Yseult Héjja-Brichard

Julien P Renoult

Tamra C Mendelson

Comparative analysis reveals assortative mate preferences in darters independent of sympatry and sex

Keywords: assortative mating preferences, sexual isolation, Etheostoma, mate preference, speciation

A preference for mating with conspecifics over heterospecifics is fundamental to the maintenance of species diversity in sexually reproducing organisms. This type of positive assortative mating results in sexual isolation, a reduction in gene flow between species due to differences in mate choice. Sexual isolation is often stronger between closely related sympatric species as compared to allopatric species, because of processes such as reinforcement. In animals, this pattern has been demonstrated across the genus Drosophila, however, most empirical evidence for stronger species-level assortative mating in sympatry comes from studies of two or only a few species. In addition, traditional theories of sexual selection suggest that because reproduction is more costly to females, they should be the choosier sex. This prediction was supported in Drosophila, but it remains unclear whether this is a general trend. We therefore conducted a comparative analysis of mate preferences in sympatric and allopatric species of darters (genus Etheostoma). We performed a meta-analysis of 16 studies, encompassing 21 species. As expected, we found stronger preferences for conspecifics over heterospecifics across studies and species. Contrary to studies in Drosophila, however, we did not find an effect of sympatry or sex on the strength of preference for conspecifics. We offer several testable hypotheses to explain the variation we observed in the strength of assortative preference.

Introduction

As evolutionary biology continues to explore the mechanisms of speciation, the processes driving reproductive isolation remain a central focus of study. One important reproductive barrier is sexual isolation, a reduction in gene flow due to differences in mate choice. Sexual isolation is a consequence of assortative mating resulting from a preference for conspecific mates [START_REF] Jiang | Assortative Mating in Animals[END_REF][START_REF] Kopp | Mechanisms of Assortative Mating in Speciation with Gene Flow: Connecting Theory and Empirical Research[END_REF]. Preference for conspecific over closely related heterospecific mates can evolve as a result of divergent natural or sexual selection in geographically isolated populations, and it can be strengthened in sympatric populations due to processes such as reinforcement [START_REF] Dobzhansky | Speciation as a Stage in Evolutionary Divergence[END_REF]Liou & Price, 1994); [START_REF] Servedio | The Role of Reinforcement in Speciation: Theory and Data[END_REF]. Understanding the evolutionary mechanisms driving the evolution of preference for conspecific mates is an active area of research in the field of speciation.

In 1989, [START_REF] Coyne | Patterns of speciation in Drosophila[END_REF], 1997) made the important discovery that prezygotic barriers in Drosophila were twice as strong in sympatric species than in allopatric species. This geographic pattern was not true for postzygotic barriers (hybrid inviability and hybrid sterility). [START_REF] Yukilevich | Asymmetrical Patterns of Speciation Uniquely Support Reinforcement in Drosophila[END_REF] later confirmed that this pattern in Drosophila is a result of mate choice and not gametic incompatibility. (Lemmon, 2009) found the same pattern at a smaller scale in two frog species of the genus Pseudacris. Females from populations in which the two species were sympatric more strongly preferred conspecific signals, compared with allopatric females, and this preference was stronger when the conspecific signal was the sympatric one (Lemmon, 2009). Comparative studies in plants also support a pattern of greater assortative mating in sympatric species, though [START_REF] Hopkins | Reinforcement in plants[END_REF] points out ways in which those studies could be strengthened.

A geographic pattern of stronger preference for conspecific mates in sympatric compared to allopatric populations can arise for a number of reasons. One is a process of reinforcement when selection against hybrids in sympatry favors increased preference for conspecific mates [START_REF] Servedio | The Role of Reinforcement in Speciation: Theory and Data[END_REF]. Another is a process of differential fusion, if geographic overlap only occurs when species have accumulated a sufficient degree of conspecific preference prior to secondary contact [START_REF] Noor | Reinforcement and other consequences of sympatry[END_REF]. A last process arises from species extinction or species sorting whereby costly sexual interaction can dramatically reduce the growth of one species leading to its extinction [START_REF] Kyogoku | Heterospecific mating interactions as an interface between ecology and evolution[END_REF]. Although the original interpretation of [START_REF] Coyne | Patterns of speciation in Drosophila[END_REF] results pointed to reinforcement as explaining the pattern of greater sexual isolation in sympatry, more recent analyses suggest that differential fusion and extinction rates might also contribute to a pattern of stronger sexual isolation in sympatry (Matute & Cooper, 2021). Thus, a stronger preference for conspecific mates in populations that are sympatric with a close congener is an important pattern that can both support and generate hypotheses about the evolutionary mechanisms driving sexual isolation.

Another important result of broad comparative studies in Drosophila is that of [START_REF] Yukilevich | The evolution of male and female mating preferences in Drosophila speciation*[END_REF], who found that for sympatric species pairs, female Drosophila had stronger preferences for conspecific males than males did for females, whereas no sex difference in preference was found in allopatric species. That result is consistent with classic sexual selection theory [START_REF] Andersson | Social polyandry, parental investment, sexual selection, and evolution of reduced female gamete size[END_REF][START_REF] Trivers | Parental Investment and Sexual Selection[END_REF], that females invest more than males in reproduction and thus represent the choosier sex. A greater strength of preference for conspecific mates in females of sympatric populations thus could suggest two things. Mating with a heterospecific in sympatry might be more costly for females than males, leading to reinforcement of females more so than male preferences; or, female preferences evolve faster than male preferences in allopatry, and they are necessary for maintaining species boundaries upon secondary contact.

Here, we focused on darters (genus Etheostoma) to perform a comparative analysis of preference for conspecific mates ("assortative preference") and to explore the role of geographic overlap and sex in predicting those preferences. Darters are a large clade of North American freshwater fishes characterized by elaborate secondary sexual traits [START_REF] Page | Peterson field guide to freshwater fishes of North America north of Mexico[END_REF]. Mate preference and mate choice have been investigated in several pairs of species of darters. Studies find that species demonstrate a varying degree of preference for conspecific over heterospecific mates (Martin & Mendelson, 2013;[START_REF] Williams | Male and female responses to species-specific coloration in darters (Percidae: Etheostoma)[END_REF][START_REF] Kopp | Mechanisms of Assortative Mating in Speciation with Gene Flow: Connecting Theory and Empirical Research[END_REF] and that assortative preference and assortative mate choice is present in both females [START_REF] Williams | Behavioral Isolation Based on Visual Signals in a Sympatric Pair of Darter Species[END_REF], 2011;Roberts et al., 2017) and males [START_REF] Ciccotto | Male Association Preference for Conspecifics in the Redband Darter, Etheostoma luteovinctum (Teleostei: Percidae) Based on Visual Cues[END_REF][START_REF] Zhou | Sexually asymmetric colour-based species discrimination in orangethroat darters[END_REF]Martin & Mendelson, 2016;Moran et al., 2017;Roberts & Mendelson, 2017;Moran & Fuller, 2018), depending on which heterospecific is presented. Moreover, hybridization has been documented in many darter species [START_REF] Bossu | Characterization of a contemporaneous hybrid zone between two darter species (Etheostoma bison and E. caeruleum) in the Buffalo River System[END_REF][START_REF] Keck | Patterns of Natural Hybridization in Darters (Percidae: Etheostomatinae)[END_REF], which could provide the substrate for reinforcement (i.e., hybrids with reduced fitness), making this clade a good system to examine geographic and sex-specific patterns of mate choice at the genus level.

Two studies of darters have tested explicitly for stronger assortative preference in sympatric populations in one or a small number of species pairs. Moran and Fuller (2018) compared male choice and aggressive behaviors in a small number of closely related species (Etheostoma caeruleum and members of the Ceasia species complex). They found that both male preferences for conspecific females and aggressive behaviors towards conspecific males were stronger in populations that were sympatric with the congener. [START_REF] Roberts | Reinforcement in the darter Etheostoma zonale: The effect of sex and sympatry on preferences[END_REF] measured the strength of assortative preference in allopatric and sympatric populations of two darter species (Etheostoma zonale and E. barrenense). They also found a stronger preference for conspecific mates in sympatric populations, but only in females, which contrasts the results of Moran and Fuller (2018), who found assortative preference only in males.

In this paper, we performed a phylogenetically informed meta-analysis to determine the extent to which increased preference for conspecific mates in sympatry is present in Etheostoma. We hypothesize that differences among species in preference for conspecifics are driven by geographic relationships, predicting that sympatric species will have a stronger preference. We also compared the strength of preference for conspecifics between males and females. Most sexual selection theory predicts that females will be choosier (Andersson, 1994;[START_REF] Darwin | The descent of man, and Selection in relation to sex[END_REF][START_REF] Trivers | Parental Investment and Sexual Selection[END_REF] but see [START_REF] Edward | The evolution and significance of male mate choice[END_REF], and [START_REF] Yukilevich | The evolution of male and female mating preferences in Drosophila speciation*[END_REF] results in Drosophila are consistent with that prediction. However, given abundant evidence of male mate choice in darters, and the contrasting results of two studies of assortative preference, the relative importance of male and female mate choice in sexual isolation, in general, remains an open question.

