Evolutionary Ecology Research, 2019, 20: 51–67

Armour plate diversity in Japanese freshwater
threespine stickleback (Gasterosteus aculeatus)
Yo Y. Yamasaki1, Seiichi Mori2, Tomoyuki Kokita3 and Jun Kitano1
1

Ecological Genetics Laboratory, National Institute of Genetics, Mishima, Shizuoka, Japan,
2
Biological Laboratory, Gifu-keizai University, Ogaki, Gifu, Japan and3
Department of Marine Bioscience, Fukui Prefectural University, Obama, Fukui, Japan

ABSTRACT
Background: Independent colonization of freshwater habitats by threespine stickleback(Gasterosteus aculeatus) offers a great opportunity to investigate the repeatability of phenotypicevolution and the genetic mechanisms underlying parallel evolution. Armour plate reduction
occurs repeatedly in North American and European freshwater populations of the threespine
stickleback. The repeated fixation of the single-origin freshwater alleles at the Ectodysplasin(Eda) locus explains the parallel evolution of plate number reduction in these populations. In
contrast, we know little about the patterns and genetic basis of armour plate diversificationin Japanese freshwater populations.
Questions: Do Japanese freshwater populations show similar patterns of armour plate
reduction as the North American and European freshwater populations? Do the same freshwater alleles underlie plate number reduction in Japanese populations?
Methods: We analysed the armour plate morphology of eleven freshwater populations andone anadromous population of threespine stickleback in Japan. We first classified each fish into
one of the three morphs: completely-, partially-, and low-plated morphs. We next measured theheights of armour plates for the completely-plated morph. We also compared the genome
sequences at the Eda locus among Japanese, North American, and European populations.
Results: Only one Japanese freshwater population was exclusively low-plated. Two freshwater
populations were a mixture of low- and partially-plated morphs. One freshwater population
was a mixture of partially- and completely-plated morphs. Seven freshwater populations andone anadromous population were exclusively completely-plated, and in four of those freshwater
populations, plate heights were reduced compared with those in the anadromous population.
Genome sequences at the Eda locus of all Japanese freshwater populations were more similar to
those of the Japanese, North American, and European anadromous and marine populations
than the freshwater alleles of the North American and European populations.
Conclusion: The parallel evolution of plate reduction occurs in some Japanese freshwater
populations, but the genetic basis for this phenotypic change differs from that of the North
American and European freshwater populations.
Keywords: convergent evolution, East Asia, freshwater adaptation, lateral plate,non-parallel genetic mechanism, scutes.
Correspondence: J. Kitano, Ecological Genetics Laboratory, National Institute of Genetics, Yata 1111, Mishima,
Shizuoka 411-8540, Japan. email: jkitano@nig.ac.jp
Consult the copyright statement on the inside front cover for non-commercial copying policies.
© 2019 Jun Kitano

