Environmental biology of fishes, tập 90, số 3, 2011

Environ Biol Fish (2011) 90:211–222
DOI 10.1007/s10641-010-9733-7

Population genetic structure, diversity and stocking effect
of the oriental weatherloach (Misgurnus anguillicaudatus)
in an isolated island
Yuichi Kano & Katsutoshi Watanabe &
Shin Nishida & Ryo Kakioka & Chris Wood &
Yukihiro Shimatani & Yôichi Kawaguchi

Received: 14 April 2010 / Accepted: 12 October 2010 / Published online: 19 November 2010
# Springer Science+Business Media B.V. 2010

Abstract Genetic endemism of island organisms and
the threat to such organisms provided by artificially
introduced genes are aspects of major interest in
evolutionary and conservation studies of fishes. In
this paper the genetic population structure of the
oriental weatherloach, Misgurnus anguillicaudatus, in
Sado Island of Japan was elucidated by phylogeographic analysis based on partial mitochondrial
control region sequences. The specimens were sampled at 62 sites in Sado Island and 14 sites on the
mainland close to the island. We found various
haplotypes of different origins, most of which had
already been reported from the mainland and other
places of Japan. This suggests that the loach has been
historically introduced to the island from various

regions of Japan. Of the 62 sites on the island,
cultured/nonnative individuals were confirmed to
have been stocked at eight specific sites for feeding

of re-introduced Japanese crested ibis (Nipponia
nippon). By a Mantel test, geographical and genetic
distances were not significantly correlated among the
local populations in Sado Island. However a significant correlation was found when the eight stocked
local populations were excluded from the analysis.
This implied that the genetic distribution pattern of
the loach on the island has been disturbed by the
stocking. In addition, the nucleotide diversity values
of stocked local populations were significantly higher
than those of other local populations, also a likely
outcome of the stocking. In conclusion, the loach on

Y. Kano (*) : Y. Shimatani
Department of Urban and Environmental Engineering,
Graduate School of Engineering, Kyushu University,
Motooka, Nishi-ku,
Fukuoka 819-0395, Japan
e-mail:

C. Wood
Department of Biology, Faculty of Sciences,
Kyushu University,
Higashi-ku,
Fukuoka 812-8581, Japan

K. Watanabe : R. Kakioka
Department of Zoology, Division of Biological Science,
Graduate School of Science, Kyoto University,
Kitashirakawa-Oiwakecho, Sakyo-ku,
Kyoto 606-8502, Japan

S. Nishida
Department of Biodiversity Sciences, Graduate School
of Social and Cultural Studies, Kyushu University,
Motooka, Nishi-ku,
Fukuoka 819-0395, Japan

C. Wood
College of Life Sciences, Zhejiang University,
Zijingang Campus, 338 Yuhangtang Road,
Hangzhou 310058 Zhejiang Province,
People’s Republic of China
Y. Kawaguchi
Laboratory of Ecosystem Management,
Division of Ecosystem Design, Institute of Technology
and Science, The University of Tokushima,
2-1 Minami-Josanjima,
Tokushima 770-8506, Japan


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the island likely had their origins in multiple historical
introductions and colonizations, where more recent
stocking for the ibis has caused further genetic
disturbance to their local populations.
Keywords Genetic disturbance . Japanese crested
ibis . Mantel test . Mitochondrial DNA .
Reintroduction . Sado Island

Introduction

For wild organisms the isolation of species in islands
often results in a higher likelihood of endemism
where original species or subspecies are sometimes
generated (Grant 1998; Avise 2000). This holds true
for the freshwater environment where island freshwater
endemism is notable in many geographical regions. For
example the islands of the Greater Antilles (including
Cuba, Jamaica, Hispaniola and Puerto Rico) are noted
to each have one to several endemic species of
freshwater crabs (Cook et al. 2008). From New
Zealand comes a similar report of an endemic species
of freshwater fish Galaxias gollumoides in Stewart
Island (McDowall and Chadderton 1999). Possibly
most remarkably, a United Nations Environment
Programme Report on Madagascar highlighted that,
of the island of Madagascar’s 140 known species of
freshwater fish, 93 (66% at a species level) were
endemic to the island (UNEP 2010).
Such taxonomic or genetic endemism is, however,
sometimes disturbed by artificially introduced genes.
Human-mediated genetic disturbance is now a serious
issue in wild animal/plant conservation (Hindar et al.
1991; Rhymer and Simberloff 1996; Beaumont et al.
2001; Randi and Lucchini 2002; Larsen et al. 2005).
In fish, one fairly extensively studied species that has
been investigated for the genetic impact of the
introduction of non-indigenous fish-farm bred individuals is the cast of the brown trout Salmo trutta.
Results were mixed. Studies in two countries, Norway
(Skaala et al. 1996) and Spain (Cagigas et al. 1999;
Almodovar et al. 2001), considered that such introductions had indeed resulted in a significant genetic

impact on wild populations. However other studies
from Norway (Heggenes et al. 2002) and Portugal
(Santos et al. 2006) conversely argued that wild
populations of the trout had not been genetically
influenced by the artificially introduced individuals.

Environ Biol Fish (2011) 90:211–222

In a Japanese example the main reason of drastic
diminution of the Japanese rosy bitterling Rhodeus
ocellatus kurumeus was elucidated to be due to their
suffering hybridization with non-indigenous rosy bitterlings Rhodeus ocellatus ocellatus which had been
introduced from the Eurasian continent (Kawamura et
al. 2001). Prevention of releases of domesticated or
exogenous individuals, whether intentional or accidental, is now universally recognized as an essential
element in nature conservation programs (Allendorf et
al. 2001).
The oriental weatherloach, Misgurnus anguillicaudatus (Cantor 1842), is a one of the most abundant
fish species found in traditional paddy fields in Japan
and other East Asian regions (Saitoh et al. 1988;
Tanaka 1999). The loach typically lives in ditches and
streams around the paddy fields, migrating into the
water-laden paddies to lay its eggs in the summer
(Saitoh et al. 1988; Tanaka 1999). Previous genetic
studies elucidated the phylogeny and phylogeography
of the loach in Japan by analysis of mitochondrial
DNA (mtDNA), revealing the existence of the two
major clades, clades A and B (Morishima et al. 2008;
Koizumi et al. 2009). Clade A is mostly distributed on
northern part of Japan and is closely-related to

Misgurnus fossilis and Misgurnus mizolepis that live
in Europe and China, respectively (Morishima et al.
2008; Koizumi et al. 2009). Clade B can be further
divided into two subclades: subclade B1 is widely
distributed all over Japan and seems to be indigenous
to Japan. Subclade B2 is distributed around Kanto
region (middle of Japan) and haplotypes of the same
lineage have been found in China (Morishima et al.
2008; Koizumi 2009). Clade A is genetically closer to
related species such as M. fossilis and M. mizolepis as
apposed to Clade B, and thus the two clades were
suggested as different species (Morishima et al. 2008;
Arias-Rodriguez et al. 2009). On the other hand
natural triploid and asexually reproducing clonal
diploid loaches have been reported at low rates in
Japan (0–16%; average: 1.4%) (Zhang and Arai
1999). These were indicated to be due to abnormal
meiosis originating from the intercross between
different lineages (Morishima et al. 2008).
With the exceptions of Japan’s four main islands,
Sado Island is the second largest island in Japan. On
Sado Island rice farming has a long history and paddy
fields are still spreading throughout the lowlands of
the island. The oriental weatherloach is thus one of


