Speciation Genetics: Evolving Approaches

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Speciation genetics: evolving approaches

Mohamed A. F. Noor* and Jeffrey L. Feder‡ Abstract | Much progress has been made in the past two decades in understanding Darwin’s mystery of the origins of species. Applying genomic techniques to the analysis of laboratory crosses and natural populations has helped to determine the genetic basis of barriers to gene flow which create new species. Although new methodologies have not changed the prevailing hypotheses about how species form, they have accelerated the pace of data collection. By facilitating the compilation of case studies, advances in genetic techniques will help to provide answers to the next generation of questions concerning the relative frequency and importance of different processes that cause speciation.

Gene flow What types of genetic change bring about speciation is sophisticated methods has led to finer dissection of the The movement of alleles one of the most basic questions in biology. Speciation is a genetic origins of species down to the level of the indi- between local populations that fundamental outcome of life. Given metabolism, repro- vidual loci and eventually nucleotides. Technical and is due to the migration of duction, mutation, heredity, and the spatial–temporal statistical advances are also extending laboratory-based individuals. subdivision of the environment and of individuals into discovery to natural populations, allowing researchers Hybrid zone populations, new species form through time, increasing to investigate barriers to gene flow, genomic interactions A location where the hybrid biodiversity. The evolution of this biodiversity can only and the genetic permeability of species boundaries in offspring of two divergent, be fully explained if we identify the heritable underpin- hybrid zones where differentiated taxa overlap and inter- partially geographically nings of species formation and the forces responsible breed. Whole-genome surveys can provide representative overlapping groups are prevalent. Hybrid zones are for their origins. snapshots of differentiation across the entire genomes sometimes stable for many Here, we review how recent advances in molecular of model genetic systems. Ultimately this progress is generations and there is often and genomic techniques are helping to achieve a greater leading to large-scale comparative genomic analysis variation in the fitness of understanding of the genetics of speciation. For the of entire taxonomic groups (model and emerging- hybrids within the zone. purpose of this review, we focus on technical advances model systems alike) from which general patterns and Genomic conflict rather than theoretical concepts, which are discussed rules might emerge. Competition within a genome extensively elsewhere1. We define speciation for sexually We conclude by discussing what the new methods for transmission to or success reproducing organisms as the transformation of within- have and potentially will reveal about the genetics of of gametes. population variation into taxonomic differences through speciation. We argue that the new technology is not the evolution of inherent barriers to gene flow. This necessarily providing results that are inherently dif- definition is not universally accepted, but it remains the ferent from those of earlier studies. Rather, the new *DCMB Group/ Biology Department, Duke University, most commonly used by students of speciation and is of methodology is adding detail by accelerating the rate BOX 91000, Durham, North the greatest utility to dissecting the genetics of the proc- and ease of data collection on a genome-wide scale. Carolina 27708, USA. ess1. We discuss whether and how molecular techniques We contend that this aspect of technology will have the ‡Department of Biological are helping to discern the genetic bases and evolutionary greatest immediate impact — it will allow us to move Sciences, University of Notre origins of barriers that contribute to population diver- from isolated case studies to compilations of results Dame, Galvin Life Science Center, PO BOX 369, Notre gence. We present some recent discoveries from labora- for representative groups to answer relative frequency Dame, Indiana 46556-0369, tory and field studies that apply molecular and genomics questions about processes and factors that contrib- USA. techniques to the speciation question. Our examples are ute to speciation. For example, how often do natural e-mails: [email protected]; chosen to illustrate some of the breadth of approaches selection, sexual selection, genomic conflict and eco- [email protected] doi:10.1038/nrg1968 that are used to tackle this exciting question. logical interactions drive divergence? What proportion Published online We begin by framing the problem from a historical of genetic change is regulatory versus functional? 3 October 2006 perspective, tracing how the development of ever-more- How important is chromosomal change in speciation?

