Host species of a sexual-parasite do not differentiate between clones of Amazon mollies

Host species of a sexual-parasite do not differentiate between clones of Amazon mollies Abstract A major mechanism of prezygotic isolation is the ability for individuals to recognize conspecifics. In gynogenetic species complexes, the sexual host species occur in syntopy with the unisexual species that relies on the sexuals’ sperm for reproduction, which provide an excellent opportunity for the evolution of fine-tuned species recognition capabilities. Here, we examined if males and females from both parental species (sailfin molly, Poecilia latipinna, and Atlantic molly, P. mexicana) can distinguish between different clonal lineages of the hybrid, all-female Amazon molly (P. formosa). Both males and females were presented with the choice of 2 different pairings of Amazon mollies: 1) a sympatric female and an allopatric female; and 2) 2 sympatric females. We found that neither males nor females of sailfin or Atlantic mollies show a preference for a clone type. These results suggest that either the parental species do not have the ability to recognize different Amazon molly clones or they recognize but do not have a preference for a specific Amazon molly clone. INTRODUCTION Finding suitable mates is critical to all animals. The process of mate selection is often viewed as a cascading selection process starting with species recognition and ending with selecting a specific mate (Ryan and Rand 1993). Species recognition should evolve in most species as a mechanism of prezygotic isolation, effectively preventing costly hybridization events (Coyne and Orr 2004). In vertebrates, all known clonal species originated from such hybridization events between closely related species (Avise 2008) suggesting there is some evolutionary importance of speciation through hybridization (Bullini 1994). Nonetheless, the majority of these resulting hybrid species seem to be either gynogenetic or hybridogenetic making them obligate sperm parasites. These unisexual females require sperm of closely related species to pseudo-fertilize their unreduced ova, and thus are forced to occur in syntopy with their parasitized hosts. Theory would predict that the clonal species should out compete the sexual species by producing 2 daughters for every one their sexual counterparts (Maynard-Smith 1978), which would drive the extinction of the sexual species with the sperm-dependent clones soon following (McKay 1971). To avoid this, the sexual hosts should be under selection to recognize the gynogenetic forms, and discriminate against them to avoid costly interactions with them. Males benefit from avoiding mating with gynogens. For instance, in a related sexual/unisexual mating system, males of both parental species (Poeciliopsis lucida and Po. monacha) actively avoid copulating with either of its gynogens (Po. monacha-2 lucida and Po. 2 monacha-lucida), seeking out only conspecific females when given a choice (McKay 1971). Similar occurrences have been documented in several studies across different species complexes (Ambystoma jeffersonianum complex of salamanders: Dawley and Dawley 1986; sailfin and Amazon mollies: Gumm et al. 2006; sexual and unisexual Eucypris virens: Schmit et al. 2013). Indeed, species recognition among unisexual species and their sexual hosts can be extremely important in sexual-unisexual mating systems where there are multiple gynogenetic forms which resulted from ancestral hybridization and back crossing events within species clusters (i.e., Ambystoma salamanders contain multiple unisexual biotypes along with multiple parental hosts: A. jeffersonianum, A. laterale, A. texanum, and A. trigrinum) (Hedges et al. 1992). Although males benefit from recognizing and discriminating against their gynogenetic clones, females should also benefit from being able to recognize gynogens because they compete with them directly for resources, including males. But how fine-tuned is this recognition? A recent study in Amazon mollies (Poecilia formosa) showed that at least 6 different clonal lineages can distinguish between clones that are genetically very similar (Makowicz et al. 2016a, and because clones may differ in how they interact with sexual females (Keegan-Rogers and Schultz 1988), it might be adaptive for sexual males and females to evolve similar recognition capabilities. In Po. lucida, males which have learned to discriminate against one clone type, do not necessarily discriminate against other “novel” clones (hemiclones vs. diploid clones vs. triploid clones) (Keegan-Rogers and Schultz 1988). This suggests that sexual hosts may be able to discriminate against different clonal lineages within the different unisexual clone types. Furthermore, with this trait present in clonal females the question of its phylogenetic origin is raised because the clonal unisexuals are of hybrid origin, and lack of the trait in the ancestral species, might indicate that the trait evolved in the clonal females. To address this, we use the gynogenetic Amazon molly, which is a small livebearing fish that is common in fresh and brackish waters throughout central Texas and northeastern Mexico. It is a natural hybrid of the sailfin molly (P. latipinna; paternal ancestor) and the Atlantic molly (P. mexicana; maternal ancestor) that originated about 120000 generations ago near Tampico, Mexico (Schartl et al. 1995; Stöck et al. 2010). In this sexual-unisexual mating complex, the females of all 3 species are similar in size, fecundity, and their preferences for males (Heubel 2004; Schlupp 2009; Schlupp et al. 2010). Because Amazon mollies are syntopic with at least one of their host species, it may be unsurprising that these species are very similar in their ecology, too; they have the same parasites (Tobler and Schlupp 2005), use the same food resources (Scharnweber et al. 2011), and have the same predators (Fischer and Schlupp 2008). This strong similarity predicts that the sexual species should evolve species recognition to manage their interactions, and this has been confirmed in several studies (Schlupp et al. 1994; Gumm and Gabor 2005; Gumm et al. 2006; Schlupp 2009). Males typically prefer conspecific females to heterospecific Amazon mollies (Schlupp 2009), especially if the males are from allopatric populations without Amazon mollies (Gabor and Ryan 2001). This indicates that at least sympatric individuals are capable of species recognition of very closely related species. The preference for conspecific females is further reflected in the amount of sperm transferred to the females (Riesch et al. 2012). Sexual females are more likely to have sperm in their genital tract and to be pregnant when compared to sympatric Amazon mollies (Riesch et al. 2008; Riesch et al. 2012). Finally, the size of the females tends to play an important role in male preferences, with males tending to prefer larger females (Schlupp 2009). In a recent study (Makowicz et al. 2016a), it was found that females of the Amazon molly are capable of very fine-tuned recognition. Despite minimal phenotypic differences between individual clones, they recognize individuals of other clones and prefer full clonal sisters. This ability is used in regulating aggression