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Courtship Sounds Advertise Species Identity and Male
Quality in Sympatric
Pomatoschistus
spp. Gobies
Silvia S. Pedroso
1
, Iain Barber
2
, Ola Svensson
3
, Paulo J. Fonseca
4
, Maria Clara P. Amorim
1
*
1Unidade de Investigac¸a
˜o em Eco-Etologia, Instituto Superior de Psicologia Aplicada – Instituto Universita
´rio, Lisbon, Portugal, 2Department of Biology, College of
Medicine, Biological Sciences and Psychology, University of Leicester, Leicester, United Kingdom, 3Department Biology and Environmental Sciences, University of
Gothenburg, Gothenburg, Sweden, 4Departamento de Biologia Animal e Centro de Biologia Ambiental, Faculdade de Cie
ˆncias da Universidade de Lisboa, Lisbon,
Portugal
Abstract
Acoustic signals can encode crucial information about species identity and individual quality. We recorded and compared
male courtship drum sounds of the sand goby Pomatoschistus minutus and the painted goby P. pictus and examined if they
can function in species recognition within sympatric populations. We also examined which acoustic features are related to
male quality and the factors that affect female courtship in the sand goby, to determine whether vocalisations potentially
play a role in mate assessment. Drums produced by the painted goby showed significantly higher dominant frequencies,
higher sound pulse repetition rates and longer intervals between sounds than those of the sand goby. In the sand goby,
male quality was predicted by visual and acoustic courtship signals. Regression analyses showed that sound amplitude was
a good predictor of male length, whereas the duration of nest behaviour and active calling rate (i.e. excluding silent periods)
were good predictors of male condition factor and fat reserves respectively. In addition, the level of female courtship was
predicted by male nest behaviour. The results suggest that the frequency and temporal patterns of sounds can encode
species identity, whereas sound amplitude and calling activity reflects male size and fat reserves. Visual courtship duration
(nest-related behaviour) also seems relevant to mate choice, since it reflects male condition and is related to female
courtship. Our work suggests that acoustic communication can contribute to mate choice in the sand goby group, and
invites further study.
Citation: Pedroso SS, Barber I, Svensson O, Fonseca PJ, Amorim MCP (2013) Courtship Sounds Advertise Species Identity and Male Quality in Sympatric
Pomatoschistus spp. Gobies. PLoS ONE 8(6): e64620. doi:10.1371/journal.pone.0064620
Editor: A. Peter Klimley, University of California Davis, United States of America
Received December 4, 2012; Accepted April 16, 2013; Published June 5, 2013
Copyright: ß2013 Pedroso et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: SSP, PJF and MCPA acknowledge the financial support of the Portuguese Science and Technology Foundation (project PTDC/MAR/68868/2006,
pluriannual programs UI&D 331/94 and UI&D 329, grants SFRH/BPD/41489/2007) and of an ASSEMBLE grant (no. 227799). IB received financial support from the
Fisheries Society of the British Isles (a research grant) and from Banco Santander (a travel grant). OS was funded by the Centre for Marine Evolutionary Biology,
University of Gothenburg, Sweden. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: IB has received funding (a travel grant) from Banco Santander. There are no patents, products in development or marketed products to
declare. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.
* E-mail: amorim@ispa.pt
Introduction
Acoustic signals convey crucial information on species, sex and
individual identity as well as on individual motivation and quality
[1]. As such, acoustic communication can provide a pre-zygotic
isolating barrier and contribute to speciation in vocal taxa
including birds [2,3], anurans [4,5] and insects [6,7]. Variation
in acoustic signals related to individual quality such as size,
condition or other quality traits is also used in mate choice by
different animals [8,9,10].
Among teleost fish, there is growing recognition of the role
played by acoustic signals among the increasing number of
species known to use acoustic communication to gain access to
limited resources, such as food, territories or mates [11,12,13].
In comparison to tetrapods, fish have relatively simple central
and peripheral vocal mechanisms and thus typically lack the
ability to emit complex frequency-modulated calls [14]; for
exception see [15,16]. Consistent with this, comparative analyses
of the acoustic signals of fishes has revealed that fine-scale
temporal patterns – such as pulse number and pulse rate – are
capable of encoding species identity ([17,18,19,20,21], but see
[12]). In addition, calling rate, sound dominant frequency (the
frequency with most acoustic energy), and the duration of pulses
in a sound have been shown to advertise body size and
condition [22,23,24,25], while calling rate drives reproductive
success in at least one fish species [13].
The relative simplicity of fish sounds and the possible lack of the
confounding effects of learning [26,27], make fish potentially
useful models for studying the evolution of acoustic signalling, and
for examining the extent to which sounds convey information
relevant for species recognition and mate choice. Yet despite their
utility, such studies are scarce in the literature. In this study we first
aimed to compare acoustic mating signals in two congeneric,
sympatric marine gobies – the sand goby (Pomatoschistus minutus)
and the painted goby (P. pictus) – that exhibit similar life histories
and breeding ecologies that overlap both spatially and temporally.
