially described specimens of the zebra sole and Chang, 1965). A total of 22 nominal species have been de- from Tranquebar, east coast of India, as Pleuronectes zebra [here scribed and included in Zebrias (Bloch, 1787; Richardson, 1846; Zebrias zebra]. Later, three other species were described on the Temminck and Schlegel, 1846; Basilewsky, 1855; Kaup, 1858; basis of similar specimens from seas of the Northwest Pacific: Bleeker, 1860; Macleay, 1882; Alcock, 1890; Regan, 1903; Gil- Solea zebrina (Temminck and Schlegel, 1846) [here Z. zebrinus, christ, 1906; Jenkins, 1910; McCulloch, 1916; Cheng and Chang, type locality: Nagasaki, Japan]; Solea ommatura (Richardson, 1965; Whitley, 1966; Rama-Rao, 1967; Talwar and Chakrapany, 1846) [type locality: Canton (presently Guangdong Province, 1967; Joglekar, 1976; Oommen, 1977; Seigel and Adamson, 1985; China)], and Solea fasciata (Basilewsky, 1855) [here Z. fasciatus, Gomon, 1987; Randall, 1995), and they are widely distributed type locality: Shantung (i.e., Shandong Province, China)]. Spe- in the Indian Ocean and western central Pacific Ocean. Zebrias cies delineation of the zebra sole has long been a controversial zebra, commonly called the zebra sole, is a kind of small flatfish issue. Some authors argued that two distinct species existed: living in shallow waters throughout the coast of the Indo-West the northern species named as Z. fasciatus, and the southern Pacific. It is characterized by having the following features: 11 one as Z. zebrinus (Jordan and Metz, 1913; Ochiai, 1955; Naka-

Foundation item: The Key Innovation Project of Chinese Academy of Sciences under contract No. KSCX2-YW-Z-0929; the National Natural Science Foundation of China under contract Nos 30870283 and 31071890. *Corresponding author, E-mail: [email protected] WANG Zhongming et al. Acta Oceanol. Sin., 2014, Vol. 33, No. 8, P. 44–54 45

bo, 2002) or Z. zebra (Ochiai, 1963, 1984; Yamada and Okamura, d1-8, d4-6, d4-8, d6-7, d6-8, d6-9, d6-10, d8-9, d8-10, and d9- 1986). A majority of researchers held that the zebra sole repre- 10 (Fig. 1). All measurements were expressed as proportions of sented a single species, but two specific names were used for the standard length, except for d6-7 and d6-8, and those related it. This species was identified either as Z. zebra (Günther, 1862 with the head, such as ED, SE, d1-2, and d1-3, which were given as Synaptura zebra; Jordan and Evermann, 1902; Hubbs, 1915; as proportions of the head length. Subsequently, SPSS v.16.0 Chu, 1931; Wu, 1932; Wang, 1933; Cheng, 1955, 1962; Tchang was employed for the basic statistical analysis of morphological and Wang, 1963; Cheng and Chang, 1965; Li, 1987; Shen, 1993; data. Principal component analysis (PCA) was also performed

Li and Wang, 1995; Shen and Wu, 2011) or as Z. zebrinus (Jordan on the covariance-variance matrix of the log10-transformed and Starks, 1906; Jordan and Hubbs, 1925; Evermann and Shaw, measurements (Xie et al., 2003). 1927; Fowler, 1929; Wu, 1929). Apparently, there are controver- The examined specimens from the coastal waters of China sies over how many zebra sole species occur in China’s seas and were housed in the Marine Biodiversity Collections of South which name(s) are available. The present investigation tries to China Sea, Chinese Academy of Sciences, Guangzhou, Guang- provide morphological and molecular evidence to confirm the dong Province, China. Type specimens of Z. zebra and Z. zebri- presence of a single zebra sole species in China’s seas, and com- nus are located in the Museum für Naturkunde, Germany (ZMB ment on the available name for this species. 2423) and Netherlands Centre for Biodiversity Naturalis (RMNH D.1308, RMNH D.1306 and 1307), respectively. 2 Materials and methods 2.3 Molecular analysis 2.1 Sample collection The sequences of mitochondrial COI gene and control re- Specimens of the zebra sole were collected from local fish- gion (D-loop) were used for molecular analysis. Sample infor- ing harbors by benthic trawling. A total of 1 107 specimens were mation is shown in Table 2. Total genomic DNA was extracted obtained from 15 sites along the China’s coast (Table 1 and using marine animal tissue DNA kits (TIANGEN Biotech, Fig. S1). Fresh specimens were stored in ice after collection and Beijing, China) following the manufacturer’s protocol. Prim- then kept in 95% ethanol or frozen at −20°C in the laboratory. ers for amplifications of mitochondrial DNA sequences were as follows: R-COI-6754 (CTAAGCCATCCTACCTGTG) and F- 2.2 Morphological analysis COI-8350 (TCAACTCCTCCCTTTCTCG) for COI; and L-17114 Measurements and counts were made on the ocular side of (RCGCCCAAAGCTAGDATTC), F-95 (GACAGTAAAGTCAGGAC- individuals. Each measurement was taken from point to point CAAGCCTTTGTGC), and F-2753 (TAGATAGAAACTGACCTG- with digital calipers and the data were recorded with precision of GATTACTCCGGT) for D-loop. The PCR was performed in a 25 0.1 mm. Meristic counts mainly followed those of Ochiai (1955) μL reaction volume containing 0.2 mmol/L dNTP, 0.5 μmol/L and Cheng and Chang (1965), including dorsal-fin rays (D), of each primer, 1 U/25 μL LA Taq (Takara, Dalian, China), 2.5 μL anal-fin rays (A), lateral-line scales (LLS, from above the pos- 10 × LA Taq Buffer II (Mg+ Plus), and approximately 50 ng DNA terior margin of operculum to caudal-fin base) and vertebrae template. PCR cycling conditions included an initial denatur- number. Additional counts were pectoral-fin rays (P). Twenty- ation at 95°C for 3 min, followed by 35 cycles of a denaturation one measurements were made, including six morphometric at 95°C for 30 s, an annealing at 48°C for 40 s, and elongation at measurements: standard length (SL), head length (HL), eye 68°C for 1−2 min, with a final extension at 72°C for 10 min. The diameter (ED, the horizontal diameter of the lower eye), space PCR products were detected in 1.0% agarose gels and purified between eyes (SE, minimal distance between the two eyes), using a Takara Agarose Gel DNA Purification Kit (Takara, China), pectoral-fin length (PL), space between band pairs (SB, the first and sequenced in both directions using the ABI 3730 Genetic space between band pairs just behind the pectoral-fin base). Analyzer (Invitrogen Corporation). Fragments that could not be There were 15 truss network measurements made between 10 directly sequenced were inserted into the PMD18-T vector (Ta- landmarks set on the ocular side: d1-2, d1-3, d1-4, d1-5, d1-6, kara, China), and transformed into E. coli competent cells and