between unisexual and sexual females, with aggressiveness increasing from clonal sisters to competing sexual females. This led us to hypothesize that not only the Amazon mollies, but also the sexual individuals that coexist with them would be able to discriminate between clones. However, it is yet unknown if these sexual species have evolved the ability to recognize individuals in the population on a much finer-tuned scale, such as individuals within the unisexual species. Furthermore, sexual species may have coevolved with local unisexuals, allowing them to recognize sympatric but not allopatric heterospecifics. Alternatively, clonal recognition might not be present in the sexual species and could have evolved in the Amazon molly. Specifically, we examined if males and females from either of the parental species, the sailfin and Atlantic molly, can distinguish between different clonal lineages of Amazon mollies. We allowed males and females of each species a choice between a sympatric and an allopatric Amazon molly female (i.e., presenting 2 Amazon mollies that represent the most variation between the different lineages). We predict that if the sexuals have evolved the ability to discriminate between clonal lineages, they will prefer to spend more time with sympatric Amazon mollies, which may be more familiar (Griffiths 1997; Barber and Wright 2001), compared to allopatric females. Alternatively, if sexual individuals prefer allopatric Amazon mollies to sympatric ones, this would be evidence for the rare-female advantage hypothesis (Keegan-Rogers 1984; Keegan-Rogers and Schultz 1988). Males and females may also respond differently, as females may prioritize familiar shoaling partners (the former prediction) and males favoring sexual selection and novel females (latter prediction). METHODS Populations The sailfin mollies were laboratory-reared offspring of wild caught individuals that originated from Weslaco, Texas (26°7′14.52 N, 97°57′41.44 W). The Atlantic mollies were laboratory-reared offspring that originated from the Río Purificacíon, Barretal, Mexico (24°4′42.85 N, 99°7′21.76 W). The Amazon mollies originated from 2 monoclonal lineages, which were laboratory-reared offspring that originated from a population in Texas and a population in Mexico. More specifically, Amazon mollies that represent “Clone 1” stimulus females originated from a sympatric population with sailfin mollies in Weslaco, Texas. Amazon molly females that represented “Clone 2” stimulus females originated from a sympatric population with Atlantic mollies in Barretal, Mexico. Each Amazon molly lineage had a genetic identity of 1, the relatedness coefficient (R) between the 2 clonal lineages was −0.684 (i.e., as related to each other as 2 random sexual individuals; Tiedemann et al. 2011), and have shown the ability to discriminate between clonal sisters and nonsisters using both visual and/or chemical cues (Makowicz et al. 2016a). We chose two of our most distantly related monoclonal lineages that differ both genetically and in population origin to increase the likelihood of heterospecific discrimination. All fish were maintained at the University of Oklahoma in 12 light:12 dark lighting conditions and fed commercial fish flakes (TetraMin®) and bloodworms ad libitum daily. Prior to the actual experiment, individuals were brought into the laboratory, and groups of focal males and females were isolated from each other, by species, in 38l tanks for a minimum of 2 weeks prior to testing. Experimental procedure A standard, binary choice test was used, allowing male and female sailfin and Atlantic mollies to choose between females in 2 treatments: 1) a female from Clone 1 and a female from Clone 2; and 2) 2 females from a sympatric (either Clone 1 for sailfin mollies or Clone 2 for Atlantic mollies). Size matched stimulus Amazon molly females were placed in clear, perforated Plexiglas cylinders that were located on either side of the experimental tank, which allowed for both chemical and visual signals (Makowicz et al. 2016). The focal individual was placed in a clear Plexiglas cylinder in the center of the tank and allowed to acclimate for 10 min. After acclimation, the focal individual was gently released from the cylinder and once it was swimming freely in the tank, the association time (s) the focal individual spent with each stimulus female was recorded for 10 min. Time spent with each female was recorded using Viewer (Biobserve GmbH), a video tracking software, which recorded how much time (s) an individual spent near each stimulus. After the allotted time, the focal fish was placed into the cylinder again, and the stimulus females were switched to detect any side bias. We tested the focal fish in both treatments, randomizing the order in which the treatments were offered, until each focal individual was presented with all 3 treatments. Strength of preference (SOP) scores were calculated, then √arcsine transformed to normalize the data. We used a repeated-measures general linear model (GLM) to compare the normalized preference scores of each treatment for each species/sex in SPSS (ver. 24). Sample sizes for each of the focal species/sex included Atlantic mollies: females (N = 10) and males (N = 26), and sailfin mollies: females (N = 16) and males (N = 27). For our model, we used the SOP scores as the dependent variable, species, sex and the treatment as the independent variables, and included Bonferroni-corrected pair-wise comparisons. There was an unusually large number of individuals showing side biases (>85% of the time spent on one side of the experimental tank) found in both species and sexes, and consequently we analyzed the results both with and without the side-biased individuals. There was little to no statistical difference if we included or removed these side-biased individuals (Table 1), therefore we interpreted side-biased individuals as “unresponsive” to either stimuli, or showing no preference. Finally, we calculated Cohen’s d and the effect-size correlation (rYλ) between the 2 different clone types for each sex and species to understand the standardized difference between the SOP scores of one clone compared to the other. Table 1 The average ± standard deviation of the time (s) individuals spent with each Amazon molly clone type for both data sets (with side-biased individuals and without side-biased individuals)   Treatment 1: Clone 1 and Clone 2  With side-biased individuals  Without side-biased individuals  Clone 1  Clone 2  Clone 1  Clone 2  Sailfin molly female  201.13 ± 149.45  289.92 ± 138.72  198.13 ± 181.76  313.43 ± 183.62  Sailfin molly male  265.76 ± 152.10  239.82 ± 148.09  288.70 ± 174.50  245.27 ± 179.29  Atlantic molly female  301.51 ± 111.92  224.56 ± 118.15  323.96 ± 142.14  179.06 ± 133.85  Atlantic molly male  246.34 ± 151.26  265.23 ± 163.56  216.38 ± 194.67  294.27 ± 212.73    Treatment 2: Sympatric Clone  With side-biased individuals  Without side-biased individuals  Clone a  Clone b  Clone a  Clone b  Sailfin molly female  240.26 ± 152.05  245.59 ± 166.04  232.34 ± 174.94  232.30 ± 191.80  Sailfin molly male  243.10 ± 174.23  280.37 ± 178.85  235.42 ± 202.34  293.13 ± 200.81  Atlantic molly female  182.33 ± 94.93  320.95 ± 122.86  145.43 ± 83.05  342.74 ± 159.90  