The ecological and developmental foundations of brood parasitism in a catfish

Domain: biology
Published: 2026-03-31
Editorial outcome: minor_revision

Abstract
Interspecific brood parasitism has evolved repeatedly from parental care. However, many non-avian brood-parasitic lineages have ancestors lacking parental care, leaving the foundations of brood parasitism in these lineages enigmatic. We examine ecological, reproductive, and developmental data from the Lake Tanganyika radiation of Synodontis catfishes, where one species, the cuckoo catfish, exhibits brood parasitism of mouthbrooding cichlid fishes. Our comparative analyses, coupled with experimental parasitism, suggest that a combination of ancestral traits (large eggs and rapid embryo development) enabled the origin of brood parasitism. Evolutionary innovations then presumably enhanced the success of brood parasitism after it emerged. Novel traits comprise frequent production of small clutches to effectively utilize host availability, the evolution of egg mimicry to facilitate host egg adoption, and modifications to development to enhance the performance of catfish embryos in the host’s buccal cavity. Interestingly, despite the distinct ecological and life history contexts of the origin of catfish brood parasitism, its evolutionary and developmental outcomes align with those of canonical avian brood parasites. This suggests that general patterns are repeated in the evolution of brood parasitism, despite disparate starting conditions.

Introduction
A loudly begging cuckoo chick, tirelessly fed by a pair of tiny songbird hosts, serves as a compelling illustration of brood parasitism. Obligate brood parasites—species that usurp the parental care of other species—have independently evolved seven times in birds, with over 100 bird species classified as obligate brood parasites 1. Theoretical and empirical insights from research on brood parasitism have influenced our understanding of coevolutionary dynamics 1–6 and life history evolution 7,8, though this research has been largely confined to avian model systems where parental care is ancestral to brood parasitism 6. However, interspecific brood parasitism is more ubiquitous in nature 1,6, with one-third (19 of 59) of cases found in lineages lacking parental care, with a transition to brood parasitism instead preceded by trophic ecological relationships such as saprophagy, scavenging and parasitoidy 9. Hence, to better understand the conditions that facilitate the evolution and persistence of brood parasitism, it is imperative to examine the origins and early stages of brood parasitism in lineages that lack parental care.

In this study, we combined ecological, reproductive, and developmental datasets across the Lake Tanganyika (LT) Synodontis catfish (Siluriformes, Mochokidae) radiation along with additional riverine Synodontis species to examine the origin and early evolution of brood parasitism in the cuckoo catfish, Synodontis multipunctatus. While other Synodontis species scatter their eggs over the substrate without parental care 10, the cuckoo catfish is an obligate brood parasite of mouthbrooding LT cichlid fishes 11. Many LT cichlids tend their eggs and embryos in their buccal cavity (termed mouthbrooding) 12. Cuckoo catfish exploit this advanced mode of parental care by intruding on spawning cichlid pairs and spawn themselves 13,14. As adult cuckoo catfish are also vigorous egg predators 15, the spawning female cichlid quickly responds to the risk of egg predation by hastily picking up her eggs, inadvertently collecting some eggs deposited by the intruding cuckoo catfish 16. Cuckoo catfish offspring is then brooded in safety alongside the cichlid eggs in the female’s mouth. The catfish eggs develop faster than those of the host and, after hatching, feed on the host’s offspring 17,18. This system resembles the relationship between cuckoos and their hosts, with the host deceived into caring for the cuckoo's offspring and incurring a significant cost to its own reproductive success. Given that adult cuckoo catfish are potent egg predators, we hypothesized that the origin of brood parasitism in the cuckoo catfish has primarily arisen from the predation of adult catfish on cichlid eggs during spawning 19. Here, we investigate the ecological, reproductive, and life history background of the LT Synodontis to identify traits associated with the origin and evolution of brood parasitism in Synodontis.

The cuckoo catfish is part of a small radiation of LT Synodontis. This lineage consists of two clades (Fig. 1a). The first (Clade I) comprises the cuckoo catfish (S. multipunctatus) and deepwater (depth >40 m) S. granulosus. The second (Clade II) includes S. petricola, S. irsacae, and S. polli, all commonly found in rocky habitats at depths from 5 m alongside the cuckoo catfish 22,24. The age of the LT Synodontis radiation has been estimated to be 2.7 (1.7–3.9) million years ago (Mya), with similar dating for further diversification within Clade I (1-2 Mya) and Clade II (0.9-1.9 Mya) 25.

We demonstrate that cuckoo catfish are trophic generalists rather than specialized egg predators. Their reproductive traits combine ancestral and novel characteristics that facilitate brood parasitism and consist of small batches of large, non-adhesive eggs that appear to mimic the large yellow eggs of their hosts. Developmental adaptations of the cuckoo catfish include enlarged jaws, pharyngeal teeth and associated skeletal supports, as well as accelerated mineralization of their dentition and supporting skeleton, enabling them to grasp and puncture host offspring and feed on their nutrient-rich yolk inside the cichlid buccal cavity. Many of these traits likely evolved in other contexts but were later co-opted to enhance the brood parasitic strategy of the cuckoo catfish.

Results
Brood parasitism in the cuckoo catfish is not associated with trophic specialization

Brood parasitism may evolve from a trophic relationship between parasitic and host species, including the exploitation of host eggs 9. Furthermore, many evolutionary radiations originated from dietary specialization 12,26,27. Thus, we examined gastrointestinal tracts and employed stable isotopic analysis to investigate whether the cuckoo catfish occupies a specific trophic niche within LT Synodontis. Considering that host egg predation is a feasible stepping stone to interspecific brood parasitism 9,19, we hypothesized that the cuckoo catfish specializes to feed on nutritionally rich cichlid eggs.

First, we analyzed the diets of all five LT Synodontis species collected in the lake. Dissecting the gastrointestinal tracts of 122 specimens and calculating Bray-Curtis dissimilarities (BCDs) based on volumetric estimates of their diet revealed that the primary differences among LT Synodontis species were associated with the relative contribution of sponges, benthic insect larvae, fish tissue, and algae (Fig. 1b, Likelihood Ratio tests: Table S1, SIMPER analysis: Table S2). The diet of the cuckoo catfish quantitatively differed from those of all other LT Synodontis species (multivariate GLM, P < 0.001; Table S2), and uniquely amongst LT Synodontis, the gut of one (of 30 examined) cuckoo catfish indeed contained fish eggs. However, the small size (<1 mm), spherical shape and high abundance (n = 257) of consumed eggs clearly indicated the eggs did not belong to any cichlid species confirmed to be host of the cuckoo catfish 28. The cuckoo catfish is obviously not a specialized egg predator. Instead, its diet is very generalized; its gastrointestinal tracts contained a mix of dietary components, featuring a high proportion of benthic aquatic invertebrates from soft sediments (Fig. 1b). This is despite that three other LT Synodontis species are clear dietary specialists (Fig. 1b); the variation in their within-species BCD values is negligible compared to between-species BCDs (<0.32, Fig. 1c, Table S4). The diet of S. granulosus consists solely of fish tissue (bones and ctenoid scales of either cichlids or Lates, Fig. S1) 29,30, typical of predators and scavengers. In contrast, S. polli is an herbivore, feeding on algae and plant detritus. The third specialist (S. irsacae) fed primarily on sponges (Porifera) (Fig. 1b, c). Like the cuckoo catfish, S. petricola is identified as a dietary generalist (Fig. 1b, c; Table S4).

The examination of gastrointestinal tracts offers only a short time window for food consumption. To compare time-integrated trophic ecology, we analyzed signatures from stable isotopic (SI) ratios of δ13C and δ15N in muscle tissues from 198 catfish. The results confirmed substantial trophic niche separation among LT Synodontis (Fig. 1d, Table S5). All species differed in their trophic position, except for the similar SI signatures observed in the cuckoo catfish and S. petricola (PERMANOVA: Table S6), two trophic generalists. The trophic level of S. granulosus was higher than that of a specialist predatory cichlid (Fig. 1e), suggesting that S. granulosus is a fish scavenger rather than a predator 31. The herbivorous S. polli encompassed the combined niches of two herbivorous cichlids 12 and sponge-eating S. irsacae occupied an extreme dietary niche with no known LT cichlid equivalent (Fig. 1e). The SI trophic niche of the cuckoo catfish was broadest of all LT Synodontis species (Fig. 1d). When compared to the trophic niche space of the cichlid radiation in Lake Tanganyika, which is strongly underpinned by trophic ecology 12, the cuckoo catfish largely overlapped with the SI space of several cichlid species (Fig. 1e). This further discounts the hypothesis of its trophic specialization on fish eggs, which would be associated with a higher δ15N signature 32.

Both diet and SI analyses consistently demonstrate a trophic component in the LT Synodontis radiation. However, the generalized diet of the cuckoo catfish and the rare occurrence of fish eggs in its diet undermine our hypothesis that the cuckoo catfish is a specialized egg predator. While the detection window for eggs in the gastrointestinal tract is short 33 and may lead to underestimation, the gastrointestinal tracts of West African Synodontis species frequently contain fish eggs 34. Therefore, strict trophic specialization is not a key driver in the origin of brood parasitism in this lineage.