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INTRODUCTION
Parallel evolution, the evolution of similar phenotypic traits in phylogenetically independent lineages inhabiting similar environments, offers a great opportunity to investigate
the repeatability and predictability of evolution (Darwin, 1859; Schluter, 2000; Losos, 2011). Parallelism
in trait evolution suggests adaptation plays a role in phenotypic evolution (Schluter, 2000).
Although parallel evolution has been widely observed across diverse taxa (reviewed in Elmer andMeyer, 2011; Rosenblum et al., 2014), recent studies have shown variation in the magnitude anddirectionality of parallelism among different study systems (Kaeuffer et al., 2012; Oke et al., 2017;
Stuart et al., 2017; reviewed in Bolnick et al., 2018). This variation can be caused by several factors, suchas differences in the microenvironment (Stuart et al., 2017), the age of populations (Berner et al., 2010;
Lucek et al., 2014), and the genetic basis of the trait (Leinonen et al., 2012). For example, when two
lineages use different genetic mechanisms (i.e. different genes or different mutations), eventhe same selective pressures can cause different patterns of phenotypic divergence becauseof different physiological and pleiotropic effects of the causative mutations (Hoekstra andNachman, 2003; Elmer and Meyer, 2011; Losos, 2011; Leinonen et al., 2012; Rosenblum et al., 2014).
Plate number reduction in the freshwater threespine stickleback (Gasterosteus aculeatus)
is a textbook example of parallel evolution (Bell, 2001). Glacial cycles created many freshwater
habitats during the Quaternary period, into which ancestral marine threespine sticklebacks
colonized in multiple regions of the Northern Hemisphere. In North America and Europe,
many freshwater populations repeatedly experienced reductions in the number of armour
plates (Wootton, 1984; Bell and Foster, 1994). There have been many hypotheses for why the loss ofarmour plates might be an adaptation to freshwater environments. A reduction in armour
could be adaptive by increasing growth through allocation of calcium to somatic growth incalcium-deficient environments (Giles, 1983; Marchinko and Schluter, 2007; Spence et al., 2012), enhancing
defensive ability against predatory invertebrates (Reimchen, 1994; Marchinko, 2009), improving burst
swimming ability by increasing body flexibility (Bergstrom, 2002), and/or increasing buoyancy by
the removal of heavy armour (Myhre and Klepaker, 2009). Previous genetic studies have identified
Ectodysplasin (Eda) as a major gene that explains variation in plate number (Colosimo et al.,2005). All low-plated freshwater threespine stickleback populations in North America andEurope examined thus far have the identical-by-descent freshwater type allele (low Eda
allele), whereas the ancestral marine or anadromous populations carry a different allele(complete Eda allele) (Colosimo et al., 2005; Raeymaekers et al., 2007; Kitano et al., 2008; Bell et al., 2010; Jones et al.,2012; Ravinet et al., 2014; O’Brown et al., 2015). The low Eda allele is maintained within marine andanadromous populations at a low frequency by gene flow from freshwater populations,and newly colonized freshwater populations use the pre-existing allelic variant for a rapid
reduction in armour plates (Schluter and Conte, 2009).
The evolutionary trajectories of armour plates in freshwater populations if the founderpopulations did not carry the low Eda allele remain largely unknown. One possible answer
is the reduction of armour plate size using different genes or mutations. Some European
freshwater threespine stickleback populations did not reduce the number of lateral plates;
however, they did show a reduction in plate size (Leinonen et al., 2012). The low frequency of the
low Eda allele in nearby marine populations is thought to constrain the reduction in plate
number (Leinonen et al., 2012). Another possible outcome is new mutations at the Eda locus,
leading to plate number reduction. A Japanese freshwater population from Gifu carries an
Eda allele similar to the North American and European complete Eda alleles (Colosimo et al.,2005); however, these fish are low-plated (Ikeda, 1933; Mori, 1987). Previous complementation tests