Both species have a short life span, are polygamic and show
exclusive paternal care [28]. Also, males of both species use low-
frequency pulsed acoustic signals to lure the females into the nest
for spawning [29,30]. Second, we examined which acoustic
features are related to male length and condition in the sand
goby, which is an increasingly popular fish model in studies of
sexual selection [31].
PLOS ONE | www.plosone.org 1 June 2013 | Volume 8 | Issue 6 | e64620
Methods
Ethics Statement
All experimental and animal care procedures comply with
Swedish animal welfare laws, guidelines and policies and all efforts
were made to maximize animal welfare. We caught fish with hand
trawls from shallow bays near to the research station and
immediately sorted them by species and gender into stock tanks,
which were provided with a sand substrate and a continuous
supply of fresh surface sea water. The sand goby males that
produced sounds were euthanized with an excessive dose of
anaesthetics (MS222, tricaine methane sulphonate; Pharmaq,
Norway) and kept frozen (280uC) until lipid quantification.
Ethical permit 143–2011 from the Animal Ethics Committee of
Gothenburg covered all experiments procedures reported here
including fish collection.
Study Species
The sand goby Pomatoschistus minutus and the painted goby P.
pictus are small coastal species with a 1–2 years life span [28] that
often live in sympatry. They typically breed for one season only,
during which males and females spawn sequentially with different
mates [32]. Mating occurs in nests that males build by excavating
underneath bivalve shells that they also cover with sand. Females
are then attracted for spawning with both conspicuous visual and
acoustic signals [29,30], (Video S1, S2). Parental care is provided
solely by the male, who defends the nest from egg predators and
tends the eggs until hatching [32].
Breeding colouration and courtship differ slightly between these
two species. Most sand goby males present a black rimmed anal
fin, darkened tail, pelvic and dorsal fins, an iridescent blue band
inside the black edge of the anal fin and a blue and black spot on
the first dorsal fin [33]. Breeding painted goby males exhibit a
darkened chin, black rimmed pelvic, anal and caudal fins, and the
species-characteristic rows of dark spots of the first and second
dorsal fins become more conspicuous than in non-breeding
specimens. Most courtship interactions in the painted goby takes
place outside the nest where males approach the female, make
quiver displays, lead the female to the nest, nudge the female flank,
and perform rapid eight swimming manoeuvres; but males also
court the females from inside the nest with quivering displays [30].
Painted goby males produce two courtship acoustic signals: drums,
which consist of low-frequency pulsed sounds (i.e. trains of pulses;
Fig. 1a) associated with quivering displays and are made either
outside or inside the nest, and short low-frequency non-pulsed
thumps, which are associated with nest displays [30]. In the sand
goby, courtship outside the nest mainly consists of males briefly
approaching and displaying erected fins to the female before
attempting to lead her to the nest. Sand goby males tend to spend
longer periods at the nest, either remaining motionless with the
head protruding at the nest entrance (Fig. S1). or in active nest
displays, i.e. quivering and producing drum sounds [29]. Drums
(Fig. 1b) are the only sound type in the sand goby, usually made
from within the nest [29].
Study Design
The study was conducted in May-June at the Sven Love´n
Centre for Marine Sciences, at Fiskeba¨ckskil on the Swedish west
coast (58u159N, 11u289E). We caught fish with hand trawls from
shallow bays near to the research station and sorted them by
species and gender into stock tanks, which were provided with a
sand substrate and a continuous supply of fresh sea surface water.
The water fed to stock and experimental tanks was kept at a
constant temperature (16uC) by a heat exchanger device. Fish
were exposed to a natural photoperiod and fed daily ad libitum with
chopped blue mussels.
We ran experiments in 26 l (painted goby) and 35 l (sand goby)
aquaria divided in three compartments by transparent acrylic
partitions. We placed a male in each lateral compartment with a
shelter (Fig. 1c) and one gravid female centrally. We only used
males that presented breeding colouration and constructed nests
during an acclimatisation period, which lasted at least 24 h.
Approximately 15 min before recordings we stopped water
circulation and any noise source. We started trials by removing
one partition, allowing one male to interact with the female for
20 min, while continuously recording all acoustic (see below) and
visual (only in the sand goby, camcorder Sony DCR-SX65)
behaviours. The video signal and a synchronized audio signal
(derived from the audio recording chain) obtained in the sand
goby trials were digitized with Pinnacle Dazzle DVD Recorder
Plus (Pinnacle Systems, Mountain View, USA) to the laptop used
for audio recordings. We used males (and females) in a maximum
of two recording sessions to obtain enough sounds for analysis.
