Zimmermann et al. BMC Evolutionary Biology
https://doi.org/10.1186/s12862-019-1528-7
(2019) 19:200
RESEARCH ARTICLE
Open Access
Nest defense in the face of cuckoldry:
evolutionary rather than facultative
adaptation to chronic paternity loss
Holger Zimmermann1, Karoline Fritzsche1,2, Jonathan M. Henshaw1,2, Cyprian Katongo3, Taylor Banda4,
Lawrence Makasa4, Kristina M. Sefc1* and Aneesh P. H. Bose1,5*
Abstract
Background: Raising unrelated offspring is typically wasteful of parental resources and so individuals are expected
to reduce or maintain low levels of parental effort when their parentage is low. This can involve facultative, flexible
adjustments of parental care to cues of lost parentage in the current brood, stabilizing selection for a low level of
paternal investment, or an evolutionary reduction in parental investment in response to chronically low parentage.
Results: We studied parental care in Variabilichromis moorii, a socially monogamous, biparental cichlid fish, whose
mating system is characterized by frequent cuckoldry and whose primary form of parental care is offspring defense.
We combine field observations with genetic parentage analyses to show that while both parents defend their nest
against intruding con- and hetero-specifics, males and females may do so for different reasons. Males in the study
group (30 breeding pairs) sired 0–100% (median 83%) of the fry in their nests. Males defended less against immediate
threats to the offspring, and more against threats to their territories, which are essential for the males’ future
reproductive success. Males also showed no clear relationship between their share of defense and their paternity of the
brood. Females, on the other hand, were related to nearly all the offspring under their care, and defended
almost equally against all types of threats.
Conclusion: Overall, males contributed less to defense than females and we suggest that this asymmetry is
the result of an evolutionary response by males to chronically high paternity loss in this species. Although
most males in the current study group achieved high parentage in their nests, the average paternity in V.
moorii, sampled across multiple seasons, is only about 55%. We highlight the importance and complexity of
studying nest defense as a form of parental care in systems where defense may serve not only to protect
current offspring, but also to ensure future reproductive success by maintaining a territory.
Keywords: Parental care, Multiple paternity, Cichlid, Paternal care adjustment, Parental investment,
Variabilichromis moorii
Background
Parental care has evolved in many species as a way for
parents to increase their own reproductive success by
enhancing the survival of their offspring. Because parental care demands time and energy it can incur fitness
costs to the caregiver (i.e. parental investment [1]), and
so parental care also presents an optimality problem [2].
* Correspondence: kristina.sefc@uni-graz.at; abose@orn.mpg.de;
aphbose@gmail.com
1
Institute of Biology, University of Graz, Universitätsplatz 2, 8010 Graz, Austria
Full list of author information is available at the end of the article
A parent’s optimal investment into care should depend
on the value of the current brood relative to the parent’s
expected future reproductive success [3]. Brood value
depends on numerous factors [4], including brood size
[5–8], offspring survival prospects [9, 10], and parentoffspring relatedness [11]. Variable and uncertain parentage is common and taxonomically widespread [12,
13], and parents may use either direct cues (e.g. olfactory
or visual) or indirect cues (e.g. social context) to assess
their level of kinship to their broods [14]. A facultative
reduction in parental investment is one way to mitigate
© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
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Zimmermann et al. BMC Evolutionary Biology
(2019) 19:200
the damage of compromised parentage, and such adjustments of parental care in response to cues of lost
parentage have been described in some species (e.g.
birds: reed buntings [15], blue tits [16], fishes: bluegill
sunfish [17], pumpkinseed sunfish [18], plainfin midshipman fish [19], insects: taurus scarab [20]), but not in
others [21–23]. Additionally, in species where paternity
is often shared among multiple males, selection may act
to reduce average paternal investment over evolutionary
timescales [24–27].
A great deal of previous empirical research, largely
conducted on birds, has investigated how parental care
responds to reduced parentage. Typically, the rate of offspring provisioning – a highly demanding parental task
[28] – is used to quantify parental care and act as a
proxy for parental investment. Yet in many non-avian
taxa, parental care rarely involves offspring provisioning,
and in this respect fishes offer an interesting contrast to
birds. With few exceptions, parental care in fishes does
not involve provisioning young with food, but instead
consists of fanning and cleaning eggs and defending
young from predators [29, 30]. Parental care in fishes
can still incur costs in terms of energetic expenditure
(e.g. ref. [18, 31]), missed foraging opportunities (e.g. ref.
[32]), exposure to predation risk (e.g. ref. [33]), and
forfeiture or postponement of additional reproductive
opportunities (e.g. ref. [34]), all of which are likely to
affect future reproductive success. To date, most assays
of parental defense behaviors in fish have involved
presenting caregivers with a threat (i.e. a live or a model
predator or competitor) and then monitoring the intensity of the caregivers’ response (e.g. ref. [18, 35–37]).