Armour plate diversity in Japanese freshwater threespine stickleback

53

and genetic analyses showed that this population likely acquired new mutations at the Eda
locus (Schluter et al., 2004; Colosimo et al., 2005; O’Brown et al., 2015). Thus, freshwater populations that
do not carry the low Eda allele offer a great opportunity to investigate how different geneticarchitectures can influence the patterns of parallel evolution.
Japan is geographically distant from North America and Europe, and more than ten
Japanese freshwater threespine stickleback populations have been identified (Ikeda, 1933; Kitanoand Mori, 2016). This distinction allows us to examine whether the patterns of parallel armour
plate evolution are shared across a species range of threespine sticklebacks. Ikeda (1933)
investigated the plate numbers of eight Japanese freshwater populations in addition toseveral anadromous populations. He reported that Japanese freshwater populations in the
Nobi Plain and east coast of Lake Biwa, known as ‘Hariyo’ in Japan, are low-plated(range = 3–13 plates), whereas other freshwater populations are completely-plated (range =30–34 plates) or partially-plated (range = 18–33 plates). Although there have been several
subsequent studies on the development of the lateral plates in the Japanese populations(Ikeda, 1934; Igarashi, 1964, 1965; Mori, 1987), variations in plate sizes have never been reported.
Furthermore, several new habitats have been found since these earlier studies (Kitano and Mori,
2016); however, their plate morphology has never been reported.
In the present study, we first investigated the armour plate morphology of Japanese
freshwater threespine stickleback populations and characterized their diversity. Specifically,
we determined whether the patterns of armour plate reduction in the Japanese freshwater
populations are similar to those in North American and European freshwater populations.
Second, we obtained sequences of the Eda locus from Japanese freshwater populations
and examined their phylogenetic relationships with Eda alleles from North American andEuropean populations. Specifically, we tested whether the same freshwater Eda alleles
underlie plate number reduction in the Japanese freshwater populations.
MATERIALS AND METHODSMorphological analysis
For morphological analysis, we used eleven freshwater populations and one anadromous
population (Table 1). Five sampling locations (Aizu, Gifu, Ono, Shiga_Jizo, and Shiga_Niu)
overlap with those reported in Ikeda (1933). The Gifu, Shiga_Jizo, and Shiga_Niu populations are called ‘Hariyo’ in Japan (Watanabe et al., 2003; Kitano and Mori, 2016). Specimens of two
freshwater populations (Towada and Gensui) and one anadromous population (Akkeshi)
have been used in previous analyses and the results reported elsewhere (Kitano et al., 2007a; Adachi
et al., 2012; Kume et al., 2018). A previous study demonstrated that the Lake Towada population
showed rapid morphological and ecological shifts (Adachi et al., 2012); therefore, we analysedsamples collected in 1985 and 1997 for this population. For statistical analysis, Lake
Towada samples collected in different years were treated as different groups (Towada1985
and Towada1997). Samples from two of the freshwater populations (Nasu and Aizu) werecollected by Seiji Igarasi in 1968 and 1969 respectively, and have been stored at the Fukui
Prefectural Educational Research Institute. Because these two populations are now considered severely endangered (Kitano and Mori, 2016), we refrained from additional sampling for thepresent study. The other seven freshwater populations were sampled using minnow traps or
hand nets by one of the authors (Table 1; Fig. 1). Because males and females often differ inexternal morphology (Kitano et al., 2007a), we used only females for the morphological analysis.

Ecotype

StreamStreamStreamStreamStreamStreamLake
Lake
StreamStreamLake
Lake
Anadromous

Population

Gifu
Shiga_Jizo
Shiga_Niu
Nasu
Aizu
Ono
Chimikeppu
OhnumaNishitappu
GensuiTowada1985
Towada1997
AkkeshiTable 1. Plate morph variations

1982
1985
1985
1968
1969
2002/2006
2011
1992
2013
2012
1985
1997
2003

Year
15
10
8
11
11
11
30
15
19
11
15
29
18

N
15
7
2
0
0
0
0
0
0
0
0
0
0

Low-plated
0
3
6
6
0
0
0
0
0
0
0
0
0

Partially-plated
0
0
0
5
11
11
30
15
19
11
15
29
18

Completely-plated

5.87 ± 0.74
6.90 ± 0.88
9.38 ± 1.92
28.64 ± 1.91
32.09 ± 0.54
31.36 ± 0.51
33.50 ± 0.73
33.20 ± 0.56
33.89 ± 0.81
33.64 ± 0.51
32.33 ± 1.23
33.31 ± 0.66
32.28 ± 0.57

No. of plates (mean ± SD)

Armour plate diversity in Japanese freshwater threespine stickleback

55

Fig. 1. Sampling locations in Japan and representative photographs of alizarin red stained specimens.
Blue and red circles indicate freshwater populations and an anadromous population, respectively.