Atlantic molly male  272.24 ± 163.42  248.86 ± 136.83  255.53 ± 211.76  231.08 ± 203.16    Treatment 1: Clone 1 and Clone 2  With side-biased individuals  Without side-biased individuals  Clone 1  Clone 2  Clone 1  Clone 2  Sailfin molly female  201.13 ± 149.45  289.92 ± 138.72  198.13 ± 181.76  313.43 ± 183.62  Sailfin molly male  265.76 ± 152.10  239.82 ± 148.09  288.70 ± 174.50  245.27 ± 179.29  Atlantic molly female  301.51 ± 111.92  224.56 ± 118.15  323.96 ± 142.14  179.06 ± 133.85  Atlantic molly male  246.34 ± 151.26  265.23 ± 163.56  216.38 ± 194.67  294.27 ± 212.73    Treatment 2: Sympatric Clone  With side-biased individuals  Without side-biased individuals  Clone a  Clone b  Clone a  Clone b  Sailfin molly female  240.26 ± 152.05  245.59 ± 166.04  232.34 ± 174.94  232.30 ± 191.80  Sailfin molly male  243.10 ± 174.23  280.37 ± 178.85  235.42 ± 202.34  293.13 ± 200.81  Atlantic molly female  182.33 ± 94.93  320.95 ± 122.86  145.43 ± 83.05  342.74 ± 159.90  Atlantic molly male  272.24 ± 163.42  248.86 ± 136.83  255.53 ± 211.76  231.08 ± 203.16  Average time spent with each stimulus did not differ strongly between the 2 data sets, and there was consistently high variation within each data set. View Large RESULTS Overall, we found no significant effects of clone type SOP in both the GLM including the side-biased individuals (F1 = 1.184, P = 0.278, Figure 1) or the one excluding the side-biased individuals (F1 = 0.351, P = 0.555). Further, there was there no interaction between clone type SOP and sex (including side-biased individuals: F1 = 0.121, P = 0.728; excluding side-biased individuals: F1 = 0.156, P = 0.694), clone type SOP and species (including side-biased individuals: F1 = 0.017, P = 0.896; without side bias: F1 = 0.026, P = 0.872), clone type SOP and treatment (including side-biased individuals: F1 = 0.230, P = 0.632; excluding side-biased individuals: F1 = 0.102, P = 0.751), clone type SOP, sex, and species (including side-biased individuals: F1 = 0.022, P = 0.882; excluding side-biased individuals: F1 = 0.032, P = 0.858), clone type SOP, sex, and treatment (including side-biased individuals: F1 = 0.243, P = 0.623; excluding side-biased individuals: F1 = 0.600, P = 0.441), or clone type SOP, species, and treatment (including side-biased individuals: F1 = 1.248, P = 0.266; excluding side-biased individuals: F1 = 0.309, P = 0.580). There was a significant effect in the 4-way interaction among clone type SOP, sex, species, and treatment when the side-biased individuals were excluded (F1 = 5.090, P = 0.027), although this effect was nonsignificant when side-biased individuals were included (F1 = 3.426, P = 0.066). Figure 1 View largeDownload slide The average ± SE of the time (s) spent with the 2 different Amazon molly clone types (Clone 1: Light blue; Clone 2: Red) and between 2 identical, sympatric clones (Individual 1: Light blue; Individual. 2: Dark blue) including the individuals with side bias. There is no significant difference between the treatments in the males or females of either species. Although, there is more variation in the females’ response, this might be indicative of the low sample size. Figure 1 View largeDownload slide The average ± SE of the time (s) spent with the 2 different Amazon molly clone types (Clone 1: Light blue; Clone 2: Red) and between 2 identical, sympatric clones (Individual 1: Light blue; Individual. 2: Dark blue) including the individuals with side bias. There is no significant difference between the treatments in the males or females of either species. Although, there is more variation in the females’ response, this might be indicative of the low sample size. Out of the 10 Atlantic molly females, 6 females did not exhibit a side bias (60%), and of the 4 females that did, only 2 of these females remained on the same side of the experimental tank for the entire time (i.e., they never visited both sides of the tank). From the 26 males we tested, 12 males did not show any side bias (46%), and of the 14 males that did, 10 of them failed to visit at least one side of the tank. When we compared the mean SOP scores between Clone 1 and Clone 2 (treatment 1) we found that for females Cohen’s d = 0.669 (effect-size correlate = 0.317), and males Cohen’s d = −0.120 (effect-size correlate = −0.060). We tested 16 sailfin molly females and 8 of those females did not show side bias (50%), of the 8 females that did, only 4 of these females did not visit the other side of the experimental tank. Out of the 27 sailfin molly males we tested, we found that 17 males did not show side bias (63%), and only 10 of the males that did, failed to visit the other side of the experimental tank. When we compared the mean SOP scores between Clone 1 and Clone 2 (treatment 1) for females Cohen’s d = −0.616 (effect-size correlate = −0.294), and males Cohen’s d = 0.173 (effect-size correlate = 0.086). DISCUSSION Our initial prediction that the sexual ancestors of the Amazon molly, the Atlantic and sailfin molly, would show discrimination between clones of the Amazon molly was not supported. Our prediction was based on 2 facts: both ancestral species show species recognition and a preference for conspecifics, and Amazon mollies, the hybrid of the Atlantic and sailfin molly, does show clonal recognition. We found, however, that neither males nor females of sailfin or Atlantic mollies showed a preference for either a familiar clone or a novel clone type, despite a known ability for species recognition. Based on our analysis of effect sizes we feel confident that this truly reflects an absence of a preference. There are 3 likely explanations for this result: 1) They may not have the ability to recognize different Amazon molly clones; 2) They recognize clones, but do not show a preference for a specific Amazon molly clone; or 3) The experimental design does not allow for us to detect a preference for a specific clone type. First, the sexual species may not have the ability to discriminate between the 2 clonal types of Amazon mollies because the recognition mechanisms the Amazon molly females use are intraspecific. This may have some implications for the evolution of the unique ability for kin recognition in this sexual-unisexual mating complex. Amazon mollies are a relatively young species (ca. 100000–120000 generations ago; Schartl et al. 1995; Stöck et al. 2010). If clonal recognition indeed evolved after the formation of this species, the process was speedy. Poschadel et al. (2009) reported of divergent mating preferences in Amazon mollies, which, in addition to clonal recognition, might also have evolved after the hybridization event that led to the Amazon molly. So far, however, it is unclear if the parental species have the ability to recognize kin from non-kin, and also if kin recognition evolved in the Amazon mollies themselves or if it was inherited from one or both of their paternal species. Second, individuals may be able to discriminate between Amazon mollies; yet they do not show a preference for a particular clone type. For males, due to the lack of known genetic benefits for mating with the Amazon molly females, there may not be any adaptive value in order to evolve the ability to distinguish between the different clonal lineages in the first