DNA Barcoding Reflects the Diversity and Variety of Brooding Traits of Fish

Fisheries Research 185 (2017) 137–144

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Fisheries Research

j ournal homepage: www.elsevier.com/locate/fishres

Full length article

DNA barcoding reﬂects the diversity and variety of brooding traits of

ﬁsh species in the family Syngnathidae along China’s coast

∗

Yan-Hong Zhang, Geng Qin, Hui-Xian Zhang, Xin Wang, Qiang Lin

CAS Key Laboratory of Tropical Marine Bio-Resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou,

Guangdong 510301, China

a r a

t b

i c s t

l e i n f o r a c t

Article history: DNA barcoding offers a rapid and accurate assessment for species labelling and identiﬁcation. Here, we

Received 22 April 2016

investigated the performance of DNA barcodes in a sample of Syngnathidae, a unique group of ﬁsh with

Received in revised form 9 September 2016

male pregnancy. A total of 1002 DNA barcodes using 649 base-pair fragments of the cytochrome c oxi-

Accepted 15 September 2016

dase subunit I (COI) gene were generated. All species were associated with distinct DNA barcode and

Handled by Prof. George A. Rose

could be readily distinguished; seven of the COI barcode clusters represented the ﬁrst species records

Available online 20 September 2016

submitted to the Barcode of Life Data Systems (BOLD) and GenBank databases. In the Neighbor-joining

tree of COI sequences, two major clusters (Gastrophori and Urophori) were displayed, which could also be

Keywords:

Seahorse classiﬁed by their brood pouches. However, the tail-brooding Hippichthys cyanospilus and trunk-brooding

Pipeﬁsh Syngnathoides biaculeatus were reverse-clustered together with the Gastrophori and the Urophori, respec-

DNA barcoding tively. Both seahorse and pipeﬁsh sequences showed high frequencies of nucleotide substitutions. The

COI probability of nucleotide substitutions, those in pipeﬁsh were higher than that of all seahorses. However,

Conservation we identiﬁed no signal for positive selection based on the COI gene in any of the data sets. Our results

supported DNA barcoding as an efﬁcient molecular tool for achieving better monitoring, conservation,

and management of ﬁsheries.

1. Introduction exploitation of seahorses, in addition to their unique reproduc-

tive system and morphology, have led researchers to pay increased

The family Syngnathidae (seahorses, pipeﬁsh, and seadragons) attention to the large gaps in knowledge of wild seahorse biology

is the sole vertebrate group in which embryonic development and ecology (Woods, 2002).

occurs within a special pouch in males (Herald, 1959). Seamoths There is taxonomic ambiguity for several genera and species.

may be the primitive sister group of the seahorses, pipeﬁsh and The seahorse and pipeﬁsh were originally placed in the same

seadragons (Pietsch, 1978). About 41valid seahorse species and lineages based on morphological and biological characteristics

over 400 pipefish species have been described along the major- (Wilson et al., 2001). More recently, the identification of fish in the

ity inhabiting shallow seabed of the Indo-central western Paciﬁc family Syngnathidae has primarily been based on morphological

◦

Oceans (below latitude 26 N) (Dawson, 1985; Koldewey and recognition; however, the considerable degree of skill and taxo-

Martin-Smith, 2010; Lourie et al., 2016). Seahorses sold as tradi- nomic expertise required often complicate species identiﬁcation

tional Chinese medicine, ornaments and aquaria presentation have (Gutiérrez et al., 2014). As an alternative, molecular methods like

shown increasing value in recent years (Koldewey and Martin- DNA barcoding were utilised to explore taxonomic issues. DNA

Smith, 2010; Lin et al., 2008). At least 77 countries trade seahorses barcoding is one potentially important technique used to identify

in high volumes, meaning that various species of seahorse are being species and determine biological diversity (Hebert et al., 2003a).

harvested on a large scale (McPherson and Vincent, 2004). Sea- The effectiveness of this technique relies on the relatively short

horses are frequently taken in as trawl by catch. This process makes universal molecular tag (approximately 650 bp from the 5 region

their habitats vulnerable to degradation and destruction, which of the mitochondrial cytochrome oxidase I [COI] gen), which is

has led to a sharp decline in wild seahorse stocks. Unsustainable substantially greater between than within species. Therefore, this

approach has been widely applied across phylogenetically distant

animal groups (Hajibabaei et al., 2006; Hebert et al., 2004). Notably,

∗ the development of DNA barcode libraries is based on community

Corresponding author.