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How often does speciation occur in the face of peri- Classical approaches odic or regular gene flow, generating mosaic patterns Descriptive and conceptual. Although he entitled his of genomic differentiation? It is not clear that the new famous book On the Origin of Species, Darwin2 had only methodology will lead to a major shift in our think- vague insights into the speciation process itself, viewing ing about the ways in which speciation can occur. We it as a later stage in a continuum from adaptive diver- propose that this limitation is not technical, but mostly gence among ‘varieties’ within species. The Russian rests in the imagination of students in the field. geneticist Theodosius Dobzhansky and the German systematist Ernst Mayr instilled broader excitement in the field of speciation genetics with three conceptual advances in the 1930s and 1940s. First, Dobzhansky3 Box 1 | Classical approaches to the study of speciation genetics and Mayr4 compiled lists of traits that prevent gene H G F E D C B A flow between species, such as habitat divergence and hybrid sterility (here, we refer to them collectively as Inverted in AR barriers). They noted that some of these barriers prevent the formation of hybrid offspring, whereas others prevent the success and propagation of hybrid offspring once formed. Second, Dobzhansky and Mayr argued AR ST A that species can be defined in the context of these traits. A A This second advance identified a means for the study of speciation genetics: the genetics of barriers acting to reduce gene flow in nature indicates the genetics B of speciation itself. Third, they studied these barriers B B directly: Mayr defined which ones operate in nature and Dobzhansky determined their genetic basis in C H the laboratory through controlled crosses (BOX 1). The D C implications of this last endeavour were profound, dem- C onstrating that barriers are traits that can be genetically H G D D mapped and that species boundaries can be quantified F E H genetically. G Despite the dramatic advances in molecular and E genomic techniques, the ‘old-school’ approaches of F G E Dobzhansky and Mayr for studying the genetics of spe- F ciation still apply today5 (BOX 1). Since that time, simi- Genetic studies of speciation, which date back to the early twentieth century, lar laboratory and field studies have become plentiful attempt to identify the number, distribution and type of genes that contribute to enough for elegant meta-analyses of broader taxonomic phenotypes that prevent gene flow between species. They also examine patterns of data sets to be carried out. These meta-analyses have differentiation between the genomes of diverging taxa. found, unsurprisingly, that barriers between long- Laboratory or garden hybridization studies diverged species are typically much stronger or more Laboratory or garden hybridization studies have traditionally had a central role in effective than barriers between very recently diverged understanding the genetic basis of barrier (speciation-related) traits. Examining species (for example, see REFS 6–11). Therefore, genetic the distribution of phenotypes over one or two generations of controlled crosses divergence is associated with the accumulation of more has been used to infer the contributions of the sex chromosomes versus autosomes, (and/or stronger) barriers, irrespective of how genetic identify patterns of dominance and provide estimates of the minimum number of genes that affect the speciation-related traits. An advance was made with the first divergence is measured. true genetic mapping efforts, when associations were assessed between genetic Echoing the differences in Dobzhansky’s and Mayr’s approaches, a slight divide has nonetheless emerged (now, often molecular) markers and barrier traits in F2 or other hybrids. For example, Dobzhansky133 correlated hybrid testis size (a proxy for fertility) with within the speciation community. At one extreme are genotype at seven mutant markers in a cross between races of Drosophila the researchers who investigate barriers to gene flow pseudoobscura (now, described as separate species). In principle, this is no different using laboratory crosses of well-established model from modern QTL mapping studies. species, often focusing on easily scored phenotypes Genetic diversity within and between species such as hybrid sterility or inviability. They have been Speciation genetics has also been explored using studies of genetic diversity successful in identifying genes that contribute to these within and between species. Hybridization or differentiation between two species traits (see below). However, the specific traits or genes can be inferred from the ratios of shared alleles to ‘fixed’ differences. Even before identified might not directly reduce or have previously DNA was known to be the hereditary material, studies of genetic diversity within restricted gene flow between the focal species in nature and between species were carried out using either phenotypes or polytene (indeed, they often do not, as several species studied in chromosome arrangements (see figure, which shows a chromosomal heterozygote this way do not occur together in nature). Moreover, for the standard (ST) and arrowhead (AR) arrangements in D. pseudoobscura). These were succeeded in the 1960s by protein electrophoretic approaches to the these genes could contribute to reproductive isolation study of diversity and speciation, and today by the use of DNA-based genetic in the laboratory, but if they arose after gene flow was markers (BOX 2) and DNA sequences. essentially complete between species in nature, they These approaches form much of the foundation for many modern studies of would not represent ‘speciation’ loci in the strict sense. speciation genetics. Nonetheless, these studies have provided valuable

