The ability to perceive and recognise a reflected mirror image as self (mirror self-recognition, MSR) is considered a hallmark of cognition across species. Although MSR has been reported in mammals and birds, it is not known to occur in any other major taxon. Potentially limiting our ability to test for MSR in other taxa is that the established assay, the mark test, requires that animals display contingency testing and self-directed behaviour. These behaviours may be difficult for humans to interpret in taxonomically divergent animals, especially those that lack the dexterity (or limbs) required to touch a mark. Here, we show that a fish, the cleaner wrasse Labroides dimidiatus, shows behaviour that may reasonably be interpreted as passing through all phases of the mark test: (i) social reactions towards the reflection, (ii) repeated idiosyncratic behaviours towards the mirror, and (iii) frequent observation of their reflection. When subsequently provided with a coloured tag in a modified mark test, fish attempt to remove the mark by scraping their body in the presence of a mirror but show no response towards transparent marks or to coloured marks in the absence of a mirror. This remarkable finding presents a challenge to our interpretation of the mark test—do we accept that these behavioural responses, which are taken as evidence of self-recognition in other species during the mark test, lead to the conclusion that fish are self-aware? Or do we rather decide that these behavioural patterns have a basis in a cognitive process other than self-recognition and that fish do not pass the mark test? If the former, what does this mean for our understanding of animal intelligence? If the latter, what does this mean for our application and interpretation of the mark test as a metric for animal cognitive abilities?Editor’s noteThis Short Report received both positive and negative reviews by experts. The Academic Editor has written an accompanying Primer that we are publishing alongside this article (https://doi.org/10.1371/journal.pbio.3000112). The linked Primer presents a complementary expert perspective; it discusses how the current study should be interpreted in the context of evidence for and against self-awareness in a wide range of animals.

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Cooperative breeding has been studied intensively in many species of birds and mammals but remain less well studied in fish. We report a remarkable new example of a cooperatively breeding cichlid from Lake Tanganyika, Neolamprologus obscurus. Using field observations and microsatellite DNA analyses, we studied group structure, helping behavior, relatedness, and dispersal of this species. We present four major observations. First, large territorial breeding males mated with one to eight breeding females, each of which was territorial and unrelated to another. Second, one to ten smaller fish ("subordinates") of both sexes were allowed to stay inside the breeding females' territories. Subordinates were often highly related to both the respective breeding male and female and performed territory defense and shelter maintenance, which is regarded as helping behaviors. Third, one to three subordinate males, similar in size to breeding females, were allowed to stay inside a breeding male's territory but were not tolerated in the breeding females' territories. Pairwise relatedness suggests these individuals are usually sons of the respective breeding male. Fourth, pairwise relatedness estimates suggest that juveniles delay dispersal and assist their mothers in raising offspring. As female subordinates grow up, they leave the father's territory and disperse into other groups. In contrast, male subordinates leave their mother's territory but remain within the territory of their father. The described social system makes N. obscurus a promising new model species to study the evolution of cooperative breeding.

Unravelling the evolution of complex social organization in animals is an important aim, not least because it helps to understand the evolutionary roots of human sociality. Recent advances in comparative methods allow to approach this question in a phylogenetic context. The validity of such comparative approaches depends strongly on the quality of information regarding the behaviour, sociality, and reproduction of animals in natural systems, and on the quality of the phylogenetic reconstruction. Applying a novel comparative approach, a recent study of Dey et al. (, Nature Ecology & Evolution, 1, 137) concluded that evolutionary transitions to cooperative breeding in cichlid fishes were not associated with the social mating pattern. Here we argue that this result was adversely affected by equivocal classifications of mating patterns, and inadequate phylogenetic data. In order to illustrate the impact of the mating system misclassifications, we scored mating patterns as reported in the original literature and re‐analysed the dataset based on Dey et al.’s tree topology. The result suggests that the mating system does in fact significantly explain the evolutionary transition to cooperative breeding in lamprologine cichlids, but we submit that a reliable conclusion cannot be reached before improving the behavioural information and the underlying phylogenetic reconstruction. The problems identified in this case study are not unique and we urge caution in the interpretation of results from comparative phylogenetic studies in general. We do agree with Dey et al. () though that the lamprologine cichlids of Lake Tanganyika may constitute a fundamental test case for the theory of social evolution, but better information on their behaviour and phylogenetic relationships is needed to allow meaningful analyses.

Cooperatively breeding animals, in which helpers may participate in reproduction with dominant breeders, are ideal species for examining intraspecific variation in testis size because they often exhibit both monogamous breeding (low risk of sperm competition) and polyandrous breeding (high risk) within a population. However, little is known about testis investment as a result of sperm competition in these animals. The substrate-brooding cichlid fish Julidochromis ornatus has a cooperatively breeding system, in which some males mate monogamously and other males reproduce as dominant breeders or helpers within cooperatively breeding groups, in which male helpers frequently sire young. We examined the relationship between testis investment and male social status in relation to the risk of sperm competition. As predicted from sperm competition models, in groups with male helpers, both the male breeders and the male helpers invested more in testes mass, compared to breeding males without male helpers. We also found a positive relationship between the testes mass of male breeders and their male helpers, suggesting that males increase their investment in reproductive capability under the risk of sperm competition. Sperm competition models also predict that larger testes are associated with increased siring success. Our paternity analysis supported this prediction; we found a positive relationship between testis investment by male helpers and the number of offspring they sired.

