F I S H and F I S H E R I E S , 2013, 14, 60–76

A comparative analysis of chemically induced sex reversal in

teleosts: challenging conventional suppositions

Alistair M Senior & Shinichi Nakagawa

Department of Zoology, University of Otago, PO Box 56, Dunedin 9054, New Zealand

Abstract Correspondence:
Environmental sex reversal (ESR) occurs when extreme environmental factors Alistair M Senior,
Department of
overpower predetermined sexual development. Scientific theory is beginning to
Zoology, University of
acknowledge the potential roles of sex-reversed individuals in influencing population Otago, PO Box 56,
dynamics and driving the evolution of sex-determination mechanisms. ESR is a Dunedin 9054, New
phenomenon that has been widely observed in fish and can be induced by exposing Zealand
individuals to exogenous hormones. However, reports of the susceptibility of fish to Tel.:
+64 (0) 3 479 5046
hormonally induced ESR vary greatly – a concept we termed ‘inducibility’. It has been
Fax:
suggested that variation in inducibility can be attributed to biological differences +64 (0) 3 479 7584
among species of different taxonomic groups (i.e. phylogenetic effects are present). E-mail:
Here, we provide the first quantitative test of this theory, which was achieved by senal826@student.
combining published data with phylogenetic trees, using phylogenetically controlled otago.ac.nz

comparative analysis. Our results confirm that a great amount of variation exists in Received 21 Jul 2011
the reported inducibility of fish. However, species and taxonomic relationships were Accepted 24 Oct 2011
responsible for trivial portions of variation. Rather, we found that sampling
(measurement) errors in combination with methodological differences across studies
accounted for much more variation in inducibility than taxonomy did. Given that our
analysis contains representatives from over 25% of all teleost orders, we conclude
that inducibility is not a taxonomically constrained trait in teleosts. Therefore, we
suggest that the sex-determination mechanisms of most fish are uniformly plastic.
Further, we propose that ESR occurs relatively regularly over evolutionary time in
many teleost species, playing a vital role in the maintenance of homomorphic sex
chromosomes in this taxonomic group.

Keywords Endocrine-disrupting chemicals, environmental sex reversal, hormone,

meta-regression, phylogenetic signal, sex determination

Introduction 61
Methods 62
Data collection 62
Estimating inducibility from experiments 63
Coding estimates of I for PMMs 66
Coding explanatory variables for PMMs 66
Phylogeny construction 66
Collating and analysing estimates of inducibility 67
Calculating measurement error, variance components and phylogenetic heritability 67
Results 68

60 DOI: 10.1111/j.1467-2979.2011.00446.x  2011 Blackwell Publishing Ltd

 A comparative study of sex reversal A M Senior and S Nakagawa
Variance components and phylogenetic analyses
Model estimates
Discussion
Phylogenetic insights
Wider evolutionary insights
Ecological insights
Acknowledgements
References
Supporting information
Appendix
Introduction
In many fish species, as in mammals and birds, sex is
determined by somatic chromosomes (Devlin and
Nagahama 2002). However, unlike mammals and
birds, in fish, it is possible for environmental factors
to overpower the sex-determining effects of somatic
chromosomes, leading to environmental sex reversal
(ESR). In some instances, ESR may be invoked by a
naturally occurring environmental factor, for exam-
ple, an unusually high temperature (Baroiller et al.
1995, 1999; Grossen et al. 2011). Alternatively,
ESR can result from environmental extremes that
have a purely anthropogenic cause, such as artifi-
cially manipulated temperature or the presence of
high levels of exogenous hormones and endocrine-
disrupting chemicals (EDCs). Inducing sex reversal in
fish via chemical exposure has been exploited by
both the aquaculture industry and research scien-
tists for many years (Pandian and Sheela 1995;
Devlin and Nagahama 2002; Pandian and Kiran
Kumar 2002; Cnaani and Levavi-Sivan 2009).
The aquaculture industry uses ESR to maintain
single-sex populations of fish, which are favourable
for two reasons: (i) some species such as Nile
Tilapia (Oreochromis niloticus, Cichlidae) grow more
rapidly if kept in single-sex populations and (ii)
reproduction is far easier to control if males and
females are segregated (Cnaani and Levavi-Sivan
2009). Additionally, in some ornamental species
such Siamese fighting fish (Betta splendens, Osphro-
nemidae), individuals of one sex are more valuable
than the other (Piferrer and Lim 1997; Devlin and
Nagahama 2002). Physiologists have also found it
useful to exploit the phenomenon of ESR in fish.
Sex reversal leads to an unusual combination of
opposed genetic and phenotypic sex, which may be
exploited to infer the mechanisms underlying sex
 2011 Blackwell Publishing Ltd, F I S H and F I S H E R I E S , 14, 60–76
68
68
69
69
71
72
72
72
74
75
determination within a species (Yamamoto and
Kajishima 1968; Mair et al. 1987; Liu et al. 2008;
Orban et al. 2009).
In most cases, the induction of sex reversal via
exposure to environmental hormones or EDCs is
favourable so long as sex reversal is complete (i.e.,
all the individuals within the treated population
become one sex). However, the procedure of induc-
ing sex reversal itself is not straightforward. For
example, suboptimal or excessive hormonal dosages
may result in the occurrence of intersex individuals
(individuals with malformed/mixed gonads) or high
levels of mortality (Pandian and Sheela 1995;
Devlin and Nagahama 2002). As a result, in an
attempt to optimize the procedure of sex reversal,
hundreds of studies testing the sex-reversing effects
of varying hormonal dosages in numerous fish
species have been published (Devlin and Nagahama
2002; Cnaani and Levavi-Sivan 2009). Yet, many
of these published studies report conflicting results
about the susceptibility of test species to ESR
induced by the application of exogenous chemicals
– a concept that will be referred to as inducibility
throughout this article.
This vast array of published material has
spawned several qualitative reviews on the subject
of induced sex reversal in fish (Pandian and Sheela
1995; Devlin and Nagahama 2002; Pandian and
Kiran Kumar 2002; Cnaani and Levavi-Sivan
2009). In their review, Pandian and Sheela
(1995) suggest that inducibility (susceptibility to
sex reversal by exogenous hormones) is a taxonom-
ically constrained trait, i.e. inducibility varies more
between, than within taxonomic families. Specifi-
cally, they suggest that the intensity of treatment
required to induce sex reversal resulting in a single-
sex population increases in the following taxonomic
order: Cichlidae < Cyprinodontidae < Anabantidae
61
A comparative study of sex reversal A M Senior and S Nakagawa

