Aquatic Invasions (2013) Volume 8, Issue 2: 219–229
doi: http://dx.doi.org/10.3391/ai.2013.8.2.09

Open Access© 2013 The Author(s). Journal compilation © 2013 REABIC

Research Article

Predicting potential hybridization between native and non-native Rhodeus
ocellatus subspecies: the implications for conservation of a pure native
population in northern Kyushu, Japan
Norio Onikura1*, Takuya Miyake2, Jun Nakajima3, Shinji Fukuda4, Tomonori Kawamato 1
and Koichi Kawamura 2
1 Fishery Research Laboratory, Kyushu University, 4-46-24 Tsuyazaki, Fukutsu, Fukuoka 811-3304, Japan
2 Graduate School of Bioresources, Mie University, 1577 Kurimamachiya-cho, Tsu, Mie 514-8507, Japan
3 Fukuoka Institute of Health and Environmental Sciences, 39 Mukaizano, Dazaifu, Fukuoka 818-0135, Japan
4 Institute of Tropical Agriculture, Kyushu University, 6-10-1 Hakozaki, Fukuoka 812-8581, Japan
E-mail: onikura@agr.kyushu-u.ac.jp (NO), spriggan@sannet.ne.jp (TM), cyprin@kyudai.jp (JN), shinji-fkd@agr.kyushu-u.ac.jp (SF),s0740510@agr.kyushu-u.ac.jp (TK), kawa-k@bio.mie-u.ac.jp (KK)

*Corresponding author
Received: 11 October 2012 / Accepted: 13 May 2013 / Published online: 20 May 2013
Handling editor: Kit Magellan

Abstract
Hybridization between native and non-native fishes is a serious global problem. Thus, there is a need to develop monitoring methods forpredicting potential hybridization to evaluate the risk of genetic introgression and to identify important areas for conservation of pure native
populations. Here, we developed a prediction model for intersubspecific hybridization, based on distribution and genetic data. We selected
Rhodeus ocellatus kurumeus and R. ocellatus ocellatus as the native and non-native subspecies, respectively. First, we developed
generalized linear models (GLMs) for the species habitat requirements by using presence/absence data and environmental variables. The
best-fit models showed river length gave conflicting effects for the two subspecies. Next, we developed a GLM using the ratio of non-native
haplotypes in mitochondrial DNA as an objective variable, with the predicted probabilities of the occurrence of each fish and spatial
information as explanatory variables. The best-fit model selected the distance from the center of native distribution and the non-native habitat
requirement as key factors. Our findings indicate that hybridization occurs highly and/or initially near the margin of native distribution where
non-native habitat requirements are available. Our model could identify sites in native habitats with very low potential risk for genetic
invasion as important areas for conservation of pure native populations.
Key words: Cyprinidae; bitterling; genetic introgression; habitat use; prediction model

Introduction
The introduction and establishment of non-native
species have negative effects on individuals,
populations, and communities of native species,
and introduced species have contributed to thedecline or extinction of native species worldwide(Coblentz 1990; Lodge 1993; Townsend 2003).
Hybridization is one of the most detrimental
impacts of non-native species on native species(Sato et al. 2010a). Numerous cases of hybridization
have been documented in freshwater fishes (e.g.,
Rhymer and Simberloff 1996; Epifanio andNielsen 2000; Kawamura et al. 2001; Konishi etal. 2003). Nevertheless, in terms of legislation
and regulation little attention has been paid tothe severity of ecological impact from hybridization
despite the fact that hybridization can cause
extinction of a species, subspecies, or population
by genetic extermination with genetic introgression(Rhymer and Simberloff 1996; Allendorf et al.
2001; Sato et al. 2010a).
We can classify the hybridizations of freshwater
fishes in Japan into three levels: interspecific,
intersubspecific, and intraspecific hybridization.
There are reports of interspecific hybridization
between the Shinai-motsugo, Pseudorasbora
pumila pumila Miyadi, 1930, and the topmouth
gudgeon, P. parva Temminck et Schlegel, 1846
(Konishi et al. 2003), as well as intersubspecifichybridization between the Japanese rosy
bitterling, Rhodeus ocellatus kurumeus Jordan219

