View source for WikiJournal Preprints/Speciation by reinforcement

{{Article info
|journal=WikiJournal of Science
|last1=Colvin
|first1=Andrew Z.
|et_al=true
|correspondence={{nospam|azcolvin429|gmail.com}}
|license={{CC BY SA work}}
|submitted=2020-12-22
|w1=Reinforcement (speciation)
|keywords=Speciation, Reinforcement, Secondary contact, Low-fitness hybrids, Wallace effect, Reproductive isolation, Speciation by natural selection
|abstract=

'''Reinforcement''' is a process of speciation where natural selection increases the reproductive isolation between populations of a species. This occurs as a result of selection acting against the production of hybrid individuals of low fitness. The idea was originally developed by Alfred Russel Wallace and is sometimes referred to as the Wallace effect. The modern concept of reinforcement originates from Theodosius Dobzhansky. He envisioned a species separated allopatrically, where during  secondary contact, individuals from the two populations mate, producing hybrids with lower fitness. Natural selection results from the hybrid's inability to produce viable or fertile offspring; thus members of one species who do not mate with members of the other have greater reproductive success. This favors the evolution of greater pre-zygotic isolation (differences in behavior or biology that inhibit the formation of hybrid zygotes). Reinforcement is one of the few cases in which selection can favor an increase in pre-zygotic isolation, influencing the process of speciation directly.<ref name="Modes and Mechanisms of Speciation">{{Citation|title=Modes and Mechanisms of Speciation|author1=Hannes Schuler |author2=Glen R. Hood|author3=Scott P. Egan|author4=Jeffrey L. Feder|journal=Reviews in Cell Biology and Molecular Medicine|year=2016|volume=2|issue=3|pages=60–93}}</ref> This aspect has been particularly appealing among evolutionary biologists.<ref name="Marshalletal2002">{{Citation|title=Reinforcement: the road not taken|author1=Jeremy L. Marshall|author2=Michael L. Arnold|author3=Daniel J. Howard|journal=Trends in Ecology & Evolution|year=2002|volume=17|issue=12|pages=558–563|doi=10.1016/S0169-5347(02)02636-8}}</ref> 

The scientific support for reinforcement has fluctuated since its inception, and  terminological confusion and differences in usage over history have led to multiple meanings. Various objections have been raised by evolutionary biologists as to the plausibility of its occurrence. Since the 1990s, data from theory, experiments, and nature have overcome many of the past objections, rendering reinforcement widely accepted,<ref name="Speciation">{{Citation |title=Speciation |author1=Jerry A. Coyne |author2=H. Allen Orr |date=2004 |pages=1–545 |publisher=Sinauer Associates |isbn=978-0-87893-091-3 }}</ref>{{rp|354}} though its overall prevalence in nature remains unknown.<ref name="Servedio&Noor">{{Citation |title=The Role of Reinforcement in Speciation: Theory and Data | author1=Maria R. Servedio |author2=Mohamed A. F. Noor | journal=Annual Review of Ecology, Evolution, and Systematics | year=2003 | volume=34 | pages=339–364 | doi=10.1146/annurev.ecolsys.34.011802.132412 }}</ref><ref name=Barrientos2009>{{Citation |title=The Genetics and Ecology of Reinforcement: Implications for the Evolution of Prezygotic Isolation in Sympatry and Beyond | author1=Daniel Ortíz-Barrientos|author2=Alicia Grealy|author3=Patrik Nosil | journal=Annals of the New York Academy of Sciences | year=2009 | volume=1168 | pages=156–182 | doi=10.1111/j.1749-6632.2009.04919.x| pmid=19566707| s2cid=4598270 | url=https://semanticscholar.org/paper/412984a2b2c3f7a34275b64af0c774c3886bcdca }}</ref>

Numerous models have been developed to understand its operation in nature—most relying on several facets: genetics, the structure of populations, the influence of selection, and mating behaviors. Empirical support for reinforcement exists, both in the laboratory and in nature. Documented examples are found in a wide range of organisms: both vertebrates and invertebrates, fungi, and in plants. The secondary contact of originally separated incipient species (the initial stage of speciation) is increasing due to human activities such as the introduction of invasive species or the modification of natural habitats.<ref name="Servedio2004">{{Citation |title=The What and Why of Research on Reinforcement | author=Maria R. Servedio | journal=PLOS Biology | year=2004 | volume=2 | issue=12 | pages=2032–2035 | doi=10.1371/journal.pbio.0020420 | pmid=15597115 | pmc=535571 }}</ref> This has implications for measures of biodiversity and may become more relevant in the future.<ref name="Servedio2004"/>
}}

== History and terminology ==
Reinforcement has endured a complex history with its popularity among scholars having changed over time.<ref name="Noor1999">{{Citation |title=Reinforcement and other consequences of sympatry |author1=Mohamed A. F. Noor|journal=Heredity|year=1999|volume=83|issue= 5|pages=503–508|doi= 10.1038/sj.hdy.6886320|pmid=10620021|s2cid=26625194 }}</ref><ref>{{Citation |title=Coupling, Reinforcement, and Speciation |author1=Roger K. Butlin|author2=Carole M. Smadja |journal=The American Naturalist |year=2018 |volume=191 |issue=2 |pages=155–172 |doi=10.1086/695136 |pmid=29351021 |url=http://eprints.whiterose.ac.uk/127342/1/695136.pdf }}</ref> Jerry Coyne and H. Allen Orr contend that the theory of reinforcement went through three phases of historical development:<ref name="Speciation"/>{{rp|366}}
# plausibility based on unfit hybrids
# implausibility based on hybrids having some fitness
# plausibility based on empirical studies and biologically complex and realistic models
Sometimes called the Wallace effect, reinforcement was originally proposed by Alfred Russel Wallace in 1889.<ref name="Speciation"/>{{rp|353}} His hypothesis differed markedly from the modern conception in that it focused on post-zygotic isolation, strengthened by group selection.<ref>M. J. Littlejohn (1981). Reproductive isolation: A critical review. In W. R. Atchley and D. S. Woodruff (eds) ''Evolution and Speciation'', Cambridge University Press, Pp. 298–334.</ref><ref>{{Citation | title=Natural Selection: Methods and Applications | author=Mario A. Fares | date=2015 | pages=3 | publisher=CRC Press | isbn=9781482263725 }}</ref><ref name="Speciation"/>{{rp|353}} Dobzhansky was the first to provide a thorough description of the process in 1937,<ref name="Speciation"/>{{rp|353}} though the term itself was not coined until 1955 by W. Frank Blair.<ref>{{Citation |title=Mating call and stage of speciation in the ''Microhyla olivacea-M. carolinensis'' complex | author=Blair, W. Frank | journal=Evolution | year=1955 | volume=9 | issue= 4| pages=469–480 | doi= 10.1111/j.1558-5646.1955.tb01556.x| s2cid=88238743 }}</ref> In 1930, Ronald Fisher laid out the first genetic description of the process of reinforcement in ''The Genetical Theory of Natural Selection'', and in 1965 and 1970 the first computer simulations were run to test for its plausibility.<ref name="Speciation"/>{{rp|367}} Later population genetic<ref>{{Citation |title=On the evolution of behavioral reproductive isolation: The Wallace effect | author1=Stanley Sawyer|author2=Daniel Hartl | journal=Theoretical Population Biology | year=1981 | volume=19 | issue=1 | pages=261–273 | doi=10.1016/0040-5809(81)90021-6 }}</ref> and quantitative genetic<ref>{{Citation |title=A Two-Sex Polygenic Model for the Evolution of Premating Isolation. I. Deterministic Theory for Natural Populations | author=J. A. Sved | journal=Genetics | year=1981 | volume=97 | issue=1 | pages=197–215 | pmid=17249073 | pmc=1214384 |doi=10.1093/genetics/97.1.217}}</ref> studies were conducted showing that completely unfit hybrids lead unequivocally to an increase in pre-zygotic isolation.<ref name="Speciation"/>{{rp|367}}

Dobzhansky's idea gained significant support; he suggested that it illustrated the final step in speciation, for example after an allopatric population comes into secondary contact.<ref name="Speciation"/>{{rp|353}} In the 1980s, many evolutionary biologists began to doubt the plausibility of the idea,<ref name="Speciation"/>{{rp|353}} based not on empirical evidence, but largely on the growth of theory that deemed it an unlikely mechanism of reproductive isolation.<ref name="Marshalletal2002"/> A number of theoretical objections arose at the time and are addressed in later sections below.

By the early 1990s, reinforcement saw a revival in popularity among evolutionary biologists; due primarily from a sudden increase in data—empirical evidence from studies in labs and largely by examples found in nature.<ref name="Speciation"/>{{rp|354}} Further, computer simulations of the genetics and migration patterns of populations found, "something ''looking'' like reinforcement".<ref name="Speciation"/>{{rp|372}} The most recent theoretical work on speciation has come from several studies using highly complex computer simulations; all of which came to similar conclusions: that reinforcement is plausible under several conditions, and in many cases, is easier than previously thought.<ref name="Speciation"/>{{rp|374}}

Confusion exists around the meaning of the term reinforcement.<ref name="eLS2012">{{cite book |author=Glenn-Peter Sætre |publisher=John Wiley & Sons|chapter=Reinforcement |title=eLS |date=2012 |doi=10.1002/9780470015902.a0001754.pub3 |isbn=978-0470016176 }}</ref> It was first used to describe the observed mating call differences in ''Gastrophryne'' frogs within a secondary contact hybrid zone.<ref name="eLS2012"/> The term secondary contact has also been used to describe reinforcement in the context of an allopatrically separated population experiencing contact after the loss of a geographic barrier.<ref>{{Cite book |title=Genetics and the Origin of Species |first1=Theodosius |last1=Dobzhansky|year=1937 |publisher=Columbia University Press }}</ref> The term "the Wallace effect" is similar to reinforcement, but is rarely used.<ref name="eLS2012"/> Roger K. Butlin demarcated incomplete post-zygotic isolation from complete isolation, referring to the evolution of premating reproductive from incomplete isolation as reinforcement and from completely isolation as reproductive character displacement.<ref name="Butlin1989">{{cite book |first=Roger K. |last=Butlin |year=1989 |chapter=Reinforcement of premating isolation |editor-last=Otte |editor-first=D. |editor2-last=Endler |editor2-first=John A. |title=Speciation and its Consequences |publisher=Sinauer Associates |pages=[https://archive.org/details/speciationitscon0000unse/page/158 158–179] |isbn=978-0-87893-657-1 |chapter-url=https://archive.org/details/speciationitscon0000unse/page/158 }}</ref> Daniel J. Howard considered reproductive character displacement to represent either assortive mating or the divergence of traits for mate recognition (specifically between sympatric populations).<ref name="eLS2012"/> Reinforcement, under his definition, included pre-zygotic divergence and complete post-zygotic isolation.<ref name="Howard1993">{{cite book |first=Daniel J. |last=Howard |year=1993 |chapter=Reinforcement: origin, dynamics and fate of an evolutionary hypothesis |editor-last=Harrison |editor-first=R. G. |title=Hybrid Zones and the Evolutionary Process |publisher=Oxford University Press |pages=46–69 |isbn=978-0-19-506917-4 }}</ref> Maria R. Servedio and Mohamed A.F. Noor include any detected increase in pre-zygotic isolation as reinforcement, as long as it is a response to selection against mating between two different species.<ref name="Servedio&Noor"/> Coyne and Orr contend that, "true reinforcement is restricted to cases in which isolation is enhanced between taxa that can still exchange genes".<ref name="Speciation"/>{{rp|352}}

