The Past and Future of Experimental Speciation
Authors: Nathan J. White, Rhonda R. Snook, Isobel Eyres
Journal: Trends in Ecology & Evolution (2019)

Experimental speciation is an excellent complement to snapshot studies of natural populations because it can disentangle recurring problems that confound studies of natural populations.Experimental speciation made early significant contributions to understanding evolutionary processes mediating the evolution of reproductive isolation.Over the past decade, speciation genomics has provided better predictions on how barrier loci spread in the genome and how speciation-with-gene-flow can occur.These developments remain difficult to test in natural populations and have not been widely adopted in experimental speciation research.Future integration of genomic tools in an experimental speciation framework will provide a step-change to understanding these outstanding speciation questions. Speciation is the result of evolutionary processes that generate barriers to gene flow between populations, facilitating reproductive isolation. Speciation is typically studied via theoretical models and snapshot tests in natural populations. Experimental speciation enables real-time direct tests of speciation theory and has been long touted as a critical complement to other approaches. We argue that, despite its promise to elucidate the evolution of reproductive isolation, experimental speciation has been underutilised and lags behind other contributions to speciation research. We review recent experiments and outline a framework for how experimental speciation can be implemented to address current outstanding questions that are otherwise challenging to answer. Greater uptake of this approach is necessary to rapidly advance understanding of speciation. Speciation is the result of evolutionary processes that generate barriers to gene flow between populations, facilitating reproductive isolation. Speciation is typically studied via theoretical models and snapshot tests in natural populations. Experimental speciation enables real-time direct tests of speciation theory and has been long touted as a critical complement to other approaches. We argue that, despite its promise to elucidate the evolution of reproductive isolation, experimental speciation has been underutilised and lags behind other contributions to speciation research. We review recent experiments and outline a framework for how experimental speciation can be implemented to address current outstanding questions that are otherwise challenging to answer. Greater uptake of this approach is necessary to rapidly advance understanding of speciation. The progression and outcome of speciation (see Glossary) depend on interactions between evolutionary forces [1Ravinet M. et al.Interpreting the genomic landscape of speciation: a road map for finding barriers to gene flow.J. Evol. Biol. 2017; 30: 1450-1477Crossref PubMed Scopus (256) Google Scholar] that act with varying importance over space and time to either facilitate or impede the evolution of reproductive isolation (RI) [2Abbott R. et al.Hybridization and speciation.J. Evol. Biol. 2013; 26: 229-246Crossref PubMed Scopus (1312) Google Scholar]. RI may arise through the action of genetic drift and/or divergent natural selection, may depend on gene flow via continuous migration or secondary contact, is impacted by population size and structure, and influenced by genomic properties such as mutation and recombination rates [3Ortiz-Barrientos D. James M.E. Evolution of recombination rates and the genomic landscape of speciation.J. Evol. Biol. 2017; 30: 1519-1521Crossref PubMed Scopus (6) Google Scholar]. Understanding the relative contributions of these processes to the evolution of RI is the focus of speciation research. A classic and highly successful approach to studying speciation involves identifying a phenotypically divergent trait and testing its association with the level of RI between extant populations [4McKinnon J.S. et al.Evidence for ecology's role in speciation.Nature. 