Environ Biol Fish (2011) 90:211–222

the main freshwater fishes on the island (Honma
1961). On the other hand, Sado Island has been

famous for being one of the last wild habitats for the
Japanese crested ibis (Nipponia nippon Temminck
1835). Hence Sado Island was the site chosen for ibis
reintroduction and the species was reintroduced to the
island in a state-level restoration program in autumn
2008 and 2009. To aid the bird’s survival in the wild,
many restoration initiatives have been implemented
on the island. In particular, artificial biotopes and
organic-farmed paddy fields have been provided in
various locations of the island to provide enhanced
feeding opportunities for the ibis. The preferred food
type for the ibis is loach (Chikatsuji 2002; Tei 2007),
and therefore cultured individuals were sometimes
stocked in the biotopes and paddy fields in Sado
Island (Kano et al. 2010). This had previously been
the case in the Chinese restoration where such
stocking was considered essential to aid the recovery
of the crested ibis in central China. Therefore, for the
20 years since restoration began in China, loach have
been purchased every spring and placed into the rice
fields to help breeding ibis raise their chicks. Even so,
in China, the expanding population still faced food
shortage problems (Xi et al. 1997; Wood et al. 2010).
Furthermore, there remains a concern for such
indiscreet stocking, which had a risk to cause
genetic/ecological disturbances in the loach (Koizumi
et al. 2009).
Previously, Koizumi (2009) noted that samples
from Sado Island belonged to the subclade B1.
However further detailed information, such as sampling places, frequencies and sequences, were not

detailed in this study. Thus it is necessary to further
clarify the detail information regarding the population
genetic aspects of the loach in Sado Island. As large
genetic divergences within freshwater fish species
were found in various other species (Avise 2000;
Watanabe et al. 2006) and such divergences are likely
to be accelerated in isolated habitats like islands
(Grant 1998), there is a possibility that the loach in
the island has some evolutionary/genetic uniqueness,
which should therefore be protected as an evolutionary significant unit (Waples 1991). On the other hand,
Honma (1961) indicated that most of the freshwater
fishes in the island, including the loach, had been
artificially introduced from the mainland by humans.
However Honma’s study failed to provide significant
evidence or strong supporting details for this scenario.

213

In this paper we determined 838 bp of control
region sequences on the mitochondrial DNA
(mtDNA) of 295 individuals, which were sampled
from throughout Sado Island and from adjacent areas
of the mainland. These sites included 8 areas where
non-native individuals had been known to have been
intentionally stocked. Based on the results obtained
from phylogeographic/population genetic analyses,
we discuss the genetic identity of the loach in the
island, as well as the effects of the loach stocking
associated with ibis reintroduction.

Materials and methods
Sampling and DNA analysis
Sado Island (area: 855 km2; coastline: 277 km) is
located in the Sea of Japan, 40 km off the coast of the
mainland of Niigata Prefecture in Japan (Fig. 1). The
Island is roughly divided into three regions, namely,
Osado, Kuninaka and Kosado (Fig. 1). The Osado
region is characterized by high mountains and small
rural communities interspersed with paddy fields
along the coastline. The Kuninaka region, by contrast,
is flatland with paddy fields extending over almost the
entire region. In the Kosado region low mountains
and shallow sloping valleys are found throughout,
with rural communities and local towns scattered in
the mountains and along the coastline.
The specimens were collected at 62 paddy field
sites on the island and 14 similar sites in the Niigata
region of the mainland (Fig. 1), in 2–15 July 2008 and
1–4 June 2009. In each site, 2 to 6 individuals were
net captured within a 50 m2, and preserved in absolute
ethanol for mtDNA analysis. Simultaneously, information of any loach stocking at the sampling sites
was collected through local records, interviews with
people and government contacts.
In total, 295 specimens were used for the DNA
analysis. Using DNA extraction kits (AquaPure
Genomic DNA Isolation Kit; BioRad Laboratory,
Hercules, CA, USA), total genomic DNA was isolated
from a piece of the pectoral fin of the loach.
Polymerase chain reaction (PCR) amplification was
carried out using the primer pair of L15923 (5′TTAAAGCATCGGTCTTGTAA-3′) (Iguchi et al.

1997) and 12SAR-H (5′-ATARTRGGGTATC
TAATCCYAGTT-3′) (Martin et al. 1992). The PCR


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Environ Biol Fish (2011) 90:211–222

Fig. 1 Map of Sado Island and adjacent Niigata region in the
mainland of Japan, and haplotype compositions of the loach
Misgurnus anguillicaudatus local populations. A sixty degree
sector of a pie chart indicates one individual (full circle:
6 individuals) with its black, white and grey patterns
corresponding to the clades/subclades A, B1 and B2, respec-

tively (see also Fig. 2). The loach stocking was conducted at the
points flagged with the star sign (S1–S8); black, white and grey
stars indicate that the stocked individuals were introduced from
Sado Island, mainland and unknown places, respectively (see
also Fig. 3). The grey pattern on the map shows the land use of
paddy fields as potential loach habitat

condition consisted of 30 cycles of denaturation
(72°C, 15 s), annealing (47°C, 15 s), and extension
(72°C, 30 s) on a PC808 thermal cycler (ASTEC,
Fukuoka, Japan). After purifying the PCR products
by treatment with ExoSAP-It (USB Corp., Cleveland,
OH, USA), they were sequenced on an automated
DNA sequencer (Applied Biosystems 3130xl Genetic
Analyzer; Applied Biosystem, Foster City, CA, USA)

using amplification primers and the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems).

were edited with MEGA4 (Tamura et al. 2007) and
aligned manually (838 bp of the 3′half of the mtDNA
control region). The nucleotide sequences were deposited in DDBJ/EMBL/GenBank (Accession numbers:
AB543940–AB543971). The information of the local
populations was also deposited in GEDIMAP (http://
gedimap.zool.kyoto-u.ac.jp; Watanabe et al. 2009)
(Population ID: P1087–P1162).
Phylogenetic reconstructions were carried out
using the following three methods: maximum likelihood (ML) in PhyML ver. 3.0 (Guindon and Gascuel
2003), neighbor-joining (NJ) (Saitoh and Nei 1987) in
MEGA ver. 4.0.2 and the Bayesian method with
MrBayes ver. 3.1.2 (Ronquist and Huelsenbeck 2003).
To identify the best model of evolutionary changes for
the ML and Bayesian analyses, the data set was

Phylogenetic analysis
The phylogenetic analysis was conducted using the
overlapped part of our sequences above and those
published in Morishima et al. (2008). The sequences


Environ Biol Fish (2011) 90:211–222

subjected to the Akaike information criterion (AIC;
Akaike 1974) and was calculated using MrModeltest
ver. 2.3 (Nylander 2004); which is a modified version
of Modeltest version 3 (Posada and Crandall 1998).
The GTR (Yang 1994) + I + Γ was selected. The

Tamura-Nei (Tamura and Nei 1993) model was applied
for NJ method, because the GTR model is not mounted
in the MEGA program. ML heuristic searches were
performed using an SPR (subtree pruning and regrafting) search from 10 random starting trees with four rate
categories. Bootstrap analyses were performed with
10,000 replicates for NJ and 100 replicates for ML
trees. MrBayes uses a MCMCMC algorithm that runs
four Markov chains simultaneously. The Markov
chains began from a random tree and ran for
13,000,000 generations, with sampling every 100
generations, to yield a posterior probability distribution. The first 10,001 samples were discarded as burnin. The appropriateness of burn-in values and the
convergence of chains were evaluated using TRACER
1.5.3 ( The
remaining trees after burn-in were used to generate a
50% majority rule consensus tree.
The net average distances between clades A and B,
clade A and Misgurnus spp. (M. mizolepis and M.
fossilis), clade B and M. spp., and subclades B1 and
B2 are also computed by MEGA 4.
Hierarchical genetic structure
To know the genetic structure of the loach in Sado Island
and Niigata, hierarchical analysis of molecular variance
(AMOVA: Excoffier et al. 1992) was performed using
Arlequin ver. 3.1 (Excoffier et al. 2005). The variations
were partitioned into three levels: among geographical
groups (Sado Island and Niigata), among local
populations within groups, and within local populations. In this analysis, clade A and B were treated
separately as different biological units (Morishima et
al. 2008). The Tamura-Nei model was used as the
genetic distance. Statistical tests of Φ-statistics were

performed at 10 000 permutations. Because individuals
of clade A were not obtained from Niigata, this analysis
was done for the clade B data.
Genetic isolation by distance
A mantel test was conducted for clade B in both Sado
Island and Niigata to test the genetic isolation by

