ANDREAS F. KAUTT*, GONZALO MACHADO-SCHIAFFINO*, JULIAN TORRES-DOWDALL & AXEL MEYER (*equal contribution)
Manuscript in preparation for submission
Abstract
Understanding when and how population divergence in the absence of geographic barriers can occur is a central objective in speciation research. Especially the earliest stages of intrapopulation divergence may be informative on the processes that constrain or facilitate speciation. Midas cichlid fish in Crater Lake Asososca Managua in Nicaragua represent a promising system in which to investigate the earliest stages of ecological sympatric speciation: the small and extremely young lake is protected from its surroundings and inhabited by a single endemic species of Midas cichlids, Amphilophus tolteca. Individuals are known to specialize along the benthic-limnetic axis, yet it remained unclear whether individuals would indeed differentially use the two habitats, which can have strong implications for the likelihood of speciation in sympatry. In this study we investigate for the first time whether fish of a single species of Midas cichlids differentially use the limnetic and benthic habitats in a crater lake in Nicaragua. We found that fish from the open-water habitat (middle of the lake) were indeed significantly more elongated than fish from the benthic habitat (shore). Stable isotope analyses confirmed that the former also exhibit a more limnetic lifestyle. Yet, neither microsatellite markers nor more than 10 000 SNP markers revealed any genetic clustering in the population. Split-brood design experiments in the laboratory suggest that the observed differences in body elongation are, however, indeed mostly genetically determined and not merely phenotypically plastic. Demographic inferences suggest that the apparent lack of genetic differentiation could be simply due to a lack of time, as intrapopulation competition may only have been strong enough to exert a considerable disruptive selection pressure a few hundred generations ago.
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Introduction
Under which conditions speciation with gene flow can and cannot occur remains a key question in evolutionary biology. Especially the earliest stages of population divergence may be informative about the conditions that promote or stall the process of speciation (Via 2009). In this regard, theoretical and empirical case studies that have elucidated the factors that impede speciation (Matessi et al. 2001; Bolnick 2011; Martin 2013; Comeault et al. 2015) have proofed just as informative as those in which further population divergence and speciation may be expected (Filchak et al. 2000; Hawthorne & Via 2001; Turner & Hahn 2007). Generally, theory has revealed that sympatric speciation requires a form of frequency-dependent disruptive selection and assortative mating to overcome the homogenizing force of gene flow and recombination (Gavrilets 2003; Gavrilets 2004). Disruptive selection can arise form intrapopulation competition, which may be apparent by individual specialization to certain food resources (Martin & Pfennig 2009). If this specialization is genetically determined and leads to assortative mating due to the spatial or temporal segregation (habitat isolation) of mating pools or influences the propensity of individuals to mate with others of a similar phenotype (mate preference) population divergence in sympatry is greatly facilitated (Servedio et al. 2011; Smadja & Butlin 2011). Nonetheless, the conditions for sympatric speciation are very restricted (Gavrilets 2005) and more empirical case studies investigating especially the earliest stages of speciation in sympatry are sorely needed (Bolnick & Fitzpatrick 2007; Bird et al. 2012).
Freshwater fish make for excellent systems to study the early stages of divergence-with-gene-flow due to the often young age and isolated nature of lakes. A common theme in the diversification of freshwater fishes is an initial divergence along the open-water (limnetic) and shore-associated (benthic) habitats (Robinson & Wilson 1994). Limnetic ecomorphs exhibit characteristic elongated / fusiform body shapes in comparison to the rather high-bodied benthic ecomorphs. Eco-morphological divergence along the benthic-limnetic axis is exemplified in a variety of fish taxa inhabiting postglacial lakes (Schluter 1996). The fact that this has happened in a similar direction in so many different taxa and often multiple times within the same species provides strong support for a role of natural selection and character displacement (Schluter & McPhail 1992, 1993). Yet, the commonality of this parallel benthic-limnetic divergence is anything but universal. Although fish have diversified along this axis in a considerable number of cases, this number is overshadowed by the cases where fish have not diversified and a single generalist population occupies a lake (Bolnick 2011). Identifying the conditions that have facilitated or hindered population divergence and speciation remains thus a key objective in enhancing our ability to predict evolutionary processes. The young age and replicated manner of postglacial lakes
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in the temperate zone has made them excellent study systems to study the early stages of population divergence and speciation.