efforts, and the use of the Barcode of Life Data Systems (BOLD)

E-mail address: [email protected] (Q. Lin).

http://dx.doi.org/10.1016/j.ﬁshres.2016.09.015

138 Y.-H. Zhang et al. / Fisheries Research 185 (2017) 137–144

specimen and was preserved in 95% ethanol until DNA isolation;

then, all of the live seahorse specimens were released back into

the sea. Vouchers were morphologically identiﬁed to identiﬁca-

tion reliability level two, as described by the Fish-BOL collaborators’

protocol (Steinke and Hanner, 2011), namely, ‘specimen identiﬁed

by a trained identiﬁer who had prior knowledge of the group in the

region or used available literature to identify the specimen’. Ref-

erences in the literature used in our study are as follows: Cheng

(1962), Lourie et al. (1999), Zheng (1987) and Zhu et al. (1963).

2.2. DNA extraction

A dorsal ﬁn from each specimen was removed and macerated

using phosphate-buffered saline (PBS) buffer for extraction. The

macerating tissue from the specimens was frozen in liquid nitrogen

and then ground into powder. Genomic DNA was extracted using

an AxyPrep Multisource Genomic DNA Miniprep Kit (Axygen Bio-

sciences, USA) following the manufacturer’s protocol with minor

◦

modiﬁcations: the tissue homogenate was incubated at 56 C for

2 h during cell lysis with Proteinase K. All DNA samples were stored

◦

at −80 C until polymerase chain reaction (PCR) ampliﬁcation.

2.3. PCR and DNA sequencing

Fragments of the mitochondrial COI gene were ampliﬁed using

the following universal ﬁsh barcoding primers: forward Fish-F2

5 -TCGACTAATCATAAAGATATCGGGAC-3 and reverse Fish-R2 5 -

ACTTCAGGGTGACCGAAGAATCAGAA-3 (Ward et al., 2005). All PCR

reactions were conducted in a total volume of 50 ␮l, utilising 3 ␮l

(10–100 ng) DNA, 0.25 ␮l Taq DNA polymerase (5 U/␮l, TaKaRa,

China-Japan Joint Company, Dalian, China), 1 ␮l of each primer

(10 ␮M), 4 ␮l dNTP Mixture (2.5 mM), 5 ␮l Ex Tag Buffer (10×),

and 35.75 ␮l ddH2O. The thermocycling sequence was performed

® ◦

Fig. 1. Map generated using Surfer for Windows showing the sampling locations

with an initial step of 94 C for 3 min, followed by 35 cycles at

for ﬁshes along China’s coast. Sample sizes per site are given in parentheses. ◦ ◦ ◦

94 C for 30 s, annealing at 52 C for 30 s, 72 C for 1 min, and a ﬁnal

◦

step at 72 C for 10 min. The ampliﬁed PCR products were checked

has resulted in DNA barcoding technology being commonly viewed for optimal fragment size on 1.5% agarose gels. The puriﬁcation

as the gold standard in species identiﬁcation. To date, many pub- of PCR products for sequencing was conducted using an E.Z.N.A.

lished papers have explicitly shown that COI barcoding can be used Gel Extraction Kit (Omega, USA). COI genes were commercially

to effectively discriminate between ﬁsh species (KeskIn and Atar, sequenced using PCR-puriﬁed products (BGI, China).