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genetic and evolutionary insights that can be applied In this regard, analysis of natural populations can to naturally hybridizing but less tractable species, and broaden our surveys by providing entries for identi- have identified the rate of accumulation of alleles that fying candidate speciation genes in non-model genetic cause hybrid incompatibilities in isolated species. At the organisms20–22. In hybrid zones, genomic regions that other extreme are naturalists who study very recently are relatively impermeable to introgression (admixture) diverged, often hybridizing populations. Although their and display enhanced differentiation probably contain studies are directly applicable to natural divergence in genes that generate barriers between populations23–28. its early stages, the populations might be ephemeral and In addition, QTL analysis in natural hybrid zones can never actually speciate: such taxa could eventually fuse look for correlations of markers with phenotypic traits (for example, see REF. 12). So, an unproven assumption of that distinguish taxa, several of which could be associ- eventual speciation underlies these studies. The authors ated with barriers29,30. Therefore, hybrid zones not only of this review were trained at opposite ends of this divide. provide valuable genetic information on the natural We argue that complementary insights are yielded history of speciation and its causes, but can also be used by these two approaches, and genetic and genomic to help to move from broad-scale characterization of techniques have facilitated progress in both arenas. genomic architecture to the specific genes responsible for barriers20,31. In the past decade, we have witnessed Laboratory-crossable species. Genetic crosses of labo- the transformation of many non-model genetic organ- ratory-amenable organisms have been a touchstone isms, such as Heliconius butterflies and Gasterosteus for dissecting the genetics of speciation since the early sticklebacks, into ‘emerging’ model systems that are of Introgression 1930s, providing information on the minimum number particular interest because they integrate complementary The movement of alleles from of genes and the relative contributions of different chro- information on speciation from the field and laboratory one species into the gene pool mosomes (especially the sex chromosomes) to barriers to provide a fuller understanding of the process. of another through repeated backcrossing of an interspecies to gene flow between taxa. Although the basic protocol As with genetic crosses, genetic studies of samples hybrid with one of the parent for gene mapping remains the same, several technical from natural populations have greatly benefited from species. and methodological advances have increased the pace technical advances in the sensitivity, speed, types and of discovery since Dobzhansky’s13 initial study of hybrid numbers of marker that can be scored. Sequence-based Backcross male sterility in Drosophila pseudoobscura ‘races’. These studies of multiple nuclear loci are now standard and The mating of an individual with its parent, or with an individual advances include improvements in molecular genotyp- combinations of different types of marker are being used of the same genotype as its ing (for example, see REFS 14,15; also see below), statisti- to investigate different aspects of the problem of gene parent, to follow the inheritance cal advances in detecting association between markers flow32,33 (see also Luikart et al.34 and BOX 2 for a descrip- of alleles and phenotypes. and traits (for example, see REFS 16,17) and improved tion of the types of molecular genetic marker used in REF. 18 Transplant studies genetic cross methodologies (for example, see ). population genomics studies). Analyses of hybrid zones Studies in which organisms Repeated backcrosses have been used to introgress small have yielded estimates of the number of genes that con- are moved from a native to an and well-defined segments from one species into another tribute to barriers, insights into how the balance between introduced setting to examine to determine the effects of single loci. For example, selection and migration shapes gene frequency clines, the effects of the environment in a tour-de-force, True et al.19 inserted transposable and assessments of the relative importance of inherent or of related species. P-elements into 87 positions in the genome of Drosophila genomic incompatibilities, population demography Admixture mauritiana, and introgressed each of these segments into and ecology in maintaining the genetic integrity of The mixing of genetically Drosophila simulans by backcrossing for 15 generations taxa20–22,30,35–45. In addition, an increasing (although differentiated groups. to determine the genetic positions of hybrid-sterility- still limited) number of case studies provide evidence divergence-with-gene-flow speciation Cline conferring loci. Even now, a decade later, this represents for (for example, The gradual change of a an impressive accomplishment. see REFS 33,46–49) and related phenomena, including genotype or phenotype in a sympatric speciation through genic mechanisms that do not species over a geographical Data from nature. One can only learn so much by study- involve changes in ploidy number50–52, and hybrid speciation area that is often associated ing model organisms in the laboratory. One problem and the creative role of introgression in divergence1,53–55. with an environmental gradient. is that a reproductive barrier between geographically Many of the case studies cited are not universally Divergence-with-gene-flow isolated populations that is identified on the basis of accepted1. Nonetheless, the modern approaches we speciation laboratory crosses might not be an effective barrier to discuss below can be and have been used to bolster the Speciation that progresses gene flow in the field if and when these populations evidence for these controversial hypotheses in particular without the complete absence of gene exchange between encounter each other. Surveys of natural hybrid zones cases, such as sympatric speciation. diverging taxa. This type of and/or transplant studies in the field are therefore needed speciation includes but is not to complement laboratory-based studies to establish the Modern approaches limited to sympatric speciation. significance and strength of specific barriers in nature. Recreation or dissolution of speciation events. Genetic Another problem is the lack of taxonomic representation. studies of speciation often suffer from the problem Sympatric speciation Speciation through divergence Successful mapping studies have, to date, been mainly that they are investigating a process that is either in geographically overlapping limited to model systems such as Drosophila species, complete or nearly so. One innovative yet relatively taxa. where detailed linkage maps and tools such as deletions, underused empirical solution to this problem involves inbred lines, genomic sequences and transformation sys- the experimental recreation of new species and hybrid Hybrid speciation Hybridization between two tems are available. These model systems might not be zones in nature. Applying molecular techniques to the species gives rise to a new, representative of the types of trait or genetic architecture traditional experimental hybrid approach has been pure-breeding taxon. involved in speciation across taxonomic groups. fruitful in studies of hybrid speciation. Using Helianthus

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Box 2 | Molecular genetic markers used in population genetic and genomic studies The types of molecular marker used in population genomics have been reviewed elsewhere34, but this box presents a few examples of some of the most popular markers. AFLPs Amplified fragment length polymorphisms (AFLPs) are produced by selective PCRs that amplify many segments of the genome, and variability is scored by the presence or absence of particular amplicons. The advantage of this approach is that it can be high-throughput and fast; the disadvantages are that the markers are dominant (heterozygotes cannot be differentiated from homozygotes) and there can be some problems with reproducibility. Microsatellites These are arrays of 1–6 bases of repetitive sequence. They are often highly polymorphic, and are co-dominant markers. However, development of these markers is more expensive, and fewer can be scored simultaneously than with AFLPs. Additionally, their mutational properties are such that independent mutations often recreate the same allele, so identity does not indicate co-ancestry (a problem called homoplasy). Sequence data Sequence data are the ultimate source of all variation. Sequence variation could be in the form of SNPs or possibly gross features such as insertions or deletions. Direct examination is generally the most rigorous approach and can be done on a large scale when sequenced genomes are available, but, at present, this is often too laborious and expensive for non-model species. SNPs These are often misleadingly referred to as a class of genetic marker, but the means whereby SNPs are identified and/or scored are variable. They can be identified by techniques such as those using RFLPs (restriction fragment length polymorphisms), direct sequencing of alleles and comparing published DNA sequences. They can be subsequently scored by the same methods or by many others including pyrosequencing, real-time PCR and microspheres. Developing SNPs as markers can be quick if genome sequences or other sequence databases are available. Transposable elements Transposable elements are genomic mobile genetic elements. Some of these, such as SINEs (short interspersed elements), have been used for population genetics or phylogenetics because the probability of independent insertions into a particular spot is near zero, suggesting essentially no homoplasy.