While such methods can be useful for gauging a parent’s
instantaneous motivation to defend, they lose ecological
relevance because in the wild, parents rarely have a
single threat to contend with and must partition their
defense efforts against multiple simultaneous or consecutive intrusions. This highlights a research need to
better understand defense as a component of parental
care under natural conditions. Indeed, whereas behaviors
such as offspring provisioning or fanning are largely
depreciable forms of care determined by the needs and
value of a particular brood, defense is typically nondepreciable and must additionally respond to the level of
predation pressure in the environment. Consequently,
optimal defense effort is challenging to predict based on
brood value or parentage alone.
Many caregiving fishes are also highly territorial, with
individuals competing for and defending territories that
are subsequently used for rearing offspring in addition
to attracting mates, foraging, and sheltering [29]. The
behaviors involved in territory and brood defense can
overlap, or even exactly coincide, because both involve
driving territory intruders away. The study of how
Page 2 of 11
defense varies with brood value is thus complicated both
by the dual purpose of defense behaviors and by
temporal and spatial variation in intruder pressure. Furthermore, in species with biparental care, the amount of
care provided by one parent may respond to the state
and behavior of the other parent [38–40]. Sexual conflict
between parents is expected to arise because each parent
benefits when the other takes on a larger portion of the
parental workload [41]. Sexual conflict can also be exacerbated when the value of the current offspring differs
greatly between the parents. Such asymmetries in brood
value occur, for example, when males are cuckolded
[42], which is common in many fish mating systems
[13]. Asymmetries in brood value could potentially lead
to divergent evolutionary motivations for each parent to
provide defense. For example, defense performed by
females may serve primarily to protect young, while
defense by males may be primarily to retain the territory
and protect it from exploitation.
Here, we investigate how male brood defense relates
to paternity in a sexually monomorphic, socially monogamous and biparental cichlid fish, Variabilichromis
moorii, from Lake Tanganyika, East Africa. V. moorii is a
herbivorous substrate-breeder [43, 44] and is very abundant (> 6 adults per 10m2) along rocky shorelines in
southern Lake Tanganyika [45, 46]. Solitary individuals
and mated pairs of the small (< 10 cm), dark-colored
cichlid defend their territories against other members of
the species-rich littoral fish community that compete
with them for food and space. Additionally, nonterritorial V. moorii cruise the rocky littoral, snatch up
food and on occasion participate in the spawning of the
mated pairs [47, 48]. Spawning females attach their
clutches of up to > 100 eggs onto vertical rock surfaces.
Free swimming larvae appear 4–5 days post spawning
and are guarded by the parents for a period of up to
100 days, i.e. until grown to > 3 cm [44]. V. moorii
broods have remarkable levels of extra-pair paternity,
with brood-tending males siring an average of ~ 55% of
the fry on their territories (range = 0–100%) and with 10
or more sires contributing to some broods [47, 49]. Females, on the other hand, have nearly absolute (~ 96%)
maternity of the fry on their territories (small numbers
of foreign fry, related to neither parent, are occasionally
found within broods [47]). Despite this sex-asymmetry
in parentage, both sexes defend the brood against intruding con- and heterospecifics [50–52]. In our study,
we observed and scored defense behaviors of male and
female parents on their territories under natural conditions, where intrusions by fry predators and territory
competitors occur frequently [43, 50–52]. This allowed
us to collect observational data on territory and brood
defence from 31 breeding pairs without experimental
manipulation of the nest environment. Paternity shares
Zimmermann et al. BMC Evolutionary Biology
(2019) 19:200
Page 3 of 11
of the parental males were assessed based on microsatellite genotyping. We hypothesized that asymmetries between the parents in their defense labour (i.e. the
number of defensive actions aimed towards intruders)
would reflect asymmetries in brood value, which is
greatly influenced by paternity in our study system. We
also considered the possibility that the defense offered
by each sex might differ depending on whether intruders
represented threats primarily to the offspring (i.e. fry
predators) or to the territory (i.e. risk of territory takeover or exploitation).
parents (8.32 ± 0.34 cm; Spearman rank correlation, rS =
0.42, p = 0.019, n = 31 territories). Paternity shares (relative to maternal brood size) of the territory-holding
males ranged from 0% (in 1 territory) to 100% (in 12
territories), with a median of 82.8% (IQR = 42.4%, n = 30
territories; Fig. 1). The number of extra-pair sires per
brood ranged from 0 to 8 (median = 1 extra-pair sire,
IQR = 1.75 extra-pair sires, n = 30 territories).