The reproductive traits of cuckoo catfish combine ancestral and novel characteristics that facilitate brood parasitism

One of the biggest challenges for brood parasites is to synchronize their reproduction with that of their hosts and to induce them to accept parasitic eggs. We predicted that the cuckoo catfish produces multiple small clutches to match the continuous breeding season of their hosts 35 and thus be ready to capitalize on forthcoming opportunities for parasitism. We also predicted that cuckoo catfish have evolved egg characteristics that facilitate host acceptance through mimicry in terms of egg size and coloration. The usual Synodontis spawning is seasonal and involves egg scattering, during which the eggs may adhere to a substrate 10. Since egg adherence may hinder collection by host cichlids and might compromise developmental performance in the host’s buccal cavity, we predicted decreased egg adhesiveness of cuckoo catfish eggs. We used hormonally-induced reproduction in five LT Synodontis and three additional riverine Synodontis species to collect data on standardized clutch and egg characteristics. We further obtained histological data on the gonads of four LT Synodontis species from individuals collected in the wild at the start and end of the rainy season (n = 239).

Notably, the clutch size of the cuckoo catfish (mean 28, range 12-45 eggs) was an order of magnitude smaller than that of all other Synodontis species (means of 208-767 eggs) (LM, P <0.001, Fig. 2a, Table S7). The size of individual cuckoo catfish eggs (2.7 mm) was approximately double that of other LT (1.3-1.8 mm) and riverine (1.3-1.5 mm) Synodontis (LM, P <0.001), except for its sister species, S. granulosus, which produced slightly larger eggs (2.8 mm) (Fig. 2b, Table S8 Synodontis egg size was not related to body size, as the cuckoo catfish (female total length 83 ± 1.9 mm) and S. petricola (87 ± 1.9 mm) were smaller than S. irsacae (99 ± 3.0 mm), S. polli (120 ± 1.7 mm) (Fig. 2c, d) and riverine Synodontis 10, as well as S. granulosus (131 mm), the largest LT Synodontis species. The color of cuckoo catfish eggs was distinctly shifted towards a yellow hue compared to the more transparent eggs of all other LT Synodontis and the opaque brownish eggs of riverine Synodontis (LM, yellow-blue CIE_B axis: P <0.001; red-green CIE_A axis: P ≤ 0.03; Fig. 2e, Table S9). This modification of the cuckoo catfish egg color and size appears to imitate notably larger, yellow eggs of host cichlids 35,37. Finally, while the eggs of three LT Synodontis were highly adhesive (50-95% remained adhered to a vertical surface), the cuckoo catfish and S. granulosus laid eggs with considerably lower adhesiveness (20-25% adhered; ordered-beta GLM, P <0.001). Low adhesiveness was not exclusive to the cuckoo catfish and S. granulosus, as it was also recorded in some riverine Synodontis (Fig. 2f, Table S10). We also conducted ancestral trait reconstructions and calculated evolutionary signal metrics (Pagel's λ and Blomberg's K) for reproductive traits. Significant phylogenetic signal was detected in egg coloration along the red–green axis, but not the yellow–blue axis, and moderate phylogenetic signal was observed for clutch size (Fig. S2).

Reproductive seasonality, based on the histological examination of the gonads of fish collected in the lake, was observed in a single species, S. irsacae, with 0% of females and 40% of males exhibiting ripe gonads in November and 93% and 100% in March (n=56; 2 tests: P <0.001 and P=0.003). In contrast, other LT Synodontis had 67-90% of fish with ripe gonads across sexes and species in November, and 94-100% in March (n=182 across three species; Table S11). The relative female investment in reproduction, calculated as ovary mass relative to body size in fish with ripe gonads, was highest in the seasonally spawning S. irsacae (GLM, P < 0.05), but comparable among other LT Synodontis, including the cuckoo catfish (Fig. 2c). Relative testis mass did not differ among species (GLM, P = 0.185; Fig. 2d).

The analysis of reproductive traits thus revealed that cuckoo catfish produce small batches of large, non-adhesive eggs that mimic the large yellow eggs of their hosts 35 which likely facilitates egg adoption by the hosts. Further, the large cuckoo catfish eggs contain large yolk (nutrients) reserves to support rapid early development 38. The regular production of small egg batches uniquely enables the cuckoo catfish to spawn every few days 14, while other traits conducive to brood parasitism (large egg size, low egg adhesiveness, and lack of reproductive seasonality) are not specific adaptations to a parasitic strategy but rather preceded its origin.

The early development of the cuckoo catfish has evolved to exploit the host clutch

In the host buccal cavity, cuckoo catfish hatch early and, upon consumption of their own yolk sac, prey on the host clutch 17. We predicted that cuckoo catfish embryos possess adaptations for predation on host offspring inside the cichlid buccal cavity, specifically in terms of the accelerated development of their dentition and jaw apparatus 17. We hormonally induced the reproduction of all five LT and two riverine Synodontis species and produced a standardized fine-scale ontogenetic series following in vitro fertilization. We then compared early growth and development, with particular emphasis on the oral and pharyngeal dentition.

All Synodontis species hatched within the first 2 days post-fertilization (dpf) and consumed their embryonic yolk sac at 6 dpf (Fig. S3). The cuckoo catfish and S. granulosus exhibited higher post-hatching growth rates than all other species (Fig. 4a). The offspring of these two species were nearly twice as large (LM, P <0.001, Table S12) and considerably more developmentally advanced at the age of 10 dpf (Fig. 4b). However, only the cuckoo catfish possessed a well-mineralized dentition at 6 dpf (Fig. 4c) when they started to consume host offspring 17,18. The cuckoo catfish also initially possessed larger jaws (Fig. 4c), pharyngeal teeth, and associated skeletal support (Fig. 4d, Fig. S4), as well as accelerated mineralization of its dentition and supportive skeleton (Table S13) in comparison to other LT Synodontis, including S. granulosus. Synodontis granulosus initially had a slightly larger pharyngeal dentition than other species (Fig. S4, Table S13), but the pace of its development and mineralization did not differ from the other species.

Comparison with the rate of dental development in other teleost fishes clarified that the development of the cuckoo catfish’s dentition and supportive skeleton is strongly accelerated 39,40. We propose that the cuckoo catfish has evolved a uniquely rapid post-hatching development, which enables its embryos to grasp and puncture host offspring to feed on their nutrient-rich yolk. These adaptive modifications of the cuckoo catfish derive from pre-existing traits, such as larger eggs and a more rapid post-hatching growth, which it shares with its sister species S. granulosus. However, S. granulosus do not possess dental and skeletal adaptations to feed on host embryos. These observations corroborate our hypothesis that the embryo development of the cuckoo catfish is under strong selection to enhance its ability to consume host clutches, resulting in the rapid development of embryonic dentition.

Potential for brood parasitism in S. granulosus

Considering that large egg size and rapid embryo growth are shared between the cuckoo catfish and its sister species S. granulosus, it might be speculated that S. granulosus represents an unrecognized brood parasite. Unlike all other LT Synodontis species, which are commonly bred in captivity and documented as egg scatterers 10,23, there is a lack of data on the reproduction of deepwater S. granulosus. Hence, we examined whether S. granulosus eggs and embryos can survive, develop, and feed in the buccal cavity of a host cichlid and repeated the same experiment with S. polli as a non-parasitic species control.

We experimentally inoculated cichlids 13 with S. granulosus eggs and tested their survival over the incubation period of 4, 7, 12 and 14 days, using the cuckoo catfish eggs inoculated into other mouthbrooding females as controls. First, we used a Lake Victoria cichlid (Haplochromis sp. CH44), a non-sympatric cichlid species which has not evolved egg rejection of the cuckoo catfish 13. We found that the survival rate of S. granulosus in mouthbrooding Haplochromis was high (75% of 56 eggs inoculated across 15 mouthbrooding host females and all incubation periods), and consistent with survival of the cuckoo catfish embryos (73.2% of 56 eggs across 14 mouthbrooding hosts; Binomial GLMM, P = 0.873) (Fig. 4a, b). The ability to survive incubation in cichlid mouth is not a universal feature of LT Synodontis; no embryos of non-parasitic S. polli were recovered from Haplochromis hosts (20 eggs across 5 mouthbrooding hosts) after 7 days of incubation, whereas 93% (26 of 28 inoculated eggs across 7 mouthbrooding hosts) cuckoo catfish control embryos survived that period (Fig. 4c). Cuckoo catfish embryos grasp and pierce host embryos to suck their yolk sacs (Fig. 4d, e) from day 4 post-hatching and no cichlid embryos were found in experimentally parasitized females (n = 13) at age of 12-14 days. Despite having a comparable body size at that age (Fig. 3a, b), the dentition of S. granulosus embryos is relatively undeveloped (Fig. 3c, d), and the alimentary tract of S. granulosus offspring recovered from host buccal cavity contained only traces of material resembling yolk (Fig. 4f) while host offspring also disappeared (n = 9 host clutches).

When S. granulosus eggs were inoculated into the buccal cavity of a natural cuckoo catfish host (Shuja horei) 28 (which can effectively decrease the load of cuckoo catfish eggs 41), none of the 18 eggs (across 6 mouthbrooding S. horei) survived 4 or 7 days of incubation, compared to a 30% survival rate of cuckoo catfish eggs (6 of 20 eggs across 4 of 6 mouthbrooding S. horei) (Fig. 4g). This finding suggests that the apparent exaptations (i.e., adaptations originally evolved outside parasitism context) to brood parasitism in S. granulosus are sufficient to survive 14 days of incubation (i.e. 8 days after yolk sac consumption) in an evolutionarily naive host, although their capacity to extract resources from the host clutch was inefficient. However, these putative exaptations to brood parasitism failed to support survival in the more challenging environment of LT cichlid which is a common host of the cuckoo catfish 28 and has evolved defenses against parasitism 17.