2013; Knebelsberger et al., 2015, 2014; Ribeiro et al., 2012; Ward

et al., 2005; Zhang and Hanner, 2012). 2.4. Data analyses

This data release presents the results of a DNA barcoding study of

the seahorse and pipeﬁsh of China. China has a long distance coast- Nucleotide sequences were assembled and edited using BioEdit

line, and the constant ocean area along the mainland crosses an 7.0.9.0 (Hall, 1999) and then aligned using ClustalW (Larkin

oriental zone, warm-temperature zone, and tropical zone (Briggs, et al., 2007). The sequences were submitted to GenBank (acces-

1995), which leads to wide distributions and abundances for some sions KP139900-KP140670 and KT355036 – KT355266), and the

ﬁsh of the family Syngnathidae (Zhang et al., 2014). In the present sequences and specimen details were submitted to BOLD (see Sup-

study, we applied DNA barcoding for the identiﬁcation of this group plementary Table A online). The numbers of haplotypes for each

of ﬁsh along China’s coast, which is regarded as an important branch species were estimated using the software DnaSP 5.10.00 (Librado

of the marine biodiversity center of the Indo-West Paciﬁc Ocean and Rozas, 2009). Nucleotide compositions and nucleotide pair and

(Barber et al., 2000). The DNA barcoding data generated in this study codon usage frequencies were calculated using MEGA 6.0 soft-

will be used as an efﬁcient molecular tool for achieving better mon- ware (Tamura et al., 2013). Sequence divergences were calculated

itoring, conservation, and management of the family Syngnathidae. using the Kimura 2-Parameter (K2P) distance (Kimura, 1980). The

intraspeciﬁc distances, interspeciﬁc values within the same genus,

2. Material and methods and interspeciﬁc values between genera within the same fam-

ily were so calculated, respectively. This system usually makes a

2.1. Sample collection suitable metric model when genetic distances are small (Nei and

Kumar, 2000). An unrooted neighbor-joining (NJ) tree was cre-

A total of 485 seahorses, 459 pipeﬁsh and 58 seamoths were ated based on K2P distances using MEGA software (version 6.0)

collected from 31 locations along China’s coast (Fig. 1) (collec- with bootstrap tests of 1000 replicates (Tamura et al., 2013). Pega-

tion information available at http://www.barcodinglife.org/). The sus volitans and P. laternarius were used as outgroups. To identify

sampling location map was created using Surfer for Windows the variable selective pressures in COI, we employed the CODEML

(Keckler, 1997). Most specimens were collected by researchers algorithm from the PAML package (Yang, 2007). The improved

on board trawl boats, and a few were obtained with the help of branch-site model (Yang and Nielsen, 2000; Zhang et al., 2005)

local ﬁshermen and buyers. A dorsal ﬁn was removed from each was used to estimate the ratio (␻) of the rates of nonsynonymous

Y.-H. Zhang et al. / Fisheries Research 185 (2017) 137–144 139

Fig. 2. Neighbor-joining (NJ) tree based on COI barcodes from 237 ﬁsh haplotypes in Syngnathiformes along China’s coast. Numbers in brackets indicate the number

of specimens analysed per species. The numbers next to the branches indicate bootstrap values >50%. Brood-pouch cross-sections illustrate pouch variation [ ,

fully enclosed pouch; , enclosed pouch (inverted); , enclosed pouch (semi); , rudimentary pouch with plates and skin-folds; , individual egg

compartments; , free spawning] among the major lineages of trunk- (Gastrophori) and tail-brooding (Urophori) species.

(Ka) to synonymous (Ks) substitutions (␻ = Ka/Ks). To avoid local 2.5. Ethics statement

optimal maximum likelihood iterations, three initial omega values

were used (omega = 0.5, omega = 1, and omega = 2). We used the The Seahorse Project is a key study in the South China Sea

likelihood ratio test to determine signiﬁcant differences between Institute of Oceanology and a key research focus of the Chinese

the null and alternative models (Yang, 1998). Academy of Sciences. The seahorses used in this experiment were

approved for use in research, the sampling areas in our study are

140 Y.-H. Zhang et al. / Fisheries Research 185 (2017) 137–144

public, and there are no special policies protecting the seahorses.

Some research in our laboratory aims to obtain detailed informa-

tion regarding wild seahorses and then to provide data that may

lead to the protection of seahorses in some areas. All seahorse

samples utilised in this study are in accordance with animal ethics

approval for experimentation granted by the Chinese Academy of

Sciences. All experimental protocols were approved by the Com-

mittee on the Ethics of Animal Experiments of the Chinese Academy

of Sciences.