After each experiment we removed the subject male and replaced
the female. Males were weighed to the nearest 0.01 g (fresh
weight, W) and measured to the nearest mm (standard length, SL).
The sand goby males that produced sounds were euthanized with
an excessive dose of anaesthetics (MS222, tricaine methane
sulphonate; Pharmaq, Norway) and kept frozen (280uC) until
lipid quantification (see below). Silent sand goby males, all painted
goby males and all females were returned to their natural habitat
after trials.
Experimental aquaria used for the sand goby were insulated
from room floor born noise by two layers of rubber foam shock
absorbers located between the table and each of two wooden
Figure 1. Courtship drums and recording setup. Oscillograms of
courtship drums made by (a) the painted goby (Pomatoschistus pictus)
and (b) the sand goby (P. minutus). The long line indicates sound
duration whereas the short line depicts the interval between two
consecutive pulses. (c) Setup used for the sand goby recordings
illustrating the outer compartments occupied by territorial males and a
female in the central compartment and the position of the two
hydrophones. Trials began by removing one partition (illustrated by the
arrow) which allowed interaction between one male and a female.
doi:10.1371/journal.pone.0064620.g001
Species and Male Quality Encoded by Fish Sounds
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boards (1.5 cm thick). On top of these boards a styrofoam 4 cm
thick layer supported the tanks. Sand goby male sounds were
registered with a custom-made hydrophone [34] (frequency
response within 63 dB from 20 Hz to 1 kHz) and a High Tech
94 SSQ hydrophone (High Tech Inc., Gulfport, MS, USA;
sensitivity 2165 dB re1V/mPa; frequency response within 61dB
from 30 Hz to 6 kHz), both connected to an A/D converter
device (Edirol UA-25, Roland, Japan; 16 bit, 8 kHz) controlled by
a laptop through Adobe Audition 3.0 (Adobe Systems Inc.,
Mountain View, CA, USA), allowing simultaneous two-channel
recordings. The custom-made hydrophone was housed inside the
male’s nest chimney while the other was placed in the central
compartment. In the case of painted goby sound recordings,
experimental aquaria were placed on top of a 7 cm thick
styrofoam board and recordings were obtained only when external
noise was minimal. Sounds were registered with a ITC hydro-
phone (ITC 8073, ITC, Santa Barbara, USA; sensitivity 167 dB
re1V/mPa, frequency response within 61.5 dB from 20 Hz to
2 kHz), suspended just above the male’s nest, connected to a
Marantz PRC660 solid state recorder (Marantz, Eindhoven,
Netherlands).
We analysed drum acoustic features with Raven 1.2.1 for
Windows (Bioacoustics Research Program, Cornell Laboratory of
Ornithology, Ithaca, NY, USA) following the methodology of [21]
and [30]. We measured in both species sound duration (ms),
number of pulses, pulse repetition rate (number of pulses divided
for the sound duration, Hz), dominant frequency (the frequency
where the sound has maximum energy, measured from power
spectra: FFT size 8192 points, time overlap 60.0%, Hamming
window, Hz) and drum interval (peak-to-peak interval between the
last and the first pulses of two consecutive drums, ms). In the sand
goby only we also measured sound pressure level (SPL, calibrated
average RMS, dB re 1 mPa) from sounds registered in the nest
where the hydrophone was fixed approximately 2 cm away from
the male that keeps a rather stable position relative to the nest
entrance. Note that SPL measurements should be taken as
approximate values since not only recordings were carried out in
the limited water body of the aquarium but also, and more
importantly, any variation in the distance between the fish and the
hydrophone can affect these measurements. The ratio between the
average peak-to-peak interval of the 13
th
–16
th
and the 3
rd
–6
th
pulses was calculated to examine the existence of sound
production fatigue in the sand goby (i.e. the decrease in pulse
emission rate due to fatigue) and to check if the ability to sustain
the rate of sound pulse emission depends on fish condition/quality.
For the sand goby only, because sounds are emitted in rapid
sequences (bursts), we further quantified drum burst duration (the
duration of a sequence of drums, s; see [29] for details), burst
interval (time interval between drum bursts, s; see [29] for details),
as well as drum calling rate (drum min
21
), active calling rate
(considering only the min when fish vocalised, drum min
21
) and
calling effort (percentage of min spent calling). Drum calling rate,
active calling rate and calling effort were calculated for the total
20 min of only one trial (the one with most acoustic/visual
activity).
Twenty six painted goby males were tested for sound
production. Of these only 16 out of 23 vocal males produced
enough sounds to be included in the analysis. Vocal (analysed)
painted goby males averaged 40 mm (6SD, range: 62.8, 36–
45 mm) in SL and 0.82 g (60.15, 0.50–1.05 g) in W. We analysed
an average of 67638.3 (6–145) sounds for each painted goby
male.