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distance. The geographical distance (direct distance)
and genetic distance (DA with Tamura-Nei distance)
were calculated by ArcGIS 9.3 (ESRI Japan, Tokyo)
and MEGA4, respectively, for all possible pairs of the
local populations within the groups. The Mantel test
was implemented using the Arlequin ver. 3.1 with
10,000 permutations. For Sado Island, the test was
further implemented by eliminating the local populations that suffered any known stocking event (S1–S8)
in order to isolate such an effect. The significance
level of P values was compensated by the false
discovery rate (FDR) (Benjamini and Hochberg
1995). The test was not conducted for clade A
because of the deficiency of data.
Genetic identity of stocked populations
We tried to clarify the genetic identity of the eight
local populations in stocked sites by comparing other
genetic groups (Sado Island and Niigata). In each
clade (A and B), the individuals were pooled together
into the Osado, Kuninaka, Kosado or Niigata groups,
with the exception of individuals from the stocked
sites which were grouped independently. Pairwise DA

values were calculated to estimate the genetic distance
between the groups by MEGA4. According to this
genetic distance data, the neighbor-joining trees
among the regional groups were constructed using
Phylip ver. 3.68 (Felsenstein 2010) for respective
clades.
The net average distances between meta-groups
Sado (Osado, Kuninaka, Kosado and S7) and Mainland (Niigata, S2, S3, S4 and S8), and between Osado
and Kuninaka, Osado and Kosado, and Kuninaka and
Kosado were calculated by MEGA 4 in respective
clades.
Nucleotide diversity and stocking effect
We examined a hypothesis that the nucleotide
diversity (π) would be higher in the places where
the stocking had been unambiguously implemented,
as in such cases the original and introduced haplotypes would be likely to be mixed together. The
values of nucleotide diversity of clade B within the
local populations were obtained by the MEGA4 with
the Tamura-Nei distance method. The diversity values
were compared between stocked (S1–S8) and other
local populations using the Mann–Whitney U-test.


216
Table 1 Stocking information of the oriental weatherloach (Misgurnus
anguillicaudatus) in Sado
Island

Environ Biol Fish (2011) 90:211–222
Site

Abundance

Period

Source

Note

S1

>2,000

2007–2008

Unknown

Purchased from a local supermarket

S2

>10,000

1970s

Mainland

Purchased from a fish farm in the mainland

S3

2,000–3,000

2006–2007

Mainland

Purchased from a fish farm in the mainland

S4

ca. 5,000

2003–2006

Mainland

Purchased from a fish farm in the mainland

S5

>5,000

2006–2009

Unknown

S6

300–400

2007

Unknown

S7

30–50

2007

Sado Island

Captured in Kuninaka Region

S8

ca. 4,000

2004–2007

Mainland

Purchased from a fish farm in the mainland

The test was not conducted for clade A because of the
deficiency of data.

Results
Loach stocking

The loach stocking was confirmed to have been
conducted on at least 8 sites in Sado Island (Fig. 1;
S1–S8). Details are shown in Table 1. There were no
formal records regarding the stocking and we had no
other option other than to depend on local people’s
memory. The exact abundance, period and source of
the stocking therefore, remained rather ambiguous.
Phylogenetic analysis
Analyses based on ML, NJ and Bayesian method
yielded very similar topologies (Fig. 2), which were
approximately consistent to the result of Morishima et
al. (2008). Four haplotypes of clade A were revealed,
all found exclusively on Sado Island. None of these
haplotypes were identical to any from the Niigata
region or to those reported by Morishima et al.
(2008). However, the four haplotypes were not
monophyletic but were scattered in a major subgroup
of clade A. As for clade B, 25 haplotypes were found
in Sado Island, of which 19 haplotypes were identical
to those obtained from Niigata region or those in the
study of Morishima et al. (2008). On the other hand,
15 haplotypes were found from the Niigata region, of
which 12 and 10 haplotypes were identical to those
obtained from Sado Island and Morishima et al.
(2008), respectively. As in the case of clade A, the
haplotypes of clade B on the island were also widely

scattered throughout the tree without any apparent
cohesiveness (Fig. 2).
The net average distances between clades A and B,

clade A and Misgurnus spp., clade B and M. spp., and
subclades B1 and B2 are 0.083, 0.051, 0.087, and
0.025, respectively. The distance between clades A
and B was much higher than that between clade A and
M. spp.
Population genetic analyses
The AMOVA analyses (Table 2) indicated that the
largest genetic variance occurred within local
populations (47.9%) and that the lowest value was
among the local populations within groups (24.4%).
Significant differentiation was found in all levels
(P<0.001).
Table 3 showed the results of the Mantel test for
genetic isolation by distance. There was no significant
relationship between geographical and genetic distances in clade B of Sado Island (R2 =0.012; P>0.05),
while there was a significant relationship in the case
where the eight stocked local populations were
excluded from the analysis (R2 =0.048; P<0.033).
The clade B in Niigata also demonstrated a significant
Fig. 2 The phylogenetic tree of Misgurnus anguillicaudatus„
constructed by ML method. The numbers along each branches
show bootstrap percentages for ML/NJ, and Bayesian posteriorprobability. Bootstrap values of over 80% and posteriorprobability of over 0.95 are shown. The haplotype names in
parentheses correspond to those in Morishima et al. (2008) (The
duplications were compiled by the underbar). Solid, white, grey
circles show haplotypes found exclusively in Sado Island,
outside Sado Island, and both in and outside Sado Island,
respectively. The clades A, B1 and B2 correspond to Morishima
et al. (2008). The DDBJ accession numbers are shown in
brackets

Environ Biol Fish (2011) 90:211–222

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218

Environ Biol Fish (2011) 90:211–222

Table 2 A hierarchical AMOVA of the mtDNA control region in clade B of Misgurnus anguillicaudatus
Variance components

Percentage of variation

8 -statistics

Between groups

1.63

27.7%

0.28*

Among population within groups

1.44

24.4%

0.34*

Within populations

2.83

47.9%

0.52*

Subdivision

Source

Sado Island population
versus Niigata population

*P<0.001; Population subdivision was set by two groups: Sado Island and Niigata in Japan. Tamura-Nei was used as distance method.
Significance tests of 8 -statistics were performed 10,000 permuations

trend (R2 =0.190; P<0.017) with the lowest P and
highest R2 values.
Figure 3 demonstrates the NJ trees showing the
genetic relationship of the stocked local populations
with other local population groups in respective
clades. In clade A, the three groups of Sado Island
(Osado, Kuninaka and Kosado) made a meta-group
from which the externally stocked local population
(S8) was separated. In clade B, origin-unknown (S1,

S5 and S6) and intra-island (S7) stocked local
populations were included into the meta-group of
Sado Island. However, the externally stocked local
populations (S2, S3, S4 and S8) were situated
peripherally around the Niigata group or at the root
of the meta-group of Sado Island. The net average
distances between Sado and Mainland, Osado and
Kuninaka, Osado and Kosado, and Kuninaka and
Kosado were 0.00709, 0.00052, 0.00007 and
0.00137, respectively in clade A, and 0.00840,
0.00027, 0.00086 and 0.00060, respectively in clade
B. In both clades, the distance between Sado and
Mainland was higher than those between Osado and
Kuninaka, Osado and Kosado, and Kuninaka and
Kosado.
The nucleotide diversity of stocked local populations
(N=8; median: 0.0067; min: 0.0042; max: 0.0260) was
significantly higher than that of other local populations
(N=42; median: 0.00071; min: 0.0000; max: 0.0225)
(Mann–Whitney U=53, P<0.01).

