2 Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China;
3 Fisheries College, Zhejiang Ocean University, Zhoushan 316022, China
Syngnathus, a species-rich genus with 56 recognized species in family Syngnathidae (Dawson, 1985; Kuiter, 2000; Koldewey and Martin-Smith, 2010; Lourie et al., 2016; Shao, 2016), was particularly common and widely distributed in the Atlantic Ocean (Dawson, 1985; Riley et al., 1986; Rogers and Millner, 1996; Rogers et al., 1998; Kuiter, 2001; de Lussanet and Muller, 2007). As for Chinese coastal waters, abundant Syngnathus individuals also existed in this coastline, and these common individuals were identified as S. acus by native ichthyologists (Zhang et al., 1955, 1962; Meng et al., 1962; Feng and Cao, 1979; Shen, 1984; Zhu et al., 1984; Cheng and Zhou, 1997; Li, 2011, 2015). Apart from S. acus, another rare species identified as S. schlegeli was also once collected in this area. According to the original descriptions of this species, it's SL/HL (SL: standard length; HL: head length) was clearly distinguished from that of S. acus. Only when the SL/HL ranged from 4.8 to 7.2, could a Syngnathus individual be counted as S. schlegeli. Because except these two original descriptions themselves, other known records didn't satisfy this unique range (Zhu et al., 1963; Sun and Chen, 2013), S. schlegeli was long-termly thought an uncommon occurrence with quite difficult finds and the dominating Syngnathus species seen every day in the coastal waters of China was S. acus (Zhang et al., 1955, 1962; Meng et al., 1962; Feng and Cao, 1979; Shen, 1984; Zhu et al., 1984; Cheng and Zhou, 1997; Li, 2011, 2015).
Although only S. acus and S. schlegeli have been reported in the coastal waters of China (Shao, 2016; Zhang et al., 2017), in our opinion, taxonomic problems were not fully resolved within these two mentioned species (Zhang et al., 1955, 1962; Zhu et al., 1963; Shen, 1984, 1993; Kuiter, 2000; Shao, 2015). In such a context, the first confusing phenomenon was the fact that the type species and central distribution area of S. acus were located in Europe (Linnaeus, 1758; Dawson, 1985; Riley et al., 1986; Rogers and Millner, 1996; Rogers et al., 1998; Kuiter, 2001; Mwale, 2005; de Lussanet and Muller, 2007). It was also reported that Syngnathus had a pretty week diffusion ability to spread worldwide (Mwale, 2005). Furthermore, in the view of Japanese ichthyological systematics, only one Syngnathus species S. schlegeli rather than S. acus existed in Japanese coastal waters (see Tokiharu, 1986; Nakabo, 2000, but also Dawson, 1985; Tokiharu, 1986; Nakabo, 2000; Mwale et al., 2013; Shao, 2015). Spreading from the North-eastern Atlantic Ocean to million miles-away Chinese coastal waters but not to the approximately same far-away Japanese waters, the identification of S. acus in China was really doubtful.
Besides, European taxonomic researchers have also largely studied local S. acus (Linnaeus, 1758; Dawson, 1985; Riley et al., 1986; Rogers and Millner, 1996; Rogers et al., 1998; Kuiter, 2001; Mwale, 2005; de Lussanet and Muller, 2007). Even if the taxonomic status of Chinese S. acus proved to be true, compared to European abundant introductions, the morphological description about Chinese native S. acus was rough and deficient (Zhang et al., 1955, 1962, 1997; Zhu et al., 1963). Neither traditional medical books nor contemporary taxonomic publications of fishes gave a sufficiently and authoritatively morphological description about this pipefish species (Zhang et al., 1955, 1962, 2017; Meng et al., 1962; Feng and Cao, 1979; Shen, 1984; Zhu et al., 1984; Cheng and Zhou, 1997; Li, 2011; Li et al., 2015). They just underlined the preponderant distribution of a conspecific Syngnathus species and took it as S. acus habitually. Such deficient introduction made the identification of S. acus in the coastal waters of China lack conclusively morphological evidence despite lots of literature records (Zhang et al., 1997; Sadovy and Cornish, 2000; Clarke, 2002; Wu et al., 2009; Cheng and Ken, 2012). According to our present information, local S. acus distributed in China was also conflict with some newly increased descriptions about European S. acus (Zhang et al., 1955, 1962; Nijssen and Buizer, 1983; Tokiharu, 1986; Nakabo, 2000; Wu, 2002; Mwale, 2005; Hu, 2005; Sun and Chen, 2013).