place. Female social behaviors might receive little, if any, benefits for evolving this particular discrimination. Sexual females should be equally aggressive towards and shoal equally with all Amazon mollies. If, however, particular clonal lineages possessed specific characteristics (i.e., more aggressiveness, faster growth rates, lower parasite load, etc.) there may be possible benefits for sexual females in differentiating between the clonal lineages. For instance, if one clonal lineage were more aggressive towards sexual females, sexual females would benefit from avoiding these aggressive clones due to the high cost of female–female aggression in these species (Makowicz and Schlupp 2015). It has been shown that Amazon mollies show individual behavioral phenotypes both among and within clonal lineages (Bierbach et al. 2017). However, it is yet unknown if specific clonal lineages are more prone to behave in particular ways compared to another, although this could easily be tested. However, in the Po. monacha-lucida complex, one clone type did behave more aggressively towards host males than 5 other clone types (Keegan-Rogers and Schultz 1988); although in this sexual-unisexual mating complex there have been many hybridization and backcrossing events leading to multiple clone types unlike the sailfin-Amazon molly complex, this still remains a possibility. On the other hand, the ability for sexual individuals to recognize different clonal lineages may be a learned trait. For instance, after a female interacts with a clone that is highly aggressive, it may learn to recognize and avoid being near that clone in the future, similar to male Po. lucida (Keegan-Rogers and Schultz 1988). All our sexual fish were inexperienced with these specific clonal lineages of Amazon mollies because the Amazon mollies were never kept in mixed stock tanks. However, if there are such specific characteristics of clonal lineages, this is yet unknown. Finally, it is possible that the experimental design was not efficient enough to detect a preference for either clone type. The methods used in this study are similar to others to detect both male and female preferences in these species (Tobler et al. 2005; McCoy et al. 2008; Joachim and Schlupp 2012; Makowicz et al. 2016a, b). These studies suggest that both males and females exhibit various social preferences, including mate, and shoaling preferences, and that these preferences are detectable using standard binary choice tests. Similarly, Amazon mollies have also been tested in such a design to determine if they were able to differentiate between the different clonal types. Amazon molly females are able to detect clonal sisters from nonsisters, in both laboratory and field studies using multiple sensory channels (Makowicz et al. 2016a). We allowed both visual and chemical cues to be present to allow for individuals to utilize either or both mechanisms for recognition. Regardless, although this is a possibility, the likelihood of the design not being effective enough is small. Interestingly, we found extremely high rates of side bias in this study, which is uncommon in these fish. The nonconservative (>85%) side bias criterion used in this study was identical to other studies (McCoy et al. 2008; Makowicz et al. 2016a), and less stringent than in other studies (Schlupp et al. 1994; Schlüter et al. 1998). It is likely that the elevated levels of side biases are due to males and females of both species being nonresponsive to either the stimulus females, almost as if they cannot answer the question they were asked in our experiment. Indeed, to humans, Amazon mollies are extremely difficult to differentiate between different clonal lineages. Sexual individuals may not receive a benefit from swimming towards a stimulus that was identical to the stimulus they were already near. Yet, of the individuals that were categorized as side-biased, less than a quarter of the individuals (19 out of 82 fish) never visited the second stimulus. Nonetheless, when we evaluated effect sizes using Cohen’s d, for both species of males the means of the 2 different clone types were essentially identical, suggesting that there is actually no discrimination. There were moderate differences in female preference between the 2 clones but this may be due to the smaller sample size as compared to males. This suggests that there may not have been enough detectable difference between the stimulus females, and thus, individuals did not respond. Although our data do not support the notion that species and kin recognition are continuous in the Atlantic and sailfin molly, clearly more work needs to be done to fully test this. The origin of the unisexual species may play an important role in the ability for sexual individuals to discriminate among different clonal lineages. There are only few ways gynogenetic females can increase genetic diversity leading to distinct clonal lineages: mutation, genetic conversion, partial introgression of the male genome or full introgression resulting in increased ploidy level (Schlupp 2009). Hybridogenetic females (e.g., found in both Pelophylax esculenta complex of European frogs (Biriuk et al. 2016) and Po. monacha-lucida complex of fish), however, are unique as each sister is genetically distinct from one another in one way: they each have a father. These females reproduce clonally by discarding the paternal set of chromosomes in their gametes, resulting in identical ova of the maternal chromosomes, which duplicate, undergo meiosis and are then fertilized by another male, creating a lineage of maternally inherited genes (Avise 2008). Furthermore, for both gynogens and hybridogens, whether they resulted from a single (e.g., the Amazon molly) or multiple hybridization events (e.g., A. jeffersonianum complex of salamanders), from one or multiple back crossing events (e.g., Po. monacha-lucida mating complex), or the time since the original hybridization event may be extremely important for the sexual species to evolve the ability to recognize clone types (i.e., gynogens vs. hybridogens, diploid vs. triploid vs. tetraploid, etc.) and different clonal lineages within each clone type. It is also important to mention that some sexual-unisexual species complexes have a combination of all the above, like the Po. monacha-lucida complex which includes gynogens, hybridogens, diploids and triploid, and have originated from multiple hybridization and backcrossing events. Hence, the evolutionary history of each sexual-unisexual mating complex is essential to the evolution of recognition capabilities. In conclusion, neither host species (sailfin mollies or Atlantic mollies), nor sex (males or females) showed a detectable preference between different clonal lineages in a single-origin, gynogenetic species. This suggests there may not be an evolutionary benefit associated with the recognition of individual clones in either of the Amazon molly’s host species. Data accessibility: Analyses reported in this article can be reproduced using the data provided by Makowicz et al. (2017). We would like to thank M. Tobler, M.A. Spikes, and 2 anonymous reviewers for their input on previous versions of this manuscript. This research was approved by the Institutional Animal Care and Use Committee of the University of Oklahoma (R13-006). A.M.M. would like to thank Graduate Assistance in Areas of National Need (GAANN) Fellowship for financial support. REFERENCES Avise JC. 2008. Clonality: the genetics, ecology, and evolution of sexual abstinence in vertebrate animals . New York: Oxford Univ. Press. 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For permissions, please e-mail: journals.permissions@oup.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Behavioral Ecology Oxford University Press