Results
Maternal brood sizes ranged from 5 to 94 fry (median =
22 fry, interquartile range (IQR) = 20.5 fry, n = 31 territories); additionally, 9 of the nests contained 1–7 foreign
fry. The total length of the fry ranged from 9 to 28 mm
(mean ± s.d. 16.9 ± 5.0 mm, n = 31 territories). Brood
sizes were not correlated with fry length, which we use
here as a proxy for fry age (Spearman rank correlations;
maternal brood size: rS = − 0.05, p = 0.787; total brood
size: rS = 0.10, p = 0.592; n = 31 territories). Male total
length (mean ± s.d. 8.3 ± 0.4 cm) was correlated with female total length (mean ± s.d. 8.4 ± 0.4 cm) within social
pairs (Spearman rank correlation, rS = 0.54, p = 0.0032,
n = 31 territories), though neither sex was consistently
larger than the other (paired Wilcoxon signed rank test,
V = 134, p = 0.174, n = 31 territories). The within-pair
size differences (female total length – male total length)
ranged from − 0.7 cm to 0.8 cm (mean = 0.09 cm ± s.d.
0.37 cm, n = 28 pairs). Maternal brood sizes were positively correlated with the average total length of the
The observed pairs (n = 31) performed a median of 1.53
(range = 0.58–5.53, IQR 0.98) defense behaviors per minute. Since V. moorii consistently defended against unfamiliar, approaching fish, we used the total number of
defense behaviors performed by the pair as a proxy for
the intrusion pressure at that territory. Defense against
brood predators (median of defense behaviors per min =
0.20, range = 0.07–2.13, IQR = 0.29) occurred less often
than against territory competitors (median = 1.17,
range = 0.40–4.00, IQR = 0.87, GLMM: est. = − 1.47, se =
0.088, z = − 16.68, p < 0.0001) (Fig. 2a). Intrusion pressure varied between the two intruder types depending
on fry length (interaction, est. = 0.30, se = 0.092, z = 3.24,
p = 0.0012) and average parental total length (interaction, est. = − 0.36, se = 0.11, z = − 3.45, p = 0.0006).
Because of this, we present the results of two separate
models, one for each intruder type, in Table 1. In brief,
larger parents experienced more intrusion pressure from
territory competitors. Intrusion pressure from territory
competitors also decreased with increasing numbers of
Intrusion pressure by brood predators versus territory
competitors
Fig. 1 Brood parentage. For each genotyped brood, bars illustrate the proportions of within-pair offspring (light grey), offspring sired by extra-pair
males (dark grey) and foreign fry (black), relative to total brood size. Note that paternity shares were calculated relative to maternal brood sizes,
which do not include foreign fry
Zimmermann et al. BMC Evolutionary Biology
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(2019) 19:200
Fig. 2 a Intrusion pressure from brood predators was lower than that from territory competitors. b Males contributed relatively less than their
female partners to defense against brood predators than to defense against territory competitors. Horizontal dashed line indicates 0.5 (i.e. an
egalitarian split of defense against intruders). Both (a) and (b) depict values per territory summed across the three observation periods
Table 1 Effects of brood, parental, and territory variables on intrusion pressure by territory competitors and brood predators.
Intrusion pressure was quantified as the number of defense behaviors performed by the breeding pair of V. moorii against each
intruder type. Significant p-values (p < 0.05) are in bold
Estimate
Std. Error
2.68
0.052
z
P
Intrusion pressure from territory competitors
(Intercept)
52.0
< 0.0001
Depth
0.085
0.055
1.54
0.12
Fry length
−0.002
0.054
− 0.04
0.97
Total brood size
−0.150
0.057
−2.63
0.0085
Average parent body size
40.291
0.061
4.83
< 0.0001
1.16
0.130
8.87
< 0.0001
Depth
−0.084
0.136
− 0.62
0.54
Fry length
0.331
0.132
2.52
0.012
Intrusion pressure from brood predators
(Intercept)
Total brood size
0.037
0.119
0.32
0.75
Average parent body size
−0.095
0.130
− 0.74
0.46
Zimmermann et al. BMC Evolutionary Biology
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(2019) 19:200
fry on a territory. Intrusion pressure from brood predators increased with increasing fry length.
Relative contribution of males and females to defense
against intruders
During the 45 min of cumulative observation time per
territory, males and females were observed to engage in
0.07 (median, range = 0.00–0.47, IQR = 0.15) and 0.16
(median, range = 0.02–1.73, IQR = 0.22) defense behaviors per minute against brood predators respectively.
Against territory competitors, on the other hand, males
and females respectively engaged in 0.56 (median,
range = 0.04–1.87, IQR = 0.51) and 0.60 (median, range =
0.18–2.53, IQR = 0.42) defense behavious per minute.
When we pooled defense behaviors against brood predators with those against territory intruders, only the
intercept term was significant, indicating a lower male
share of defense relative to females (est. -0.347, se = 0.119,
z = − 2.924, p = 0.0035; Additional file 1: Table S1). Paternity share, maternal brood size and the size difference between the caregivers had no detectable effect on male
share of defense (Additional file 1: Table S1).
Male share of defense was significantly lower against
brood predators (31% of the breeding pairs’ defense
behaviors) than against territory competitors (47%; adding ‘intruder type’ as a predictor to above GLMM: est.