Table 1. Dependent variables and predictor used in the linear regression models.
Dependent variables Predictors
male SL* or
Acoustic variables
male K condition factor or drum duration* (ms)
male K condition factor number of pulses*
pulse repetition rate (Hz)
dominant frequency (Hz)
sound pressure level (SPL, dB re 1 mPa)
sound production fatigue
drum interval (ms)
drum burst duration* (s)
burst interval* (s)
drum calling rate* (drum min2
1
)
active calling rate (drum min2
1
)
calling effort*
Visual behaviour variables
nest behaviour duration (nest quiver and rest)*
total no. of courtship events performed outside the nest*
Number of female courtship events* all acoustic variables (as above)
all visual behaviour variables (as above)
male SL (mm)
male K condition factor
male relative lipid content (g)
*Data were log
10
-transformed to meet the linear regression model assumptions. Abbreviations and units are shown between brackets.
doi:10.1371/journal.pone.0064620.t001
Species and Male Quality Encoded by Fish Sounds
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Thirty two sand goby males were tested from which 21
vocalised, and we analysed an average of 28625.0 (6–108) sounds
per vocal male. Vocal males averaged 44 mm in SL (63.2, 39–
49 mm) and 0.95 g (60.22, 0.60–1.40 g) in W. Non-vocal sand
goby males (N = 11) were similar sized to vocal males and
averaged 46 mm (6SD, range: 66.8, 38–45 mm) in SL and
1.14 g (60.58, 0.70–1.10 g) in W.
We analysed sand goby behaviour from videos using EthoLog
v.2.2 [35] and measured: duration of nest behaviour, i.e. resting
with the head protruding from the nest entrance and nest
quivering (s), total frequency of courtship outside the nest
including approach, quiver outside the nest and lead (see above).
We also quantified the latency for females to enter the male’s nest
(s) and the frequency of female courtship behaviour, a character-
istic bobbing movement exhibited by sexually receptive sand goby
females in front of the male also accompanied by blackening of the
eyes [33]. We only used one trial per male (as above, the one with
most acoustic/visual activity) for behaviour analysis and only
considered 10 min of video starting from the first male-female
interaction.
Lipid Quantification
We used both the condition factor (Fulton’s K, where K = W/
SL
3
) [36] and body lipid content as metrics of male condition.
Lipid content was measured in 21 sand goby males after [37]. In
short, defrosted males were desiccated at 60uC for 24 h and
weighed individually on a scale (Sartorius LE26P, Go¨ttingen,
Germany) to the nearest 0.001 mg. Lipids were extracted in
100 ml of petroleum ether (Sigma-Aldrich, St. Louis, USA) for
8 h. Body lipid content of each male was considered as the
difference in dry weight before and after lipid extraction, and
expressed as the lipid content relative to 100 g of fresh tissue.
Data Analysis
We compared drum duration, number of pulses per drum, pulse
repetition rate, dominant frequency and sound interval between
species with ttests. As multiple statistical tests can increase type I
errors (rejecting H
0
when H
0
is true) we chose an experimentwise
error rate higher than the usual 5% (i.e. of 1%) in order to reduce
the probability of type I errors while not compromising excessively
statistical power, i.e. while avoiding increased chances of
performing type II errors (not rejecting H
0
when H
0
is false) [13].
Drum interval was compared between species with a factorial
ANOVA using sound interval classes as a factor with 7 levels (5
classes of 100 ms below 0.5 s, plus 0.5–1 s and .1 s), and species
as a factor with two levels. We quantified the percentage of
occurrences in each sound interval class and square root arcsin-
transformed the data to meet the ANOVA assumptions, as
percentages typically follow a binomial distribution [38]. As
acoustic features were not related with male SL in either species
(Pearson correlation, P. pictus: N = 15, R = 20.32–0.10, P.0.05; P.
minutus: N = 21, R = 20.37–0.39, P.0.05) we did not control
comparisons between species for male size.
We used multiple linear regression analyses (Table 1) to
determine if acoustic and visual behavioural variables were good
predictors of male quality using a stepwise selection procedure
(P#0.05 to add and P$0.10 to remove). We used male SL, K
condition factor and relative lipid content as dependent variables
in three regression models. We included as possible predictors the
male mean values for both acoustic and visual behaviour
parameters. We considered all acoustic parameters plus nest
behaviour duration (nest quiver and rest) and total number of
courtship events performed outside the nest. We used log
10
-
transformation [38] to normalise male SL and the predictors drum
duration, number of pulses, sound burst duration, burst interval,
drum rate, calling effort, nest behaviour and courtship outside the
nest.