Host species of a sexual-parasite do not differentiate between clones of Amazon mollies

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Abstract

Abstract A major mechanism of prezygotic isolation is the ability for individuals to recognize conspecifics. In gynogenetic species complexes, the sexual host species occur in syntopy with the unisexual species that relies on the sexuals’ sperm for reproduction, which provide an excellent opportunity for the evolution of fine-tuned species recognition capabilities. Here, we examined if males and females from both parental species (sailfin molly, Poecilia latipinna, and Atlantic molly, P. mexicana) can distinguish between different clonal lineages of the hybrid, all-female Amazon molly (P. formosa). Both males and females were presented with the choice of 2 different pairings of Amazon mollies: 1) a sympatric female and an allopatric female; and 2) 2 sympatric females. We found that neither males nor females of sailfin or Atlantic mollies show a preference for a clone type. These results suggest that either the parental species do not have the ability to recognize different Amazon molly clones or they recognize but do not have a preference for a specific Amazon molly clone. INTRODUCTION Finding suitable mates is critical to all animals. The process of mate selection is often viewed as a cascading selection process starting with species recognition and ending with selecting a specific mate (Ryan and Rand 1993). Species recognition should evolve in most species as a mechanism of prezygotic isolation, effectively preventing costly hybridization events (Coyne and Orr 2004). In vertebrates, all known clonal species originated from such hybridization events between closely related species (Avise 2008) suggesting there is some evolutionary importance of speciation through hybridization (Bullini 1994). Nonetheless, the majority of these resulting hybrid species seem to be either gynogenetic or hybridogenetic making them obligate sperm parasites. These unisexual females require sperm of closely related species to pseudo-fertilize their unreduced ova, and thus are forced to occur in syntopy with their parasitized hosts. Theory would predict that the clonal species should out compete the sexual species by producing 2 daughters for every one their sexual counterparts (Maynard-Smith 1978), which would drive the extinction of the sexual species with the sperm-dependent clones soon following (McKay 1971). To avoid this, the sexual hosts should be under selection to recognize the gynogenetic forms, and discriminate against them to avoid costly interactions with them. Males benefit from avoiding mating with gynogens. For instance, in a related sexual/unisexual mating system, males of both parental species (Poeciliopsis lucida and Po. monacha) actively avoid copulating with either of its gynogens (Po. monacha-2 lucida and Po. 2 monacha-lucida), seeking out only conspecific females when given a choice (McKay 1971). Similar occurrences have been documented in several studies across different species complexes (Ambystoma jeffersonianum complex of salamanders: Dawley and Dawley 1986; sailfin and Amazon mollies: Gumm et al. 2006; sexual and unisexual Eucypris virens: Schmit et al. 2013). Indeed, species recognition among unisexual species and their sexual hosts can be extremely important in sexual-unisexual mating systems where there are multiple gynogenetic forms which resulted from ancestral hybridization and back crossing events within species clusters (i.e., Ambystoma salamanders contain multiple unisexual biotypes along with multiple parental hosts: A. jeffersonianum, A. laterale, A. texanum, and A. trigrinum) (Hedges et al. 1992). Although males benefit from recognizing and discriminating against their gynogenetic clones, females should also benefit from being able to recognize gynogens because they compete with them directly for resources, including males. But how fine-tuned is this recognition? A recent study in Amazon mollies (Poecilia formosa) showed that at least 6 different clonal lineages can distinguish between clones that are genetically very similar (Makowicz et al. 2016a, and because clones may differ in how they interact with sexual females (Keegan-Rogers and Schultz 1988), it might be adaptive for sexual males and females to evolve similar recognition capabilities. In Po. lucida, males which have learned to discriminate against one clone type, do not necessarily discriminate against other “novel” clones (hemiclones vs. diploid clones vs. triploid clones) (Keegan-Rogers and Schultz 1988). This suggests that sexual hosts may be able to discriminate against different clonal lineages within the different unisexual clone types. Furthermore, with this trait present in clonal females the question of its phylogenetic origin is raised because the clonal unisexuals are of hybrid origin, and lack of the trait in the ancestral species, might indicate that the trait evolved in the clonal females. To address this, we use the gynogenetic Amazon molly, which is a small livebearing fish that is common in fresh and brackish waters throughout central Texas and northeastern Mexico. It is a natural hybrid of the sailfin molly (P. latipinna; paternal ancestor) and the Atlantic molly (P. mexicana; maternal ancestor) that originated about 120000 generations ago near Tampico, Mexico (Schartl et al. 1995; Stöck et al. 2010). In this sexual-unisexual mating complex, the females of all 3 species are similar in size, fecundity, and their preferences for males (Heubel 2004; Schlupp 2009; Schlupp et al. 2010). Because Amazon mollies are syntopic with at least one of their host species, it may be unsurprising that these species are very similar in their ecology, too; they have the same parasites (Tobler and Schlupp 2005), use the same food resources (Scharnweber et al. 2011), and have the same predators (Fischer and Schlupp 2008). This strong similarity predicts that the sexual species should evolve species recognition to manage their interactions, and this has been confirmed in several studies (Schlupp et al. 1994; Gumm and Gabor 2005; Gumm et al. 2006; Schlupp 2009). Males typically prefer conspecific females to heterospecific Amazon mollies (Schlupp 2009), especially if the males are from allopatric populations without Amazon mollies (Gabor and Ryan 2001). This indicates that at least sympatric individuals are capable of species recognition of very closely related species. The preference for conspecific females is further reflected in the amount of sperm transferred to the females (Riesch et al. 2012). Sexual females are more likely to have sperm in their genital tract and to be pregnant when compared to sympatric Amazon mollies (Riesch