An animal that tries to remove a mark from its body that is only visible when looking into a mirror displays the capacity for mirror self-recognition (MSR), which has been interpreted as evidence for self-awareness. Conservative interpretations of existing data conclude that convincing evidence for MSR is currently restricted to great apes. Here, we address proposed shortcomings of a previous study on MSR in the cleaner wrasse Labroides dimidiatus, by varying preexposure to mirrors and by marking individuals with different colors. We found that (1) 14/14 new individuals scraped their throat when a brown mark had been provisioned, but only in the presence of a mirror; (2) blue and green color marks did not elicit scraping; (3) intentionally injecting the mark deeper beneath the skin reliably elicited spontaneous scraping in the absence of a mirror; (4) mirror-naive individuals injected with a brown mark scraped their throat with lower probability and/or lower frequency compared to mirror-experienced individuals; (5) in contrast to the mirror images, seeing another fish with the same marking did not induce throat scraping; and (6) moving the mirror to another location did not elicit renewed aggression in mirror-experienced individuals. Taken together, these results increase our confidence that cleaner fish indeed pass the mark test, although only if it is presented in ecologically relevant contexts. Therefore, we reiterate the conclusion of the previous study that either self-awareness in animals or the validity of the mirror test needs to be revised.

Summary 1.Communities of different species are often structured according to niche differentiation associated with competitive interactions. We show that similar principles may apply on an ecological time-scale when individuals of a species having a wide size variation compete for resources, using the Lake Tanganyika cichlid Lobochilotes labiatus (5-30 cm). This species has a mouth especially adapted to suck up invertebrates from rock crevices. 2. Individuals defended feeding territories against similar-sized conspecifics, but not against differentsized ones. Thus, territories of similar-sized fish rarely overlapped, but up to a total of seven individuals (of seven size-ranks) had broadly overlapping territories with dissimilar-sized individuals. Comparison with expectation from the null model demonstrated clearly that observed size ratios between adjacent size rank were determined non-randomly regardless of sexual combinations. 3. Larger individuals took larger prey types of larger average size, but more importantly used wider rock crevices from which to suck food than smaller individuals. We calculated pairwise values of Schoener's index of diet overlap C d and the values of Levin's index of diet breadth B d (prey type and prey size) and the same for the width of the rock crevices used for foraging ( C r and B r ). C d remained high among all combinations of the seven ranks. In contrast, C r declined strongly in combinations of adjacent ranks (to 0·27), and was low or zero among further different size ranks. This shows that fish with overlapping territories divided the food resources largely through foraging site partitioning. Accordingly, B d did not depend on the size difference to the nearest two coinhabiting fish, whereas B r did. 4. We conclude that this L. labiatus community is structured non-randomly: body size-dependent effects on foraging site usage result in competition with, and territorial exclusion of, similar-sized individuals, but not of dissimilar-sized individuals that were accepted as coinhabitants. Accordingly, mean body size ratios (large/small) between two adjacent ranks were consistently approximately 1·28 [standard deviation (SD) = 0·07, n = 104], while approximately 1·34 from the null model (SD = 0·34, n = 10 400 simulations). We discuss our results as an example of Hutchinson's rule, applied originally to size ratios of different species.

SummaryParental roles and the amount of care in bi-parental fish have been assumed to be determined by sex. We studied the parental behaviour in a substrate brooding cichlid Julidochromis ornatus (40-90 mm in total length) in which both parents participate in care of eggs and young. In the study population, ca 80% of paired females were larger than their partners and pairs mated assortatively for size. Males spent more time with their offspring in female-largest pairs, whereas the opposite was found for male-largest pairs. These differences in the amount of care were more conspicuous when differences in body size were greater, whereas similar sized pairs shared parental tasks. These results suggest that the amount of parental care is largely affected by the relative size within pairs independent of sex in J. ornatus. However, the frequencies of defensive behaviours were not different in both female-largest and malelargest pairs. This indicates that parental roles would not completely change as the change of the body size difference within pairs. The larger parents were socially dominant over the partners regardless of sex, and observations of aggressive behaviours within pairs suggest that the larger fish are likely to make the partners perform parental care. Higher frequencies of aggressive encounters were observed in the similar-sized pairs than in the different-sized ones. Higher costs associated with frequent aggressive behaviours in the similar-sized pairs may be related to their small brood size, and may be partly responsible for the size-assortative mating with sexual size difference in this fish.