Table 1. Specimen information of zebra sole along the coastal waters of China in this study Groups Localities (abbreviation) Time Sample size Standard length/mm 1 Wendeng (WD) Shandong Province May 2010 80 118.78–237.35 2 Qingdao (QD) Jun. 2008–May 2010 100 114.23–231.82 3 Rizhao (RZ) Dec. 2009 100 106.27–230.34 4 Lianyungang (LY) Jiangsu Province Dec. 2009–May 2010 100 153.31–263.01 5 Dafeng (DF) May 2010 100 118.99–254.87 6 Lüsi (LS) May 2008 25 162.47–219.86 7 Zhoushan (ZS) Zhejiang Province Sep. 2010 100 84.44–244.80 8 Shitang (ST) Oct. 2010 89 151.12–227.57 9 Pingtan (PT) Fujian Province Oct. 2010 91 92.90–146.13 10 Dongshan (DS) Oct. 2009 72 152.28–249.61 11 Shenzhen (SZ) Guangdong Province Oct. 2009–Aug. 2010 40 102.21–204.90 12 Zhapo (ZP) Sep. 2009–Apr. 2011 49 96.41–167.98 13 Zhanjiang (ZJ) Jun. 2009–Apr. 2011 37 100.31–191.13 14 Beihai (BH) Guangxi Autonomous Jun. 2009–May 2010 100 98.12–173.53 15 Qisha (QS) Region May 2010 24 119.50–149.68 46 WANG Zhongming et al. Acta Oceanol. Sin., 2014, Vol. 33, No. 8, P. 44–54

1 cm

Fig.1. Truss network measurements taken on the zebra sole. The numbers mark the position of the landmarks: 1. tip of mouth cleft; 2. midpoint of anterior margin of upper eye; 3. midpoint of anterior margin of lower eye; 4. upper origin of pectoral-fin base; 5. origin of anal fin; 6. base of the 30th ray of the dorsal fin; 7. cross point on the lateral line from base of the 30th ray of the dorsal fin to base of the 18th ray of the anal fin; 8. base of the 18th ray of the anal fin; 9. dorsal origin of caudal fin; and 10. ventral origin of caudal fin.

Table 2. Information of the zebra sole samples along coastal waters of China for molecular analysis COI D-loop Species Localities (abbreviation) Individuals Accession No. Individuals Accession No. Zebra sole Qingdao, Shandong Province (QD) 3 KF142433 9 KF142451 KF142432 KF142452 KF142431 KF142453 KF142454 KF142455 KF142456 KF142457 KF142458 KF142425 Dafeng, Jiangsu Province (DF) 2 KF142439 1 KF142448 KF142438 Zhoushan, Zhejiang Province (ZS) 1 KF142426 1 KF142421 Shitang, Zhejiang Province (ST) 1 KF142428 1 KF142422 Pingtan, Fujian Province (PT) 2 KF142435 1 KF142450 KF142434 Dongshan, Fujian Province (DS) 2 KF142437 1 KF142449 KF142436 Zhanjiang, Guangdong Province (ZJ) 1 KF142427 — — Beihai, Guangxi Autonomous Region (BH) — — 6 KF142442 KF142443 KF142444 KF142445 KF142446 KF142447 Qisha, Guangxi Autonomous Region (QS) 2 KF142429 2 KF142423 KF142430 KF142424 Zebrias quagga Zhanjiang, Guangdong Province (ZJ) 3 KF142469 — — KF142440 KF142441 Zebrias japonicus Beihai, Guangxi Autonomous Region (BH) 2 KF142467 — — KF142468 Zebrias crossolepis Beihai, Guangxi Autonomous Region (BH) 3 KF142464 — — KF142465 KF142466 Heteromycteris japonicus Macun, Hainan Province (MC) 1 KF142460 — — WANG Zhongming et al. Acta Oceanol. Sin., 2014, Vol. 33, No. 8, P. 44–54 47