Discussion
The details of loach’s population genetic characteristics
in Sado Island were elucidated by our study. A total of
29 haplotypes, including two highly distinctive lineages (clades A and B), were found in Sado Island
(Figs. 1 and 2), of which 16 haplotypes were identical
to those found in other places of Japan. Furthermore,
we found no distinct group that consisted only of the
Sado Island individuals as all the haplotypes belonged
to previously known lineage groups (as detailed in

Morishima et al. 2008). Therefore it would be natural
to suggest that these genes had been introduced from
various places around mainland Japan rather than
conclude that these lineages are historically native to
the island. Further, the result of AMOVA showed the
highest variance at the within-population level, suggesting haplotypes of different origins were included in
single local populations. We discuss the possible
biological entities of clade A and B later.
Minato (1967) reconstructed a paleogeographical
map showing the Japanese Archipelago during the
Last Glacial Maximum (0.020–0.018 mya) on the
premise of –140 m sea level, in which Sado Island
and the mainland had been connected. On the other
hand, Ohshima (1990) illustrated that the sea level in
the Last Glacial Maximum had been −80 m sea level,
in which case Sado Island and the mainland would
have remained isolated by the sea channel. Ohshima

Table 3 Results of Mantel test for clade B of Misgurnus anguillicaudatus
Test group

No. of populations

Correlation coeffecient

R2 value

Mantel P value

vs.

FDR P value

Sado Island

50

0.111

0.012

0.083

>

0.050

Sado Island without S1–S8

42

0.219

0.048

0.019

<

0.033

Niigata

14

0.435

0.190

0.004

<<

0.017

FDR P values of significance level were calculated according to Benjamini and Hochberg (1995)


Environ Biol Fish (2011) 90:211–222

Fig. 3 Neighbor-joining tree based on the pairwise distances
(DA) among groups in each clades (A and B). The trees were
rooted at the middle points. The individuals of Osado,
Kuninaka and Kosado in Sado Island (solid circle) and Niigata
(white circle) were each pooled together except the individuals
of the local population that suffered recent stocking events (star
sign; S1–S8). Color of the star signs indicate the origin of the
stocked individuals; black: Sado Island; white: mainland; grey:
unknown

(1990) also suggested that Sado Island and the
mainland had been separated from the time of the
middle Pleistocene (0.8–0.2 mya), though further
supporting details were not provided. However,
genetic studies on the shrew mouse Sorex caecutiens
(Ohdachi et al. 2001), the wild boar Sus scrofa
(Watanobe et al. 2004) and the wood mouse Apodemus specious (Tomozawa et al. 2010) are congruent
with Ohshima’s description showing their colonizations were likely prior to the middle/late Pleistocene
as suggested from the genetic data. The ground beetle
Damaster blaptoides capito (Lews 1881), the land
snail Euhadra sadoensis (Pilsbry and Hirase 1903),
the wrinkled frog Rana sp. (or Rana rogosa ssp.)
(Miura et al. 2004) and the longhorn beetle Mesechthistatus binodosus insularis (Nakamine and Takeda
2008) were also known as endemic species/subspecies
of the island, whereas the endemism of the “Sado
salamander” Hynobius sadoensis (Sato 1940) was
rejected by Matsui et al. (1992), who suggested a
human introduction for this species.
From the results of this study we have no explicit
evidence whether or not genetically endemic loach
populations have previously existed in Sado Island.
Humans have lived in Sado Island at the very least
since the early Jomon Era (approx. 5,000 years ago)
(Nakagawa et al. 1989). Such people have been
known to be actively commuting between the island
and mainland of Japan since ancient times (Kitami

219

and Komura 1987; Tanaka and Nishigaki 1987).

Thus, through such historical human activities, there
is a high possibility over the period that various
stocks of loach on occasion may have been introduced from multiple locations within mainland Japan.
Whether or not native loach populations had existed
in the island before such introductions remains
unclear. As the loach can survive for periods in moist
soil (Kubota 1961), it could have been possible, even
in ancient times, to bring the loach as a food across
the sea. The famous Japanese writer Kōyō Ozaki
described an old story of the Sado loach in his travel
report (Ozaki 1904), in which the Emperor Juntoku-tei
(1197–1242) ate loach soup in Sado Island. If this is
true, it follows that the loach had inhabited the island
for at least 800 years. Detection of isolation by distance
in Sado Island, despite being at a weaker level than that
of Niigata, may be an indication confirming just such a
historical presence of loach on the island (Table 3). As
for other primary freshwater fishes, crucian carp
Carassius auratus, common carp Cyprinus carpio,
catfish Silurus asotus, ricefish Oryzias latipes and
sculpin Cottus pollux were also recorded to be
historically distributed on the island (Honma 1961),
though the exact origins remained unknown due to
lack of genetic studies. Future studies may elucidate
the identities of these primary freshwater fishes,
including the loach, on the island.
We did find some evidence of genetic disturbance
by the stocking. There was no significant relationship
between geographical and genetic distances in the
clade B local populations in Sado Island. However, in

case where the obviously stocked local populations
(S1–S8) were excluded from the data, a positive
significant correlation was then revealed (Table 3).
This would indicate that the geographical distribution
of clade B genes in the island was disturbed by the
stocking. In fact, the inter-island stocked local
populations (S2, S3, S4 and S8) were genetically
outliers when compared with the whole Sado gene
pool (Fig. 3), with only the intra-island stocked local
population (S7) categorized within the meta-group of
Sado Island. We suspect that the three originunknown stocked local populations (S1, S5 and S6)
were also brought from somewhere else within the
island because of their similarity to the Sado metagroup. Additionally, the nucleotide diversities of
stocked local populations were significantly higher
than those of other local populations. In invasion


220

biology, there has been a genetic paradox regarding
introduced populations. How do introduced populations, whose genetic variation has likely been decreased by population bottleneck/founder effect,
persist and adapt to a new habitat? In Kolbe et al.
(2004)’s report regarding introduced lizards, as an
explanation of this paradox, the nucleotide diversity
of the lizards was high in the newly introduced
populations because of cases of multiple introductions. In our case, the situation would be a little
different because original individuals and stocked
individuals may have been mixed. It will be much
more obvious that the nucleotide diversity is high in
such an apparently mixed population. While we have

clarified the stocking effects to a certain level, more
sensitive molecular markers, e.g., microsatellite DNA,
should be used in future studies, especially to further
examine population genetic information such as allele
richness and gene flow.
In this study, we also met some difficulties and
unsolved problems. First, natural triploid and asexually reproducing clonal diploid loaches have been
reported at low rates (0–16%; average: 1.4%) in Japan
(Zhang and Arai 1999). In this study we did not
discriminate between these and normal diploid loaches. However, the present results should not be
significantly affected by this in that the triploid
individuals, if included, would be likely to occur at
the low frequency rate as has been reported from
other regions. Second, how the clades A and B
(Fig. 2) should be treated was a key question. No
definite study has yet concluded whether these two
clades are from a same species or not. However, at
least at this stage, Arias-Rodriguez et al. (2009)
showed that there existed significant difference
between the two in microsatellite-centromere map
distances on chromosomes. In this study we presumed
the two clades as separated biological units according
to Morishima et al. (2008) and Arias-Rodriguez et al.
(2009). Future studies may formally divide the loach
into two species. Third, there was difficulty in
obtaining sufficient information regarding stocking.
The memory of the local people seemed, at times, a
little unclear and some seemed unwilling to talk about
stocking for fear of blame for genetic disturbance. We
suspect there may well be more stocked populations

than identified here, and that the effects of the
stocking were much more considerable than those
detected in our analysis.