< Poeciliidae < Salmonidae < Cyprinidae (Pandian number or as a percentage of males to females
and Sheela 1995). accompanied by a sample size to which this
Given advancements in comparative and analyt- percentage applies, and (iv) ESR must be induced
ical techniques, coupled with the publication of via either a dietary (chemical mixed with food) or a
numerous teleost phylogenies (Mank et al. 2006; bath (chemical mixed with tank water) route of
Fraga et al. 2007; Li et al. 2008; Mayden et al. chemical administration.
2009), it is now possible to combine the vast wealth We then extracted data from each experiment in
of published experimental data on induced ESR in the remaining 155 papers (see Supporting Infor-
fish. Here, we provide the first quantitative test of mation for a complete list). Our experimental level
the theory put forward by Pandian and Sheela was defined as follows. If a study exposed a single
(1995). Our primary aim is to use phylogenetically species to one chemical using one method of
controlled comparative analysis (or phylogenetic exposure (e.g., Nile Tilapia exposed to testosterone
mixed-effects meta-regression; Hadfield and Nakag- via immersion in tanks containing dilutions of the
awa 2010) to examine whether published data hormone) and the experimental populations are
support Pandian and Sheela’s suggestion, i.e. induc- exclusively composed of single or mixed genotypic
ibility is a taxonomically constrained trait. sex, then this is a single experiment; otherwise, the
paper contains multiple experiments (see Fig. 1 for
a schematic diagram of our definition). Splitting
Methods
data down to this experimental level provided us
with a total of 246 experiments (each of which
Data collection
was become a single data point in our final
We began by collecting studies that examined the analysis, i.e. the experimental level was the unit
sex-reversing effects of a varying range of ESR- of analysis).
inducing chemical dosages on teleosts from two
main sources. Firstly, Devlin and Nagahama (2002)
provide a list of 155 studies examining sex reversal
in fish. We took this list to be a comprehensive list of
publications on the subject of experimentally
induced fish sex reversal up to and including
2001. Secondly, we carried out an online search
using the ISI Web of Knowledge database with the
search terms ‘sex reversal’, ‘fish’ and ‘teleost’ for
papers published between 2002 and 2011. This
search produced a total of 98 additional studies, one
of which was a review paper (Orban et al. 2009).
Searching Orban et al.’s (2009) references lead us to
six further studies (total number of studies
found = 259).
Of these 259 studies, we rejected 104 that did not
meet the following four inclusion criteria: (i) the
paper must include a controlled experimental study
on a captive group, either in a laboratory or natural
setting, which attempted to induce ESR in a teleost
species by administering a single sex reversal-
inducing chemical (we rejected combinations of
chemicals, unless the mixture was used to aid
administration; e.g. hormone with ethanol), (ii) the
paper must specify the chemical, fish species, route
of chemical administration and the length of time
over which the chemical was administered, (iii) the Figure 1 Conceptual flow diagram depicting how
paper must report the sex ratio of the experimental the experimental level within an article is defined in our
and control groups after treatment, either as a direct analyses.

62  2011 Blackwell Publishing Ltd, F I S H and F I S H E R I E S , 14, 60–76

 A comparative study of sex reversal A M Senior and S Nakagawa
Splitting studies under these criteria still allowed
some variables to vary at the experimental level. For
example, within an experiment, the number of days
for which fish were exposed to a chemical could vary
(e.g., 10, 15, 20 days of exposure). In such cases,
when data were collated using phylogenetic meta-
regression models (PMMs), the mean value of vari-
ables, which vary at the experimental level, was used.
Our procedure of extracting data at the experi-
mental level also meant that it was necessary for
some experiments to share control data. For exam-
ple, a study may have compared the effects of two
steroid hormones (e.g. testosterone and estradiol) on
sex ratio in a single species but only had a single
control group. In this case, because the study varied
the chemical, it would be split into two experiments
(one with the results of testosterone on sex ratio and
the other with the effects of estradiol on sex ratio).
This control data point would be in our analysis
twice (once for each experiment, it should be noted
that pseudo-replication originating from this proce-
dure was controlled in our models). Control data
were shared between experiments in this way if the
study was split due to using different chemicals or
routes of chemical administration. However, if the
study was split because it varied the subject species
or subjected both populations of single and mixed
genotypic sex to induced sex reversal, control data
were not shared. In these cases, it is conceivable
that control groups would produce different sex
ratios between different species, and control groups
would certainly produce different sex ratios between
mono and mixed genotypic sex populations.
Estimating inducibility from experiments
To estimate inducibility (I), we reanalysed the data
(the number of males and females in control and
treatment groups) from each published experiment
separately. Using the R (version 2.13.0, R Develop-
ment Core Team 2011) function brglm in the
package brglm (Kosmidis 2007), we fitted a bias-
reduced binomial generalized linear model (GLM)
with a logit-link function. The response variable
was the proportion of males in the exposed group at
the end of the treatment; the explanatory variable
was the log10 dosage of the sex-reversing chemical
that the group was exposed to (or more precisely,
log10 (x + 1) with x being the original dosage). We
used the parameter estimate for the effect of dosage
on sex ratio and its SE (both on the logit scale), as
the estimate of I and its uncertainty, respectively for
 2011 Blackwell Publishing Ltd, F I S H and F I S H E R I E S , 14, 60–76
each experiment (essentially a dosage response).
Box provides an example of this process, based on
simplified data for clarity.
Box
Below is an example of some hypothetical data,
to clarify our procedures for extracting data. Box
Table 1 contains some hypothetical data typical
of the results reported by the published studies
that we collected. Presented are the results of
three experiments that tested increasing dosages
of a masculinizing chemical on groups of fish
where each group contains ten individuals.
Experiments 1 and 2 are examples of mixed
genotypic sex experiments. At dosage zero, both
groups have equal numbers of males and
females. Experiment 3 is an example of data
from a mono-genotypic sex experiment and at
dosage zero, this experiment reports only female
fish. In all experiments, as the level of hormone a
group of fish is exposed to increases, the number
of males in the group increases. It is obvious
from visual inspection of the data that Experi-
ment 1 reports a greater inducibility (I) than
Experiment 2, because complete sex reversal of
the group is reported at a dosage of 1 in
Experiment 1, but not until dosage 10 in
Experiment 2.
To quantify the differences in reported I, we
model the results of each experiment individu-
ally in the statistical environment R (package
brglm) using a binomial generalized linear model
with a logit-link function. Essentially, we fit the
sex ratio as the response variable and the log10
dosage as the explanatory variable. Specified in
R as follows:
brglm ðcbind ðMales; FemalesÞ
 log10 ðDosage þ 1Þ; family ¼ binomialÞ
This analysis produces a single slope estimate
for each experiment, which is the response of the
group sex ratio to exposure to an increasing
dosage of chemical (essentially a dosage re-
sponse). Of the two mixed-sex experiments, this
estimate is largest for Experiment 1 (GLM
est. = 10.3), rather than for Experiment 2
(GLM est. = 2.9). However, overall the largest
estimate is that for experiment 3 (GLM
est. = 92). The reason Experiment 3 has such
a large estimate of I is because the intercept
63
A comparative study of sex reversal A M Senior and S Nakagawa