If S. granulosus is indeed an unrecognized brood parasitic species parasitizing deep-water cichlids, it possesses many traits of the generalized Synodontis life history. First, its very large clutches exceed the capacity of host buccal cavity by more than an order of magnitude. Second, the eggs do not visually imitate host eggs. Third, their ovulation frequency is not adapted to the opportunistic exploitation of unpredictable opportunities to parasitism. Finally, its embryonic dentition is not adapted to efficient extraction of resources from host embryo, as seen in the cuckoo catfish. Nevertheless, by using hosts from shallow-water habitats in our experiment, we cannot rule out the possibility that S. granulosus has specialized to parasitise deep-water cichlids. Irrespectively of breeding strategy of S. granulosus, its reproductive and developmental characteristics appear to represent ancestral conditions within this small clade and perhaps exemplify the transient state, with many traits which have evolved in a different context, and from which brood parasitism in cuckoo catfish might have evolved.

Methods
All research methods and protocols adhered to national and institutional animal care and use guidelines, administered under permit No. CZ62760203 and approved by ethical boards of the Institute of Vertebrate Biology and the Czech Academy of Sciences (approval No. 32-2019). Fieldwork and sample export have been approved by research permits (Zambia: K-4335/18 KA/K.48/18 and individual study permits of MR, HZ, RB, MP, VB and SK), Tanzania (TAFIRI/HQ/RES.CLEARANCE/82, Costech: 2022-603-NA-2022-228). Import of samples to EU has been approved by Czech State Veterinary Administration (Zambia: SVS/2024/064505-G, Tanzania: SVS/2024/064505-G).

Adult fish collection

Adult Synodontis and cichlids were collected during nine field expeditions to Zambia and Tanzania between 2019 and 2024. Fish were collected during Scuba dives using hand nets, stop nets, and baited minnow traps and brought to the surface for further processing. Additional samples (of deepwater S. granulosus) were obtained from deep-water gill nets deployed by local fishermen.

Fish were typically processed within the first two hours after collection. They were euthanized with an overdose of clove oil, and a piece of fin was stored in 96% ethanol for DNA barcoding and further genetic analyses. The fish were measured, weighed, labelled, fixed in 6% formaldehyde, and subsequently transferred to 70% ethanol for long-term storage. A piece of muscle from the fish flank (dorsolateral side) was taken and stored in ethanol for the analysis of stable isotope ratios. Vouchers are stored at the Natural History Museum in Vienna, and collections are held at the Institute of Vertebrate Biology, Czech Academy of Sciences 49. Morphological identifications were confirmed by DNA barcoding using the cytochrome c oxidase I gene 22.

Phylogeny

A detailed ddRADseq-based phylogeny with broad geographic representation has been published elsewhere 22. To visualize the phylogenetic background of our study species, we selected one individual for each study LT Synodontis species and one riverine outgroup (Synodontis nigrita). The tree was inferred using a maximum likelihood approach in IQ-TREE v.2.0.7 and a concatenated alignment of 6536 ddRADseq loci. We used the same nucleotide substitution model (TVM+F+R2) as in 22 and 5000 ultrafast bootstrap replicates.

Dietary data and analyses

A diet analysis was conducted on samples collected from the southern tip of the lake in Zambia, specifically at Mpulungu (Chituta Bay, Kalambo Lodge, Mutondwe Island, Mpulungu) and Ndole Bay (Chimba Rocks, Mpende Fisheries, Katete, and Cape Kachese), where all examined fish species co-occur locally. All primary sample data are georeferenced 49. For the diet analysis, we included only fish processed within two hours of their collection. Individuals with unidentified digested matter (i.e. with fish bait used in the traps) in their gastrointestinal tract were excluded from further analyses.

In the laboratory, all specimens were measured for total length (TL) to the nearest 1 mm and weighed (W) to the nearest 0.01 g. Gut contents were weighed to the nearest 0.001 g and stored in glycerin. Food items were subsequently identified to the lowest practical taxon (i.e., to order, family, genus, or species) under a stereomicroscope (Nikon SMZ1000, Japan) and counted, or their counts estimated in cases of high abundance of particular prey items. The total number and biomass (to the nearest 0.1 mg) of each prey type were recorded for every fish specimen. Prey items were combined by taxon and quantified by frequency of occurrence and percentage of biomass. Proportions of diet were calculated from the contents of the gastrointestinal tracts, excluding the category ‘animal remains’ which were decayed beyond a state to identify it. Chironomidae, Ephemeroptera, Trichoptera, Coleoptera, and Heteroptera were pooled as ‘benthic insects‘. All other diet categories were kept separate, as we could not unambiguously distinguish their ecological origins (i.e., benthic vs. planktonic, surface-bounded vs. free roaming). We analyzed 122 fish (30 S. multipunctatus, 3 S. granulosus, 41 S. irsacae, 9 S. polli, and 39 S. petricola).

To test species differences in diet composition, we used Multiple Response Permutation Procedure (MRPP). We calculated the average within- and between-species differences (Bray-Curtis dissimilarities, BCDs) in diet compositions (meandist; package ‘vegan’) 50. Dietary specialists are predicted to have small average within-species BCDs, whereas the average within-species BCDs of dietary generalists is the same as or higher than the average between-species BCDs. We then examined the overall species effect on the diet using a Multivariate Generalized Linear Model (multivariate GLM) with a negative binomial error distribution (manyglm; package ‘mvabund’) 51. This approach addressed the challenge of interpreting statistical significance in distance-based multivariate comparisons, which is complicated by the confounding effect of abundance on variance in the data. Subsequently, an ANOVA was conducted to determine the importance of specific food categories in the separation across Synodontis species using the Likelihood Ratio statistic (LRT) (Table S1). Only four food categories (algae, sponges, zoobenthic insects, Copepoda, and fish scales) had a significant effect on the model fit (Table S1). We then employed SIMPER analysis to assess the importance of individual food categories in the pairwise contrasts among Synodontis species. Food categories contributing cumulatively to >80% dissimilarity within each species pair are listed in Table S2. Finally, a multivariate PCoA (Principal Coordinates Analysis) was applied to represent the BCD values in Euclidean space. Since the variables ‘locality’, ‘sex’, and ‘body size’ did not significantly contribute to the multivariate GLM, only the variable ‘species’ was included in the final analysis.

Stable isotope data and analyses

To estimate the extent of trophic niche separation among LT Synodontis, we measured the stable carbon (δ13C) and nitrogen (δ15N) isotope composition of 198 Synodontis specimens (5 species), 62 cichlids (8 species), and 34 baseline items from various trophic levels. A piece of fish muscle (or entire individuals for the baseline items) was initially stored in ethanol and dried in the laboratory. To account for a shift in the values of δ13C and δ15N caused by preservation in ethanol 52, we collected a subsample of each species as dried samples paired with the ethanol-based sample. The reported stable isotope (SI) values were corrected using the average shift in SI values for each species separately. Samples (2 mg of dry mass) were analyzed by Iso-Analytical Ltd. (Crewe, UK) using the Europa Scientific 20-20 IRMS elemental analyzer calibrated against international standards. The δ13C values were corrected for lipid content following established protocols 53. Isotope values of baseline items were corrected for trophic enrichment according to specified methods 54 and organized into informative categories (i.e., macrophyta: a combination of 4 plant species). Full details are provided in the source data 49.

The δ13C and δ15N values were represented as biplots. Baseline and Synodontis values were overlaid with stable isotope signatures of cichlids (8 species with well-defined trophic positions 12, encompassing a large isospace). This allowed us to compare the degree of overlap in trophic positions of LT Synodontis with the locally coexisting cichlid trophic guilds. The δ15N values indicate sample positions in the food chain; δ15N enrichment increases with trophic levels. The δ13C values help to indicate primary producers at the start of the food chain and differentiate between littoral and pelagic environments; higher values suggest shallow (littoral) sources, while lower values indicate deeper (pelagic) habitats 31,55.

We employed permutation-based analysis of variance (PERMANOVA) to examine differences in SI signatures among Synodontis species using the function ‘pairwise.adonis’ (R package pairwiseAdonis) 56, which acts as a wrapper for the ‘adonis’ function from the R package ‘vegan’, with 10,000 permutations. We applied the Benjamini-Hochberg procedure to account for multiple testing.

Reproductive traits

Gonads were dissected from formalin-fixed specimens (127 females, 114 males) of Synodontis collected at the beginning (November 2021) and final part (March 2022) of the rainy season in the Zambian region of the lake (Mpulungu area). The fish were dissected in the laboratory, the gonads were separated, weighed to the nearest 0.01 g, immersed in 70% ethanol and processed by Histoplast (Sigma). For each gonad, 60–80 semi-serial sections were examined using hematoxylin and eosin (H&E) staining under light microscopy to detect the presence of mature oocytes and spermatozoa 57. We identified a season-specific proportion of sexually mature individuals, namely fish with mature oocytes (indicated by the presence of at least some gametes at the two latest stages of vitellogenesis or spermiogenesis) 57,58. We compared the proportion of ripe gonads between the beginning and end of the rainy season for each species and sex separately, using chi-squared tests.

Investment in gonads was modeled as gonad mass (mg) relative to body size (TL, mm) for females and males separately. Gonad mass was log-transformed before analysis and modeled (Linear Model) as a function of body size (TL), species, and their interaction. When the interaction between TL and species was not significant (i.e., the slope of the relationship between gonad mass and TL was not species-specific), a simplified model (without interaction) was employed, and its fit was compared with the more complex model using ANOVA. Tukey tests were used for pairwise differences when species significantly differed in their relative investment in reproduction. Two female S. irsacae were excluded from the statistical analysis as major outliers (ovaries of 8.03 and 8.58 g compared to <2.7 g for all other females). Including these two females in the dataset would add stronger support to the outcome of larger ovaries in S. irsacae. The TL measured on this set of fish was used to compare body size across Synodontis species.