-0.580, se = 0.171, z = − 3.390, p = 0.0007; Fig. 2b). We
next investigated defense against each intruder type separately. Male share of defense against territory competitors was not significantly related to paternity, maternal
brood size, or the size difference between the breeding
partners (Table 2a), and there was no difference between
male and female shares in defense against territory competitors (intercept term, Table 2a). Male share of defense
against brood predators was similarly not significantly
related to paternity, maternal brood size, or the size difference between the partners (Table 2b), but males did
engage in significantly fewer defense behaviors against
brood predators than their female partners (intercept
term, Table 2b).
Discussion
In our study, females contributed significantly more than
males to defense against brood predators (i.e. egg and
fry predators as well as piscivorous species; henceforth
called brood defense). In contrast, defense against territory competitors (mostly algae-eaters and zooplanktivores; henceforth called territory defense) was shared
more equally between both parents (Fig. 2). Males participated more in territory defense than brood defense,
but the relationships between either form of male
defense and paternity or the other proxies of brood value
were statistically unclear (sensu ref. [53]). We interpret
these results as evolutionary responses to low paternity
shares and different levels of motivation for brood and
territory defense (see below). We also show that V.
moorii parents face greater intrusion pressure from territory competitors than from brood predators. Intrusion
pressure from brood predators increased with fry size,
possibly because older fry are more mobile and conspicuous. Intrusion pressure from territory competitors
increased with parent size, potentially because larger individuals hold larger and more valuable territories (but
see ref. [50, 52]). Territory competitors also intruded
more when brood sizes were small (Table 1), a pattern
that could arise if algal cover in territories diminishes
with the number of offspring grazing there, thus
Table 2 Effects of paternity on male share of defense against territory competitors and brood predators. Maternal brood size and
the size difference between females and males were included as additional factors that may affect male share of defense and/or
male paternity share (see Methods). Significant intercept terms indicate non-egalitarian defense behaviors between males and
females (note that parameter estimates are on the scale of the logit-link function). (a) Results for the model fit to intrusion pressure
from territory competitors. (b) Results for the model fit to intrusion pressure from brood predators. Significant p-values (p < 0.05)
are in bold
Estimate
Std. Error
−0.175
0.125
z
P
(a) Intruder type: territory competitors
(Intercept)
−1.398
0.162
Paternity
0.439
0.487
0.901
0.368
Maternal brood size
0.001
0.124
0.010
0.992
Female – Male size difference
0.089
0.139
0.638
0.523
(Intercept)
−1.004
0.268
−3.749
0.0002
Paternity
0.138
0.947
0.146
0.884
(b) Intruder type: brood predators
Maternal brood size
0.180
0.239
0.752
0.452
Female – Male size difference
−0.283
0.269
−1.051
0.293
Zimmermann et al. BMC Evolutionary Biology
(2019) 19:200
enhancing the foraging attractiveness (for herbivores) of
territories with smaller broods. All of these possibilities
require experimental verification in future studies.
Page 6 of 11
however, that sex biased parental care has even been observed in systems with lifelong genetic monogamy where
brood value and reproductive opportunities do not differ
between males and females (e.g. ref. [66]).
Males defend less than females against brood predators
In comparison to other fish species, the paternity of
pair-bonded V. moorii males is especially low and variable [54– 59]. Pair-bonded, territory-holding male V.
moorii achieve an average (± SD) of 57 ± 33% paternity
in their broods (values collated from ref. [47] and unpublished data). Note, however, that the average
paternity was higher in the subset of males considered in
this study (72%, see Results section for details). In agreement with a previous study [52], male V. moorii contributed less than females to the total defense behaviors
exibited by the pair. Here, we showed that this nonegalitarian share of defense is due to a deficit in male
defense against brood predators specifically. The low
participation of males in brood defense (31%) relative to
females may thus represent an evolutionary response to
chronically low average paternity in territory-holding V.
moorii. Over evolutionary time, low average brood value
can reduce paternal investment and/or maintain a low
level of investment [22, 24, 26, 27, 60].
Sex-specific parental roles are common in biparental
brood-caring fishes and have often been interpreted as
division of labor associated with sexual size dimorphism;
the larger individual, typically the male, emphasizes
defense against intruders, while the smaller individual,
typically the female, remains close to the brood and
provides direct brood care such as fanning or cleaning
(e.g. ref. [61–65]). V. moorii, however, are sexually
monomorphic and social pair formation occurs between
similarly-sized individuals (ref. [52]; our data). We could
not find any statistically clear correlation between male
share of defense and the body size differences seen
within pairs. Though we cannot rule out whether other
phenotypic traits that differ between the sexes (unrelated
to body size) lead females to be more specialized in
defense, both sexes appear similarly capable of performing
defense behaviors. Overall, this supports the idea that
differential brood defense between the sexes is linked to
the asymmetry in average brood value. Data from recent
studies suggest that in V. moorii, paired males likely do
not have a higher expected future reproductive success
than their female partners, which could have otherwise
explained the lower degree of male parental expenditure.