Material and Methods

Inclusion criteria

We conducted our meta-analysis on studies that investigated mate preference in Etheostoma in a comparable manner over the past twelve years (2010)(2011)(2012)(2013)(2014)(2015)(2016)(2017)(2018)(2019)(2020)(2021)(2022)(2023). We included 12 published papers (indicated with an asterisk in the reference section) and five unpublished datasets, encompassing 21 species of Etheostoma distributed across the phylogeny of the genus (Near et al., 2011), arranged in 14 different pairs of species.

All studies used a dichotomous mate preference paradigm where individuals do not have physical access to one another. The main measure of preference is the time the focal fish spent in an association zone adjacent to either a conspecific or a heterospecific individual of the opposite sex (Figure 1). Most studies allowed only visual cues, with focal and stimulus fish separated into different tanks. Two studies (O'Rourke & Mendelson, 2010 and Barber & Mendelson, unpublished) used partitions to separate focal and stimuli fish that were not water-tight, thus potentially allowing exchanges of olfactory cues.

This conservative choice of experimental design removes aggressive physical interactions among members of the same sex as an explanation for the behavior of the focal fish. It also emphasizes mate preference rather than mate choice, as choice is better measured by allowing physical access to potential mates (as in e.g. Moran et al., 2017;[START_REF] Zhou | Sexually asymmetric colour-based species discrimination in orangethroat darters[END_REF]. Studies in our analysis were conducted by different lead authors in different physical lab spaces but at the same university, using similar protocols. This similarity in design is well suited for meta-analyses, which often suffer from discrepancies in experimental design across studies [START_REF] Dougherty | The effect of experimental design on the measurement of mate choice: A meta-analysis[END_REF].

Effect size calculation

All included studies measured the time spent in the association zones. Sample sizes, means and standard deviations were extracted from each paper and when those variables were not available, we contacted corresponding authors to obtain the raw data. The effect size was calculated as a Pearson's correlation coefficient r of times spent in conspecific association zones minus heterospecific association zones for each tested species and sex of each study (Equation 1):

, (Equation 1) sample size of tested individuals, which is identical for both conspecifics and heterospecifics in our case. We did not Z-transform our correlation coefficient as this can affect the accuracy of statistical models (see [START_REF] Janicke | Assortative Mating in Animals and Its Role for Speciation[END_REF] for a similar decision).

𝑟 = 𝑑 𝑑 2 + 1 𝑝*(1-𝑝)

Moderators and their rationale

We sought to determine which factors might influence the strength of preference for conspecifics over heterospecifics, as represented by the effect size. We selected two 'natural' factors: geography and sex of the tested individual, and three 'experimental' factors: the size of the association zones, stimulus type, and recording duration times.

Geography:

We predicted the geographic relationship between the two species in a pair to influence the strength of preference for conspecifics, with a stronger preference in sympatric species. For each study, we determined whether species pairs were allopatric or sympatric [START_REF] Etnier | Fishes Of Tennessee[END_REF][START_REF] Lee | Phylogeny and Temporal Diversification of Darters (Percidae: Etheostomatinae)[END_REF][START_REF] Page | Handbook of Darters (First Edition)[END_REF] and included this variable as a moderator. Some species pairs consist of both allopatric and sympatric populations (i.e., incomplete range overlap); pairs were scored according to the population of origin.

Sex: Although classical sexual selection theory predicts a stronger preference in females, some studies have found the opposite pattern, with a stronger preference for conspecifics in males compared to females (e.g., [START_REF] Kopp | Mechanisms of Assortative Mating in Speciation with Gene Flow: Connecting Theory and Empirical Research[END_REF]Moran & Fuller, 2018). Our dataset includes as many male-focal individuals as females, which allows us to compare assortative preferences between the sexes.

Experimental factors: Previous work showed that experimental design impacts mating preference outcomes [START_REF] Dougherty | The effect of experimental design on the measurement of mate choice: A meta-analysis[END_REF]. Moreover, response to video playbacks compared to live fish varies with at least one of the tested species (Roberts et al., 2017) and could potentially be affected by the sex of the focal individual. We thus included three moderators to reflect the variability in experimental setups. Namely, we included the size of the association zones (5 or 10 cm), the stimulus type (behaving fish, motorised model, fish animation, video playbacks), and recording duration times (5, 10, 15, or 20 min) as experimental factors in our model. We predict that a larger association zone and longer recording duration will result in stronger effect sizes as more data are included. We expect some differences between species due to variation in stimulus type (e.g., live versus video playback).

Phylogeny

To control for the non-independence of the strength of preference due to a shared evolutionary ancestry that varies between species pairs, we included phylogenetic information in our statistical models, using Near and colleagues' (2011) phylogeny. Their phylogeny is based on the cytochrome b mitochondrial gene and two nuclear gene sequences, the S7 ribosomal protein (first intron) and the recombination activating gene-1 (RAG1, exon 3). We retrieved the data file (Nexus format) from TreeBase. We pruned the phylogenetic tree with the drop.tip function from the ape R package [START_REF] Paradis | ape 5.0: An environment for modern phylogenetics and evolutionary analyses in R[END_REF] to keep only our 21 species of interest. In the case where several individuals per species were available, we kept the individual from the closest geographical population to the population studied in our meta-analysis. The resulting tree (Figure 4) was converted into a matrix of phylogenetic distance that was included in our meta-regression models.

Statistical analyses

All statistical analyses were carried out in R version 4.2.0 (R Core Team, 2022). We used the package metafor [START_REF] Viechtbauer | Conducting Meta-Analyses in R with the metafor Package[END_REF] to perform the meta-analysis modelling. To determine the overall mean effect size, we ran a first multi-level meta-analysis model fitted via restricted maximum likelihood ("REML") estimation with the function rma.mv. We included study identity as a random effect to account for the non-independence of effect sizes. We removed species as an additional random effect as this variable explained 0% of the variance and removing it slightly decreased the AIC score of the model. Phylogeny was included in all our models as a variance-covariance matrix estimated from the phylogenetic tree. To assess the respective influence of our different moderators (i.e. explanatory factors) on the mean effect size, we ran meta-regression models for each moderator separately (function rma.mv with the 'mods' parameter). We calculated the level of heterogeneity across all effect sizes using the I² statistic to determine how generalizable our findings are [START_REF] Higgins | Measuring inconsistency in meta-analyses[END_REF]. Script for statistical analyses and data are available on OSF [https://osf.io/hnf8m/].

Results

In total, we extracted 68 effect sizes from 16 studies investigating 21 focal species of darters. This includes 32 effect sizes for males and 36 for females, 42 for allopatric and 22 for sympatric populations.

We found no publication bias in our dataset as shown by a roughly symmetrical funnel plot (Figure 2). This was further confirmed by Egger's regression test (Z = -0.2687, p = 0.7881).

Our multi-level meta-analysis model revealed an overall effect size of medium strength (r = 0.3213, p = 0.0006, CI = 0.1382 -0.5044), corresponding to positive preferences for conspecifics. A negative effect size would correspond to preferences biased towards heterospecifics, and an effect size not different from 0 to an absence of assortative mate preference. The total heterogeneity across effect sizes (I²) amounts to 30.08% (14.15% from the study identity and 15.93% from phylogeny).

Our investigation of the respective influence of our moderators with separate meta-regression models showed that none of them impacted variation in effect sizes. We found no overall difference in preference strength between males and females, no difference between sympatric and allopatric populations, and no effect of any of the experimental factors that we included (size of the association zones, stimulus type, and recording duration times). However, given the highly unbalanced sample sizes for those experimental factors, conclusions should be carefully drawn. The results of our meta-regression models are summarised in Table 1.

Discussion

Our meta-analysis of 16 datasets encompassing 21 species of darters revealed a positive mean effect size of preference for conspecific mates when using a dichotomous paradigm based on visual cues. This result suggests that most species included in our analysis prefer conspecifics of the opposite sex over heterospecifics. Our investigation of potential moderators of the strength of this preference suggests that neither geographic relationships (i.e., sympatry versus allopatry), nor sex, nor any of the three experimental factors (association zone size, stimulus type, and recording time durations) predictably influence the strength of preference for conspecifics.

An overall preference for conspecifics is consistent with previous meta-analyses conducted at higher taxonomic levels [START_REF] Janicke | Assortative Mating in Animals and Its Role for Speciation[END_REF][START_REF] Jiang | Assortative Mating in Animals[END_REF]. These analyses investigated assortative mating across species of birds, insects, fishes, amphibians, gastropods, and crustaceans and found similar trends for overall positive assortative mating. Janicke et al ( 2013) investigated the strength of assortative mating in 307 species in 130 families across these taxa and found an overall effect size of 0.36, independent of species richness. They confirmed the results of [START_REF] Jiang | Assortative Mating in Animals[END_REF], who obtained an overall effect size of 0.26 across 254 species in five animal phyla, though that study did not correct for phylogenetic non-independence. If we assume that assortative preference is a logical precursor to assortative mating, then our results showing assortative preference across Etheostoma demonstrate overall assortative mating at the genus level, with an effect size of 0.32.