3. Results

A total of 1002 COI sequences were obtained, representing 29

species, 12 genera and two families (Syngnathidae and Pegasidae)

from 31 locations along China’s coast. A total of 237 unique haplo-

types were generated. Syngnathus schlegeli had the most haplotypes

(30) in the data set. The number of specimens clustered under the

same species is indicated in Fig. 2. All sequences were trimmed

to a consensus length of 649 bp. Codon usage was investigated to

detect any possible stop codons; however, none were observed.

The mean nucleotide compositions for the complete data set were

as follows: 25.14% adenine (A), 31.83% thymine (T), 25.26% cytosine

centage of G-C (51.08%) was detected in Solegnathus spinosissimus,

whereas the lowest (39.18%) was in Doryichthys boaja. Within the

649-bp nucleotide sequences for the complete data set, there were

371 conserved sites, 278 variable sites, 273 parsimony informative

sites, and ﬁve singleton sites, accounting for 57.16%, 42.84%, 42.06%,

and 0.77% of the length of the sequence, respectively. Transitional

Fig. 3. The distribution of K2P distances at COI sequences. (a) Box plots of K2P dis-

pairs (si = 69) were present in greater numbers than transversional tances at different taxonomic levels. The box represents the 25–75th percentiles of

the data set, and the horizontal line represents the median. The grey bar indicates

pairs (sv = 42). The ratio of si/sv (R) was 1.62 for the data set.

the ‘barcoding gap’ between the intra- and interspeciﬁc distances. (b) The frequency

The NJ tree revealed that all of the individuals of the same species

distributions of 1560 intraspeciﬁc and 26,406 interspeciﬁc pairwise genetic K2P dis-

clustered together (Fig. 2). No single haplotype was shared between

tances. The enlarged area displays the ‘barcoding gap’ (grey) between the intra- and

species in our data set, and no taxonomic deviation was detected interspeciﬁc distances.

at the species level. The rapid diversiﬁcation of Syngnathidae was

clear from the DNA barcoding analysis, and this analysis clariﬁes the

basal branching of the trunk-brooding lineages (Gastrophori) from tative COI sequences for 10 seahorses, 17 pipeﬁsh and 2 seamoths

the tail-brooding lineages (Urophori) (Fig. 2). The tail-brooding Hip- were compared with the existing BOLD and GenBank databases,

pichthys cyanospilus clustered together with Gastrophori species, 18 species (H. barbouri, H. comes, H. histrix, H. ingens, H. kelloggi,

suggesting that tail brooding has evolved independently in this lin- H. kuda, H. mohnikei, H. spinosissimus, H. trimaculatus, Doryrham-

eage. This study also supported a close evolutionary relationship phus excisus, D. japonicas, D. dactyliophorus, H. cyanospilu, Microphis

between Syngnathoides biaculeatus and the Urophori. All seahorse brachyurus, S. biaculeatus, S. acus, S. schlegeli and P. volitans) showed

species uniformly formed a subfamily of Hippocampus, with a very high species-speciﬁc similarities (97–100%) in both databases.

bootstrap value of 100% on the NJ tree. There were close rela- For example, S. biaculeatus had 100% maximum identity in Gen-

tionships between Hippocampus, Corythoichthys ﬂavofasciatus and Bank, whereas the percentage similarity in the BOLD database

Trachyrhamphus serratus. H. casscsio and H. kuda formed a cohesive for this species was 99.84%. The remaining one species of sea-

group with a moderately signiﬁcant bootstrap value of 82%. horse, four species of pipeﬁsh and one species of seamoth were

Genetic distances increased from lower to higher taxonomic lev- submitted to the BOLD databases for the ﬁrst time. Notably, this

els (Fig. 3a). Intraspeciﬁc K2P distances ranged from 0.15% to 1.57%, study also highlighted shortcomings in this aspect of the BOLD

and most of the intraspeciﬁc genetic distances were below 1%. The and GenBank databases. For example, GenBank failed to discrim-

mean intraspeciﬁc genetic distance was 0.49%. The mean genetic inate S. hardwickii from S. dunckeri. At the time of analysis, the

distances between congeneric and confamilial species were 12.53% top GenBank hit using our S. hardwickii sequences was a single S.