All specimens were stained with alizarin red as described previously (Peichel et al., 2001). The
lateral plates were counted under a dissecting microscope (SZ61, Olympus, Tokyo, Japan).
This was done on the right side of the fish except for one anadromous fish from Akkeshi,
which had damage to its right side; thus, the left side was measured. We first classified eachfish into one of three morphs: completely-, partially-, or low-plated as described previously(Hagen and Gilbertson, 1972; Bell, 2001; Kitano et al., 2008). Fish with only anterior plates and lacking akeel were classified as low-plated morphs; fish with both anterior plates and a caudal keelwith a gap of two or more unplated myomeres were classified as partially-plated morphs;
and fish with complete rows of lateral plates with no or just one unplated myomere wereclassified as completely-plated morphs.
The standard length (SL) of all specimens was measured using a digital calliper with aresolution of 0.01 mm. For completely-plated fish, the heights of plates 8–24 were alsomeasured using the same digital calliper. The heights of the first seven plates, which form asolid structural unit with the pelvic girdle and dorsal spines (Reimchen, 1983), cannot bemeasured with precision, and thus their heights were not taken into account. For partiallyplated fish, we measured the height of the 8th plate: plate height was measured at thisposition in a previous genetic study of variation in plate height (Colosimo et al., 2004). Bothmeasurements were natural log (ln)-transformed before statistical analysis.
For plate number, we performed an analysis of variance (ANOVA). For plate height,
we first performed a principal components analysis using only completely-plated fish tosummarize the 17 ln-transformed plate heights. Then, PC1, which explained 97.61% of thevariance, was used as a plate height trait (plate height PC1) for subsequent analysis (forcomponent loadings, see evolutionary-ecology.com/data/3158Appendix.pdf). Because plate

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Yamasaki et al.

height PC1 is correlated with body size, we performed an analysis of covariance (ANCOVA)
of PC1 with ln-transformed SL (lnSL) as a covariate to account for the effects of body sizeon plate height. First, we tested for an interaction between lnSL and plate height PC1.
When the interaction was not significant, we tested the effect of population on plate height
PC1 with lnSL as a covariate excluding the interaction term. Both the ANOVA and
ANCOVA were followed by Tukey-Kramer post hoc tests to locate pairs that were significantly different from each other after the correction of multiple comparisons. Next,
we similarly conducted ANCOVA for ln-transformed height of the 8th plate using bothpartially- and completely-plated fish with lnSL as a covariate.
Phylogenetic and SNP analysis of the Eda locus
The genomic sequences of the Eda locus were obtained from whole genome sequence
data (Table 2). For phylogenetic analysis, data for 17 populations of G. aculeatus and one
population of G. nipponicus were obtained from a publicly available database (accession
numbers are given in Table 2). We excluded two German freshwater populations whoseplate morphs were polymorphic (Feulner et al., 2015). For the Nishitappu, Chimikeppu, Ono,and Gifu populations, sequence libraries of eight individuals per population were preparedusing a NEBNext Ultra DNA Library Prep Kit for Illumina (Illumina, San Diego, CA).
Whole genome sequencing was performed on three lanes of the Illumina HiSeq X system in
150-base pair (bp) paired-end mode with each individual fish being distinguished by uniqueindex barcodes (NEBNext Multiplex Oligos for Illumina). The obtained reads were
deposited in the DNA Data Bank of Japan (DRA007515) (Table 2).
Sequence reads of each fish were mapped to the repeat sequence-masked Broad S1stickleback reference genome using CLC Genomics Workbench 8.0 (Qiagen, Hilden,
Germany) as described previously (Yoshida et al., 2014), or BWA-MEM (Li, 2013) with default
settings. SNP calling was performed using samtools 1.7 and bcftools 1.6 (Li et al., 2009),
producing vcf files. From the vcf files, we used the VCFtools 0.1.15 software program(Danecek et al., 2011) to extract genomic sequences of the Eda locus (Chromosome IV: positions
12,800,220–12,810,446 bp in the Ensembl BROAD S1 coordinate). This region contains
all exons and introns of Eda. Only sites with eight or more read depths were used. Sites
with gaps or low coverage were masked with N for each individual: the average count of
N per sequence was 2881 bp. We assigned exons and introns of Eda and searched for thebest partitioning scheme and the best substitution model for phylogenetic analysis using
the PartitionFinder2 software program (Lanfer et al., 2017). The AICc scores between the
GTRGAMMA and GTRGAMMAI models were compared; the GTRGAMMAI model
had a better AICc score than the GTRGAMMA model. In the best model, the sequence
was partitioned into five subsets:• Subset1 (7818–7988, 9435–9695, and 1–315 bp);
• Subset2 (316–5762 bp);
• Subset3 (8339–8373 and 5763–5862 bp);
• Subset4 (8554–9013, 8374–8456, and 5863–7817 bp); and• Subset5 (9145–9434, 7989–8338, 9014–9144, and 8457–8553 bp).
RAxML 8.2.4 (Stamatakis, 2014) was then used to construct a maximum likelihood tree using
the GTRGAMMAI model. Statistical support of clades was calculated by 1000 bootstrap