then sequenced. Primers used in sequencing were the same as The studied specimens of zebra sole were analyzed with PCA those used for PCR, and new primers were designed for walking on the covariance-variance matrix of log10-transformed mea- sequencing. The sequenced fragments were assembled using surements (Table 4). A scatterplot graph was drawn based on DAMBE V 5 (Xia and Xie, 2001) and BioEdit V 7.0.1 (Hall, 1999). the factor scores of the first two PCs (Fig. 3). It showed that in- Sequences were aligned in ClustalX V 1.8.3. Intra- and inter- dividuals of zebra sole were relatively concentrated into groups species/group genetic distances were calculated in MEGA V 5.0. related to their localities both on PC 1 axis and PC 2 axis, but Transitions and transversions were plotted against Tamura-Nei there were overlaps between neighboring groups that could (TN93) genetic distances in DAMBE V 5 to test for substitu- not be divided into apparent partitions. PC 1 (the first principal tion saturation. With Heteromycteris japonicus as the outgroup, component), considered the general size axis (Marcus, 1990), cluster analysis was conducted among the zebra sole and three did not separate the groups of zebra sole from neighboring lo- closely-related congeners, Zebrias quagga, Zebrias japonicus, calities. PC 2 (the main shape axis; James and McCulloch, 1990) and Zebrias crossolepis using COI gene sequences by Bayesian and PC 3 (another shape axis) did not allow the separation ei- inference (BI) in MrBayes V 3.0. ther, indicating that the differences in the morphological mea- The best-fit models of nucleotide substitution were used surements between groups of zebra sole were size-related. with MrModeltest 2.1 (Nylander et al., 1999). Bayesian phylogenetic analyses were performed using general Lset values (e.g., 3.2 Molecular analysis nst and rates), allowing the program to converge on the best es- The COI gene sequences from the zebra sole and Z. quagga, timates of the model parameters. Other parameter settings were Z. japonicus, Z. crossolepis, and Heteromycteris japonicus were as follows: each Markov chain was started from a random tree determined, and the length was 1 478 bp after alignment. The and run for 3×105 generations with every 100th generation sam- D-loop sequences of 22 zebra sole individuals were 1 073–1 074 pled, with 3 001 trees finally obtained. Four chains, three heated bp. The K2P genetic distances based on COI sequences of 14 ze- (temperature = 0.5˚C) and one cold, were simultaneously run bra sole individuals ranged from 0% to 0.8% with an average of using Metropolis-coupled Markov chain Monte Carlo (MCMC) 0.4% (Table S2), which were significantly smaller (by about 50 to enhance the mixing capabilities of the Markov chains. The times) than those of inter-species (19.9%−21.8%) within the ge- Bayesian analysis was repeated twice, aged samples (Burnin = nus Zebrias (Table S3). The intra-species K2P genetic distances 7 500) were discarded, and the remaining samples were used to of four other sole were all smaller than 1%, ranging from 0.09% construct the consensus tree. (Z. quagga) to 0.54% (Z. japonicus) (Table S3). In addition, those based on 22 D-loop sequences of the zebra sole ranged from 3 Results 0.5% to 1.1% with an average of 0.8% (Table S4). A phylogenetic 3.1 Morphological analysis tree was constructed by Bayesian inference based on COI se- The maximums, minimums, and means of meristic counts quences of the zebra sole and Z. quagga, Z. japonicus, and Z. and ratios of morphometric characters for the zebra sole from crossolepis, with H. japonicus as the outgroup. The scatterplot of 15 successive localities along the China’s coast were computed transitions and transversions against genetic distances showed (Table S1). Frequency distributions of vertebrae number for 66 that nucleotide substitution did not reach saturation. GTR + I + individuals are shown in Table 3. Ranges of all meristic charac- G was selected as the best evolutionary model by MrModeltest. ters overlapped among neighboring groups without obvious The topology showed that individuals clustered into species- break points to separate them. Number ranges of D, A, P, LLS of based clades. All zebra sole individuals clustered into one clade the ocular side, and the ratio of HL/SL were plotted to show the with a bootstrap value of 100%, but they did not form mono- variation (Fig. 2). phyletic groups according to their localities (Fig. 4).

Table 3. Frequency distributions of vertebrae number of zebra sole along the China’s coast Vertebrae number Localities Sample size 48 49 50 51 52 53 54 Wendeng, Shandong Province (WD) 1 3 1 5 Qingdao, Shandong Province (QD) 2 2 Rizhao, Shandong Province (RZ) 1 4 5 Lianyungang, Jiangsu Province (LY) 1 1 2 Dafeng, Jiangsu Province (DF) 3 3 6 Lüsi, Jiangsu Province (LS) 1 1 2 Zhoushan, Zhejiang Province (ZS) 1 3 1 5 Shitang, Zhejiang Province (ST) 1 1 3 5 Pingtan, Fujian Province (PT) 1 2 2 5 Dongshan, Fujian Province (DS) 1 1 3 1 6 Shenzhen, Guangdong Province (SZ) 5 1 6 Zhapo, Guangdong Province (ZP) 3 3 6 Zhanjiang, Guangdong Province (ZJ) 1 1 1 3 Beihai, Guangxi Autonomous Region (BH) 4 2 6 Qisha, Guangxi Autonomous Region (QS) 2 2 Total 8 8 16 9 16 7 2 66 48 WANG Zhongming et al. Acta Oceanol. Sin., 2014, Vol. 33, No. 8, P. 44–54

100 100 95 95 90 90 85 85 80 80 75 75 Anal-fin rays Dorsal-fin ray s 70 70 65 65 60 60 55 55 12345678910 11 12 13 14 15 123456789101112131415 Groups Groups

140 12 11 130 10

s 9 120 8 7 110 6 5

Lateral-line scale 100 4 3 90 2 Pectoral-fin rays on the eye-side 1 80 0 123456789101112131415 1 23456789101112131415 Groups Groups

0.20

0.16 HL/S L

0.12

0.08 123456789101112131415 Groups

Fig.2. The maximums, minimums, and means of four meristic characters and the ratio of HL/SL for the 15 groups of zebra sole along the coastal waters of China.

4 Discussion each meristic count varied in a large range, they considered that the two species were the same one and tentatively regarded 4.1 Single or multiple species of zebra sole along the China’s Z. fasciatus as a synonym of Z. zebra. In addition, specimens coast? examined were limited in the two studies, without successive Many previous investigations focused on the species delin- sampling on a large scale in its known range. Since then, no fur- eation of the zebra sole based on external morphology, espe- ther work on this issue was conducted. cially the meristic counts. For example, Ochiai (1955) found that Actually, ranges of the morphological characters overlapped there were differences in the numbers of D, A, LLS, and verte- in Ochiai’s specimens except that the vertebrae counts had sig- brae counts, and in the ratio of HL/SL between Z. fasciatus and nificant differences: 44–46 versus 51–53. In this study, however, Z. zebrinus. Cheng and Chang (1965) reported that ranges of the ranges of meristic characters and ratios from morphometric five morphological characters utilized by Ochiai to distinguish and truss network measurements overlapped between neigh- between Z. fasciatus and Z. zebra (under which Z. zebrinus was boring groups, respectively; in particular, the number of ver- synonymized) overlapped based on specimens from the Yellow tebrae was not less than 48 in any individual (Table 3). Zebra and Bohai Seas, East China Sea, and South China Sea. Although sole from the sampling region could not be divided into two or WANG Zhongming et al. Acta Oceanol. Sin., 2014, Vol. 33, No. 8, P. 44–54 49

4 DF 3 ST BH QS LY 2 PT 1

0 ZP QD −1 ZS SZ

Factor score of PC 2 ZJ DS −2 LS −3 RZ

−4 WD −5

−3 −2 −1 0123 Factor score of PC1

Fig.3. Scatterplot graph based on factor scores of the first two principal components from morphometric and truss network measurements of zebra sole along the coastal waters of China.

