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SHORT REPORT
No genetic deviation between two morphotypes of the snipefishes
(Macroramphosidae: Macroramphosus) in Japanese waters
Taisuke Noguchi •Kay Sakuma •Tomo Kitahashi •
Hajime Itoh •Yasunori Kano •Gento Shinohara •
Jun Hashimoto •Shigeaki Kojima
Received: 11 February 2014 / Revised: 8 September 2014 / Accepted: 29 September 2014 / Published online: 29 October 2014
ÓThe Ichthyological Society of Japan 2014
Abstract We examined morphological and molecular
characteristics of individuals of Macroramphosus in Japa-
nese waters (the East China Sea and the northwestern
Pacific). Two morphotypes (M.scolopax-type and
M.gracilis-type) that were differentiated based on 10
quantitative morphological characters were not supported
by molecular analyses using nuclear and mitochondrial
DNA markers, while a genetic deviation was observed
between populations of Macroramphosus from the north-
western Pacific and the northeastern Atlantic. Macroram-
phosus scolopax-type and M.gracilis-type individuals are
thought to be intraspecific morphotypes adapted to plank-
ton and benthos feeding, respectively.
Keywords Macroramphosus Japanese waters
Morphotype Molecular phylogeny
Introduction
Snipefishes of genus Macroramphosus Lacepe
`de 1803
(Gasterosteiformes: Syngnathoidei: Macroramphosidae)
are laterally compressed, streamlined fish with a long
second spine on the dorsal fin and a tiny mouth at the tip of
a greatly elongated snout. They are distributed at ocean
depths less than 600 m in tropical and subtropical areas
around the world (Ehrich 1976; Bilecenoglu 2006). Mohr
(1937) suggested that only two of the 17 nominal species of
Macroramphosus were valid: Macroramphosus scolopax
(Linnaeus 1758), which is characterized by a deep body
with a long second dorsal fin spine; and Macroramphosus
gracilis (Lowe 1839), which has a slender body with a
short dorsal fin spine. Based on Japanese specimens, Okada
and Suzuki (1951) stated that this genus was monotypic
based on large overlaps of morphological characters
between them. Ehrich (1976) arrived at the same conclu-
sion based on the existence of individuals with intermedi-
ate morphologies in the northeastern Atlantic and thought
that M. gracilis-type individuals were juveniles of M.
scolopax. However, Miyazaki et al. (2004) showed that
they can be differentiated even at the larval stage based on
specimens collected off Kochi in southern Japan. Presently,
most researchers consider M. scolopax and M. gracilis as
valid species (Bilecenoglu 2006).
Using nucleotide sequences of the mitochondrial control
region and a nuclear DNA marker (the first intron of the S7
This article was registered in the Official Register of Zoological
Nomenclature (ZooBank) as DDF0EB17-0748-49F5-8E5D-
C1E95ACF1669.
This article was published as an Online First article on the online
publication date shown on this page. The article should be cited by
using the doi number.
T. Noguchi H. Itoh S. Kojima (&)
Graduate School of Frontier Sciences, The University of Tokyo,
1-5-1 Kashiwanoha, Kashiwa, Chiba 277-8563, Japan
e-mail: kojima@aori.u-tokyo.ac.jp
K. Sakuma
National Research Institute of Far Seas Fisheries, Fisheries
Research Agency, 5-7-1 Orido, Shimizu, Shizuoka,
Shizuoka 424-0902, Japan
T. Kitahashi Y. Kano
Atmosphere and Ocean Research Institute, The University of
Tokyo, 1-5-1 Kashiwanoha, Kashiwa, Chiba 277-8564, Japan
G. Shinohara
National Museum of Nature and Science, 4-1-1 Amakubo,
Tsukuba, Ibaraki 305-0005, Japan
J. Hashimoto
Faculty of Fisheries, Nagasaki University, 1-14 Bunkyo,
Nagasaki, Nagasaki 852-8521, Japan
123
Ichthyol Res (2015) 62:368–373
DOI 10.1007/s10228-014-0443-6
ribosomal protein gene), Robalo et al. (2009) analyzed the
phylogenetic relationships among specimens of M. scol-
opax,M. gracilis, and specimens with intermediate mor-
phologies collected off the coast of Portugal (northeastern
Atlantic) and suggested that individuals of Macroram-
phosus in the northeastern Atlantic are monotypic. How-
ever, too few genetic variations were detected in the
nuclear DNA region to determine the genetic differences
between M.scolopax-type and M.gracilis-type individuals.