N. Onikura et al.

and Thompson, 1914 (Rok), and the Chinese rosy
bitterling, R. o. ocellatus Kner, 1866 (Roo) (Kanoh
et al. 2005). The intraspecific hybridizations
between different regional populations have been
demonstrated in a number of species, including
the zezera, Biwia zezera Ishikawa, 1895
(Horikawa and Mukai 2007; Horikawa et al. 2007).
Rok and P. pumila are designated as “criticallyendangered” in the Japanese Red Data Book
(Kawamura 2003), mainly because of hybridization
with alien subspecies and closely-related species.
By contrast, several alien species, subspecies andpopulations, including Roo and P. parva, areclassified as "invasive with high risk" (Onikura
et al. 2011), by the Fish Invasiveness Scoring Kit(Copp et al. 2005).
Anthropogenic hybridization between nativeand non-native fishes in Japan has been
confirmed by morphological and genetic studies(Kawamura et al. 2001; Konishi et al. 2003; Konishi
and Takata 2004a; Kanoh et al. 2005; Koga andGoto 2005), validation of crossing experiment inaquaria (Konishi and Takata 2004b; Kawamura
2005; Kawamura et al. 2009), analysis of
distribution patterns and habitat uses in native
and alien subspecies and their hybrids (Miyake
et al. 2008; Konishi et al. 2009; Onikura et al.
2012), and evaluation of risk of genetic invasion(Onikura et al. 2011). However, to date no-one hasdeveloped monitoring methods for predicting
hybridization, or elucidated environmental requirements for genetic introgression from native to nonnative subspecies.
Here, we address the hybridization between
Rok (native subspecies) and Roo (non-native
subspecies) as a serious problem. Rok, which isan endemic cyprinid species of Japan, was widelydistributed in creeks, ponds, and small rivers inwestern Japan prior to the 1940s (Figures 1-2,
Kawamura 2005). By contrast, Roo, which belongs
to a sister subspecies of Rok, was accidentally
introduced into Japan from mainland China in
the 1940s (Nakabo 2002; Matsuzawa and Senou
2008). Hybridization between Rok and Roo is amajor factor contributing to the loss of Rok
populations (Kawamura 2003). Currently, Rooand hybrid populations are expanding throughout
Japan, excluding Kyushu Island (Figure 2). The
process of genetic invasion of Roo into Rok in
Honshu and Shikoku islands is believed to haveoccurred as follows: occurrence of Rok mainly in
man-made ponds; progression of inbreeding
depression within each population in eachfragmented habitat; inferior fitness level of each220

population; invasion of non-native Roo populations;and rapid subspecies hybridization (Kanoh et al.
2005; Kawamura 2005; Kawamura et al. 2009).
By contrast, Rok populations in northern
Kyushu Island have high genetic variationcompared with Rok populations in Honshu andShikoku islands (Kawamura 2005). In Kyushu
Island, Rok populations inhabit open-water
systems (Kawamura 2005), such as agricultural
canal networks and rivers (Miyake et al. 2008;
Onikura et al. 2007, 2012) and have a wide rangeof habitats (Miyake et al. 2008; Onikura et al.
2012). Kawamura (2005) suggested that the highgenetic variation of Rok population in this area
should be maintained by the superior population
size and habitat conditions compared to those in
Honshu and Shikoku islands.
On the basis of previous reports on the
distributions of Rok, Roo, and hybrid populations
in western Japan, we conclude that the preventionof genetic introgression by Roo in northern
Kyushu Island is the key to conserve the Rok
population of Japan. The aims of this study wereto evaluate the risk of genetic introgression by
Roo and to identify important areas for Rok
conservation in this island. Our previous studies
have shown the distributions of Rok, Roo, andtheir hybrid populations in this island (Figure 3,
Miyake et al. 2008; Onikura et al. 2012). Here, wedeveloped a predictive model of genetic
introgression by the nonnative subspecies based on
genetic and distribution data.
Methods
Study design
We selected northern Kyushu Island of Japan as
our survey area according to Miyake et al.
(2008) and Onikura et al. (2012) where Rok, Rooand their hybrids are well distributed. Twoestimations were used in developing a genetic
invasion model. One estimated habitat requirementsof the two subspecies and the other estimated theease of hybridization in those areas. In theformer, the hybrid population was expected tooccur in the overlapping area of Rok and Roo
distributions based on their habitat requirements.
We predicted the distributions of Rok and Roo innorthern Kyushu by using models with highaccuracy and landscape parameters as explanatory
variables in our previous study (Onikura et al.
2012), which indicated that the overlapping areasof distribution of the two subspecies were in
limited areas that contained the habitat