This is because males and females evidently engage in
long-term pair-bonds and males apparently do not compensate for their lower parentage by reproducing outside
of their pairs [47]. Differential brood defense also raises
the question of how divergent evolutionary interests shape
sex differences in parental care activities even in species
without pronounced sexual dimorphism. We note,
Males and females defend to similar extents against
territory competitors
While brood defense in V. moorii was female-biased,
territory defense was shared in a more egalitarian manner, suggesting that both parents benefit similarly from
protecting against territory competitors. However, unlike
brood defense, territory defense is complicated by the
fact that it may be motivated by multiple factors. On the
one hand, territory defense may serve to protect against
territory takeovers and exploitation. This may be a
powerful motivator for defending, as the reproductive
success of individuals is greatly enhanced by territory
ownership [47]. On the other hand, territory defense still
benefits current offspring because almost all the species
we oberved, regardless of trophic specialization, consume fry opportunistically. Furthermore, offspring can
suffer indirectly if territory competitors are permitted to
take over the territory or graze the algae on which V.
moorii feed. This highlights a challenging question: Why
do parents evolve to defend against territory competitors
– to protect the territory, the offspring, or both? Broadly,
parental care is defined as a suite of parental traits
(behavioral or non-behavioral) that has evolved and is
currently maintained for the purpose of increasing offspring fitness [67]. It is therefore unclear how well territory defense fits the definition of parental care as such
defense may enhance the probability of future reproductive success (by retaining the territory for future use)
while also, perhaps incidentally, benefitting any current
offspring. Future research focusing on teasing apart the
relative importance of these factors will be important.
Paternity does not relate to male brood defense
On average, males contributed less to brood defense
than did females, which mirrors the males’ lower average
relatedness to the broods (i.e. low average paternity but
high maternity [47, 49]). However, when we looked at
patterns of paternity and defence within the current
study group, we did not find the expected positive correlation between an individual male’s brood paternity
and his share of brood defense, which would indicate a
facultative adjustment of paternal investment into the
current brood.
It is important to consider how well V. moorii males
satisfy the requirements under which a facultative adjustment of care to current paternity would be predicted
[21, 22]. First, such adjustments are only predicted if
males expect higher paternity in future broods. Overall,
Zimmermann et al. BMC Evolutionary Biology
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(2019) 19:200
V. moorii broods show exceptionally variable paternity,
both within and between seasons [47, 49]. Within-male
variation in brood paternity can also be substantial: over
3 years of intensive sampling of a wild breeding population of V. moorii (5 field excursions in total), we
obtained genetic paternity data from the broods of seven
individual males that were recaptured on multiple occasions. The paternity of these males varied extensively
across broods (average within-male standard deviation
in brood paternity was 30.1%, see Additional file 2: Table
S2). Thus, males faced with low current paternity can indeed expect higher success in future broods.
Second, reliable cues of parentage must be readily
available for brood care to be adjusted in response to
variable paternity. It is currently unclear whether V.
moorii males have access to reliable cues, direct or indirect, of paternity loss. Males could foreseeably use the
number of potential cuckolders intruding on a spawning
event as a gauge of sperm competition intensity [17, 20].
Indeed, in our current study, sire number was strongly
correlated with paternity of the paired male (Spearman
correlation coefficient r = − 0.834). However, the number
of rivals present during a spawning becomes an increasingly unreliable measure as the number of unsuccessful
cuckolders in the group increases and becomes more
variable [48]. Direct observations of V. moorii spawning
events will be valuable for assessing the utility of this
paternity cue. The use of direct offspring cues (e.g. olfactory kin recognition) can affect brood care decisions of
cuckolded males [17] and has been demonstrated in several fish species, including cichlids [68–71]. However, it
is currently unknown whether V. moorii males are sensitive to such direct cues. Future studies taking a more
targetted approach at investigating the mechanisms of
kin recognition in this species would be valuable.
Third, if care is to be adjusted in response to variable
parentage, providing care must be costly in terms of
reducing the parent’s residual reproductive value. While
brood defense necessarily involves energetic expenditure
[18, 32, 72], brood predators in this system pose little
threat of predation or injury to the parents (i.e. negligible ‘risk investment’). Thus, it is possible that an evolutionary reduction in paternal investment in response to
chronic paternity loss [24] may have rendered the costs
of brood defense too low to warrant clear adjustments in
care.
Conclusions
High rates of cuckoldry and low average paternity over
evolutionary time in V. moorii may have selected for a
low baseline level of paternal relative to maternal investment. This was most evident for brood defense, in which
males showed reduced participation in comparison to
females. However, males engaged in similar amounts of
territory defense as their female partners. Such results
are in line with the idea that brood value is generally
lower for males, but both partners benefit from longterm retention of their territory. We highlight the
challenges of studing territory defense from the perspective of parental investment theory and emphasize the
importance of uncovering the factors that motivate
defense against territory competitors.