We further investigated the effect of geographic overlap and sex on the strength of assortative preference, as both variables are thought to modulate assortative mating. Based on the findings of [START_REF] Coyne | Patterns of speciation in Drosophila[END_REF] in Drosophila, whose estimation of prezygotic isolation for sympatric pairs was at least twice as large as for allopatric pairs, as well as other studies of single species pairs showing a similar pattern [START_REF] Höbel | Reproductive character displacement in the acoustic system of green tree frogs (Hyla cinerea)[END_REF][START_REF] Pauers | Female preferences for conspecific males indicate reproductive isolation between sympatric Labeotropheus Ahl from Lake Malaŵi[END_REF], we predicted stronger assortative preference in sympatric species of Etheostoma. Our statistical analyses did not support this prediction. Rather, out of the four sympatric populations included in the meta-analysis, two showed a stronger preference for conspecifics (E. zonale and E. barrenense), one had no assortative preference (E. flabellare) and one, E. olmstedi, showed unexpectedly disassortative preference, with individuals from sympatric populations showing a stronger preference for heterospecifics (Figure S1). Interestingly, the allopatric populations of E. olmstedi that were also included in the analysis did not show such a reverse pattern but rather a stronger preference for conspecifics (Figure S2). This variation across species in the relationship between geographic overlap and assortative preference might suggest that species, and populations within species, differ in the costs and benefits of mating with heterospecifics. The classic cost of mating with heterospecifics is a reduction in offspring fitness, and the strength of this postzygotic barrier is likely to vary across species pairs. Alternatively, this variation might reflect differences in the extent of syntopy between nominally sympatric species. For example, E. barrenense and E. zonale, a species pair in the analysis in which both species exhibit strong assortative preference, are notably syntopic where they overlap. E. barrenense is more strongly associated with bedrock substrate and E. zonale with vegetation [START_REF] Greenberg | Habitat use and feeding behavior of thirteen species of benthic stream fishes[END_REF][START_REF] Hlohowskyj | Substrate Selection by Fantail (Etheostoma flabellare), Greenside (E. blennioides), and Rainbow (E. caeruleum) Darters[END_REF]), yet both are commonly found and collected together over bedrock or coarse gravel (pers obs).

Given evidence of reduced survival in the hybrids of this species pair (Williams & Mendelson, 2014), assortative preference between these species may have arisen due to reinforcement (see [START_REF] Roberts | Reinforcement in the darter Etheostoma zonale: The effect of sex and sympatry on preferences[END_REF]. In contrast, E. olmstedi and E. flabellare, between which preference is either absent or disassortative, are known to exhibit distinct habitat and foraging preferences [START_REF] Greenberg | Habitat use and feeding behavior of thirteen species of benthic stream fishes[END_REF][START_REF] Proulx | A Study of Darter (Percidae) Assemblages in Several Tributaries of the Ottawa River[END_REF] and may represent micro-allopatric species. Thus, the conditions that predict stronger assortative preference in sympatry, including the opportunity for maladaptive hybridization, may not hold for this species pair.

As such, our results do not support the prediction of overall greater assortative preference in sympatric populations or species. Rather, they highlight variation among species that can help explain when and why assortative preferences evolve, for example, due to variation in the costs and benefits of interspecific mating, or in the extent of true geographic overlap. Additional factors that might affect the strength of assortative preference include the presence or absence of nuptial coloration. Assortative mating based on color pattern differences has been shown in a number of animal taxa, including Midas cichlid fish [START_REF] Elmer | Color assortative mating contributes to sympatric divergence of neotropical cichlid fish[END_REF], Heliconius butterflies [START_REF] Jiggins | Reproductive isolation caused by colour pattern mimicry[END_REF], and strawberry poison frogs (e.g., [START_REF] Summers | Visual mate choice in poison frogs[END_REF]. Notably, two species in our analysis that show strong assortative preference are strikingly colorful, and previous studies show that both males and females of these species preferentially associate with conspecific color patterns [START_REF] Williams | Female preference for male coloration may explain behavioural isolation in sympatric darters[END_REF][START_REF] Hopkins | Reinforcement in plants[END_REF]. In contrast, the species in our analysis that lacks assortative preference (E. flabellare), and the species for which assortative preference varies across populations (E. olmstedi), are largely achromatic. Adding additional darter species that vary in chromatism therefore could allow us to test whether the relationship between assortative preference and geographic overlap is predicted by the presence of nuptial coloration. Alternatively, species might differ in temporal patterns of mating, with some mating earlier or later in the season. Like micro-allopatry, this type of temporal reproductive isolation precludes maladaptive hybridization and thus precludes selection against hybrids leading to increased assortative preference in sympatry. A broader survey of species could allow us to include these and other additional factors, with multiple species pairs representing each factor, and thus evaluate whether they modulate the relationship between geography and assortative preference.

In addition to geography, we investigated the effect of sex on the strength of preference for conspecifics. Classical sexual selection theory predicts that females will be choosier than males, as the cost of investment is expected to be skewed towards females [START_REF] Andersson | Social polyandry, parental investment, sexual selection, and evolution of reduced female gamete size[END_REF][START_REF] Trivers | Parental Investment and Sexual Selection[END_REF]. In our meta-analysis, sex did not have a significant effect on the strength of preference for conspecifics. Although counter to conventional sexual selection theory, this result is consistent with the natural history of darters. Male darters invest considerably in reproduction, with energetic courtship displays, nuptial coloration, and in some species, paternal care (e.g. [START_REF] Kelly | Diversification of egg-deposition behaviours and the evolution of male parental care in darters (Teleostei: Percidae: Etheostomatinae)[END_REF][START_REF] Kopp | Mechanisms of Assortative Mating in Speciation with Gene Flow: Connecting Theory and Empirical Research[END_REF]. Indeed, similar to poison frogs [START_REF] Summers | Phenotypic and genetic divergence in three species of dart-poison frogs with contrasting parental behavior[END_REF], darter species that exhibit paternal care are mostly achromatic, suggesting a potential trade-off in energy allocation for reproduction.

The absence of an effect of sex on assortative preference is also consistent with several previous studies in darters. For example, assortative preference was stronger in males than in females when secondary contact was simulated in artificial streams for allopatric species of darters in the earliest stages of divergence [START_REF] Kopp | Mechanisms of Assortative Mating in Speciation with Gene Flow: Connecting Theory and Empirical Research[END_REF]. Similarly, for the darter species E. caeruleum and members of the Ceasia (E. spectabile) species complex, only males of sympatric species showed greater preference for conspecifics (Moran & Fuller, 2018). Yet in other studies, female darters exhibited stronger assortative preference than males, as in E. nigripinne [START_REF] O'rourke | Male and female preference for conspecifics in a fish with male parental care (Percidae: Catonotus)[END_REF]. Thus, the absence of an effect of sex on the strength of preference seems to accurately reflect the diversity of reproductive behaviors and preferences in this genus. Including even more species in the analysis could allow us to identify groups of species (e.g., subgenera) in which males are choosier than females or vice versa. Such a pattern could further elucidate the factors or conditions driving sex differences in assortative preference.

Beyond the biological implications of our results, we also sought to determine whether experimental factors could influence effect sizes when comparing multiple studies. In terms of the type of stimuli, i.e., whether stimulus fish were live, model (dummy), video playback, or computer animation, we found that it did not affect the strength of preference for conspecific stimuli. This result is an important validation of the use of artificial stimuli in mate choice research [START_REF] Chouinard-Thuly | Technical and conceptual considerations for using animated stimuli in studies of animal behavior[END_REF][START_REF] Powell | What artifice can and cannot tell us about animal behavior[END_REF], in that artificial stimuli appear to be as useful as live animals in detecting mate preference. Artificial stimuli allow researchers to replace and reduce the number of live animals in experiments, as encouraged by ethical animal use protocols. Results of [START_REF] Williams | Female preference for male coloration may explain behavioural isolation in sympatric darters[END_REF] corroborate our finding, showing equivalent responses to live and dummy fish in Etheostoma zonale and E. barrenense. Roberts and colleagues (2017) found equivalent responses to live and video playback stimuli in E. zonale, but not in E. barrenense. Thus the type of stimulus may have some effect on the strength of preference, but this appears to be typeand species-specific. We note that most studies in our analysis used live stimuli. Nonetheless, we continue to explore the efficacy of various stimulus types in testing mate preference in darters.

Although we did not expect a significant effect of recording time duration, we found a tendency for longer durations to have smaller effect sizes. It might thus be important to keep the duration of observation short (i.e. under 10 min) as the expression of preference may begin to taper after a few minutes as the focal individual loses interest. Besides mean durations in association zones, additional measures of fish preference could provide important information. Additional behavioral measures might include the fish's head orientation or line of sight, and pursuit behaviors, to quantify interest in the presented stimuli. For instance, two studies in darters reported glass jabbing behavior as a measure of a female's mating interest and of a male's aggressive behavior [START_REF] Soudry | Male rainbow darters (Etheostoma caeruleum) prefer larger conspecific females[END_REF][START_REF] Williams | Male and female responses to species-specific coloration in darters (Percidae: Etheostoma)[END_REF]. One study also reports the number of times a fish visits an area [START_REF] Soudry | Male rainbow darters (Etheostoma caeruleum) prefer larger conspecific females[END_REF], which could indicate exploratory differences between species or sexes that may reflect preference.