et al. 2008; Riesch et al. 2012). Finally, the size of the females tends to play an important role in male preferences, with males tending to prefer larger females (Schlupp 2009). In a recent study (Makowicz et al. 2016a), it was found that females of the Amazon molly are capable of very fine-tuned recognition. Despite minimal phenotypic differences between individual clones, they recognize individuals of other clones and prefer full clonal sisters. This ability is used in regulating aggression between unisexual and sexual females, with aggressiveness increasing from clonal sisters to competing sexual females. This led us to hypothesize that not only the Amazon mollies, but also the sexual individuals that coexist with them would be able to discriminate between clones. However, it is yet unknown if these sexual species have evolved the ability to recognize individuals in the population on a much finer-tuned scale, such as individuals within the unisexual species. Furthermore, sexual species may have coevolved with local unisexuals, allowing them to recognize sympatric but not allopatric heterospecifics. Alternatively, clonal recognition might not be present in the sexual species and could have evolved in the Amazon molly. Specifically, we examined if males and females from either of the parental species, the sailfin and Atlantic molly, can distinguish between different clonal lineages of Amazon mollies. We allowed males and females of each species a choice between a sympatric and an allopatric Amazon molly female (i.e., presenting 2 Amazon mollies that represent the most variation between the different lineages). We predict that if the sexuals have evolved the ability to discriminate between clonal lineages, they will prefer to spend more time with sympatric Amazon mollies, which may be more familiar (Griffiths 1997; Barber and Wright 2001), compared to allopatric females. Alternatively, if sexual individuals prefer allopatric Amazon mollies to sympatric ones, this would be evidence for the rare-female advantage hypothesis (Keegan-Rogers 1984; Keegan-Rogers and Schultz 1988). Males and females may also respond differently, as females may prioritize familiar shoaling partners (the former prediction) and males favoring sexual selection and novel females (latter prediction). METHODS Populations The sailfin mollies were laboratory-reared offspring of wild caught individuals that originated from Weslaco, Texas (26°7′14.52 N, 97°57′41.44 W). The Atlantic mollies were laboratory-reared offspring that originated from the Río Purificacíon, Barretal, Mexico (24°4′42.85 N, 99°7′21.76 W). The Amazon mollies originated from 2 monoclonal lineages, which were laboratory-reared offspring that originated from a population in Texas and a population in Mexico. More specifically, Amazon mollies that represent “Clone 1” stimulus females originated from a sympatric population with sailfin mollies in Weslaco, Texas. Amazon molly females that represented “Clone 2” stimulus females originated from a sympatric population with Atlantic mollies in Barretal, Mexico. Each Amazon molly lineage had a genetic identity of 1, the relatedness coefficient (R) between the 2 clonal lineages was −0.684 (i.e., as related to each other as 2 random sexual individuals; Tiedemann et al. 2011), and have shown the ability to discriminate between clonal sisters and nonsisters using both visual and/or chemical cues (Makowicz et al. 2016a). We chose two of our most distantly related monoclonal lineages that differ both genetically and in population origin to increase the likelihood of heterospecific discrimination. All fish were maintained at the University of Oklahoma in 12 light:12 dark lighting conditions and fed commercial fish flakes (TetraMin®) and bloodworms ad libitum daily. Prior to the actual experiment, individuals were brought into the laboratory, and groups of focal males and females were isolated from each other, by species, in 38l tanks for a minimum of 2 weeks prior to testing. Experimental procedure A standard, binary choice test was used, allowing male and female sailfin and Atlantic mollies to choose between females in 2 treatments: 1) a female from Clone 1 and a female from Clone 2; and 2) 2 females from a sympatric (either Clone 1 for sailfin mollies or Clone 2 for Atlantic mollies). Size matched stimulus Amazon molly females were placed in clear, perforated Plexiglas cylinders that were located on either side of the experimental tank, which allowed for both chemical and visual signals (Makowicz et al. 2016). The focal individual was placed in a clear Plexiglas cylinder in the center of the tank and allowed to acclimate for 10 min. After acclimation, the focal individual was gently released from the cylinder and once it was swimming freely in the tank, the association time (s) the focal individual spent with each stimulus female was recorded for 10 min. Time spent with each female was recorded using Viewer (Biobserve GmbH), a video tracking software, which recorded how much time (s) an individual spent near each stimulus. After the allotted time, the focal fish was placed into the cylinder again, and the stimulus females were switched to detect any side bias. We tested the focal fish in both treatments, randomizing the order in which the treatments were offered, until each focal individual was presented with all 3 treatments. Strength of preference (SOP) scores were calculated, then √arcsine transformed to normalize the data. We used a repeated-measures general linear model (GLM) to compare the normalized preference scores of each treatment for each species/sex in SPSS (ver. 24). Sample sizes for each of the focal species/sex included Atlantic mollies: females (N = 10) and males (N = 26), and sailfin mollies: females (N = 16) and males (N = 27). For our model, we used the SOP scores as the dependent variable, species, sex and the treatment as the independent variables, and included Bonferroni-corrected pair-wise comparisons. There was an unusually large number of individuals showing side biases (>85% of the time spent on one side of the experimental tank) found in both species and sexes, and consequently we analyzed the results both with and without the side-biased individuals. There was little to no statistical difference if we included or removed these side-biased individuals (Table 1), therefore we interpreted side-biased individuals as “unresponsive” to either stimuli, or showing no preference. Finally, we calculated Cohen’s d and the effect-size correlation (rYλ) between the 2 different clone types for each sex and species to understand the standardized difference between the SOP scores of one clone compared to the other. Table 1 The average ± standard deviation of the time (s) individuals spent with each Amazon molly clone type for both data sets (with side-biased individuals and without side-biased individuals)   Treatment 1: Clone 1 and Clone 2  With side-biased individuals  Without side-biased individuals  Clone 1  Clone 2  Clone 1  Clone 2  Sailfin molly female  201.13 ± 149.45  289.92 ± 138.72  198.13 ± 181.76  313.43 ± 183.62  Sailfin molly male  265.76 ± 152.10  239.82 ± 148.09  288.70 ± 174.50  245.27 ± 179.29  Atlantic molly female  301.51 ± 111.92  224.56 ± 118.15  323.96 ± 142.14  