Methods
Field observations of brood defense and sampling
Field work took place in October and November 2015 at
a study quadrat (area ≈ 1600m2 depth = 1.7–5.8 m) by
Mutondwe Island, Zambia (8°42′29.4″S 31°07′18.0″E).
The quadrat contained 85 territories with breeding V.
moorii, from which we randomly selected 31 territories
with free-swimming fry for behavioral observations (offspring spend most of the brood care period as freeswimming fry [44]). The two breeding adults from each
territory were captured while on SCUBA, fin-clipped,
sexed by examination of their genital papillae [52], and
released. V. moorii adults are sexually monomorphic and
so in order to distinguish between the male and female
parent at each territory during subsequent observations,
we clipped the caudal fins of the males and females
differently (dorsal/ventral tip of the fin for males/females
respectively). The fin clips were stored in 99.9% ethanol
and later used for DNA-based parentage analyses (see
below).
We measured the total length of the breeding fish to
the nearest 0.1 cm. Two males and one female escaped
after fin-clipping in the field before their length could be
measured. However, given that V. moorii pair sizeassortatively (ref. [43], current study) and that we were
able to catch and measure their social partners, we
linearly interpolated these missing body lengths (using
the function ‘approx’, R package ‘stats’). This allowed us
to preserve the sample size in all our analyses including
parent sizes, although we note that omitting the territories with missing size data produced qualitatively similar
results (Additional file 3: Table S3).
We started behavioral observations between 1 and 3
days after taking the fin clips. Fin-clipped individuals recover quickly, resuming parental care within 3 min after
clipping (personal observations HZ, KF, JH, AB), and so
the handling of the fish for clipping is unlikely to affect
our observations, which occurred at least 24 h later. The
same scuba diver (HZ) observed each territory for 15
min on each of three consecutive days (i.e. three 15-min
observation sessions per territory). All observations took
place before noon to avoid time-related differences in
the behavior of the focal individuals as well as in the
abundance of intruders. In two cases, the territoryholding male was absent for one of the three observation
Zimmermann et al. BMC Evolutionary Biology
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(2019) 19:200
intervals. In one of these cases, the female was observed
driving off her male partner repeatedly during the observation interval on the third day; in the other, the male
was missing on the first day of observation. Since we
were interested in the parental care decisions of the
males, we avoided scoring behaviors that may not have
been under the males’ control and omitted these 2 days
from the analyses of parental care. During each observation interval, we tallied the defense behaviors of male
and female territory-holders, which consisted of displays
and overt aggression against fish intruding into the
territory. We counted lateral displays (fish present their
lateral side and erect fins to an intruder), frontal displays
(fish face the intruder with lifted gill covers but not
necessarily with erect fins) and overt aggression (attacks
against intruders, which typically involved darting towards an intruder to chase it off). All intruders were
identified to the species level, except for two species
(Telmatochromis dhonti and T. temporalis) that could
not be reliably distinguished.
After behavioral observations were completed, we
collected all fry (882 fry from 31 territories), euthanized
them in a bath of MS-222 (1 g / 1 L lake water), and preserved them in 99.9% ethanol for parentage analyses (see
below). We estimated average fry length per brood by
measuring the total length of four randomly chosen fry
to the nearest mm. Here, fry length was used as a proxy
for fry age. When broods contained fry of two or more
different size classes, which occurs rarely due to foreign
fry living on non-natal territories [47], we took measurements from four individuals in each size class, and then
calculated a weighted average across all fry classes in the
brood.
In our analyses, we distinguish between total brood
size (all fry collected from a territory, including foreign
fry in some territories [27 out of 882 fry in total were
foreign]) and maternal brood size (fry attributable to the
female territory-holder based on genetic parentage analyses). We also took fin clips for DNA extraction from
an additional 158 adults captured within the quadrat
(n = 219 total) to increase our sample size of adult V.
moorii for estimating population allele frequencies. The
fieldwork was carried out with the permission of the
Fisheries Department of Zambia and under a study
permit issued by the government of Zambia.