(a) (b)
1.0

4
Proportion of Males (logit scale)

0.8

Proportion of Males
0.6

0.4

–2

0.2

–4

0.0

0.0 0.5 1.0 1.5 2.0 0.0 0.5 1.0 1.5 2.0
log10 Dosage +1 log10 Dosage +1

Box Figure 1 The response of group sex ratio to exposure to an increasing dosage of chemical in Experiment 1 (solid line),
Experiment 2 (dashed line) and Experiment 3 (dotted line), as estimated by a binomial generalized linear model (GLM).
These estimates are our measures of inducibility (I). (a) Model estimates directly plotted on the logit scale as given by
the GLM output, these are the values of I that we used in our analyses, (b) the same model estimates back-transformed to
the original scale (proportion of males).

Box (Continued) Box Table 1 Hypothetical data, typical of that reported

by the studies included in our comparative analyses. It
value (number of males at zero) is much lower shows the number of males and females in a group of
than it is in other experiments. This difference in fish exposed to a dosage of masculinizing chemical.
slope estimates owing to differing intercept
values, highlights the importance of splitting Experiment Dosage Males Females
up data at our experimental level (see Methods).
When these model estimates are graphed on the
1 0 5 5
logit scale (Box Fig. 1a), the differences between 0.1 7 3
them are clearly visualized. The estimates of sex 1 10 0
ratio response to increasing dosage on the logit 10 10 0
scale are our estimation of I reported by each 2 0 5 5
experiment. For our real dataset, we repeated 0.1 6 4
this process for all 222 experiments, compress- 1 7 3
ing the data from each experiment to a model 10 10 0

slope to obtain 222 separate estimates of I. 3 0 0 10

When estimates are back-transformed (Box 0.1 7 3
Fig. 1b), it becomes clear how these estimates 1 10 0
10 10 0
reflect the difference in I reported by the exper-
iments.

I), suggesting that the model had not converged.

Of the 246 experiments, which we reanalysed, 24
were removed because the data provided were
insufficient or the sample size was below our cut-
off (n = 3) for analysis using a GLM. Thus, these
data produced an extremely large slope (estimate of
Once these experiments had been removed, we were
left with 222 estimates of I, across 52 species within
11 orders (see Fig. 2), from 139 published studies
(see Data S1). Finally, we split our 222 estimates
into those experiments that induce ESR via a dietary

64  2011 Blackwell Publishing Ltd, F I S H and F I S H E R I E S , 14, 60–76

 (a) (Dietary) (b) (Bath)
N N
Family Family
Huso huso x A. ruthenus 2 Cobitidae Misgurnus mizolepis 1
Acipenseridae
Acipenser brevirostrum 1
Anguilla anguilla 4 Puntius conchonius 1
Anguillidae
Anguilla japonica 1 Cyprinidae
Cobitidae M. anguillicaudatus 1 Carassius auratus grandoculis 1
Tinca tinca 2
Danio rerio 3 Heteropneustidae Heteropneustes fossilis 4
Cyprinidae Puntius gonionotus 1
Cyprinus carpio 15 Ictaluridae Ictalurus punctatus 1
Carassius a. auratus 2
Bagridae Pelteobagrus fulvidraco 2 Salmo salar 1
Ictalurus punctatus 5
Ictaluridae
Ictalurus furcatus 2 Salvelinus alpinus 1
Salmo salar 6
Salvelinus namaycush 3 Oncorhynchus kisutch 3
Salvelinus fontinalis 1 Salmonidae
Salmonidae
Salvelinus alpinus 1 Oncorhynchus tshawytscha 3
Oncorhynchus masou 1
Oncorhynchus mykiss 19 Oncorhynchus masou 1
Esox masquinongy 1
Esocidae
Esox lucius 2 Oncorhynchus mykiss 4
Sebastidae Sebastes schlegeli 2
Cyclopteridae Cyclopterus lumpus 1 Osphronemidae Betta splendens 2
Osphronemidae Betta splendens 5
C. nigrofasciatum 2 Cichlasoma nigrofasciatum 1
Tilapia zillii 1

 2011 Blackwell Publishing Ltd, F I S H and F I S H E R I E S , 14, 60–76

Oreochromis aureus 10 Pseudocrenilabrus multicolor 3
Cichlidae O. mossambicus x O. niloticus 1 Cichlidae
Oreochromis mossambicus 11 Oreochromis aureus 1
Oreochromis niloticus 22
Oreochromis spilurus 2 Oreochromis niloticus 7
Perca flavescens 2
Percidae
Sander lucioperca 1 Pomoxis nigromaculatus 3
Serranidae Epinephelus malabaricus 1 Centrarchidae
Pomoxis nigromaculatus 2 Lepomis macrochirus 1
Centrarchidae Micropterus salmonidae 4
Lepomis macrochirus 2 Moronidae Morone chrysops x Morone saxatilis 2
Moronidae Dicentrarchus labrax 10
Siganidae Siganus guttatus 1 Paralichthydae Paralichthys olivaceus 1
Paralichthydae Paralichthys olivaceus 9
Xiphophorus helleri 3 Poeciliidae Poecilia reticulata 4
Poeciliidae Poecilia sphenops 2
Poecilia reticulata 3 Adrianichthyidae Oryzias latipes 4

–2 0 4 –6 0 22
Estimate of Inducibility (I) Estimate of Inducibility (I)

Figure 2 Forest plots of mode inducibility (I) per species (filled circles, random effects) and the meta-analytic mean I across all species (filled squares, fixed effect). (a) The dietary
administration dataset as given by ModelD0. (b) The bath administration dataset as given by ModelB0. Horizontal bars for each mode represent 95% credible intervals (CI). The number of
experiments that estimate is based on (N) and the phylogeny included in the analysis of that dataset are presented alongside each estimate.