StreamStreamStreamStreamStreamLake
StreamLake
StreamLake
StreamLake
Stream
Lake
River
River
River
Lake
Anadromous
AnadromousMarineAnadromous
Anadromous
Brackish

Gifu
Shiga_Edaore
Nasu
Aizu
Ono
Chimikeppu
Nishitappu
Towada2010
Little Campbell River
Bear Paw Lake
Little Meadow Creek
Long Lake
Misty Stream Inlet
Misty Lake
Malenter Au
Eider
Orraelva
Skogseidvatnet
Akkeshi
Little Campbell River
North Sea
G. nipponicus
G. wheatlandi
P. pungitius

Low
Low/partial
Partial/complete
Complete
Complete
Complete
Complete
Complete
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Complete
Complete
Not describedComplete
Low/Partial
PartialPlate morph
Japan
Japan
Japan
Japan
Japan
Japan
Japan
Japan
CanadaUSA
USA
USA
CanadaCanadaGermany
Germany
NorwayNorwayJapan
CanadaDenmark
Japan
USA
Japan

Country
8
1
1
1
8
8
8
4
1
1
1
1
1
1
1
1
1
1
8
1
5
8
1
1

N
DRA007515
DRA004954
DRA004954
DRA004954
DRA007515
DRA007515
DRA007515
DRA005065
DRA004949
Ensembl
PRJEB5198
PRJEB5198
PRJEB5198
PRJEB5198
PRJEB5198
PRJEB5198
PRJEB5198
PRJEB5198
DRA001136
DRA004937
PRJEB2954
DRA001136
DRA001086
DRA001085

Accession #This study
Yoshida et al. (2019)
Yoshida et al. (2019)
Yoshida et al. (2019)
This study
This study
This study
Yoshida et al. (2019)
Ishikawa et al. (2017)
Jones et al. (2012)
Feulner et al. (2015)
Feulner et al. (2015)
Feulner et al. (2015)
Feulner et al. (2015)
Feulner et al. (2015)
Feulner et al. (2015)
Feulner et al. (2015)
Feulner et al. (2015)
Yoshida et al. (2014)
Ishikawa et al. (2017)
Feulner et al. (2013)
Yoshida et al. (2014)
Yoshida et al. (2014)
White et al. (2015)

Reference

Note: For the Nasu population and G. wheatlandi, partially-plated individuals were used for sequencing. The plate morph of a Shiga-Edaore fish used for sequencing is
unknown.

EcotypePopulation

Table 2. Populations used for Eda sequence analysis

58

Yamasaki et al.

replications. A phylogenetic tree was drawn using the Figtree 1.3.1 software program(Rambaut, 2009). A phylogenetic analysis using only sites where all individuals were genotyped(1084 bp) gave rise to qualitatively similar results.
We additionally checked the genotypes at position 12811481 on Chromosome IV in theEnsembl BROAD S1 coordinate, where nucleotide substitution between T and G issuggested to be responsible for plate morph variation (O’Brown et al., 2015), and several nearby
sites differing in nucleotides between the low and complete Eda alleles (O’Brown et al., 2015)(Chromosome IV: 12800508, 12808630, 12811933, 12813328, 12813394, 12815024,
12815027, 12816201, 12816202, 12816360, 12816402, and 12816464 in the Ensembl
BROAD S1 coordinate). A single nucleotide polymorphism at 12808303 of Chromosome
IV was analysed previously [position 5 in O’Brown et al. (2015)], but was located in the region
that was masked in the repeat-masked reference sequence, so we did not analyse this SNP.
We also investigated SNPs of two other stickleback species, G. wheatlandi and Pungitius
pungitius, at these loci using previously determined whole genome sequences (Yoshida et al., 2014;
White et al., 2015) (Table 2).
RESULTS
Diversity in lateral plate morphology
Only one population, that from Gifu, which provided 15 specimens, was exclusively
low-plated (Fig. 2). The Shiga_Jizo population contained a mixture of seven low-plated

Fig. 2. A violin plot of plate number. Letters above the plots indicate the results of the Tukey-Kramer
post hoc test. Populations with the same letters indicate there is no significant difference in plate
number between them.