Meanwhile, it was reported that S. schlegeli spread from eastern Russia southward to Taiwan Island (Masuda et al., 1984), including the Japanese coast, Korean Peninsula, Ryuku Islands and Bonin Islands (Masuda et al., 1984; Dawson, 1985; Kim et al., 2005). It was noteworthy that the unique SL/HL criterion of S. schlegeli proposed by Chinese gaugers was not adopted in any Japanese ichthyological systematics; thus, if we do not take SL/HL into consideration, an obviously morphological overlaps between Chinese S. acus and S. schlegeli will be found (Zhang et al., 1955, 1962; Meng et al., 1962; Feng and Cao, 1979; Shen, 1984; Zhu et al., 1984; Cheng and Zhou, 1997; Li, 2011, 2015). The descriptions of Chinese S. acus were synchronously consistent with the characteristics of Japanese S. schlegeli.
In present study, we first performed a survey on morphological characters of Syngnathus collected from four locations, with the aim of providing plentifully morphological characters of this widelydistributed species and determining whether it was misidentified in China. A comparison among morphological data belonging to typical S. acus, S. schlegeli and specimens we collected was conducted consequently. At the same time, a mitochondrial DNA barcoding approach was also employed by means of cytochrome oxidase subunit 1 (COI) gene sequencing, in order to solve the chaotically taxonomic problems of these specimens at genetic level. COI gene variability highly diverges between species but little within a conspecifics (Hajibabaei et al., 2007; Gao et al., 2011; Gross, 2012). It is suitable for identifying animal species (Bian et al., 2008; Puckridge et al., 2013). When an appropriate fragment of the COI gene is used to identify controversial fish species, whether formerly recorded or uncovering cryptic ones, it has widely been proved effective and credible (Zemlak et al., 2009; He et al., 2011).2 MATERIAL AND METHOD 2.1 Sampling
Specimens were collected from the coastal waters of Dandong, Yantai, Qingdao, and Zhoushan from August 2014 to October 2014 (Fig. 1). All 357 individuals were identified based on morphological characteristics commonly-used by Herald (1941), Dawson(1985, 1986) and Mwale (Mwale, 2005; Mwale et al., 2013). A piece of freezed body tissue from each individual was then obtained and preserved in 95% ethanol. All examined specimens were preserved at the Fishery Ecology Laboratory, Fisheries College, Ocean University of China in Qingdao.2.2 Morphological study
Counts and measurements followed the standard methods given by Dawson (1985) and Herald (1941). 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 and the rays of both pectoral fins were counted. Some fin-ray counts could not be determined because of the poor condition of this specimen. In such instances the reported values were taken from the original description (for holotypes) or the specimen was discarded from analysis. The number of subdorsal rings was the sum of subdorsal trunk and tail rings (Dawson, 1986; Mwale, 2005; Mwale et al., 2013). Table 1 and Fig. 2 show the meristic and morphometric characters examined for specimens.2.3 DNA extraction and sequencing
After morphometric measurements of all 357 specimens, 4 individuals from Yantai, 5 from Qingdao, 4 from Zhoushan and all from Dandong were randomly 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 μmol/L), reverse primer (R, 1 μL, 10 μmol/L), dNTPs (2 μL, 2.5 mmol/L each), EasyTaq DNA Polymerase (0.15 μL, 5 U/μL) and 10× PCR buffer (2.5 μL, 25 μmol/L). A Biometra thermal cycler (Göttingen, Germany) with the following given procedure: one initial denaturation (95℃, 5 min), thirty-five cycles consisting of denaturation (94℃, 50 s), annealing (54℃, 50 s) and extension (72℃, 48 s), and one final extension (72℃, 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.2.4 COI analysis