In this study, we conducted molecular phylogenetic
analyses using mitochondrial and nuclear DNA markers
with sufficient variability in Japanese individuals of Mac-
roramphosus from the East China Sea and northwestern
Pacific to evaluate the genetic differences between mor-
photypes (M.scolopax-type and M.gracilis-type). In
addition, we analyzed the phylogenetic relationships
among Japanese and Portuguese individuals of
Macroramphosus.
Materials and methods
A total of 124 specimens of Macroramphosus were sam-
pled at 11 sites (31°09.90–32°29.80N, 127°30.80–
129°09.60E, 134 to 333 m deep) using an otter trawl or a
beam trawl in the East China Sea during 10 cruises of the
training vessel (T/V) Nagasaki-maru of Nagasaki Univer-
sity. Two specimens were collected using an otter trawl at
two sites (36°29.90N, 140°57.10E, 150 m and 37°36.90N,
141°35.80E, 210 m) off the Pacific coast of northeastern
Japan during two cruises of the research vessel (R/V)
Wakataka-maru of Tohoku National Fisheries Research
Institute, Fisheries Research Agency. Sixteen frozen
specimens collected off Kochi and northwestern Pacific
were provided by Prof. K. Sasaki, Kochi University.
Information about the sampling sites is not available
because these specimens were obtained from a fish market.
The specimens were classified based on the qualitative
characters described by Miyazaki et al. (2004): M. scol-
opax-type having red-orange bodies and second dorsal fin
spines with roughly serrated posterior margins, M. gracilis-
type having dark or brownish bodies and second dorsal fin
spines with smooth posterior margins, and intermediate-
type individuals. A small piece of muscle tissue taken from
each specimen was stored in a freezer (-30 °C) until use.
The remaining parts were fixed in 10 % seawater formalin
and preserved in 70 % ethanol.
Following Bilecenoglu (2006), the snout length, eye
diameter, postocular head length, head length, postoper-
cular body length, length of the dorsal spine, distance
between the two dorsal fins, maximum body depth, length
before the dorsal spine, length after the dorsal spine, and
standard length of fixed specimens were measured using a
vernier caliper (0.05 mm accuracy). A non-metric multi-
dimensional scaling (nMDS) analysis was performed
using the Bray–Curtis similarity index based on the first
10 length values divided by standard length. Permuta-
tional multivariate analysis of variance (PERMANOVA)
was used to examine the statistical significance of dif-
ferences. All tests were conducted using R, version 3.1.0
(R Development Core Team 2008) and the package vegan
(Oksanen et al. 2008).