== Models ==
[[File:Speciation by Reinforcement Schematic.svg|right|thumb|upright=2|Reinforcement assists speciation by selecting against hybrids upon the secondary contact of two separated populations of a species.]]
One of the strongest forms of reproductive isolation in nature is sexual isolation: traits in organisms involving mating.<ref name="HoskinHiggie2010">{{Citation |title=Speciation via species interactions: the divergence of mating traits within species | author1=Conrad J. Hoskin|author2=Megan Higgie | journal=Ecology Letters | year=2010 | volume=13 | issue=4 | pages=409–420 | doi=10.1111/j.1461-0248.2010.01448.x | pmid=20455922 | s2cid=16175451 | url=https://semanticscholar.org/paper/6b93769cb9901a0e8c15b802f1daa81962f6549f }}</ref> This fact has led to the idea that, because selection acts so strongly on mating traits, it may be involved in the process of speciation.<ref name="HoskinHiggie2010"/> The influence of natural selection against hybrids on the evolution of premating isolation is reinforcement, and it can happen under any mode of speciation<ref name="Speciation"/>{{rp|355}} (''e.g.'' allopatry, parapatry, sympatry, ecological, etc.)<ref>{{Citation |title=Reinforcement during ecological speciation | author=Mark Kirkpatrick | journal=Proceedings of the Royal Society B | year=2001 | volume=268 | issue=1473 | pages= 1259–1263| doi=10.1098/rspb.2000.1427 | pmid=11410152 | pmc=1088735 }}</ref> It necessitates two forces of evolution that act on mate choice: natural selection and gene flow.<ref name="K&S1999">{{Citation |title=The reinforcement of mating preferences on an island | author1=Mark Kirkpatrick|author2=Maria R. Servedio | journal=Genetics | year=1999 | volume=151 | issue=2 | pages=865–884 | pmid=9927476| pmc=1460501|doi=10.1093/genetics/151.2.865}}</ref> Selection acts as the main driver of reinforcement as it selects against hybrid genotypes that are of low-fitness, regardless if individual preferences have no effect on survival and reproduction.<ref name="K&S1999" /> Gene flow acts as the primary opposing force against reinforcement, as the exchange of genes between individuals leading to hybrids cause the genotypes to homogenize across the incipient species.<ref name="K&S1999"/> Butlin laid out four primary criteria for reinforcement to be detected in natural or laboratory populations:<ref name="Butlin1989"/>
*Gene flow between two taxa exists or can be established to have existed at some point.
*There is divergence of mating-associated traits between two taxa.
*Patterns of mating are modified, limiting the production of low fitness hybrids.
*Other selection pressures leading to divergence of the mate-recognition system have not occurred.

After speciation by reinforcement occurs, changes after complete reproductive isolation (and further isolation thereafter) are a form of reproductive character displacement.<ref name="Liou&Price">{{Citation |title=Speciation by reinforcement of premating isolation | author1=Lily W. Liou|author2= Trevor D. Price | journal=Evolution | year=1994 | volume=48 | issue=5 | pages=1451–1459 | doi= 10.1111/j.1558-5646.1994.tb02187.x| pmid=28568419 | s2cid=22630822 }}</ref> A common signature of reinforcement's occurrence in nature is that of reproductive character displacement; characteristics of a population diverge in sympatry but not allopatry.<ref name="Servedio2004"/><ref name="Barrientos2009"/> One difficulty in detection is that ecological character displacement can result in the same patterns.<ref name="MCSN2012">{{Citation |title=What do we need to know about speciation? | author=The Marie Curie SPECIATION Network | journal=Trends in Ecology & Evolution | year=2012 | volume=27 | issue=1 | pages=27–39| doi=10.1016/j.tree.2011.09.002 | pmid=21978464 | url=http://www.cell.com/trends/ecology-evolution/abstract/S0169-5347(11)00261-8 }}</ref> Further, gene flow can diminish the isolation found in sympatric populations.<ref name="MCSN2012"/> Two important factors in the outcome of the process rely on: 1) the specific mechanisms that causes pre-zygotic isolation, and 2) the number of alleles altered by mutations affecting mate choice.<ref>{{Citation |title=Can reinforcement complete speciation? | author1=Claudia Bank|author2=Joachim Hermission|author3=Mark Kirkpatrick | journal=Evolution | year=2012 | volume=66 | issue=1 | pages=229–239 | doi=10.1111/j.1558-5646.2011.01423.x | pmid=22220877 | s2cid=15602575 | url=https://semanticscholar.org/paper/b80a3665dd3acf0d50df8f5708be9a1e7e25c0e7 }}</ref> In instances of peripatric speciation, reinforcement is unlikely to complete speciation in the case that the peripherally isolated population comes into secondary contact with the main population.<ref name=S&M1997/> In sympatric speciation, selection against hybrids is required; therefore reinforcement can play a role, given the evolution of some form of fitness trade-offs.<ref name="Modes and Mechanisms of Speciation"/> In sympatry, patterns of strong mating discrimination are often observed—being attributed to reinforcement.<ref name="Noor1999"/> Reinforcement is thought to be the agent of gametic isolation.<ref>{{Citation |title=Reinforcement of Gametic Isolation in ''Drosophila'' |author=Daniel R. Matute |journal=PLOS Biology |year=2010 |volume=8 |issue=6 |pages= e1000341|doi=10.1371/journal.pbio.1000341 |pmid= 20351771|pmc=2843595 }}</ref>

=== Genetics ===
The underlying genetics of reinforcement can be understood by an ideal model of two haploid populations experiencing an increase in linkage disequilibrium. Here, selection rejects low fitness <math>Bc</math> or <math>bC</math> allele combinations while favoring combinations of <math>BC</math> alleles (in the first subpopulation) and <math>bc</math> alleles (in the second subpopulation). The third locus <math>A</math> or <math>a</math> (the assortive mating alleles) has an effect on mating pattern but is not under direct selection. If selection at <math>B</math> and <math>C</math> cause changes in the frequency of allele <math>A</math>, assortive mating is promoted, resulting in reinforcement. Both selection and assortive mating are necessary, that is, that matings of <math>A \times A</math> and <math>a \times a</math> are more common than matings of <math>a \times A</math> and <math>A \times a</math>.<ref name="Felsenstein1981">{{Citation |title=Skepticism Towards Santa Rosalia, or Why are There so Few Kinds of Animals? | author=Joseph Felsenstein | journal=Evolution | year=1981 | volume=35 | issue=1 | pages=124–138 | doi=10.2307/2407946 | pmid=28563447 | jstor=2407946 }}</ref> A restriction of migration between populations can further increase the chance of reinforcement, as it decreases the probability of the differing genotypes to exchange.<ref name="eLS2012"/> An alternative model exists to address the antagonism of recombination, as it can reduce the association between the alleles that involve fitness and the assortive mating alleles that do not.<ref name="eLS2012"/> Genetic models often differ in terms of the number of traits associated with loci;<ref name="Theory&Sp2001">{{Citation |title=Theory and speciation | author1=Michael Turelli |author2=Nicholas H. Barton |author3=Jerry A. Coyne| journal=Trends in Ecology & Evolution | year=2001 | volume=16 | issue=7 | pages=330–343 | doi= 10.1016/S0169-5347(01)02177-2| pmid=11403865 }}</ref> with some relying on one locus per trait<ref name="S&M1997"/><ref name="Servedio2000"/><ref name="Cain1999">{{Citation |title=Reinforcing selection is effective under a relatively broad set of conditions in a mosaic hybrid zone | author1=Michael L. Cain|author2=Viggo Andreasen|author3=Daniel J. Howard | journal=Evolution | year=1999 | volume=53 | issue=5 | pages=1343–1353 | doi=10.1111/j.1558-5646.1999.tb05399.x | pmid=28565558| s2cid=31107731 }}</ref> and others on polygenic traits.<ref name="Liou&Price"/><ref name="K&S1999"/><ref>{{Citation |title=Reinforcement and divergence under assortive mating | author=Mark Kirkpatrick | journal=Proceedings of the Royal Society B | year=2000 | volume=267 | issue= 1453| pages=1649–1655  | doi=10.1098/rspb.2000.1191 | pmid=11467428 | pmc=1690725 }}</ref>

=== Population structures ===
[[File:Reinforcement Parameter Space (annot).png|right|thumb|upright=1.2|A parameter space representing the conditions in which speciation by reinforcement can occur. Here, three outcomes can arise: 1) extinction of one of the initial populations; 2) the initial populations can hybridize; 3) the initial populations can speciate. The outcomes are determined by both initial divergence and level of fitness of the hybrids.<ref name="Liou&Price"/>]]
The structure and migration patterns of a population can affect the process of speciation by reinforcement. It has been shown to occur under an island model, harboring conditions with infrequent migrations occurring in one direction,<ref name="K&S1999"/> and in symmetric migration models where species migrate evenly back and forth between populations.<ref name="S&M1997">{{Citation |title=The effects of gene flow on reinforcement | author1=Maria R. Servedio|author2=Mark Kirkpatrick | journal=Evolution | year=1997 | volume=51 | issue=6 | pages=1764–1772 | doi=10.1111/j.1558-5646.1997.tb05100.x | pmid=28565111 | s2cid=12269299 }}</ref><ref name="Servedio2000">{{Citation |title=Reinforcement and the genetics of nonrandom mating | author=Maria R. Servedio | journal=Evolution | year=2000 | volume=54 | issue=1 | pages=21–29 | doi= 10.1111/j.0014-3820.2000.tb00003.x| pmid=10937179| s2cid=12563023 }}</ref> Reinforcement can also occur in single populations,<ref name="Theory&Sp2001"/><ref name="Liou&Price"/> mosaic hybrid zones,<ref name=Cain1999/> and in parapatric populations with narrow contact zones.<ref>{{Citation |title=Can gene flow prevent reinforcement?| author=Neil Sanderson | journal=Evolution | year=1989 | volume=43 | issue=6 | pages=1223–1235 | doi=10.2307/2409358 | pmid=28564502 | jstor=2409358 }}</ref> Population densities are an important factor in reinforcement, often in conjunction with extinction.<ref name="Liou&Price"/> It is possible that, when two species come into secondary contact, one population can become extinct—primarily due to low hybrid fitness accompanied by high population growth rates.<ref name="Liou&Price"/> Extinction is less likely if the hybrids are inviable instead of infertile, as fertile individuals can still survive long enough to reproduce.<ref name="Liou&Price"/>