Heteromycteris japonicus (GZ) Table 4. Loadings for each measurement on the first two prin- Zebrias crossolepis (YL1) cipal components 1.00 Zebrias crossolepis (YL2) Zebrias crossolepis (YL3) Measurements PC1 PC2 PC3 Zebrias quagga (EM1) HL 0.892 0.233 0.147 1.00 Zebrias quagga (EM3) ED 0.776 0.209 0.155 Zebrias quagga (EM2) SE 0.678 0.201 0.260 the zebra sole (DF1) 1.00 the zebra sole (ZJ) PL 0.105 0.162 0.973 0.98 the zebra sole (PT2) SB 0.350 0.849 0.225 0.93 the zebra sole (QS2) d1−2 0.874 0.162 0.179 the zebra sole (QS1) d1−3 0.742 −0.190 0.174 the zebra sole (DF2) 1.00 the zebra sole (ZS) d1−4 0.927 0.233 0.146 the zebra sole (QD2) d1−5 0.893 0.302 0.114 0.93 the zebra sole (DS1) d1−6 0.924 0.305 0.125 the zebra sole (PT1) d1−8 0.913 0.322 0.131 the zebra sole (QD3) 0.97 the zebra sole (ST) d4−6 0.920 0.325 0.107 the zebra sole (DS2) d4−8 0.899 0.334 0.122 the zebra sole (QD1) d6−7 0.932 0.293 0.091 1.00 Zebrias japonicus (RB1) d6−8 0.937 0.291 0.087 Zebrias japonicus (RB2) d6−9 0.948 0.250 0.049 0.05 d6−10 0.949 0.253 0.050 d8−9 0.946 0.261 0.043 Fig.4. BI phylogenetic tree based on COI sequences of d8−10 0.946 0.258 0.040 zebra sole and three other soles, with Heteromycteris ja- d9−10 0.870 0.301 0.087 ponicus as the outgroup. Numbers on the branches indi- Total variance explained/% 71.98 10.15 6.49 cate Bayesian posterior probabilities. more distinctive groups or species by morphological charac- based on D-loop sequences were a little higher than those of ters, though ranges of some meristic characters were somewhat COI, but could be regarded in the range of intra-species level, large from north to south. because the D-loop region has a higher rate of molecular evolu- In a COI-based identification system, genetic distances tion than that in the COI gene in the fish mitogenome (Clayton, of the intra-species are commonly lower than 1% and rarely 1982). Furthermore, from the topology of the BI phylogenetic higher than 2%, while those of the inter-species are usually 10 tree, all zebra sole individuals were clustered into a clade and times higher (Hebert et al., 2003a, b). In this study, the genetic could not be distinguished by the localities. Therefore, it could distances between zebra sole individuals were about 50 times be concluded that they did not have a significant genetic differ- lower than those of the inter-species and fell into the range of entiation over their distribution range (localities). the intra-species level. On the other hand, genetic distances Based on the results of morphological and molecular analy- 50 WANG Zhongming et al. Acta Oceanol. Sin., 2014, Vol. 33, No. 8, P. 44–54