Classification of intruders as brood predators and
territory competitors
Breeding V. moorii defended against a wide variety of
species, suggesting that defense may be motivated by
both brood care and territoriality. In an attempt to
distinguish between brood defense and territoryoriented defense, we classified the intruders into two
groups (Table 3). Brood predators include well-
Table 3 Classification of species into territory competitors and
brood predators. Total number of defense actions against each
species during the observation of the focal territories is given in
parentheses
Territory competitors
Brood predators
Variabilichromis moorii (657)
Telmatochromis vittatus (177)
Telmatochromis temporalis & T. dhonti
(219)
Neolamprologus fasciatus
(145)
Eretmodus cyanostictus (172)
Lamprologus callipterus (17)
Neolamprologus modestus (104)
Ctenochromis horei (15)
Julidochromis ornatus (84)
Lepidiolamprologus elongatus
(12)
Neolamprologus caudopunctatus (50)
Mastacembelus sp. (11)
Neolamprologus savoryi (45)
Altolamprologus compressiceps
(5)
Lobochilotes labiatus (13)
Lepidiolamprologus attenuatus
(1)
Lamprichthys tanganicanus (6)
Ophthalmotilapia ventralis (45)
Neolamprologus pulcher (31)
Xenotilapia spilopterus (28)
Neolamprologus tetracanthus (27)
Tropheus moorii (22)
Petrochromis famula (11)
Aulonocranus dewindti (7)
Interochromis loocki (7)
Neolamprologus sexfasciatus (6)
Neolamprologus mustax (3)
Neolamprologus prochilus (2)
Petrochromis polyodon (2)
Simochromis diagramma (2)
Haplotaxodon microlepis (1)
documented egg and fry predators as well as piscivorous species [43, 51, 73]. The remaining species, many
of which are algae-eaters or zooplanktivores, have
been observed to hold territories similar to those of
V. moorii and to occasionally take over territories
from V. moorii (personal observation KF, HZ). These
species were classified as territory competitors. Although most species will opportunistically consume
eggs and fry, the species grouped together as brood
predators are more specialised in doing so than any
of the species within the territory competitor group.
The observer (HZ) did not notice any attempts made
by territory competitors to eat the defended fry,
whereas they did observe this from brood predators,
though none of the observed predation attempts were
successful. None of the observed intruders pose a
threat to adult V. moorii.
Zimmermann et al. BMC Evolutionary Biology
(2019) 19:200
Genetic parentage analysis
DNA extraction from fin clips of adult V. moorii
followed an ammonium acetate precipitation protocol
[74]. Genetic paternity could only be determined for 30
territories, because one breeding male could not be captured for fin-clipping. We followed a standard Chelex
protocol [75] for extracting DNA from fry tissue (n =
882). After centrifugation for 5 min at 4000*g, tubes
were stored at − 20 °C until PCRs were performed.
All samples were genotyped at 14 microsatellite loci in
2 multiplex reactions as described in Bose et al. [47].
The expected heterozygosity ranged from 0.700 to 0.946
(mean ± sd, 0.879 ± 0.077) and no deviation from HardyWeinberg equilibrium could be detected (for details see
ref. [47]). We estimated population allele frequencies
from the sample of 219 adult V. moorii, and parentage
analyses were carried out for each brood separately with
the help of COLONY (v 2.0.6.1, [76]). We estimated paternity share (percentage of brood sired by the broodtending males), the absolute number of fry sired by the
brood-tending male, and the number of extra-pair sires.
Paternity share was estimated as the number of withinpair fry divided by the maternal brood size. We carefully
checked the COLONY output for cases in which extrapair fry were proposed on the basis of only one to three
allele mismatches with the brood-tending males. Most of
these cases could be resolved as genotyping errors by rescoring electropherograms or repeating the PCR. In the
few remaining cases, mismatches at one or two loci were
considered to be due to mutations or unrecognized
genotyping errors, whereas mismatches at more than
two loci were assumed to indicate a different sire than
the brood-tending male. We also identified cases in
which COLONY presumed that there were two separate
extra-pair males, each having sired a single fry, despite
the two fry sharing the same mother. We saw no compelling reason to reject the more parsimonious assumption of one shared father in these cases, and corrected
the estimated number of extra-pair sires accordingly.
Statistical analyses
We used R v. 3.4.4 (R Development Core Team) for all
statistical analyses. For the statistical models that follow,
all continuous predictor variables as well as brood sizes
(count variables ranging from 5 to 94 fry) were mean-centered and scaled by dividing by their standard deviations prior to analyses. Proportion variables (i.e.
paternity) were mean-centered.
Since each unfamiliar (i.e. non-neighboring) fish that
approached the V. moorii territory elicited a single
defense response by one of the two breeding individuals,
we used total defense (i.e. the sum of male and female
defense actions) as a proxy for the intrusion pressure on
the territory. We first tested whether intrusion pressure
Page 9 of 11
from brood predators differed from that of territory
competitors and whether these pressures varied with
properties of the broods or territories. To do this, we fit
generalized linear mixed-effects models (GLMM, R
package lme4 [77]) with poisson error distributions. We
included total defense behaviors performed by the
breeding pair per 15-min observation period versus each
intruder type (count) as our response variable. We included intruder type (categorical variable: brood predator vs. territory competitor), total brood size, average fry
length, territory depth, and average total length of the
parental individuals as predictor variables. To account
for non-independence due to the temporal grouping of
observations, we also fit ‘territory ID’ nested within the
‘date of the territory’s first observation’ as a random
effect. We also fit an observation-level random intercept
to account for overdispersion [78]. We included the
number of fry, as well as the sizes of the fry and parents,
as predictor variables because of the possibility that
larger individuals or groups are more conspicuous to
intruders. We included territory depth in our model
because predator density has previously been suggested
to increase with depth [52]. Next, we focused on each
intruder type separately and fit two GLMMs, as just
described, for territory competitors and for brood predators independently.