2004; 429: 294-298Crossref PubMed Scopus (381) Google Scholar, 5Le Pennec G. et al.Adaptation to dislodgement risk on wave-swept rocky shores in the snail Littorina saxatilis.PLoS One. 2017; 12e0186901Crossref PubMed Scopus (25) Google Scholar, 6Huber S.K. et al.Reproductive isolation of sympatric morphs in a population of Darwin's finches.Proc. R. Soc. B Biol. Sci. 2007; 274: 1709-1714Crossref PubMed Scopus (116) Google Scholar]. The increasing application of high-throughput genomic data to address speciation genomics questions (Box 1) is used to reconstruct population history (e.g., demography) and infer the evolutionary processes leading to speciation, often over a long timescale [1Ravinet M. et al.Interpreting the genomic landscape of speciation: a road map for finding barriers to gene flow.J. Evol. Biol. 2017; 30: 1450-1477Crossref PubMed Scopus (256) Google Scholar, 3Ortiz-Barrientos D. James M.E. Evolution of recombination rates and the genomic landscape of speciation.J. Evol. Biol. 2017; 30: 1519-1521Crossref PubMed Scopus (6) Google Scholar, 7Wolf J.B.W. Ellegren H. Making sense of genomic islands of differentiation in light of speciation.Nat. Rev. Genet. 2017; 18: 87-100Crossref PubMed Scopus (241) Google Scholar]. This approach is analogous to the use of forward genetics to study the function of a gene, but applied to the study of RI. Here, the study of speciation begins with a phenotype (RI) and proceeds to identify the potential evolutionary processes that caused RI to build up between diverged populations. Many studies support the success of this approach [1Ravinet M. et al.Interpreting the genomic landscape of speciation: a road map for finding barriers to gene flow.J. Evol. Biol. 2017; 30: 1450-1477Crossref PubMed Scopus (256) Google Scholar, 4McKinnon J.S. et al.Evidence for ecology's role in speciation.Nature. 2004; 429: 294-298Crossref PubMed Scopus (381) Google Scholar, 5Le Pennec G. et al.Adaptation to dislodgement risk on wave-swept rocky shores in the snail Littorina saxatilis.PLoS One. 2017; 12e0186901Crossref PubMed Scopus (25) Google Scholar, 6Huber S.K. et al.Reproductive isolation of sympatric morphs in a population of Darwin's finches.Proc. R. Soc. B Biol. Sci. 2007; 274: 1709-1714Crossref PubMed Scopus (116) Google Scholar, 7Wolf J.B.W. Ellegren H. Making sense of genomic islands of differentiation in light of speciation.Nat. Rev. Genet. 2017; 18: 87-100Crossref PubMed Scopus (241) Google Scholar]. However, this forward method of studying speciation is actually backward looking, reflecting a static snapshot of the processes that contributed to divergence. Realistically, signals of early barriers to gene flow are likely erased or overwritten as speciation progresses. Thus, such studies are challenged to deduce the action of multiple evolutionary processes impacting phenotypic and genomic factors that influence speciation, either sequentially or simultaneously, either in the same or different directions, inferred over long evolutionary histories.Box 1Speciation GenomicsThe reduced cost of genomics has expanded the ability to address outstanding questions in speciation [1Ravinet M. et al.Interpreting the genomic landscape of speciation: a road map for finding barriers to gene flow.J. Evol. Biol. 2017; 30: 1450-1477Crossref PubMed Scopus (256) Google Scholar, 4McKinnon J.S. et al.Evidence for ecology's role in speciation.Nature. 2004; 429: 294-298Crossref PubMed Scopus (381) Google Scholar, 6Huber S.K. et al.Reproductive isolation of sympatric morphs in a population of Darwin's finches.Proc. R. Soc. B Biol. Sci. 2007; 274: 1709-1714Crossref PubMed Scopus (116) Google Scholar, 25Nosil P. et al.Ecological explanations for (incomplete) speciation.Trends Ecol. Evol. 2009; 24: 145-156Abstract Full Text Full Text PDF PubMed Scopus (513) Google Scholar]. Of interest is how barrier loci are distributed across genomes and how they evolve during population divergence. Predicted genomic patterns are based on whether speciation proceeds between geographically separated populations without gene flow, or with gene flow occurring either during initial divergence or following secondary contact. In allopatry, divergence is not substantially constrained by the extent of genetic linkage and recombination relative to the strength of either selection or drift producing RI. In contrast, during speciation-with-gene-flow, selection for divergence is opposed by the processes of both gene flow and recombination that