ses, we drew the conclusion that there is a single species of the on specimens from Canton. This species has long been syn- zebra sole along the China’s coast without significant differen- onymized with either Z. zebra by Günther (1862) or Z. zebrinus tiation. by Jordan and Starks (1906) since its original description. Two syntypes of this species located in the Cambridge Philosophi- 4.2 Available names for the zebra sole along the China’s coast cal Society have been destroyed (Whitehead, 1969). Richard- The zebra sole from coastal waters of China has been record- son’s original description indicated that the three vertical fins ed either as Z. zebra (Z. zebrinus as a synonym) or as Z. zebrinus blended together, the body was crossed by vertical bars, with a (Z. zebra as a synonym), with Z. fasciatus and S. ommatura as peculiar eye-like mark on the caudal fin formed by several yel- synonyms, or Z. fasciatus as a valid species. Given the occur- low spots; and the meristic counts were D 70, A 60, P 11, and V rence of a single species of zebra sole along the China’s coast, 3 vel 4. These characters were present in our specimens from it is necessary to determine which name is available for it. In Guangdong Province. Thus, we followed the arrangement that the original description of Pleuronecte zebra (Bloch, 1787) [= Z. Solea ommatura was a junior synonym of Z. zebrinus. zebra], the lateral line is straight (Fig. S2a) and meristic counts There are still 18 other available nominal species in the ge- are P 4, V 6, A 48, C 10, and D 71. The type specimen of P. zebra, nus Zebrias. They are all different from the zebra sole along the located in the Museum für Naturkunde, is inaccessible because China’s coast (here Z. zebrinus) by the following characters: (1) it is lost to science. For this reason, ZMB 2423 (83 mm SL) was Z. maculosus has no vertical bars but dark patches and spots designated as the lectotype by Paepke (1999). We examined this on the ocular side, with fewer anal fin rays (50–54 vs. 60–88); lectotype, and found no transverse band pairs on the ocular (2) Z. captivus and Z. quagga have orbital tentacles never seen side and yellow marks on the caudal fin (Fig S2b). The speci- in other congeners; (3) the caudal fin is not fully confluent with mens from coastal waters of China had more than 60 anal-fin other vertical fins in Z. annandalei, Z. callizona, Z. cochinensis, rays and 16–18 caudal-fin rays, and the minimum number of Z. crossolepis, Z. japonicus, Z. keralensis, Z. lucapensis, Z. regani, anal-fin rays was 56 in the literature record (Ochiai, 1984). How- and Z. synapturoides; (4) pectoral fins are of equal or subequal ever, no specimens with 48 anal-fin and 10 caudal-fin rays had size in Z. altipinnis, Z. cancellata, and Z. craticula, and interor- been encountered. In addition, our specimens had a curved su- bital space of the latter two species is naked; (5) all rays in the praorbital line and yellow marks on the caudal fin. All these dif- dorsal and anal fins are unbranched in Z. scalaris (formerly Syn- ferences indicated that our examined specimens of zebra sole aptura fasciata Macleay) and Z. penescalaris; and (6) the caudal were not conspecific of Pleuronecte zebra. fin is mostly white and pectoral fin on the blind side is longer As for another available name for Z. zebrinus, Temminck than the other in Z. munroi. and Schlegel (1846) first described Solea zebrina [= Z. zebrinus] based on specimens from Nagasaki, Japan (Fig S2c). The meris- Acknowledgements tic counts were D 77, A 66, P 10, V 5, and C 15, all falling within The authors are very grateful to Ma Sheng (Ocean University the ranges for the specimens from coastal waters of China. We of China), Cheng Hanliang (Huai Hai Institute of Technology) examined three type specimens, RMNH D.1308 (lectotype; Fig. and Yang Jinquan (Shanghai Ocean University) for their assis- S2d) and RMNH D.1306 and 1307 (paralecotypes, see Boese- tance in sample collection. We also thank Christa Lamour (Mu- man, 1947), located in the Netherlands Centre for Biodiversity seum für Naturkunde, Germany) and Ronald de Ruiter (Neth- Naturalis (formerly the Rijks Museum van Natuurlijke Historie, erlands Centre for Biodiversity Naturalis) for their help with Leiden). This species had 11 pairs of transverse bands on the examination of type specimens. ocular side, with yellow marks on the caudal fin that were completely confluent with other vertical fins. These characters were shared with our specimens (Fig. 1); hence the available specific References name for zebra sole from coastal waters of China is Z. zebrinus Alcock A W. 1890. On some undescribed shorefishes from the Bay of (Temminck and Schlegel, 1846). Bengal. Annals and Magazine of Natural History, 6: 425–443 Basilewsky (1855) initially described Solea fasciata (=Z. fas- Basilewsky S. 1855. Ichthyographia Chinae borealis. Nouveaux mé- moires de la Société impériale des naturalistes de Moscou, 10: ciatus) based on specimens from Shantung. The original de- 215–263 scription was vague, and provided no useful information for Bleeker P. 1860. Zesde bijdrage tot de kennis der vischfauna van Japan. species identification except for the transverse bands on the oc- Acta Societatis Regiae Scientiarum Indo-Neêrlandicae, 8(1): ular side. The type specimens of Z. fasciatus cannot be tracked 1–104 down (Eschmeyer, 2012), so the validity of this species remains Bloch M E. 1787. Naturgeschichte der ausländischen Fische, vol 3. Ber- unresolved. Ochiai (1955, 1984) regarded Z. fasciatus as a valid lin: Aus Kosten der Verfassers, und in Commission in der Buch- species based on specimens from Japan’s seas. This taxonomic handlung der Realschule, 1–146 Boeseman M. 1947. Revision of the fishes collected by Burger and Von treatment was followed by Yamada and Okamura (1986) and Siebold in Japan. Zoologische Mededelingen, 28: 1–242 Nakabo (2002). All counts used in their study, including D 86– Cheng Paoshan. 1955. Pleuronectiformes. In: Tchang Tchunglin, 92, A 73–80, LLS 107–123, and vertebrae 51–53, fall within the Cheng Chingtai, Cheng Paoshan, et al., eds. Fishes of the Yellow ranges of the counterparts of our specimens (D 79–98, A 69–84, Sea and Pohai, China (in Chinese). Beijing: Science Press, 1−362 LLS 105–138, and vertebrae 51–54) from Wendeng, Qingdao, Cheng Paoshan. 1962. Pleuronectiformes. In: Institute of Zoology CAS, and Rizhao, in the coastal area of Shandong Province, China. It Institute of Oceanology CAS, Shanghai Fisheries College, eds. is likely that Z. fasciatus is a junior synonym of Z. zebrinus, but Fishes of the South China Sea (in Chinese). Beijing: Science Press, 1−1184 the taxonomic status requires further confirmation with com- Cheng Paoshan, Chang Youwei. 1965. Studies on the Chinese soleoid parison between specimens of Z. fasciatus (Basilewsky) from fishes of the genus Zebrias, with description of a new species Japan and Korea. from the South China Sea. Acta Zootaxonomica Sinica (in Chi- S. ommatura was described by Richardson (1846) based nese), 2(4): 267−278 WANG Zhongming et al. Acta Oceanol. Sin., 2014, Vol. 33, No. 8, P. 44–54 51

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110° 115° 120° 125° E

40° N

QD WD RZ

35° LY a

DF ellow Se Y

a 30° ZS

East China Se 25° PT

DS SZ QSBH ZP ZJ 20° South China Sea

Fig.S1. Sample collection sites (•) of the zebra sole along coastal waters of China. The site abbreviations for each locality: Wendeng (WD), Qingdao (QD), Rizhao (RZ), Shandong Province; Liangyungang (LY), Dafeng (DF), Lüsi (LS), Jiangsu Province; Zhoushan (ZS), Shitang (ST), Zhejiang Province; Pingtan (PT), Dongshan (DS), Fujian Province; Shenzhen (SZ), Zhapo (ZP), Zhanjiang (ZJ), Guangdong Province; Beihai (BH), Qisha (QS), Guangxi Autonomous Region.