We further tested which factors influenced female courtship
behaviour, i.e. female courtship and latency to enter the nest, using
linear regression analysis (Table 1). As female courtship was
correlated with latency to enter the nest (Pearson correlation,
R = 0.59, P= 0.005) we only carried out this analysis using female
courtship as a dependent variable (log
10
-transformed). We used as
predictors all the acoustic and visual behaviour variables as above
and male quality features, SL, K and lipids. We further correlated
female courtship with female SL and K condition factor (measured
before trials) to ascertain if female size and K (a proxy for
roundness) are related to female courtship behaviour.
All final regression models complied with all assumptions of
multiple linear regression. All model residuals were normally
distributed. We assessed autocorrelation of residuals and multi-
collinearity of predictors with Durbin–Watson statistics and with
the variance inflation factor (VIF). We performed further residual
analysis by visually inspecting residual plots.
Figure 2. Comparison of courtship drums produced by males
of
Pomatoschistus minutus
and
P. pictus
.Symbols show means for
pulse repetition rate and dominant frequency and error bars represent
61 standard deviation.
doi:10.1371/journal.pone.0064620.g002
Species and Male Quality Encoded by Fish Sounds
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We conducted statistical analyses with SPSS for Windows (20.0,
SPSS Inc., New York, USA) and Statistica (10, Statsoft Inc., Tulsa,
USA).
Results
Differences between Species
Both sand and painted gobies made low frequency drums (main
frequencies below 300 Hz) lasting less than 1 s and composed of c.
24 pulses (Fig. 1; Table 2). Drums did not differ significantly
between species in sound duration (ttest, t= 1.75, df = 28.8,
P.0.05) or number of pulses (t= 0.43, df = 35, P.0.05) (Table 2).
Both pulse repetition rate and dominant frequency were signifi-
cantly higher in the painted than in the sand goby (pulse repetition
rate: t=23.92, df = 35, P,0.001; dominant frequency:
t=212.26, df = 35, P,0.001; Fig. 2). A comparison of sound
intervals between species using sound interval class and the species
as factors showed that, although there were no significant
differences between species in the mean sound interval, the sand
goby produced sounds with intervals between 0.1 and 0.3 s
significantly more frequently than the painted goby whereas the
painted goby drummed with intervals larger than 0.5 s more often
than the sand goby (ANOVA, interaction F
5,210
= 1.33, P,0.001;
sound interval class F
5,210
= 0.80, P,0.001; species F
1,210
= 0.11,
P.0.05; Fig. 3).
Predictors of Size and Condition in the Sand Goby
Vocal males ranged in size between 39–49 mm SL (mean 6
SD = 43.663.2 mm, cv = 0.07), in condition factor between 0.9–
1.4 (1.160.15, cv = 0.13) and showed a large individual variability
in relative lipid content: 1.1–3.9 g (2.360.75 g, cv = 0.32).
Likewise, males varied extensively in their levels of acoustic
(Table 2) and visual courtship activity. Calling rate varied between
0.2–4.3 drum min
21
(1.761.36 drum min
21
, cv = 0.79) and active
calling rate, that accounted only for the time spent calling, ranged
between 1.9–15.5 drum min
21
(7.463.64 drum min
21
, cv = 0.49).
Nest behaviour (quiver and rest) lasted from 0–533.7 s
(131.36173.7 s, cv = 1.32) whereas males performed between 0–
11 courtship displays outside the nest (4.563.37, cv = 0.75). All but
one vocal male (i.e. 20 males) succeeded in attracting the female
into the nest and only 3 vocal males did not receive any eggs.
Silent males were not successful in obtaining eggs in the nest.
The best regression model showed that only drum SPL was a
good predictor of male length with louder males having a higher
SL. Drum SPL accounted for 36% of the variation in body length
(Table 3; Fig. 4). Male condition variables were significantly
predicted by male visual and acoustic behaviour, with males
exhibiting shorter periods of nest behaviour and higher calling
rates having a higher K condition factor and relative higher lipid
Table 2. Acoustic features of drums produced during courtship by males of Pomatoschistus pictus and P. minutus.