=== Selection ===
Speciation by reinforcement relies directly on selection to favor an increase in pre-zygotic isolation,<ref name="Modes and Mechanisms of Speciation"/> and the nature of selection's role in reinforcement has been widely discussed, with models applying varying approaches.<ref name="Theory&Sp2001"/> Selection acting on hybrids can occur in several different ways. All hybrids produced may be equality low-fitness,<ref name="Liou&Price"/> conferring a broad disadvantage. In other cases, selection may favor multiple and varying phenotypes<ref name="S&M1997"/> such as in the case of a mosaic hybrid zone.<ref name="Cain1999"/> Natural selection can act on specific alleles both directly or indirectly.<ref name="Theory&Sp2001"/><ref name="K&S1999"/><ref>{{Citation |title=Beyond reinforcement: The evolution of premating isolation by direct selection on preferences and postmating, prezygotic incompatibilities | author=Maria R. Servedio | journal=Evolution | year=2001 | volume=55 | issue=10 | pages=1909–1920 | doi= 10.1111/j.0014-3820.2001.tb01309.x| pmid=11761053| s2cid=25296147 }}</ref> Under direct selection, the alleles causing increased premating isolation diverge because they directly affect viability; the evolution of premating isolation in this case can mimic reinforcement but is arguably a different process. In cases where an allele is indirectly selected, its frequency increases due to linkage disequilibrium; this is the case under more standard concepts of reinforcement.<ref name="eLS2012"/> The condition of the hybrids under selection can play a role in post-zygotic isolation, as hybrid inviability and sterility prohibit gene flow between populations.<ref name="Noor1999"/> Selection against the hybrids can even be driven by any failure to obtain a mate, as it is effectively indistinguishable from sterility—each circumstance results in no offspring.<ref name="Noor1999"/>

=== Mating and mate preference ===
Some initial divergence in mate preference must be present for reinforcement to occur.<ref name="Noor1999"/><ref name="Liou&Price"/><ref>{{Citation |title=Speciation by reinforcement: a model derived from studies of Drosophila | author1=J. K. Kelly|author2= Mohamed A. F. Noor | journal=Genetics | year=1996 | volume=143 | issue=3 | pages=1485–1497 | pmid=8807317| pmc=1207414|doi=10.1093/genetics/143.3.1485 }}</ref> Any traits that promote isolation may be subjected to reinforcement such as mating signals (''e.g.'' courtship display), signal responses, the location of breeding grounds, the timing of mating (''i.e.'' allochrony), or even egg receptivity.<ref name="eLS2012"/> Individuals may also discriminate against mates that differ in various traits such as mating call or morphology.<ref name="Hoskin2005">{{Citation |title=Reinforcement drives rapid allopatric speciation | author1=Conrad J. Hoskin|author2=Megan Higgie|author3=Keith R. McDonald|author4=Craig Moritz | journal=Nature | year=2005 | volume=437 | issue= 7063| pages=1353–1356 | doi=10.1038/nature04004 | pmid=16251964| bibcode=2005Natur.437.1353H | s2cid=4417281 }}</ref> Many of these examples are described below.

== Evidence ==
Evidence for speciation by reinforcement has been gathered since the 1990s, and along with data from comparative studies and laboratory experiments, has overcome many of the objections to the theory.<ref name="Servedio&Noor"/><ref name="Barrientos2009"/><ref name="Speciation"/>{{rp|354}} Reinforcement can be shown to be occurring (or to have occurred in the past) by measuring the strength of pre-zygotic isolation in a sympatric population in comparison to an allopatric population of the same species.<ref name="Speciation"/>{{rp|357}} Comparative studies of this allow for determining large-scale patterns in nature across various taxa.<ref name="Speciation"/>{{rp|362}} Mating patterns in hybrid zones can also be used to detect reinforcement.<ref name="Howard1993"/> Reproductive character displacement is seen as a result of reinforcement,<ref name="Noor1999"/> so many of the cases in nature express this pattern in sympatry. Reinforcement's prevalence is unknown,<ref name="Servedio&Noor"/> but the patterns of reproductive character displacement are found across numerous taxa (vertebrates, invertebrates, plants, and fungi), and is considered to be a common occurrence in nature.<ref name="Howard1993"/> Plants are thought to provide suitable conditions for reinforcement to occur.<ref name="Barrientos2009" /> This is due to a number of factors such as the unpredictability of pollination, pollen vectors, hybridization, and hybrid zones.<ref name="Barrientos2009" /> The study of plants experiencing speciation by reinforcement has largely been overlooked by researchers;<ref name="Speciation" />{{rp|364}} however, there is evidence of its occurrence in them.<ref name="Hopkins2013">{{Citation|author=Robin Hopkins|title=Reinforcement in plants|journal=New Phytologist|volume=197|issue=4|pages=1095–1103|year=2013|doi=10.1111/nph.12119|pmid=23495388}}</ref> It has been argued that reinforcement is extremely common in birds and has been documented in a wide range of bird species.<ref>{{Citation|author1=Emily J. Husdon |author2=Trevor D. Price|title=Pervasive Reinforcement and the Role of Sexual Selection in Biological Speciation|journal=Journal of Heredity|volume=105|pages=821–833|year=2014|doi=10.1093/jhered/esu041|pmid=25149257|doi-access=free}}</ref> Studies of reinforcement in nature often prove difficult, as alternative explanations for the detected patterns can be asserted.<ref name="Speciation"/>{{rp|358}} Nevertheless, empirical evidence exists for reinforcement occurring across various taxa<ref name="Noor1999"/> and its role in precipitating speciation is conclusive.<ref name="eLS2012"/>

{| class="wikitable sortable" style="margin-left: auto; margin-right: auto; border: none;"
|+ Table of examples of reinforcement occurring in nature
|-
!Organism group
!''Genus or species''
!Description
|-
| rowspan="6" |Amphibians
|''Litoria''
|The two frog species ''Litoria ewingi'' and ''L. verreauxii'' live in southern Australia with their two ranges overlapping. The species have very similar calls in allopatry, but express clinal variation in sympatry, with notable distinctness in calls that generate female preference discrimination.<ref name="eLS2012" /> The zone of overlap sometimes forms hybrids and is thought to originate by secondary contact of once fully allopatric populations.<ref name="eLS2012" />