Environ Biol Fish (2011) 90:211–222

In conclusion, our study provides details of the
genetic properties of the loach in Sado Island and
suggests the possibility that the loach has been
historically introduced from other places beyond the
island while recent stocking for the ibis has caused
further genetic disturbance to their local populations.
Although the majority of the loach populations in the
island may not be “genuinely native”, nevertheless
our results do not actively justify loach stocking
hereafter. Fish stocking in the wild should always be
conducted as a cautious biological procedure (IUCN/
SSC 2010; The Ichthyological Society of Japan 2010)
and the action carries with it key questions of
philosophical biodiversity values such as “intrinsic
value”, “existence value”, “food value” and so on
(Koricheva and Siipi 2004; Oksanen 2004). It also
brings to question how we go about defining the words
such as “native” and “endemic”. How do we treat the
populations that were introduced by humans, for
example, 800 years ago? Can we equate them with
those recently introduced? The decisions for these
issues are beyond the reach of the current study. Either
way the most essential action for the restoration of the
ibis would be to create an environment itself where

native species can naturally propagate rather than to
increase the biomass temporarily by an artificial means
(Ministry of Environment, Government of Japan
2010). If a critical situation for the ibis demands a
stop-gap measure of provisioning, then highly localized provisioning should be conducted together with
measures in place to attempt to prevent or minimize the
stocked loach migrating to other areas.
Acknowledgements We are grateful for assistance provided by
S. Ikematsu, J. Nakajima, M. Sato, W. Tanaka and T. Yamashita.
This work was supported by the Global Environment Research
Fund of the Ministry of the Environment, Japan (Subject No.
F-072, representative: Y. Shimatani), Grant-in-Aid for Young
Scientists B of the Ministry of Education, Culture, Sports, Science
and Technology (Subject No. 19710027, representative: Y.
Kawaguchi), Global COE Programs (Center of excellence for
Asian conservation ecology as a basis of human-nature mutualism,
representative: T. Yahara; and Formation of a Strategic Base for
Biodiversity and Evolutionary Research: from Genome to
Ecosystem, representative: K. Agata, Kyoto University), MEXT,
Japan.

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Environ Biol Fish (2011) 90:223–233
DOI 10.1007/s10641-010-9734-6

Diet of age-0 tarpon (Megalops atlanticus)

in anthropogenically-modified and natural nursery
habitats along the Indian River Lagoon, Florida
Zachary R. Jud & Craig A. Layman &
Jonathan M. Shenker

Received: 5 January 2010 / Accepted: 12 October 2010 / Published online: 3 November 2010
# Springer Science+Business Media B.V. 2010

Abstract As human development in coastal areas
increases, the role of anthropogenically-created habitats in the life history of marine organisms is
becoming increasingly important. We examined the
diet of age-0 tarpon, Megalops atlanticus, in and
around man-made mosquito control impoundments
along the Indian River Lagoon in east-central Florida,
with a particular focus on identifying dietary patterns
associated with tarpon size and nursery habitat type
(i.e., between perimeter pool habitats in established
impoundments and newly-created restoration marsh
habitats). Age-0 tarpon were found to consume a wide
variety of prey organisms, and exhibited considerable
dietary variation among study sites. Smaller juvenile
tarpon consumed a limited number of small prey taxa,
while larger individuals fed on a greater range of prey
taxa and sizes. Overall, copepods and fishes were the
dominant prey items; however, the consumption of
these organisms varied considerably among size
classes and sites. There was no clear difference in
Z. R. Jud (*) : C. A. Layman
Department of Biological Sciences, Marine Sciences
Program, Florida International University,

3000 NE 151st Street,
North Miami, FL 33181, USA
e-mail:
J. M. Shenker
Department of Biological Science,
Florida Institute of Technology,
150 W University Blvd,
Melbourne, FL 32901, USA

tarpon diet between the two types of habitat we
examined. The ability of juvenile tarpon to utilize
such a diverse range of prey organisms may allow
populations to inhabit a variety of habitats, including
man-made marshes. When natural systems have been
degraded or destroyed, human-altered habitats can
assume a nursery role for the species.
Keywords Dietary plasticity . Habitat fragmentation .
Mosquito control impoundments . Optimal foraging .
Predator-prey interactions . Mangrove marsh
restoration

Introduction
Florida’s coastal wetlands play an important role in
the recruitment of tarpon, Megalops atlanticus (Wade
1962; Crabtree et al. 1995; Shenker et al. 2002). The
most important nursery habitats for tarpon in the
continental United States are located in Florida, as it
is the only state where winter water temperatures
consistently remain within the species’ tolerance
range (Howells 1985). Because adult tarpon are

highly valued by the sport fishing industry, most of
the research conducted on the species has been
targeted at mature individuals. Much less is known
about the early life history stages, when tarpon rely on
threatened coastal wetlands. In particular, little is
known about the diet of juvenile tarpon following
settlement. A better understanding of how certain


224

early life history traits develop across a range of
nursery habitats is critical for the conservation of the
species, as natural nursery habitats are being modified
by humans at a rapid rate.
Along the east coast of Florida, man-made mosquito control impoundments have become increasingly important to tarpon recruitment because a large
percentage of the region’s natural marshes were
impounded for mosquito control purposes from the
1950s to 1970s (Brockmeyer et al. 1997; Poulakis et
al. 2002; Stevens et al. 2007). Historically, mosquito
control impoundments were highly fragmented ecosystems that lacked connections with the surrounding
marine environment. In recent decades, hydrologic
connectivity has been restored to most of Florida’s
impounded marshes (Brockmeyer et al. 1997). Transient fish species begin to utilize impoundments
immediately following reconnection (Vose and Bell
1994; Llanso et al. 1998; Poulakis et al. 2002), and in
many ways, reconnected impoundments appear to
function much like natural marshes (Stevens et al.
2006; Lewis and Gilmore 2007). In addition to
reconnecting existing mosquito control impoundments, state and regional agencies have started

creating man-made restoration marshes (Lewis and
Gilmore 2007). Juvenile tarpon have been observed in
these newly-created marsh habitats less than 1 year
after construction (Z. R. Jud, unpubl. data).
Our objective was to characterize the diet of age-0
tarpon in established mosquito control impoundment
habitats and newly-constructed restoration marshes.
Although the limited body of work on the trophic
ecology of age-0 tarpon identified considerable
variability in diet (Harrington and Harrington 1960,
1961; Catano and Garzon-Ferreira 1994; ChaconChaverri 1994), it is not clear how these dietary
patterns are affected by anthropogenic habitat modification. Prey species composition, size, and diversity
were compared among different size classes of age-0
tarpon. We then made dietary comparisons among
several study sites, and specifically examined diet
differences between mosquito control impoundment
and restoration marsh habitats.

Methods
Between September and November of 2007, 15 eastcentral Florida mosquito control impoundments and

Environ Biol Fish (2011) 90:223–233

restoration marshes were examined daily for the
presence of age-0 tarpon. These wetlands, located in
Indian River and St. Lucie counties, are hydrologically connected to the Indian River Lagoon, a barbuilt estuary that parallels Florida’s Atlantic coast.
Age-0 tarpon were captured at a total of seven study
sites (Table 1). Tarpon Hole (TH) and Site Six (#6)
were characterized as perimeter pool habitats within
established mosquito control impoundments (Fig. 1a,

b). Perimeter pools are mangrove-lined depressions
scoured into the marsh surface by the pumps that
were historically used to fill impoundments. These
habitats are decades old, have well-established plant
and animal communities, and are surrounded by a
complex network of mature red mangrove (Rhizophora mangle) prop roots. Perimeter pool habitats
remain inundated during all tidal stages, and serve as
an important refuge for fish during periods of low
water.
Three additional collection sites, Restored Creek
(RC), Restored Creek North (RCN), and Beach Pool
(BP) were located in newly-constructed restoration
marsh habitats (Fig. 1c, d). These man-made
marshes had been constructed during the previous
year by the St. Lucie County Mosquito Control
District in an effort to provide increased marsh
habitat for fishes and wildlife. The restoration
marshes were dug using heavy equipment, and were
designed to mimic the structure and hydrology of
natural mangrove creek systems. The absence of
mature red mangrove trees and prop roots, coupled
with smooth, muddy shorelines, resulted in low
structural complexity compared to perimeter pool
habitats. All of these man-made marshes were
hydrologically connected to the Indian River Lagoon
or existing mosquito control impoundments.
Age-0 tarpon were also opportunistically collected
at two natural (i.e., not anthropogenically-created)
sites in the Indian River Lagoon. These sites (Tarpon
Hole East—THE, and Blind Creek—BC) were

located in close proximity to perimeter pool and
restoration marsh habitats, and were included to
provide some comparison between man-made and
natural habitats. Although tarpon from these two sites
were not used in our primary analysis (i.e., comparing
diet between perimeter pool and restoration marsh
habitats), they were included in the general description of tarpon diet and the analysis of diet vs. tarpon
length.