P. pictus P. minutus
Acoustic parameters Mean SD Range Range
abs
Mean SD Range Range
abs
Drum duration (ms) 628.8 171.8 358.0–1118.8 29–4288 797.2 395.4 387.4–1914.1 108–8044
No. of pulses 23.3 6.5 11.2–38.0 2–139 24.8 12.1 12.2–62.7 3–229
Pulse repetition rate (Hz) 38.4 6.7 27.2–52.1 19.5–64.7 31.5 3.8 25.0–39.3 15.2–47.6
Dominant frequency (Hz) 229.8 18.0 194.2–255.0 144–266 151.9 19.6 126.7–187.9 94.7–236
Drum interval (s) 2.27 0.74 1.24–3.29 – 0.95 0.69 0.16–2.90 0.06–0.63
Number of sounds in a burst – – – – 3.5 1.58 1.1–7.0 1–12
Drum burst duration (s) – – – – 2.5 0.98 0.98–4.85 0.14–9.75
Burst interval (s) – – – – 81.3 71.14 5.8–289.4 0.5–867.3
Drum calling rate
(drum min
21
)
– – – – 1.7 1.36 0.2–4.3 –
Active calling rate
(drum min
21
)
– – – – 7.4 3.64 1.9–15.5 –
Calling effort – – – – 16.2 10.12 6.9–42.9 –
Sound pressure level
(dB re 1 mPa)
– – – – 90.7 5.11 83.8–98.1 78.5–137.3
Sound production fatigue – – – – 1.1 0.09 0.9–1.3 0.8–1.9
Descriptive statistics is based on male means except for absolute range values (range
abs
) that concern all data (P. pictus: 1073 drums and 884 sound intervals from 16
males; P. minutus: 580 drums and 594 sound intervals from 21 males). Sound intervals only concern interval up to 10 s.
doi:10.1371/journal.pone.0064620.t002
Figure 3. Frequency of occurrence of time intervals between
consecutive drums in
P. minutus
and
P. pictus
.Drum intervals were
square root arcsin-transformed. Note that in the shaded area broader
interval classes than the ones represented on the left area of the graph
are shown. Symbols show means and error bars depict 95% confidence
intervals.
doi:10.1371/journal.pone.0064620.g003
Species and Male Quality Encoded by Fish Sounds
PLOS ONE | www.plosone.org 5 June 2013 | Volume 8 | Issue 6 | e64620
levels respectively. Variation in nest behaviour (quiver display and
rest) significantly explained 29% of the observed variability in K
condition factor and active calling rate significantly explained 38%
of lipid content variability (Table 3; Fig. 4). Our model predicts
that males differing in active calling rate by 10 drums min
21
(e.g.
12 vs. 2 drum min
21
) would indicate a difference of 1.2 g in their
relative lipid content.
Factors Influencing Female sand Goby Courtship
Behaviour
Females took between 13.1–600 s (165.66138.4 s, cv = 0.84) to
enter the males’ nest and one female did not enter the nest
(latency = 600 s) although she courted the male four times and the
male performed nest behaviour for 421 s from which 55% was
spent actively courting the female, i.e. quivering. Fourteen out of
21 females courted the males. Female courtship events ranged
from 0–14 (2.863.8, cv = 1.36), with 86% of the variation in the
number of female courtship events being explained by male nest
behaviour (quiver and rest) and vocal activity. Nest behaviour
Figure 4. Relation between visual and acoustic predictor variables and male quality parameters in
P. minutus
.Predictors included in
the final linear regression models were male nest behaviour duration, sound pressure level (SPL) and active drum rate (drums min
21
). Regression lines
and 95% confidence interval bands are shown.
doi:10.1371/journal.pone.0064620.g004
Table 3. Table for predictors of male sand goby P. minutus standard length (SL) and condition (K condition factor and body lipid
content).
Dependent
variable
Included
predictor
B
S.E.M.
tPrF
Model
significance R
2
DW VIF
SL Intercept 0.31 0.11 2.92 0.009
SPL 0.004 0.01 3.16 0.005 0.60 F
1,19
= 10.00 P= 0.005 0.36 2.75 1.0
K Intercept 1.25 0.05 24.00 ,0.001
Nest 20.08 0.03 22.70 0.015 20.54 F
1,19
= 7.31 P= 0.015 0.29 2.2 1.0
Lipid Intercept 1.37 0.29 4.75 ,0.001
Active drum rate 0.18 0.04 3.34 0.004 0.62 F
1,19
= 11.17 P= 0.004 0.38 1.9 1.0
Nest behaviour duration (quiver and rest) and SL were log
10
-transformed to meet the linear regression model assumptions. r – partial correlation between the
dependent variable and the predictor, controlling for the effects of the other predictors in the model. DW - Durbin Watson statistics.
doi:10.1371/journal.pone.0064620.t003
Species and Male Quality Encoded by Fish Sounds
PLOS ONE | www.plosone.org 6 June 2013 | Volume 8 | Issue 6 | e64620
alone explained 78% of the observed variation in female courtship
and the number of drums in a burst further explained 8% of data
variability (Table 4). Nest behaviour was positively related to
female courtship whereas the number of sounds in a burst
correlated negatively (Fig. 5). Female courtship was not related
with female size (Pearson correlation, N =20, R =20.23, P.0.05)
or K condition factor before mating (R = 20.18, P.0.05).
Discussion
Species recognition and mate choice critically important in
animal communication and in making the ‘correct’ choices often
relies on the female’s response to variation in male traits [39]. Yet
there are few studies in the literature that simultaneously explore
female mate recognition and quality evaluation in the same fish
species [18,20]. Here we show that the simple drums produced by
male sand gobies while courting the females potentially contain
important information in both species recognition and mate
assessment. We also show that male visual behaviour influences
female courtship, exhibited by sexually receptive sand goby
females [33].