and 24.06%, respectively. The mean pairwise genetic distance was dunckeri sequence (97% identity). Further investigation and con-

estimated at 20.02% among 1002 COI sequences. Overall, the mean sistent sequences from multiple positively identiﬁed S. hardwickii

genetic distance between confamilial species was nearly 49-fold samples led us to conclude that our pipeﬁsh specimen truly repre-

higher than that among individuals within species. Comparisons sents S. hardwickii. Similarly, the morphological characteristics of

of K2P distances displayed a gap of 1% between intraspeciﬁc and H. casscsio were different from those of H. kuda (data not shown);

interspeciﬁc distances within our sequence library (Fig. 3b). Inter- however, H. casscsio sequences in GenBank and BOLD also appeared

speciﬁc K2P distances ranged from 2.58% to 29.31%. The lowest to be mislabelled. GenBank included a COI sequence for T. serratus,

genetic distance (with the exception of intraspeciﬁc distance) and we recorded 99% maximum identity using our T. serratus sam-

(2.58%) was calculated between H. kuda and H. casscsio, and the ples; however, the BOLD Identiﬁcation System (BOLD-IDS) lacked a

highest genetic distance (29.31%) was calculated between Duncke- legitimate barcode. Further, both BOLD-IDS (species-level and pub-

rocampus dactyliophorus and Pegasus volitans. lic data records) and the GenBank database were unable to identify

Table 1 shows the comprehensive barcoding identiﬁcation C. ﬂavofasciatus, D. boaja, M. retzii, Oostethus manadensis and P.

results based on GenBank and BOLD databases. When represen- laternarius. No match was garnered for these species from BOLD-

Y.-H. Zhang et al. / Fisheries Research 185 (2017) 137–144 141

Table 1

Summary of identiﬁcation based on each species barcode sequence using the BOLD-IDS and BLASTN search from GenBank.

Species studied BOLD-IDS GenBank (BLASTN)

Species identiﬁcation % Species identiﬁcation % Max

Hippocampus barbouri Hippocampus barbouri 100 Hippocampus barbouri 100

Hippocampus comes Hippocampus comes 100 Hippocampus comes 99

Hippocampus histrix Hippocampus histrix 99.84 Hippocampus histrix 99

Hippocampus ingens Hippocampus ingens 100 Hippocampus ingens 100

Hippocampus kelloggi Hippocampus kelloggi 100 Hippocampus kelloggi 100

Hippocampus kuda Hippocampus kuda 100 Hippocampus kuda 100

Hippocampus mohnikei Hippocampus mohnikei 97.33 Hippocampus mohnikei 98

Hippocampus spinosissimus Hippocampus spinosissimus 100 Hippocampus spinosissimus 99

Hippocampus trimaculatus Hippocampus trimaculatus 100 Hippocampus trimaculatus 100

∗ ∗

Hippocampus casscsio Hippocampus kuda 99.84 Hippocampus kuda 99

Corythoichthys ﬂavofasciatus No match 0 Notoscopelus resplendens 83

Doryichthys boaja No match 0 Microphis brachyurus 86

Doryrhamphus excisus Doryrhamphus excisus 99.84 Doryrhamphus excisus 99

Doryrhamphus japonicus Doryrhamphus japonicus 99.8 Doryrhamphus japonicus 99

Dunckerocampus dactyliophorus Dunckerocampus dactyliophorus 99.8 Dunckerocampus dactyliophorus 99

Hippichthys cyanospilus Hippichthys cyanospilus 99.8 Hippichthys cyanospilus 99

Microphis brachyurus Microphis brachyurus 99.84 Microphis brachyurus 99

Microphis leiaspis Microphis leiaspis 99.8 Entelurus aequoreus 82

Microphis retzii No match 0 Microphis brachyurus 83

Oostethus manadensis No match 0 Niviventer sp. 82

∗

Solegnathus hardwickii No match 0 Solegnathus dunckeri 97

Solegnathus lettiensis Solegnathus lettiensis 97.3 Solegnathus dunckeri 94

Solegnathus spinosissimus Solegnathus spinosissimus 100 Solegnathus dunckeri 86

Syngnathoides biaculeatus Syngnathoides biaculeatus 99.84 Syngnathoides biaculeatus 100

Syngnathus acus Syngnathus acus 99.84 Syngnathus acus 99

Syngnathus schlegeli Syngnathus schlegeli 99.21 Syngnathus schlegeli 99

Trachyrhamphus serratus No match 0 Trachyrhamphus serratus 99

Pegasus laternarius No match 0 Acanthopagrus berda 83

Pegasus volitans Pegasus volitans 98.91 Pegasus volitans 98

Asterisks with bolded words correspond to problematic identiﬁcations of species in this study using either one or both of the databases.