All 16 individuals' original sequences were successfully obtained and revised by DNASTAR software (DNASTAR Inc., Madison, WI, USA). One COI sequence of Hippocampus trimaculatus and 15 sequences of Syngnathus were also downloaded from NCBI for phylogenetic tree study (Table 2). These 32 COI sequences were then aligned using the above DNASTAR software. MEGA 5.0 (Tamura et al., 2011) was used to construct maximum likelihood tree.3 RESULT 3.1 Morphological characters
No individual with SL/HL ranging from 4.8 to 7.2 was found. The generally morphological features were shown in Fig. 3. Body and head were moderately compressed. The specimens were mainly brownish, usually mottled with pale. Encased in a series of bony rings without any scales, these pipefish were typically slender and elongate. Their superior ridges of trunk and tail were not conjoined, but the inferior were. As for lateral trunk ridge, it extended alone the trunk and eventually disappeared in the vicinity of anal ring. Sometimes the confluent ends were not straight but upward-sloping, even connected to the extension line of superior ridges of tail. Besides, the ends of lateral trunk ridge were located in the last trunk ring or the first tail ring. No pipefish in this study had any lateral tail ridges. Median dorsal ridge of snout was entire and low, although there was no lateral spine on snout. In contrast to the result that no median dorsal body ridge had been found in any samples, a median ridge could be seen in ventral trunk. Opercle ridges were usually prominent and complete in early juveniles but typically incomplete or vestigial in sub-adult individuals. All ridges present in specimens were smooth.
In addition to 18–20 trunk rings and 39–43 tail rings, the specimens had 10 caudal-fin rays, 36–46 dorsal-fin rays, 12–14 pectoral-fin rays, and 2–4 anal–fin rays. Dorsal fin base usually started from the last trunk ring, rarely from the first tail ring or the penultimate trunk ring. The number of trunk rings covered by the DFB was 1 mainly, 0 or 2 rarely, and that of tail rings covered by the DFB was 9 mostly, 8 or 10 scarcely. Each snout was slender and longer, its length was 1.61–2.08 in head length and 0.43–0.67 in post-orbital length. Besides, snout depth was 5.23–9.33 in snout length. Head length was 2.05–2.68 in trunk length, 7.25–8.93 in standard length, 0.25–0.31 in post–orbital length, and 3.09–3.59 in pre–dorsal fin distance. Standard length ranged from 117 mm to 213 mm with an average length of 180.3 mm and was 0.11–0.14 dorsal-fin length. Snout depth was 5.28–9.33 in snout length and 1.67–3.33 in trunk depth. Trunk depth was 0.37–0.67 in pectoral–fin length and 0.26–0.47 in caudal-fin length.
Table 3 shows summaries of selected morphological characters of specimens in present and previous studies.3.2 Sequence analysis of the COI gene
Sixteen 651-bp-long sequences of COI gene fragments were obtained. After combined the downloaded COI sequences of Syngnathus, a total of 32 sequences were used for analysis. Table 4 reported the genetic distances between specimens. The mean distance within individuals and among species was 0.52% and 13.88% respectively. Genetic distance between our 16 specimens and S. schlegeli downloaded from NCBI was only 0.49%. Among specimens in present study and other Syngnathus species, genetic distances ranged from 12.98% to 20.79%, which vastly exceeded the threshold of species delimitation (~2%).