erode associations between genes under selection [82Nosil P. Feder J.L. Genomic divergence during speciation: causes and consequences.Philos. Trans. R. Soc. B Biol. Sci. 2012; 367: 332-342Crossref PubMed Scopus (255) Google Scholar]. The genic view of speciation-with-gene-flow posits that speciation is initiated by selection acting against gene flow at specific targets of selection, and speciation genomics is interested in how barriers to gene flow initiate and facilitate (through the build-up of linkage disequilibrium) RI, including subsequent genomic divergence that is dependent on genomic architecture [25Nosil P. et al.Ecological explanations for (incomplete) speciation.Trends Ecol. Evol. 2009; 24: 145-156Abstract Full Text Full Text PDF PubMed Scopus (513) Google Scholar, 26Nosil P. Harmon L. Niche dimensionality and ecological speciation.in: Butlin R.K. Speciation and Patterns of Diversity. Cambridge University Press, 2009: 127-154Crossref Google Scholar, 83Wu C.-I. The genic view of the process of speciation.J. Evol. Biol. 2001; 14: 851-865Crossref Scopus (910) Google Scholar, 84Turner T.L. et al.Genomic islands of speciation in Anopheles gambiae.PLoS Biol. 2005; 3e285Crossref PubMed Scopus (550) Google Scholar]. Patterns of divergence are predicted to be different depending on whether gene flow is primary or secondary [85Richards E. et al.Searching for sympatric speciation in the genomic era.bioRxiv. 2019; (Published online January 28, 2019. https://doi.org/10.1101/367623)Google Scholar].Speciation genomics has begun to address these issues by identifying barrier loci evolving in response to selection or drift, their effect sizes, genomic distribution, and associations, and how this builds up as RI increases, along with inferring demographic history and gene flow [86Ellegren H. et al.The genomic landscape of species divergence in Ficedula flycatchers.Nature. 2012; 491: 756Crossref PubMed Scopus (421) Google Scholar, 87Nadeau N.J. et al.Genomic islands of divergence in hybridizing Heliconius butterflies identified by large-scale targeted sequencing.Phil. Trans. R. Soc. B. 2012; 367: 343-353Crossref PubMed Scopus (235) Google Scholar, 88Marques D.A. et al.Genomic landscape of early ecological speciation initiated by selection on nuptial colour.PLoS Genet. 2016; 12e1005887Crossref PubMed Scopus (153) Google Scholar, 89Smadja C.M. et al.Large-scale candidate gene scan reveals the role of chemoreceptor genes in host plant specialization and speciation in the pea aphid.Evolution (N. Y). 2012; 66: 2723-2738Google Scholar]. However, there are well-reviewed confounding factors influencing genome heterogeneity that are unrelated to speciation (e.g., population history, gene flow over time, variation in strength, and timing of selection [7Wolf J.B.W. Ellegren H. Making sense of genomic islands of differentiation in light of speciation.Nat. Rev. Genet. 2017; 18: 87-100Crossref PubMed Scopus (241) Google Scholar, 9Noor M.A.F. Bennett S.M. Islands of speciation or mirages in the desert? Examining the role of restricted recombination in maintaining species.Heredity (Edinb). 2009; 103: 439-444Crossref PubMed Scopus (266) Google Scholar, 10Cruickshank T.E. Hahn M.W. Reanalysis suggests that genomic islands of speciation are due to reduced diversity, not reduced gene flow.Mol. Ecol. 2014; 23: 3133-3157Crossref PubMed Scopus (619) Google Scholar]), and disentangling these factors remains challenging in studies of natural populations. Models of the rate, direction and magnitude of gene flow through time tend to rely on summary measures or comparing limited sets of hypothesised scenarios. Additionally, the impact of selection on divergence can sometimes be clearly identified [5Le Pennec G. et al.Adaptation to dislodgement risk on wave-swept rocky shores in the snail Littorina saxatilis.PLoS One. 2017; 12e0186901Crossref PubMed Scopus (25) Google Scholar, 90Jiggins C.D. Ecological speciation in mimetic butterflies.Bioscience. 2008; 58: 541-548Crossref Scopus (100) Google Scholar, 91Le Rouzic A. et al.Strong and consistent natural selection associated with armour reduction in sticklebacks.Mol. Ecol. 2011; 20: 2483-2493Crossref PubMed Scopus (56) Google Scholar, 92Forbes A.A. et al.Revisiting the particular role of host shifts in initiating insect speciation.Evolution (N. Y). 