Fig.S2. Plates in the original description and lectotypes of Pleuronectes zebra and Solea zebrine. a. The original plate of Pleuronectes zebra (Bloch, 1787, Pl. 187); b. ZMB 2423, the lectotype of Pleuronectes zebra; c. the original plate of Solea zebrina (Temminck and Schlegel, 1846, Pl. 95, fig. 1); and d. RMNH.D.1308, the lectotype of Solea zebrina. WANG Zhongming et al. Acta Oceanol. Sin., 2014, Vol. 33, No. 8, P. 44–54 53 6–9 60–73 68–84 Qisha, 98–114 Guangxi 0.147–0.175 0.116–0.206 0.054–0.106 0.057–0.168 0.019–0.031 0.229–0.318 0.085–0.178 0.326–0.380 0.127–0.152 0.269–0.348 0.375–0.479 0.155–0.212 0.364–0.426 0.430–0.501 0.607–0.661 0.398–0.475 0.621–0.674 0.614–0.692 0.596–0.671 0.047–0.063 7.833±0.943 0.161±0.007 0.159±0.019 0.081±0.015 0.101±0.027 0.025±0.003 0.279±0.023 0.127±0.022 0.352±0.012 0.138±0.007 0.308±0.020 0.440±0.027 0.172±0.014 0.396±0.016 0.459±0.014 0.630±0.014 0.435±0.022 0.644±0.015 0.654±0.021 0.634±0.021 0.055±0.004 65.750±3.620 77.583±3.999 107.833±4.580 5–10 60–74 70–88 Beihai, 91–119 Guangxi 0.129–0.168 0.131–0.191 0.064–0.167 0.031–0.155 0.017–0.039 0.246–0.384 0.067–0.227 0.306–0.383 0.122–0.157 0.247–0.351 0.393–0.484 0.145–0.188 0.356–0.436 0.406–0.489 0.588–0.680 0.375–0.495 0.599–0.694 0.589–0.695 0.566–0.679 0.047–0.076 7.630±0.913 0.143±0.007 0.163±0.012 0.105±0.019 0.090±0.029 0.024±0.004 0.310±0.023 0.150±0.024 0.343±0.014 0.139±0.007 0.300±0.017 0.443±0.017 0.166±0.010 0.396±0.017 0.446±0.017 0.632±0.016 0.428±0.019 0.647±0.016 0.650±0.018 0.628±0.018 0.059±0.005 66.970±3.002 79.080±3.657 104.980±4.966 6–9 63–81 74–90 100–128 Zhanjiang, Guangdong 0.145–0.185 0.137–0.210 0.063–0.140 0.061–0.141 0.016–0.026 0.236–0.349 0.101–0.201 0.316–0.366 0.131–0.162 0.267–0.332 0.418–0.473 0.150–0.190 0.359–0.404 0.429–0.488 0.596–0.660 0.397–0.466 0.611–0.676 0.603–0.681 0.582–0.661 0.049–0.064 7.757±0.819 0.162±0.010 0.174±0.015 0.089±0.013 0.099±0.019 0.021±0.003 0.283±0.029 0.147±0.023 0.342±0.011 0.144±0.008 0.297±0.014 0.448±0.014 0.166±0.011 0.382±0.013 0.460±0.016 0.632±0.016 0.426±0.017 0.648±0.016 0.642±0.017 0.623±0.018 0.057±0.004 70.054±4.261 81.784±3.625 111.595±6.441 6–11 64–76 75–89 Zhapo, 104–129 Guangdong 0.124–0.163 0.146–0.206 0.081–0.128 0.063–0.139 0.015–0.028 0.257–0.356 0.091–0.177 0.324–0.376 0.120–0.151 0.273–0.339 0.423–0.476 0.145–0.191 0.359–0.430 0.422–0.494 0.611–0.664 0.396–0.475 0.629–0.678 0.604–0.674 0.581–0.654 0.052–0.068 7.898±0.909 0.144±0.008 0.171±0.011 0.106±0.011 0.108±0.012 0.020±0.003 0.302±0.025 0.144±0.021 0.344±0.012 0.135±0.008 0.301±0.015 0.449±0.010 0.160±0.010 0.392±0.015 0.450±0.015 0.637±0.013 0.424±0.017 0.653±0.013 0.644±0.015 0.623±0.016 0.060±0.004 70.408±2.547 82.571±3.037 114.020±5.165 6–9 64–76 73–89 103–125 Shenzhen, Guangdong 0.137–0.168 0.145–0.195 0.069–0.126 0.037–0.152 0.015–0.028 0.236–0.335 0.091–0.190 0.319–0.392 0.127–0.162 0.269–0.333 0.401–0.474 0.136–0.184 0.361–0.430 0.405–0.532 0.576–0.676 0.382–0.473 0.592–0.690 0.600–0.689 0.579–0.670 0.046–0.066 0.153±0.009 7.975±0.758 7.975±0.758 0.173±0.013 0.103±0.012 0.105±0.022 0.021±0.003 0.293±0.022 0.142±0.023 0.342±0.015 0.143±0.009 0.296±0.016 0.445±0.017 0.162±0.012 0.398±0.014 0.456±0.021 0.635±0.018 0.423±0.020 0.650±0.018 0.643±0.019 0.623±0.019 0.056±0.004 70.925±3.012 70.925±3.012 82.675±3.594 82.675±3.594 113.825±4.878 113.825±4.878 7–10 63–77 74–93 99–126 Zhejiang Dongshan, 0.118–0.177 0.114–0.187 0.065–0.166 0.042–0.135 0.013–0.028 0.225–0.364 0.086–0.196 0.302–0.388 0.117–0.160 0.275–0.335 0.419–0.482 0.138–0.175 0.360–0.452 0.407–0.496 0.598–0.675 0.392–0.456 0.616–0.689 0.613–0.686 0.592–0.666 0.037–0.067 0.143±0.014 7.903±0.690 7.903±0.690 0.151±0.016 0.104±0.016 0.090±0.017 0.020±0.003 0.306±0.031 0.150±0.025 0.346±0.014 0.132±0.008 0.304±0.014 0.455±0.014 0.158±0.008 0.400±0.017 0.445±0.018 0.644±0.014 0.425±0.016 0.658±0.014 0.653±0.014 0.633±0.014 0.056±0.005 70.917±3.183 70.917±3.183 82.542±3.678 82.542±3.678 111.861±5.311 111.861±5.311 6–10 62–79 77–92 Fujian 101–128 Pingtan, 0.126–0.164 0.120–0.187 0.057–0.142 0.032–0.147 0.015–0.037 0.228–0.369 0.092–0.208 0.124–0.160 0.263–0.339 0.421–0.488 0.142–0.200 0.350–0.448 0.405–0.481 0.590–0.682 0.394–0.482 0.606–0.697 0.312–0.368 0.586–0.678 0.565–0.656 0.050–0.073 0.145±0.007 7.648±0.830 0.152±0.013 0.099±0.016 0.107±0.018 0.022±0.004 0.300±0.025 0.156±0.022 0.340±0.012 0.141±0.007 0.297±0.015 0.452±0.012 0.167±0.010 0.391±0.017 0.443±0.014 0.637±0.016 0.428±0.016 0.652±0.016 0.646±0.017 0.625±0.017 0.060±0.004 71.604±3.254 83.462±3.668 111.209±4.871 6–10 64–83 78–98 106–130 Shitang, Zhejiang 0.114–0.146 0.116–0.205 0.047–0.135 0.028–0.114 0.013–0.030 0.256–0.415 0.086–0.193 0.113–0.143 0.268–0.345 0.421–0.493 0.133–0.174 0.393–0.465 0.387–0.476 0.622–0.708 0.371–0.464 0.637–0.722 0.309–0.382 0.631–0.709 0.610–0.688 0.047–0.070 0.130±0.006 0.151±0.015 