A maximum likelihood (ML) phylogenetic tree was constructed using MEGA 5.0 (Fig. 4). Hippocampus trimaculatus was chosen as the outgroup to root the tree. All COI sequences of specimens in present study clustered in the same group, and 11 haplotypes were defined. All haplotype sequences were submitted to GenBank with the following accession numbers: KR152210–KR152220. The haplotype 4 (Hap-4) was shared by 1 specimen from Yantai and 3 from Qingdao. The haplotype 8 (Hap-8) were shared by 2 specimens from Dandong and the haplotype 2 (Hap-2) by 2 individuals from Zhoushan. The remaining haplotypes were unique and each of them was shared by one specimen. At the same time, a large genetic distance (13.17%) between specimens and S. acus indicated that they couldn't be the same species.4 DISCUSSION
The morphological characters of specimens used in this study were photographed, counted and compared with previously representative records on Table 3. These individuals were characterized by 18– 20 trunk rings, 39–43 tail rings, 10 caudal-fin rays, 36–46 dorsal-fin rays, 12–14 pectoral-fin rays and 2–4 anal-fin rays. These phenotypic traits were consistent with the descriptions of S. schlegeli from Japan (Kaup, 1856; Dawson, 1986; Tokiharu, 1986; Nakabo, 2000; Mwale et al., 2013) and S. acus from Europe (Linnaeus, 1758; Mwale, 2005). Besides, by reviewing all known references including our present results, it could be obviously found that S. schlegeli and S. acus were very similar in meristic values. Almost all morphological characteristics such as SL/ HL were overlapped (Linnaeus, 1758; Kaup, 1856; Dawson, 1985, 1986; Tokiharu, 1986; Nakabo, 2000; Mwale et al., 2013). Such overlaps appeared to be very common among Syngnathus that were either closely related or lived in similar habitats (Herald, 1965; Fritzsche, 1980; Kuiter, 2000).
Although S. schlegeli was amazingly similar with S. acus and long-termly misidentified (Linnaeus, 1758; Kaup, 1856; Zhang et al., 1955, 1962; Meng et al., 1962; Zhu et al., 1963, 1984; Herald, 1965; Feng and Cao, 1979; Fritzsche, 1980; Cheng and Zheng, 1987; Cheng and Zhou, 1997; Kuiter, 2000; Li, 2011, 2015), there were still some difference between these two species. The first distinguishable character was body color: S. acus had distinctive body rings, colored sandy brown with darker bars all along its body (Picton and Morrow, 2015). However, S. schlegeli was mainly brownish, usually mottled with pale (Shao, 2015). The second distinction was body length: total length of specimens measured in this study ranged from 117 mm to 213 mm with an with an average 180.3 mm but that of adult S. acus was generally 300 mm to 400 mm with a reported maximum length of 470 mm (Nakabo, 2000; Mwale, 2005; Picton and Morrow, 2015; Shao, 2015). The maximum total length of S. schlegeli up to now was only 300 mm (Shao, 2015). In fact, the English name of S. acus was "greater pipefish" (Linnaeus, 1758; Herald, 1941; Dawson, 1985). As the comparative word "greater" indicated, S. acus was not only longer but also thicker much than most other species among Syngnathus. The third difference was S. schlegeli with the name "Pacific seaweed pipefish" had a single northwest-Pacific distribution (Tokiharu, 1986; Nakabo, 2000). S. acus, however, was distributed in waters along the Eastern Atlantic Ocean including the Mediterranean and Black seas (Dawson, 1986; Mwale, 2005; Shao, 2015). These three difference could separate S. schlegeli from S. acus easily when identifying.
COI sequence was recognized as an effective and reliable method for species identification (Hebert et al., 2003; Domingues et al., 2013; Qin et al., 2013). Our analysis indicated that S. schlegeli specimens in this study could be morphologically different from other Syngnathus species. Moreover, the genetic distance between specimens and two Syngnathus species, S. acus and S. schlegeli, was 13.17% and 0.49% respectively. Taxonomic identifications as well as COI-sequence accuracy of European S. acus and Japanese S. schlegeli have already been recognized for many years. This reality provided our DNA barcoding studies a strongly genetic level support that the species S. schlegeli in the coastal waters of China was misidentified.