2017; 71: 1126-1137Google Scholar], but it is frequently challenging to characterise selection pressures – increasingly so the further selection is traced back through history. Thus, understanding the role of ecological differentiation, isolation, and genomic differentiation in response to specific evolutionary processes is difficult to reconstruct [93Langerhans R.B. Predictability and parallelism of multitrait adaptation.J. Hered. 2017; 109: 59-70Crossref PubMed Scopus (34) Google Scholar]. Alongside the development of models which can coestimate demography and selection, the ability to directly observe these processes during experiments designed to track such interactions will provide powerful data to apply to natural systems where direct observation during the evolution of RI is unavailable. The reduced cost of genomics has expanded the ability to address outstanding questions in speciation [1Ravinet M. et al.Interpreting the genomic landscape of speciation: a road map for finding barriers to gene flow.J. Evol. Biol. 2017; 30: 1450-1477Crossref PubMed Scopus (256) Google Scholar, 4McKinnon J.S. et al.Evidence for ecology's role in speciation.Nature. 2004; 429: 294-298Crossref PubMed Scopus (381) Google Scholar, 6Huber S.K. et al.Reproductive isolation of sympatric morphs in a population of Darwin's finches.Proc. R. Soc. B Biol. Sci. 2007; 274: 1709-1714Crossref PubMed Scopus (116) Google Scholar, 25Nosil P. et al.Ecological explanations for (incomplete) speciation.Trends Ecol. Evol. 2009; 24: 145-156Abstract Full Text Full Text PDF PubMed Scopus (513) Google Scholar]. Of interest is how barrier loci are distributed across genomes and how they evolve during population divergence. Predicted genomic patterns are based on whether speciation proceeds between geographically separated populations without gene flow, or with gene flow occurring either during initial divergence or following secondary contact. In allopatry, divergence is not substantially constrained by the extent of genetic linkage and recombination relative to the strength of either selection or drift producing RI. In contrast, during speciation-with-gene-flow, selection for divergence is opposed by the processes of both gene flow and recombination that erode associations between genes under selection [82Nosil P. Feder J.L. Genomic divergence during speciation: causes and consequences.Philos. Trans. R. Soc. B Biol. Sci. 2012; 367: 332-342Crossref PubMed Scopus (255) Google Scholar]. The genic view of speciation-with-gene-flow posits that speciation is initiated by selection acting against gene flow at specific targets of selection, and speciation genomics is interested in how barriers to gene flow initiate and facilitate (through the build-up of linkage disequilibrium) RI, including subsequent genomic divergence that is dependent on genomic architecture [25Nosil P. et al.Ecological explanations for (incomplete) speciation.Trends Ecol. Evol. 2009; 24: 145-156Abstract Full Text Full Text PDF PubMed Scopus (513) Google Scholar, 26Nosil P. Harmon L. Niche dimensionality and ecological speciation.in: Butlin R.K. Speciation and Patterns of Diversity. Cambridge University Press, 2009: 127-154Crossref Google Scholar, 83Wu C.-I. The genic view of the process of speciation.J. Evol. Biol. 2001; 14: 851-865Crossref Scopus (910) Google Scholar, 84Turner T.L. et al.Genomic islands of speciation in Anopheles gambiae.PLoS Biol. 2005; 3e285Crossref PubMed Scopus (550) Google Scholar]. Patterns of divergence are predicted to be different depending on whether gene flow is primary or secondary [85Richards E. et al.Searching for sympatric speciation in the genomic era.bioRxiv. 2019; (Published online January 28, 2019. https://doi.org/10.1101/367623)Google Scholar]. Speciation genomics has begun to address these issues by identifying barrier loci evolving in response to selection or drift, their effect sizes, genomic distribution, and associations, and how this builds up as RI increases, along with inferring demographic history and gene flow [86Ellegren H. et al.The genomic landscape of species divergence in Ficedula flycatchers.Nature. 