The rainforests of northeast Queensland, Australia were separated into north and south refugia by climate fluctuations of the Pliocene and Pleistocene.<ref>{{Citation|author1=C. J. Schneider|author2=M. Cunningham|author3=C. Moritz|title=Comparative phylogeography and the history of endemic vertebrates in the Wet Tropics rainforests of Australia|journal=Molecular Ecology|volume=7|issue=4|pages=487–498|year=1998|doi=10.1046/j.1365-294x.1998.00334.x|s2cid=84601584}}</ref> About 6500 years ago, the rainforests reconnected, bringing the diverged, incipient populations of ''Litoria genimaculata'' into secondary contact. The species contact zones exhibit, "strong post-zygotic selection against hybrids" and enhanced isolation from differences in mating call.<ref name="Hoskin2005">{{Citation |title=Reinforcement drives rapid allopatric speciation | author1=Conrad J. Hoskin|author2=Megan Higgie|author3=Keith R. McDonald|author4=Craig Moritz | journal=Nature | year=2005 | volume=437 | issue= 7063| pages=1353–1356 | doi=10.1038/nature04004 | pmid=16251964| bibcode=2005Natur.437.1353H | s2cid=4417281 }}</ref>
|-
|''Gastrophryne''
|Allopatric populations of ''Gastrophryne olivacea'' and ''G. carolinensis'' have recently come into secondary contact due to forest clearing.<ref name="Butlin1987">{{Citation|author=Roger Butlin|title=Speciation by Reinforcement|journal=Trends in Ecology & Evolution|volume=2|issue=1|pages=8–13|year=1987|doi=10.1016/0169-5347(87)90193-5|pmid=21227808}}</ref> The calls that the males make to attract females differ significantly in frequency and duration in the area where the two species overlap, despite them having similar calls where they do not.<ref name="Speciation" />{{rp|359}} Further, the hybrids that form in sympatry have calls that are intermediate between the two.<ref name="Butlin1987" />
|-
|''Hyla''
|Patterns of reproductive character displacement involving acoustic displays have been found in ''Hyla cinerea'' and ''H. gratiosa'', with greater female preference for conspecific males in areas of sympatry.<ref>{{Cite journal|author1=Gerlinde Höbel |author2= H. Carl Gerhardt|title=Reproductive character displacement in the acoustic communication system of green tree frogs (''Hyla cinerea'')|journal=Evolution|volume=57|issue=4|pages=894–904|year=2003|doi=10.1111/j.0014-3820.2003.tb00300.x |pmid=12778558}}</ref>
|-
|''Lithobates''
|Three species of true frogs (''Lithobates sphenocephalus'', ''L. berlandieri'', and ''L. blairi'') are temporally isolated in that their breeding seasons are spaced out in areas where they live in sympatry, but not where they live in allopatry.<ref name="Hillis1981">{{Citation|author=David M. Hillis|title=Premating Isolating Mechanisms Among Three Species of the ''Rana pipiens'' Complex in Texas and Southern Oklahoma|journal=Copeia|volume=1981|issue=2|pages=312–319|year=1981|doi=10.2307/1444220|jstor=1444220}}</ref> Selection against interspecific mating due to low hybrid fitness and low hybrid fertility has reinforced the observed character displacement of breeding times.<ref name="Hillis1981" />
|-
|''Anaxyrus''
|An alternative to detecting reproductive character displacement in populations that overlap in sympatry is measuring rates of hybridization in contact zones.<ref name="Butlin1987" /> The frog species ''Anaxyrus americanus'' and ''A. woodhousii'' have shown a decrease in hybridization from 9%–0% over approximately 30 years.<ref>{{Citation|author=J. Michael Jones|title=Effects of thirty years hybridization on the toads ''Bufo americanus'' and ''Bufo woodhousii fowleri'' at Bloomington, Indian|journal=Evolution|volume=27|issue=3|pages=435–448|year=1973|doi=10.1111/j.1558-5646.1973.tb00690.x|pmid=28564913|s2cid=39042605}}</ref><ref name="Butlin1987" /> 
|-
|''Spea''
|Sympatric spadefoot toads ''Spea multiplicata'' and ''S. bombifrons'' have hybridized with decreasing frequency over a 27-year period (about 13 generations).<ref>{{Citation|author=Karin S. Pfennig|title=A test of alternative hypotheses for the evolution of reproductive isolation between spadefoot toads: Support for the reinforcement hypothesis|journal=Evolution|volume=57|issue=12|pages=2842–2851|year=2003|doi=10.1554/03-228|pmid=14761062|s2cid=198152266}}</ref>
|-
| rowspan="3" |Birds
|''Ficedula''
|The ''Ficedula'' flycatchers exhibit a pattern that suggests premating isolation is being reinforced by sexual selection.<ref name="Sætre et al 1997">{{Citation|author1=Glenn-Peter Sætre|author2=T. Moum|author3=S. Bures|author4=M. Kral|author5=M. Adamjan|author6=J. Moreno|title=A sexually selected character displacement in flycatchers reinforces premating isolation|journal=Nature|volume=387|issue=6633|pages=589–592|year=1997|bibcode=1997Natur.387..589S|doi=10.1038/42451|s2cid=4363912}}</ref> The pied flycatcher (''F. hypoleuca'') has brown females, brown males, and black-and-white males. The related collard flycatcher (''F. albicollis'') has brown females and only black-and-white males. The two species exist in separate populations that overlap in a zone of sympatry.<ref name="Sætre et al 1997" /> In the range of overlap, only brown males of ''F. hypoleuca'' exist and are thought to have evolved the brown plumage to prevent hybridization.<ref>{{Citation|author1=R. V. Alatalo|author2=L. Gustafsson|author3=A. Lundberg|title=Hybridization and breeding success of collared and pied flycatchers on the island of Gotland|journal=Auk|volume=99|pages=285–291|year=1982}}</ref> Mating choice tests of the species find that females of both species choose conspecific males in sympatry, but heterospecific males in allopatry.<ref name="Sætre et al 1997" /> The patterns could suggest mimicry, driven by interspecific competition;<ref name="Speciation" />{{rp|361}} however, song divergence has been detected that shows a similar pattern to the mating preferences.<ref>{{Citation|author=Lars Wallin|title=Divergent character displacement in the song of two allospecies: the pied flycatcher ''Ficedula hypoleuca'' and the collared flycatcher ''Ficedula albicollis''|journal=Ibis|volume=128|issue=2|pages=251–259|year=1986|doi=10.1111/j.1474-919X.1986.tb02672.x}}</ref>
|-
|''Geospiza''
|''Geospiza fulginosa'' and ''G. difficilis'' males on the Galápagos Islands show a noted preference for conspecific females where they meet in sympatry, but not in allopatry.<ref>{{Citation|author1=L. M. Ratcliffe|author2=Peter R. Grant|title=Species recognition in Darwin's finches (''Geospiza'', Gould). II. Geographic variation in mate preference|journal=Animal Behaviour|volume=31|issue=4|pages=1154–1165|year=1983|doi=10.1016/S0003-3472(83)80022-0|s2cid=53178974}}</ref>
|-
|''Aechmophorus''
|The dark and light subspecies of the western grebe (''Aechmophorus occidentalis'') show enhanced pre-zygotic isolation.<ref name="Ratti, J. T. 1979">{{Citation|title=Reproductive Separation and Isolating Mechanisms between Sympatric Dark- and Light- Phase Western Grebes|author=John T. Ratti|journal=The Auk|year=1979|volume=93|issue=3|pages=573–586|doi=}}</ref>
|-
|Crustaceans
|''Faxonius''
|Reproductive character displacement in body size was detected in sympatric populations of '' Faxonius rusticus'' and ''F. sanbornii''.<ref>{{Citation|author=Mark J. Butler IV|title=Evaluation of Possible Reproductively Mediated Character Displacement in the Crayfishes, ''Orconectes rusticus'' and ''O. sanbornii''|journal=Ohio Journal of Science|volume=88|issue=3|pages=87–91|year=1988}}</ref>
|-
| rowspan="2" |Echinoderms
|''Arbacia''
|An example of gametic isolation involves the allopatric sea urchins (''Arbacia'') have minimal bindin differences (bindin is a protein involved in the process of sea urchin fertilization, used for species-specific recognition of the egg by the sperm) and have insufficient barriers to fertilization.<ref name="Speciation" />{{rp|243}} Comparison with the sympatric species ''Echinometra'' and ''Strongylocentrotus'' of the Indo-Pacific finds that they have significant differences in bindin proteins for fertilization and marked fertilization barriers.<ref>{{Citation|author1=Edward C. Metz|author2=Gerardo Gómez-Gutiérrez|author3=Victor D. Vacquier|title=Mitochondrial DNA and Bindin Gene Sequence Evolution Among Allopatric Species of the Sea Urchin Genus ''Arbacia''|journal=Molecular Biology and Evolution|volume=15|issue=2|pages=185–195|year=1998|doi=10.1093/oxfordjournals.molbev.a025914|pmid=9491615|doi-access=free}}</ref>
|-
|''Echinometra''
|Laboratory matings of closely related sea urchin species ''Echinometra oblonga'' and ''E. sp. C'' (the species is unnamed, dubbed ''C'') produce fertile and viable hybrids, but are unable to fertilize eggs of the parent species due to divergence of the alleles that code for bindin proteins: an example of post-zygotic isolation.<ref name="Speciation" />{{rp|343–344}} Populations in sympatry manifest this difference in bindin protein versus those in allopatry.<ref name="Speciation" />{{rp|343–344}} Selection actively acts against the formation of hybrids in both nature (as no documented cases of hybrids have been found) and in the laboratory.<ref name="GeyerPalumbi2003">{{Citation|author1=Laura B. Geyer|author2=Stephen R. Palumbi|title=Reproductive character displacement and the genetics of gamete recognition in tropical sea urchins|journal=Evolution|volume=57|issue=5|pages=1049–1060|year=2003|doi=10.1111/j.0014-3820.2003.tb00315.x |pmid=12836822}}</ref> Here, the evolution of female egg receptors is thought to pressure bindin evolution in a selective runaway process.<ref name="GeyerPalumbi2003" /> This example of reproductive character displacement is highly suggestive of being the result of—and has been cited as strong evidence for—reinforcement.<ref name="GeyerPalumbi2003" /><ref name="Speciation" />{{rp|343–344}}
|-
| rowspan="2" |Fish
|''Gasterosteus''
|In British Columbia, benthic and limnetic morphs of ''Gasterosteus aculeatus'' exist together in sympatry in some lakes, while containing only one morph in other lakes.<ref name="RundleSchluter1998">{{Citation|author=Howard D. Rundle|title=Reinforcement of stickleback mate preferences: Sympatry breeds contempt|journal=Dolph Schluter|volume=52|issue=1|pages=200–208|year=1998|doi=10.1111/j.1558-5646.1998.tb05153.x|pmid=28568163|hdl=2429/6366|s2cid=40648544|hdl-access=free}}</ref> Female benthic morphs in sympatric populations actively discriminate against limnetic males, resulting in low rates of crossing (some gene flow has occurred between the morphs) and low fitness hybrids.<ref name="Speciation" />{{rp|360}} 
|-
|''Etheostoma''
|Both selection against hybrids and reproductive character displacement in egg fertilization is observed in ''Etheostoma lepidum'' and ''E. spectabile''.<ref>{{Citation|author=C. Hubbs|title=Duration of sperm function in the percid fishes ''Etheostoma lepidum'' and ''Etheostoma spectabile'', associated with sympatry of the parent populations|journal=Copeia|volume=1960|issue=1|pages=1–8|year=1960|doi=10.2307/1439836|jstor=1439836}}</ref>
|-
|Fungi
|''Neurospora''
|A strong case of reinforcement occurring in fungi comes from studies of ''Neurospora''.<ref name="Dettman2003">{{Citation|author1=Jeremy R. Dettman|author2=David J. Jacobson|author3=Elizabeth Turner|author4=Anne Pringle|author5=John W. Taylor|title=Reproductive isolation and phylogenetic divergence in ''Neurospora'': Comparing methods of species recognition in a model eukaryote|journal=Evolution|volume=57|issue=12|pages=2721–2741|year=2003|doi=10.1554/03-074|pmid=14761052|s2cid=198153854}}</ref> In crosses between different species in the genera, sympatric pairs show low reproductive success, significantly lower than allopatric pairs.<ref name="Dettman2003" /> This pattern is observed across small and large geographic scales, with distance correlating with reproductive success.<ref name="Dettman2003" /> Further evidence of reinforcement in the species was the low fitness detected in the hybrids create from crosses, and that no hybrids have been found in nature, despite close proximity.<ref name="Dettman2003" />
|-
|Mammals
|''Peromyscus''
|The white-footed mouse (''Peromyscus leucopus'') and cotton mouse (''P. gossypinus'') exhibit reproductive character displacement in mating preferences, with heterospecific matings taking place between the species.<ref>{{Citation|author=H. MacCarley|title=Ethological isolation in the cenospecies ''Peromyscus leucopus''|journal=Evolution|volume=18|issue=2|pages=331–342|year=1964|doi=10.1111/j.1558-5646.1964.tb01605.x|s2cid=84959325}}</ref>
|-
| rowspan="8" |Insects
|''Aedes''
|Ethological isolation has been observed between some mosquito species in the Southeast Asian ''Aedes albopictus'' group, suggesting—from laboratory experiments of mating trials—that selection against hybrids is occurring, in the presence of reproductive character displacement.<ref>{{Citation|author=Denson Kelly McLain |author2=Karamjit S. Rai|title=Reinforcement for ethological isolation in the southeast asian ''Aedes albopictus'' subgroup (Diptera: Culicidae)|journal=Evolution|volume=40|issue=60|pages=1346–1350|year=1986|doi=10.1111/j.1558-5646.1986.tb05759.x|pmid=28563509|s2cid=26849954}}</ref>
|-
|''Timema''
|Female mate discrimination is increased with intermediate migration rates between allopatric populations of ''Timema cristinae'' compared to high rates of migration (where gene flow impedes selection) or low rates (where selection is not strong enough).<ref>{{Citation|author=Patrik Nosil|author2=Bernard J. Crespi|author3=Cristina P. Sandoval|title=Reproductive isolation driven by the combined effects of ecological adaptation and reinforcement|journal=Proceedings of the Royal Society B|volume=270|issue=1527|pages=1911–1918|year=2003|doi=10.1098/rspb.2003.2457|pmc=1691465|pmid=14561304}}</ref><ref>{{Citation|author=Patrik Nosil|author2=Bernard J. Crespi|author3=Regine Gries|author4=Gerhard Gries|title=Natural selection and divergence in mate preference during speciation|journal=Genetica|volume=129|issue=3|pages=309–327|year=2007|doi=10.1007/s10709-006-0013-6|pmid=16900317|s2cid=10808041}}</ref>
|-
|''Magicicada''
|Where the ranges of the cicada species ''Magicicada tredecim'' and ''M. neotredecim'' overlap (where they are sympatric), the pitch of ''M. neotredecim'' male calling songs is roughly 1.7&nbsp;kHz compared to 1.1&nbsp;kHz for those of ''M. tredecim'', with corresponding female song pitch preference differences.<ref name="Cooley et al 2001">{{Citation|author=John R. Cooley|author2=Chris Simon|author3=David C. Marshall|author4=Karen Slon|author5=Christopher Ehrhardt|title=Allochronic speciation, secondary contact, and reproductive character displacement in periodical cicadas (Hemiptera: ''Magicicada'' spp.): genetic, morphological, and behavioural evidence|journal=Molecular Ecology|volume=10|issue=3|pages=661–671|year=2001|doi=10.1046/j.1365-294x.2001.01210.x|pmid=11298977|s2cid=24760583}}</ref> In allopatric ''M. neotredecim'' populations, the mating call pitch is 1.3–1.5&nbsp;kHz.<ref name="Cooley et al 2001" /> The biogeography of the cicadas suggests that ''M. neotredecim'' originated after the retreat of the last glacial advance in North America.<ref name="Cooley et al 2001" />
|-
|''Laupala''
|The song differences of ''Laupala'' crickets on the Hawaiian Islands appear to exhibit patterns consistent with character displacement in sympatric populations.<ref name="Butlin1989"/> A similar pattern exists with ''Allonemobius fasciatus'' and ''A. socius'', species of ground crickets in eastern North America.<ref name="Benedix&Howard1991">{{Citation|author=J. H. Benedix Jr. |author2=Daniel J. Howard|title=Calling song displacement in a zone of overlap and hybridization|journal=Evolution|volume=45|issue=8|pages=1751–1759|year=1991|doi=10.1111/j.1558-5646.1991.tb02685.x|pmid=28563959|s2cid=46400422}}</ref>
|-
|''Allonemobius''
|A similar pattern as ''Laupala'' crickets exists with ''Allonemobius fasciatus'' and ''A. socius'', species of ground crickets in eastern North America.<ref name="Benedix&Howard1991"/>
|-
|''Calopteryx''
|Males in sympatric populations of the damselflies ''Calopteryx maculata'' and ''C. aequabilis'' are able to discriminate between females of different species better than those in allopatric populations; with females of ''C. aequabilis'' in sympatric populations exhibiting lighter wing colors compared to allopatric females—an illustration of reproductive character displacement.<ref>{{Citation|author=Jonathan K. Waage|title=Reproductive Isolation and the Potential for Character Displacement in the Damselflies, ''Calopteryx Maculata'' and ''C. Aequabilis'' (Odonata: Calopterygidae)|journal=Systematic Biology|volume=24|issue=1|pages=24–36|year=1975|doi=10.1093/sysbio/24.1.24}}</ref><ref>{{Citation|author=Jonathan K. Waage|title=Reproductive character displacement in ''Calopteryx'' (Odonata: Calopterygidae|journal=Evolution|volume=33|issue=1|pages=104–116|year=1979|doi=10.1111/j.1558-5646.1979.tb04667.x|pmid=28568062|s2cid=43039210}}</ref>
|-
|''Agrodiaetus''
|Fifteen species of sympatrically distributed ''Agrodiaetus'' butterflies with pronounced differences in wing color pattern likely arose as a result of speciation by reinforcement.<ref>{{Citation|author=C. D. Jiggins|title=Reinforced butterfly speciation|journal=Heredity|volume=96|issue=2|pages=107–108|year=2006|doi=10.1038/sj.hdy.6800754|pmid=16222327|s2cid=24389006}}</ref> Phylogenetic patterns indicate the differences arose in allopatry and were reinforced when the distributions came into secondary contact.<ref>{{Citation|author=Vladimir A. Lukhtanov|author2=Nikolai P. Kandul|author3=Joshua B Plotkin|author4=Alexander V. Dantchenko|author5=David Haig|author6=Naomi E. Pierce|title=Reinforcement of prezygotic isolation and karyotype evolution in ''Agrodiaetus'' butterflies|journal=Nature|volume=436|issue=7049|pages=385–389|year=2005|bibcode=2005Natur.436..385L|doi=10.1038/nature03704|pmid=16034417|s2cid=4431492}}</ref>
|-
|''Drosophila''
|''Drosophila'' is one of the most studied species in speciation research.<ref>{{Citation|author=James Mallet |title=What does Drosophila genetics tell us about speciation?|journal=Trends in Ecology & Evolution|volume=21|issue=7|pages=386–393|year=2006|doi=10.1016/j.tree.2006.05.004|pmid=16765478}}</ref> Dobzhansky and Koller were the first to study isolation between ''Drosophila'' species.<ref name="Speciation" />{{rp|358}} Since then, other studies of natural populations such as the ''D. paulistorum'' races exhibiting stronger isolation in sympatry versus allopatry,<ref>{{Citation|author=Lee Ehrman|title=Direct Observation of Sexual Isolation between Allopatric and between Sympatric Strains of the Different ''Drosophila paulistorum'' Races|journal=Evolution|volume=19|issue=4|pages=459–464|year=1965|doi=10.2307/2406243|jstor=2406243}}</ref> or the enhanced isolation found in sympatric populations of ''D. mojavensis'' and ''D. arizonae'' in southwest America.<ref>{{Citation|author=Marvin Wasserman|author2=H. Roberta Koepfer|title=Character displacement for sexual isolation between ''Drsophila mojavensis'' and ''Drosophila arizonensis''|journal=Evolution|volume=31|issue=4|pages=812–823|year=1977|doi=10.1111/j.1558-5646.1977.tb01073.x|pmid=28563708|s2cid=36693544}}</ref> Rare, sterile hybrids form between ''D. pseudoobscura'' and ''D. persimilis'', with sympatric ''D. pseudoobscura'' females discriminating against ''D. persimilis'' males; more so than allopatric populations.<ref name="Noor1995">{{Citation|author=Mohamed A. F. Noor|title=Speciation driven by natural-selection in ''Drosophila''|journal=Nature|volume=375|issue=6533|pages=674–675|year=1995|bibcode=1995Natur.375..674N|doi=10.1038/375674a0|pmid=7791899|s2cid=4252448}}</ref> Other ''Drosophila'' research on reinforcement has been from laboratory experiments and is discussed below. On the east coast of Australia, ''D. serrata'' shares a zone of sympatric overlap with the closely relates species ''D. birchii''.<ref>{{Citation|author=Megan Higgie |author2= Mark W. Blows|title=Are Traits That Experience Reinforcement Also Under Sexual Selection?|url=http://espace.library.uq.edu.au/view/UQ:129790/UQ129790_OA.pdf|journal=The American Naturalist|volume=170|issue=3|pages=409–420|year=2007|doi=10.1086/519401|pmid=17879191}}</ref> The species exhibits reproductive character displacement, with sexual selection operating on the hydrocarbons of the flies cuticle.<ref>{{Citation|author=Megan Higgie|author2=Steve Chenoweth|author3=Mark W. Blows|title=Natural Selection and the Reinforcement of Mate Recognition|url=https://espace.library.uq.edu.au/view/UQ:10714/Higgie_et_al_200.pdf|journal=Science|volume=290|issue=5491|pages=519–521|year=2000|bibcode=2000Sci...290..519H|doi=10.1126/science.290.5491.519|pmid=11039933}}</ref> Reinforcement appears to be driving their speciation in nature, supported by simulated experimental laboratory populations.<ref>{{Citation|author=Megan Higgie |author2=Mark W. Blows|title=The Evolution of Reproductive Character Displacement Conflicts with how Sexual Selection Operates within a Species|journal=Evolution|volume=62|issue=5|pages=1192–1203|year=2008|doi=10.1111/j.1558-5646.2008.00357.x|pmid=18298640|s2cid=333466}}</ref><ref name="HoskinHiggie2010"/>
|-
| rowspan="5" |Molluscs
|''Partula''
|''Partula suturalis'' is polymorphic for shell chirality in that it has two forms: sinistral (left-handed) and dextral (right-handed) shells, unlike other monomorphic species on the island of Mo'orea which have only one form (with the exception of ''P. otaheitana'').<ref name="Johnson1982">{{Citation|author=Michael S. Johnson|title=Polymorphism for direction of coil in ''Partula suturalis'': Behavioral isolation and positive frequency dependent selection|journal=Heredity|volume=49|issue=2|pages=145–151|year=1982|doi=10.1038/hdy.1982.80|doi-access=free}}</ref> This polymorphic trait has a direct effect on mate choice and mating behavior; as shown in laboratory mating tests that opposite-coil pairs mate much less often.<ref name="Johnson1982" /> In areas where ''P. suturalis'' lives sympatrically with other sinistral and dextral ''Partula'' species, the opposite ''P. suturalis'' morph is typically present.<ref name="Butlin1987" /> Butlin succinctly describes one example of this unique pattern:<blockquote>''P. suturalis'' is sympatric with the dextral ''P. aurantia'' and sinistral ''P. olympia'', whose ranges abut but do not overlap; ''P. suturalis'' is sinestral in the range of ''P. aurantia'' and dextral in the range of ''P. olympia'' and does not normally hybridize with either species. However, where their ranges meet there is a sharp transition in the coil of ''P. suturalis'' and in this transition zone it hybridizes with both ''P. aurantia'' and ''P. olympia''.<ref name="Butlin1987" /></blockquote>