Crater lakes in Africa and Nicaragua inhabited by cichlid fishes represent a similar setting. Moreover, crater lakes form usually remote and isolated water sheds that make multiple invasions rather unlikely (but see Martin et al. 2015). Thus, in contrast to fish in postglacial lakes in which divergence may have happened due to character displacement after a secondary invasion (Schluter 1996), divergence in crater lake cichlids is often assumed to have happened in sympatry. Similarly to fish in postglacial lakes, however, the evolutionary outcome varies. Haplochromine cichlids in Uganda, for example, have repeatedly evolved an overall more limnetic body shape without diversifying after the colonization of crater lakes (Machado-Schiaffino et al. 2015). Midas cichlids (belonging to the Amphilophus sp. species complex) that have independently colonized several crater lakes in Nicaragua from the same source population of the great lakes Nicaragua and Managua show a variable pattern (Elmer et al. 2010b): they have independently evolved into one limnetic and several benthic species in the two crater lakes Apoyo and Xiloá (Barluenga et al. 2006; Kautt et al. 2012; Elmer et al. 2014) whereas in other crater lakes they have not diversified along the benthic-limnetic axis (Elmer et al. 2010b). The variability in body elongation in Midas cichlids seems to be correlated to the mean depth of the respective crater lake (Recknagel et al. 2014), which might influence the potential for intrapopulation competition and thus frequency-dependent disruptive selection. Consequently, Crater Lake As. Managua - the focal lake of this study- has a mean depth (54.3 m) similar to that of L.
Xiloá (60 m) and its population exhibits a large morphological variance in body shape elongation (Kusche et al. 2014).
Lake As. Managua is the youngest crater lake known to harbor Midas cichlids (Elmer et al. 2010b) and is inhabited by a single endemic species of Midas cichlids, Amphilophus tolteca (Recknagel et al. 2013b). Individuals specialize along the benthic-limnetic axis, that is, individual body shapes are correlated to their lower pharyngeal jaw shapes and their feeding ecology, yet no population divergence and genetic structuring could be detected (Kusche et al. 2014). This makes crater lake As. Managua an interesting system to study whether the conditions for further divergence are given and, if so, why population divergence is lacking. A main prediction is that individuals are not only specialized, but do indeed differentially use the limnetic and benthic habitats. In other words, we would expect fish using the open-water habitat to exhibit on average a more elongated body shape and limnetic lifestyle (diet) compared to individuals from the shore. So far, this prediction has never been tested in A. tolteca, or Midas cichlids in general, as sampling of fish usually occurred exclusively in the benthic habitat (close to the shore). Differential habitat use is a
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key factor that may increase the likelihood of population divergence, as the spatial segregation could automatically lead to assortative mating, that is habitat isolation (Servedio et al. 2011). In fact, habitat isolation is a necessary assumption in a model of sympatric speciation tailored to Midas cichlids in L. Apoyo (Gavrilets et al. 2007). We acknowledge that the limnetic and benthic habitats in a lake are not discrete and treating them as two distinct habitats is certainly an oversimplification, yet this distinction is a common and fairly reasonable assumption (Bolnick 2011). Another factor that had so far not been tested is the role of phenotypic plasticity in the variation of body shape elongation. While adaptive phenotypic plasticity might increase the potential for ecological speciation in the long-term by allowing for the colonization of new environments and population persistence, it will usually impede population divergence in sympatry (Bolnick 2011; Thibert-Plante & Hendry 2011). Finally, the demographic history of a population will determine what levels of population divergence can be expected. At the very earliest stages of intrapopulation divergence there might simply not have been enough time for significant genetic differentiation at the genomic level to build up. The apparent lack of genetic differentiation can thus not be readily interpreted as a lack of population divergence in extremely young populations such as crater lake cichlids, since genetic differentiation will lag behind population divergence and only build up with time.
In this study, we were for the first time able to obtain samples from the open-water habitat (the middle of the lake) and test whether fish in L. As. Managua differentially use the limnetic and benhtic habitats. In this regard, we used an integrative approach by investigating the morphological differentiation, long-term diet differences, and genetic differentiation of fish captured in two different habitats. In addition, we performed experiments in the laboratory to test whether differences in body shapes could be explained by phenotypic plasticity. Finally, we reconstructed the demographic history of the population in L. As. Managua using coalescent simulations and the site frequency spectrum in order to put our results of the putative stage of population divergence in perspective to demographic correlates.