2012; 491: 756Crossref PubMed Scopus (421) Google Scholar, 87Nadeau N.J. et al.Genomic islands of divergence in hybridizing Heliconius butterflies identified by large-scale targeted sequencing.Phil. Trans. R. Soc. B. 2012; 367: 343-353Crossref PubMed Scopus (235) Google Scholar, 88Marques D.A. et al.Genomic landscape of early ecological speciation initiated by selection on nuptial colour.PLoS Genet. 2016; 12e1005887Crossref PubMed Scopus (153) Google Scholar, 89Smadja C.M. et al.Large-scale candidate gene scan reveals the role of chemoreceptor genes in host plant specialization and speciation in the pea aphid.Evolution (N. Y). 2012; 66: 2723-2738Google Scholar]. However, there are well-reviewed confounding factors influencing genome heterogeneity that are unrelated to speciation (e.g., population history, gene flow over time, variation in strength, and timing of selection [7Wolf J.B.W. Ellegren H. Making sense of genomic islands of differentiation in light of speciation.Nat. Rev. Genet. 2017; 18: 87-100Crossref PubMed Scopus (241) Google Scholar, 9Noor M.A.F. Bennett S.M. Islands of speciation or mirages in the desert? Examining the role of restricted recombination in maintaining species.Heredity (Edinb). 2009; 103: 439-444Crossref PubMed Scopus (266) Google Scholar, 10Cruickshank T.E. Hahn M.W. Reanalysis suggests that genomic islands of speciation are due to reduced diversity, not reduced gene flow.Mol. Ecol. 2014; 23: 3133-3157Crossref PubMed Scopus (619) Google Scholar]), and disentangling these factors remains challenging in studies of natural populations. Models of the rate, direction and magnitude of gene flow through time tend to rely on summary measures or comparing limited sets of hypothesised scenarios. Additionally, the impact of selection on divergence can sometimes be clearly identified [5Le Pennec G. et al.Adaptation to dislodgement risk on wave-swept rocky shores in the snail Littorina saxatilis.PLoS One. 2017; 12e0186901Crossref PubMed Scopus (25) Google Scholar, 90Jiggins C.D. Ecological speciation in mimetic butterflies.Bioscience. 2008; 58: 541-548Crossref Scopus (100) Google Scholar, 91Le Rouzic A. et al.Strong and consistent natural selection associated with armour reduction in sticklebacks.Mol. Ecol. 2011; 20: 2483-2493Crossref PubMed Scopus (56) Google Scholar, 92Forbes A.A. et al.Revisiting the particular role of host shifts in initiating insect speciation.Evolution (N. Y). 2017; 71: 1126-1137Google Scholar], but it is frequently challenging to characterise selection pressures – increasingly so the further selection is traced back through history. Thus, understanding the role of ecological differentiation, isolation, and genomic differentiation in response to specific evolutionary processes is difficult to reconstruct [93Langerhans R.B. Predictability and parallelism of multitrait adaptation.J. Hered. 2017; 109: 59-70Crossref PubMed Scopus (34) Google Scholar]. Alongside the development of models which can coestimate demography and selection, the ability to directly observe these processes during experiments designed to track such interactions will provide powerful data to apply to natural systems where direct observation during the evolution of RI is unavailable. Laboratory experimental evolution (EE) experiments can address these challenges by manipulating evolutionary processes thought to generate RI over many generations and then testing the outcome on the evolution of RI. Experimental speciation (ES) is analogous to the use of reverse genetics to study gene function. It begins with the putative evolutionary processes and proceeds to identify the conditions leading to and maintaining RI. This approach is experimental and therefore directly identifies the evolutionary processes and circumstances for the evolution of RI. ES complements snapshot studies (Table 1) but is also a stand-alone powerful approach because it reveals speciation processes in real time. ES has been implemented for several decades and when its influential contribution was last reviewed, 10 years ago by Fry [8Fry J.D. Laboratory experiments on speciation.in: Garland T.J. Rose Michael R. Experimental Evolution. University of California Press, 2009: 631-656Crossref Google Scholar], the technique seemed poised to exponentially accelerate understanding the evolution of RI. Fry also outlined neglected speciation questions that ES was well suited to answer. Since Fry's review, speciation theory has advanced to incorporate more sophisticated ideas on genomic conditions and constraints impacting the evolution of RI. Snapshot studies have widely adopted a genetic approach to identifying signatures of RI. However, these conventional studies are vexed with inference problems, limiting understanding of speciation [9Noor M.A.F. Bennett S.M. Islands of speciation or mirages in the desert? Examining the role of restricted recombination in maintaining species.Heredity (Edinb). 