The reversal in chirality to sinistrality must have evolved as an isolating mechanism,<ref>{{Citation|author=J. Murray |author2= B. Clarke|title=The genus ''Partula'' on Moorea: speciation in progress|journal=Proceedings of the Royal Society B|volume=211|issue=1182|pages=83–117|year=1980|bibcode=1980RSPSB.211...83M|doi=10.1098/rspb.1980.0159|s2cid=85343279}}</ref> with patterns of reproductive character displacement suggesting speciation by reinforcement.<ref name="Johnson1982"/>
|-
|''Satsuma''
|''Satsuma largillierti'' lives on the western half of Okinawa Island while ''S. eucosmia'' lives on the eastern half. Both snail populations overlap in sympatry along the middle of the island, where the penis length of the species differs significantly in sympatry (a case of reproductive character displacement)<ref>{{Citation|author=Carl T. Bergstrom |author2= Lee Alan Dugatkin|title=Evolution|date=2016|pages=508–509|edition=2nd|publisher=W. W. Norton & Company|isbn=9780393937930}}</ref>), but not in allopatry.<ref>{{Citation|author=Yuichi Kameda|author2=Atsushi Kawakita|author3=Makoto Kato|title=Reproductive Character Displacement in Genital Morphology in ''Satsuma'' Land Snails|journal=The American Naturalist|volume=173|issue=5|pages=689–697|year=2009|doi=10.1086/597607|pmid=19298185}}</ref>
|-
|''Lymnaea''
|A similar pattern as in ''Satsuma'' has been found with ''Lymnaea peregra'' and ''L. ovata'' snails in the Swiss lake Seealpsee; with mating signal acting as the sympatrically displaced trait.<ref>{{Citation|author=Esther B. Wullschleger|author2= Jürgen Wiehn|author3=Jukka Jokela|title=Reproductive character displacement between the closely related freshwater snails ''Lymnaea peregra'' and ''L. ovata''|journal=Evolutionary Ecology Research|volume=4|pages=247–257|year=2002}}</ref>
|-
|''Haliotis''
|The abalone genus ''Haliotis'' has 19 species that occur in sympatry and one that occurs in allopatry. Of the sympatric species, they all contain sperm lysin that drives gamete isolation, but the allopatric species does not.<ref>{{Citation|author=Y. H. Lee|author2=T. Ota|author3=V. D. Vacquier|title=Positive selection is a general phenomenon in the evolution of abalone sperm lysin|journal=Molecular Biology and Evolution|volume=12|issue=2|pages=231–238|year=1995|doi=10.1093/oxfordjournals.molbev.a040200|pmid=7700151|doi-access=free}}</ref><ref name="Speciation" />{{rp|343}}
|-
|''Mytilus''
|A pattern of sperm lysin differentiation (like that found in ''Haliotis)''  is documented in the mussel species ''Mytilus galloprovincalis'' and ''M. trossulus'' and has likely occurred within the last 200 years due to human-mediated distribution by ships.<ref name="Speciation" />{{rp|343}}
|-
| rowspan="8" |Plants
|''Phlox''
|In the Texas wildflower ''Phlox drummondii'', cis-regulatory mutations of genes that code for anthocyanin pigmentation have caused genetic divergence of two populations.<ref name="HopkinsRausher2011">{{Citation|author=Robin Hopkins |author2= Mark D. Rausher|title=Identification of two genes causing reinforcement in the Texas wildflower ''Phlox drummondii''|journal=Nature|volume=469|issue=7330|pages=411–414|year=2011|bibcode=2011Natur.469..411H|doi=10.1038/nature09641|pmid=21217687|s2cid=205223257}}</ref> Hybrids (between ''P. drummondii'' and ''P. cuspidata'') with maladaptive, intermediate characteristics are under-pollinated; increasing reproductive isolation through reinforcement.<ref name="HopkinsRausher2011" /> The maintenance of the ancestral flower color in the allopatric population is favored weakly by selection, where the derived color in the sympatric population is being driven by strong selection.<ref>{{Citation|author=Rob Hopkins|author2= Rafael F. Guerrero|author3=Mark D. Rausher|author4=Mark Kirkpartrick|title=Strong Reinforcing Selection in a Texas Wildflower|journal=Current Biology|volume=24|issue=17|pages=1995–1999|year=2014|doi=10.1016/j.cub.2014.07.027|pmid=25155503|doi-access=free}}</ref> 