A comparison of egg traits (size, adhesiveness, coloration) among five LT species and three riverine species of Synodontis was conducted on eggs obtained following hormonal stimulation of fish (Ovaprim, a salmon gonadotropin-releasing hormone analog, Syndel Laboratories Ltd., Canada) 59. Females, under clove oil anesthesia, were injected with 0.5 μL/g of Ovaprim, and ripe eggs were gently stripped out after 12 to 24 hours onto glass Petri dishes. After 3 to 5 minutes, the dishes containing the eggs were carefully transferred into a larger glass tank filled with water and positioned vertically (at 90°). Egg adhesion was determined as the proportion of eggs remaining on the glass surface after 5 seconds compared to the total number of eggs initially placed on the dish. Eggs that did not adhere to the glass surface fell away within this 5-second period. Egg adhesion was modelled as the proportion of eggs adhered to the Petri dish in a vertical position relative to the total number of eggs on the dish, using a Generalized Linear Model (GLM) with ordered beta distribution and a logit link. The egg adhesion of the cuckoo catfish was set as the reference value, and contrasts with the egg adhesion of other species were tested.

To compare egg coloration, a subsample of eggs from each female was placed on a separate glass Petri dish. Standardized pictures (RAW format) were taken over a standard grey card (18% standard reflectance, KELDAN grey card including a scale tape) from each subsample of eggs immediately after the test for egg adhesiveness. Raw pictures were adjusted to the grey background in the Mica ToolBox (ImageJ plugin, 60 and converted into a linear multispectral image. The linear images were transformed into cone-catch images (representing the human visible range in daylight) by using a custom-prepared cone-catch model for the digital camera used (Canon 7D). This was carried out by taking a RAW image of the KELDAN color checker under natural daylight, preparing a linear multispectral image of the color checker, and following the standard protocol and the built-in algorithm of the Mica ToolBox to generate a cone mapping model (using the CIE 1931 400–700 nm, D65 400–700 nm configuration). The cone-catch images were then further transformed into CIELab color space within the Mica ToolBox. Seven to ten eggs were measured per individual clutch (except for only three eggs in a S. macrostigma clutch). The average values for each clutch were used in further analysis. Egg coloration was quantified by CIE_A and CIE_B axes. The CIE_A represents the red-green continuum (positive values: red, negative values: green, zero: grey). The CIE_B represents the yellow-blue continuum (positive values: yellow, negative values: blue, zero: grey). A Gaussian Linear Model was used to compare the CIE_B and CIE_A among species, with the cuckoo catfish set as the reference species. The contrasts between the cuckoo catfish and the other species are reported.

To analyze egg size, the images taken for color comparison (which included a scale) were used, and the egg diameters were measured using ImageJ 1.53t. A Linear Mixed Model (LMM) with Gaussian error distribution compare egg sizes across species. Up to 10 eggs were measured from the same clutch, with clutch ID modelled as a random intercept to account for the lack of independence. The cuckoo catfish served as the reference species.

Clutch size was estimated from the same set of clutches as other traits (i.e., following hormonal stimulation). The eggs were counted on Petri dishes. A linear model using log-transformed data was employed to calculate interspecific contrasts. We also compared the clutch sizes obtained during natural egg maturation and following hormonal stimulation in the cuckoo catfish (where comparison was feasible), finding no difference (Linear Model, P = 0.728, N = 9 and 10 clutches for hormonally induced and natural clutches, respectively). All statistical analyses were conducted using glmmTMB 61. We tested model dispersion, zero inflation, and model misspecification using the DHARMa package 62. Post-hoc pairwise comparisons were calculated using emmeans 63 with Benjamini-Hochberg procedure for multiple testing.

We then used the phytools package 64 to reconstruct ancestral character states and the geiger package 65 to calculate evolutionary signal metrics. Blomberg's K measures the relative strength of phylogenetic signal, estimating how closely related species resemble each other compared with expectations under Brownian Motion (BM) evolution. We used Pagel's λ to estimate the extent to which the phylogeny explains trait covariance. Synodontis nigriventris and S. macrostigma were used as outgroups.

Developmental traits

To compare the growth and development of LT Synodontis, we conducted in vitro fertilization in the laboratory to raise the offspring under standardized conditions. Wild-caught adult fish were treated with Ovaprim, according to 59, with species-specific modifications in timing. The eggs and sperm were extracted from the adult fish and mixed for a minimum of 10 min. The fertilized eggs were placed in a water recirculation system and maintained at 27 ± 0.5 °C. One day post-fertilization, all non-developing (i.e., unfertilized) eggs were removed. Embryo samples were collected at 24-h intervals until the age of 10 days post fertilization and stored in 4% PFA at 4 °C.

The body size of fish was measured from the snout to the end of the caudal fin (Total Length, TL) to the nearest 0.1 mm. Growth rate was modelled using a Gaussian Linear Model on log-transformed TL, with species, age, and their interaction as fixed factors in the glmmTMB package. Species-specific contrasts were also analyzed at 10 dpf.

To assess the development of dentition, PFA-stored embryos of all LT and two outgroup Synodontis species were washed in PBS for 10 min at room temperature (RT) and then bleached in a solution containing 1% KOH and 3% hydrogen peroxide (1:1) under light. Subsequently, the specimens were washed in distilled water for 30 min at RT and placed in a saturated sodium tetraborate (Sigma-Aldrich: cat. No. 71996) solution overnight. Further staining for mineralized tissues was carried out in Alizarin Red S (Thermo Scientific Chemicals, cat. No. 400481000) solution in 1% KOH overnight, followed by washing in distilled water for 30 min. Embryos were then transferred to 1% trypsin (Sigma-Aldrich, cat. No. T4799) in 2% sodium tetraborate solution for 1-3 days (depending on the embryo size). After that, specimens were moved into glycerol through a series of glycerol in 1% KOH solution (25%, 50%, 75%) with each step performed overnight. Once in glycerol, the specimens were squeezed and coverslipped. Photographs were taken using an Olympus BX51 microscope under fluorescent light with an Olympus DP74 camera, employing the cellSens software. Final images were composed in Photoshop 2023 from photos of varying focus distances; contrast and colors were adjusted.

For skeletal development, staining followed the protocol by 66. Photographs were taken using an Olympus SZX12 stereoscopic microscope equipped with an Olympus u-tv0.5xc-3 camera and QuickPhoto Micro (ver. 2.3) software, during which Z-stacking was also performed. Final images were edited in Photoshop to smooth the background and minimize distraction.

Potential for S. granulosus parasitism

Based on the reproductive and developmental traits of S. granulosus, we experimentally assessed whether the existing set of traits enables its offspring to survive in the buccal cavity of a mouthbrooding cichlid. Six pairs of wild-caught adult S. granulosus were hormonally stimulated (Ovaprim, as detailed above) for in vitro fertilization to obtain viable embryos. The fertilized eggs were then placed in tumblers with recirculating incubation water 28. Water for incubation was prepared using reverse osmosis water with 11 g/L Cichlid Lake Salts (Seachem Laboratories Inc., USA). The pH was adjusted to 8.5–9 using Tanganyika Buffer (Seachem Laboratories Inc., USA), and the temperature was set to 27 (± 0.5) °C. The eggs of S. multipunctatus (used as parasitic controls) were obtained from 10 pairs and treated in the same manner as the eggs of S. granulosus. The eggs of non-parasitic control S. polli were obtained from 9 pairs of hormonally stimulated parents. As the experiments with S. granulosus and S. polli were conducted at different time, a separate control using S. multipunctatus eggs (obtained from 10 parental pairs) was completed during S. polli experiment. After 24 hours of incubation, the experimental eggs were examined under a binocular microscope to confirm their proper morphological development. The same water quality was used for incubation and subsequent experiments.

To maximize the simultaneous availability of freshly spawned mouthbrooding females, we separated the sexes of both host species (S. granulosus experiment: 54 females and 19 males of Haplochromis sp. CH44; 30 females and 15 males of S. horei for two weeks; S. polli experiment: 69 females and 16 males of Haplochromis sp. CH44 for three weeks). Subsequently, we housed females and males together for three days and used any freshly mouthbrooding cichlid female from the two species for inoculation. Haplochromis sp. CH44 is a species from Lake Victoria, known to be susceptible to experimental brood parasitism by S. multipunctatus eggs and embryos 13 and producing relatively small eggs (2.5 mm). Shuja horei is a natural host of S. multipunctatus, which likely possesses defense mechanisms 41 and produces larger eggs (4.7 mm). We used two or four developing Synodontis eggs at 24 hours post-fertilization to artificially inoculate a mouthbrooding female. The eggs were collected into a Pasteur pipette, which was then gently inserted into the mouths of the brooding cichlid female and released 13.