Finally, the last experimental factor that varied between studies was the size of the association zones. In their study comparing several pairs of darter species, Mendelson and colleagues (2018) reported results for zone sizes of both 5 and 10 cm. We found no difference between 5 and 10 cm in terms of effect size neither in their study (t = -0.6319, df = 53.624, p = 0.5301) nor in our meta-analysis (data for 5cm was included in the meta-analysis, but including the data for 10cm instead made no difference for effect sizes). However, effect sizes tended to be larger for wider association zones, which is logical, since a bigger area of the tank can be occupied for a longer period of time. To avoid inflating results with larger association zones, we adjusting the size of the association zone to reflect the visual acuity of the tested species [START_REF] Caves | Visual acuity in ray-finned fishes correlates with eye size and habitat[END_REF].

Conclusion

Conducting a meta-analysis of dichotomous mate preference trials in darter fish (Etheostoma), we found an overall effect size of medium strength, indicating a preference for conspecific over heterospecific mates across the genus, with no significant difference between males and females. Comparing sympatric and allopatric populations, we found no effect of geographic overlap on the strength of preference. Our results suggest that the link between assortative mating and geography is not as straightforward as might be predicted from hypotheses of reinforcement, and the link between assortative mating and sex is not as straightforward as classical interpretations of sexual selection might suggest. We postulate several factors that could modulate the strength of preference for conspecifics in the genus Etheostoma and explain the lack of evidence for an overall effect of geography and sex. Our results thus highlight the complexity of assortative mating and the need for further study. Given the diversity of the genus, Etheostoma is a great system to investigate such questions.

Figures Figure 1: Mate preference experimental

Legend: Illustration of a dichotomous mate preference paradigm. The main measure is the time that the focal fish (circled) spends in either association zone adjacent to either a conspecific or a heterospecific individual of the opposite sex. The exact design varies between studies as mate options can be live fish, motorised models, videos or computer animations displayed on a monitor. Created with BioRender.

3: Forest plot of effect sizes

Legend: Forest plot showing mean effect sizes (± CIs) calculated using Equation 1 of the main moderators: sex and geographic relationship, as well as an average of all the moderators included in the analysis. Freshwater Fishes of Maryland and Southeastern Fishes Council Proceedings,1(11). https://trace.tennessee.edu/sfcproceedings/vol1/iss11/2 Lemmon, E. M. ( 2009

Figure 2 :

 2 Figure 2: Funnel plot to test for publication bias

Figure 4 :

 4 Figure 4: Phylogeny of the focal included in the meta-analysis

  ). Diversification of Conspecific Signals in Sympatry: Geographic Overlap Drives Multidimensional Reproductive Character Displacement in Frogs.Evolution, 63(5), 1155-1170. https://doi.org/10.1111/j.1558-5646.2009.00650.x Liou, L. W., & Price, T. D. (1994). Speciation by Reinforcement of Premating Isolation.Evolution, 48(5), 1451-1459. https://doi.org/10.2307/2410239 * Martin, M. D., & Mendelson, T. C. (2013). Incomplete behavioural isolation and asymmetric female preference in darter sister species (Percidae: Etheostoma). Journal of Fish Biology, 83(5), 1371-1380. https://doi.org/10.1111/jfb.12239 * Martin, M. D., & Mendelson, T. C. (2016). Male behaviour predicts trait divergence and the evolution of reproductive isolation in darters (Percidae: Etheostoma). Animal Behaviour, 112, 179-186. https://doi.org/10.1016/j.anbehav.2015.11.027 * Mattson, C. L., Roberts, N. S., & Mendelson, T. C. (2020). Male preference for conspecific females depends on male size in the splendid darter, Etheostoma barrenense. Animal Behaviour, 165, 89-96. https://doi.org/10.1016/j.anbehav.2020.04.022 Matute, D. R., & Cooper, B. S. (2021). Reinforcement alone does not explain increased reproductive isolation in sympatry (p. 2021.05.06.442525). bioRxiv. https://doi.org/10.1101/2021.05.06.442525 * Mendelson, T. C., Gumm, J. M., Martin, M. D., & Ciccotto, P. J. (2018). Preference for conspecifics evolves earlier in males than females in a sexually dimorphic radiation of fishes. Evolution, 72(2), 337-347. https://doi.org/10.1111/evo.13406 Moran, R. L., & Fuller, R. C. (2018). Male-driven reproductive and agonistic character displacement in darters and its implications for speciation in allopatry. Current Zoology, 64(1), 101-113. https://doi.org/10.1093/cz/zox069 Moran, R. L., Zhou, M., Catchen, J. M., & Fuller, R. C. (2017). Male and female contributions to behavioral isolation in darters as a function of genetic distance and color distance. Evolution, 71(10), 2428-2444. https://doi.org/10.1111/evo.13321

Figure S2 :

 S2 Figure S2: Effect sizes for the allopatric populations in the meta-analysis (alphabetical order).

Supplementary material

Figure S1: Effect sizes for the sympatric populations included in the meta-analysis order). r gives the effect size value for the sympatric population of the species. N indicate the number of effect sizes (i.e. number of experiments) that were included per population.


Erwan Harscouet-Commecy

Rita Covas

H Dieter Oschadleus

Pierre Colencon

Matthieu Pelte

Paul Dufour

Staffan Andersson

Julien P Renoult

Claire Doutrelant

Pierre Colençon

Comparative evidence for sexual selection on nest weave pattern in weaverbirds

Keywords: Ploceidae, extended phenotype, comparative analysis, fractals, scale-invariance, computer vision, bird nest, sensory drive, quality indicator, Fourier slope

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Introduction

Extended phenotypes are traits produced outside the body of the individual carrying the genes coding for their expression [START_REF] Dawkins | The extended phenotype: the gene as the unit of selection[END_REF]. Examples from a wide diversity of taxa include spider webs, beaver dams, armour of caddisfly larvae, bowerbirds' display arenas, animal nests or burrows and human houses. The concept of extended phenotypes was introduced almost four decades ago [START_REF] Dawkins | The extended phenotype: the gene as the unit of selection[END_REF], but few studies have investigated their adaptive functions, diversification and associated life histories. As a consequence, the evolutionary mechanisms underlying the striking diversity of these traits are still poorly understood. While it is likely that many extended phenotypes were initially selected for their protective or resource-gathering function [START_REF] Hansell | Animal Architecture[END_REF], some may have evolved additional functions, such as sexual signalling [START_REF] Schaedelin | Extended phenotypes as signals[END_REF]. To date, this hypothesis has remained poorly investigated.

Nests are prominent examples of extended phenotypes that may serve multiple functions.

Their primary function is to ensure the protection of offspring during development [START_REF] Hansell | Bird Nests and Construction Behaviour[END_REF]. However, in some species, nests are associated with ostentatious signals and thus they can draw predators' or competitors' attention. For instance, in some fishes like sticklebacks [START_REF] Östlund-Nilsson | The artistic three-spined stickleback (Gasterosteous aculeatus)[END_REF], or birds like starlings [START_REF] Rubalcaba | Sexual and natural selection in the evolution of extended phenotypes: the use of green nesting material in starlings[END_REF] and kites [START_REF] Sergio | Raptor Nest Decorations Are a Reliable Threat Against Conspecifics[END_REF], males decorate nests with coloured algae, green aromatic material and white plastic, respectively. These observations seem at odds with the primary protective function of nests. The current explanation to this paradox proposes that these conspicuous nests could secondarily act as sexual signals [START_REF] Schaedelin | Extended phenotypes as signals[END_REF]. This is further supported by evidence that some quantitative or qualitative aspects of nest architecture contribute to determine mating success [START_REF] Jordan | The social and ecological costs of an 'over-extended' phenotype[END_REF].

Among birds, weaverbirds (Ploceidae) are famous for the highly visible and intricately elaborate nests they construct [START_REF] Crook | The Evolution of Social Organisation and Visual Communication in the Weaver Birds (Ploceinae)[END_REF]. They have developed a weaving technique in which strands of material are inserted, recovered and reinserted repeatedly in several ways leading to impressive woven nests [START_REF] Del Hoyo | Handbook of the birds of the world. vol. 15. weavers to new world warblers[END_REF] (Fig. S1A). The nests can take various forms such as ball, mass, retort, or sock, and are made from different plant materials.

In many weaver species, nests are built almost entirely by males, with females adding only the inner lining to the bottom of the egg chamber. Weaverbirds are a diverse family containing 121 species organized in 15 genera distributed across Sub-Saharan Africa, Indo-Malayan region and Indian Ocean islands [START_REF] De Silva | Phylogenetic relationships of weaverbirds (Aves: Ploceidae): A first robust phylogeny based on mitochondrial and nuclear markers[END_REF]. They present various mating strategies, some species being polygamous and others monogamous. Weaverbirds also show between-species variation in sexual dimorphism [START_REF] Del Hoyo | Handbook of the birds of the world. vol. 15. weavers to new world warblers[END_REF][START_REF] Song | Evolution of Social Organization: Phylogenetic Analyses of Ecology and Sexual Selection in Weavers[END_REF]. Finally, in some species, the nest seems to be part of the male courtship display, while in others, the males display far from the nest, sometimes even before its construction [START_REF] Crook | The Evolution of Social Organisation and Visual Communication in the Weaver Birds (Ploceinae)[END_REF]. This family thus presents a large variation in both nest design, intensity of sexual selection and association with the nest while displaying, and therefore represents a unique setup in which to investigate the influence of sexual selection on the evolution of elaborate nests.