8.202±0.782 0.097±0.015 0.073±0.016 0.020±0.004 0.306±0.028 0.146±0.022 0.127±0.006 0.302±0.014 0.456±0.013 0.153±0.008 0.427±0.016 0.429±0.019 0.664±0.019 0.419±0.017 0.680±0.018 0.344±0.015 0.673±0.018 0.652±0.018 0.058±0.005 74.596±3.186 86.753±3.661 116.719±5.336 6–10 68–84 80–95 108–132 Zhejiang Zhoushan, 0.118–0.167 0.105–0.216 0.055–0.127 0.049–0.113 0.010–0.028 0.239–0.371 0.101–0.214 0.111–0.163 0.263–0.341 0.422–0.473 0.135–0.187 0.377–0.458 0.389–0.485 0.593–0.711 0.381–0.466 0.687–0.022 0.303–0.387 0.627–0.713 0.605–0.694 0.049–0.065 0.134±0.009 0.158±0.017 8.380±0.858 0.093±0.014 0.079±0.014 0.019±0.004 0.298±0.021 0.150±0.022 0.132±0.009 0.294±0.015 0.450±0.011 0.154±0.010 0.424±0.016 0.420±0.018 0.672±0.022 0.415±0.018 0.608±0.723 0.333±0.014 0.678±0.019 0.657±0.019 0.058±0.004 76.130±3.062 87.900±3.300 120.930±5.456 8–9 Lüsi, 70–81 79–92 Jiangsu 112–132 0.122–0.170 0.112–0.184 0.069–0.150 0.036–0.088 0.013–0.029 0.257–0.363 0.105–0.221 0.112–0.153 0.252–0.348 0.397–0.475 0.134–0.174 0.379–0.464 0.380–0.483 0.618–0.713 0.356–0.464 0.631–0.726 0.304–0.376 0.637–0.727 0.616–0.708 0.046–0.060 0.142±0.011 0.155±0.019 0.104±0.021 8.200±0.400 0.069±0.013 0.019±0.004 0.306±0.025 0.169±0.027 0.131±0.010 0.299±0.022 0.441±0.017 0.154±0.011 0.421±0.020 0.432±0.022 0.666±0.023 0.413±0.024 0.679±0.022 0.336±0.016 0.674±0.022 0.655±0.023 0.053±0.004 77.080±2.513 87.560±2.772 122.920±4.525 7–11 69–84 81–96 Jiangsu Dafeng, 107–136 0.115–0.170 0.120–0.201 0.071–0.140 0.034–0.102 0.010–0.031 0.214–0.352 0.088–0.187 0.110–0.149 0.255–0.342 0.407–0.480 0.124–0.176 0.384–0.464 0.390–0.484 0.649–0.712 0.360–0.450 0.663–0.725 0.304–0.369 0.643–0.721 0.624–0.703 0.045–0.067 0.137±0.014 0.159±0.017 0.101±0.014 8.350±0.753 0.068±0.013 0.018±0.003 0.294±0.027 0.138±0.021 0.127±0.007 0.296±0.015 0.441±0.016 0.152±0.009 0.418±0.017 0.419±0.018 0.677±0.014 0.408±0.016 0.690±0.014 0.329±0.013 0.685±0.017 0.665±0.017 0.055±0.004 76.180±2.744 87.550±3.176 122.760±5.671 7–10 69–88 81–98 Jiangsu 110–138 0.115–0.149 0.128–0.218 0.060–0.143 0.031–0.100 0.010–0.032 0.247–0.359 0.110–0.333 0.113–0.148 0.244–0.341 0.411–0.492 0.126–0.171 0.370–0.451 0.390–0.459 0.641–0.714 0.355–0.466 0.656–0.727 0.297–0.361 0.635–0.716 0.611–0.694 0.043–0.073 0.131±0.007 0.165±0.015 0.097±0.015 8.560±0.726 0.066±0.014 0.018±0.003 0.299±0.023 0.156±0.028 0.128±0.007 0.292±0.017 0.449±0.017 0.149±0.008 0.411±0.017 0.421±0.014 0.669±0.012 0.407±0.019 0.683±0.012 0.330±0.011 0.676±0.017 0.657±0.017 0.056±0.004 76.700±2.883 88.340±3.141 Lianyungang, 123.020±6.005 7–11 70–81 82–95 Rizhao, 110–137 Shandong 0.114–0.155 0.104–0.205 0.069–0.150 0.025–0.109 0.012–0.027 0.259–0.379 0.100–0.211 0.115–0.154 0.259–0.329 0.412–0.480 0.132–0.175 0.363–0.468 0.392–0.463 0.629–0.700 0.364–0.450 0.641–0.715 0.296–0.366 0.623–0.714 0.602–0.696 0.046–0.064 0.132±0.008 0.164±0.018 0.100±0.014 0.069±0.019 8.820±0.767 0.018±0.003 0.303±0.024 0.155±0.023 0.131±0.008 0.291±0.016 0.445±0.013 0.152±0.009 0.407±0.019 0.423±0.015 0.663±0.016 0.410±0.018 0.677±0.016 0.330±0.012 0.673±0.019 0.653±0.019 0.055±0.004 76.520±2.700 88.020±2.874 124.180±6.615 7–10 69–84 79–98 105–136 Qingdao, Shandong 0.111–0.155 0.109–0.173 0.067–0.131 0.027–0.123 0.011–0.032 0.234–0.348 0.102–0.213 0.107–0.151 0.252–0.327 0.423–0.469 0.138–0.180 0.370–0.440 0.391–0.483 0.601–0.696 0.377–0.468 0.618–0.709 0.308–0.362 0.603–0.695 0.587–0.677 0.042–0.064 0.140±0.008 0.151±0.012 0.094±0.013 0.082±0.017 0.019±0.003 8.260±0.610 0.299±0.025 0.161±0.022 0.137±0.008 0.294±0.017 0.446±0.010 0.163±0.009 0.400±0.013 0.433±0.017 0.650±0.018 0.421±0.018 0.664±0.017 0.336±0.013 0.656±0.018 0.638±0.019 0.053±0.004 75.590±3.112 86.910±3.558 120.520±6.057 7–11 70–84 81–98 Ranges, means and standard deviations of meristic characters and ratios of morphometric and truss network measurements of the zebra sole along China’s coast sole along China’s of the zebra of morphometric and truss and ratios measurements deviations of meristic network characters means and standard Ranges, 115–138 Wendeng, Shandong 8.600±0735 0.109–0.166 0.123–0.185 0.041–0.116 0.019–0.083 0.007–0.024 0.224–0.355 0.065–0.197 0.106–0.152 0.245–0.318 0.423–0.479 0.140–0.176 0.358–0.411 0.397–0.459 0.621–0.708 0.348–0.449 0.637–0.722 0.307–0.363 0.629–0.721 0.611–0.704 0.048–0.069 0.144±0.013 0.153±0.016 0.085±0.015 0.049±0.013 0.015±0.003 0.283±0.029 0.144±0.026 0.131±0.008 0.284±0.015 0.444±0.010 0.155±0.008 0.386±0.012 0.430±0.014 0.658±0.017 0.402±0.018 0.673±0.017 0.331±0.012 0.670±0.018 0.652±0.018 0.056±0.004 77.675±2.863 88.488±3.122 125.525±4.741 P A D LLS PL/SL SB/SL HL/SL SE/HL ED/HL d1-4/SL d4-8/SL d1-5/SL d6-8/SL d1-6/SL d6-9/SL d1-8/SL d4-6/SL d8-9/SL d1-2/HL d1-3/HL d6-10/SL d8-10/SL d9-10/SL d6-7/d6-8 Table S1. Table 54 WANG Zhongming et al. Acta Oceanol. Sin., 2014, Vol. 33, No. 8, P. 44–54