sunflower species, Rieseberg and colleagues56–58 were (but incomplete) spore viability. This experiment showed able to recreate the complex phenotypes of ancient a direct contribution of the genome rearrangement to hybrid species from early generation synthetic hybrids. hybrid spore viability, but also demonstrated that genic Consistent with expectations, the same combinations of effects must also contribute. parental chromosomal segments required to generate Despite past imperfections in design, Rieseberg extreme phenotypes in synthetic hybrids are those found and colleagues47,67 concluded from a review of the col- in the natural hybrids. lective experimental hybrid literature that most traits Experimental hybridization has also proved use- that differentiate species seem to be under selection ful for reconstituting possible sequential phenotypic in the wild, that hybrid fitness tends to be contingent steps from ancestral to derived states in the evolution on both hybrid genotype and the habitat into which of barriers between taxa, such as in the study of flower they are placed, that intrinsic isolating factors are not colour and shape in monkeyflowers59,60 and wing pat- necessarily more stable and irreversible than extrinsic, terns in Heliconius butterflies61. These experiments are ecologically related barriers, and that hybrid incompat- natural extensions of some of the classical work that ibilities could be quickly purged in both experimental Pyrosequencing selected for reduced gene flow in maize62 and Drosophila and natural hybrid zones. These conclusions demon- A method for DNA sequencing, 63,64 65 in which the inorganic species . In a recent example, Leu and Murray iden- strate that the coupling of experimental hybridization pyrophosphate that is tified the genetic basis for altered mate preference by with genomics holds great promise for understanding released from a nucleoside selection for increased assortative mating between two the speciation process. triphosphate on DNA chain yeast strains. Although this study did not yield informa- elongation is detected by a bioluminometric assay. tion on particular examples from nature, it provided a Empirical and statistical analyses for introgressive hypothesis for one physiological means of assortative hybridization. The new molecular approaches are hav- Microspheres mating in yeasts. ing a major impact on resolving the genetic architecture (Also known as microparticles Yeast have also been used in the experimental ‘de- and permeability of species boundaries. In the past or microbeads). Small evolution’ of a species. In an elegant study that exam- decade, we have witnessed a shift in surveys of natural 1–100 μm diameter particles that are used as solid supports ined the effect of genome rearrangements on hybrid populations from studies that are based on mitochon- 66 in bioassays. They can carry sterility, Delneri et al. experimentally reconfigured the drial sequences to studies of multiple nuclear loci to a probe or primer, and can Saccharomyces cerevisiae genome to make it collinear high-throughput scans of entire genomes for detect- contain internal magnetic with that of its relative Saccharomyces mikatae. Hybrids of ing introgression and regions of higher differentiation compounds to allow magnetic separation or internal these two species normally produce only inviable spores, (‘islands of speciation’) between diverging taxa. It is now fluorescent compounds whereas hybrids of the experimentally manipulated possible to use microarray hybridization techniques for labelling. S. cerevisiae with S. mikatae produced hybrids with some on samples from natural populations to rapidly paint

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a broad view of genomic differentiation. Follow-up the Wang–Wakeley–Hey test of shared polymorphism sequence analysis is used to finely map the boundaries to fixed differences48,82, the linkage disequilibrium of introgressed versus diverged regions and to quantify test of gene flow83, the isolation with migration model the extent of differentiation for these genes. devised by Nielsen and Wakeley84 and its multilocus Genetic data alone do not answer the question of extension85, and the relative node depth approach23,86,87, whether taxa are or have undergone introgressive hybridiz- provide more sophisticated approaches to test for possible ation unless the results are viewed in the context of an introgression (BOX 3). appropriate evolutionary model for rigorous statistical Recent analyses suggest that many evolutionarily testing. Concomitant with the advances in technology, related, geographically overlapping taxa exchange genes more sophisticated analytical methods are also being through introgressive hybridization1,55,88,89. Moreover, developed to discern whether shared variation is due to these taxa commonly possess mosaic (composite) introgression from a related species or represents the per- genome structures88. One particularly relevant finding sistence of ancestral polymorphism. These methods can is that genomic regions that are relatively impermeable be categorized as being based on either summary popula- to many instances of introgression have been associ- tion genetic statistics or phylogenetic gene-tree-building ated with low recombination rates, such as is caused by approaches. Gene-tree approaches can reveal alleles chromosomal rearrangement. Studies in sunflowers30, that show discordant phylogenetic patterns (paraphyly) the D. pseudoobscura subgroup83,90–92 and Rhagoletis that could indicate introgression68–71, but could also be pomonella23,93 found evidence for greater introgression explained by incomplete lineage sorting. Gene trees have (lower divergence) in collinear segments of the genome also been used to distinguish historical processes through than in inverted segments. Other regions of restricted nested clade analysis72,73. However, because it is difficult to recombination, such as pericentromeric regions or devise powerful statistical tests of competing hypotheses translocated/inverted regions, have also been implicated for gene-tree-based methodologies such as nested clade in divergence between races and species (for example, analysis74, one can incorrectly accept a best-fit model see REFS 27,28,94). The continued application of new for a process that never occurred. So, gene trees could molecular approaches coupled with powerful statistical find their most practical application as a first, qualitative analyses promises to lead to even greater insights into approach to identify loci that might have introgressed in the relationship between genome structure and the the past or to identify taxa of possible hybrid origin. persistence of species despite gene flow.