Predicting potential hybridization between native and non-native subspecies

Figure 1. A male of Rhodeus ocellatus kurumeus from
Tsuyazaki creek in northern Kyushu Island, Japan (A); a maleof Rhodeus ocellatus ocellatus from the Tiaoxi River,
Yangtze River basin, China (B); a male of the hybrid between
Rhodeus ocellatus kurumeus and Rhodeus ocellatus
ocellatus from Kashima creek in northern Kyushu, Japan (C).
A, C – photographs by Dr. J. Nakajima; B - photograph by Dr.
Y. Kano, Kyushu University, Japan.

requirements of the two subspecies. Therefore,
we calculated the probability of occurrence ofeach subspecies in each site and used theseprobabilities as explanatory variables of a genetic
invasion model.
To estimate the ease of hybridization, we firstexamined previous studies on the mechanism of
hybridization. Rok and Roo show gradual premating reproductive isolation in culture experiments(Kawamura et al. 2009). However, Roo and hybrid
populations have been expanding throughout
Japan since the 1940s (Nakabo 2002; Matsuzawa
and Senou 2008). One reason for this expansion

Figure 2. The distribution of Rhodeus ocellatus subspecies inJapan in 1940 and 2010. The native and non-native subspecies are
Rhodeus ocellatus kurumeus (Rok) and R. o. ocellatus (Roo),respectively. Hybrid populations between these subspecies occur.
The distribution of hybrid populations is included in the Roo
distribution.

is the progression of inbreeding depression
within Rok populations in fragmented habitats(Kanoh et al. 2005; Kawamura 2005; Kawamura etal. 2009). Before the 1940s, Rok inhabited small
isolated ponds in western Japan, excluding northern
Kyushu Island, and the small populations in eachpond showed low genetic variation and inferior
fitness levels because of excessive inbreeding.
When Roo invaded Rok habitat, introgressive
hybridization between the two subspecies
progressed rapidly. Therefore, fragmented habitats
and small Rok population sizes led to the ease of
introgressive hybridization by Roo. In northern221

N. Onikura et al.

Figure 3. The current
presence/absence of Rhodeus
ocellatus subspecies innorthern Kyushu, Japan. The
distribution data for Rok, Roo,and hybrid populations are
summarized in detail in
Appendix 1.

Kyushu Island, Rok is widely distributed in openwater systems and there are few fragmented areas
near the margin of the distribution (Figure 3). In
addition, Rok population sizes near the marginsof their distribution are smaller than those near
the center (Figure 4), and small Rok populations
may lack genetic stability. We, therefore, added
the distance from the center of the Rok distribution
to each site as an explanatory variable indeveloping a genetic invasion model.
Roo was accidentally introduced into Japan
from mainland China in the 1940s. Since then,this subspecies has expanded its distribution
throughout Japan by commingling with otheraquatic species in aquaculture and game fishing(Kawanabe et al. 1989; Nakabo 2002; Matsuzawa
and Senou 2008). The same phenomenon occurs
with alien fishes introduced from domestic
regions. When the juveniles of Ayu, Plecoglossus
altivelis Temminck et Schlegel, 1846, from Lake
Biwa located in central Honshu Island werereleased into rivers throughout Japan, several
fish species commingled with them and havebeen expanding throughout Japan (Matsuzawa
and Senou 2008). The Japanese crucian carp,
Carassius cuvieri Temminck and Schlegel, 1846,
222