2009; 103: 439-444Crossref PubMed Scopus (266) Google Scholar, 10Cruickshank T.E. Hahn M.W. Reanalysis suggests that genomic islands of speciation are due to reduced diversity, not reduced gene flow.Mol. Ecol. 2014; 23: 3133-3157Crossref PubMed Scopus (619) Google Scholar, 11Westram A.M. Ravinet M. Land ahoy? Navigating the genomic landscape of speciation while avoiding shipwreck.J. Evol. Biol. 2017; 30: 1522-1525Crossref PubMed Scopus (3) Google Scholar]. ES provides a potent method to test speciation theory by controlling and/or testing genomic factors and environmental conditions thought to influence speciation, factors that forward speciation approaches cannot disentangle (see 'A Selection of New Challenges That Experimental Speciation Can Address').Table 1ES and Studies Using Natural Populations Are Highly ComplementaryaSeveral advantages (bold) and limitations of each approach have been matched to illustrate their complementary nature.Laboratory-based ESComparative methods with natural populationsRare (but important) serendipitous events are likely to be missed unless the experiment is largeBetter represents the importance of a given process, rather than just its occurrenceStarting population characteristics and genome are defined or quantified a priori by the researcherIn most cases, it is challenging to reconstruct ancestral populations and their genomesTypically reliant upon standing variation aloneGreater potential for de novo mutation or introgression from other populations to play a roleEnvironment is controlled and can be kept constant or manipulated in a controlled manner, throughoutOften difficult to determine ancestral environment required to delineate the role of geography in restricting gene flowMany initial effects may be due to laboratory adaptation. If laboratory adaptation has occurred pre-EE, genetic diversity will be lowerPopulations are typically close to equilibrium in the wildEvolutionary responses are replicated over a series of lines to robustly link conditions to responsesNo true replication. Lack of parallelism may create uncertainty that a phenotypic change is a direct response to a given variableEvolution of traits is limited to what can be performed in culture conditions. Low niche dimensionality means only simple contrasts can be madeA much wider range of traits can be selected upon or ariseGene flow can be more accurately and reliably determined from highly controlled migration levels, and measures of local adaptation and RIDifficult to determine level of ongoing gene flowLimited to a subset of organisms suitable for EECan study any diverging populationsEasy to separate intrinsic and extrinsic forms of RIDifficult to disentangle intrinsic from extrinsic RILaboratory settings may exclude many of the ecological aspects that separate speciesCan assess the full range of isolating mechanisms found in the wildPhenotypic and genomic data can be collected with high temporal resolution providing estimates of phenotypic change and evolutionary hindsight of underlying genomic changesEven if ancestral genomes can be reconstructed, phenotype data is typically only a single snapshot, so cannot be matched to genomic dataExperiments can only cover short timescales and subsets of the speciation processLong timescales of divergence can be studied (although histories must be inferred)a Several advantages (bold) and limitations of each approach have been matched to illustrate their complementary nature. Open table in a new tab Here, we review ES studies over the past decade to examine progress on Fry's original neglected speciation questions. We identify areas of speciation research that have progressed since that review, such as