Genetic assimilation and the evolution of direction of genital asymmetry in anablepid fishes

1. Introduction

The role of genetic assimilation, the transition of phenotypic variation from being environmentally or stochastically determined to becoming genetically determined [1–3], has been debated for several decades [4,5]. In the last 20 years, the concept has regained interest owing to the accumulation of theoretical models [6] and empirical examples [7,8] that support genetic assimilation as a plausible evolutionary outcome of natural selection [4] and emphasize its potential role in driving diversification [9,10]. Empirical support for genetic assimilation of phenotypes has come from phylogenetic comparative studies of bilateral asymmetries (e.g. paired structures that differ in size or shape between sides, or medial structures that deflect or rotate to one side) [11,12]. In a number of cases, antisymmetry (defined as left- and right-sided morphs being at equal frequencies in a population) was evolutionarily ancestral to directional asymmetry (defined as almost all individuals in a population being either left- or right-sided) [11]. These evolutionary transitions from antisymmetry to directional asymmetry are considered examples of genetic assimilation [11,13] because the direction of asymmetry is environmentally or stochastically determined in almost all studied antisymmetric species (e.g. [14–16]), but genetically determined in directionally asymmetric species [11,12,17].

Animal genitalia are known to be evolutionarily highly labile [18], and this even includes the evolution of asymmetry in genitalia [19]. In most known cases, directional asymmetry in genitalia evolved from a symmetric ancestral condition, but taxa with antisymmetric genitalia are exceptionally rare [19]. Those few lineages that include species with both antisymmetric and directionally asymmetric genitalia provide the rare opportunity to determine where the evolution of directionality falls on the continuum between a phenotype-precedes-genotype mode, compatible with genetic assimilation, and the more classical genotype-precedes-phenotype evolutionary mode (sensu [11]). Fish of the family Anablepidae represent one such lineage [19–21]. This family comprises three extant genera: Oxyzygonectes (white-eye fish, 1 species), Anableps (four-eyed fishes, 3 species) and Jenynsia (one-sided livebearers, 15 species), and three extinct genera: Tucumanableps, Sanjuanableps and Sachajenynsia (each with 1 species) [22–24]. Whereas Oxyzygonectes uses external fertilization and lays eggs, all species of the other two extant genera are viviparous and the anal fin of males has been modified into a gonopodium, an intromittent organ for internal fertilization (figure 1) [25,26]. Molecular and fossil data suggest that the gonopodium of anablepid fishes evolved once in the common ancestor of Anableps and Jenynsia [22,27]. In both genera, the gonopodium is asymmetric and the tip bends either to the left or to the right in approximately equal proportions in the species that have been studied so far (i.e. the three species of Anableps and Jenynsia lineata) [20,21]. Additionally, Anableps females also possess an asymmetric structure that laterally covers the gonopore, called a foriculum [20]. The antisymmetric pattern in males is associated with an apparent lack of heritability for bending sidedness, as the direction of asymmetry was found to be expressed stochastically in anablepid fishes [28]. However, some populations have been reported to be monomorphic in terms of genital sidedness [29,30]. This could indicate possible transitions between antisymmetry and directional asymmetry, which would suggest genetic assimilation of this character because of its lack of heritability [28]. Yet, these reports are anecdotal and not associated with particular populations or species [29,30]. Additionally, the phylogenetics of the family Anablepidae has been in flux [23,31], limiting our ability to understand the evolution of genital asymmetry in these fishes and the corresponding role that genetic assimilation might play in the evolution of this character.

To study the evolution of genital asymmetry in Anablepidae, we first determined the variation in the type of genital asymmetry in anablepids (i.e. antisymmetry, directional asymmetry), by phenotyping almost 2000 individuals (including all but two species with gonopodia) for direction of asymmetry. Second, to map this trait onto a phylogeny, we reconstructed a time-calibrated phylogeny of the family Anablepidae based on genome-wide single nucleotide polymorphism (SNP) data from 114 individuals comprising 17 out of the 19 extant species, as well as calibration times inferred from fossils of extinct species in this family. Finally, we tested if offspring genital asymmetry resembles their parental phenotype by conducting common garden breeding experiments in Jenynsia tucumana, a species that shows an excess of left-sided individuals in the wild. Combined, our three-pronged approach highlights the possible role of genetic assimilation in shaping sidedness of male asymmetric genitalia in anablepid fishes.

2. Material and methods

3. Results

4. Discussion

Genital asymmetry appears to be evolutionarily labile (like other morphological characteristics of animal genitalia) [18], with multiple transitions from symmetry to asymmetry across lineages [51–54]. In anablepid fishes, the transition from symmetry to asymmetry appears to have occurred twice: once in males after the evolution of the male gonopodium and again in females of the genus Anableps. Sachajenynsia pacha, an extinct species from the middle Miocene found in northern Argentina and sister taxa to all Jenynsia + Anableps, has a simple, symmetric gonopodium (figure 1) [22]. Antisymmetry is likely the ancestral state in the two extant genera with gonopodia (figure 2). Previously, we predicted that the probabilities of observing transitions from antisymmetry to directional asymmetry were low, considering the small additive genetic variation observed for the direction of genital asymmetry [28]. Here, we show that in the family Anablepidae the transition from antisymmetry to directional asymmetry indeed occurred multiple times, suggestive of recurrent genetic assimilation of the direction of asymmetry in this group of fishes. Additionally, our breeding experiment indicates that the sidedness of genital asymmetry cannot be predicted based on the morphotype of the sire, whereas the probability of expressing one morph over the other one might be predictable.