Similarly, in ''P. pilosa'' and ''P. glaberrima'', character displacement of petal color has been driven by selection, aided by pollen discrimination.<ref>{{Citation|author=Donald A. Levin |author2= Harold W. Kerster|title=Natural selection for reproductive isolation in ''Phlox''|journal=Evolution|volume=21|issue=4|pages=679–687|year=1967|doi=10.1111/j.1558-5646.1967.tb03425.x|pmid=28563087|s2cid=13457880}}</ref> 
|-
|''Solanum''
|Displacement in flower size has also been observed in the nightshade species ''Solanum grayi'' and ''S. lymholtzianum'' in sympatry as well as ''S. rostratum'' and ''S. citrullifolium''.<ref>{{Citation|author=Michael D. Whalen|title=Reproductive Character Displacement and Floral Diversity in Solanum Section ''Androceras''|journal=Systematic Biology|volume=3|issue=1|pages=77–86|year=1978|doi=10.2307/2418533|jstor=2418533}}</ref>
|-
|''Pinus''
|The bishop pine (''Pinus muricata'') is divided into two populations distinguished by monoterpene, stomata, and alloenzyme differences; flowering time; and needle color: blue foliage in the northern population and green foliage in the southern populations in California.<ref>{{Citation|author=Constance I. Millar|title=A steep cline in ''Pinus muricata''|journal=Evolution|volume=37|issue=2|pages=311–319|year=1983|doi=10.1111/j.1558-5646.1983.tb05541.x|pmid=28568365|s2cid=34080334}}</ref> A small region exists where the species meet in a cline—sustained by selection due to a flowering time divergence, thought to represent reinforcement taking place.<ref name="Butlin1987" />
|-
|''Gilia'' and ''Gossypium''
|A unique case of post-zygotic instead of pre-zygotic isolation has been observed in both ''Gilia''<ref>{{Citation|author=Verne Grant|title=The selective origin of incompatibility barriers in the plant genus ''Gilia''|journal=The American Naturalist|volume=100|issue=911|pages=99–118|year=1966|doi=10.1086/282404}}</ref> and ''Gossypium'' suggesting that in plants, post-zygotic isolation's role in reinforcement may play a larger role.<ref name="Speciation" />{{rp|361}}
|-
|''Juncus''
|Sympatric populations of ''Juncus effusus'' exhibits genetic differentiation of plants that flower at different times preventing hybridization.<ref>{{Citation|author=Stefan G Michalski |author2= Walter Durka|title=Separation in flowering time contributes to the maintenance of sympatric cryptic plant lineages|url=|journal=Ecology and Evolution|volume=5|issue=11|pages=2172–2184|year=2015|doi=10.1002/ece3.1481|pmc=4461419|pmid=26078854}}</ref> Allochrony may play a role.<ref name="Taylor&Frieson2017">{{Citation|author=Rebecca S. Taylor |author2= Vicki L. Friesen|title=The role of allochrony in speciation|url=https://onlinelibrary.wiley.com/doi/epdf/10.1111/mec.14126|journal=Molecular Ecology|volume=26|issue=13|pages=3330–3342|year=2017|doi=10.1111/mec.14126|pmid=28370658|s2cid=46852358}}</ref>
|-
|''Agrostis''
|The grass species ''Agrostis tenuis'' grows on soil contaminated with high levels of copper leeched from an unused mine. Adjacent is the non-contaminated soil. The populations are evolving reproductive isolation due to differences in flowering time (allochrony) with isolation reinforced by unfit hybrids.<ref name="McNeillyAntonovics1968">{{Citation|author=Thomas McNeilly |author2= Janis Antonovics|title=Evolution in closely adjacent plant populations. IV. Barriers to gene flow|journal=Heredity|volume=23|issue=2|pages=205–218|year=1968|doi=10.1038/hdy.1968.29|doi-access=free}}</ref>
|-
|''Anthoxanthum''
|The grass species ''Anthoxanthum odoratum'' grows on soil contaminated with high levels of lead and zinc leeched from an unused mine. Adjacent is the non-contaminated soil. The populations are evolving reproductive isolation due to differences in flowering time (allochrony) with isolation reinforced by unfit hybrids.<ref name="McNeillyAntonovics1968" />
|-
|''Costus''
|''Costus allenii'', ''C. laevis'', and ''C. guanaiensis'';<ref>{{Citation|author=Douglas W. Schemske|title=Floral convergence and pollinator sharing in two bee-pollinated tropical herbs|journal=Ecology|volume=62|issue=4|pages=946–954|year=1981|doi=10.2307/1936993|jstor=1936993}}</ref><ref>{{Citation|author=Kathleen M. Kay and Douglas W. Schemske|title=Pollinator assemblages and visitation rates for 11 species of neotropical ''Costus'' (Costaceae)|journal=Biotropica|volume=35|issue=2|pages=198–207|year=2003|doi=10.1646/02159|s2cid=198158311}}</ref> ''C. pulverulentus'' and ''C. scaber''.<ref>{{Citation|author=Kathleen M. Kay |author2= Douglas W. Schemske|title=Natural selection reinforces speciation in a radiation of neotropical rainforest plants|journal=Evolution|volume=62|issue=10|pages=2628–2642|year=2008|doi=10.1111/j.1558-5646.2008.00463.x|pmid=18637960|s2cid=205781802}}</ref> All species been shown to maintain isolation due to the production of unfit hybrids.
|}

{{Gallery
|mode=Packed-overlay
|title=Some of the species studied for patterns of reinforcement
|height=200
|align=center
|footer=
|File:Brown Tree Frog 2.jpg
|The southern brown tree frog, ''Litoria ewingi''
|File:Geospiza fuliginosa.jpg
|The small ground finch, one of Darwin's finches of the Galápagos
|File:Faxonius rusticus-male dorsal.jpg
|The rusty crayfish ''Faxonius rusticus'' (male)
|File:Arbacia lixula (oursin noir).JPG
|The sea urchin ''Arbacia lixula''
|File:Gasterosteus aculeatus.jpg
|''Gasterosteus aculeatus'', the three-spined stickleback
|File:White-footed Mouse, Cantley, Quebec.jpg
|White-footed mouse ''Peromyscus leucopus''
|File:Distribution magicicada usa fixed.png
|Distribution of the periodical cicadas in the U.S. The yellow area corresponds to the sympatric overlap of ''Magicicada neotredecim'' (blue) with ''M. tredecim'' (red)
|File:CDC-Gathany-Aedes-albopictus-1.jpg
|''Aedes albopictus'' mosquito
|File:Drosophila melanogaster - side (aka).jpg
|''Drosophila'' fruit fly
|File:Ebony Jewelwing, male, Gatineau Park.jpg
|The ebony jewelwing damselfly ''Calopteryx maculata''
|File:Neptunea - links&rechts gewonden.jpg
|Some gastropod shells, like those of ''Partula'' can coil in sinistral and dextral directions such as these, ''Neptunea angulata'' (left) and ''N. despecta'' (right) shells.
|File:Phlox pilosa Central Basin TN.jpg
|''Phlox pilosa'' plant
|File:Juncus effuses.jpg
|''Juncus effusus''
}}