179.06 ± 133.85  Atlantic molly male  246.34 ± 151.26  265.23 ± 163.56  216.38 ± 194.67  294.27 ± 212.73    Treatment 2: Sympatric Clone  With side-biased individuals  Without side-biased individuals  Clone a  Clone b  Clone a  Clone b  Sailfin molly female  240.26 ± 152.05  245.59 ± 166.04  232.34 ± 174.94  232.30 ± 191.80  Sailfin molly male  243.10 ± 174.23  280.37 ± 178.85  235.42 ± 202.34  293.13 ± 200.81  Atlantic molly female  182.33 ± 94.93  320.95 ± 122.86  145.43 ± 83.05  342.74 ± 159.90  Atlantic molly male  272.24 ± 163.42  248.86 ± 136.83  255.53 ± 211.76  231.08 ± 203.16    Treatment 1: Clone 1 and Clone 2  With side-biased individuals  Without side-biased individuals  Clone 1  Clone 2  Clone 1  Clone 2  Sailfin molly female  201.13 ± 149.45  289.92 ± 138.72  198.13 ± 181.76  313.43 ± 183.62  Sailfin molly male  265.76 ± 152.10  239.82 ± 148.09  288.70 ± 174.50  245.27 ± 179.29  Atlantic molly female  301.51 ± 111.92  224.56 ± 118.15  323.96 ± 142.14  179.06 ± 133.85  Atlantic molly male  246.34 ± 151.26  265.23 ± 163.56  216.38 ± 194.67  294.27 ± 212.73    Treatment 2: Sympatric Clone  With side-biased individuals  Without side-biased individuals  Clone a  Clone b  Clone a  Clone b  Sailfin molly female  240.26 ± 152.05  245.59 ± 166.04  232.34 ± 174.94  232.30 ± 191.80  Sailfin molly male  243.10 ± 174.23  280.37 ± 178.85  235.42 ± 202.34  293.13 ± 200.81  Atlantic molly female  182.33 ± 94.93  320.95 ± 122.86  145.43 ± 83.05  342.74 ± 159.90  Atlantic molly male  272.24 ± 163.42  248.86 ± 136.83  255.53 ± 211.76  231.08 ± 203.16  Average time spent with each stimulus did not differ strongly between the 2 data sets, and there was consistently high variation within each data set. View Large RESULTS Overall, we found no significant effects of clone type SOP in both the GLM including the side-biased individuals (F1 = 1.184, P = 0.278, Figure 1) or the one excluding the side-biased individuals (F1 = 0.351, P = 0.555). Further, there was there no interaction between clone type SOP and sex (including side-biased individuals: F1 = 0.121, P = 0.728; excluding side-biased individuals: F1 = 0.156, P = 0.694), clone type SOP and species (including side-biased individuals: F1 = 0.017, P = 0.896; without side bias: F1 = 0.026, P = 0.872), clone type SOP and treatment (including side-biased individuals: F1 = 0.230, P = 0.632; excluding side-biased individuals: F1 = 0.102, P = 0.751), clone type SOP, sex, and species (including side-biased individuals: F1 = 0.022, P = 0.882; excluding side-biased individuals: F1 = 0.032, P = 0.858), clone type SOP, sex, and treatment (including side-biased individuals: F1 = 0.243, P = 0.623; excluding side-biased individuals: F1 = 0.600, P = 0.441), or clone type SOP, species, and treatment (including side-biased individuals: F1 = 1.248, P = 0.266; excluding side-biased individuals: F1 = 0.309, P = 0.580). There was a significant effect in the 4-way interaction among clone type SOP, sex, species, and treatment when the side-biased individuals were excluded (F1 = 5.090, P = 0.027), although this effect was nonsignificant when side-biased individuals were included (F1 = 3.426, P = 0.066). Figure 1 View largeDownload slide The average ± SE of the time (s) spent with the 2 different Amazon molly clone types (Clone 1: Light blue; Clone 2: Red) and between 2 identical, sympatric clones (Individual 1: Light blue; Individual. 2: Dark blue) including the individuals with side bias. There is no significant difference between the treatments in the males or females of either species. Although, there is more variation in the females’ response, this might be indicative of the low sample size. Figure 1 View largeDownload slide The average ± SE of the time (s) spent with the 2 different Amazon molly clone types (Clone 1: Light blue; Clone 2: Red) and between 2 identical, sympatric clones (Individual 1: Light blue; Individual. 2: Dark blue) including the individuals with side bias. There is no significant difference between the treatments in the males or females of either species. Although, there is more variation in the females’ response, this might be indicative of the low sample size. Out of the 10 Atlantic molly females, 6 females did not exhibit a side bias (60%), and of the 4 females that did, only 2 of these females remained on the same side of the experimental tank for the entire time (i.e., they never visited both sides of the tank). From the 26 males we tested, 12 males did not show any side bias (46%), and of the 14 males that did, 10 of them failed to visit at least one side of the tank. When we compared the mean SOP scores between Clone 1 and Clone 2 (treatment 1) we found that for females Cohen’s d = 0.669 (effect-size correlate = 0.317), and males Cohen’s d = −0.120 (effect-size correlate = −0.060). We tested 16 sailfin molly females and 8 of those females did not show side bias (50%), of the 8 females that did, only 4 of these females did not visit the other side of the experimental tank. Out of the 27 sailfin molly males we tested, we found that 17 males did not show side bias (63%), and only 10 of the males that did, failed to visit the other side of the experimental tank. When we compared the mean SOP scores between Clone 1 and Clone 2 (treatment 1) for females Cohen’s d = −0.616 (effect-size correlate = −0.294), and males Cohen’s d = 0.173 (effect-size correlate = 0.086). DISCUSSION Our initial prediction that the sexual ancestors of the Amazon molly, the Atlantic and sailfin molly, would show discrimination between clones of the Amazon molly was not supported. Our prediction was based on 2 facts: both ancestral species show species recognition and a preference for conspecifics, and Amazon mollies, the hybrid of the Atlantic and sailfin molly, does show clonal recognition. We found, however, that neither males nor females of sailfin or Atlantic mollies showed a preference for either a familiar clone or a novel clone type, despite a known ability for species recognition. Based on our analysis of effect sizes we feel confident that this truly reflects an absence of a preference. There are 3 likely explanations for this result: 1) They may not have the ability to recognize different Amazon molly clones; 2) They recognize clones, but do not show a preference for a specific Amazon molly clone; or 3) The experimental design does not allow for us to detect a preference for a specific clone type. First, the sexual species may not have the ability to discriminate between the 2 clonal types of Amazon mollies because the recognition mechanisms the Amazon molly females use are intraspecific. This may have some implications for the evolution of the unique ability for kin recognition in this sexual-unisexual mating complex. Amazon mollies are a relatively young species (ca. 100000–120000 generations ago; Schartl et al. 1995; Stöck et al. 2010). If clonal recognition indeed evolved after the formation of this species, the process was speedy. Poschadel et al. (2009) reported of divergent mating preferences in Amazon mollies, which, in addition to clonal recognition, might also have evolved after the hybridization event that led to the Amazon molly. So far, however, it is unclear if the parental species have the ability to recognize kin from