DOI: 10.5061/dryad.q83bk3jsx
title: Dataset of diverse evolutionary pathways shape cichlid egg size in Lake Tanganyika

description: Optimal egg size is a classic and important concept in life history
 theory. Here, we examined the ecological factors affecting egg size, using
 a comparative analysis of 83 cichlid species from Lake Tanganyika, which
 employ either a mouth-brooding or substrate-brooding. The two strategies
 differ substantially in regard to spatial limitations and food
 availability for offspring, potentially leading to divergent responses to
 the ecological factors influencing egg size. We observed a strong negative
 correlation between egg size and egg number in species that exhibit
 mouth-brooding, but not in substrate-brooding cichlids. Our results
 suggest that the importance of the relationship between clutch size and
 egg size varies substantially between these two types of parental care.
 Interestingly, in mouth-brooding species, we found that egg size increases
 when offspring exhibit external feeding behaviors (grazing behavior). We
 also demonstrate that substrate-brooding species with shorter periods of
 parental care typically produced larger eggs, compared to species with
 relatively longer periods of parental care. Overall, our results
 demonstrate that behavioral differences, including parental care type and
 duration, play an important but often overlooked role in the evolution of
 egg size across species.
description: Egg size, clutch size (i.e., the number of eggs released during a
 single spawning event), and adult female body mass were measured in 47
 mouth-brooding (belonging to 11 tribes) and 40 substrate-brooding cichlid
 species (belonging to 2 tribes), all endemic to Lake Tanganyika, in
 East-Central Africa. The sexually mature female specimens were obtained
 mainly from local fishermen and fish markets (n = 62 species, n = 161
 individuals) and from field sampling using a fine-mesh gill net, hand
 nets, and SCUBA (n = 19 species, n = 73 individuals). The captured fish
 were euthanized by overdosing with anesthetic (MS-222, Wako Pure Chemical
 Industries) and stored in a freezer until measurements were taken. Given
 the relatively small sample size of females carrying mature eggs collected
 during the field survey, we also used samples stored at Osaka Metropolitan
 University (n = 10 species, n = 22 individuals) and the Hokkaido
 University Museum (n = 7 species, n = 13 individuals). Standard length (in
 mm), body mass (i.e., gonad-free body weight to 0.001 g), and ovary mass
 (to 0.001 g) were measured. Mature eggs were distinguishable from immature
 eggs in the ovary based on coloration and size and were counted using a
 tally counter and preserved in 5% formalin. Immature eggs found in the
 same ovary were not included in the total and omitted from the analyses.
 In each clutch we randomly selected five eggs or used all eggs if the
 clutch size was smaller than five, to measure the mean egg size. Eggs were
 photographed using a digital camera equipped with a stereomicroscope
 (HD212; AMscope, USA), and the long and short diameter was measured on
 each egg to an accuracy of 0.01 mm using Image J ver. 1.52q software
 (Schneider et al. 2012). The mean value of the long and short diameter was
 used to define the mean egg size. In total, measurements were taken from
 1326 eggs from 269 female individuals representing 87 species.
   <em>Literature
 survey</em> Information on parental care, i.e.,
 brooding type - mouth or substrate-brooding, care type- maternal parental
 or biparental care, duration of parental care, and presence or absence of
 grazing behavior, was collected from the literature.
 For 44 of 47 mouth-brooding species, we collated data on care
 type (biparental or maternal care) and grazing behavior (present or
 absent; Yanagisawa 1985, 1986; Kuwamura 1988b, 1997). We predicted that
 the acquisition of external food resources by offspring (i.e., grazing
 behavior) may allow for a reduction in egg yolk sac investment and
 consequently a reduction in egg size. Although we gained life history
 trait data from the field for <em>Baileychromis
 centropomoides</em>, <em>Paracyprichromis
 nigripinnis</em>, and <em>Xenotilapia nigrolabiata,</em>
 we were unable to determine care type or the presence/absence of grazing
 behavior due to limited ecological descriptions of these species in the
 literature. For 39 of 40 substrate-brooding species, we
 collected information on care type (biparental or maternal care) and the
 duration of parental care (short or long). In this study, we defined the
 parental care period as days from the spawning event to dispersal and
 broadly classified the duration of parental care as either “shorter” or
 “longer”, based on the literature. Some cichlid species in Lake Tanganyika
 exhibit extremely short periods of parental care, often less than 20 days
 after hatching, while others have comparatively longer periods, ranging
 from 66–130 days . As far as we are aware, an intermediate period of
 parental care, between 20 and 65 days, has not been reported in any Lake
 Tanganyikan substrate-brooding species. Therefore, we defined species
 exhibiting parental care behaviors for ≤ 20 days as having “shorter” (n =
 7 species) and &gt; 20 days as having “longer” (n = 32 species)
 parentalcare duration. Information on parental care duration was lacking
 in one species <em>Neolamprologus mustax</em> again due to
 limited ecological descriptions. <em>Phylogenetic
 tree</em> To account for the effects of
 phylogeny, we included in our analyses a modified time calibrated
 phylogenetic tree of the cichlids that have radiated in Lake Tanganyika
 (Ronco <em>et al</em>. 2021). We pruned the Ronco et al.