Environ Biol Fish (2011) 90:223–233
Table 1 Description of age0 tarpon collection sites,
where n=the number of
tarpon retained for dietary
analysis

225

Site Name

Abbreviation

Habitat Type

Location

n

Site Six

#6

Perimeter Pool

27º21′49″ N
80º14′59″ W

8

Tarpon Hole

TH

Perimeter Pool

27º48′42″ N
80º25′36″ W

30

Restored Creek

RC

Restoration Marsh

27º24′48″ N
80º16′24″ W

20

Restored Creek North

RCN

Restoration Marsh

27º24′49″ N
80º16′24″ W

6

Beach Pool

BP

Restoration Marsh

27º22′41″ N
80º15′15″ W

21

Tarpon Hole East

THE

Natural Indian River Lagoon

27º48′42″ N

80º25′35″ W

5

Blind Creek

BC

Natural Indian River Lagoon

27º22′42″ N
80º15′16″ W

7

In order to reduce the potential influence of
seasonal variation, sampling was restricted to a
2 month period in the fall of 2007. This time frame
was selected to coincide with the end of the peak
recruiting period for tarpon (Harrington 1958;
Shenker et al. 2002), when maximum numbers of
age-0 fish would be expected to be present in the
mosquito control impoundments. Tarpon were captured using cast nets (4.3 m diameter, 13 mm mesh)
and seines (15.2×1.8 m, 3 mm mesh). Standard
Fig. 1 Photographs of
study sites, illustrating perimeter pool and restoration
marsh habitats. a Site Six
(perimeter pool). b Tarpon
Hole (perimeter pool). c
Beach Pool (restoration

marsh). d Restored Creek
(restoration marsh)

lengths (SL) were measured to the nearest 1.0 mm,
and the fish were weighed to the nearest 0.01 g.
Tarpon were then euthanized and the entire digestive
tract was immediately removed and placed into 10%
formalin. All tarpon greater than 215 mm SL were
released alive, as they could have been either age-0 or
age-1 individuals (Harrington 1958; Chacon-Chaverri
1994; Crabtree et al. 1995; Zerbi et al. 2001).
In the laboratory, tarpon digestive tracts were
removed from 10% formalin after one week and


226

placed into 96% ethanol. Stomachs were processed
individually under a stereomicroscope. Before each
stomach was dissected, visceral fat deposits (an
indicator of nutrition) were noted on a presence/
absence basis. Prey organisms contained in the
stomachs were identified to the lowest possible
taxonomic level. Prey items were counted and
weighed to the nearest 0.001 g on a digital balance
(wet weight). Some microscopic prey taxa were too
abundant to count individually; in these cases, prey
abundance was estimated by counting individuals in a
diluted sub-sample on a 1.0 ml Sedgwick-Rafter
counting cell at 40 X magnification. For organisms

that were too small to be weighed individually, entire
samples were filtered out of solution and weighed as a
group.
To obtain an overall description of juvenile tarpon
diet, the following values were calculated for each
prey taxon: the percent frequency of occurrence of
prey taxon i among all stomachs in the sample (%Oi =
percent frequency of occurrence), the proportion of
the number of prey items present in a prey taxon i to
the total number of prey items present in all stomachs
(%Ni = percent of total diet by number), and the
proportion of the aggregate wet weight of a prey
taxon i to the total wet weight of all prey items
(%Wi = percent of total diet by weight). Based on
these values, an Index of Relative Importance (IRI)
was calculated for each prey taxon i, where
IRIi ¼ %Oi ð%Ni þ %Wi Þ. The IRI is a compound
index that incorporates quantity, weight, and frequency of occurrence into a single numerical measure,
facilitating dietary comparisons and providing a more
accurate estimate of “dietary importance” of prey
items (Hynes 1950; Hyslop 1980; Cortes 1997;
Treloar et al. 2007; Adams et al. 2009). To standardize comparisons among prey taxa, we converted IRIi
values into %IRIi by dividing each taxon’s IRIi value
by the sum of IRIi values for all prey taxa combined
(Cortes 1997).
Juvenile tarpon were pooled across all sites to
determine how dietary composition may have
changed through ontogeny. All tarpon that had
identifiable food items in their guts were assigned to
20 mm size classes. For all size classes, %Oi, %Ni, %

Wi, and %IRI were calculated for each prey taxon. We
used a 1-way analysis of similarities (ANOSIM) to
test for significant differences in dietary composition
among size classes (PRIMER v6.1.9 software). Prey

Environ Biol Fish (2011) 90:223–233

weight values used for ANOSIM were fourth root
transformed to down-weight abundant prey taxa. To
determine whether different size classes of age-0
tarpon consumed different-sized prey items, the
median (± median absolute deviation) of mean
weights of individual prey items consumed by each
tarpon were compared among size classes using a
Kruskal-Wallis test. A Kruskal-Wallis test was also
used to determine whether different size classes of
age-0 tarpon exhibited variation in the number of
individual prey taxa consumed (median ± median
absolute deviation).
Stomach contents were analyzed to determine
whether dietary composition of age-0 tarpon varied
among study sites, as well as between established
perimeter pool habitats and newly-constructed restoration marsh habitats. To avoid confounding siterelated dietary differences with size-related dietary
differences, we tested whether tarpon from each site
were significantly different in length. A KruskalWallis test was conducted to determine whether
median tarpon size varied among sites. For each site,
%Oi, %Ni, %Wi, and %IRI were calculated for all
prey taxa. A 1-way ANOSIM (Bray-Curtis similarity
matrix, fourth root transformed prey weight values)
was then used to test whether the diet of age-0 tarpon

varied among all sites. Finally, a 2-way nested
ANOSIM was used to test whether the dietary
composition of age-0 tarpon differed within and
between perimeter pool habitats (mosquito control
impoundments) and restoration marsh habitats, where
sites were nested within the two habitat types.

Results
A total of 366 tarpon were captured during the fall of
2007. Ninety-seven of these fish were ≤215 mm and
were retained for stomach content analysis. The
remaining fish were released unharmed. The harvested fish ranged in standard length from 64 to
215 mm, with a mean (± SD) of 143.8±35.5 mm.
Seventy-one of the 97 collected stomachs contained
identifiable prey items, representing 22 different taxa
(Fig. 2). Only five prey taxa were found in more than
10% of the tarpon. Fishes (Teleostei) occurred with
the greatest frequency, followed by copepods (Copepoda), water boatmen (Corixidae), shrimp (Caridea),
and backswimmers (Notonectidae). Eleven of the 22


Environ Biol Fish (2011) 90:223–233
Fig. 2 Frequency of occurrence of 22 prey taxa in 71
tarpon that contained
identifiable prey items

227
25

% of tarpon

20

15

10

5

Syrphidae larvae

Coleoptera larvae

Isopoda

crab megalopae

caprellid amphipods

Odonata

Orthoptera

crabs

coryphaeid amphipods

Ostracoda

gammarid amphipods #2

ants

crab zoea

mysids

Coleoptera adults

Chironomidae larvae

gammarid amphipods #1

shrimp

Notonectidae

Corixidae

copepods

fishes

0

Prey category

prey categories were identified in three or fewer
individual tarpon.
Copepods were the numerically dominant prey

taxon, accounting for 97% of the prey consumed by
number. More than 300,000 copepods were identified
in the 19 tarpon stomachs that contained this prey
item. The remaining 21 prey taxa combined contributed to less than 3% of tarpon diet by number. By
weight, fishes (51%), shrimp (20%) and copepods
(18%) were the three most dominate prey items.
These were the only prey taxa that exceeded 3% of
tarpon diet by weight. When taking into account
quantity, weight, and frequency of occurrence, copepods had the highest relative IRI value, followed by
fishes, shrimp, and corixids. The remaining prey taxa
had %IRI values lower than 0.5%.
Analysis of %IRI values for the gut contents of
age-0 tarpon revealed patterns of prey consumption
related to tarpon length (Fig. 3). Some prey taxa (e.g.,
copepods) dominated the diets of smaller tarpon,
while other prey taxa (e.g., shrimp) were only
consumed by larger tarpon. Although the smallest
size class of tarpon (i.e., 60–79 mm) only consumed
copepods, this prey item remained an important
dietary component in tarpon up to the 160–179 mm
size class, and was consumed to a lesser degree by
tarpon as large as 183 mm. Some individual fish
consumed copepods in huge numbers (i.e., >50,000/
tarpon). Fishes were consumed by all but the smallest