=== Laboratory experiments ===
Laboratory studies that explicitly test for reinforcement are limited.<ref name="Speciation"/>{{rp|357}} In general, two types of experiments have been conducted: using artificial selection to mimic natural selection that eliminates the hybrids (often called "destroy-the-hybrids"), and using disruptive selection to select for a trait (regardless  of its function in sexual reproduction).<ref name="Speciation"/>{{rp|355–357}} Many experiments using the destroy-the-hybrids technique are generally cited as supportive of reinforcement; however, some researchers such as Coyne and Orr and William R. Rice and Ellen E. Hostert contend that they do not truly model reinforcement, as gene flow is completely restricted between two populations.<ref name="RiceHostert1993">{{Citation|title=Laboratory Experiments on Speciation: What Have We Learned in 40 Years?| year=1993 | author=William R. Rice |author2= Ellen E. Hostert | journal=Evolution | volume=47 | issue=6 | pages=1637–1653 | doi=10.1111/j.1558-5646.1993.tb01257.x | pmid=28568007| s2cid=42100751 }}</ref><ref name="Speciation"/>{{rp|356}} The table below summarizes some of the laboratory experiments that are often cited as testing reinforcement in some form.
{| class="wikitable sortable" style="margin-left: auto; margin-right: auto; border: none;"
|+Table of laboratory studies of reinforcement<ref name="RiceHostert1993"/><ref name="Speciation"/>{{rp|354–357}}
!Species
!Experimental design
!Result
!Year
|-
|''D. paulistorum''
|Destroyed hybrids
|Pre-zygotic isolation
|1976<ref>{{Citation|title=Partially Successful Attempt to Enhance Reproductive Isolation Between Semispecies of ''Drosophila paulistorum''|year=1976|author=T. Dobzhansky|author2= O. Pavlovsky|author3=J. R. Powell|journal=Evolution|volume=30|issue=2|pages=201–212|doi=10.2307/2407696|pmid=28563045|jstor=2407696}}</ref>
|-
|''D.pseudoobscura &''
''D. persimilis''
|Destroyed hybrids
|Pre-zygotic isolation; reproductive character displacement
|1950<ref>{{Citation |title=Natural Selection for Reproductive Isolation Between ''Drosophila pseudoobscura'' and ''Drosophila persimilis'' | author=Karl F. Koopman | journal=Evolution | year=1950 | volume=4 | issue=2 | pages=135–148 | doi=10.2307/2405390 | jstor=2405390 }}</ref>
|-
|''D. melanogaster''
|Destroyed hybrids
|Pre-zygotic isolation; reproductive character displacement
|1974<ref>{{Citation |title=Changes in Mating Behavior Produced by Selection for Ethological Isolation Between Ebony and Vestigial Mutants of ''Drosophila melanogaster'' | author=Stella A. Crossley | journal=Evolution | year=1974 | volume=28 | issue=4 | pages=631–647 | doi= 10.1111/j.1558-5646.1974.tb00795.x| pmid=28564833 | s2cid=35867118 }}</ref>
|-
|''D. melanogaster''
|Destroyed hybrids
|Pre-zygotic isolation; reproductive character displacement
|1956<ref>{{Citation |title=Selection for sexual isolation within a species | author=G. R. Knight|display-authors=etal| journal=Evolution | year=1956 | volume=10 | pages=14–22 | doi=10.1111/j.1558-5646.1956.tb02825.x| s2cid=87729275}}</ref>
|-
|''D. melanogaster''
|Destroyed hybrids
|No pre-zygotic isolation detected
|1970<ref>{{Citation|title=Selection for sexual isolation in Drosophila melanogaster by a modification of Koopman's method|year=1970|author=A. Fukatami |author2= D. Moriwaki|journal=The Japanese Journal of Genetics|volume=45|issue=3|pages=193–204|doi=10.1266/jjg.45.193|doi-access=free}}</ref>
|-
|''D. melanogaster''
|Destroyed hybrids
|Pre-zygotic isolation
|1953<ref>{{cite journal | author = Wallace B | year = 1953 | title = Genetic divergence of isolated populations of ''Drosophila melanogaster'' | journal = International Congress of Genetics, Proceedings | volume = 9 | pages = 761–764 }}</ref>
|-
|''D. melanogaster''
|Destroyed hybrids
|Pre-zygotic isolation
|1974<ref>{{Citation|title=Effects of population size and selection intensity on responses to disruptive selection in ''Drosophila melanogaster''|year=1974|author=J. S. F. Barker |author2= L. J. E. Karlsson|journal=Genetics|volume=78|issue=2|pages=715–735|doi=10.2307/2407287|jstor=2407287|pmid=4217303|pmc=1213230}}</ref>
|-
|''D. melanogaster''
| rowspan="3" |Allopatric populations in secondary contact
|N/A
|1982<ref>{{Citation |title=''Drosophila melanogaster'' populations selected for resistances to NaCl and CuSO<sub>4</sub> in both allopatry and sympatry| author=B. Wallace | journal=Journal of Heredity | year=1982 | volume=73 | issue= 1| pages=35–42 | doi= 10.1093/oxfordjournals.jhered.a109572| pmid=6802898 }}</ref>
|-
|''D. melanogaster''
|N/A
|1991<ref>Lee Ehrman, Marney A. White, and B. Wallace. 1991. A long-term study involving ''Drosophila melanogaster'' and toxic media. Pp. 175-209 in M. K. Hecht, B. Wallace, and R. J. Maclntyre, eds. Evolutionary biology, vol. 25. Plenum Press, New York.</ref>
|-
|''D. melanogaster''
|No pre-zygotic isolation detected
|1966<ref>{{Citation |title=A test of sexual isolation in ''Drosophila'' | author=Forbes W. Robertson | journal=Genetical Research | year=1966 | volume=8 | issue=2 | pages=181–187 | doi=10.1017/S001667230001003X | pmid=5922518 }}</ref><ref>{{Citation|title=The ecological genetics of growth in ''Drosophila'' 8. Adaptation to a New Diet|year=1966|author=Forbes W. Robertson|journal=Genetical Research|volume=8|issue=2|pages=165–179|doi=10.1017/S0016672300010028|pmid=5922517}}</ref>
|-
|''D. melanogaster''
|Allowed gene flow between populations
|No pre-zygotic isolation detected
|1969<ref>{{Citation|title=Disruptive and stabilizing selection on the" escape" behavior of ''Drosophila melanogaster''|year=1969|author=B. S. Grant |author2= L. E. Mettler|journal=Genetics|volume=62|issue=3|pages=625–637|pmid=17248452|pmc=1212303|doi=10.1093/genetics/62.3.625}}</ref>
|-
|''D. melanogaster''
|N/A
|No pre-zygotic isolation detected
|1984<ref>{{Citation|title=Still another attempt to achieve assortive mating by disruptive selection in ''Drosophila''|year=1984|author=E. B. Spiess |author2= C. M. Wilke|journal=Evolution|volume=38|issue=3|pages=505–515|doi=10.1111/j.1558-5646.1984.tb00316.x|pmid=28555983|s2cid=19161954}}</ref>
|-
|''D. melanogaster''
|Destroyed some hybrids
|No pre-zygotic isolation detected
|1983<ref name="HarperLambert1983">{{Citation|title=The population genetics of reinforcing selection | year=1983 | author=A. A. Harper |author2= D. M. Lambert | journal=Genetica | volume=62 | issue=1 | pages=15–23 | doi=10.1007/BF00123305| s2cid=7947934 }}</ref>
|-
|''D. melanogaster''
|Disruptive selection
|Pre-zygotic isolation; assortive mating; all later replications of the experiment failed
|1962<ref>{{Citation |title=Isolation by disruptive selection | author=J. M. Thoday |author2= J. B. Gibson | journal=Nature | year=1962 | volume=193 | issue= 4821| pages=1164–1166 | doi= 10.1038/1931164a0| bibcode=1962Natur.193.1164T | pmid=13920720 | s2cid=5156234 }}</ref>
|-
|''D. melanogaster''
|N/A
|N/A
|1997<ref>{{Citation|title=Reinforcement: a new perspective on an old controversy|year=1997|author=Ellen E. Hostert|journal=Evolution|volume=51|issue=3|pages=697–702|pmid=28568598|doi=10.1111/j.1558-5646.1997.tb03653.x|s2cid=21054233}}</ref>
|-
|''D. melanogaster''
|Destroyed hybrids
|Pre-zygotic isolation
|1971<ref>{{Citation|author=Lee Ehrman|title=Natural selection and the origin of reproductive isolation|journal=American Naturalist|volume=105|issue=945|pages=479–483|year=1971|doi=10.1086/282739}}</ref>
|-
|''D. melanogaster''
|Destroyed hybrids
|Pre-zygotic isolation
|1973<ref>{{Citation|author=Lee Ehrman|title=More on natural selection and the origin of reproductive isolation|journal=American Naturalist|volume=107|issue=954|pages=318–319|year=1973|doi=10.1086/282835}}</ref> 
|-
|''D. melanogaster''
|Destroyed hybrids
|Pre-zygotic isolation
|1979<ref>{{Citation|author=Lee Ehrman|title=Still more on natural selection and the origin of reproductive isolation|journal=American Naturalist|volume=113|pages=148–150|year=1979|doi=10.1086/283371}}</ref>
|-
|''Zea mays''
|Destroyed hybrids
|Pre-zygotic isolation; reproductive character displacement
|1969<ref>{{Citation|author=E. Paterniani|title=Selection for Reproductive Isolation between Two Populations of Maize, ''Zea mays'' L|journal=Evolution|volume=23|issue=4|pages=534–547|year=1969|doi=10.1111/j.1558-5646.1969.tb03539.x|pmid=28562870|s2cid=38650254}}</ref>
|}

=== Comparative studies ===
[[File:Enhanced pre-zygotic isolation in Drosophila (allopatric & sympatric plots-vert).png|right|thumb|upright=1.2|Pre-zygotic isolation in allopatric (red) and sympatric (blue) species pairs of ''Drosophila''. Gradients indicate the predictions of reinforcement for allopatric and sympatric populations.<ref>{{Citation |title="Patterns of Speciation in ''Drosophila''" Revisited | author=Jerry A. Coyne |author2= H. Allen Orr | journal=Evolution | year=1997 | volume=51 | issue=1 | pages=295–303 | doi=10.1111/j.1558-5646.1997.tb02412.x | pmid=28568795| s2cid=40390753 }}</ref>]]

Assortive mating is expected to increase among sympatric populations experiencing reinforcement.<ref name="eLS2012"/> This fact allows for the direct comparison of the strength of pre-zygotic isolation in sympatry and allopatry between different experiments and studies.<ref name="Speciation"/>{{rp|362}} Coyne and Orr surveyed 171 species pairs, collecting data on their geographic mode, genetic distance, and strength of both pre-zygotic and post-zygotic isolation; finding that pre-zygotic isolation was significantly stronger in sympatric pairs, correlating with the ages of the species.<ref name="Speciation"/>{{rp|362}} Additionally, the strength of post-zygotic isolation was not different between sympatric and allopatric pairs.<ref name="eLS2012"/>

This finding lends support to the predictions of speciation by reinforcement and correlates well with another later study by Howard.<ref name="Speciation"/>{{rp|363}} In his study, 48 studies with observed reproductive character displacement (including plants, insects, crustaceans, molluscs, fish, amphibians, reptiles, birds, and mammals) were analyzed.<ref name="Howard1993"/> The cases met several criteria of reinforcement such as the trait in question serving as a reproductive barrier and if there existed clear patterns of sympatry versus allopatry.<ref name="Howard1993"/> Out of the 48 candidates, 69 percent (33 cases) found enhanced isolation in sympatry, suggesting that the pattern predicted by reinforcement is common in nature.<ref name="Howard1993"/> In addition to Howard's comparative study, he guarded against the potential for positive-result publication bias by surveying 37 studies of hybrid zones. A prediction of reinforcement is that assortive mating should be common in hybrid zones; a prediction that was confirmed in 19 of the 37 cases.<ref name="Howard1993"/>

A survey of the rates of speciation in fish and their associated hybrid zones found similar patterns in sympatry, supporting the occurrence of reinforcement.<ref name="McCuneLovejoy1998">A. R. McCune and N. R. Lovejoy. (1998). The relative rate of sympatric and allopatric speciation in fishes. In D. J. Howard and S. H. Berlocher (eds) ''Endless Forms: Species and Speciation'', Oxford University Press, pp. 172–185.</ref> One study in the plants ''Glycine'' and ''Silene''; however, did not find enhanced isolation.<ref>{{Citation |title=Patterns of reproductive isolation in three angiosperm genera |author=Leonie C. Moyle|author2= Matthew S Olson|author3= Peter Tiffin |journal=Evolution |year=2004 |volume=58 |issue=6 |pages=1195–1208 |doi=10.1554/03-511 |pmid=15266970 |s2cid=198156807 }}</ref>

== Alternative hypotheses ==
Various alternative explanations for the patterns observed in nature have been proposed.<ref name="Speciation"/>{{rp|375}} There is no single, overarching signature of reinforcement; however, there are two proposed possibilities:<ref name="Speciation"/>{{rp|379}} that of sex asymmetry (where females in sympatric populations are forced to become choosy in the face of two differing males)<ref>L. Partridge and G. A. Parker. (1999). Sexual conflict and speciation. In A. E. Magurran and R. M. May (eds) ''Evolution of Biological Diversity''. Oxford University Press, pp.130–159</ref> and that of allelic dominance: any of the alleles experiencing selection for isolation should be dominant.<ref name="Noor1999"/> Though this signature does not fully account for fixation probabilities or ecological character displacement.<ref name="Speciation"/>{{rp|380}} Coyne and Orr extend the sex asymmetry signature and contend that, regardless of the change seen in females and males in sympatry, isolation is driven more by females.<ref name="Speciation"/>{{rp|380}}