Several intraspecific studies have linked weaverbirds' nest characteristics to mate choice, thus suggesting that they can act as a sexual signal. For instance, in red bishops (Euplectes orix ), village weavers (Ploceus cucullatus) and baya weavers (Ploceus philippinus), duration, strength of materials and location of the nest influenced female choice [START_REF] Metz | Male nest-building behaviour and mating success in the red bishop (Euplectes orix )[END_REF][START_REF] Collias | Nest and mate selection in the village weaverbird (Ploceus cucullatus)[END_REF][START_REF] Quader | What Makes a Good Nest? Benefits of Nest Choice to Female Baya Weavers (Ploceus Philippinus)[END_REF]. In addition, in southern masked weavers (Ploceus velatus), nest dimensions were partially repeatable, suggesting a possible heritability which would allow this trait to evolve [START_REF] Walsh | Repeatability of nest morphology in African weaver birds[END_REF]. A notable component of weavers' nests is the elaborate weave, which creates a striking visual pattern. In village weavers, nests showed an individual signature in the weave pattern [START_REF] Bailey | Image analysis of weaverbird nests reveals signature weave textures[END_REF]. However, whether and how the weave pattern and its visual appearance differ between species, and whether it could evolve through sexual selection has never been investigated.

Two main types of explanations are proposed for the origin of preferences for visual patterns.

First, some patterns can be preferred if they are quality indicators [START_REF] Pérez-Rodríguez | Shape matters: animal colour patterns as signals of individual quality[END_REF], correlating with the value or quality of the signaller, and hence affecting also the fitness of the receiver that chooses those patterns [START_REF] Cotton | Do sexual ornaments demonstrate heightened condition-dependent expression as predicted by the handicap hypothesis?[END_REF][START_REF] Smith | Animal Signals[END_REF]. For example, in paper wasps the patterning of the black facial patch is related to survival and dominance [START_REF] Tibbetts | Socially selected ornaments and fitness: Signals of fighting ability in paper wasps are positively associated with survival, reproductive success, and rank[END_REF]. Another example is the bib of red-legged partridges, where the scale-invariance of the patterning is correlated to immune response and body weight [START_REF] Pérez-Rodríguez | Fractal geometry of a complex plumage trait reveals bird's quality[END_REF]. Scale-invariance describes the regularity of a pattern, or the degree to which a pattern looks similar at a close or distant range (similar when 'zooming' in and out).

The well-studied fractals are a particular case of scale-invariant patterns. In medical research, scale-invariance has been shown to correlate with the healthy functioning of many physiological systems [START_REF] Korolj | A healthy dose of chaos: Using fractal frameworks for engineering higher-fidelity biomedical systems[END_REF], suggesting that developmental homeostasis might explain how scale-invariant patterns could be used as quality indicators in animal communication [START_REF] Pérez-Rodríguez | Shape matters: animal colour patterns as signals of individual quality[END_REF].

Second, a pattern preference might arise from a pre-existing preference. This occurs when a preference has first evolved in a context unrelated to mate choice, and sexual signals have then aligned with the pre-existing preference [START_REF] Ryan | The Sensory Basis of Sexual Selection for Complex Calls in the Túngara Frog, Physalaemus Pustulosus (sexual Selection for Sensory Exploitation)[END_REF]. For example, experimental and comparative works suggest that the breast spots of Estrildid finches have been selected because they mimic seeds to which conspecifics are attracted (Mizuno andSoma, 2021, 2022).

A particular case of exploitation of pre-existing preferences is the sensory drive, in which preexisting preferences are determined by the physical characteristics of the habitats through their effect on sensory systems [START_REF] Endler | Sensory ecology, receiver biases and sexual selection[END_REF]. The sensory drive hypothesis is supported by several studies on sexual colour signals [START_REF] Seehausen | Speciation through sensory drive in cichlid fish[END_REF][START_REF] Fleishman | Can Sensory Drive Explain the Evolution of Visual Signal Diversity in Terrestrial Species? A Test with Anolis Lizards[END_REF]. Recently, this hypothesis was applied to patterns by using two main findings in human psychology and neuroscience [START_REF] Renoult | Processing bias: extending sensory drive to include efficacy and efficiency in information processing[END_REF]. First, research in humans shows that stimuli that are efficiently processed (detected more quickly, memorized for longer and more accurately) tend to be perceived as attractive [START_REF] Endler | Sensory ecology, receiver biases and sexual selection[END_REF], a phenomenon known as 'a processing bias' [START_REF] Renoult | Processing bias: extending sensory drive to include efficacy and efficiency in information processing[END_REF]. For example, scale-invariant patterns such as fractals would be visually attractive because their spatial regularity makes them efficiently processed in the visual system [START_REF] Graf | A Dual-Process Perspective on Fluency-Based Aesthetics: The Pleasure-Interest Model of Aesthetic Liking[END_REF][START_REF] Reber | Effects of Perceptual Fluency on Affective Judgments[END_REF]. Second, what determines the efficiency of information processing is the sensitivity of neurons. A number of studies, both in humans and non-human animals, have documented that the sensitivity of many neurons in the visual system is tuned to the spatial statistics that are dominant in visual natural environments [START_REF] Barlow | Possible Principles Underlying the Transformations of Sensory Messages[END_REF]. Integrating these findings in psychology and neuroscience into the sensory drive hypothesis, it was thus predicted that stimuli mimicking the spatial statistics of natural habitats are efficiently processed, and thus are visually attractive [START_REF] Renoult | Processing bias: extending sensory drive to include efficacy and efficiency in information processing[END_REF].

In this study, we investigate whether the scale-invariance of weaverbirds' nests is linked to proxies of sexual selection across the weaver family. Scale-invariance is typically measured using the slope of the Fourier power spectrum (hereafter 'Fourier slope') [START_REF] Winkielman | Prototypes Are Attractive Because They Are Easy on the Mind[END_REF][START_REF] Hyvärinen | Natural Image Statistics: A Probabilistic Approach to Early Computational Vision[END_REF][START_REF] Pérez-Rodríguez | Shape matters: animal colour patterns as signals of individual quality[END_REF]. In humans, for both abstract and representational images, there is a preference for Fourier slopes close to -2 [START_REF] Spehar | Beauty and the beholder: the role of visual sensitivity in visual preference[END_REF][START_REF] Juricevic | Visual Discomfort and Natural Image Statistics[END_REF][START_REF] Taylor | Fractal Fluency: An Intimate Relationship Between the Brain and Processing of Fractal Stimuli[END_REF]. This value of -2 describes the maximum possible scale-invariance, as reached by perfect fractals [START_REF] Graham | Statistical regularities in art: Relations with visual coding and perception[END_REF][START_REF] Pouli | Image Statistics in Visual Computing[END_REF]. But -2 is also the expected average Fourier slope of images of natural habitats or landscapes [START_REF] Redies | Fractal-like image statistics in visual art: similarity to natural scenes[END_REF]. Therefore, signals with a Fourier slope close to -2 could be preferred either because (i) scale-invariance indicates higher qualities of the signaler ('quality indicator hypothesis'), (ii) scale-invariant nests are intrinsically efficiently processed ('universal preference hypothesis', caused by a processing bias independent of natural habitats) or (iii) they are more similar to the average scale-invariance of natural habitats (to which visual systems have adapted, 'sensory drive hypothesis'). However, beyond the average expected value of -2, there may be some variation in the Fourier slope among different natural habitats. Under the hypothesis of sensory drive, this variation could result in variation in preferences and therefore in the design of sexual signals, which are expected to co-vary with habitats [START_REF] Hulse | Sexual signaling pattern correlates with habitat pattern in visually ornamented fishes[END_REF].

Hence, it is important to calculate the scale-invariance of the habitat in which species live and not to consider by default that all natural habitats have a scale-invariance of -2 when testing the sensory drive hypothesis.

Here, we first measured the Fourier slope in images of nests and of different natural habitats. Then, we calculated (i) the difference between the nests' Fourier slope and -2 (the deviation to perfect scale-invariance) and (ii) the difference between the nests' Fourier slope and the Fourier slope of the surrounding habitat in which the nests were found. Finally, we used comparative methods to relate these differences to two main proxies of sexual selection: mating system [START_REF] Shuster | Sexual selection and mating systems[END_REF] and dimorphism [START_REF] Szekely | Sexual size dimorphism in birds[END_REF]. We tested two predictions. First (P1), if the weave is sexually selected because it indicates builder's quality or it matches a universal preference bias for efficient processing, then species under strong sexual selection (polygamous and dimorphic species) should exhibit a nest Fourier slope close to -2. Second (P2), if nest weave is sexually selected by sensory drive, then species under strong sexual selection should exhibit a nest Fourier slope close to the Fourier slope of the corresponding surrounding habitat.

The validation of either of these predictions would support the hypothesis of sexual selection on the weave of weaverbird nests.

Methods

Digital photos collection

We gathered a total of 2,829 digital photos of weaver nests (Fig. S1A) from various sources: the Natural History Museum of Tring virtual database (https://data.nhm.ac.uk/dataset/, 438 images), the Photos of Weaver Nests (PHOWN) virtual database (http://weavers.adu.org.za/, 514 images), photos taken by us in South Africa in January 2021 (1,048 images), and images from birdwatchers contacted individually (928 images). From this raw data collection, we kept 1,291 images of high quality (sharp image with good exposure, displaying a nest of size > 250 pixels) for the analyses, corresponding to 873 nests belonging to 61 species (mean = 14.3 nests per species, s.d. = 26.1).