Inoculated host females were transferred to 60 L housing tanks, which were equipped with a perforated false bottom (mesh size 0.5 cm), an air-driven sponge filter, and a clay pot serving as a shelter. The tanks were dimmed to minimize external disturbances. The females were allowed to incubate their clutches for 4 days (S. horei host: 3 clutches per Synodontis species; Haplochomis host: 2 clutches of S. granulosus) and 7 days (S. horei host: 3 clutches per Synodontis species; Haplochomis host in S. granulosus experiment: 4 clutches of S. granulosus and 7 clutches of S. multipunctatus; Haplochomis host in S. polli experiment: 5 clutches of S. polli and 7 clutches of S. multipunctatus). Because S. polli embryos had zero survival in the benign Haplochromis hosts, we did not inoculate their eggs into more challenging host (Tanganyikan S. horei). In contrast, the high survival rate of S. granulosus in Haplochomis hosts led us to extend the inoculation of 3 clutches to 12 days to test whether the offspring of S. granulosus can survive in the host's buccal cavity after all resources from their own yolk sac were depleted (which occurs at day 6 post-fertilization) and their survival depended on extracting resources from the host clutch. Finally, we further extended the duration of incubation to 14 days, which covers the period of host offspring release. At the end of incubation (day 4, 7 or 12 post-fertilization), mouthbrooded clutches were flushed from the female’s mouth using a Pasteur pipette, and surviving Synodontis offspring were counted. For replicates with 14 days post-fertilization, all live offspring retained in the tank (protected under the false perforated bottom) were counted and scored as surviving.

The success or failure of development of S. granulosus in cichlid buccal cavities was tested using GLMM with binomial error distribution, with Synodontis species (S. granulosus as treatment, S. multipunctatus as control), duration of incubation and their interaction (to account for potential species-specific survival) as fixed factors. The interaction term was not significant (P = 0.635) and removed from the final model. Host female identity was used as random intercept to account for non-independence of groups of 2 or 4 Synodontis eggs incubated in the same host. The analyses of experiments with S. granulosus and Shuja horei and with S. polli and Haplochomis did not converge, likely due to zero survival in the treatment groups (0 of 18 S. granulosus eggs compared to 6 or 20 control cuckoo catfish eggs; and 0 of 20 S. polli eggs compared to 26 of 28 control cuckoo catfish eggs, respectively). While Fisher’s exact test demonstrated significantly lower survival of S. granulosus in S. horei (P = 0.021) and of S. polli in Haplochromis (P < 0.0001), we do not report these analyses in the main text as the input data were more structured (grouped by host female individuals) than Fisher’s exact test can handle. Instead, we declare zero survival of treatment embryos in mouthbrooding hosts which strongly contrasts with common survival of their respective controls.