Figure 4. The relationship between the distance from each site to
the center of Rok distribution (Rok-DIS) and the population sizeof pure native subspecies (Rok). The logarithmic curve wasderived using data from 59 sites with uniform fishing effort.

Predicting potential hybridization between native and non-native subspecies

from Lake Biwa were released throughout Japanfor game fishing, several fishes also commingledwith it (Kawanabe et al. 1989). More than 10
alien fish species/subspecies that have been
intentionally or accidently introduced from othercountries or other domestic regions are currently
in northern Kyushu Island (Nakajima et al. 2008).
The number of alien fish species may have aneffect on the success of Roo introduction. We
included the number of other alien species (NAS)as an explanatory variable substitute for thefrequency of accidental introduction.
Redevelopment of habitat models
Although our previous study reported predicted
Rok and Roo distributions in northern Kyushu
Island (Onikura et al. 2012), it was necessary to
redevelop the Rok and Roo habitat models todevelop a genetic invasion model. We previously
analyzed Rok distribution by using native Rok
populations as presence data and other populations
as absence data (Onikura et al. 2012). However,
we assumed that most sites with hybrid
populations were originally Rok habitats prior to
the invasion by Roo. Thus, in the present study,
our presence data included both native Rok
populations and hybrid populations. However,some sites with hybrid populations were outside
the natural distribution area of Rok. These
populations were likely introduced to the sites
after hybridization occurred in other regions ofJapan and were, therefore, excluded from the
Rok presence data. As a result, we used 171/782
sites for presence/absence data (Figure 3). In theanalysis of Roo habitat, we used all Rhodeus
ocellatus populations, excluding native Rok
populations, and 56 presence and 897 absence
sites (Figure 3).
In our previous study (Onikura et al. 2012),
we developed the Rok and Roo habitat models in
the western region of northern Kyushu Islandand evaluated the performance of each model inthe eastern region. In this study, we redeveloped
the habitat models by analyzing all the data. In
addition, we defined biogeographical information(BGI, 1/0: western/eastern regions) as a dummy
variable because we previously demonstrated asmall difference in Roo distribution patterns
between western and eastern regions (Onikura etal. 2012).
We also changed some of the explanatory
variables for the habitat models. In our previous
study (Onikura et al. 2012), elevation (EL, m)and river gradient (RG) were highly correlated,
and EL was excluded to prevent multicollinearity
between predictor variables from affecting theanalyses. Information extraction of RG, which
was calculated from the distance and thedifference of elevation between 2 points, was socomplex and difficult that we used EL in thisstudy. The canal network index (CNI, Fukuda etal. 2011) used in our previous study was excluded
for the same reason as RG and this study used thenumber of river to canal connections (RC) instead of
CNI. Other explanatory variables used werelandscape variables: length of main river (LMR,km), river width (RW, m), area of paddy fields(AP, km2), and residential area (RA, km2). LMR
and EL are needed to develop the habitat models
because the distributions of freshwater fishes
inhabiting Kyushu Island are influenced by river
length and elevation (Nakajima et al. 2006;
Onikura et al. 2008). Land use information,
including AP and RA, is also needed for model
development because Rok mainly inhabit
floodplains (Nakajima et al. 2010). Details of themeasurements of landscape parameters aredescribed in previous reports (LMR and RW:
Onikura et al. 2012; RC, AP, and RA: Fukuda et al.
2011). All data were derived from Digital Map
25,000 (Japan Map Center, Tokyo), edited by the
Geographical Survey Institute, Japan. We
overlaid digital elevation data on the Map Imageand measured environmental characteristics foreach site with GIS software KASHIMIR 3D
version 8.0.9 (http://www.kashmir3d.com). We
prepared a correlation matrix, and confirmed theabsence of multicollinearity among all variables(Table 1).
We used a GLM (McCullagh and Nelder 1989)
to develop the model for Rok and Roo habitats.
We conducted a logistic regression for all possible
sets of predictor variables, from a null model
including no predictors to a full model including
all predictors. We used the Akaike Information
Criterion (AIC; Akaike 1974) for model
selection in which the model with the lowest
AIC was defined as the best-fit model. To
evaluate the model, we performed a receiveroperating characteristic (ROC) analysis between
actual and predicted fish distribution (Manel etal. 2001; Akoberg 2007). An area under the ROC
curve (AUC) reflects how well the model
distinguishes between the presence and absenceof a species: an AUC greater than 0.9 has highaccuracy and 0.7–0.9 has moderate accuracy,
whereas 0.5–0.7 indicates low accuracy223