65
A comparative study of sex reversal A M Senior and S Nakagawa
A comparative study of sex reversal A M Senior and S Nakagawa
route of administration (n = 172) and those that
use a bath treatment (n = 50). These two data sets
were analysed separately. Bath dosages (mL L)1 of
water) and dietary dosages (mg kg)1 of food) are
not directly comparable as the ratio of dosage units
is non-commensurate i.e. mg kg)1 and mL L)1 are
not equivalent. That is, the interval between values
of the explanatory variable over which our initial
GLMs were fitted is much larger for bath treatments
and thus we would expect a greater estimate of I
from bath treatments.
We also collected the following parameters asso-
ciated with each experiment: (i) the article from
which that experiment originated, (ii) the species, (iii)
the type of chemical used, (iv) the direction of
induced change, (v) whether the exposed population
was of mixed or single genotypic sex, (vi) the length of
exposure (number of days), (vii) the age at which fish
received their first treatment and (viii) the frequency
with which fish were exposed to hormones.
Coding estimates of I for PMMs
Each estimate of I was analysed as a single data
point in our PMMs. Because we fitted number of
males relative to females as the response variable, in
our initial analysis, studies that induced feminiza-
tion produced negative slopes. Therefore, all slopes
associated with a feminization study were multiplied
by negative one, making the slopes for masculini-
zation and those of feminization comparable.
Coding explanatory variables for PMMs
Each estimate of I in our final analysis was
associated with eight explanatory variables (or
predictors), which were fitted as fixed or random
factors in our analyses. Those variables that were
fitted as random categorical factors were: (i) the
article from which the experiment originated (to
control for multiple experiments from within the
same article) and (ii) the species. The fixed factors
that we fitted were: (i) the type of chemical used,
coded as hormone, inhibitor or other, (ii) the
direction of change, (iii) whether the exposed
population was mixed or mono-sex, (iv) the number
of days fish were exposed to chemicals for, (v) the
age at which fish received their first treatment and
(vi) the frequency with which fish were exposed to
chemicals.
Fixed variables 1, 2 and 3 were fitted as categor-
ical and variables 4, 5 and 6 were fitted as
66
continuous fixed effects. Variable 6 was coded as
the number of exposures per day i.e. thrice
daily = 3, daily = 1, every 2 days = 0.5, weekly =
0.14 and so forth. In some instances, authors did
not state the frequency of exposures; in such cases,
we assumed that administration frequency was
daily. The age of fish at the beginning of treatment
was reported as both categorical (e.g. juvenile) and
continuous (14 days post-hatching) forms in the
studies in our data set. Therefore, we initially chose
to record all ages in categorical form (egg, sac-fry,
swim-up fry, fry, juvenile and adult) and then
convert them to a continuous variable with four
values, where 0 = egg (or gravid female for live
bearing species), 0.33 = fry (of any kind),
0.66 = juvenile and 0.99 = adult.
Phylogeny construction
Pagel’s k is an estimate of the amount of variation
within a trait, which can be accounted for by the
shared evolutionary history of species in an analysis
(Pagel 1999) and hence is the estimate we use in our
comparative study. To estimate Pagel’s k, we
constructed two phylogenies of the 52 species left
in our analysis, one for the dietary dataset (see
Fig. 2a) and one for the bath dataset (see Fig. 2b).
These phylogenies were produced by combining
published phylogenies, to work out the taxonomic
relationships across all of the species in our analysis.
We obtained studies, which had phylogenetic rela-
tionships of species at a genus level and used these
papers to construct species level relationships (Lyde-
ard et al. 1995; Aoyama et al. 2001; Ruber et al.
2004; Takehana et al. 2005; Mank and Avise
2006a; Borsa et al. 2007; Koedprang et al. 2007).
The relationships among genus were then con-
structed using studies containing the information on
family-level relationships (Birstein and DeSalle
1998; Crespi and Fulton 2004; Near et al. 2004,
2005; Orrell and Carpenter 2004; Sloss et al. 2004;
Sparks and Smith 2004; Mank and Avise 2006b;
Craig and Hastings 2007; Ku et al. 2007; He et al.
2011). Finally, once we had constructed trees at
these lower taxonomic order, we grafted together
our family-level trees in Newick format using the
topology from papers, which specify the relation-
ships between teleost families at the order level
(Dettai and Lecointre 2005; Mank et al. 2005; Mank
and Avise 2006a; Saitoh et al. 2006; Sullivan et al.
2006; Chen et al. 2007a,b; Fraga et al. 2007;
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 A comparative study of sex reversal A M Senior and S Nakagawa

(a) (b)
40 40

30 30
Precision (1/SE)

Precision (1/SE)
20 20

10 10

0 0
–60 –40 –20 0 20 40 60 –60 –40 –20 0 20 40 60
Estimate of inducibility (I) via diet Estimate of inducibility (I) via bath

Figure 3 Funnel plots of the estimates of inducibility (I), (given by our initial re-analysis of published experiments
using a binomial GLM) plotted against the precision of that estimate (calculated as 1/SE given by a binomial GLM).
(a) Estimates of I from dietary treatments. (b) Estimates of I from bath treatments. Although these funnel plots appear to
display publication bias, they cannot be interpreted in a conventional manner (see Results for an explanation.