Armour plate diversity in Japanese freshwater threespine stickleback

59

individuals (range = 6–7 plates) and three partially-plated individuals (range = 8–9 plates).
The Shiga_Niu population also contained a mixture of two low-plated individuals (eachwith 7 plates) and six partially-plated individuals (range = 9–13 plates). Plate number in the
Shiga_Niu population was significantly higher than that in the other two populations(Shiga_Niu vs. Gifu, Padj < 0.001; Shiga_Niu vs. Shiga_Jizo, Padj < 0.001; and Gifu vs.
Shiga_Jizo, Padj = 0.231). The Nasu population had a mixture of six partially-plated individuals (range = 26–29 plates) and five completely-plated individuals (range = 29–32 plates).
The other seven freshwater populations and one marine population were exclusively
completely-plated.
The interaction between population and lnSL was significant for both plate height PC1
and the height of the 8th plate when all populations were included in the ANCOVA (PC1,
F9,141 = 4.33, P < 0.001; 8th plate, F9,150 = 2.94, P < 0.01). When we removed the Aizu
population, which had a different slope from the other populations (Fig. 3), the interaction became non-significant in the ANCOVA (PC1, F8,132 = 1.46, P = 0.177; 8th plate,
F8,141 = 1.72, P = 0.099). For the remaining populations – excluding Aizu – both plate height
PC1 and the height of the 8th plate varied significantly among populations (PC1,
F8,140 = 51.42, P < 0.001; 8th plate, F8,149 = 27.52, P < 0.001). The Tukey-Kramer post hoc
test for plate height PC1 showed that four of the freshwater populations (Ono, Chimikeppu,
Ohnuma, and Towada1997) had significantly lower plate heights than the anadromous
population (Fig. 3A). Of these four populations, the Chimikeppu population had the lowest
value (Fig. 3A). The Tukey-Kramer post hoc test for height of the 8th plate did not detect
any freshwater populations with a significantly lower plate height at this position than theanadromous population (Fig. 3B).
Phylogeny and SNPs of the Eda locus
We obtained 9695 bp aligned sequences for the phylogenetic analysis of the Eda locus. Eda
alleles show low-plated populations from North America and Europe make up a different
monophyletic group from that containing the North American and European completelyplated marine or anadromous populations (Fig. 4). Eda alleles from all Japanese freshwater
populations, including those with low-plated morphs, were included in the clade containing
the completely-plated anadromous or marine populations of North America, Europe, andJapan (Fig. 4). None of them clustered with the Eda alleles from low-plated freshwater
populations of North America and Europe.
SNP analysis confirmed that all threespine stickleback populations with low-plated
morphs from any region, including Japan (Gifu and Shiga_Edaore), have G at 12811481 of
Chromosome IV (box in Fig. 5). Interestingly, other stickleback species examined, including
G. nipponicus, G. wheatlandi, and P. pungitius, also had G at this position, althoughG. nipponicus individuals are completely-plated (Kitano et al., 2007b). Other nearby SNPs were
divergent between low- and completely-plated populations in North America and Europe.
In contrast, all Japanese freshwater populations, including low-plated populations(Gifu and Shiga_Edaore), were homozygotes of marine alleles at all examined sites except
position 12811481 (see nucleotides shown in red in Fig. 5).

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Yamasaki et al.

Fig. 3. Plate height variations among populations. Plate height PC1 (A) and ln-transformed height of
the 8th lateral plate (B) are plotted against ln-transformed standard length (SL). Letters at the rightside of the population names indicate the results of the multiple comparison tests. Populations withthe same letters indicate no significant difference in plate height PC1 or the height of the 8th plate
between them. Aizu was removed from the ANCOVA because of heterogeneity of slopes.