N. Onikura et al.

Table 1. Correlation matrix of explanatory variables used during model selection on the habitat models (Pearson’s correlation coefficient).
Variables (Acronyms)
1. Length of main river (LMR)

1

2

3

4

5

6

-

2. Elevation (EL)

0.110

-

3. River width (RW)

0.047

-0.078-

4. Number of river to canal connections (RC)

0.119

-0.2750.108-

5. Area of paddy field (AP)

0.178

-0.463-0.1270.229-

6. Residential area (RA)-0.125-0.428

0.201

0.172

-0.083-

Table 2. Correlation matrix of explanatory variables used during model selection on the genetic invasion model (Pearson’s correlation
coefficient).
Variables (Acronyms)

1

2

3

1. Probability of occurrence of native Rok (Rok-HR)-

2. Probability of occurrence of native Roo (Roo-HR)-0.022

3. Distance from central distribution of Rok (Rok-DIS)-0.1980.277-

4. Number of alien species (NAS)

0.438-0.244-0.205(Akobeng 2007). We used the base package in R
v. 2.11.1 (http://www.R-project.org) and SPSS v.
15 (SPSS Japan Inc., Tokyo, Japan) for ouranalyses.
Genetic invasion model
We used previously derived genetic data for Rok
and Roo in 68 sites (Miyake et al. 2008), andthese specimens have been preserved in the
KyushuUniversity
Museum(KYUM-PI00002235–00002314). These data were based on
mitochondrial DNA analysis of 10 to 20
individuals per site, and we indicated the ratio of
Roo haplotypes (Roo-GI) in each site in theprevious study (Miyake et al. 2008). The
frequency of Roo mt-DNA haplotypes in each
population increases with increasing genetic
introgression of Roo (Kawamura et al. 2001).
Therefore, the frequency in each site can be used
to estimate genetic invasion by Roo. We used
Roo-GI indicated in previous study as an
objective variable (i.e., the measure for genetic
disturbance).
For the genetic invasion model, we calculated
the probability of occurrence of the two
subspecies in each site, by using the best-fit
habitat models for Rok and Roo (Rok-HR and
Roo-HR). In addition, we computed the average
latitude and longitude of the sites with Rok
224

4

-

presences in the western and eastern regions. We
defined the mean value as the center of Rok
distribution in each region, and calculated the
distances from the value to each site. We thendivided the distance in each site by the maximum
distance for the Rok presence in each region andset the obtained value as the indicator for themarginality of Rok distribution (Rok-DIS). We
prepared a correlation matrix for Rok-HR, RooHR, Rok-DIS, and NAS, and confirmed theabsence of multicollinearity among all variables(Table 2).
We also used a GLM for the model on thegenetic invasion by Roo into Rok. We defined
BGI (1/0: western/eastern regions) as a dummy
variable again, and constructed a GLM as a
Gaussian distribution for all possible sets ofpredictor variables, from a null model including
no predictors to a full model including all
predictors. We defined the model with the lowest
AIC as the best-fit model. For model evaluation,
we performed a linear regression analysis
between actual and predicted genetic invasion
ratios. We used the base package in R v. 2.11.1
and SPSS v. 15 for our analyses. After the model
development, we predicted potential hybridization
between Rok and Roo by using the best-fit
model. We estimated potential risk in each site
as very low risk when Roo-GI predicted by using
the model was under 1%.