Total DNA was extracted from frozen tissue using the
DNeasy Tissue Extraction Kit (Qiagen, Valencia, CA,
USA) following the manufacturer’s instructions. The
mitochondrial DNA fragment, including the 50part of the
control region was amplified by polymerase chain reaction
(PCR) using primers L-Pro1 (50-ACT CTC ACC CCT
AGC TCC CAA AG-30) and H-DL1 (50-CCT GAA GTA
GGA ACC AGA TGC CAG-30) (Ostellari et al. 1996). The
PCR conditions were as follows: incubation at 94 °C for
2 min, followed by 30 cycles at 94 °C for 40 s, 50 °C for
60 s, and 72 °C for 90 s. The nuclear DNA fragment,
including the first intron of the S7 ribosomal protein gene,
was amplified by PCR using primers S7RPEX1F (50-TGG
CCT CTT CCT TGG CCG TC-30) and S7RPEX2R (50-
AAC TCG TCT GGC TTT TCG CC-30) (Chow and Haz-
ama 1998). The PCR conditions were as follows: incuba-
tion at 94 °C for 3 min, followed by 35 cycles at 94 °C for
45 s, 58 °C for 45 s, and 72 °C for 60 s, and a final
extension at 72 °C for 10 min. To degrade the remaining
primers and nucleotides, 5 ll of PCR products was mixed
with 1 ll of ExoSAP-IT (United States Biochemical,
Cleveland, OH, USA) and incubated at 37 °C for 15 min
followed by 80 °C for 15 min. Each purified PCR product
was used in direct cycle sequencing reactions with corre-
sponding primers and BigDye Terminator Cycle
Sequencing Kit, version 3.0 (Applied Biosystems, Foster
City, CA, USA). The nucleotide sequences were deter-
mined bi-directionally using an ABI 3130 automated DNA
sequencer (Applied Biosystems). The determined nucleo-
tide sequences were deposited in the DDBJ/EMBL/Gen-
Bank databases under the accession numbers
AB826127–826155 (control region) and AB856541–
856547 (S7).
The sequences were aligned using Clustal W
(Thompson et al. 1994) in the MEGA version 5.0 Beta
software package (Tamura et al. 2007) under default
settings. Additionally, the alignments were manually
adjusted where needed. Phylogenetic trees were recon-
structed using Bayesian inference, maximum likelihood
(ML), and neighbor-joining (NJ) methods. The seahorse
Hippocampus kuda Bleeker 1852 was used as the out-
group (GenBank Accession number NC010272). Bayes-
ian inference was performed using MrBayes 3.1.2
(Ronquist and Huelsenbeck 2003). HKY (Hasegawa et al.
Morphological polymorphism of Macroramphosus 369
123
1985)?I?G model was selected through hierarchical
likelihood ratio tests implemented in Modeltest 3.7
(Posada and Crandall 1998). Shape, proportion of
invariant sites, state frequency, and substitution rate
parameters were estimated empirically from the data in
the Bayesian analysis. Two parallel runs were made for
1910
7
generations (with a sample frequency of 1,000)
and under the default value of four Markov chains. The
first 5,000 trees for each run were discarded to ensure that
the four chains reached stationarity by referring to the
average standard deviation of split frequencies. The
consensus tree and posterior probabilities were computed
from the remaining 10,000 trees (5,000 trees 9two runs).
Posterior probabilities C95 % were considered to be
significant. The ML analysis was performed using RAx-
ML 7.2.8 on the Black Box webserver (Stamatakis 2006;
Stamatakis et al. 2008). Bootstrap runs consisted of 500
pseudoreplicates with the default GTR (Rodrı
´guez et al.
1990)?G setting, following the software manual. The NJ
analysis was performed using PAUP* 4.0b10 (Swofford
2002) based on HKY85 model with 1,000 bootstrap
pseudoreplicates. Bootstrap probabilities C75 % were
considered to be significant. Haplotype networks were
constructed with the median-joining method using Net-
work computer program, version 4.5.1.6 (Bandelt et al.
1999) based on differences in nucleotide sequences.
Results
Based on the qualitative characters shown in Miyazaki
et al. (2004), the specimens of Macroramphosus collected
in the East China Sea were classified into M. scolopax-type
(n=78), M. gracilis-type (n=42), and intermediate-type
(n=6) individuals. Specimens collected off northeastern
Japan (n=2) were classified as M. scolopax-type and
those off Kochi were classified as M. scolopax-type
(n=9), M. gracilis-type (n=1), and intermediate-type
(n=6) individuals. One M. gracilis-type specimen col-
lected off Kochi was badly damaged and could not be used
for further morphological examination. Intermediate-type
individuals showed various combinations of four diagnos-
tic characteristics. The M. scolopax-type and M. gracilis-
type individuals included both males and females.