Variation among loci in summary FST values of inter- 75,76 population differentiation and their multi-allele GST Use of whole-genome sequence assemblies. Whole- extension77 has also been used to test for introgression genome sequence assemblies have been completed (for example, see REFS 26,78). The basis for the test is for a number of eukaryotes, including several closely that divergent selection pressures on a trait can produce related taxa that can then be studied in a comparative large between-population allele frequency differences framework. Coupling whole-genome sequences with for genetic markers that encode or are linked to the trait, functional studies can yield important insights into the

and these differences are detectable by high, outlier FST genetic changes that underlie speciation. One recent values for the markers79. The test has been applied in example involves comparisons among three distantly an analogous manner to interspecific comparisons. In related yeasts. Roughly 100 million years ago, a yeast 95 this case, outlier FST values point to genomic regions ancestor experienced a whole-genome duplication . that might be relatively impermeable to introgression Subsequently, alternative copies of the duplicated loci, between taxa, presumably owing to association with a including several essential genes, were reciprocally lost barrier. Although informative as a descriptive measure, in different yeast lineages96. If two taxa were to hybridize, Nested clade analysis FST comparisons have their shortcomings. First, they do then for every reciprocally deleted gene, 25% of the A coalescent approach to not fully use the genealogical information inherent in resulting hybrid’s spores would lack a functional copy disentangling the effects of DNA sequences. Second, F values can be affected by of the gene96, providing a simple mechanism for hybrid long-term population history ST 97 from gene flow by historical and stochastic population processes that are dysfunction . Although this study fails to identify a par- reconstructing the sequence of unrelated to introgression, one of the most problematic ticular speciation event that is facilitated by this process, it events that have generated the being differences in mutation rate among genes or is possible that such reciprocal gene loss contributed to at current genetic pattern among genomic regions. F and G values should therefore be least some barriers to gene flow between yeast species. populations within a species. ST ST adjusted by appropriate estimates of mutation rate when The completion and assembly of several eukaryotic testing for introgression, which are based on either levels genome sequences has also been a boon for specia- FST A measure of population of intraspecific polymorphism or interspecific diver- tion research in several indirect ways. With respect to subdivision that is based on gence derived from outgroup comparisons. Modified genetic mapping studies, researchers can now select genetic polymorphism data approaches such as the analysis of molecular varia- markers at any location in the genome to help pin- derived from comparing the 80 81 genetic variability within and tion and the use of GST values have been developed point genes that contribute to barriers (for example, between populations. to ameliorate some of these problems. Accompanying see REF. 98). Similarly, whole-genome sequences allow biogeographical information can also strengthen the for the construction of microarrays and other genetic G ST case for differential introgression; for example, through tools to assess the expression of all known and inferred An extension of the F measure ST comparisons of cline width in hybrid zones among loci24. transcripts for differences between species or races and for multiple alleles, where GST is equal to the weighted average However, new generation methods for DNA sequence strains (for example, see REF. 99). We discuss these of FST for all alleles. data that are based on the coalescent theory, such as advances in the next section.

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Box 3 | Several methods used to infer introgression between taxa using genetic data Direct gene-tree comparisons Gene trees are inspected to detect alleles or loci that have discordant genealogical patterns which could be indicative of introgression. Generally speaking, this is a qualitative approach, although tests can be devised that are based on the coalescent theory134 or variations of the Shimodaira–Hasegawa incongruence test135 to provide a statistical framework. Assignment tests Multilocus genotypes are used to assess the number of possible populations that are represented in a sample of individuals and to assign individuals to these populations, based on maximum likelihood or Bayesian approaches136. This method could be used to identify potential hybrid individuals with roughly equal probabilities of belonging to alternative taxa, implying ongoing gene flow. Nested clade analysis Haplotype gene trees are used to define a nested series of branches (clades), allowing an evolutionary analysis of the spatial distribution of genetic variation75,76. Nested clade analysis can be used to qualitatively detect possible gene flow. For example, when taxa overlap in a portion of their range, a pattern in which populations are genealogically more similar in sympatry than allopatry would imply introgression. Outlier loci 75,76 77 Summary statistics such as FST values or their multi-allele GST extensions are calculated to identify outlier loci that have reduced inter-taxa differentiation compared with other genes. Analysis of molecular variation is more informative for DNA 80 81 sequence data and a new GST value has been developed that ameliorates some of the problems affecting estimates that are due to differences in mutation rate (polymorphism levels) among loci. Relative node depth Node depths (coalescence times) between taxa relative to an outgroup species are compared among loci for evidence of significantly greater variation than predicted by the coalescent theory, suggesting introgression23,86,87,137. Wang–Wakeley–Hey An isolation model of no gene flow between taxa since their time of separation is evaluated through comparisons of shared nucleotide polymorphisms with exclusive polymorphisms and fixed differences. Gene flow will increase the numbers of the shared polymorphisms and reduce the numbers of the fixed polymorphisms relative to the null neutral expectations of the coalescent theory. Furthermore, if gene flow occurs at some loci and not at others, it will increase the variance among loci in numbers of shared polymorphisms and fixed differences. The Wang–Wakeley–Hey test uses the difference between the highest and lowest counts of shared polymorphisms among a set of loci plus the difference between the highest and lowest counts of fixed differences observed over the same group of loci and compares the observed value with a simulated distribution. Alternatively, one can use a χ2 statistic to measure the overall fit of the data to the isolation model46. Linkage disequilibrium The difference (X) between the average linkage disequilibrium among all pairs of shared polymorphisms between taxa for a locus (DSS) and the average among all pairs of sites for which one member is a shared polymorphism and the other is an exclusive polymorphism (DSX) is calculated90. If polymorphisms are shared owing to gene flow, then DSS should tend to be positive, DSX negative, and X should be positive and large. However, in the absence of gene flow, there should be relatively little linkage disequilibrium for all cases and X should be small and close to zero. Isolation with migration model The isolation with migration model is a Markov chain Monte Carlo method that was developed by Nielsen and Wakeley84 for estimating the relative effects of migration and isolation on genetic diversity in a pair of populations from DNA sequence data. The method allows for the joint estimation of multiple demographic parameters in either a Bayesian or a likelihood framework, including the migration rate for each population, the divergence time from a common ancestor, and the relative sizes of the ancestral and two current populations. The method was originally based on data from a single non-recombining locus and has been extended to multiple loci by Hey and Nielsen85.