IDS; GenBank returned top hits for the related species Notoscopelus and GenBank databases shows that seven of the 29 species in

resplendens (83% identity), M. brachyurus (86% identity), M. brachyu- our data set returned low or no similarity matches with the two

rus (83% identity), Niviventer sp. (82% identity) and Acanthopagrus databases; thus, these species were being barcoded for the ﬁrst

berda (83% identity), respectively. time. The remaining species matched the reference sequences in

We tested for signals of nucleotide mutations in the sequences both databases. Using our S. hardwickii in queries against the BOLD-

of each hierarchical clade, including seahorses, pipeﬁsh and IDS returned “no match.” However, 97% of matches were found

seamoths. Both seahorse and pipeﬁsh sequences showed obvious with S. dunckeri sequences in GenBank. This result likely reﬂects

signals indicating nucleotide mutations (Fig. 4). The nucleotide mislabelling in GenBank, as these two species are morphologically

mutation frequencies in the sequences of all of the pipeﬁsh were (and genetically) similar. A similar situation was previously docu-

higher than those of all of the seahorses. Furthermore, the proba- mented for the two catﬁshes Clarias macrocephalus and C. batrachus

bilities of nucleotide substitutions in pipeﬁsh at positions 185, 332, (Wong et al., 2011). BOLD-IDS validates its identiﬁcation search

371, 500, 524, 534 and 620 were extremely high (probabilities >0.6) only if the species in the reference database has at least three bar-

(Fig. 4). coded specimens and identiﬁes the query sequences if it matches

We also tested for positive COI gene selection between Gas- the reference sequence within the conspeciﬁc distance of less than

trophori and Urophori using the modiﬁed branch-site model. 1% (Ratnasingham and Hebert, 2007). Therefore, correct species

Amino acid sequences were identical in the species of Gastrophori labelling, morphological taxonomy, and voucher documentation

and Urophori; therefore, they showed no sign of positive selection should be prioritised in cases in which the reassessment of spurious

within either clade (␻ < 1, P > 0.05) (Fig. 5). We then tested for a sig- data is necessary (Ward et al., 2005).

nal of the positive selection on the COI sequences from the clades of There were no stop codons in these sequences, which indicated

the enveloped brood pouch, the half-enveloped brood pouch and that there were no NUMTs (Nuclear Mitochondrial DNA: nuclear

the naked brood pouch. We identiﬁed no signal of positive selection DNA sequences originating from mitochondrial DNA sequences) in

for the COI gene in these lineages (␻ < 1, P > 0.05) (Fig. 5). our data set, a result in agreement with those of previous studies

(Ward et al., 2005).

An average G-C of 43.03% was detected in our data set. Ward

4. Discussion et al. reported that the average G-C content of Australia’s ﬁsh

species was 47.1% (Ward et al., 2005), which was congruent with

The morphological study of the specimens raised questions the content in the DNA barcoding of Indian marine ﬁsh (Lakra

regarding the observed features versus the described features. In et al., 2011). The G-C content was usually higher in the teleosts.

a few cases, morphological species keys were difﬁcult to discern Nucleotide changes primarily occurred at the 3rd codon position

(Bhattacharjee et al., 2012). The DNA barcoding approach resolved and then the 1st codon position. The mean nucleotide diversity (Pi)

some identiﬁcation issues and explained the actual species com- among all pipeﬁsh was higher (0.17303) than that of all seahorses

position of the region. A total of 1002 COI sequences with a length (0.0.09291); however, the haplotype diversity (Hd) among all sea-

of 649 bp were obtained from 10 species of seahorses, 17 species horses, pipeﬁsh and seamoths was the same (1.000). The results

of pipefish and two species of seamoths. As shown in Table 1, a indicated that lineages diversified more quickly within pipefish

comparison of our results with the COI sequences in the BOLD

142 Y.-H. Zhang et al. / Fisheries Research 185 (2017) 137–144

Fig. 5. Sites showing signals of positive selection in the ancestral branches of all ﬁsh

species in Syngnathidae. The position stands for the codon position on the cDNA

sequence.