It should be noted that a S. acus COI sequence (JF494653) from GenBank did not cluster with other two S. acus sequences. JF494653 was most likely wrong. As we have mentioned above, S. acus was mainly existent along the Eastern Atlantic Ocean (Dawson, 1986; Mwale, 2005; Shao, 2015). Individuals of GQ502180 and KJ128631 were consistent with this distribution. Kuiter (2000) has also pointed out that the pipefish long-termly regarded as S. acus in southern Africa differed considerably from the true S. acus in Mediterranean and Northeastern Atlantic. Local long-snout pipefish of southern Africa was later re-identified as S. temminckii (Dawson, 1985, 1986; Kuiter, 2000; Mwale et al., 2013). Thus, the individual of sequence JF494653 collected from Agulhas should be S. temminckii as well. The misidentification of S. schlegeli in China and S. temminckii in southern Africa once again manifested that Syngnathus appeared to be a genus complex and easily confusable that required carefully taxonomic revision (Fritzsche, 1980; Kuiter, 2000).
Attention should be paid to the standard lengths of Zhoushan specimens. Because from Table 3, we can obviously see that these specimens were much smaller than other three populations. The standard lengths of specimens recorded in Fishes of East China Sea ranged from 102 mm to 138 mm (Zhu et al., 1963), which were consistent with our Zhoushan population and also shorter than those of Dandong, Yantai, Qingdao specimens. Based on the observation that Zhoushan specimens lack brood pouch trace and were colored little flaxen meaning immaturation in S. schlegeli (Shao, 2015), these specimens were thought juvenile individuals. However, it was pretty strange that not only us but also others didn't once collect adult specimens (Zhu et al., 1963; Wu, 2002, 2005; Sun and Chen, 2013). Furthermore, populations of larger size were found in colder environments, and specimens of smaller size were found in warmer regions. Bergmann's rule has been proved by a mass of studies of terrestrial vertebrates (Rypel, 2014). According to a latest study, this science does also apply to some fishes (Rypel, 2014). Body size in our present study correlated positively with latitude. This may be another reason why Zhoushan specimens seemed so short.
It was also reported that Syngnathus was widely distributed in the coastal waters of China including the South China Sea (Zhang et al., 1955, 1962, 2017; Meng et al., 1962; Feng and Cao, 1979; Shen, 1984; Zhu et al., 1984; Cheng and Zhou, 1997; Li, 2011, 2015). We have also tried our best to collect the specimens from southern regions. But it seemed that fishermen and researchers could hardly see the occurrence of Syngnathus species in the South China Sea. The inundant distribution in Shandong province and infrequent presence in the South China Sea made a sharp contrast. Fishes of Taiwan has also stated it clearly that people in Taiwan Island scarcely witnessed S. schlegeli (Shen, 1984, 1993). This Syngnathus species may prefer cold water and live in higher latitude areas. Further domestic and overseas specimen collection is also indispensable in order to define its clearly geographic limits.5 CONCLUSION
The morphological characters and COI sequence analysis demonstrated there were significant differences between specimens and other species of the same genus, which revealed that specimens collected from the coastal waters of China were S. schlegeli in reality. S. schlegeli was recorded as S. acus for a long time. Our present study clarified this previous species misidentification. We hope this study will not only promote the sustainable exploitation, biodiversity conservation and fisheries management of Syngnathus but also contribute to species identification within this genus in the future.6 ACKNOWLEDGEMENT
Mrs. FANG Y. L. and Mrs. LIU L. contributed much to this paper. Thanks were also given to Mr. CHENG P. and Mr. LI C. for sample collection.
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