Predicting potential hybridization between native and non-native subspecies

Results
Redevelopment of habitat models for native andnon-native subspecies
The best-fit model for the native subspecies Rok
selected four variables. Of these, LMR, RC, andAP were positively correlated, while EL wasnegatively correlated (Appendix 2). The AUC
value of the best model was 0.874. The best-fit
model for the non-native subspecies Roo
selected five variables. Of these, AP was
positively correlated, while LMR, EL, and RW
were negatively correlated. The model included
BGI as one of explanatory variables. The AUC
value of the best model was 0.912.
Model for genetic invasion
The best-fit model for genetic introgression of
non-native Roo into native Rok selected threevariables including BGI. Of these, Roo-GI
increased with increasing Roo-HR and Rok-DIS(Appendix 3). The linear regression formula
between the actual and predicted genetic
invasion ratio was as follows:
Predicted = 0.5265 × Actual (r = 0.514, p <0.01, Figure 5).
In the western and eastern regions, the numberof actual non-native and hybrid distribution sites
was 53 and 4, respectively (Figure 6A), while thenumber of sites with 1% or more in Roo-GI by
predicting hybridization using the best-fit model
was 203 and 87, respectively (Figure 6B).
Assuming Roo and hybrid populations invade thesites with potential risk (Roo-GI ≥ 1%) in the
future, our prediction indicated that theirdistributions will increase from 57 to 290 sites.
In contrast, the number of actual native
distribution sites was 95 in the western region
and 40 in the eastern region (Figure 6A), while
the number of sites with very low risk (Roo-GI <1%) based on the prediction was 45 and 5,
respectively (Figure 6B). Our prediction indicatedthat the number of sites with only pure Rok
populations will decrease from 135 to 50.
In addition, the enclosure areas of actual
native distribution were approximately 1,640
km2 and 770 km2 in the western and easternregions respectively (Figure 6A), while the predicted
native distribution areas were approximately 680
and 12 km2 respectively (Figure 6B). Our
prediction indicated a decrease in native Rok
distribution areas from 2,410 to 692 km2.

Figure 5. The relationship between actual and predicted valuesof the ratio of non-native haplotypes of mitochondrial DNA (RooGI) in each population. Data are plotted using the best-fit genetic
invasion model.

Discussion
Several studies have shown the potential
distributions, habitats, or niches of alien fishes
by developing models based on abiotic
environmental information (e.g., Iguchi et al.
2004; Céréghino et al. 2005; Spens et al. 2007; Satoet al. 2010b). Such models show proactive analyses,
such as predicting occurrences and distribution,
are particularly important in preventing further
invasion and range expansion of introduced
fishes (Sato et al. 2010a). However, there aredifferences between such models and the modelin this study. The former are used to estimate
potential presence of each species, while thelatter are used to estimate potential hybridization
between native and non-native subspecies. As far
as we know, this is the first report on a genetic
invasion model that predicts potential hybridizationfrom landscape parameters. Hybridization cancause extinction of a species, subspecies, or
population by genetic introgression (Rhymer andSimberloff 1996; Allendorf et al. 2001; Sato et al.
2010a). Therefore, the environmental requirement
for genetic introgression from native to nonnative must be elucidated to preserve native
genetic resources. The results of this study cancontribute to the management of native fish
conservation and the prevention of hybridization
with non-native fish.
225

N. Onikura et al.

Figure 6. Maps of the actual distribution of Rhodeus ocellatus
subspecies (A) and the predicted ratio of Roo haplotypes of
mitochondrial DNA (Roo-GI) in each population (B). The
enclosed Rok distribution areas (A) were predicted using the bestfit Rok habitat model and Roo-GI (B) was predicted using thebest-fit genetic invasion model.