High-throughput approaches for genotyping or expression of these array-based genotyping methods require a priori analysis. Methods that allowed for high-throughput sequence information, a few, such as DArT101, do not. genotyping have dramatically increased the speed and High-throughput approaches can also be used to precision with which one can localize genes that confer look for divergence between species or races. Most barriers between species. Until recently, the bulk of studies have examined genetic divergence either across molecular genotyping used in genetic mapping relied on the genome using the single genome sequence assem- electrophoretic separation of PCR products or proteins. blies for each species (for example, see REF. 102) or using DArT Capillary-based approaches have also been available, multiple individuals of each species but typically only Short for ‘diversity arrays but these are still rather limited in throughput capa- 30 or fewer loci (for example, see REFS 46,103–107). The technology’; this is a technique bility. Several microarray-based marker methods for former gives a genome-wide view but lacks the ability for analysing DNA scoring SNPs have been developed that can allow one to distinguish divergence from polymorphism within polymorphism, which is based on hybridization to microarrays, to genotype literally thousands of markers simulta- species, whereas the opposite is true for the latter that does not require DNA neously from one individual with a small quantity approach. With newly available genomic tools, one sequence information. of DNA (for example, see REF. 100). Although most can get a glimpse of divergence between species using

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multiple individuals in a high-throughput format. A D. simulans. As such, while Ods probably contributes recent example is the study by Turner et al.28, in which to hybrid fertility, insertion of a foreign allele alone is samples of genomic DNA from seven strains of each not sufficient to cause sterility. of two hybridizing Anopheles gambiae mosquito races The above example could have suffered an addi- were hybridized to oligonucleotide microarrays. The tional complication if the native copies of Ods in the researchers identified three regions of the genome hybrid genome altered the phenotypic effect of that bore significant differentiation between the two the inserted copy. In a similar study, Greenberg et al.121 races, suggesting that genes responsible for ecological investigated the effect of the desaturase2 (desat2) and behavioural differentiation are likely to be located gene on various adaptive differences between two there. These results were generally (albeit not com- D. melanogaster populations. They used the elegant pletely) consistent with other studies that use single gene-replacement technique of Rong and Golic122, marker scoring approaches27. which, unlike many transgenic methods, leaves only Oligonucleotide and cDNA microarrays are also a single (transgenic) copy of the target gene of interest useful for rapidly assessing differences in gene expres- per genome. Using this technique on desat2, the authors sion for thousands of loci simultaneously; these tools found differences in cold tolerance and starvation might provide insights into divergent or disrupted genes susceptibility between geographical alleles. Although that could be associated with barriers. Gene expression these elegant manipulations showed much potential, often evolves quickly between related species108,109, and their result was questioned by a subsequent study disruptions in transcriptional regulation could contrib- using much larger sample sizes and greater numbers of ute to reduced fitness in sterile or inviable hybrids110. replicates123. Disruptions of gene expression (defined as levels that Another possibility is to knock out the function of a par- are higher or lower than in both parental strains) have ticular candidate gene, and then show by complementation been documented in interspecies hybrids (for example, that it conferred the barrier of interest. Presgraves et al.124 see REFS 111–113), and have at least sometimes been mapped a hybrid inviability gene using overlapping associated with sterility in hybrids (for example, see chromosomal deficiencies from D. melanogaster to a REFS 114,115). If disruptions of gene expression are the particular cytological region. They then tested inves- cause (and not the consequence) of some hybrid incom- tigator-generated loss-of-function mutations at 12 loci patibilities, then high-throughput ‘reverse-genetics’ that span the region, testing individual D. melanogaster approaches have the potential to quickly identify can- mutations for their ability to uncover hybrid lethality didate genes or candidate pathways that contribute to when heterozygous with the D. simulans wild-type speciation. That said, the challenge then is to determine allele. They found that mutant alleles at only one locus whether the association is through causation. If hybrids failed to complement the D. simulans hybrid lethal have severely underdeveloped gonads, then sterility and factor, and thereby confirmed their candidate hybrid underexpression of gonad-specific transcripts would inviability locus125. both result, but the underexpression says nothing of For emerging model systems in which such tests are the underlying causation. more difficult, in vitro tests of gene function can add support to a proposed candidate gene’s involvement for Direct gene manipulations or assays. For all candidate a barrier that separates two species. Such studies have genes, the final standard of proof for causality rests on been done using interpopulation hybrids of Tigriopus direct genetic manipulations. One can insert the can- californicus copepods, which showed reduced per- didate gene of species A into species B to demonstrate formance in several fitness-related traits relative to that a barrier is formed or disrupted by this insertion. their parents’. Rawson and Burton126 proposed that this Recent advances in technologies for gene manipula- hybrid breakdown was associated with co-adaptation tions can be broadly categorized as transposon-based, between cytochrome c and cytochrome c oxidase. Using reverse-genetics approaches (for example, replacement in vitro assays of enzyme activity, they observed that by homologous recombination) or transgenic116. These the cytochrome c variants isolated from two different approaches have been applied to several genes that populations each had significantly higher activity with confer barriers between species. For example, hybrids the cytochrome c oxidase derived from their respective of D. simulans and D. mauritiana are sterile, and one source populations, providing a mechanistic explana- contributor might be the putative hybrid sterility gene tion for the observed hybrid fitness reduction. In a sub- Odysseus (Ods). This gene was initially identified by sequent study, Harrison and Burton127 used site-directed traditional mapping and introgression approaches117–119. mutagenesis to construct cytochrome c variants and When full-length Ods cDNA from each of the two showed that interpopulation hybrid breakdown can be species was injected into a fertile hybrid line of these attributed to a single, naturally occurring amino-acid