non-kin, and also if kin recognition evolved in the Amazon mollies themselves or if it was inherited from one or both of their paternal species. Second, individuals may be able to discriminate between Amazon mollies; yet they do not show a preference for a particular clone type. For males, due to the lack of known genetic benefits for mating with the Amazon molly females, there may not be any adaptive value in order to evolve the ability to distinguish between the different clonal lineages in the first place. Female social behaviors might receive little, if any, benefits for evolving this particular discrimination. Sexual females should be equally aggressive towards and shoal equally with all Amazon mollies. If, however, particular clonal lineages possessed specific characteristics (i.e., more aggressiveness, faster growth rates, lower parasite load, etc.) there may be possible benefits for sexual females in differentiating between the clonal lineages. For instance, if one clonal lineage were more aggressive towards sexual females, sexual females would benefit from avoiding these aggressive clones due to the high cost of female–female aggression in these species (Makowicz and Schlupp 2015). It has been shown that Amazon mollies show individual behavioral phenotypes both among and within clonal lineages (Bierbach et al. 2017). However, it is yet unknown if specific clonal lineages are more prone to behave in particular ways compared to another, although this could easily be tested. However, in the Po. monacha-lucida complex, one clone type did behave more aggressively towards host males than 5 other clone types (Keegan-Rogers and Schultz 1988); although in this sexual-unisexual mating complex there have been many hybridization and backcrossing events leading to multiple clone types unlike the sailfin-Amazon molly complex, this still remains a possibility. On the other hand, the ability for sexual individuals to recognize different clonal lineages may be a learned trait. For instance, after a female interacts with a clone that is highly aggressive, it may learn to recognize and avoid being near that clone in the future, similar to male Po. lucida (Keegan-Rogers and Schultz 1988). All our sexual fish were inexperienced with these specific clonal lineages of Amazon mollies because the Amazon mollies were never kept in mixed stock tanks. However, if there are such specific characteristics of clonal lineages, this is yet unknown. Finally, it is possible that the experimental design was not efficient enough to detect a preference for either clone type. The methods used in this study are similar to others to detect both male and female preferences in these species (Tobler et al. 2005; McCoy et al. 2008; Joachim and Schlupp 2012; Makowicz et al. 2016a, b). These studies suggest that both males and females exhibit various social preferences, including mate, and shoaling preferences, and that these preferences are detectable using standard binary choice tests. Similarly, Amazon mollies have also been tested in such a design to determine if they were able to differentiate between the different clonal types. Amazon molly females are able to detect clonal sisters from nonsisters, in both laboratory and field studies using multiple sensory channels (Makowicz et al. 2016a). We allowed both visual and chemical cues to be present to allow for individuals to utilize either or both mechanisms for recognition. Regardless, although this is a possibility, the likelihood of the design not being effective enough is small. Interestingly, we found extremely high rates of side bias in this study, which is uncommon in these fish. The nonconservative (>85%) side bias criterion used in this study was identical to other studies (McCoy et al. 2008; Makowicz et al. 2016a), and less stringent than in other studies (Schlupp et al. 1994; Schlüter et al. 1998). It is likely that the elevated levels of side biases are due to males and females of both species being nonresponsive to either the stimulus females, almost as if they cannot answer the question they were asked in our experiment. Indeed, to humans, Amazon mollies are extremely difficult to differentiate between different clonal lineages. Sexual individuals may not receive a benefit from swimming towards a stimulus that was identical to the stimulus they were already near. Yet, of the individuals that were categorized as side-biased, less than a quarter of the individuals (19 out of 82 fish) never visited the second stimulus. Nonetheless, when we evaluated effect sizes using Cohen’s d, for both species of males the means of the 2 different clone types were essentially identical, suggesting that there is actually no discrimination. There were moderate differences in female preference between the 2 clones but this may be due to the smaller sample size as compared to males. This suggests that there may not have been enough detectable difference between the stimulus females, and thus, individuals did not respond. Although our data do not support the notion that species and kin recognition are continuous in the Atlantic and sailfin molly, clearly more work needs to be done to fully test this. The origin of the unisexual species may play an important role in the ability for sexual individuals to discriminate among different clonal lineages. There are only few ways gynogenetic females can increase genetic diversity leading to distinct clonal lineages: mutation, genetic conversion, partial introgression of the male genome or full introgression resulting in increased ploidy level (Schlupp 2009). Hybridogenetic females (e.g., found in both Pelophylax esculenta complex of European frogs (Biriuk et al. 2016) and Po. monacha-lucida complex of fish), however, are unique as each sister is genetically distinct from one another in one way: they each have a father. These females reproduce clonally by discarding the paternal set of chromosomes in their gametes, resulting in identical ova of the maternal chromosomes, which duplicate, undergo meiosis and are then fertilized by another male, creating a lineage of maternally inherited genes (Avise 2008). Furthermore, for both gynogens and hybridogens, whether they resulted from a single (e.g., the Amazon molly) or multiple hybridization events (e.g., A. jeffersonianum complex of salamanders), from one or multiple back crossing events (e.g., Po. monacha-lucida mating complex), or the time since the original hybridization event may be extremely important for the sexual species to evolve the ability to recognize clone types (i.e., gynogens vs. hybridogens, diploid vs. triploid vs. tetraploid, etc.) and different clonal lineages within each clone type. It is also important to mention that some sexual-unisexual species complexes have a combination of all the above, like the Po. monacha-lucida complex which includes gynogens, hybridogens, diploids and triploid, and have originated from multiple hybridization and backcrossing events. Hence, the evolutionary history of each sexual-unisexual mating complex is essential to the evolution of recognition capabilities. 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Behavioral EcologyOxford University Press

Published: Mar 1, 2018

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