Based on our phylogenetic analyses of genome-wide SNP data, we infer three independent transitions from antisymmetry to biased antisymmetry or directional asymmetry in the genitalia of anablepids. Our phylogenetic hypothesis places J. obscura as the earliest lineage to split from the other species in the genus (figure 3), which differs from previous phylogenetic hypotheses that placed this species in the subgenus Jenynsia (sensu [48]) and as sister species to J. tucumana (e.g. [23,47]). These previous hypotheses would indicate only two transitions from antisymmetry to directional asymmetry or biased antisymmetry in Jenynsia. Previous phylogenetic hypotheses of the family were based on 71 morphological characters. In these datasets, J. obscura often had a large number of autapomorphies (e.g. [47,55]), and its placement was only poorly supported (e.g. [23]). All the synapomorphies for the subgenus Jenynsia proposed by Ghedotti [48] and Aguilera et al. [56] are recovered in J. obscura. However, this species also shows the synapomorphies proposed for the subgenus Plesiojenynsia based on additional material examined (G.A. 2021, unpublished data). Phylogenetic analyses of mitochondrial markers again recovered J. obscura as sister to all other Jenynsia species (electronic supplementary material, figure S8). These results support the placement of J. obscura as the sister group to all other Jenynsia species and indicate that multiple independent transitions from genital antisymmetry to an excess of left-sided individuals have occurred in the Anablepidae. Alternatively, it is plausible that a left-biased asymmetry might have evolved once in the most recent common ancestor to all Jenynsia species and was later lost independently and repeatedly in three lineages (first in J. unitaenia, J. diphyes and J. eirmostigma, then in J. darwini, J. lineata, J. luxata and J. onca (but two species in this clade show a marginally significant left-biased asymmetry, figure 3), and third in J. alternimaculata, J. maculata and J. sulfurica). The placement of J. obscura (94% left-sided males) as the first split in Jenynsia makes it difficult to differentiate these two alternatives. A more thorough evaluation of other species might help resolve this issue, especially given that some of the species without an overall departure from a 1 : 1 ratio might contain particular populations with a significant bias toward one direction [21] (see also electronic supplementary material, table S1). Similarly, a larger sample size for the genus Anableps could help, as there are data suggesting that some populations show some bias in female sidedness [20,30].

The inference of multiple transitions in the direction of male genitalia highlights the question of what, if any, is the genetic basis of genital sidedness in anablepid fishes. Previous breeding experiments with two antisymmetric species of anablepids (i.e. J. lineata and A. anableps) [28] indicated that the direction of the gonopodium is expressed stochastically. The new breeding experiment in J. tucumana, a species showing an excess of left-sided males, also suggests a stochastic determination of the direction of the gonopodium, but in this case with a bias towards left-sided offspring. Assuming morphological asymmetries to be threshold traits [12,57,58] allows us to hypothesize how phenotypic variation in the direction of genital asymmetry is assimilated in anablepid fishes (figure 4): under the assumption that variation in a liability factor (e.g. morphogens) is mostly due to developmental stochasticity, individuals with a level of the liability factor above a certain threshold will develop right-sided gonopodia, and below this threshold, left-sided gonopodia. In antisymmetric species, the threshold is expected to coincide with the centre of the liability factor distribution (figure 4a). Biased antisymmetry (e.g. J. obscura and J. tucumana) or directional asymmetry (e.g. J. sanctaecatarinae) can evolve if the threshold is displaced from the mean of the distribution (figure 4b). This mechanism has been proposed to explain genetic assimilation of other polyphenisms [59] and might also explain the inheritance of sidedness in the polymorphic starry flounder (Platichthys stellatus) [12,60]. The hypothesis of mostly stochastically determined liability levels and a displaced threshold is also in line with the results of our breeding experiment, which shows an excess of left-sided male offspring, independent of sire morph. However, one caveat to this interpretation is that the contribution of the mother's genotype to the phenotypic variation in gonopodium sidedness of male offspring in unknown. Multiple generation breeding crosses that track the ancestry of the female might be needed to clarify their contribution. This will be less relevant if the loci coding for the phenotype is on sex chromosomes, but unfortunately sex determination mechanisms in anablepids remain unknown. Also, we do not make any assumption about the genetic architecture of the threshold, which could vary from simple to very complex. This would mostly impact the width of the threshold, affecting the variation within broods, but would not conceptually change the model. To sum up, this model might help reconcile the lack of heritability of sidedness (e.g. [28,61]), and the lack of response to artificial selection on the direction of asymmetry (e.g. [14,15,62,63]), with the fact that the direction of asymmetry certainly evolves [11,17]. Thus, future studies might benefit from focusing on the heritability of the threshold, which is manifested in the proportion of offspring of each morph, rather than in the heritability of sidedness per se.