tarpon (i.e., 60–79 mm), but %IRI values for fishes
varied considerably among tarpon size classes.
The results of a 1-way ANOSIM identified
significant differences in prey composition among
size classes (R=0.07, p=0.02). There was no significant difference in the mean weight of individual prey

items consumed per tarpon among size classes
(Kruskal-Wallis; H=13.2, p=0.07) or the number of
prey taxa consumed per tarpon among size classes
(Kruskal-Wallis; H = 12.6, p = 0.08). However, as
tarpon length increased, there was an increase in the
variation (median absolute deviation) of prey weight
and the absolute number of prey taxa consumed per
fish (Fig. 4). Larger tarpon fed on a wide range of
prey sizes, whereas smaller tarpon typically consumed
only small prey items. Additionally, larger tarpon fed
on a more diverse range of prey organisms. The
stomach contents of all tarpon <100 mm contained no
more than one prey taxon, while individuals ≥100 mm
contained from one to five prey taxa, with a mean
(± SD) of 1.98 ± 1.3 prey taxa per tarpon.
Since the above analyses established that diet
varied with tarpon size, comparisons among sites
would be most relevant if all sites contained tarpon of
similar size. The median size of tarpon from Tarpon
Hole was significantly smaller than four of the other
sites (Kruskal-Wallis; H=46, p<0.001), but there was
no significant difference in tarpon size among the
remaining six sites. Dietary differences identified at


228

Environ Biol Fish (2011) 90:223–233

Fig. 3 Percent of diet by

IRI (%IRI) for all tarpon
that contained identifiable
prey items. Data are presented based on 20 mm size
classes. Only %IRI values
greater than 0.9% are shown

100%

n=4

n=5

n=6

n=15

n=17

n=10

n=10

n=4

80%

crab zoea
Corixidae
Syrphidae larvae

% of diet by IRI

60%

Coleoptera adults
coryphaeid amphipods
gammarid amphipods #2
gammarid amphipods #1
shrimp
fishes

40%

copepods

20%

20
0-2
19

18
0-1
99

16
0-1
79

14

0-1
59

12
0-1
39

10
0-1
19

80
-99

60
-79

0%

Tarpon size classes (mm)

Tarpon Hole must be viewed with caution since the
smaller size of tarpon at this site may have influenced
diet, independent of habitat type.
Dietary patterns varied significantly among the
seven study sites (1-way ANOSIM; R=0.24, p=
0.001), including pairs of sites separated by ≤25 m.
Even within a single site on a single sampling date,
the diets of individual tarpon were often dominated by
different prey taxa, suggesting high levels of withinsite variation in prey consumption. None of the 22

prey taxa we identified were present in tarpon diets at
all seven sites. Some prey taxa (e.g., crab zoea) were
only found in tarpon diets at one or two sites and may
have represented a locally available food source.

Other prey categories (e.g., fishes) were consumed
at nearly all of the study sites.
Tarpon diets at Site Six, a perimeter pool site, were
dominated by shrimp and amphipods (Fig. 5). Although 14 different prey taxa were consumed at the
Restored Creek restoration marsh site (more than any
other site), copepods were the dominant dietary item,
with the remaining 13 taxa being consumed in low
quantities. At Restored Creek North, a restoration
marsh habitat located just 25 m from Restored Creek,
crab zoea and amphipods were the dominant prey
items. Diets at the third restoration marsh site, Beach
Pool, were dominated by copepods, corixids, and
fishes. At Tarpon Hole, the perimeter pool habitat


Environ Biol Fish (2011) 90:223–233

a

0.04
0.035

Mean prey weight (g)

Fig. 4 Length-based dietary characteristics of age-0

tarpon. a Tarpon length vs.
the mean weight of each
prey item consumed (median ± median absolute deviation). b Tarpon length vs.
the number of different prey
taxa consumed per tarpon
(median ± median absolute
deviation). In both cases,
there was no significant
difference among size classes, but variation tended to
increase with tarpon length

229

0.03
0.025
0.02
0.015
0.01
0.005
0
60-79
n =4

80-99
n =5

100-119
n =6

120-139

n =15

140-159
n =17

160-179 180-199
n =10
n =10

200-219
n =4

Tarpon size classes (mm)

b

6

# of prey taxa consumed

5
4
3
2
1
0
60 -79
n =4

80 -99

n =5

100 -11 9
n =6

120 -13 9
n =15

140 -15 9
n =17

160 -17 9
n =10

180 -19 9
n =10

200 -21 9
n =4

Tarpon size classes (mm)

with smaller tarpon, copepods and fishes were the
dominant prey taxa.
Age-0 tarpon from the two natural IRL sites had
different dietary patterns than individuals from the
perimeter pool or restoration marsh sites. Diets of
tarpon at Blind Creek were almost entirely composed
of fishes, while at Tarpon Hole East, shrimp were the
most important prey taxon. These were the only two

sites where diets were dominated by large prey
organisms. At the time of dissection, visceral fat
deposits were noted in 18 of 21 tarpon (86%) from
Beach Pool and six of seven tarpon (86%) from Blind
Creek. Only two of the 69 tarpon (3%) captured at the
remaining five sites had visceral fat deposits.
A 2-way nested ANOSIM was used to determine
whether diets differed within and between the two
main habitat types: 1) perimeter pools in established
mosquito control impoundments, and 2) newlyconstructed restoration marshes. A significant differ-

ence in diets was identified among sites within the
two habitat types (R=0.26, p=0.001); however, there
was no significant difference between the two habitat
types (R =0.08, p=0.4). Variation in tarpon diet
among sites within the two habitats was greater than
variation between the two habitats.

Discussion
Age-0 tarpon exhibited considerable dietary variability among size classes and study sites. The size-based
dietary shifts we observed in age-0 tarpon were likely
caused by an increase in prey capture ability resulting
from changes in morphology or feeding kinematics
(Guigand and Turingan 2002). A reduction in gape
limitation and an increase in swimming speed and
efficiency may allow larger age-0 tarpon to consume
larger, more evasive (i.e., harder to capture) prey


230

organisms. Previous studies identified a shift from
copepod consumption to piscivory as juvenile tarpon
reached 75–100 mm (Harrington and Harrington
1960; Catano and Garzon-Ferreira 1994; ChaconChaverri 1994). We observed a similar dietary shift in
tarpon from mosquito control impoundments and
restoration marshes; however, the shift occurred at
larger body sizes, and a variety of other prey taxa (i.e.,
not just fishes) were consumed by these larger
individuals (Fig. 3). The consumption of copepods by
relatively large juvenile tarpon was unexpected, and
may be unique to the man-made habitats we studied.
The presence of huge quantities of copepods in the
diets of some age-0 tarpon suggests that the fish may
employ a combination of suction and ram feeding
(Guigand and Turingan 2002) while rapidly swimming
through swarms of copepods, essentially filtering prey
out of the water column. Filter feeding has not
previously been described in juvenile tarpon; however,
the presence of long, tightly-spaced gill rakers supports
dietary observations that suggest this feeding strategy
may be employed (Langeland and Nøst 1995).
The rapid growth experienced during the juvenile
phase in many organisms can lead to a continuouslyshifting niche (Werner and Gilliam 1984). In the case
of age-0 tarpon, changes in the composition and size
of prey items consumed as body size increases may
function to reduce intraspecific competition, particularly when prey abundance is limited. Since recruitment of juvenile tarpon occurs in pulses throughout
the summer (Shenker et al. 2002), it is not uncommon
to find several different size classes of tarpon
simultaneously occupying the same nursery habitat.