Jondeung et al. 2007; Holcroft and Wiley 2008; Li and a thinning interval of 1000, which gave us
et al. 2008; Mayden et al. 2009). 1000 estimates from each MCMC chain. An inverse
gamma prior for random effects and residuals was
specified for all of our analyses (in MCMCglmm,
Collating and analysing estimates of inducibility
V = 1, nu = 0.002 with V being variance and nu
Our 222 estimates of I were analysed using the R the degree of belief parameter in an inverse Wishart
function MCMCglmm in the package MCMCglmm distribution; Hadfield 2010). We ran three indepen-
(Hadfield 2010). We carried out phylogenetic dent chains for each model. Of the three chains, we
mixed-effects meta-regression (PMMs; Hadfield and present parameter estimates, from that which pro-
Nakagawa 2010) by fitting estimates of I (model duced the lowest DIC.
slopes from binomial GLM) as the response variable To test our model and parameter convergence,
and exploring our eight explanatory variables, to we ran model diagnostics on all PMMs. We began
see which best explain the variation in I estimates by checking for autocorrelation between subse-
observed between experiments. In our PMMs, each quent samples within each MCMC chain for both
estimate of I was weighted based on its precision random and fixed effects. We found that autocor-
(measurement error variance, which was the square relation between subsequent lags was consistently
of SE procured by the GLMs). low (<0.1), suggesting that our MCMC chains
Our initial intercept models (ModelD0 for dietary mixed well. To check the consistency of estimates
data and ModelB0 for bath data) consisted of three produced by our models, we also used Gelman-
random factors alone (article of origin, test species Ruben diagnostics on the three independent
and phylogeny; see Table 1). We then ran a full model MCMC chains run for each model and found
for each data set (ModelDFULL and ModelBFULL), which that results were consistent (point estimates of
contained all of our recorded fixed effects alongside the potential scale reduction factor < 1.1), sug-
the three random effects in the intercept models. gesting that our three independent chains had
Finally, we explored models with combinations of converged.
those fixed effects to find the model favored by
deviance information criterion (DIC); models with
Calculating measurement error, variance
the lowest DIC are preferred (Lunn et al. 2000).
components and phylogenetic heritability
Interactions between fixed effects were not explored
owing to the number of variables present in the In our PMMs, a weight was assigned to each
dataset. estimate of I in the form of sampling (measurement)
We ran all PMMs for fifteen million iterations, error variance. Sampling error variance was calcu-
with a burn-in period of fourteen million iterations lated by squaring the SE of each estimate of I, which

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A comparative study of sex reversal A M Senior and S Nakagawa
was produced by the initial modelling of published
results.
The proportion of variation explained by various
model components (see Table 1), was calculated as
follows. We began by calculating the total amount
of variance in each dataset, then subtracted the
percentage accounted for by sampling error, fixed
effects and partitioned the remaining variation
among the random effects and the residual variation
as estimated by each model (see Table A2).
Pagel’s k is an estimate of the degree of phylo-
genetic signal present within a given trait (Pagel
1999). Pagel’s k, which is equivalent to phyloge-
netic heritability H2 (Lynch 1991), was calculated
as given by Equation (1), where r2Phylogeny is the
model estimate for the phylogeny, r2Species is the
model estimate for species ID, r2Article is the model
estimate for the Article ID and r2Residual is the model
estimate for residual variance.
r2Phylogeny
Pagel’s; k ¼
r2Phylogeny þ rSpecies þ r2Article
2 þ r2Residual
ð1Þ
Results
Variance components and phylogenetic analyses
Our initial reanalysis of estimates of I using bino-
mial GLMs showed considerable variation among
the published data from both dietary and bath
datasets, some of which were associated with a large
SE (Fig. 3). If conventionally interoperated, the
funnel plots (Fig. 3) appear to show a sign of
publication bias (i.e. asymmetries in funnels; Egger
et al. 1997). Because these plots display model
slopes (I) estimates (rather than conventional effect
sizes), we expect these values to be positive. Initially,
negative slopes resulted from feminization experi-
ments where the exposed population became
female-skewed. However, we multiplied these values
by negative one to make them comparable to
masculinization experiments. In this instance, neg-
ative slopes (i.e., I) can be interpreted as an
incidence of ‘counterintuitive sex change’. For
example, an androgen (e.g., testosterone) is applied,
but the exposed population becomes female-biased.
We began by constructing an intercept model for
each dataset (diet data = ModelD0 and bath
data = ModelB0), which fitted article identity, spe-
cies identity and our phylogeny as random effects
(see Table 1). If a relationship between taxonomic
68
family and I exists, then species ID and/or phylog-
eny would account for a large proportion of the
variation in estimates of I. That is, we would detect
a phylogenetic signal. However, both intercept
models suggest that species and phylogeny account
for <1% of the variation in estimates of I respec-
tively, hence little phylogenetic signal exists (Pagel’s
k: dietary data < 0.001 and bath data < 0.001;
Pagel 1999; see Table 1). Therefore, there appears
to be little or no difference in the inducibility of the
different teleost species in our analyses (see Fig. 2).
Rather than biological differences accounting for
variance in I, it appears that sampling error
variance accounts for most of the variation in the
published literature (see Table 1). Thus, the varia-
tion in I that we report is primarily driven by
differences in experimental design between studies
(i.e., variation in sample size and the number of
different dosages (e.g. 0, 0.1 or 0, 0.1, 1) experi-
mentally tested).
Model estimates
Our intercept model (ModelD0) suggests that, on
average, fish have an inducibility of 1.741 via
dietary treatments (ModelD0 I: mode = 1.741,
mean = 1.706, 95% credible interval (CI) =
1.199–2.129, see Fig. 2). This estimate means that,
on average, if we expose a mixed-sex population
(1:1 males/females) to an increase in dosage of an
N
Mono Sex 32
Mixed Sex 140
Contrasts
(Mono - Mixed)
0 6
Estimate of Inducibility (I)
Figure 4 The mode estimates of inducibility (I) and 95%
credible interval (CI) of mono and mixed genotypic sex
groups of fish based on our dietary dataset, estimated
from the DIC favoured model (ModelDDIC), alongside the
number of experiments that the estimate is based on.
The contrast (pair-wise comparison) between estimates is
also presented.
 2011 Blackwell Publishing Ltd, F I S H and F I S H E R I E S , 14, 60–76
 A comparative study of sex reversal A M Senior and S Nakagawa

Table 1 The proportion of total variance (r2) in estimates of I explained by fixed effects (r2FE ), random effects (r2Article ,
r2Species , and r2Phylogeny ) and sampling error variance (r2SE ) explained by our null, DIC favored and full models.