In the nMDS ordination space based on 10 quantitative
morphological characters, M. scolopax-type and M.
gracilis-type individuals formed groups separated from
each other, while the intermediate-type individuals were
plotted between them (Fig. 1). No separation was
observed between individuals of the same type from other
sea areas. Differences in spatial distributions of the three
types in the nMDS ordination space were significant
(PERMANOVA, P\0.05).
Nucleotide sequences of the 50part of the mitochondrial
control region (381 base pairs, bp) were determined for
specimens of Macroramphosus from the East China Sea
(n=26), off northeastern Japan (n=2), and off Kochi
(n=15). No indels were detected, and 29 types of
sequences (haplotypes) were identified. The most dominant
haplotype was obtained from individuals from all three
geographic areas as well as from all three morphotypes.
Phylogenetic relationships among the Japanese and
Portuguese individuals were analyzed based on 345-bp-
long sequences of mitochondrial DNA, which were com-
mon to both the present and previous studies (Robalo et al.
2009). A single deletion was detected between one Portu-
guese haplotype and all other haplotypes. The Japanese
specimens were retrieved as a robust clade with a Bayesian
posterior probability of 0.96 and bootstrap values of 97 %
and 100 % under ML and NJ criteria, respectively (Fig. 2).
None of the M. scolopax-type, M. gracilis-type, or inter-
mediate-type individuals formed subclades within the
Japanese clade. The Portuguese fishes consisted of two
genetically distinct lineages, as shown in Robalo et al.
(2009). The relationship between the Japanese clade and
Portuguese lineages was not resolved with a meaningful
support value either in the Bayesian analysis, resulting in
basal trichotomy, or in the ML or NJ analysis. The average
genetic distance between Japanese and Portuguese fishes
(0.090 under HKY85 model) was larger than that between
the two Portuguese groups (0.058).
Fig. 1 The non-metric multi-dimensional scaling (nMDS) ordination
of individuals of Macroramphosus from the East China Sea and
northwestern Pacific based on 10 quantitative morphological charac-
ters. Circles and triangles indicate specimens from the East China Sea
and the northwestern Pacific, respectively, and black,gray, and white
symbols indicate M. scolopax-type, intermediate-type, and M. grac-
ilis-type individuals, respectively
370 T. Noguchi et al.
123
Although we directly sequenced PCR products contain-
ing the first intron of the gene encoding the S7 ribosomal
protein for 64 specimens of Macroramphosus from the East
China Sea (n=46), off northeastern Japan (n=2), and off
Kochi (n=16), those from only 10 individuals (five M.
scolopax-type, two M. gracilis-type, and two intermediate-
type individuals from the East China Sea, and one M.
scolopax-type individual from off Kochi) could be deter-
mined, because they showed one or two sequences that were
differentiated by a single substitution. Most individuals in
all three morphotypes had two heterozygous sequences of
different lengths. Lengths of the obtained sequences were
either 528 or 531 bp and an indel of three nucleotides was
detected among them. The length of sequences from
individuals collected off the coast of Portugal (Robalo et al.
2009) corresponded to the length of our short sequences.
However, more than five nucleotide substitutions were
detected between Portuguese and Japanese individuals.
Seven haplotypes were identified in Japanese individuals.
Haplotypes obtained from neither M. scolopax-type, M.
gracilis-type, nor intermediate-type individuals formed an
exclusive cluster in a haplotype network (Fig. 3).
Discussion
As the present results showed no genetic deviation
between the two morphotypes for either mitochondrial or
Fig. 2 Phylogenetic
relationships among Japanese
and Portuguese individuals of
Macroramphosus inferred from
Bayesian analyses based on
partial (345 base pairs)
nucleotide sequences of the
mitochondrial control region.
Black,gray, and white circles
indicate haplotypes obtained
from M. scolopax-type,
intermediate-type, and M.
gracilis-type individuals,
respectively. Morphotypes of
Portuguese individuals were
determined by Robalo et al.
(2009). The tree was rooted with
a seahorse Hippocampus kuda.