All statistical tests were two-tailed. All individual measurements were taken from distinct individuals. Any covariates dropped form the final models are reported in the methods.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript by Reichard and colleagues describes an investigation of the evolution of brood parasitism in a species of African Rift Lake catfish, Synodontis multipunctatus, that manipulates mouth-brooding cichlids into caring for its young, with the catfish larvae additionally feed on the host’s own offspring. As the authors point out, their system involves the evolution of brood parasitism in a lineage with an ancestral condition of no parental care. This situation contrasts with more extensively studied examples in birds, where the ancestral condition was the presence of parental care.\nThe authors first test the hypothesis that the evolution of brood parasitism in S. multipunctatus was facilitated by an initial stage of adults feeding on the cichlid eggs; such trophic interactions have been documented in other systems of brood parasitism. They document the gut contents of wild-caught specimens of five species of Lake Tanganyika Synodontis, finding variation in diet among species and that S. multipunctatus has a generalized diet, rather than being a specialized egg predator. This result was further supported by stable isotope analysis of muscle tissue from the Synodontis species.\nThe authors next induced reproduction in the lab of the same five Lake Tanganyika Synodontis species along with three riverine outgroups in the genus. They compared reproductive characteristics, as well as embryonic and larval development, to determine whether several proposed adaptations to brood parasitism arose before or after the divergence of S. multipunctatus from its egg-scattering relatives. Among the reproductive characteristics of S. multipunctatus, large egg size, low adhesiveness of eggs, and lack of spawning seasonality appeared to have been present before the evolution of brood parasitism. In contrast, egg color mimicking that of the host cichlids as well as the frequent production of small clutches of eggs appear to be unique to S. multipunctatus.\nSimilar analysis of embryonic and larval development suggested that the rapid larval growth of S. multipunctatus is shared with its sister species S. granulosus, while rapid mineralization of oral and pharyngeal teeth and jaws (enabling feeding on cichlid eggs) is unique to the former species.\nFinally, the authors tested whether larvae of S. granulosus could survive in the mouths of cichlids and/or feed on the young of the host. They found that they could survive, but their ability to feed on eggs was significantly lower than that of S. multipunctatus. Such survival was also greater in a cichlid from a different lake compared to that in a sympatric cichlid that has likely evolved defenses against parasitism by S. multipunctatus.\nThis is a well-designed and executed study, and the manuscript is clearly written. The S. multipunctatus system is a fascinating one that I believe will be of wide interest. The finding that some of the likely adaptations for brood parasitism were present before its evolution is also likely to be of general interest to evolutionary biologists\nI have only minor suggestions for improvement as follows.\n1) Evolution of reproductive parasitism from species with no parental care brought to my mind the “nest associates” that spawn in the pebble mounds of the North American chubs of the genus Nocomis. (Betts et al 2024. Freshwater Biology, 69, 450–459. https://doi.org/10.1111/fwb.142242). Is this of any relevance to the Synodontis system? If not, there is no need to comment on it in the manuscript.\n2)The authors find that the diet of S. granulosus consisted exclusively of fish tissue. Can they speculate on whether this is the ancestral condition of the S. multipunctatus lineage, and if so, whether this might be the trophic interaction between catfishes and cichlids that preceded brood parasitism?\n3) It appears from Figure 2 (e and f) that the larger egg size of S. multipunctatus is not associated with larger adult body size. Can the authors comment explicitly on the adult size of the five Lake Tanganyika species?\n4) In line 224, it is indicated that host offspring disappeared in the presence of inoculated S. granulosus. Was this disappearance complete in both S. multipunctatus and S. granulosus, or was it less in the latter species?\n5) In lines 254-255 the authors suggest that “Occasional interactions with cichlid hosts may have been sufficient to trigger the origin of obligate brood parasitism …” Can they speculate on what these interactions might be? Do the cichlids occupy areas that would be attractive for egg scattering? Is the spatial overlap between S. multipunctatus and the host cichlids known when the former is not spawning?", "reviewer_2": "The manuscript “The ecological and developmental foundations of brood parasitism in a catfish” presents a comprehensive and well-written study that aims to shed light on the origin and early evolution of brood parasitism within the Lake Tanganyika Synodontis radiation. It seems this system is particularly intriguing because it offers a rare opportunity to study the evolution of brood parasitism in a lineage that lacks parental care.\nThe authors adopt an integrative approach, which I find highly commendable, combining multivariate ecological quantification, various reproductive life-history traits, developmental series (covering growth, dental development rates), and an experimental brood parasitism assay. The scope of the data collection is impressive and I really enjoyed reading the manuscript!\nIn summary, the main findings indicate that the cuckoo catfish differs from its closest relatives in numerous traits, particularly in egg coloration, clutch size, egg size, and the timing of jaw development. Furthermore, both the focal species and its sister species exhibit elevated growth rates and reduced egg adhesion. Taken together, the suite of traits observed in the focal species align well with characteristics expected to facilitate brood parasitism.\nThe breadth of data surely provides valuable context for discussing the evolutionary foundations of brood parasitism from multiple perspectives, which I consider a major strength of the work. However, I have a few concerns regarding the extent to which the analyses fully address the central evolutionary question, as well as some minor specific comments:\n1. The conclusive power of the model system\nAll statistical tests in the manuscript address whether the cuckoo catfish S. multipunctatus differs from other species. While valid in that context, this does not answer the broader question implied by the authors: what traits relate to the origin and evolution of brood parasitism? Addressing that question would require phylogenetic comparative analyses – which are absent from the manuscript.\nUnfortunately, the nature of the system offers very limited statistical power, as it effectively has a comparative sample size of N = 1 for brood parasitism (since the trait appears to have evolved only once within the clade). Given this limitation, can we realistically identify traits that are statistically linked to the origin and evolution of brood parasitism?\nUndoubtedly, the observed trait shifts make intuitive sense in light of brood parasitism and generate intriguing hypotheses; nevertheless, the work remains primarily descriptive and hypothesis-generating, rather than formally detecting signals of adaptation. While this work is a valuable contribution – especially for broader comparative context or discussions across systems – the limitations should be explicitly acknowledged, and a more cautious tone adopted when interpreting patterns as adaptive responses or drivers.\n2. The experimental brood parasitism assay\nThis experiment is highly intriguing and a valuable step toward understanding potential parasitism in S. granulosus. However, its interpretative strength could be improved by including a known substrate-spawning Synodontis as a negative control.\nThe current experimental design only demonstrates that S. granulosus offspring do not survive in what they call “sympatric host” mouths. However, the absence of a non-parasitic control makes it difficult to assess whether the high survival in the naïve host (where both species performed well) is expected for a non-parasitic Synodontis or if S. granulosus falls somehow along a parasitic spectrum?\nA second concern I have is the match between S. granulosus and the tested host species S. horei. As mentioned in the manuscript, S. granulosus is a deep-water species (>40 m), whereas S. horei is a well-documented shallow-water dweller rarely encountered below 5–10 m and is hence unlikely a true sympatric host for S. granulosus (the match with no embryo survival). In contrast, S. horei is a known true sympatric host of S. multipunctatus (the match that performed very well). So, how can host-specificity within the lake be excluded as a driver of this pattern?\nMinor specific comments:\n- The use of the term Pre-adaptation: This may seem like a matter of semantics, but I would encourage the authors to reflect on their use of the term pre-adaptation. While it has been widely used historically, the term can imply evolutionary foresight. The perhaps more widely accepted term is exaptation, which conveys that a trait originally evolved for one functon(or without a specific function) and was later co-opted for another.\nRegardless of which term is chosen, I recommend briefly defining it at first use to ensure clarity.\n- L- 63: In the introduction, the hypothesis: ”We hypothesized that the origin of brood parasitism in the cuckoo catfish has primarily arisen from the predation of adult catfish on cichlid eggs during spawning” does feel somewhat abrupt since there is little lead-up to the idea that predation might be the key precursor.\n- Figure 1: The color coding of Synodontis species is inconsistent across panels (e.g., in Fig. 1a S. granulosus is dark blue, whereas in Fig. 1b it appears purple)?\n- L.93: It is mentioned that fish eggs were found in stomach contents, but I cannot locate them in the overview of dietary items shown in Fig. 1b. If they are included under a broader category, please clarify this in the text or figure legend.\n- Figure 1b: Cladocera are listed as prey items. To my knowledge, water fleas do not occur in Lake Tanganyika. Could this be a taxonomic mix-up, or is there a specific explanation for their presence? Clarifying this would be helpful.\n- L.93–94: The statement “However, the small size and spherical shape of consumed eggs clearly indicated the eggs did not belong to a mouthbrooding cichlid species.” may be a bit too general. For instance, the mouthbrooder Perissodus microlepis has very small, nearly spherical eggs. Perhaps clarify that they were evidently not from the major known host species rather than ruling out all mouthbrooders?\n- L.100–101: How was it determined whether the bones or scales belonged to cichlid or non-cichlid species?"}, "author_response": "We thank both reviewers for their thoughtful and constructive feedback, which has substantially\nimproved our manuscript. We carefully addressed every comment and revised the text for\nclarity, accuracy, and consistency. This includes refined interpretations, corrected terminology\nand clarified diet classifications. Most notably, in response to the reviewers’ suggestions, we\nperformed a new artificial parasitism experiment using a non-parasitic Synodontis species,\nwhich strengthened our key conclusions. We also conducted ancestral trait reconstruction as\nsuggested by the reviewer 2. Accordingly, we added new supporting information, updated data\nvisualizations and files submitted to Figshare repository.\nWe hope that these revisions, new analysis, and additional experiment fully addressed\nthe concerns raised by the reviewers. Our detailed responses to each point follows.\nFinally, we also added a short summary of results at the end of introduction, as requested\nfor Nature Communications paper (our paper has been transferred from Nature submission\nwhich does not include that paragraph).\nRESPONSE TO REVIEWERS’ COMMENTS\nReviewer #1 (Remarks to the Author):\nThe manuscript by Reichard and colleagues describes an investigation of the evolution of\nbrood parasitism in a species of African Rift Lake catfish, Synodontis multipunctatus, that\nmanipulates mouth-brooding cichlids into caring for its young, with the catfish larvae\nadditionally feed on the host’s own offspring. As the authors point out, their system involves\nthe evolution of brood parasitism in a lineage with an ancestral condition of no parental care.\nThis situation contrasts with more extensively studied examples in birds, where the ancestral\ncondition was the presence of parental care.\nThe authors first test the hypothesis that the evolution of brood parasitism in S.\nmultipunctatus was facilitated by an initial stage of adults feeding on the cichlid eggs; such\ntrophic interactions have been documented in other systems of brood parasitism. They\ndocument the gut contents of wild-caught specimens of five species of Lake Tanganyika\nSynodontis, finding variation in diet among species and that S. multipunctatus has a\ngeneralized diet, rather than being a specialized egg predator. This result was further\nsupported by stable isotope analysis of muscle tissue from the Synodontis species.\nThe authors next induced reproduction in the lab of the same five Lake Tanganyika\nSynodontis species along with three riverine outgroups in the genus. They compared\nreproductive characteristics, as well as embryonic and larval development, to determine\nwhether several proposed adaptations to brood parasitism arose before or after the divergence\nof S. multipunctatus from its egg-scattering relatives. Among the reproductive characteristics\nof S. multipunctatus, large egg size, low adhesiveness of eggs, and lack of spawning\nseasonality appeared to have been present before the evolution of brood parasitism. In\ncontrast, egg color mimicking that of the host cichlids as well as the frequent production of\nsmall clutches of eggs appear to be unique to S. multipunctatus.