 (2021) tree using the ‘droptip’ function in R ver 4.4.0 (R Core Team.
 2024) package ape (Paradis <em>et al.</em> 2004) down to 85
 species, excluding <em>Oreochromis tanganicae</em> and
 <em>Tylochromis polylepis</em> from our initial sample size of
 87 species. We then obtained divergence ages of <em>O.
 tanganicae</em> and <em>T. polylepis</em>, which
 secondarily invaded Lake Tanganyika from a time estimated phylogenetic
 tree targeting representatives of all cichlid tribes (Ronco et al. 2021).
 These divergence ages were integrated into the previously pruned
 phylogenetic tree, resulting in a tree representing all 87 species used in
 our study. The ancestral states of egg size were estimated in phytools
 (Revell 2024) to visualize the posterior probabilities of each trait
 condition of mean egg size of species, along the branches of the cichlid
 phylogeny in Lake Tanganyika.  
 <em>Phylogenetic signal life-history
 traits</em> We calculated phylogenetic signals
 for egg size, clutch size, and female body mass with the ‘phylosig’
 function in the R package phytools. We note that measurement error can
 lead to an underestimation of the strength of phylogenetic signals and
 potentially obscure patterns of phylogenetic correlation among species in
 univariate cases. Therefore, for the 62 species for which repeated data
 were available, the strength of the phylogenetic signal was calculated,
 considering intraspecific variation. Each trait value was
 log10-transformed and the mean for all species and standard error for
 available species were calculated and used in the analysis.
   <em>Phylogenetic comparative
 analyses</em> We used Bayesian phylogenetic mixed
 model analyses (BPMM; R package “MCMCglmm”) to investigate the effects of
 brooding strategy (i.e. mouth- and substrate-brooding) and various
 parental care traits on the trade-off between clutch size and egg size. In
 each model, we fitted the pruned phylogenetic tree with 87 species as a
 random effect. First, we used BPMM to investigate the
 effect of brooding strategy on mean egg size We fitted a model mean with
 egg size (log<sub>10</sub>-transformed) as the response
 variable and brooding type (mouth- or substrate brooding), mean clutch
 size (log<sub>10</sub>-transformed), mean female body mass
 (log10-transformed), and their interactions as fixed effects. The pruned
 phylogenetic tree was fitted as a random effect. The error distribution
 for the continuous variables followed a Gaussian distribution and were
 consequently standardized to facilitate the comparison of regression
 coefficients among the fixed effects. We used the uninformative priors for
 the random effect and the residual variance (inverse-Wishart distribution,
 nu = 1/2, V = 0.002) and non-informative priors for the fixed effects. The
 MCMC was performed with 205000 total iterations, a burn-in of 5000, and a
 thinning interval of 100 was applied. We evaluated model convergence based
 on the visual examination of trace plots. The model convergence was also
 checked by running the MCMC chain independently and using the
 Gelman-Rubin's diagnostic was less than 1.1 using the
 'gelman.diag' function in the R package coda (Plummer
 <em>et al.</em> 2006). For all fixed effects, random effects
 and residuals, the effective sample size exceeded 1000. We also computed
 the variance inflation factor (VIF) to assess collinearity between
 log-transformed clutch size and body mass. The VIF for each fixed effect
 was lower than 10 (Clutch size = 8.26, female body mass = 4.96, brooding
 type = 1.75, clutch size: brooding type = 8.45, female body mass: brooding
 type = 4.67). Next, since egg size variation was likely
 to account for different ecological conditions between mouth- and
 substrate brooders, we ran BPMMs separately for mouth-brooding and
 substrate-brooding species to investigate factors influencing variation in
 egg size within brooding strategies. For mouth-brooding species, we fitted
 a model with mean egg size as the response variable and with mean clutch
 size, mean female body mass, grazing behavior (presence or absence), and
 care type (biparental or maternal mouth-brooding) as explanatory
 variables, omitting any two-way interaction terms. For substrate-brooding
 species, we fitted a model with mean egg size as the response variable and
 with mean clutch size, mean female body mass, parental care duration
 (short or long), and care type (biparental or maternal brood guarding) as
 explanatory variables, again omitting any two-way interactions. The
 phylogenetic tree, isolated to either mouth-brooding or substrate-brooding
 species was incorporated as a random effect in each model. All other model
 components were conducted the same as described above.