The non-native subspecies Roo is expanding
its distributions throughout Japan by commingling
with other aquatic species in aquaculture andgame fishing (Kawanabe et al. 1989; Matsuzawa
and Senou 2008). Several alien fishes are becoming
established in northern Kyushu Island throughthe same process (Nakajima et al. 2008). Therefore,226

we hypothesized that the number of alien fish
species may have an effect on the success of Roo
introduction. Nevertheless, the best model ofgenetic invasion in this study did not select NAS
as an explanatory variable. Such accidental
introductions occurred mainly through the releasesof Ayu and Japanese crucian carp in Japan, andthese 2 species occur throughout Kyushu Island.
The accidental introductions of Roo may haveoccurred throughout this island at the same time,
resulting in a model that had no effect on Roo
introduction.
It is logical that the best model of genetic
invasion in this study selected Roo-HR, which
represents the habitat requirement of Roo. Oneof the other selected variables, Rok-DIS, which
represents the distance from the center of Rok
distribution, indicates a negative effect on RooGI. These results suggest that initial and/or highinvasion of non-native Roo occur at the marginof the native Rok distribution. In summary, ourresults demonstrate that genetic invasion by Roo
is influenced by the distribution of native
subspecies and the habitat requirements of nonnative subspecies.
Population size and genetic variation of native
subspecies contribute to the correlation between
Roo-GI and Rok-DIS in the genetic invasion model.
A lack of heterosis (or hybrid vigor) (Kanoh et al.
2005; Kawamura et al. 2009) and gradual premating reproductive isolation (Kawamura et al.
2009) have been confirmed as ecological andgenetic characteristics among native Rok, nonnative Roo, and hybrid populations. In Honshu,
Roo and hybrid populations invade almost all
Rok distributions, and Roo and hybrid populations
predominate ecologically (Kawamura et al. 2001)
because of their superior fitness in fragmented
and isolated habitats (Kanoh et al. 2005;
Kawamura et al. 2009). Thus, the extinction of Rok
populations results not only from hybridization,
but also from inferior fitness (Kawamura et al.
2009). However, in our study area in northern
Kyushu Island, Rok populations have highgenetic variation compared with Rok populationsin Honshu and Shikoku islands (Kawamura
2005). In addition, Rok populations inhabit
open-water systems (Kawamura 2005), such as
agricultural canal networks and rivers (Miyake etal. 2008; Onikura et al. 2007, 2012), and have awide range of habitats (Miyake et al. 2008;
Onikura et al. 2012). These results suggest thatthe high genetic variation of Rok populations inthis area are likely to be maintained by superiorPredicting potential hybridization between native and non-native subspecies