\nSimilar analysis of embryonic and larval development suggested that the rapid larval growth\nof S. multipunctatus is shared with its sister species S. granulosus, while rapid mineralization\nof oral and pharyngeal teeth and jaws (enabling feeding on cichlid eggs) is unique to the\nformer species.\nFinally, the authors tested whether larvae of S. granulosus could survive in the mouths of\ncichlids and/or feed on the young of the host. They found that they could survive, but their\nability to feed on eggs was significantly lower than that of S. multipunctatus. Such survival\nwas also greater in a cichlid from a different lake compared to that in a sympatric cichlid that\nhas likely evolved defenses against parasitism by S. multipunctatus.\nThis is a well-designed and executed study, and the manuscript is clearly written. The S.\nmultipunctatus system is a fascinating one that I believe will be of wide interest. The finding\nthat some of the likely adaptations for brood parasitism were present before its evolution is\nalso likely to be of general interest to evolutionary biologists.\nRESPONSE: Thank you very much for positive feedback and a nice summary of our study.\nI have only minor suggestions for improvement as follows.\n1) Evolution of reproductive parasitism from species with no parental care brought to my\nmind the “nest associates” that spawn in the pebble mounds of the North American chubs of\nthe genus Nocomis. (Betts et al 2024. Freshwater Biology, 69, 450–459.\nhttps://doi.org/10.1111/fwb.142242). Is this of any relevance to the Synodontis system? If not,\nthere is no need to comment on it in the manuscript.\nRESPONSE: This system appears not to be directly relevant toSynodontis brood parasitism,\nunless we address this issue in a broader context. Several instances of brood parasitism are\nknown in fishes, although the terminology distinguishing “nest association” from “brood\nparasitism” remains debated. The topic is too broad to fall into the scope of the present study.\nActually, we prepare a separate opinion paper that summarizes and reviews all documented\ncases of brood parasitism in fishes, including a discussion of which systems should (and\nshould not) be classified as true brood parasitism. Established brood parasites include the\nlineage ofPungtungia and Pseudopungtungiaminnows in East Asia, as well as recently\nidentified cases of brood parasitic catfish (other than the cuckoo catfish) from Lake Malawi\nand Lake Tanganyika. The North American minnows that act as nest associates are\nparticularly difficult to categorize, as both costs and benefits may accrue to the “host” species.\nGiven this complexity and to avoid digressing from the focus of our current study, we prefer\nnot to include this discussion in the manuscript.\n2)The authors find that the diet of S. granulosus consisted exclusively of fish tissue. Can they\nspeculate on whether this is the ancestral condition of the S. multipunctatus lineage, and if so,\nwhether this might be the trophic interaction between catfishes and cichlids that preceded\nbrood parasitism?\nRESPONSE:We emphasize that the feeding ofS. granulosuslikely reflects scavenging or\nscale-eating rather than active predation. We have clarified its classification as a fish\nscavenger when discussing the stable isotope results and, for consistency, now also briefly\nmention other Synodontis species. Although it might be possible to speculate about a link\nbetween ancestral state of consumption of (non-egg) fish tissue and the evolution of brood\nparasitism, we believe that any such association remains highly speculative and our data\ncannot illuminate the issue.\n3) It appears from Figure 2 (e and f) that the larger egg size of S. multipunctatus is not\nassociated with larger adult body size. Can the authors comment explicitly on the adult size of\nthe five Lake Tanganyika species?\nRESPONSE:Thank you for this comment.S. multipunctatus is indeed one of the\nsmallerSynodontis species in Lake Tanganyika. We have now added explicit body-size\ninformation of all LT Synodontis species (mean and s.e. of the specimens used to estimate\ngonadal investment) to the main text, in addition to the information that could previously be\ninferred from Fig. 2e and 2f (now 2c and 2d).\n4) In line 224, it is indicated that host offspring disappeared in the presence of inoculated S.\ngranulosus. Was this disappearance complete in both S. multipunctatus and S. granulosus, or\nwas it less in the latter species?\nRESPONSE:Yes, survival of host offspring was zero in bothS. multipunctatus andS.\ngranulosus. We have clarified this in the text by stating that no host embryos were present in\nexperimentally parasitized females at 12–14 days post-fertilization (forS. multipunctatus, n =\n13; forS. granulosus, n = 9).\n5) In lines 254-255 the authors suggest that “Occasional interactions with cichlid hosts may\nhave been sufficient to trigger the origin of obligate brood parasitism …” Can they speculate\non what these interactions might be? Do the cichlids occupy areas that would be attractive for\negg scattering? Is the spatial overlap between S. multipunctatus and the host cichlids known\nwhen the former is not spawning?\nRESPONSE:We now clarify that the initial interactions were most likely related to egg\npredation and have added the phrase “when feeding on their eggs.” Host cichlids and cuckoo\ncatfish broadly co-occur in rocky littoral habitats of Lake Tanganyika, and both are among the\nmost common fishes in these areas. Consequently, spatial overlap and frequent encounters\noccur even outside spawning events, making initially opportunistic interactions (such as egg\npredation) a plausible precursor to the evolution of obligate brood parasitism.\nReviewer #2 (Remarks to the Author):\nThe manuscript “The ecological and developmental foundations of brood parasitism in a\ncatfish” presents a comprehensive and well-written study that aims to shed light on the origin\nand early evolution of brood parasitism within the Lake Tanganyika Synodontis radiation. It\nseems this system is particularly intriguing because it offers a rare opportunity to study the\nevolution of brood parasitism in a lineage that lacks parental care.\nThe authors adopt an integrative approach, which I find highly commendable, combining\nmultivariate ecological quantification, various reproductive life-history traits, developmental\nseries (covering growth, dental development rates), and an experimental brood parasitism\nassay. The scope of the data collection is impressive and I really enjoyed reading the\nmanuscript!\nIn summary, the main findings indicate that the cuckoo catfish differs from its closest relatives\nin numerous traits, particularly in egg coloration, clutch size, egg size, and the timing of jaw\ndevelopment. Furthermore, both the focal species and its sister species exhibit elevated\ngrowth rates and reduced egg adhesion. Taken together, the suite of traits observed in the focal\nspecies align well with characteristics expected to facilitate brood parasitism.\nThe breadth of data surely provides valuable context for discussing the evolutionary\nfoundations of brood parasitism from multiple perspectives, which I consider a major strength\nof the work. However, I have a few concerns regarding the extent to which the analyses fully\naddress the central evolutionary question, as well as some minor specific comments:\n1. The conclusive power of the model system\nAll statistical tests in the manuscript address whether the cuckoo catfish S. multipunctatus\ndiffers from other species. While valid in that context, this does not answer the broader\nquestion implied by the authors: what traits relate to the origin and evolution of brood\nparasitism? Addressing that question would require phylogenetic comparative analyses –\nwhich are absent from the manuscript.\nUnfortunately, the nature of the system offers very limited statistical power, as it effectively\nhas a comparative sample size of N = 1 for brood parasitism (since the trait appears to have\nevolved only once within the clade). Given this limitation, can we realistically identify traits\nthat are statistically linked to the origin and evolution of brood parasitism?\nUndoubtedly, the observed trait shifts make intuitive sense in light of brood parasitism and\ngenerate intriguing hypotheses; nevertheless, the work remains primarily descriptive and\nhypothesis-generating, rather than formally detecting signals of adaptation. While this work is\na valuable contribution – especially for broader comparative context or discussions across\nsystems – the limitations should be explicitly acknowledged, and a more cautious tone\nadopted when interpreting patterns as adaptive responses or drivers.\nRESPONSE:We agree that the single origin of brood parasitism in our study system imposes\ninherent limitations on statistical inference. To address this in the revised version, we have\nconducted ancestral trait reconstructions and calculated evolutionary signal metrics (Pagel’s λ\nand Blomberg’s K) for traits related to reproduction. Given the small comparative sample\nsize, we recognize that these analyses have limited power. Accordingly, we present them\noutside the main ms (Extended Data. Fig. 1) and interpret them cautiously.\nMore broadly, we explicitly acknowledge in the revised manuscript that the “single\nevolutionary origin (of brood parasitism in this clade) without a replication” necessarily\nrestricts comparative power. As a result, our study cannot provide conclusive tests of\ncorrelated evolution. We highlight this limitation clearly (i.e., single origin) and have adjusted\nthe tone of our interpretations to reflect this limitation.\n2. The experimental brood parasitism assay\nThis experiment is highly intriguing and a valuable step toward understanding potential\nparasitism in S. granulosus. However, its interpretative strength could be improved by\nincluding a known substrate-spawning Synodontis as a negative control.\nThe current experimental design only demonstrates that S. granulosus offspring do not survive\nin what they call “sympatric host” mouths. However, the absence of a non-parasitic control\nmakes it difficult to assess whether the high survival in the naïve host (where both species\nperformed well) is expected for a non-parasitic Synodontis or if S. granulosus falls somehow\nalong a parasitic spectrum?\nRESPONSE:We fully agree with the reviewer’s suggestion and have conducted an\nadditional experiment usingS. polli as a non-parasitic, substrate-breeding control.\nTo control for potential effects of age or condition of a new host cichlid cohort used in this\nexperiment, we included a new cuckoo catfish control in the experiment. The results were\nclear: noS. polli embryos survived after 7 days of incubation in allopatric Haplochromis\nhosts, whereas cuckoo catfish embryos survived successfully (93%) over the same period,\nconsistent with prior experiments.\nWe have elaborated on this experiment in the main text and updated the Methods accordingly.\nFigure 4 has been modified to include the new dataset (panel with S. polli results) and\npresenting now results forS. granulosusand cuckoo catfish embryos separately for 4–7 days\nand 12–14 days periods (different panels). This allows direct visual comparison of embryo\nsurvival between parasitic and non-parasitic Synodontis for specific developmental periods.\nThe previous version had combined data where time periods were separated by distinctive\ndata point symbols.\nA second concern I have is the match between S. granulosus and the tested host species S.\nhorei. As mentioned in the manuscript, S. granulosus is a deep-water species (>40 m),\nwhereas S. horei is a well-documented shallow-water dweller rarely encountered below 5–10\nm and is hence unlikely a true sympatric host for S. granulosus (the match with no embryo\nsurvival). In contrast, S. horei is a known true sympatric host of S. multipunctatus (the match\nthat performed very well). So, how can host-specificity within the lake be excluded as a driver\nof this pattern?\nRESPONSE:We agree that our original wording did not clearly convey our rationale.S.\nhorei was chosen as a host because it has an evolutionary history with brood parasitic cuckoo\ncatfish, thus making it feasibly a “challenging” host (Cohen et al. 2018, Reichard et al. 2023).\nNo information is available on the possible natural host species ofS. granulosus, although\npossible candidates would be Cyphotilapia spp., Benthochromis spp., and Trematocara spp.\nThe possibility of S. granulosus embryos to survive in host cichlids only became apparent\nafter combining our results and reaching preliminary conclusions, which prompted later\nexperimental testing.\nWe have reworded instances of “sympatric” in relation toS. granulosusand cichlids to avoid\nimplying natural co-occurrence and instead refer to “Lake Tanganyika cichlids” where\nappropriate. The main logic behind usingS. horei was to test embryo survival against a\nchallenging cichlid host, not to imply host-specific sympatry. This rationale is now clearly\narticulated in the revised manuscript.\nMinor specific comments:\n- The use of the term Pre-adaptation: This may seem like a matter of semantics, but I would\nencourage the authors to reflect on their use of the term pre-adaptation. While it has been\nwidely used historically, the term can imply evolutionary foresight. The perhaps more widely\naccepted term is exaptation, which conveys that a trait originally evolved for one functon(or\nwithout a specific function) and was later co-opted for another.\nRegardless of which term is chosen, I recommend briefly defining it at first use to ensure\nclarity.\nRESPONSE: We now use the term exaptation and introduce it specifically when first\nmentioned: “…that the apparent exaptations (i.e., adaptations originally evolved outside\nparasitism context)…”\n- L- 63: In the introduction, the hypothesis: ”We hypothesized that the origin of brood\nparasitism in the cuckoo catfish has primarily arisen from the predation of adult catfish on\ncichlid eggs during spawning” does feel somewhat abrupt since there is little lead-up to the\nidea that predation might be the key precursor.\nRESPONSE: We have now reworded the statement and it starts: “Given that cuckoo catfish\nare potent egg predators, we hypothesised…”\nThe fact that the cuckoo catfish are potent egg predators is also stated earlier in the paragraph\n“As adult cuckoo catfish are also vigorous egg predators (15), ”\n- Figure 1: The color coding of Synodontis species is inconsistent across panels (e.g., in Fig.