description: ## Description of files and variables: ## File: data_cichlid.csv
 **Description:** dataset including mouth- and substrate-brooding cichlids
 (n = 83 species). ##### Variables * species: Species Code. The name
 corresponds to the phylogenetic tree data. * samplesize: Number of mature
 females we caught. * tribe: The tribe to which the species belonged. *
 broodtype: brooding type (mouth- or substrate-brooding) * care_sex: sexes
 mainly provide parental care  * parentalcare_dulation: duration of
 parental care. For mouth-brooders, data collection for parental care
 period was not conducted; therefore, missing data are indicated as NA. *
 guarding: sexes provide brood guarding (BI: biparental guarding, MG:
 maternal guarding, NG: no guarding) * bodyweight: mean body weight of
 matured females (g) * gonalweight: mean weight of ovary in matured females
 (g) * clutchsize: the mean number of eggs in ovary * eggsize: the maen
 size of matured egg in ovary (mm) * SL: mean standard length (mm) *
 gonadfreeBW: mean body size (= bodyweight-gonalweight) * logES: log 10
 transfered eggsize * logCS: log 10 transfered clutchsize * logBW: log 10
 transfered gonadfreeBW #### File: trait.csv **Description:** dataset
 including mouth- and substrate-brooding cichlids (n = 87 species). #####
 Variables * species: Species Code. The name corresponds to the
 phylogenetic tree data. * samplesize: Number of mature females we caught.
 * tribe: The tribe to which the species belonged. * broodtype: brooding
 type (mouth- or substrate-brooding) * care_sex: sexes mainly provide
 parental care. Data of some species is missing because of luck of life
 history knowledge.  * guarding: sexes provide brood guarding (BI:
 biparental guarding, MG: maternal guarding, NG: no guarding). Data of some
 species is missing because of luck of life history knowledge.  *
 bodyweight: mean body weight of matured females (g) * gonalweight: mean
 weight of ovary in matured females (g) * clutchsize: the mean number of
 eggs in ovary * eggsize: the mean size of matured egg in ovary (mm) * SL:
 mean standard length (mm) * gonadfreeBW: mean body size (=
 bodyweight-gonalweight) #### File: pop2_backbone_Tanga.tre
 **Description:** Phylogenetic tree containing all species used in the
 study #### File: use.tree **Description:** Phylogenetic tree created by
 combining pop2_backbone_Tanga.tre and b1_pruned.tre #### File:
 b1_pruned.tre **Description:** Phylogenetic tree published by Ronco et al.
 2021. This phylogenetic tree was used to create the use.tree #### File:
 lifehistorytraits_rawdata.csv **Description:** Raw data before averaging
 of cichlid fishes in Lake Tanganyika ##### Variables * name: scientific
 names * name2: Species Code. The name corresponds to the phylogenetic tree
 data. * SL_f: standard length (mm) * eggnum: the number of eggs in ovary *
 eggsize: the size of matured egg in ovary (mm) * fBW: body weight of
 matured females (g) * gonadweight: weight of ovary in matured females (g)
 * gonad_free_BW: body size (= bodyweight-gonalweight) NA indicates data
 due to measurement errors. #### File: RbS_test_for_SB.csv
 **Description:** data set for post-hoc Rate_by_state test in
 substrate-brooders.  ##### Variables * species: scientific names * egg
 size: log 10 transfered egg size * care_duration: parental care duration
 (longer: species with longer parental care, shorter: species with shorter
 parental care) #### File: RbS_test_for_MB.csv **Description:** data set
 for post-hoc Rate_by_state test in mouth-brooders.  ##### Variables *
 species: scientific names * guarding: presence or absence of grazing
 behavior (P: presence, A: absence) * egg size: log 10 transfered egg size
 #### File: MCMCGLMM(Figure3_5).R (in Zenodo) **Description:** R code to
 reproduce the analysis by Phylogenetic MCMC GLMM. This R code corresponds
 to the analysis associated with Figures 3, 4, and 5. #### File:
 RbS_test_for_SB.R (in Zenodo) **Description:** R code to reproduce the
 analysis by Rate_by_state test for substrate-brooders. #### File:
 RbS_test_for_MB.R (in Zenodo) **Description:** R code to reproduce the
 analysis by Rate_by_state test for mouth-brooders. #### File:
 other_analysis.R (in Zenodo) **Description:** R code to reproduce the
 analyses other than the MCMC and Rate_by_state tests. This code was
 accidentally deleted from Zenodo during dataset adjustments and was
 re-added on October 30. #### File: MCMCGLMM_results.pdf **Description:**
 Figures showing the results of the MCMC. We created Figures 3, 4, and 5 by
 editing these figures. ## Code/software * R (R Core Team (2024)) ## Access
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 ## Change log: Changes since October 28, 2025: the code hosted on Zenodo
 was slightly adjusted resulting in a new data version, but there are not
 meaningful changes. The README was updated to reflect this.