Inter-specific Differences
Both sand and painted goby males produce low frequency
drumming sounds, mainly while quivering in the nest, to entice the
females to enter the nest and spawn (present study and [29,40]).
Drums produced by the painted goby showed significantly higher
dominant frequencies than those produced by the sand goby.
Although higher dominant frequencies are expected in smaller
individuals of some fishes [24,41,42] we did not observe a
relationship between this acoustic parameter and fish length in
either studied species. Also, fish SL overlapped considerably in
both species (36–45 mm SL in the painted goby vs. 39–49 mm SL
in the sand goby) suggesting that size alone is not responsible for
inter-specific differences in sound frequency. Malavasi and
colleagues [21] have studied five different genera of vocalising
gobies from the Mediterranean, including Pomatoschistus, and
consistently showed a significant relationship between body size
and dominant frequency only in the grass goby Zosterissessor
ophiocephalus. Sound frequency could hence provide a reliable cue
for species recognition in Pomatoschistus spp. since it appears to be
independent of body size and falls well within their hearing
sensitivity and expected frequency discriminating abilities [43,44].
The present study also revealed that the pulse repetition rate
and sound intervals differed significantly between the two studied
species, with the painted goby showing higher pulse rates within a
sound and longer intervals between sounds. Although few
systematic comparisons of acoustic signals are available for
closely-related fish species [21], temporal characteristics of sounds
are thought to be important carriers of species-specific information
in fish. In fact distinct temporal patterns, such as pulse number
and rate, often differentiate sounds of closely-related sympatric
species such as in the Pomacentridae, Cichlidae and Mormyridae
[18,20,41,45]. Further, playback experiments testing male poma-
centrids from the genus Stegastes provided evidence that fish can
distinguish conspecific courtship sounds from those of closely-
related congenerics, based on the number of pulses and pulse rate
[17,45]. Our results are consistent with the hypothesis that the
pulse rate – and possibly also the pattern of sound emission (i.e. the
sequence considering sound intervals) – contribute to the
recognition of conspecific mates, although sound intervals show
higher intra-specific variability than pulse rate (Table 2) and thus
potentially provide less reliable species-specific acoustic cues.
Figure 5. Relation between male courtship behaviour and the
frequency of female courtship in
P. minutus
.Male courtship
behaviour predictors in the final linear regression model were nest
behaviour duration and the number of sounds in a burst. Regression
lines and 95% confidence interval bands are shown.
doi:10.1371/journal.pone.0064620.g005
Table 4. Table for predictors of female sand goby P. minutus courtship.
Dependent
variable
Included
predictor
B
S.E.M.
tP rF
Model
significance R
2
DW VIF
Female courtship Intercept 0.26 0.12 2.19 0.04
Nest 0.27 0.04 6.43 ,0.001 0.84 1.0
Sounds in burst 20.08 0.03 23.01 0.008 20.59 F
2,19
= 24.75 P,0.001 0.86 2.1 1.0
Nest behaviour duration (quiver and rest) and female courtship were log
10
-transformed to meet the linear regression model assumptions. r – partial correlation between
the dependent variable and the predictor, controlling for the effects of the other predictors in the model. DW - Durbin Watson statistics.
doi:10.1371/journal.pone.0064620.t004
Species and Male Quality Encoded by Fish Sounds
PLOS ONE | www.plosone.org 7 June 2013 | Volume 8 | Issue 6 | e64620
Acoustic and Visual Cues for Mate Choice
Sand goby males examined in the study presented a relatively
small variability (CV) in male length and K condition factor
(,15%) but showed considerable variability in their fat reserves
(32%). Further, acoustic and visual courtship behaviour was also
highly variable among males (75–132%) providing a basis for mate
assessment. All male quality parameters (SL, K and lipids) were
predicted by male visual or acoustic features. Sound amplitude
significantly explained 36% of the variation in male length.
According to the best multiple regression model, a variation of
1 cm in SL (e.g. 4–5 cm) corresponds to a change of 24 dB in
sound amplitude (see Table 3 and Fig. 4), comparable to the
findings of [29] for Baltic sea populations of sand goby and of [40]
for the painted goby. Male size is important for the sand goby as
larger males have a competitive advantage over nest sites [46,47]
and are preferred by females [48]. If females of the studied sand
goby population are selecting larger mates to increase their fitness,
then they may also use sound amplitude as a redundant signal of
male quality. In insects, anurans and birds, variation in male
sound amplitude plays an important role in both female choice
and male-male competition, and females of several species have
been found to prefer louder calls [9,49,50].