Fixed effects
Data set Model name in model r2FE (%) r2Article (%) r2Species (%) r2Phylogeny (%) r2SE (%) r2Residual (%) DIC

Diet ModelD0 NA NA 0.024 0.004 0.007 87.74 12.16 738.7

ModelDDIC Genotypic sex 0.581 0.013 0.007 0.008 87.74 11.60 721.7
ModelDFULL All 0.229 0.014 0.009 0.009 87.74 11.95 735.7

Bath ModelB0 NA NA 0.000 0.031 0.000 35.05 64.92 353.9

ModelBFULL All 8.156 0.138 0.000 0.000 35.05 56.62 339.1

ESR-inducing chemical from 0 mg kg)1 of food to
9 mg kg)1 of food, approximately 35% of the
individuals will reverse sex. Our intercept model
for bath data (ModelB0) suggests that via bath
treatments fish have an inducibility of 6.231
(ModelB0 I: mode = 6.231 mean = 7.258, 95%
CI = 1.906–12.896, see Fig. 2). This estimate can
be interpreted as on average, if we expose a mixed-
sex population (1:1 males/females) to an increase in
dosage of an ESR-inducing chemical from a con-
centration of 0 mL L)1 of water to 9 mL L)1 of
water, nearly 50% of individuals will reverse sex; i.e.
complete sex reversal (although it should be noted
that, owing to the smaller sample size of the bath
data set, there is a large credible interval surround-
ing this estimate).
With regard to the methods used to induce ESR,
for the dataset containing dietary treatments, DIC
favours a model that only contains the genotypic
sex of the exposed population as a fixed factor and
the three random effects in the intercept model
(ModelDDIC, see Table 1). The ModelDDIC suggests
that significantly more individuals switch sex in
populations of a single genotypic sex than in
populations of mixed genotypic sex, for the same
increase in the dosage of sex-reversing chemical
(see Fig. 4; ModelDDIC [MONOSEX – MIXEDSEX] I: mean =
1.692 mode = 1.567, 95% CI = 0.86–2.579, see
Table A1). Other than the difference between mixed
and mono-genotypic sex treatment groups, our full
model for the dietary dataset (ModelDFULL) did not
detect any significant differences between any
moderator variables (see Table A1).
For the dataset containing estimates of inducibil-
ity (I) via bath treatments, DIC favours the full
model that fits all fixed effects plus the three random
effects in the intercept models (ModelBFULL, see
Table 1). However, none of the fixed effects we
investigated in the bath dataset are statistically
significant. This lack of statistical significance is
owing to large CIs surrounding our estimates,
which are probably a result of the limited sample
size in the bath dataset (See Table A1).
Discussion
We set out to quantitatively test the hypothesis
proposed by Pandian and Sheela (1995) that
susceptibility to induced sex reversal by exposure
to exogenous hormones (inducibility, I) is a taxo-
nomically constrained trait within teleosts. We
combined estimates of inducibility across species,
from the published literature with a phylogenetic
tree by using phylogenetically controlled mixed-
effects meta-regressions (PMMs). Contrary to what
Pandian and Sheela (1995) proposed, our results
suggest that inducibility is a relatively uniform trait
among those taxa included in our analyses at all
taxonomic levels (Fig. 2).
Phylogenetic insights
Our results suggest that once variation in sample
size and within-study methodology has been ac-
counted for, there is little evidence for phylogenetic
signal in the inducibility of those species included in
our analyses (see Table 1). Recently, a similar
analysis was published by Guénard et al. (2011),
which looked at the relationship between phylogeny
and susceptibility of species to toxic chemicals. In
stark contrasts to what our study finds, Guénard
et al. (2011) show that strong phylogenetic inertia
is present in their dataset, with regard to suscepti-
bility to toxins. However, it is not surprising that
Guénard et al. (2011) detected phylogenetic signal
because their analysis looked at species from a wider
taxonomic background than our study (e.g. their