Sedberry, 2011; Zhang et al., 2014). In this study, some intraspeciﬁc

genetic variations were reduced to 0.15% when comparing individ-

uals from distant localities. However, some pairwise K2P distances

exceeded 1%, such as H. spinosissimus. The K2P values for conspe-

ciﬁc, congeneric and confamilial distances were 0.49%, 12.53% and

24.06%, respectively. This increase in genetic distance through the

higher taxonomic levels supports a signiﬁcant change in the genetic

divergence at species boundaries (Hubert et al., 2008; KeskIn˙ and

Atar, 2013; Lakra et al., 2011).

The NJ tree revealed a genetic relationship between species.

Fig. 4. Sites showing signals indicating nucleotide mutations in the ancestral

Each species was associated with a speciﬁc DNA barcode cluster,

branches of all seahorses (a), all pipeﬁsh (b), and all seamoths (c). The horizontal

and the relationship between these species was clearly established.

dotted line indicates the probability of nucleotide substitutions equal to 0.6.

When we examine the NJ tree, a clustering pattern was determined

that could be informative regarding the phylogenetic relationships

than within seahorses. The COI locus harbours a high mutational between conspeciﬁc, congeneric, and confamilial levels. Wilson

rate, even for mtDNA (Saccone et al., 1999). We did not detect et al. (2001) used a combined sequence of Cytb, 12S rRNA and 16S

a signature for positive selection in any species. One possible rRNA to reconstruct the evolutionary history of the Syngnathidae.

explanation for this ﬁnding could be that it represents a signal of Their study supported Duncker’s morphologically based grouping

purifying selection in the derived branches. of the Urophori and Gastrophori (Duncker, 1912). Interestingly,

The branch length between species tends to be substantially our study showed that tail brooding might have been secondarily

deeper than between conspeciﬁc individuals, leading to a barcod- acquired in H. cyanospilus, making this species of particular interest

ing gap in the distribution of intraspeciﬁc and interspeciﬁc genetic in studies aimed at understanding the functional and morphologi-

distances (Meyer and Paulay, 2005). When COI is used as a barcod- cal changes associated with the evolution of male pregnancy in this

ing marker for animal species, the intraspeciﬁc K2P distances are group. A similar result was found for trunk-brooding S. biaculeatus,

usually below 1% and rarely more than 2% (Hebert et al., 2003a). which clustered together with Urophori species, suggesting that

The mean intraspeciﬁc genetic distance ranged from 0.15% to 1.57% trunk brooding evolved independently in this lineage. Our study

for the entire data set. Biological mechanisms, water dynamics suggested closer evolutionary relationships between Hippocam-

(such as ocean currents), geographic distance, and historical events pus, C. ﬂavofasciatus and T. serratus compared with that between

may play important roles in the population genetic structures of Hippocampus and S. acus, which challenges the phylogenetic rela-

marine species (Barber et al., 2000; Nelson et al., 2000; Saarman tionship proposed by Wilson et al. (2001). However, DNA barcoding

et al., 2010). Many marine ﬁsh lack a phylogeographic structure is of limited value in elucidating phylogenetic relationships (Vences

and show genetic homogeneity within populations (Friess and et al., 2005). Further genetic analyses are required to address this

Y.-H. Zhang et al. / Fisheries Research 185 (2017) 137–144 143

issue. Seahorses have fully enclosed pouches; C. ﬂavofasciatus and T. Duncker, G., 1912. die Gattungen der Syngnathidae. Mitteilungen aus dem

Naturhistorischen Museum in Hamburg 29, 219–240.

serratus have semi-enclosed pouches. The two types of brood pouch

Foster, S., Vincent, A., 2004. Life history and ecology of seahorses: implications for

are present in the form of a single sac chamber. However, S. acus

conservation and management. J. Fish Biol. 65, 1–61.

has an inverted pouch that is morphologically similar to two sac Friess, C., Sedberry, G., 2011. Genetic evidence for a single stock of the deep-sea

teleost Beryx decadactylus in the North Atlantic Ocean as inferred from

chambers. The morphological traits also supported our molecular

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