For the habitats, we first identified all habitats occupied by at least one species of weavers [START_REF] Del Hoyo | Handbook of the birds of the world. vol. 15. weavers to new world warblers[END_REF]. We listed a series of keywords: grassland, forest, woodland, wetland, scrub and savannah corresponding to the primary natural habitat of these species. We added two habitat keywords that do not appear in [START_REF] Del Hoyo | Handbook of the birds of the world. vol. 15. weavers to new world warblers[END_REF], agricultural and city, resulting in eight categories of 'surrounding habitats' (weaver habitat). Then we identified four categories of 'foreign habitats': sandy desert, mountain, glacier and coral reef, which are not occupied by weavers (Fig. S2).

For the surrounding' and foreign habitats we gathered images from the internet by searching keywords corresponding to the twelve habitat categories in Google image and using free databases when available. We associated an image to a category based on the type and distribution of the vegetation. We selected images according to their quality (no blur, correct illumination) and angle of view (wide-angle photos depicting a landscape view of habitats as could be seen by birds in flight) (Fig. S2). The number of photos per habitat category varied from 166 to 381 (mean = 257).

Fourier slope

From each nest image, we segmented the nest out using VGG annotator [START_REF] Dutta | The VIA Annotation Software for Images, Audio and Video[END_REF], replacing the original background with a white background. Each image was then cropped to make it square and resized to 1,024 x 1,024 pixels (Fig. S1B). The Fourier slope was calculated using standard methods in empirical aesthetics [START_REF] Redies | Artists portray human faces with the Fourier statistics of complex natural scenes[END_REF][START_REF] Graham | Statistical regularities in art: Relations with visual coding and perception[END_REF], with Matlab 2021a [START_REF] Higham | MATLAB guide[END_REF]. Because we are interested in the weave pattern specifically, we excluded variation in the Fourier slope caused by the global shape of the nests (see (Fig. S1) for details). For nests with more than one image (different viewing angles), we used the mean slope value. For habitats, images were also cropped to make them square, and the Fourier slope was calculated from resized images following the same procedure as for nest images. We calculated one slope value per habitat category as the mode of the distribution of slope values of all images of that category.

Fixed predictor variables

To explore the potential role of sexual selection in the evolution of nest weave in Ploceidae, we collected data on mating system and tarsus-based sexual size dimorphism as proxies of sexual selection intensity, these were included in separate models since they are not independent. In addition, we tested the effects of nest visibility and display location and their interactions with the two proxies of sexual selection. We also included two control variables: the origin of data and the size of the image. Detailed information on the predictors is given in (Table 1).

Response variables

Deviation from the presumed selection optima

The response variables describe the deviation of each nest Fourier slope to the predicted selection optima and are calculated as the absolute difference between the nest Fourier slope and the optimum. To test the quality indicator/universal preference prediction (P1), we used a selection optimum of -2, which is the slope value corresponding to maximal scale-invariance [START_REF] Graham | Statistical regularities in art: Relations with visual coding and perception[END_REF][START_REF] Pouli | Image Statistics in Visual Computing[END_REF]. The response variable is then the distance of each nest Fourier slope to -2. For the sensory drive prediction (P2), the selection optimum varies for each nest because it corresponds to the modal Fourier slope of the habitat where the nest was found. Using the GPS coordinates of each photographed nest, we identified which weaver habitat category (grassland, forest, woodland, wetland, scrub, savannah, agricultural and city (see del Hoyo et al., 2011)) surrounded the nest based on the landscape structure as observed on Google Earth satellite images. Each individual nest was thus associated with the modal slope value of its habitat category. We associated individual nests to habitats rather than attributing a species to a habitat because most weaver species are generalists [START_REF] Del Hoyo | Handbook of the birds of the world. vol. 15. weavers to new world warblers[END_REF].

With these response variables we performed four models. Models 1.1 and 1.2 fitted the distance between the nests' Fourier slope and -2 as a response variable (P1). Model 1.1 included mating system, display location, nest visibility, image size and data origin as fixed predictors with interaction terms between mating system and display location and nest visibility. Model 1.2 was similar but used tarsus dimorphism instead of mating system. Models 2.1 and 2.2 fitted as a response variable the distance between the Fourier slope of the nest and the slope of the habitat surrounding the nest (P2). Model 2.1 and 2.2. included mating system and tarsus dimorphism, respectively.

Deviation from alternative selection optima

Any significant association between proxies of sexual selection and the nest weave deviation from presumed selection optima (of either P1 or P2) would suggest selection for matching nest weave to one of these optima. Yet to provide even stronger evidence of selection, it is necessary to show that similar associations are not found when considering alternative optima.

We therefore re-fitted the four models using different selection optima. For models 1.1 and 1.2 (P1), the response variable was the deviation of the nest Fourier slope from a range of optima located between -1.5 and -2.5 (with 0.05 step, leading to 21 models with mating system as predictor and 21 with dimorphism). For models 2.1 and 2.2 (P2), the response variable was the deviation of the nest Fourier slope from the distribution mode of the surrounding habitat and one of the four following foreign habitats: desert, mountain, glacier or coral reef (leading to a total of 10 models, five with mating system and five with dimorphism as predictors). We then examined how the model estimates of the predictors vary in each of these new models simulating alternative selection optima. Observing that the estimates of proxies of sexual selection are the highest for the optimum predicted for P1 (-2) or P2 (surrounding habitat) would thus represent additional support for a stabilizing sexual selection on the weave of weaverbird nests around this optimum.

Statistical models

For all the analyses described above, we fitted Bayesian phylogenetic generalized linear mixed models using the R-package "MCMCglmm" [START_REF] Hadfield | MCMC Methods for Multi-Response Generalized Linear Mixed Models: The MCMCglmm R Package[END_REF]. To account for phylogenetic relatedness and allow multiple data entries per species, we included the inverse of the variance-covariance matrix and species as random effects in each model. The variance-covariance matrix was built from the recently published Ploceidae phylogenetic tree [START_REF] De Silva | Phylogenetic relationships of weaverbirds (Aves: Ploceidae): A first robust phylogeny based on mitochondrial and nuclear markers[END_REF].

Model equations are detailed in supplementary material (Eqs S1-S4). We assumed that our response variables follow a Brownian model of evolution. Weak informative inverse-Wishart priors' distribution (V = 1 and nu = 0,002) were set for both random and fixed effects. We let the MCMC algorithm run for 5,000,000 iterations, with a burn-in period of 500,000 and a sampling interval of 5,000. Statistical significance of the posterior mean estimate was inferred when the 95% credible interval (CI) did not cross zero.

Sensory drive permutation tests

To further test the sensory drive hypothesis, we determined a null distribution of estimates by randomly permutating (500 times) the surrounding habitat associated to each nest and fitted models 2.1 and 2.2 described above on each permutated dataset. We then compared the real estimates to the null distributions of estimates. A predictor has a statistically significant effect on the deviation of the nest Fourier slope to the Fourier slope of the surrounding habitat if its real estimate is outside the 95% interval of the null distribution.

Results

Fourier slope of nests and habitats

All nests and habitats had a Fourier slope within the range [-1.3; -2.4] (Fig. 1). The mean Fourier slope of nests was -1.99. For habitats, the mean Fourier slope was -1.80 when considering all categories of weaver habitats (surrounding habitats), and -1.89 for habitats not occupied by weavers (foreign habitats). Mean slopes differed between surrounding and foreign habitats (t test, t = -17.4, P < 10 -3 ), across categories of foreign habitats (ANOVA test, F = 83.5, P < 10 -3 )) and across categories of weaver surrounding habitats (ANOVA test, F = 55.5, P < 10 -3 )).

Presumed selection optima

When testing the quality indicator/universal preference prediction (P1), we found significant associations between the deviation of the nest weave pattern from the maximum scale-invariance (slope -2) and both proxies of sexual selection (Fig. 2) (Fig. 3). This implies that polygamous and sexually dimorphic species build more scale-invariant nests than monogamous and monomorphic species do. Display location had a significant effect in model 1.2 (tarsus dimorphism) but not in model 1.1 (mating system) (Fig. 3). None of the other predictors were significant (Fig. S3A).

Regarding the prediction of the sensory drive hypothesis (P2), we did not find any effect of the two proxies of sexual selection (Fig. 3). Thus, polygamous and sexually dimorphic species build nests whose scale-invariance does not match that of the surrounding habitat better than the nests of monogamous and monomorphic species do. Nest visibility had a significant effect in model 2.2 (tarsus dimorphism) but not in model 2.1 (mating system) (Fig. 3). None of the other predictors were significant (Fig. S4A).

Alternative selection optima

We observed important variation in the estimates of all the tested predictors when varying the Fourier slope optimum (the reference slope to which the distance of each nest slope is calculated, Fig. 4).

