fevo-09-605943 February 5, 2021 Time: 16:59 # 1 ORIGINAL RESEARCH published: 11 February 2021 doi: 10.3389/fevo.2021.605943 Phenotypic Variation in an Asexual-Sexual Fish System: Visual Lateralization Allison D. Connelly* and Michael J. Ryan Department of Integrative Biology, The University of Texas at Austin, Austin, TX, United States Sexual reproduction is nearly ubiquitous in the vertebrate world, yet its evolution and maintenance remain a conundrum due to the cost of males. Conversely, asexually reproducing species should enjoy a twofold population increase and thus replace sexual species all else being equal, but the prevalence of asexual species is rare. However, stable coexistence between asexuals and sexuals does occur and can shed light on the mechanisms asexuals may use in order to persist in this sex-dominated world. The asexual Amazon molly (Poecilia formosa) is required to live in sympatry with one of its sexual sperm hosts –sailfin molly (Poecilia latipinna) and Atlantic molly (Poecilia mexicana)—and are ecological equivalents to their host species in nearly every way except for reproductive method. Here, we compare the visual lateralization between Amazon mollies and sailfin mollies from San Marcos, Texas. Neither Amazon mollies Edited by: nor sailfin mollies exhibited a significant eye bias. Additionally, Amazon mollies exhibited Andrea S. Aspbury, Texas State University, United States similar levels of variation in visual lateralization compared to the sailfin molly. Further Reviewed by: investigation into the source of this variation –clonal lineages or plasticity—is needed to Jonathan Mee, better understand the coexistence of this asexual-sexual system. Mount Royal University, Canada Caitlin R. Gabor, Keywords: clones, mirror image response, Poecilia formosa, Poecilia latipinna, sexual reproduction Texas State University, United States *Correspondence: Allison D. Connelly INTRODUCTION [email protected] Sexual reproduction is practiced by 99.9% of species—either exclusively or at one point in Specialty section: their life—despite the 2-fold cost of sex (Avise, 2008). Historical arguments suggest that this This article was submitted to cost is more than offset by the benefit to sex that accrues from greater genetic variance Behavioral and Evolutionary Ecology, (Williams, 1975; Maynard Smith, 1978; Maynard Smith and Szathmary, 1999). Recent studies a section of the journal further highlight the importance of breaking apart genetic associations during recombination; Frontiers in Ecology and Evolution separating deleterious alleles from beneficial alleles, as well as spreading beneficial alleles across Received: 13 September 2020 a wide genomic background increases the efficiency of selection and adaptation to a capricious Accepted: 20 January 2021 environment (Barton and Charlesworth, 1998; Otto, 2009; Sharp and Otto, 2016; McDonald Published: 11 February 2021 et al., 2016). Indeed, empirical studies now document the long-term, evolutionary benefit held Citation: by sexually reproducing populations compared to asexually reproducing populations (Poon Connelly AD and Ryan MJ (2021) and Chao, 2004; Cooper, 2007; Becks et al., 2012; Gray and Goddard, 2012; McDonald et al., Phenotypic Variation in an Asexual-Sexual Fish System: Visual 2016). Despite this, some asexual species persist (Bi and Bogart, 2010; Fradin et al., 2017; Lateralization. Warren et al., 2018), even to the point of being named “ancient” (Heethoff et al., 2009; Schön Front. Ecol. Evol. 9:605943. et al., 2009). Of particular interest are the forms of asexual reproduction—gynogenesis and doi: 10.3389/fevo.2021.605943 hybridogenesis—that require the asexual and sexual species to coexist along much of their Frontiers in Ecology and Evolution| www.frontiersin.org 1 February 2021| Volume 9| Article 605943 fevo-09-605943 February 5, 2021 Time: 16:59 # 2 Connelly and Ryan Phenotypic Variation in Asexual Fish range (reviewed in Avise, 2008; gynogenetic: Poecilia formosa, 2004; Nakajima et al., 2004). Visual lateralization is present Menidia clarkhubbsi, Phoxinus eos-neogaeus, Cobitis elongatoides- in many poeciliid species in response to some but not all taenia, Fundulus heeroclitus; hybridogenetic: Poeciliopsis stimuli, including the two species used in this study: Girardinus monacha-lucida, Rana esculenta). The aforementioned studies falcatus, Gambusia holbrooki, G. nicaraguensis, Brachyrhaphis investigated asexuals and sexuals in isolation, however, further roseni (Bisazza et al., 1997b), Poecilia latipinna, P. formosa, investigations are needed into how asexual species can persist P. mexicana, P. reticulata (Fuss et al., 2019). Additionally, alongside sexual species in light of the benefits of sex. eye bias is heritable for one poeciliid species (Bisazza et