Support indices are shown on
branches to major clades for
Bayesian posterior probabilities
above 0.5 (left) and bootstrap
frequencies above 50 % under
the likelihood and neighbor-
joining criteria (middle and
right)
Morphological polymorphism of Macroramphosus 371
123
nuclear markers, we conclude that Japanese individuals of
Macroramphosus are monotypic in spite of significant
morphological differences. Similar results were reported
for species of Macroramphosus inhabiting the North
Atlantic (Robalo et al. 2009). Genetic deviation found
between Japanese and Portuguese populations (Fig. 2)
and morphological differences between individuals from
different sea areas (Kuranaga and Sasaki 2000;Clarke
1984; Noguchi et al., unpublished data) suggest that the
genus Macroramphosus is not monotypic. However,
molecular data have been obtained only from these two
sea areas and morphological data of specimens used in
Robalo et al. (2009) are not available. Close taxonomic
examination using additional morphological and molec-
ular data sets are necessary to judge whether this genus is
monotypic or not.
Two morphotypes of Macroramphosus differ in depth
distribution and food source: namely M. gracilis-type feeds
on plankton within the water column and M. scolopax-type
feeds on benthic organisms on the sea bottom (Ehrich and
John 1973; Matthiessen et al. 2003; Miyazaki et al. 2004).
In the East China Sea, the frequency of M. gracilis-type
individuals was higher in samples collected using an otter
trawl (49.1 %) than in those collected using a beam trawl
(3.8 %) (Noguchi et al., unpublished data). Our beam trawl
collects animals within about 1 m from the sea floor, while
the sampling range of our otter trawl is much wider
(0–4 m), suggesting that M. gracilis-type individuals are
distributed at greater distances from the seafloor than M.
scolopax-type individuals. Stable isotope analyses per-
formed in our lab on the same samples (Seike et al.,
unpublished data) revealed significant differences in
nitrogen stable isotope ratios between the morphotypes,
which corroborates the differences in primary food sources.
Miyazaki et al. (2004) showed that morphotypes of
individuals of Macroramphosus are determined at the lar-
val stage, and that the body shape, body color, and
development of scutes differ between the two morphotypes
(Miyazaki et al. 2004; Bilecenoglu 2006). These results
suggest that different sets of alleles determining the feeding
habits are cooperatively expressed within each morpho-
type. Such trophic polymorphism has been shown in many
freshwater fish species (Smith and Sku
´lason 1996),
including charr (Anderson 2003), sticklebacks (Kristja
´ns-
son et al. 2002), perch (Olsson and Eklo
¨v2005), cichlids
(Swanson et al. 2003), and whitefish (Whiteley 2007).
Inland water bodies are often divided into separated
regions and connected via tectonic movements and floods,
which facilitates allopatric evolution of trophic polymor-
phism. In contrast, no clear dispersal barriers are recog-
nized in the open ocean. To the best of our knowledge,
trophic polymorphism has not been reported in other open
sea fishes. Thus, species of Macroramphosus offer a rare
case of trophic polymorphism in the open ocean.
Since the snipefishes of the genus Macroramphosus are
dominant on the continental shelves around Japan and
relatively easy to sample and breed (Kitajima, personal
communication), they may be used as a model group in
adaptive evolution studies in the open ocean. Therefore,
further comprehensive studies on their morphology,
molecular phylogeny, and ecology are necessary.
Acknowledgments We are grateful to Prof. K. Sasaki, Kochi Uni-
versity, and Mr. K. Shimizu and Mr. N. Yamawaki, Nagasaki Uni-
versity for providing the specimens. We thank the captains, officers,
and crew members of T/V Nagasaki-maru of Nagasaki University and
R/V Wakataka-maru of Tohoku National Fisheries Research Institute,
Fisheries Research Agency, for their support in specimen sampling.
We also thank Prof. J. Pabalo and the anonymous reviewers for many
helpful comments on our manuscript. All experiments comply with
the current laws of Japan.
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