Armour plate diversity in Japanese freshwater threespine stickleback

61

Fig. 4. Maximum likelihood tree of the Eda locus based on 9695 bp. Values at the nodes indicate
statistical support calculated by 1000 bootstrap replications. Only support values >70% are shown.
Blue letters and bars indicate freshwater populations, while red letters and bars indicate marine or
anadromous populations. Note that we arbitrarily set a root of the tree between the North American/
European low Eda clade and others, so we are unsure whether the low Eda allele is older than the split
between G. nipponicus and G. aculeatus.

DISCUSSION
Overall, we observed a reduction in either plate number or plate size in the Japanese freshwater threespine sticklebacks. However, the patterns in plate reduction varied considerably
among freshwater populations. Only one population was exclusively low-plated. Three
populations were polymorphic, two with low- and partially-plated morphs and one withpartially- and completely-plated morphs. The remaining seven populations were exclusively
completely-plated, with four populations having a reduction in plate size.
The prevalence of completely-plated morphs in freshwater populations has also beenreported from the east coast of Canada (Hagen and Moodie, 1982) and eastern Europe (Münzing,1963), whereas low-plated morphs are relatively common in freshwater populations of thewest coast of North America and southern Europe (Münzing, 1963; Hagen and Gilbertson, 1972;
Wootton, 1984). Factors responsible for global variation in the prevalence of low-plated freshwater populations may be both ecological and genetic. Ecologically, based on the global

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Yamasaki et al.

Fig. 5. SNPs in the regulatory region of Eda and nearby sites. SNP positions follow O’Brown et al.
(2015): position 4 (12800508), position 6 (12808630), position 7 (12811933), position 8 (12813328),
position 9 (12813394), position 10 (12815024), position 11 (12815027), position 12 (12816201), position
13 (12816202), position 14 (12816360), position 15 (12816402), and position 16 (12816464) on
Chromosome IV. A single nucleotide polymorphism located in repetitive sequences (position 5) was
not analysed here. The black box indicates the nucleotide substitution between T and G at 12311481
of Chromosome IV, where G is shared by all low-plated threespine stickleback populations examined.
Nucleotides shown in blue indicate the low Eda allele, while those in red indicate the complete Eda
allele. Nucleotides that belonged to neither are shown in black. Populations shown in blue indicate
freshwater populations, while those in red indicate marine or anadromous populations. A P. pungitius
fish used for the SNP analysis is brackish water-resident and shown in black. We found heterozygotes
at position 12311481 in one North Sea fish and at positions 8–16 in another North Sea fish, which are
not shown here.

distribution patterns of completely-plated freshwater populations, Hagen and Moodie (1982)
inferred that cold temperatures may be associated with the prevalence of the completelyplated morph, although the physiological mechanisms are unclear. Consistent with thisidea, the Japanese low-plated sticklebacks were found only in the southern part of the
distribution, where atmospheric temperature is very warm, and sticklebacks occupy only
spring-fed habitats with a water temperature of around 10–20⬚C throughout the year (Kitano

Armour plate diversity in Japanese freshwater threespine stickleback

63

and Mori, 2016). In contrast, all sticklebacks in the northern part of Japan, Hokkaido Island,