al., Coexisting asexual and sexual species often mediate direct 2000). We chose to examine lateralization using a mirror competition through niche partitioning, in which asexuals image scrutiny test (Sovrano et al., 1999; De Santi et al., occupy a narrow portion of the habitat or resource range of 2001). The mirror image scrutiny test assesses the preference their host species (MacArthur and Pianka, 1966; Vrijenhoek, for left- or right-eye use when examining a reflection. Here 1978; Fussey and Sutton, 1981; Schenck and Vrijenhoek, 1986; we compare eye use between the asexual Amazon molly and Case, 1990; Rist et al., 1997; Martins et al., 1998; Negovetic sexual sailfin molly. et al., 2001). However, not all coexisting species exhibit niche separations, so their coexistence proposes even more of a dilemma. One such species complex is the Amazon and sailfin MATERIALS AND METHODS molly system. The Amazon molly (Poecilia formosa) is an asexual fish originating from a single hybridization event between an Sampling was approved by Texas State University (permit Atlantic molly female (P. mexicana) and sailfin molly male 04-523-691) and all animal procedures were approved by (P. latipinna) over 100,000 years ago in Tampico, Mexico the University of Texas Institutional Animal Care and Use (Schartl et al., 1995b; Stöck et al., 2010; Warren et al., 2018 Committees (AUP-2018-00089). but see Alberici da Barbiano et al., 2013). Amazon mollies reproduce via gynogenesis –females usually require the sperm of a closely related species to trigger embryogenesis, but the Specimen paternal genome is usually excluded (Schlupp, 2005; occasional We collected sailfin and Amazon mollies from San Marcos, Texas − paternal introgression discussed in Schartl et al., 1995a). This (29.89, 97.93) in August 2018 and 2019 and brought them back type of reproduction requires that they occupy the range to the lab at the University of Texas in Austin. We separated of one of their host species, thus placing them in direct species into 37-liter tanks with approximately eight individuals competition. Indeed, the Amazon molly now covers a broad to a tank; male sailfin mollies were included in this total and range of habitats from Rio Tuxpan, Mexico to the Nueces the ratio of males to females was balanced for both species. River, Texas USA –as well as introduced locations in central Each tank contained gravel and air filtration and set to a 14:10 Texas, always sympatric with one of its sexual hosts (Schlupp light dark schedule. All fish were fed twice daily ad libitum with et al., 2002). Previous investigations into this asexual-sexual commercial fish flakes (Tetramin, Germany). We injected unique system found few significant differences among morphological elastomer tags into fish at least 72 h prior to experimentation, to traits, physiological traits, and ecological traits (Table 1). Their track identity throughout experimentation. We tested 25 female continued coexistence with their sexual host may rely on genetic sailfin mollies and 27 Amazon mollies in June–August 2019 (2018 and phenotypic variation. This variation may occur at the collection specimen, both species) and June 2020 (2019 collection population level, with genetic variation partitioned amongst specimen, both species). Prior to experiments, we removed fish different clonal lineages (Lampert et al., 2006; Stöck et al., from communal tanks and placed them individually in 3-L 2010; Alberici da Barbiano et al., 2013; Warren et al., 2018). isolation tanks for 24 h. Phenotypic variation can also result from behavioral plasticity. Cognitive behaviors are often the most plastic phenotype (Bell Apparatus et al., 2009). Plasticity in asexual species may provide them with The experiment consisted of two identical tanks (length × width a broad range of responses to environmental change and thus × height: 41 × 21 × 26 cm) to control for any tank effect. assist in persistence. Tanks contained 20 cm high Acrylic mirrors on the two longer In this study we focus on cognitive behaviors –which is lacking walls and white corrugated plastic board completely covering in previous comparisons—to determine if average performance the two shorter walls. Black corrugated poster board covered the or variation in performance could shed light on the coexistence exterior of the long walls to prevent visuals of the room. Blue of the asexual Amazon molly with its sexual host. We used a filter (Marineland magnum bonded pad filter media) covered well-studied cognitive task—visual lateralization (Clayton, 1993; the bottom of the tank and each tank was filled with 15 cm of McGrew and Marchant, 1999; Pascual et al., 2004; Rogers et al., treated water, aerated prior to trial. A camera (Microsoft lifecam 2004; Vallortigara and Rogers, 2005; Rogers