Size-based shifts in diet may allow multiple size
classes of age-0 tarpon to coexist in confined nursery
habitats.
A variety of factors may explain the dietary differences we observed among sites and between habitat
types. Within estuaries, prey communities can vary
considerably among different habitats (Layman and
Silliman 2002; Valentine-Rose et al. 2007). Although
we did not sample prey organisms at our study sites,
the high complexity of perimeter pool habitats may
have supported a different prey base than the
structurally-simple restoration marsh habitats. Additionally, our study sites possessed different levels of
connectivity to adjacent mangrove forests and surrounding bodies of water. Habitats with higher levels
of connectivity often support a greater variety of prey

Environ Biol Fish (2011) 90:223–233

species (Poulakis et al. 2002; Layman et al. 2007;
Lewis and Gilmore 2007). Further, physical parameters
(e.g., turbidity, salinity, temperature, dissolved oxygen
levels, etc.) can vary widely, even at small spatial
scales. In addition to affecting prey abundance and
diversity, these factors may also influence prey
selection or capture efficiency. In turbid waters, for
example, age-0 tarpon may be more successful at
capturing certain prey organisms (e.g., prey organisms
that flee at the sight of a predator), while in low
turbidity habitats, other prey organisms (e.g., cryptic
prey organisms) may be located and captured more
easily (Shoup and Wahl 2009). The interplay between
these factors is complex and difficult to quantify, but

their combined effects on prey base and prey capture
ability likely shape the diet of tarpon.
While we anticipated that the diet of tarpon within
mosquito control impoundment sites would differ
from restoration marsh sites, we found no clear
pattern between diet and habitat type. There appeared
to be obvious physical and biological differences
between the two main habitat types we examined, but
we did not quantitatively determine whether all sites
actually fit into this simple dichotomy. Our grouping
of sites together as perimeter pool habitats and
restoration marsh habitats may have underestimated
the actual biotic and abiotic variability among sites,
which, in turn, could have influenced diet.
The tarpon collected at Beach Pool, a restoration
marsh site, present one possible explanation of how
juvenile tarpon utilize newly-constructed marshes.
These tarpon had the greatest percentage of empty
stomachs out of all seven sites, yet they also possessed
visceral fat deposits. Stored fat is indicative of high
nutrition (Borcherding et al. 2007), but superficially,
this doesn’t correspond well to the empty stomachs
found at the site. The fish-dominated diets of tarpon
captured at Blind Creek, an adjacent natural Indian
River Lagoon site, may explain why the restoration
marsh fish showed signs of high nutrition, despite
having frequently empty stomachs. It appears that
tarpon periodically move through the short culvert
connecting Beach Pool to Blind Creek, leaving the
sanctuary of the nursery marsh to make brief feeding

forays into the Indian River Lagoon estuary. If manmade restoration marshes do function to provide
shelter for age-0 tarpon between feeding bouts, the
creation of these habitats may actually benefit juvenile
tarpon recruitment.


Environ Biol Fish (2011) 90:223–233
Fig. 5 Percent of diet by
IRI (%IRI) for all tarpon
that contained identifiable
prey items. Data are categorized by study site. Only
%IRI values greater than
0.9% are shown

231

100%

n =20

n =5

n =17

n =6

n =13

n =5

n =5

80%

coryphaeid amphipods
Coleoptera adults
60%

% of diet by IRI

crab zoea
crabs
gammarid amphipods #2
gammarid amphipods #1
Corixidae
shrimp

40%

fishes
copepods

20%

0%
TH

#6

RC

RCN

BP

BC

THE

Study sites

Although dietary variation can be a result of
genetic differences across a species’ range, variation
in diet for highly dispersive species like tarpon
(McMillen-Jackson et al. 2005) is likely a result of
localized feeding plasticity (Turingan et al. 1995;
Crabtree et al. 1998; Cutwa and Turingan 2000). The
ability of age-0 tarpon to feed on many different prey
organisms may allow the species to inhabit a range of
nursery habitats, including man-made marshes, despite
potential shifts in prey abundance and diversity
associated with anthropogenic disturbance. The classic

notion that pristine ecosystems are the only valuable
nursery habitats for marine organisms may not apply to
all species. We have shown that when natural systems
have been degraded or destroyed, human-altered
habitats can assume a nursery role. As coastal areas
become increasingly modified by man, the amount of
natural nursery marsh habitat available to recruiting

tarpon will continue to decline. Juvenile tarpon may
become even more dependent on man-made mosquito
control impoundments and restoration marshes as the
degradation of coastal habitats continues.


232
Acknowledgments This research was funded in part through
grants provided by Bonefish and Tarpon Trust, the Don Hawley
Foundation, and the Florida Fly Fishing Association, as well as
a teaching assistantship provided by Florida Institute of
Technology. We are particularly grateful for the generous
support and unlimited access provided by Jim David and the
St. Lucie County Mosquito Control District. The fieldwork
carried out during this study would not have been possible
without the assistance of John O’Connell, Leslie Martin, Kate
Heckman, Mike Gerlach, and Rick Oldham. Tarpon were
collected pursuant to Florida Fish and Wildlife Conservation
Commission Permit # 06SR-035.

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Environ Biol Fish (2011) 90:235–242
DOI 10.1007/s10641-010-9735-5

Pectoral fin ray aging: an evaluation of a non-lethal method
for aging gars and its application to a population
of the threatened Spotted Gar
William Roy Glass & Lynda D. Corkum &
Nicholas E. Mandrak

Received: 28 October 2009 / Accepted: 26 October 2010 / Published online: 13 November 2010
# Springer Science+Business Media B.V. 2010

Abstract Spotted Gar (Lepisosteus oculatus), a species listed as Threatened under the Canadian Species
at Risk Act (SARA) was collected during May and

June, 2007 from several sites in Rondeau Bay, a
shallow coastal wetland of Lake Erie. The first
pectoral fin ray was removed from 78 individuals to
age the fish and to determine individual growth
characteristics. To assess the validity of using pectoral
rays to age Spotted Gar, we compared techniques
(otoliths, branchiostegal rays and pectoral rays) for
ten individuals captured in southwestern Michigan.
Agreement between readers and amongst the three
structures was high; thus aging of Spotted Gar using
sectioned pectoral rays is an effective method.
Rondeau Bay specimens varied in age from 3 to
10 years and from 515 to 761 mm total length.
Regression analysis of length vs. age
À data was
Á
calculated to be y ¼ 19:217x þ 491:19 R2 ¼ 0:22 .
The low R2 value is attributed to having males and
females, which differ in growth rates, combined.
Growth rates of Rondeau Bay specimens were
W. R. Glass (*) : L. D. Corkum
Department of Biological Sciences, University of Windsor,
401 Sunset Avenue,
Windsor, Ontario N9B 3P4, Canada
e-mail:
N. E. Mandrak
Great Lakes Laboratory for Fisheries and Aquatic Sciences,
Central & Arctic Region, Fisheries and Oceans Canada,
867 Lakeshore Road,
Burlington, Ontario L7R 4A6, Canada

compared to a Louisiana population using ANCOVA.
No significant difference was found in the rate of
growth between these populations; however, condition was low as compared to a standard weight
equation. This may lead to lower fecundity, contributing to the species’ rarity in Canada.
Keywords Age . Growth . Species at risk . Pectoral
ray . Lake Erie . Spotted Gar

Introduction
The Spotted Gar (Lepisosteus oculatus) is a fish
species designated as Threatened under the Canadian
Species at Risk Act (SARA). The species is distributed
throughout the Mississippi River drainage with its
northern limit extending into Canada (Fig. 1). In
Canada, L. oculatus inhabits three coastal wetlands of
Lake Erie (Point Pelee, Rondeau Bay and Long Point
Bay) with historic records from Lake St. Clair
(COSEWIC 2005). The Threatened designation in
Canada is due to their limited distribution and
possible loss of critical habitat (COSEWIC 2005).
When preparing management strategies for species
at risk, information is needed on life history traits,
habitat associations, habitat availability and recovery
targets (Rosenfeld and Hatfield 2006). This information is lacking for the Spotted Gar in Canada. Much
of what is known about the Spotted Gar is based
mainly on data gathered in the southern portion of its