Regarding the quality indicator/universal preference hypothesis (P1), for all three predictors that were significant in the previous regressions (models 1), the estimates were the largest with a response variable describing a deviation from -2 (exactly -2 for mating system and display location, close to -2 for tarsus dimorphism, Fig. 4A-C). These results strongly suggest that the maximal possible scale-invariance corresponds to an optimum and that weaverbird nests are under sexual selection to stabilize their weave scale-invariance around this optimum. In contrast, the estimate of nest visibility which was significant in model 1.1 (but not 1.2) does not peak around -2 (Fig. 4D), which is also not the case for the other predictors (Fig. S3B).

Regarding the sensory drive hypothesis (P2), we first considered nest visibility, the only predictor significantly associated to the deviation of nest slope to the surrounding habitat slope. Fig. 4H shows that, as for the surrounding habitat, some foreign habitats, when set as an optimum, also yielded significant estimates. For the other predictors, the estimates obtained using the deviation from the surrounding habitat as a response variable were always lower than at least one estimate obtained when using the deviation from a foreign habitat (Fig. S4B).

These results confirm that the weave of weaverbird nests has probably not been selected to match the scale-invariance of the surrounding habitat.

Permutation test of sensory drive

For both mating system and tarsus size dimorphism, the observed estimates fell within the 95% limits of the distribution of estimates expected according to a random association between a nest and its habitat. All other predictors were also non-significant, except display location for model 2.1 (Fig. S5).

Discussion

Weaverbirds are widely known for their exceptional nests and intricate weave patterns. We used comparative analyses to investigate whether sexual selection contributes to explain the striking regularity of the weave pattern. We found that the Fourier slope of weaves, a measure of scaleinvariance and thus pattern regularity, varies across species within the family. More precisely, weave scale-invariance is closer to its maximal possible value (reflecting perfect regularity) for the nests built by polygamous and more dimorphic species. Using simulations of selection optima, we further showed that sexual selection is likely stabilizing the weave pattern close to the maximal possible scale-invariance. In contrast, we did not find any evidence that the scale invariance of nests covaries with that of their surrounding habitat, neither in monogamous and weakly dimorphic species, not in polygamous and highly dimorphic species. We discuss below the mechanisms that could explain these results and their implications.

Sexual selection for maximizing scale-invariance

The link documented here between the spatial regularity of the weave and mating system and dimorphism supports an effect of sexual selection on the evolution of weave pattern. Several non-mutually exclusive hypotheses can explain this sexual selection.

First, the scale-invariance of traits could indicate the quality of individuals. A link between scale-invariance and condition has been revealed in body trait [START_REF] Pérez-Rodríguez | Fractal geometry of a complex plumage trait reveals bird's quality[END_REF] but also movements [START_REF] Hausdorff | Fractal dynamics of human gait: stability of long-range correlations in stride interval fluctuations[END_REF][START_REF] Stergiou | Human movement variability, nonlinear dynamics, and pathology: Is there a connection?[END_REF][START_REF] Peng | Fractal mechanisms in neuronal control: human heartbeat and gait dynamics in health and disease[END_REF]. In sheep, goats and hens, the fractality of foraging behaviour is used to monitor welfare [START_REF] Burgunder | Fractal measures in activity patterns: Do gastrointestinal parasites affect the complexity of sheep behaviour?[END_REF][START_REF] Alados | Fractal structure of sequential behaviour patterns: an indicator of stress[END_REF][START_REF] Maria | Complexity of behavioural sequences and their relation to stress conditions in chickens (Gallus gallus domesticus): a non-invasive technique to evaluate animal welfare[END_REF] while in chimpanzees, individuals with more intense parasite infections exhibit less scale-invariant feeding patterns [START_REF] Burgunder | Complexity in behavioural organization and strongylid infection among wild chimpanzees[END_REF].

The link between scale-invariant behaviour and condition suggests a mechanism explaining how pattern regularities in extended phenotypes, such as weaver nests, could be used as indicators of quality.

Second, a link between scale-invariance of weave and male quality could arise from more scaleinvariant nests indicating male dexterity or cognitive capacities. In bowerbirds (Ptilonorhynchidae), and in manakins (Pipridae), brain size (which has been suggested to be an indicator of quality [START_REF] Sayol | Predictable evolution towards larger brains in birds colonizing oceanic islands[END_REF]) is positively correlated to the complexity of the bower [START_REF] Day | Evolution of Bower Complexity and Cerebellum Size in Bowerbirds[END_REF] and to the gestural complexity of the courtship display [START_REF] Lindsay | Acrobatic Courtship Display Coevolves with Brain Size in Manakins (Pipridae)[END_REF], respectively. In weaverbirds, it is not known whether scale-invariance in weave pattern is associated with cognitive capacities, but inter-individual differences in the ability to construct nests have been previously shown, most notably between young, non-experienced males and older, experienced males [START_REF] Collias | The Development of Nest-Building Behavior in a Weaverbird[END_REF]. Measuring the scale invariance of the nests from birds falling in these different categories would be an important step to evaluate this hypothesis.

Third, more scale-invariant nests could be more resistant, have better thermal insulation or be more waterproof. The link between scale-invariance and these properties has never been studied in bird nests, but is well established in materials physics [START_REF] Carpinteri | Fractal nature of material microstructure and size effects on apparent mechanical properties[END_REF][START_REF] Guo | A study on the effects of the fractal characteristics of aggregates on the mechanical behavior of cemented sand and gravel[END_REF][START_REF] Kong | Fractal harmonic law and waterproof/dustproof[END_REF]. In textile industry, for example, scale-invariance is frequently monitored to detect defective fabrics [START_REF] Conci | A fractal image analysis system for fabric inspection based on a box-counting method[END_REF], and fractal structures have been recently shown to increase abrasion resistance, hydrophobic properties and moisture permeability [START_REF] Zhang | Abrasion Resistant/Waterproof Stretchable Triboelectric Yarns Based on Fermat Spirals[END_REF]. If it confers resistance and isolation to nests, scale-invariance would be under both natural and sexual selection for direct (nestling protection) and indirect (parental genes) benefits.

Finally, species under strong sexual selection could build more scale-invariant nests if there is a universal preference bias for efficiently processed visual patterns. Such a preference has been evidenced in both humans [START_REF] Winkielman | Prototypes Are Attractive Because They Are Easy on the Mind[END_REF][START_REF] Reber | Processing Fluency and Aesthetic Pleasure: Is Beauty in the Perceiver's Processing Experience?[END_REF] and other animals [START_REF] Halberstadt | The Generality and Ultimate Origins of the Attractiveness of Prototypes[END_REF][START_REF] Fantz | Form preferences in newly hatched chicks[END_REF], and is qualified as bias because it is expressed toward stimuli which species have not co-evolved with [START_REF] Renoult | Processing bias: extending sensory drive to include efficacy and efficiency in information processing[END_REF]. Regarding scale-invariance, in particular, humans have a universal attraction to highly scale-invariant patterns in various inanimate (architectures [START_REF] Joye | Architectural Lessons from Environmental Psychology: The Case of Biophilic Architecture[END_REF] or paintings [START_REF] Taylor | Perceptual and Physiological Responses to Jackson Pollock's Fractals[END_REF] and animate (faces [START_REF] Menzel | Fourier power spectrum characteristics of face photographs: attractiveness perception depends on low-level image properties[END_REF]) stimuli. Stimuli with low scale-invariance also cause visual discomfort [START_REF] Penacchio | Visual discomfort and the spatial distribution of Fourier energy[END_REF]. Because high scale-invariance is intrinsically efficiently processed [START_REF] Spehar | Beauty and the beholder: the role of visual sensitivity in visual preference[END_REF], these preferences are generally explained by the universal preference bias for efficient processing. Nevertheless, the application of this preference bias to scale-invariance still needs to be tested empirically in non-human animals.

Nest scale-invariance and sensory drive

In human psychology and neuroscience, a common explanation for the preference for a Fourier slope of -2 is that this value describes the level of scale-invariance that is dominant in natural environments. Because the brain is tuned to efficiently process images of the natural environment, any stimulus with a Fourier slope of -2 also happens to be efficiently processed, and thus to be attractive [START_REF] Redies | Fractal-like image statistics in visual art: similarity to natural scenes[END_REF][START_REF] Spehar | Fractals in art and nature: why do we like them? In: Human Vision and Electronic Imaging XVIII[END_REF]. In this study, however, we found that the mode Fourier slope of natural landscape peaks around -1.8, not around -2. When simulating a selection optimum at -1.8, none of the two main proxies of sexual selection intensity could significantly explain variation in the match between nest and habitat scale-invariance.

Additionally, we found that the Fourier slope varies across natural habitats (see also [START_REF] Hulse | Sexual signaling pattern correlates with habitat pattern in visually ornamented fishes[END_REF]). Given the observed inter-habitat variation in scale-invariance, to test the sensory drive hypothesis we also compared the statistics of nest weaves to that of their surrounding habitat. Again, we found no evidence that species with more intense sexual selection construct nests that match the visual properties of the surrounding habitat better than species with less intense selection. Hence, the sensory drive hypothesis does not seem to explain the variability in weave pattern observed in this study. However, we cannot dismiss the possibility that, in our study, the spatial scale considered (the whole landscape around each nest) was too large, and that sensory drive operates at the scale of the immediate nest background (for an example of background influences attractiveness in human faces, (see [START_REF] Menzel | Fourier power spectrum characteristics of face photographs: attractiveness perception depends on low-level image properties[END_REF]). Finally, and in contrast to the previous explanation, sensory drive could operate on an evolutionary time scale, and thus the hypothesis should be tested while considering the general environment in which species have evolved rather than the surrounding habitats of each nest. Given that sensory drive relies on the tuning of neuronal sensitivities to the physical properties of habitats [START_REF] Endler | Sensory ecology, receiver biases and sexual selection[END_REF], and that this tuning is both shaped by plastic mechanisms during development and by adaptation during evolution [START_REF] Sengpiel | Influence of experience on orientation maps in cat visual cortex[END_REF], an exhaustive investigation of the sensory drive hypothesis should ideally consider all temporal and spatial interactions that have occurred between the environment and the sensory system. Such an investigation is, however, outside the scope of this study.