two species, a strong and statistically significant effect substitution. Alas, the story is more complicated, as F2 was seen which depended on which of the two parent hybrid offspring do not show consistently higher fit- species the inserted alleles came from120. This result ness when cytochrome c genotype matches maternal provides a molecular confirmation of the effect of alle- mtDNA-type in a constant 20°C environment128,129. les at this locus on hybrid fertility. However, this effect Nonetheless, seeing how potential barriers can be was only observed when the alleles were inserted into mapped to a single amino acid in non-model systems is introgression lines and not when inserted into pure impressive and promising.

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Box 4 | Examples of putative barrier genes that have been isolated to date Synthesis, advances and prospects The application of molecular genetic and genomics Several putative ‘barrier genes’ have been identified in various species, most of them techniques to the study of speciation has led to sig- in species. Below we provide several examples and their purported Drosophila nificant progress in two areas. First, technical advances barrier effects. These genes are associated with reproductive incompatibilities, but, in some cases, might not act as barrier genes in nature, as other aspects of their have facilitated the mapping and characterization of biology might already prevent gene flow between the focal species. For a more specific genes that are responsible for barriers to gene extensive discussion of these and other examples, see REFS 1,47. flow. Second, more extensive and sensitive genetic and bindin statistical surveys of natural populations have allowed Bindins are acrosomal proteins on the outside of sperm that promote adhesion to inferences to be made about the speciation process and eggs, and their role in species-specific binding has been studied extensively in the nature of species boundaries. Most, although not Echinometra sea urchins138,139. all, of this progress involves improvements in scale and cytc speed, although we have moved into a phase in which we Co-adaptation between cytochrome c, cytochrome c oxidase, and other parts of the now have several ‘barrier genes’ in hand and potentially electron transport system could mediate differences in hybrid viability or many more to come soon (BOX 4). Collectively, these loci development time among Tigriopus californicus copepod populations126,140. However, include both housekeeping and regulatory functions, and 1,130,131 F2 hybrid offspring do not have a consistent pattern of higher fitness when the frequently seem to be targets of natural selection . cytochrome c genotype matches the maternal mitochondrial DNA-type in a Nevertheless, we could be surprised in the future as more constant 20°C environment128,129. barriers are mapped and prove to involve mechanisms desat2 such as meiotic drive or genomic conflict to a greater The desaturase2 (desat2) gene is associated with female cuticular hydrocarbon extent than is currently appreciated. differences between some African and most non-African Drosophila melanogaster141. We are also gaining a clearer picture of the genetic The African allele is also associated with behavioural discrimination by African architecture of species barriers through the analysis 123,142 strains , although Caribbean strains possess the African allele while being of naturally occurring and experimentally generated behaviourally indiscriminate. There are also mixed reports of its effects on hybrids. These studies have confirmed many traditional ecological adaptation121,123. views about speciation, such as the role that geography Hmr often has in population divergence. They have also When mutated, the ( ) transcriptional regulator gene Hybrid male rescue Hmr yielded evidence supporting contentious processes, rescues male hybrids that die at the larval–pupal transition in crosses between D. melanogaster and Drosophila simulans, and restores fertility in hybrid especially in animals, related to divergence-with-gene- females143–145. flow speciation. For example, many more potential cases of sympatric speciation have been proposed50–52. lysin As a result of these discoveries, questions are now Within acrosomes of abalone (Haliotis) sperm lysin interacts with VERL (vitelline envelope receptor for lysin)146 on eggs to mediate species-specific binding of sperm shifting from whether specific types of gene and types to eggs147. Lysins could also mediate fertilization specificity in other gastropods. of process occur to what their relative frequency and importance for speciation are. Genomics is therefore Nup96 A hemizygous (and presumably homozygous) copy of the D. simulans Nup96 gene having a major impact on speciation research, not bears sequence variations that cause inviability when associated with the through a fundamental paradigm shift in theory, but D. melanogaster X chromosome125. However, the effect of this gene is only noticeable by providing rigorous confirmations of hypotheses and facilitating a methodological transition to meta-analysis. in deficiency lines, as F1 hybrids of these species are typically fully sterile. Ods The new techniques are providing the means for compil- Odysseus (Ods) is a homeobox-containing gene that is associated with hybrid male ing extensive catalogues of compelling case studies from sterility between the geographically isolated species D. simulans and representative groups to answer the next