While this model may explain the evolution of the direction of asymmetry, the question remains why there is a consistent bias towards left-sided individuals: all species of anablepids that departed from a 1 : 1 left–right ratio have an excess of left-sided males (figure 3). The low number of species with directional asymmetry or biased antisymmetry impedes strong conclusions, as the probability of three species evolving in the same direction by chance is not negligible (e.g. p = 0.125, although this probability would be reduced if we consider marginally significant species like J. darwini and J. luxata). However, this pattern is predicted by some models proposed to explain the molecular mechanisms that determine asymmetry sidedness of morphological structures [12,64,65]. The direction of asymmetry might result from intracellular or cytoplasmic chirality that biases the production of one asymmetric morph over the other one by influencing the side of expression of certain morphogens (e.g. [66–70]). Under this scenario, subcellular asymmetries might favour the shift of thresholds consistently towards one direction, resulting in one morph having higher probability of fixation (e.g. more than 90% of snail taxa are sinistral) [71]. This is further supported by the observation that some of the species that show a non-significant trend (i.e. J. darwini and J. luxata) are also left-biased (electronic supplementary material, table S1). Alternatively, there could be fitness advantages of one morph over the other due to correlated traits or constraints [72]. This topic warrants further investigation.

How often do we see evidence for genetic assimilation in the evolution of sidedness of genital asymmetry? As previously reviewed [19], genital asymmetry is rare in nature, albeit phylogenetically widespread, and most asymmetric species are directionally asymmetric. Antisymmetry occurs in only few taxa, and lineages that might be informative for the study of genetic assimilation (i.e. those with both antisymmetric and directional asymmetric species) are rare [19]. In a few of these groups directional asymmetry is ancestral to antisymmetry [54,73], a seldom-seen trend in the evolution of asymmetry sidedness [11,17]. However, as in anablepid fishes, antisymmetry is ancestral to directional asymmetry in other taxa ([11,61,74], this study), suggesting that genetic assimilation of direction of genital asymmetry occurred independently multiple times. In anablepid fishes, sidedness evolved consistently in one direction (i.e. left-sided males became more abundant or fixed), and the same was observed for earwigs [72,75,76]. However, in phallostethid fishes, entirely right-sided as well as entirely left-sided species evolved [74,77]. In summary, our study shows that asymmetric traits could evolve in a manner compatible with the postulants of genetic assimilation (reviewed in [11]), and this occurred repeatedly in different lineages. It also suggests that future studies should focus on the heritability of the proportion of offspring of each morph, rather on the heritability of sidedness per se, to better understand the evolution of directional asymmetry by genetic assimilation.

Ethics

This research was approved by the Regierungspräsidium Freiburg, Baden Württemberg, Germany (reference no.: G-17/110), the Ministerio de Ambiente y Recursos Naturales de Nicaragua (permit no. DGPN/DB-IC-015-2015), the Dirección de Flora y Fauna, Tucumán, Argentina (permit no. DFFS-1757/330-214-9), the Sistema de Autorização e Informação em Biodiversidade, Brazil (SISBIO permit no. 33278-4 and 14028) and the Dirección Nacional de Recursos Acuáticos, Uruguay (permit no. DINARA-002/2014).

Data accessibility

The short-read data and associated information have been archived in the NCBI SRA database under Bioproject accession no. PRJNA822935 (SAMN27284034-SAMN27284138). COX I sequences were deposit in Genbank (accession nos ON156456–ON156467). The data associated with the phenotyping of individuals and the breeding experiment are available in the electronic supplementary material, tables S1 and S5, respectively. The nexus and xml files used in the phylogenetic analyses are archived in the Dryad Digital Repository [78].

The data are provided in electronic supplementary material [79].

Authors' contributions

J.T.-D.: conceptualization, data curation, formal analysis, funding acquisition, investigation, methodology, project administration, resources, visualization, writing—original draft, and writing—review and editing; S.J.R.: data curation, formal analysis, and writing—review and editing; G.A.: data curation and writing—original draft; A.F.K.: data curation, and writing—review and editing; J.R.V.: formal analysis, and writing—review and editing; G.G.: data curation, resources, and writing—review and editing; A.C.P.: data curation, resources, and writing—review and editing; G.C.D.: data curation, and writing—review and editing; W.J.daG.: data curation, resources, and writing—review and editing; A.M.: conceptualization, data curation, funding acquisition, resources, and writing—review and editing.

All authors gave final approval for publication and agreed to be held accountable for the work performed herein.

Conflict of interest declaration

We declare we have no competing interests.

Funding

This work was supported by grants from the Deutsche Forschungsgemeinschaft (grant nos TO914/2-1 and ME2752/27-1) to J.T.-D. and A.M., the Zukunftskolleg, Konstanz to J.T.-D. and the Hector Fellow Academy to S.J.R. and A.M. Field collections in Nicaragua were supported by a European Research Council- Advanced Grant (ERC ‘GenAdap’ 293700) to A.M.