 2011 Blackwell Publishing Ltd, F I S H and F I S H E R I E S , 14, 60–76 69

A comparative study of sex reversal A M Senior and S Nakagawa
analysis includes amphibians and crustaceans
alongside fish). When species from very high-level
taxonomic groups are included in comparative
analysis, phylogenetic signal is very likely to be
detected because of the clear biological differences
between the different groups, even though there is
very little variation within those groups. For exam-
ple, including amphibian species in the current
analyses would have resulted in detecting strong
phylogenetic signal due to differences between the
inducibility of amphibians and fish, rather than
differences between the inducibility of fish groups.
Given that we did not detect any phylogenetic
signal in our dataset, the question becomes ‘why
not?’ There are two potential reasons: (i) it is
possible that phylogenetic signal does exist in the
dataset, but our statistical procedures did not detect
it owing to inaccuracy or poor sensitivity or
(ii) phylogenetic signal does not exist in susceptibil-
ity to induced ESR in teleosts in general. With
regard to the first point, observing a number of
expected results indicates to us that our analytical
procedures were probably sound. Firstly, our anal-
ysis (PMMs) detected the difference in I between
populations of a single genotypic sex vs. those of
mixed genotypic sex (see Fig. 4). Populations of
mixed genotypic sexes have (on average) an equal
sex ratio (1:1) at dosage zero (e.g. Komatsu et al.
2006), meaning the intercept on the natural scale is
0.5, whereas those of single genotypic sex have only
one sex at dosage zero (1:0) (e.g. Kang et al. 2008),
meaning the intercept on the natural scale is 0.
Hence, the slopes produced by our initial binomial
models should be shallower when based on data
from mixed-sex populations, than mono-sex popu-
lations (see Box). Secondly, the same can be said for
studies that induce sex reversal using bath treat-
ments as opposed to those that use dietary treat-
ments. Realistically, bath treatments expose fish to
much higher dosages of sex-reversing chemical
than dietary treatments (see Methods section
above). Therefore, the estimate of I produced from
the bath dataset should be larger than that from the
diet dataset, and we show that it is (see Fig. 2).
Given that our procedures detected these
expected differences between ESR-inducing tech-
niques, we suggest that our methodology would
have detected any phylogenetic signal if at all
present. Hence, it seems unlikely that phylogenetic
signal in I exists in the current dataset. Phylogenetic
signal will typically not be detected under two
circumstances: (i) when there are between-species
70
effects present independent of phylogeny (i.e.
between species, I varies highly regardless of phy-
logenetic relationship) or (ii) when there is little
variation among species (Losos 2011). There is a
possibility that our analysis suffers from the problem
of publication bias (Rothstein et al. 2005), which
results in between-species level effects being over-
looked. That is, our dataset may not include species
where ESR is difficult or even impossible because
such results are unlikely to be published or high
mortality causes the sample size of such studies to
fall below our cut-off (see Methods section above).
In response to this argument, we would like to
make three comments. Firstly, we acknowledge that
it is possible to find examples where sex reversal is
impossible or difficult [e.g. masculinization in the
black molly (Poecilia sphenops, Poeciliidae) (George
and Pandian 1998)].
However, given the relatively uniform estimates
of I that our analyses report, there is no reason to
believe that such between-species level effects are
widespread. Secondly, even if between-species
effects are present, it is still unlikely that they are
related to higher-level phylogenetic effects (e.g.
family-level effects) because if this was the case,
our analysis would have detected some phylogenetic
signal. Finally, we have sufficient evidence to assess
the theory proposed by Pandian and Sheela (1995),
who suggest the relationships between the relative
inducibility of six teleost families from four orders.
Of those six families, we have representatives from
four (and all orders), including the families that
Pandian and Sheela (1995) suggest are the most
inducible (Cichlidae) and the least inducible (Cyp-
rinidae), and we find no differences between the
inducibility of these families.
In all, Teleostei totals forty orders (Helfman et al.
2009) of which we have representatives from
eleven, and we would argue that representation of
over one quarter of teleost orders is reasonable.
Further, we believe that our analysis is as thorough
an analysis as is possible given the data currently
available. Based on our results and the reasonable
representation of numerous taxa, we believe it is
most likely that little variation in I exists between
teleosts. If this was the case, why would it have been
suggested that such taxonomic differences are
present (Pandian and Sheela 1995)? Our analysis
confirms that considerable variability in estimates of
I within the published literature exists, and it seems
likely that phylogenetic signal has been inferred
from this observed variation. We believe that this
 2011 Blackwell Publishing Ltd, F I S H and F I S H E R I E S , 14, 60–76
 A comparative study of sex reversal A M Senior and S Nakagawa
inference was made because, when variation is
observed in numerous studies across taxa, biological
variability is probably the most ‘satisfying’ explana-
tion available. In other words, biologists tend to
ascribe observed variation to biological rather than
statistical phenomena.
However, in the case of ESR, it seems more likely
that sampling errors (i.e. variation owing to limited
sample size and study designs) drive a large portion
of variation in published estimates of inducibility.
A further possibility is that methodological differ-
ences account for the variation in published data.
While we were able to control for many of the
differences between methodologies (e.g. type of
chemical used), it was not possible to account for
unstated aspects of the methods researchers used.
For example, some studies do not directly report the
quantity of food experimental fish are fed, reporting
that fish were fed ad libitum or to satiation (Arslan
and Phelps 2004), whereas other studies report
feeding fish at a fixed percentage of body weight
(Fujioka 2002). This inconsistency would have a
particularly strong bearing on the estimates of I
reported by the large number of studies that
administer hormones via dietary supplementation.
Wider evolutionary insights
Comparative analyses such as the current study can
provide accurate characterizations of general bio-
logical and taxonomic trends (Losos 2011). Mank
et al. (2006) used comparative methods to demon-
strate the wide diversity of sex-determining mech-
anisms in fish. In particular, Mank et al. (2006)
have suggested that the source of this diversity is
switched between somatic mechanisms of sex
determination (i.e. male heterogamety, XY, and
female heterogamety, ZW), which occur fairly
readily over evolutionary time. Further, it has been
suggested that sex reversal events, which lead to
individuals in the population that bear an opposing
sexual genotype and phenotype, can drive the
evolution of a change in sex-determining mecha-
nism (Mank and Avise 2006a; Grossen et al. 2011).
Assuming the absence of widespread between-spe-
cies level effects, our results suggest that the
mechanisms of sex determination in teleosts are
relatively plastic and open to environmental inter-
ferences, which may ultimately lead to sex reversals.
We propose that their potential ubiquity explains
the diversity of sex-determining mechanisms oper-
ating in teleosts.
 2011 Blackwell Publishing Ltd, F I S H and F I S H E R I E S , 14, 60–76
Not only is it possible that sex reversal events
could induce a transition between male and female
heterogamety, but it has also been suggested that
sex-reversed individuals play a role in maintaining
the structure of sex chromosomes in their species
(Perrin 2009; Stelkens and Wedekind 2010; Wede-
kind 2010). Perrin (2009) hypothesized that sex
reversal would allow sex chromosomes to recom-
bine in the heterogametic sex (XY or ZW). When
recombinations of this type occur, they allow the
chromosome unique to that genotypic sex (Y or W)
to be purged of deleterious mutations (Perrin 2009).
Perrin (2009) argues that ultimately such events
lead to the homomorphic sex chromosomes present
in fish and amphibian species.
To date, there has been a large body of evidence
suggesting that recombination rates between sex
chromosomes are dependent on phenotypic sex
(Perrin 2009; Wedekind 2010; Matthias et al.
2011), the first requirement for Perrin’s (2009)
hypothesis. However, the prevalence of mass sex
reversal events in wild populations is largely
unknown (Wedekind 2010), the second require-
ment for Perrin’s (2009) hypothesis. Alho et al.
(2010) demonstrated that in the common frog
(Rana temporaria, Ranidae), sex reversals do occur in
wild populations of amphibians. Given how uni-
formly plastic the mechanisms of sex determination
appear to be in fish according to our results, we
would suggest that numerous species of fish also
have the potential to undergo regular sex reversal.
Over evolutionary time, such events are likely to be
common in populations of most teleosts, ultimately
playing a role in the maintenance of homomorphic
sex chromosomes in fish lineages.
The view that biologists hold of sex as a trait is
currently evolving and it is no longer accepted that
one of two distinct phenotypes (male or female) is
determined by a single factor. It is clear that even
when sex appears to be determined by one gene on
a sex chromosome, a complex interplay between
genome and environment determines sexual char-
acteristics (Mittwoch 2006). Ultimately, sex should
be viewed as a reaction norm like many other
phenotypic traits, where the resulting phenotype is
dependent on the reaction of genes to environment
(Ah-King and Nylin 2010). The concept of sex as a
reaction norm is not immediately obvious in some
taxa such as mammals, where sex chromosomes
play a very stable role in the sex-determining
process and are unperturbed by most environmental
factors (Ah-King and Nylin 2010).
71
A comparative study of sex reversal A M Senior and S Nakagawa
However, sex determination by a gene–environ-
ment interaction is obvious in fish, where sexual
development is plastic and environment plays a
strong role in determining sexual phenotype, as also
demonstrated by our analyses. We have not dis-
cussed the adaptive or evolutionary advantage that
plasticity in sex determination offers. Nonetheless,
given the apparent prevalence of plasticity in sexual
development among teleosts, it would seem that fish
are an ideal taxa in which to explore the evolution-
ary significance of plastic sex determination.
Ecological insights
Numerous studies have reported ESR in wild fish
populations as a result of exposure to effluents
containing EDCs (Hotchkiss et al. 2008). Such
effects that pollutants could have on fish population
dynamics were modelled by Hurley et al. (2004).
They found that in systems of male heterogametic
sex determination (XY), environmental masculini-
zation could lead to population collapse, with the
population becoming dependent on the masculiniz-
ing chemical to maintain phenotypic males.
In contrast, environmental feminization in male
heterogametic systems was not quite so perilous
(the converse is true for systems of female heterog-
ametic sex determination – i.e. ZW). In the light of
our results, researchers investigating the effects of
EDCs in aquatic ecosystems should not assume that
the effects, which they observe, are limited to just
one or two fish species. Given that we observe
uniform inducibility across all teleost families
(included in our analyses), if ecologists detect
chemically induced ESR in one species, it seems
likely that other fish species are also affected.
Acknowledgements
We would like to thank Jiahui Nat Lim for his help in
constructing our phylogeny, as well as Eduardo
Santos, Manna Warburton and two anonymous
reviewers for their comments and thoughts on this
research. We would also like to thank the University
of Otago, Zoology Department and the Marsden
Fund (UOO0812), New Zealand, for funding this
research. Additionally, we would like to thank Dunja
Lamatsch, Mark Lokman and Gerry Closs for helping
to write a funding proposal, without which none of
this research would have been possible.
72
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 A comparative study of sex reversal A M Senior and S Nakagawa