We also found that male sand goby condition – an important
determinant of competitive dominance over nests sites when body
size differences are small [51] – could be predicted by male
behaviour. Indeed, 29% of variability in male K condition factor
and 38% of the variability in male fat reserves could be explained
by nest behaviour duration and active calling rate, respectively. In
general, courtship behaviour is likely to impose energy costs
[52,53]. However, the energetic costs of visual courtship in the
sand goby seem controversial [37,54,55], and in the painted goby
visual courtship does not relate to the male condition factor or fat
reserves [40]. On the other hand, calling activity in fish with
parental care appears to advertise male condition such as fat
reserves (sand goby - present study; Lusitanian toadfish [25];
painted goby [40]) that are key to prolonged nest defence and
general parental activities [56]. In these cases, calling activity is
likely under mate choice since males that vocalize more frequently
enjoy a higher reproductive success [13,40]. Interestingly, both in
the sand goby (present study) and in the painted goby [40], a
relationship between fat reserves and calling activity could be
detected even over a short (20 min) observation period, suggesting
that only males in good condition have the ability to pay the costs
of intense calling bouts. In birds, in which the sexual function of
song has been well established, male song rate may also influence
reproductive success [57,58].
Female Sand Goby Courtship Behaviour
Female courtship was positively related with the duration of
male nest behaviour, which explained most of the variability of this
female behaviour (78%). Female courtship has been documented
as a signal of sexual receptivity in the sand goby [33]. In the
present study, however, all but one of the females that did not
court spawned in the male’s nest, indicating that they were also
sexually receptive. It is possible that the relative quality of mates
may have affected courtship and assessment time leading to
variability in mutual courtship, i.e. male nest behaviour and
female courtship. Females could be assessing male condition
through nest behaviour duration since it appears to be a good
predictor of male condition factor. On the other hand, males could
also be assessing female quality including fecundity. It is becoming
clear that preference for mates may vary between and within
individuals and that the perception of variability in mate- or self-
quality can influence mating preferences and the mate assessment
process [59,60]. However this hypothesis still needs to be tested.
Gobies from the genus Pomatochistus are very similar morpho-
logically [61] and use the same sensory channels during mate
attraction [21] facing the potential costs of wasting time, energy,
nutrients, or gametes in erroneous heterospecific sexual interac-
tions [62] that may lead to fitness loss associated with reproductive
interference, i.e. the process of mate acquisition that adversely
affects the fitness of at least one of the species involved and that is
caused by incomplete species recognition (reviewed in [63]).
Reproductive interference might cause from only a slight decrease
in fitness to the displacement of one species. Hence, traits that
reduce these costs should be positively selected and lead to
reinforcement of premating barriers, such as character displace-
ment, or promote ecological segregation of species [64]. We have
shown that dominant sound frequency and temporal patterns of
vocal signals may potentially enable species recognition in two
sympatric Pomatoschistus species, consistent with other empirical
studies on sympatric closely-related fish species. In turn, sound
amplitude and active calling rate may contribute to conspecific
mate assessment and preference within the sand goby. Future
studies should test if these or other acoustic features are in fact
used in species recognition and in mate choice in fish. We propose
the sand goby as a major model species to address these fairly
unexplored questions. In particular the use of acoustic signals in
conspecific vs. heterospecific mate recognition still remains to be
demonstrated in fish.
Supporting Information
Figure S1 Pomatoschistus minutus in experimental
nest. Note the nest chimney that houses the hydrophone.
(JPG)
Video S1 Courtship behaviour in the painted goby
Pomatoschistus pictus.The male leads the female into the
nest and makes drumming sounds before and after she enters the
nest.
(MP4)
Video S2 Courtship behaviour in the sand goby Poma-
toschistus minutus.The male leads the female into the nest
and drums while she is outside the nest. The female approaches
with blackened eyes (a signal of spawning readiness) and the male
nudges the female flank. The male continues to drum after the
female enters the nest.
(MP4)
Acknowledgments
We would like to thank Bengt Lundve for his valuable assistance with
animal keeping and setup construction at the Sven Love´n Centre for
Marine Sciences, and Charlotta Kvarnemo and Ineˆs Gonc¸alves for support
and help with the lipid analyses. We are thankful to Eric Parmentier and an
anonymous referee for providing useful feedback to this paper.
Author Contributions
Conceived and designed the experiments: MCPA PJF IB. Performed the
experiments: IB SSP. Analyzed the data: IB SSP MCPA. Contributed
reagents/materials/analysis tools: OS MCPA PJF IB. Wrote the paper:
MCPA. Revised the manuscript: OS IB PJF. Supervised lipid analysis: OS.
Species and Male Quality Encoded by Fish Sounds
PLOS ONE | www.plosone.org 8 June 2013 | Volume 8 | Issue 6 | e64620
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