speciation-with-gene-flow models and genomic conditions impacting speciation, but which ES studies have not been applied. We provide a framework for using ES combined with genomics to enable rapid advances in understanding speciation. Fry's review suggested ES could address: the relative efficacy of selection and drift in generating RI; the relative rates of evolution of different types of reproductive barriers; the feasibility of sympatric and parapatric speciation; and the feasibility of reinforcement [8Fry J.D. Laboratory experiments on speciation.in: Garland T.J. Rose Michael R. Experimental Evolution. University of California Press, 2009: 631-656Crossref Google Scholar]. We summarise the limited progress on these topics in the past decade, identify new areas in which ES has been used, and argue that since Fry's review, two fundamental shifts in speciation theory and approach have occurred that have been ignored in an ES framework. To maintain differences between populations, barriers to gene flow must emerge and generate RI. Barriers can act at the prezygotic (premating and postmating, prezygotic) and/or postzygotic stage, and can be influenced by extrinsic isolation and/or intrinsic isolation. Initial ES studies found relatively strong support for divergent natural selection generating RI in allopatry, even on arbitrary traits with no clear link to an isolating mechanism [8Fry J.D. Laboratory experiments on speciation.in: Garland T.J. Rose Michael R. Experimental Evolution. University of California Press, 2009: 631-656Crossref Google Scholar]. However, under sympatric conditions, disruptive selection did not generally lead to RI, likely because many of the divergently selected traits had little relevance to fitness [8Fry J.D. Laboratory experiments on speciation.in: Garland T.J. Rose Michael R. Experimental Evolution. University of California Press, 2009: 631-656Crossref Google Scholar]. Since Fry's review, few ES studies have altered conditions for local adaptation and then tested for the evolution of RI. Most studies tested the role of sexual selection and sexual conflict in generating RI [12Parker G.A. Partridge L. Sexual conflict and speciation.Philos. Trans. R. Soc. B Biol. Sci. 1998; 353: 261-274Crossref PubMed Scopus (364) Google Scholar, 13Gavrilets S. Is sexual conflict an "engine of speciation"?.Cold Spring Harb. Perspect. Biol. 2014; 6: a017723Crossref PubMed Scopus (46) Google Scholar]. Fry found equivocal support for sexual selection generating RI [8Fry J.D. Laboratory experiments on speciation.in: Garland T.J. Rose Michael R. Experimental Evolution. University of California Press, 2009: 631-656Crossref Google Scholar]. Subsequent work on sexual selection and speciation continues to fail to find significant RI [14Gay L. et al.Does reproductive isolation evolve faster in larger populations via sexually antagonistic coevolution?.Biol. Lett. 2009; 5: 693-696Crossref PubMed Scopus (25) Google Scholar, 15Plesnar-Bielak A. et al.No evidence for reproductive isolation through sexual conflict in the bulb mite Rhizoglyphus robini.PLoS One. 2013; 8: 1-8Crossref Scopus (7) Google Scholar, 16Debelle A. et al.Sexual selection and assortative mating: an experimental test.J. Evol. Biol. 2016; 29: 1307-1316Crossref PubMed Scopus (11) Google Scholar, 17Reding L.P. et al.Sexual selection hinders adaptation in experimental populations of yeast.Biol. Lett. 2013; 9: 20121202Crossref PubMed Scopus (14) Google Scholar], even when manipulating genetic variation and population size to increase the likelihood of response [14Gay L. et al.Does reproductive isolation evolve faster in larger populations via sexually antagonistic coevolution?.Biol. Lett. 2009; 5: 693-696Crossref PubMed Scopus (25) Google Scholar] and assessing different RI barriers [15Plesnar-Bielak A. et al.No evidence for reproductive isolation through sexual conflict in the bulb mite Rhizoglyphus robini.PLoS One. 2013; 8: 1-8Crossref Scopus (7) Google Scholar]. One species, Drosophila melanogaster, has been tested independently in two laboratories but only one study found RI [18Syed Z.A. et al.Reproductive isolation through experimental manipulation of sexually antagonistic coevolution in Drosophila melanogaster.Sci. Rep. 2017; 7: 3330Crossref PubMed Scopus (