Appendix
Table A1 The modal estimates of inducibility (I) and 95% credible intervals (CI) of contrasts for factorial variables,
the modal estimates of I and 95% CI for numeric variables and the models that those estimates are calculated from.
Estimates with CIs that do not cross zero are considered statistically significant. Inhibitor chemical types were not
present in the bath data set.

Dietary data Bath data

Variable/contrast Mode I CI Model Mode I CI Model

Mono sex – mixed sex (genotypic sex) 1.567 0.860–2.579 ModelDDIC 3.406 )4.775–12.839 ModelBFULL
Hormone – inhibitor (chemical type) 0.451 )0.605–2.148 ModelDFULL NA NA NA
Hormone – ‘other’ (chemical type) 0.806 )2.418–4.912 ModelDFULL 3.592 )13.585–21.362 ModelBFULL
Inhibitor – ‘other’ (chemical type) 0.162 )3.806–4.080 ModelDFULL NA NA NA
Feminization – masculinization )0.883 )4.004–1.174 ModelDFULL 5.467 )0.698–10.691 ModelBFULL
(direction of sex change)
Age 0.188 )11.474–9.864 ModelDFULL 8.412 )15.966–24.475 ModelBFULL
Frequency 0.015 )4.098–13.382 ModelDFULL 2.164 )12.149–9.165 ModelBFULL
Length of treatment days 2.632 )16.264–24.342 ModelDFULL )0.078 )0.339–0.214 ModelBFULL

Table A2 The model mean and mode estimates of I for fixed and random effects ± 95% confidence intervals (CI), as
given by intercept, DIC favoured and Full PMMs for both dietary and bath data.

Fixed effects Mean I Mode I CI

ModelDDIC
Intercept (mono-genotypic sex groups) 3.121 2.960 2.224–3.953
Mixed genotypic sex 1.429 1.385 0.994–1.853

ModelDFULL
Intercept (hormone, mono-genotypic sex and 2.828 3.079 1.260–4.307
feminization studies)
Inhibitor 2.068 2.146 0.529–3.545
‘Other’ chemicals 1.706 1.333 )1.771–6.041
Masculinization (diff. between masculinization 0.127 0.176 )0.510–0.835
and feminization)
Mixed genotypic sex (diff. between mixed and )1.907 )1.800 )3.053–)0.967
mono-genotypic sex groups)
Length of treatment 0.002 0.003 )0.004–0.007
Age 0.598 0.460 )1.109–2.384
Frequency of exposure 0.057 )0.006 )0.126–0.259

ModelBFULL
Intercept (hormone, mono-genotypic sex and 15.52 17.82 1.503–29.09
feminization studies)
Other chemicals 12.66 9.53 )9.949–36.97
Masculinization (diff. between masculinization )5.278 )5.467 )10.69–0.698
and feminization)

 2011 Blackwell Publishing Ltd, F I S H and F I S H E R I E S , 14, 60–76 75

A comparative study of sex reversal A M Senior and S Nakagawa

Table A2 (Continued)

Fixed effects Mean I Mode I CI

Mixed genotypic sex (diff. between mixed )5.255 )3.406 )12.84–4.775

and mono-genotypic sex groups)
Length of treatment )0.056 )0.078 )0.339–0.214
Age 6.053 8.412 )15.97–24.48
Frequency of exposure )0.966 2.164 )12.15–9.165

Random effects

ModelD0
Article 0.309 0.008 3 · 10)03–1.83
Species 0.065 0.002 2 · 10)03–0.287
Phylogeny 0.076 0.004 1 · 10)03–0.367
Residual 3.445 3.357 2.063–4.471

ModelB0
Article 12.04 )0.04 2 · 10)03–53.60
Species 5.223 0.204 2 · 10)03–26.35
Phylogeny 17.22 )0.168 2 · 10)03–106.5
Residual 56.00 54.34 18.40–94.21

ModelDDIC
Article 0.178 0.006 3 · 10)03–0.698
Species 0.035 0.001 2 · 10)03–0.159
Phylogeny 0.046 0.002 3 · 10)03–0.216
Residual 3.160 3.411 1.997–4.214

ModelDFULL
Article 0.178 0.005 2 · 10)03–0.792
Species 0.040 0.002 2 · 10)03–0.170
Phylogeny 0.053 0.002 3 · 10)03–0.240
Residual 3.374 3.409 2.205–4.768

ModelBFULL
Article 37.40 0.057 3 · 10)03–98.95
Species 4.889 0.357 3 · 10)03–27.58
Phylogeny 9.854 )0.153 2 · 10)03–51.64
Residual 38.95 16.79 7.334–84.38

76  2011 Blackwell Publishing Ltd, F I S H and F I S H E R I E S , 14, 60–76

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