Next, we tested whether males varied their share of
defense in relation to brood value. We first fit a
GLMM with a binomial error distribution, in which
we ignored intruder type by pooling our data between
brood predators and territory intruders. We included
the proportion of defense behaviors, observed within
each 15-min period, that was performed by the male
(proportion based on count data) as our response
variable (“male share of defense”). We included paternity share, maternal brood size, and the size difference between the caregivers (female total length
minus male total length) as predictor variables. Since
we were interested in asymmetries between the parents, we calculated paternity share relative to maternal brood size (as adopted young do not directly
contribute to either parent’s fitness). We included
‘day of observation’ (i.e. day 1, 2 or 3) and ‘territory
ID’ as random intercepts and also included an
observation-level random intercept to account for
overdispersion [78]. To test whether males contributed differently to defense against brood predators
than they did against territory competitors, we added
‘intruder type’ as a predictor to this GLMM. Finally,
we fit two separate GLMMs, one for each intruder
type, to address differences in male behavior against
brood predators and territory competitors. Note that
the intercept terms in these GLMMs test whether
defense is shared equally between males and females.
Zimmermann et al. BMC Evolutionary Biology
Page 10 of 11
(2019) 19:200
Supplementary information
Supplementary information accompanies this paper at https://doi.org/10.
1186/s12862-019-1528-7.
Additional file 1: Table S1. Effects of paternity on male share of
defense, when pooling across intruder types. Maternal brood size and the
size difference between females and males were included as additional
factors that may affect male share of defense and/or male paternity share
(see main text). The significant intercept term indicates non-egalitarian
defense behaviors between males and females (note that parameter estimates are on the scale of the logit-link function).
Additional file 2: Table S2. Variation in within-male paternity shares.
Paternity of breeding males recaptured over three years of sampling was
determined using nine microsatellite markers (Pmv17, Pzeb3, TmoM11,
UNH2075, Hchi59, Hchi94, Ppun9, Ppun20, Ppun21; see main text), and is
given in percent of maternal brood size.
Additional file 3: Table S3. Results of statistical models for intrusion
pressure and male share in defense after excluding nests with missing
parental size values. (A) and (B) show results corresponding to those in
Tables 1 and 2 of the main text, respectively.
Additional file 4: Table S4. Nest descriptions and observed defense
behaviors.
Abbreviations
GLMM: Generalized linear mixed model; IQR: Interquartile range;
PCR: Polymerase chain reaction
Acknowledgements
We thank the Department of Fisheries in Mpulungu for kindly supporting
our research at Lake Tanganyika. We are also grateful to B. Mbao our boat
driver. Thank you to the groups of M. Taborsky and T. Takahashi for sharing
their equipment and expertise, especially to D. Josi, J. Frommen, J. Flury, F.
Heussler, and H. Tanaka (whom we miss dearly). Furthermore, thank you to
Stephan Koblmüller for sharing his knowledge on Tanganyikan cichlids with
us.
Authors’ contributions
KS, HZ, KF and AB conceived the study. HZ, JH, and KF conducted the field
work with contribution by CK, TB and LM. HZ and KF performed the
microsatellite analyses. HZ, KS, and AB analyzed the data. HZ, KS, and AB
wrote the manuscript with input from the other co-authors. All authors read
and approved the final manuscript.
Funding
This work was supported by the Austrian Science Fund (FWF, grant number
P 27605-B25 to KMS) and the Österreichischer Austauschdienst (OeADGmbH, Ernst Mach Grant to JMH). The funding bodies had no role in the design of the study and collection, analysis, and interpretation of data and in
writing the manuscript.
Availability of data and materials
The data used in this paper are available in Additional file 4: Table S4.
Ethics approval and consent to participate
The fieldwork was carried out with the permission of the Fisheries
Department of Zambia and under a study permit issued by the government
of Zambia (SP 004478). The procedures used in this study were in line with
the guidelines set by the Animal Behavior Society (Animal Behaviour, 135: IX, 2018) regarding the treatment of animals in research and teaching. Only
trained personnel handled the fish. Euthanasia of larvae followed the guidelines of the Directive 2010/63/EU. The study was carried out with the ethical
approval of the ethics committee of the University of Graz (permit number
39/50/63 ex 2018/19).
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Author details
Institute of Biology, University of Graz, Universitätsplatz 2, 8010 Graz, Austria.
2
Department of Biological Sciences, University of Idaho, 875 Perimeter MS,
Moscow, ID 3051, USA. 3Department of Biological Sciences, University of
Zambia, Great East Road Campus, P.O. Box 32379, Lusaka, Zambia. 4Lake
Tanganyika Research Unit, Department of Fisheries, Ministry of Fisheries and
Livestock, P. O. Box 420055, Mpulungu, Zambia. 5Present address:
Department of Collective Behaviour, Max Planck Institute for Ornithology,
Universitätsstraße 10, 78464 Konstanz, Germany.
1
Received: 13 March 2019 Accepted: 17 October 2019
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