\n1a S. granulosus is dark blue, whereas in Fig. 1b it appears purple)?\nRESPONSE: We have corrected all colour biases. They resulted from variable transparency\nvalues applied in some panels where overlapping data points were visualized.\n- L.93: It is mentioned that fish eggs were found in stomach contents, but I cannot locate them\nin the overview of dietary items shown in Fig. 1b. If they are included under a broader\ncategory, please clarify this in the text or figure legend.\nRESPONSE: Fish eggs were originally included within the broader “fish” category, but we\nrecognize that separating them in the text may have caused confusion. We have now modified\nFigure 1b to display fish eggs as a distinct category (brown colour) and retain this separation\nthroughout the analysis. This clarification is also reflected in the figure legend and\nrepresented the results tables.\n- Figure 1b: Cladocera are listed as prey items. To my knowledge, water fleas do not occur in\nLake Tanganyika. Could this be a taxonomic mix-up, or is there a specific explanation for\ntheir presence? Clarifying this would be helpful.\nRESPONSE:Upon revisiting our samples and notes, we confirmed that the items labelled as\nCladocera were misidentified: one instance represents Copepoda and the other Caridea.\nHowever, we note that there is at least one report of Cladocera in Lake Tanganyika (Harding,\nJ. P. (1957). Crustacea: Cladocera. Exploration hydrobiologique, 1946-1947) and we also\nobserved two confirmed cases of Cladocera in the diet ofSynodontis melanostictus, a riverine\nSynodontis occurring near the river mouths. This species was not included in our analyses and\nits diet may have included items from riverine rather than strictly lacustrine sources. We have\ncorrected the figure and text to reflect these clarifications.\n- L.93–94: The statement “However, the small size and spherical shape of consumed eggs\nclearly indicated the eggs did not belong to a mouthbrooding cichlid species.” may be a bit\ntoo general. For instance, the mouthbrooder Perissodus microlepis has very small, nearly\nspherical eggs. Perhaps clarify that they were evidently not from the major known host\nspecies rather than ruling out all mouthbrooders?\nRESPONSE: We see that the text may have been misleading. We now add egg size and their\nlarge abundance but also clearly state “did not belong to any known host cichlid species.”\nRevised text (additions in bold): “However, the small size (<1 mm) and spherical shape of\nconsumed eggs, along with their high abundance (257 eggs), clearly indicated the eggs did\nnot belong to any known host cichlid species.\n- L.100–101: How was it determined whether the bones or scales belonged to cichlid or non-\ncichlid species?\nRESPONSE:We have clarified that the scales may have also belonged to Lates species.\nIdentification was based on scale morphology: all observed scales were ctenoid, and we\ncompared them to reference collections and published descriptions of lake fish lineages\n(sardines, cyprinids, killifish, alestids, and catfish; Viertler et al., 2021; Jawad, 2015). To\nimprove transparency, we have included supplementary material illustrating the fish scales\n(and bones, otoliths and tooth) found in the stomachs ofS. granulosus (Suppl. Fig. 1)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The revised version of the manuscript addresses all of the concerns I raised in my previous review in a thorough and satisfactory manner.", "reviewer_2": "The authors have now thoroughly revised the manuscript and have addressed all of my previous comments. In particular, they have added phylogenetic comparative perspectives, included a negative control in the parasitism assay, and clarified all minor points raised.\nThese additional analyses represent a valuable extension to the manuscript. Specifically, the ancestral state reconstructions now formally document the relative timing of trait evolution, which in the previous version was only visually inferred. Likewise, the inclusion of S. polli as a negative control has clearly strengthened the experimental design. Together, these changes have substantially improved the manuscript.\nThat said, I still have two minor follow-up comments, as outlined below.\n1) ancestral trait reconstructions\nI am not sure whether I agree with the statement that these particular analyses “suffer” from the single evolutionary origin of brood parasitism (e.g. L173–174: “Despite the interpretative limitation imposed by a single evolutionary origin of brood parasitism, significant …”).\nMy original concern about the effective comparative sample size of N = 1 applies specifically to formal tests of correlated evolution between traits and parasitism. In other words, the limitation imposed by a single origin of brood parasitism constrains analyses that would ask whether parasitic versus non-parasitic lineages differ in trait values once shared ancestry is accounted for (e.g. phylogenetic ANOVA or PGLS with brood parasitism as a predictor). Such analyses are not conducted here – instead the focus is on differences between species.\nWhile I think the manuscript is in general carefully worded not to oversell it as a test for evolutionary correlations, there are (still) instances where it is implied. For example, in the reporting of statistical results in Fig. 2 the caption states: “Asterisks indicate whether clutches of the cuckoo catfish were significantly smaller than those of all other species (LM, P < 0.001).” If the question is whether S. multipunctatus differs from other species individually, we expect pairwise comparisons not one statistical test. However, if the question is whether parasitic species differ from non-parasitic species as a group, then a comparative model accounting for phylogenetic non-independence would be required. I therefore recommend revisiting the wording throughout the manuscript to ensure that this distinction is consistently maintained and that statistical tests are described in a way that matches the questions they address.\n2) experimental parasitism\nDespite the fact that S. granulosus performs more similarly to the cuckoo catfish than to the negative control in a naïve host, the interpretation of these results does not seem to have been fully adjusted. In light of these findings, one of my questions from the previous round remains: how can host specificity within the lake be excluded as a driver of the observed patterns?\nHost–parasite interactions and adaptations go both ways. The authors argue that S. horii has evolved defence mechanisms through coevolution with S. multipunctatus. It seems equally plausible, however, that S. multipunctatus is specialized on its natural shallow-water hosts (shallow-water haplochromines). We do not know whether S. multipunctatus would perform equally well in a deep-water, non-haplochromine mouthbrooder — a “challenging” but potentially not compatible host. Basically, the reciprocal scenario relevant if S. granulosus were a brood parasite of deep-water hosts.\nRelatedly, several arguments used to reject brood parasitism in S. granulosus appear to implicitly take shallow-water haplochromines as the relevant reference. For example, statements that its eggs do not visually imitate host eggs overlook the considerable variation in egg size, colour, and clutch structure among mouthbrooding cichlids. Adaptation to different hosts could plausibly result in different egg phenotypes. Similarly, arguments based on large clutch size do not fully exclude alternative scenarios, such as egg distribution across multiple hosts e.g. during lek-like spawning events.\nHaving said that, I want to emphasize that I am not arguing that S. granulosus is necessarily a brood parasite. Rather, I think the current results make it difficult to rule out host-specific parasitic strategies and suggest that traits that are expressed differently can still be relevant to brood parasitism. I therefore encourage the authors to discuss the evidence for and against parasitism in S. granulosus in a more balanced way (e.g. L 264-273)."}, "author_response": "RESPONSE TO REVIEWER S' COMMENTS\nReviewer #1 (Remarks to the Author):\nThe revised version of the manuscript addresses all of the concerns I raised in my previous\nreview in a thorough and satisfactory manner.\nReviewer #2 (Remarks to the Author):\nThe authors have now thoroughly revised the manuscript and have addressed all of my\nprevious comments. In particular, they have added phylogenetic comparative perspectives,\nincluded a negative control in the parasitism assay, and clarified all minor points raised.\nThese additional analyses represent a valuable extension to the manuscript. Specifically, the\nancestral state reconstructions now formally document the relative timing of trait evolution,\nwhich in the previous version was only visually inferred. Likewise, the inclusion of S. polli as\na negative control has clearly strengthened the experimental design. Together, these changes\nhave substantially improved the manuscript.\nThat said, I still have two minor follow-up comments, as outlined below.\n1) ancestral trait reconstructions\nI am not sure whether I agree with the statement that these particular analyses “suffer” from\nthe single evolutionary origin of brood parasitism (e.g. L173–174: “Despite the interpretative\nlimitation imposed by a single evolutionary origin of brood parasitism, significant …”).\nMy original concern about the effective comparative sample size of N = 1 applies specifically\nto formal tests of correlated evolution between traits and parasitism. In other words, the\nlimitation imposed by a single origin of brood parasitism constrains analyses that would ask\nwhether parasitic versus non-parasitic lineages differ in trait values once shared ancestry is\naccounted for (e.g. phylogenetic ANOVA or PGLS with brood parasitism as a predictor). Such\nanalyses are not conducted here – instead the focus is on differences between species.\nWhile I think the manuscript is in general carefully worded not to oversell it as a test for\nevolutionary correlations, there are (still) instances where it is implied. For example, in the\nreporting of statistical results in Fig. 2 the caption states: “Asterisks indicate whether clutches\nof the cuckoo catfish were significantly smaller than those of all other species (LM, P <\n0.001).” If the question is whether S. multipunctatus differs from other species individually,\nwe expect pairwise comparisons not one statistical test. However, if the question is whether\nparasitic species differ from non-parasitic species as a group, then a comparative model\naccounting for phylogenetic non-independence would be required. I therefore recommend\nrevisiting the wording throughout the manuscript to ensure that this distinction is consistently\nmaintained and that statistical tests are described in a way that matches the questions they\naddress.\nRESPONSE: We have deleted the first part of the sentence reporting analysis of phylogenetic\nsignal: “Despite the interpretative limitation imposed by a single evolutionary origin of brood\nparasitism,”, as suggested.\nWe have also reworked the contrast of results presented on Fig. 2 (reproductive traits). They\nwere treated as pairwise contrast of ANOVA result and we now replace it with overall post-\nhoc tests (Sidak method). This does not change the outcome at all, but we have reworded the\ninterpretation as suggested by the referee (minor changes/simplification of the main text, and\nfigure 2 caption specification, all tracked in the marked-up version of the ms).\n1\n2) experimental parasitism\nDespite the fact that S. granulosus performs more similarly to the cuckoo catfish than to the\nnegative control in a naïve host, the interpretation of these results does not seem to have been\nfully adjusted. In light of these findings, one of my questions from the previous round\nremains: how can host specificity within the lake be excluded as a driver of the observed\npatterns?\nHost–parasite interactions and adaptations go both ways. The authors argue that S. horii has\nevolved defence mechanisms through coevolution with S. multipunctatus. It seems equally\nplausible, however, that S. multipunctatus is specialized on its natural shallow-water hosts\n(shallow-water haplochromines). We do not know whether S. multipunctatus would perform\nequally well in a deep-water, non-haplochromine mouthbrooder — a “challenging” but\npotentially not compatible host. Basically, the reciprocal scenario relevant if S. granulosus\nwere a brood parasite of deep-water hosts.\nRelatedly, several arguments used to reject brood parasitism in S. granulosus appear to\nimplicitly take shallow-water haplochromines as the relevant reference. For example,\nstatements that its eggs do not visually imitate host eggs overlook the considerable variation\nin egg size, colour, and clutch structure among mouthbrooding cichlids. Adaptation to\ndifferent hosts could plausibly result in different egg phenotypes. Similarly, arguments based\non large clutch size do not fully exclude alternative scenarios, such as egg distribution across\nmultiple hosts e.g. during lek-like spawning events.\nHaving said that, I want to emphasize that I am not arguing that S. granulosus is necessarily a\nbrood parasite. Rather, I think the current results make it difficult to rule out host-specific\nparasitic strategies and suggest that traits that are expressed differently can still be relevant to\nbrood parasitism. I therefore encourage the authors to discuss the evidence for and against\nparasitism in S. granulosus in a more balanced way (e.g. L 264-273).\nRESPONSE: We have softened our arguments and mention the possibility that S. granulosus\nis specialised on deepwater cichlid hosts.\nWe thank both referees once more for constructive feedback and reviews.\nPlease note that we re-analysis of post-hoc comparisons rendered updated version of data\nanalysis, which we resubmitted to Figshare repository as a new version.\nWe append tracked version of the main ms and figure captions (including editorial request\nfor formatting changes and more details to provide) below.\nTracked version of Supplementary Material is provided as a separate file (formatting changes\nonly, large file size due to inclusion of Figures).\n2"}]

Decision letter