generation of D. mauritiana119. Its effect has been shown to require several interactors120,148. frequency issues. Additionally, the techniques can be per used in combination for a more complete understanding The period (per) gene contributes to differences in courtship song between of specific cases. For example, genetic mapping initially D. melanogaster and D. simulans that are associated with behavioural identified the Ods gene that causes hybrid sterility117,118, discrimination149,150. It also functions as a regulator of circadian rhythms. When its effect was confirmed by gene manipulation120, and transformed between species, the gene changes the important courtship song possible downstream targets or consequences of its difference151, but, curiously, no behavioural discrimination is apparent152. allelic replacement were described through microarrays Xmrk2 and real-time PCR115,132. These types of combinatorial The Xmrk2 gene is overexpressed in some hybrid classes that are produced by approach bring us closer to understanding the molecular Xiphophorus maculatus platyfish and Xiphophorus helleri swordtails, and causes mechanisms that underlie speciation. 153 invasive melanomas that can be fatal . Functional tests have confirmed the role of As we continue to move into the era of ‘comparative this gene in tumour formation, but its effects are inconsistent when different strains speciation genomics’ it is important to recognize both of platyfish are used. the strengths and limitations of genomic technologies. YUP Unquestionably, the pace and scale of data acquisition The YELLOW UPPER (YUP) region controls the presence or absence of yellow will accelerate, which is due in large part to advances in pigments in the petals of pink-flowered Mimulus lewisii, which is pollinated by sequencing technology. It is therefore not unreasonable bumblebees, and its red-flowered sister species Mimulus cardinalis, which is pollinated by hummingbirds59. This region could have a role in mediating a species to think that in the next decade a single-investigator grant barrier through differential pollinator attraction, but it has not yet been localized could propose to obtain and compare whole-genome to a single gene. sequences for multiple species or individuals. Annotations of genes will also improve such that the functional

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consequences of any observed differences could be event. Under certain instances of divergence-with-gene- determined computationally, and candidate genes for flow speciation it might be possible to discern the order a particular trait could be identified almost instanta- in which changes evolved between taxa, based on levels neously. In this regard, it is important not to neglect of neutral genetic divergence separating genes. Genes non-model organisms and to develop comparative that restricted gene flow early in a speciation event will genomics for both emerging and model systems to link tend to show greater differentiation for linked sequences field and laboratory-based studies of speciation genetics. than those that arose later. For other speciation events, The ‘genomics revolution’ is not the be-all or end-all however, the best we might be able to do is to infer the for the study of speciation, however, and the new purely possible order of change, based on information from sequence-based and/or expression-based approaches experimental hybridization studies. In addition, large- to studying speciation merely generate hypotheses to scale comparative meta-analyses of taxa in different be tested. Another liability is that our current capacity chronologically or temporally ordered stages of diver- to acquire data has exceeded our analytical ability to gence could reveal trends in which certain types of barrier interpret the results, especially with regards to micro- appear more often than not early in the speciation array experiments. Consequently, many barely inter- process relative to others (for example, see REFS 6–11). preted data sets appear in the literature that are based In conclusion, the technical advances of the past on overly simplistic models. The assumption is often that decade have facilitated and accelerated speciation with enough data, irrespective of how poorly analysed, research. However, contrary to the hype, the new someone will eventually ‘divine’ their true significance genomics approaches have not led to major conceptual and meaning. For now, the new genomic tools are most insights into how we think new species form. Nor do we, reliably used to generate hypotheses, with careful and the authors, foresee any theoretical breakthrough in the precise old-school reductionist bench work still needed near future that would be driven by technology alone. to follow up and test these hypotheses. The new methods are the tools that help to test and A second problem is establishing the chronology of shape imaginative new hypotheses — the real ‘engines’ the genetic and phenotypic changes that lead to spe- of progress. We now know much more about the genetic ciation. This problem pertains to determining both the changes that make new species and the processes that are sequence in which mutations in the same and different responsible for their evolution. One day we might even genes arose to generate a particular barrier and the order reach consensus on the nagging question of what exactly in which different barriers arose during a speciation constitutes a species.

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