=== Ecological or ethological influences ===
Ecology can also play a role in the observed patterns—called ecological character displacement. Natural selection may drive the reduction of an overlap of niches between species instead of acting to reduce hybridization<ref name="Speciation"/>{{rp|377}} Though one experiment in stickleback fish that explicitly tested this hypotheses found no evidence.<ref name="RundleSchluter1998"/>

Species interactions can also result in reproductive character displacement (in both mate preference or mating signal).<ref name="HoskinHiggie2010"/> Examples include predation and competition pressures, parasites, deceptive pollination, and mimicry.<ref name="HoskinHiggie2010"/> Because these and other factors can result in reproductive character displacement, Conrad J. Hoskin and Megan Higgie give five criteria for reinforcement to be distinguished between ecological and ethological influences:
<blockquote>(1) mating traits are identified in the focal species; (2) mating traits are affected by a species interaction, such that selection on mating traits is likely; (3) species interactions differ among populations (present vs. absent, or different species interactions affecting mating traits in each population); (4) mating traits (signal and/or preference) differ among populations due to differences in species interactions; (5) speciation requires showing that mating trait divergence results in complete or near complete sexual isolation among populations. Results will be most informative in a well-resolved biogeographic setting where the relationship and history among populations is known.<ref name="HoskinHiggie2010"/></blockquote>

=== Fusion ===
It is possible that the pattern of enhanced isolation could simply be a temporary outcome of secondary contact where two allopatric species already have a varying range of pre-zygotic isolation: with some exhibiting more than others.<ref name="Templeton1981">{{Citation |title=Mechanisms of Speciation – A Population Genetic Approach | author=Alan R. Templeton | journal=Annual Review of Ecology, Evolution, and Systematics | year=1981 | volume=12 | pages=23–48 | doi= 10.1146/annurev.es.12.110181.000323}}</ref> Those species pairs that have weaker pre-zygotic isolation will eventually fuse, losing their distinctiveness.<ref name="Noor1999"/> This hypothesis does not explain the fact that individual species in allopatry, experiencing consistent gene flow, would not differ in levels of gene flow upon secondary contact.<ref name="Noor1999"/><ref name="Noor1995"/> Furthermore, patterns detected in ''Drosophila'' find high levels of pre-zygotic isolation in sympatry but not in allopatry.<ref name="Coyne&Orr1989">{{Citation |title=Patterns of speciation in ''Drosophila'' | author1=Jerry A. Coyne |author2=H. Allen Orr| journal=Evolution | year=1989 | volume=43 | issue=2 | pages=362–381 | doi=10.1111/j.1558-5646.1989.tb04233.x | pmid=28568554 | s2cid=1678429 }}</ref> The fusion hypothesis predicts that strong isolation should be found in both allopatry and sympatry.<ref name="Coyne&Orr1989"/> This fusion process is thought to occur in nature, but does not fully explain the patterns found with reinforcement.<ref name="Speciation"/>{{rp|376}}

=== Sympatry ===
[[File:ReinforcementSympatric (patterns detections).png|right|thumb|upright=1.2|Phylogenetic signature to distinguish sympatric speciation from reinforcement. Stronger pre-zygotic isolation (indicated by the red boxes and associated arrows) should be detected between Z and Y and between Z and X if species Z sympatrically speciated (green) from the common ancestor of species Y and X. If Z, Y, and X speciated allopatrically (blue), with Z and Y experiencing secondary contact, strong pre-zygotic isolation should be found between Z and Y, but not between Z and X.<ref name="Noor1997AmNat"/>]]
It is possible that the process of sympatric speciation itself may result in the observed patterns of reinforcement.<ref name="Speciation"/>{{rp|378}} One method of distinguishing between the two is to construct a phylogenetic history of the species, as the strength of pre-zygotic isolation between a group of related species should differ according to how they speciated in the past.<ref name="Noor1997AmNat">{{Citation |title=How often does sympatry affect sexual isolation in ''Drosophila''? | author=Mohamed A. F. Noor | journal=The American Naturalist | year=1997 | volume=149 | issue=6 | pages=1156–1163 | doi=10.1086/286044 | pmid=18811269}}</ref> Two other ways to determine if reinforcement occurs (as opposed to sympatric speciation) are:
* if two recently speciated taxa do not show signs of post-zygotic isolation of both sympatric and allopatric populations (in sympatric speciation, post-zygotic isolation is not a prerequisite);<ref name="MK&VR2002">{{Citation |title=Speciation by Natural and Sexual Selection: Models and Experiments | author=Mark Kirkpatrick |author2= Virginie Ravigné | journal=The American Naturalist | year=2002 | volume=159 | pages=S22–35 | doi=10.1086/338370 | pmid=18707367| s2cid=16516804 | url=https://semanticscholar.org/paper/b76b2d680d49a8a043a8123fcdca5a5a5053c7c8 }}</ref> 
* if a cline exists between two species over a range of traits (sympatric speciation does not require a cline to exist at all).<ref>{{Citation |title=Adaptation, speciation and hybrid zones | author=N. H. Barton |author2= G. M. Hewitt | journal=Nature | year=1989 | volume=341 | issue= 6242| pages=497–503 | doi=10.1038/341497a0 | pmid=2677747 | bibcode=1989Natur.341..497B | s2cid=4360057 }}</ref>

=== Sexual selection ===
A Fisherian runaway process can occur during the process of reinforcement, increasing mate discrimination rapidly.<ref name="Noor1999"/><ref name="Coyne&Orr1989"/> Additionally, when there is a low cost to female mate preferences, changes in male phenotypes can result, expressing a pattern identical to that of reproductive character displacement.<ref>{{Citation |title=Sexual Selection and the Evolution of Costly Female Preferences: Spatial Effects | author=Troy Day | journal=Evolution | year=2000 | volume=54 | issue=3 | pages=715–730 | doi=10.1111/j.0014-3820.2000.tb00074.x  | pmid=10937247 }}</ref> Post-zygotic isolation is not needed, initiated simply by the fact that unfit hybrids cannot get mates.<ref name="Noor1999"/>

== Arguments against reinforcement ==
A number of objections were put forth, mainly during the 1980s, arguing that reinforcement is implausible.<ref name="Noor1999"/><ref name="HoskinHiggie2010"/><ref name="Speciation"/>{{rp|369}} Most rely on theoretical work which suggested that the antagonism between the forces of natural selection and gene flow were the largest barriers to its feasibility.<ref name="Speciation"/>{{rp|369–372}} These objections have since been largely contradicted by evidence from nature and additional models.<ref name="Howard1993"/><ref name="Speciation"/>{{rp|372}}

Concerns about hybrid fitness playing a role in reinforcement has led to objections based on the relationship between selection and recombination.<ref name="Barrientos2009"/><ref name="Speciation"/>{{rp|369}} That is, if gene flow is not zero (if hybrids aren't completely unfit), selection cannot drive the fixation of alleles for pre-zygotic isolation.<ref name="Felsenstein1981"/> For example: If population <math>X</math> has the pre-zygotic isolating allele <math>A</math> and the high fitness, post-zygotic alleles <math>B</math> and <math>C</math>; and population <math>Y</math> has the pre-zygotic allele <math>a</math> and the high fitness, post-zygotic alleles <math>b</math> and <math>c</math>, both <math>ABC</math> and <math>abc</math> genotypes will experience recombination in the face of gene flow. Somehow, the populations must be maintained.<ref name="Speciation"/>{{rp|369}}

In addition, specific alleles that have a selective advantage within the overlapped sympatric populations, such as alleles causing prezygotic isolation, are only useful within that population.<ref name="Moore1957">J. A. Moore. (1957). An embryologist's view of the species concept. In Ernst Mayr (eds) ''The Species Problem'', American Association for the Advancement of Science, pp. 325–338.</ref> However, if they are selectively advantageous, gene flow should allow the alleles to spread throughout both the sympatric and allopatric populations.<ref name="Moore1957"/> To prevent this, the alleles would have to be deleterious in the other population.<ref name="Speciation"/>{{rp|371}} This is not without problems, as gene flow from the presumably large allopatric regions could overwhelm the area when two populations overlap.<ref name="Speciation"/>{{rp|371}} For reinforcement to work, gene flow must be present, but very limited.<ref name="S&M1997"/><ref name="Cain1999"/>

Recent studies suggest reinforcement can occur under a wider range of conditions than previously thought<ref name="Theory&Sp2001"/><ref name="MK&VR2002"/><ref name="Speciation"/>{{rp|372–373}} and that the effect of gene flow can be overcome by selection.<ref name="Matute2020">{{Citation |title=Reinforcement Can Overcome Gene Flow during Speciation in ''Drosophila'' | author=Daniel R. Matute | journal=Current Biology | year=2010 | volume=20 | issue= 24| pages=2229–2233 | doi=10.1016/j.cub.2010.11.036 | pmid=21129972| pmc=3019097 }}</ref><ref name="Coyne2010(blog)">{{Citation |author=Jerry A. Coyne | title="Reinforcement" and the origin of species | publisher=Wordpress | date=2010 | url=https://whyevolutionistrue.wordpress.com/2010/12/08/reinforcement-and-the-origin-of-species/ }}</ref> For example, the two species ''Drosophila santomea'' and ''D. yakuba'' on the African island São Tomé occasionally hybridize with one another, resulting in fertile female offspring and sterile male offspring.<ref name="Matute2020"/> This natural setting was reproduced in the laboratory, directly modeling reinforcement: the removal of some hybrids and the allowance of varying levels of gene flow.<ref name="Coyne2010(blog)"/> The results of the experiment strongly suggested that reinforcement works under a variety of conditions, with the evolution of sexual isolation arising in 5–10 fruit fly generations.<ref name="Coyne2010(blog)"/>

In conjunction with the fusion hypothesis discussed above, reinforcement can be thought of as a race against both fusion and extinction.<ref name="Templeton1981"/> The production of unfit hybrids is effectively the same as a heterozygote disadvantage; whereby a deviation from genetic equilibrium causes the loss of the unfit allele.<ref name="HarperLambert1983">{{Citation|title=The population genetics of reinforcing selection | year=1983 | author=A. A. Harper |author2= D. M. Lambert | journal=Genetica | volume=62 | issue=1 | pages=15–23 | doi=10.1007/BF00123305| s2cid=7947934 }}</ref> This effect would result in the extinction of one of the populations.<ref>{{Citation |title=More evidence against speciation by reinforcement | author=H. E. H. Paterson | journal=South African Journal of Science | year=1978 | volume=74 | pages=369–371 }}</ref> This objection is overcome when both populations are not subject to the same ecological conditions.<ref name="Speciation"/>{{rp|370}} However, it is still possible for extinction of one population to occur, as has been shown in population simulations.<ref>{{Citation |title=A Theoretical Investigation of Speciation by Reinforcement | author=Hamish G. Spencer|author2= Brian H. McArdle|author3=David M. Lambert | journal=The American Naturalist | year=1986 | volume=128 | issue=2 | pages=241–262 | doi=10.1086/284557 }}</ref> For reinforcement to occur, pre-zygotic isolation must happen quickly.<ref name="Speciation"/>{{rp|370}}

== References ==
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