were completely-plated, a region where the surfaces of lakes and rivers freeze in winter.
In addition to ecological factors, there may be genetic constraints on the evolution of
low-plated freshwater morphs. The absence of standing allelic variation at the Eda locus in
the ancestral marine or anadromous populations could be one potential cause (Leinonen et al.,2012). We observed that all Eda alleles in the Japanese populations examined belonged to thegroup of the complete Eda alleles. Furthermore, all 198 anadromous individuals collected
in 2006 in eastern Hokkaido (Kitano et al., 2009) were completely-plated. This contrasts with theobservation that low- and partially-plated morphs were at low frequencies in North
America and Europe (Kitano et al., 2008; Leinonen et al., 2012). These data suggest that the absence ofthe pre-existing low Eda allele in the ancestral populations might constrain the Japanese
freshwater populations from reducing their plate number. Thus, it is likely that both ecological and genetic factors are responsible for variation in the frequency of differently plated
morphs in Japan. Future genotyping of Eda alleles of many Japanese Pacific Ocean marine
threespine sticklebacks will reveal how much standing variation exists at Eda around theJapanese archipelago.
A significant reduction in plate height was observed in four freshwater populations(Chimikeppu, Ohnuma, Ono, and Towada1997) when analysing plate height PC1 scores,
whereas no significant reduction was observed in height of the 8th plate in any freshwater
population. Although we could not include the Aizu population in our statistical analysis ofplate size because of heterogeneity of slopes, this population also appeared to have smaller
plates than the anadromous population (Figs. 1 and 3). Populations displaying no reductionin plate size (Nishitappu, Gensui, and Towada1985) are thought to have been introduced
only recently. Although the age of freshwater colonization is not clear for the Nishitappu
and Gensui populations, the Lake Towada population was established sometime around the1970s (Adachi et al., 2012; Yoshida et al., 2016). Lake Towada is a crater lake, where no fish were
present until the nineteenth century, and the stickleback was first reported in 1979 (Adachi
et al., 2012). In 1985, this population did not show any reduction in plate size, but by 1997 it
had done so, suggesting that plate size reduction can occur rapidly either due to phenotypic
plasticity or genetic changes.
Further research on temporal changes in plate morphology in populations other than theLake Towada population would help us to understand the ecological mechanisms underlying plate evolution over contemporary time-scales (Bell et al., 2004; Kitano et al., 2008; Bell and Aguirre,2013). Two of the Japanese freshwater populations found to display polymorphism in thepresent study (Nasu and Shiga_Jizo) were also reported to have done so in the 1930s orprior to that time (Ikeda, 1933). This indicates that polymorphism has been maintained
for several decades. Disruptive natural selection may be important for the maintenance ofpolymorphism as reported in a Canadian lake population (Marchinko et al., 2014).
Japanese freshwater stickleback populations are valuable systems to investigate thegenetic basis for parallel evolution (Elmer and Meyer, 2011; Losos, 2011; Rosenblum et al., 2014). Our
phylogenetic and SNP analysis of the Eda locus shows that all Eda alleles of Japanese
freshwater populations are more closely related to those of completely-plated anadromousor marine populations in Japan, North America, and Europe than to those of the lowplated freshwater populations of North America and Europe. Although previous studies
have indicated that de novo mutation of Eda rather than fixation of the pre-existing low Eda
alleles is responsible for plate reduction in the Gifu population (O’Brown et al., 2015), and thepresent study indicates that the Shiga_Edaore population also has the same mutation as the

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Gifu population, the genetic mechanisms behind the reduction in plate number in otherJapanese freshwater populations remain elusive. Interestingly, G. nipponicus also had G at
12311481 of Chromosome IV, despite the fact that G. nipponicus is completely-plated (Kitano
et al., 2007b; Higuchi et al., 2014). Because G. nipponicus displays a lower plate height at the caudal
part than sympatric G. aculeatus (Kitano et al., 2007b; Higuchi et al., 2014), it is possible that thismutation may play a role in plate size reduction in G. nipponicus. The genetic basis for platesize reduction in the North American and European freshwater populations is reported
to be caused by transposon insertion at the enhancer region of the Growth/Differentiation
Factor 6 (GDF6) gene (Indjeian et al., 2016). Examining whether the GDF6 locus is also responsible for plate size reduction in the Japanese freshwater populations and whether thesame transposons are inserted at the same site will provide further insights into the genetic
mechanisms of parallel evolution of plate reduction.
ACKNOWLEDGEMENTS
We thank members of the Kitano Lab and participants of Stickleback 2018 for discussion, the City of
Ono and ROIS for support of Stickleback 2018, Mark Ravinet for reading the abstract, the Fukui
Prefectural Educational Research Institute for providing the stickleback specimens, and Katie Peichel
and an anonymous reviewer for constructive comments. This study was supported by JSPS Kakenhi
17KT0028 and 15H02418 to J.K. and 18K14764 to Y.Y.Y.

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