population sizes and habitat conditions comparedto those in Honshu and Shikoku islands(Kawamura 2005). We believe that initial and/or
high genetic invasion by Roo occurs near themargin of Rok distribution because population sizes
and genetic variation are smaller and lower thannear the center. Using Rok population size datafrom 59 sites, we derived a logarithmic curveshowing significant regression between Rok-DIS
and Rok population sizes.
While anthropogenic hybridizations are
detrimental to many species through direct andindirect means (Rhymer and Simberloff 1996),
natural hybridizations play important roles in theevolution of many animal taxa (Allendorf et al.
2001). Allendorf et al. (2001) provide a
categorization of hybridization to help guidemanagement decisions: Types 1–3, shown as
natural hybridization, are a natural part of theevolutionary legacy of taxa, and Types 4–6,
shown as anthropogenic hybridization, havedifferent consequences from a conservation
perspective. Hybridizations are without backcrosses
in Type 4, with backcrosses and widespread
introgression in Type 5, and with backcrosses
and complete admixture in Type 6. Type 4 isdetrimental because there is wasted reproductive
effort rather than genetic mixing. Type 6 resultsin the extinction of pure populations. Hybridization
between Rok and Roo falls under Type 6.
Allendorf et al. (2001) mention that few, if any,
pure populations remain and there does notappear to be any selection against the hybrids inthis case. Therefore, we must make effective useof the prediction of our current model.
In Japan, the Invasive Alien Species Act(Ministry of the Environment, Japan 2004) went
into effect in June 2005. Several fish species,
such as mosquitofish (Gambusia affinis Baird etGirard, 1853), bluegill (Lepomis macrochirus
Rafinesque, 1819), and largemouth black bass
(Micropterus salmoides Lacépède, 1802), aredesignated as invasive alien species in the act,and several actions, such as cultivation andtransportation, are regulated for these species.
However, Roo is designated as an adventive
species without regulation in the act. This study
predicts a drastic increase in the number of sites
with genetic invasion (from 57 to 290), and large
decreases in the number of sites (from 135 to 50)
and area (2,410 to 692 km2) with pure native
populations. These predictions suggest very highrisk of Roo invasion. Another problem is genetic
homogenization (Olden et al. 2004). Genetic
homogenizations occur throughout Japan, andthe hybridization between Rok and Roocontributes to this homogenization (Watanabe2012). We emphasize the need for legal andmanagement actions to prevent the expansion of
Roo and hybrid populations in this area.
In this study, Rok-DIS shows significant
correlation with Rok population sizes, implying
higher potential risk than our predictions forgenetic invasion. Even if a site is near the centerof Rok distribution, high genetic invasion may
occur there via negative environmental changewith decreasing Rok population size. Recently,
in northern Kyushu, the habitat conditions ofRok, including the center of Rok distribution,
have become depleted because earthen agricultural
canals are now being replaced with concrete
revetments, and canal networks with complicatedintersections are being reconstructed to create
simple, uncrossed networks (Onikura et al. 2007).
The Rok populations in canals with concrete
revetments are smaller than those in canals withearthen revetments (Onikura et al. 2007). Rok
populations affected by such negative impacts
may be more susceptible to the genetic invasion
by Roo. We believe that large Rok populations
depend not only on the locations within the Rok
distribution area but also on habitat conditions.
The genetic invasion model could identify sites
with very low potential risk for genetic invasion.
Therefore, we recommend selecting actual Rok
habitats with very low Roo invasion risk astarget sites, and take actions to restore theirhabitats in order to maintain Rok populations inthe selected sites.ecosystems.
Acknowledgements
The authors thank Dr. K. Watanabe, Dr. N. Takeshita, Dr. T.
Mukai, and three anonymous reviewers for providing usefulcomments. This work was supported by the Environment
Research and Technology Development Fund (RF-0910 and S9)of the Ministry of the Environment, Japan. The cost of publication
was supported in part by the Research Grant for Young
Investigators of the Faculty of Agriculture, Kyushu University.

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Supplementary material
The following supplementary material is available for this article:
Appendix 1. Presence data of Rhodeus ocellatus kurumeus (Rok), R. o. ocellatus (Roo) and the hybrid populations between these
species in this study.
Appendix 2. Results of statistical analyses and selected explanatory variables of top 5 and null habitat models for native
Rhodeus ocellatus kurumeus (Rok) and non-native R. ocellatus ocellatus (Roo).
Appendix 3. Results of statistical analyses and selected explanatory variables of top 5 and null genetic invasion models fornative Rhodeus ocellatus kurumeus (Rok) by non-native Rhodeus ocellatus ocellatus (Roo).
This material is available as part of online article from:http://www.aquaticinvasions.net/2013/Supplements/AI_2013_Onikura_etal_Supplement.pdf

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