Display location, nest visibility and other factors in relation to weave pattern

Males of more sexually selected species did not build more scale-invariant nests when they display closer to their nest or when their nest is highly visible. This may be because nests function as extended signalling phenotypes even when they are not associated with males displaying at the nests and when hidden. This is the case notably in most species of Euplectes sp. weavers, which are highly polygamous and dimorphic and build highly scale-invariant nests despite usually displaying far from the nest and building nests hidden in the vegetation. As single predictors, display location and nest visibility were each found to be significant in only one of the two models, and given this inconsistency, we cannot confirm their association with scale-invariance.

Finally, it is important to note that our proxies of sexual selection did not explain all the observed variation in scale-invariance. For instance, the species that construct the more fractal nests, Ploceus pelzelni and P. xanthops, are socially monogamous and present a moderate level of sexual dimorphism. Conversely, among Euplectes species, E. capensis and E. aureus are exceptions in that they construct low fractal nests despite being polygamous and sexually dimorphic. Additional factors not included in our models thus certainly influence weave pattern in weaverbirds. One of these factors could be the material used to weave the nest, which is known to vary between species [START_REF] Del Hoyo | Handbook of the birds of the world. vol. 15. weavers to new world warblers[END_REF] and that could be constrained by the availability of plant species in the nesting habitat. Another factor may be predation by birds and snakes, which is known to influence the general architecture of weaverbird nests [START_REF] Street | Convergent evolution of elaborate nests as structural defences in birds[END_REF] and thus also possibly the weave.

Conclusion

Our comparative analysis revealed a general association between two common proxies of sexual selection, mating system and tarsus dimorphism, and the scale-invariance of the weave pattern of nests at the level of the whole weaverbird family. These results indicate that sexual selection likely influences the regularity of weave pattern in these nests. They also highlight the potential importance of scale-invariance in animal communication. If high scale-invariance provides simultaneous benefits in terms of physical properties of materials and visual attractiveness, then natural and sexual selection likely share similar optima for this trait and a pervasive use of scale-invariance in animal communication can be predicted in extended phenotypes, such as structures built by organisms. photos online. Liliana Silva for helping with the image analysis. Megan Thompson, Marwan Naciri, Pietro D'amelio and Amelie Fargeville for statistical discussions. We are grateful to Olivier Penacchio for sharing his code to estimate the Fourier slope of images. We also thanks Faansie Peacock for sharing illustrations. Last, we thanks the French ANR through project AAPG2019.

Tables Table 1: Summary of the models' fixed predictors, predictions and rational to include theses predictors in the models. All continuous variable were Z-transformed before inclusion in the models. The sample sizes for each level of categorical variable are in brakets. Source might come from HWB: Handbook of the Birds of the World (del Hoyo et al., 2011) and/or from an "expert": when collected during field observations and through literature survey by D.

Oschadleus. The literature used is included in the dataset available online. The sexual dimorphism index was calculated for each species as 1-(mean tarsus length of the larger sex/mean tarsus length of the smaller sex) [START_REF] Lovich | A review of techniques for quantifying sexual size dimorphism[END_REF]. Because in weavers, males are always larger than females (all values were less than or equal to 0) we multiplied this index by -1 to make it positive and facilitate interpretation. Predictions are formulated for either P1: nest weave is sexually selected because it indicates builder's quality or it matches a universal preference bias for efficient processing, or P2: nest weave is sexually selected by sensory drive. small bars indicate species whose nest weave has an average Fourier slope close to -2 (highly scale-invariant). The scale-invariance of the nest was corrected for its form, its origin and the dimension of the image before calculating the deviation to -2. The fine dotted line around the phylogeny corresponds to the mean deviation to -2. This graph includes six species that were not included in our models due to missing data for other predictors. (A-D) The response variable is the distance between the nest Fourier slope and a Fourier slope reference value, named simulated slope optimum, within the range [-2.5; -1.5]. These barplots allow to compare estimates when the reference value is -2 and when it is others values around -2. (E-H) The response variable is the distance between the nest Fourier slope and one reference value corresponding to the Fourier slope of the surrounding habitat or other habitats in which weaver species never lived and are totally naive (foreign habitats). Red bars correspond to positive estimates and blue bars to negative estimates. Darker bars correspond to significant effects (when lower and upper confident intervals do not cross 0). Bars with a thick outline correspond to the estimates shown in Figure 3. Dotted lines correspond to the presumed selection optima. The names of the models are written at the bottom right of each panel. In models 1, the response variable is the deviation of the nest Fourier slope to -2, while models 2, it is the deviation of the nest Fourier slope to its surrounding habitat. Models 1.1 and 2.1 include mating system as a predictor while models 1.2 and 2.2 include tarsus dimorphism.

(since the common ancestor of all sampled taxa) before the speciation event that resulted in taxa i and i ′ . Our model is also estimating three variances: σ 2 S the intraspecific variance caused by the species contingent characteristics and σ 2 P the intraspecific variance caused by evolutionary history (the phylogenetic effect). Together σ 2 S and σ 2 P accounts for the betweenspecies variability. Finally σ 2 R is the residual error corresponding to the within-species variance of the trait (but also including measurement error and environment effect). weave and the overall shape of the nest. Because we were interested in analyzing the weave pattern only, we excluded variations caused by nest shape. To do so, for each cropped nest image (B) we created a corresponding binary silhouette image in which pixels within the nest were all set to 0 (black) (C). The Fourier slope was calculated both for the nest image (B) and the silhouette image (C). We then performed a linear regression analysis with the nest Fourier slope as a response variable and the silhouette Fourier slope as predictor, and used the residuals as new values of Fourier slope for the subsequent analyses. An alternative could have been to

analyse square images randomly sampled from within the nest; however, in preliminary analyses we found that such images were too small and thus had a Fourier slope strongly influenced by artefacts (aliasing). To calculate the Fourier slope, we transformed each image into the frequency domain using a Fast Fourier Transform and we computed the rotational average of the power spectrum for each frequency. Power spectrum and frequency were analysed in the log-log plane. We performed a least squares fit of a regression line to the log-log power spectrum, fitting data points binned at regular intervals. Only the frequency range between 10 and 256 cycles per image was used for fitting. The Fourier slope corresponds to the slope of the fitted regression line.

FiguresFigure 1 :

 1 Figures

Figure 2 :

 2 Figure 2: Phylogenetic distribution of the two proxies of sexual selection and asso-

Figure 3 :

 3 Figure 3: Estimates of the predictors tested in the four models. The posterior

Figure 4 :

 4 Figure 4: Variation of model estimates when simulating different slope optima.

Figure S1 :

 S1 Figure S1: Fourier analysis pipeline. Photos of a nest of Eastern golden weaver (Ploceus

Figure S2 :

 S2 Figure S2: Photos of habitats. (A) Habitats surrounding weaver nests (left to right):

Figure S3 :

 S3 Figure S3: Relationship between the deviation of the nest Fourier slope to a Fourier

Figure S5 :

 S5 Figure S5: Density distribution of the permutation test. Density distribution of the

Acknowledgments

We would like to thank Carrie Hickman, Kyle Middleton, Stuart and Bronwyn Dunlop, Amar-Singh HSS, and the other people that send us nests photos. Thomas Behrens for collecting nests

The dataset and R scripts used to produce the analyses presented in this paper are available at: (a Dryad and Github link will be provided

Competing interests

The authors declare no competing interests.

Supplementary materials

Equations S1-S4: The four modeled equations. We modeled the distance of the nest slope to (i) -2, here y, and to (ii) the surrounding habitat mode slope, here z, of species i and individual j explained by several fixed predictors: tarsus size dimorphism d and mating system m used as proxies for sexual selection intensity, display location l, nest visibility v, tarsus dimorphism or mating system in interaction with display location and nest visibility respectively dl and dv or ml and mv. Note that m i = 0 when i is monogamous and 1 when i is polygamous, l i = 0 when i do not display at the nest and 1 when i display at the nest, v i = 0 when i construct hidden nest and 1 when i construct visible nest. We also include two additionally fixed predictors to control for the origin of the data o (o j = 0 when the nest of j was photographed in museums and 1 when the nest of j was photographed in the wild) and the dimension of the nest image n. To account for the non-independence of data collected on the same species, the multiple measurement effect, we included species identity s as random effect. Aslo, to account for the non-independence between species we add p the phylogenetic effect as random effect. The final term ϵ correspond to the residual error term. The three last terms are assumed to be normally distributed where I correspond to the identity matrix and C the phylogenetic correlation matrix where element C i,i ′ is the amount of time that elapsed