Table S2. The K2P genetic distances based on COI sequences of the zebra sole Individuals QD1 QD2 QD3 DF1 DF2 ZS ST PT1 PT2 DS1 DS2 ZJ QS1 QS2 QD1 QD2 0.005 QD3 0.006 0.003 DF1 0.006 0.003 0.004 DF2 0.006 0.003 0.004 0.004 ZS 0.005 0.002 0.003 0.003 0.002 ST 0.005 0.002 0.001 0.003 0.003 0.003 PT1 0.005 0.002 0.003 0.003 0.003 0.003 0.003 PT2 0.008 0.005 0.006 0.006 0.006 0.006 0.006 0.006 DS1 0.005 0.000 0.003 0.003 0.003 0.002 0.002 0.002 0.005 DS2 0.004 0.002 0.003 0.003 0.003 0.003 0.003 0.003 0.006 0.002 ZJ 0.006 0.003 0.005 0.001 0.005 0.004 0.004 0.004 0.006 0.003 0.004 QS1 0.008 0.005 0.006 0.005 0.005 0.005 0.005 0.005 0.006 0.005 0.005 0.006 QS2 0.006 0.003 0.004 0.003 0.004 0.003 0.003 0.003 0.004 0.003 0.003 0.004 0.003 Notes: Max=0.008; Min=0.000; Avg=0.004.

Table S3. Means of the K2P genetic distances based on COI sequences of different soles Inter-species Species Sample size Intra-species zebra sole Z. uagga Z.japonicus Z.crossolepis zebra sole 14 0.004 0 Z.quagga 3 0.202 0.000 9 Z. japonicus 2 0.199 0.212 0.005 4 Z.crossolepis 3 0.215 0.218 0.208 0.002 7 H. japonicus 1 0.224 0.262 0.212 0.232 —

Table S4. The K2P genetic distances based on D-loop sequences of the zebra sole Inter-groups QD1–9 DF1 ZS ST PT1 DS1 BH1–6 QS1–2 Intra-groups QD1–9 0.007 DF1 0.007 ZS 0.008 0.008 ST 0.005 0.007 0.007 PT1 0.009 0.007 0.007 0.008 DS1 0.006 0.006 0.006 0.005 0.007 BH1–6 0.009 0.009 0.010 0.009 0.011 0.008 0.009 QS1–2 0.008 0.008 0.008 0.008 0.009 0.007 0.008 0.008 Notes: Max=0.011; Min=0.005; Avg=0.008.