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Biological Journal of the Linnean Society, 2002, 75, 509–516. With 1 figure On the use of genetic divergence for identifying species J. WILLEM H. FERGUSON* Department Zoology and Entomology, University of Pretoria, 0002 Pretoria, South Africa Received 25 May 2001; accepted for publication 12 December 2001 Degree of genetic divergence is frequently used to infer that two populations belong to separate species, or that several populations belong to a single species. I explore the logical framework of this approach, including the following assumptions: (i) speciation takes place over very long periods of time; (ii) reproductive isolation is based on the slow accumulation many genetic differences throughout the genome; (iii) genetic divergence automatically leads to reproductive isolation between species; and (iv) pre-mating and post-mating reproductive isolation have a similar genetic basis. I argue that so many exceptions to these assumptions have been demonstrated that they cannot be used with any reliability to distinguish different species. In addition, genetic distance as a species criterion is mostly used within the framework of Mayr’s Biological Species Concept and is not free of assumptions about the nature of species or of speciation. The use of genetic distance to infer separate species (or the lack of these) is not parsimonious, its theoretical foundations are not well understood, and it cannot be applied over a wide range of plants and animals. I explore alternative approaches towards solving the species problems normally solved using genetic distance. © 2002 The Linnean Society of London, Biological Journal of the Linnean Society, 2002, 75, 509–516. ADDITIONAL KEYWORDS: genetic distance – isolating mechanisms – pre-mating isolation – post-mating isolation – speciation. INTRODUCTION Systematists attempt to describe the variation among taxa as well as the historical relationships among them. The rules used in performing this task can be complex: e.g. Johns & Avise (1998) suggested that the criteria for identifying generic and familial level distinctions in birds appear to differ from those used in other taxa. Universal criteria applicable to all taxa and at several taxonomic levels would be helpful in eliminating such inconsistencies. This ideal might perhaps be attainable if systematic techniques were independent of assumptions from theories of evolutionary processes giving rise to taxa. For instance, different approaches to species definitions have yielded quite divergent assessments of the extant biodiversity (Peterson & Navarro-Siquenza, 1999). Theory-dependent analyses would therefore limit the scope of systematic studies. Genetic distance, representing the degree of dissimilarity between the genetic compositions of taxa, therefore appears to be an ideal *E-mail: [email protected] systematic tool (Ayala, 1975). For instance, the mitochondrial cytochrome b gene (cyt b) appears to evolve at a similar rate in a wide array of organisms (Avise, 1994), about 2% per Myr (Johns & Avis, 1998). This enables phylogenetic inference in a wide array of animal taxa (e.g. mammals: Irwin, Kocher & Wilson, 1991; fishes: Lydeard & Roe, 1997; birds: Moore & de Fillipis, 1997). Indeed, since the postulation of the ‘molecular clock’ idea (Zuckerlanderl & Pauling, 1965) provisional dating of phylogenetic events has been possible. Genetic distance measures only the degree of genetic divergence between taxa and is not explicitly bound to any of the current species concepts or process-laden theories. Empirical measurements of genetic differences between closely related species frequently have DNA sequence divergences in the order of 0.2 or with a Nei’s genetic distance (Nei, 1972) D ª 0.1. Following this reasoning, several taxa have been fused into a single species, partly or wholly based on genetic distance data indicating small differences (e.g. Pasquet & Thibault, 1997; Gimnig & Eldridge, 1999; Sastad, Stenoien & Flatberg, 1999). Alternatively, single presumptive species have been split into several species, © 2002 The Linnean Society of London, Biological Journal of the Linnean Society, 2002, 75, 509–516 509 510 J. W. H. FERGUSON partly or exclusively based on genetic distance data indicating large genetic differences (e.g. Hänflig & Brandl, 1998; Paggi et al., 1998; Baric & Sturmbauer, 1999). The reasoning behind this is that, given sufficient genetic differentiation between two particular species, the genetic incompatibility is so clear that hybrids are expected to be inviable (Sasa et al., 1998; Wu & Hollocher, 1998). I have not come across a complete argument explaining the logic of genetic divergence in identifying species, but I believe that Figure 1 gives a fair representation of the reasoning behind this approach. The aim of this contribution is to test the following assumptions required for reliable species demarcations based on genetic distance. 1 There is a fairly clearly defined band of genetic distance values that demarcate species-level genetic differences between lineages. 2 Sufficient genetic distance indicates reproductive isolation between presumptive species (reproductive isolation is here taken as the combination of both pre-mating isolation and post-mating isolation in the sense of Mayr, 1963). 3 Reproductive isolation occurs because of the slowly accumulating number of genetic differences between lineages (an additive quantitative genetic model). 4 Speciation events can be detected as a consequence of the long time periods required for sufficient (A) Genetic divergence (B) Genetic incompatibility (C) Reproductive isolation (D) Lack of gene flow (E) Inference of separate species Figure 1. Visual representation of an argument for using genetic divergence for identifying separate species. genetic divergence between two or more lineages. 5 Genetic distance, as a means of identifying different species, is not theory-dependent. DISCUSSION GENETIC DISTANCES BETWEEN CONGENERIC SPECIES Genetic divergence values have been measured for a large number of species using a range of molecular markers across all the major taxa. In terms of genetic distance (Nei, 1972), within-genus D ranged from 0.01 among bird taxa (Avise & Walker, 1998) to 3.00 among salamanders (Highton & Larson, 1979) with a mean in the order of 0.2 (Nei, 1987). Avise & Aquadro (1982) surveyed genetic distances (Nei, 1972) based on protein-electrophoretic data for 44 vertebrate genera, covering all the vertebrate orders. Within-genus D ranged from ~0 to about 3.0, with significant betweenorder differences in mean values of D (e.g. amphibians around 0.8; mammals around 0.4; birds around 0.1). Johns & Avise (1998) reported on sequence divergence (p) values based on 1800 cyt b sequences. They encountered some differences in divergence ranges between vertebrate orders (birds 0–0.225; herpetofauna ~0–0.27; mammals 0–0.32; fishes 0–0.4) but concluded that the cyt b gene appears to evolve at a constant rate in all the vertebrate orders. For sister taxa, the values ranged from 0 to some 0.2 with modes at about 0.03 (birds 0.035; mammals 0.035; fishes 0.05). Given these ranges of values from both protein-electrophoretic and DNA sequence data, it appears that a clear, predictive value for separating species-level differences from population-level differences has not emerged. In a statistical sense, the standard error of the estimate of the degree of genetic divergence required for taxa to constitute separate species is so large that that it is not useful in this particular way. GENETIC DIVERGENCE AND REPRODUCTIVE ISOLATION Reproductive isolation is the keystone of the Biological Species Concept (Mayr, 1963). Accordingly, species are separated by isolating mechanisms that prevent interbreeding. These can be grouped into pre-mating isolating mechanisms (e.g. behavioural and physical incompatibility) and post-mating isolating mechanisms (e.g. genetic incompatibility resulting in death of embryos). The literature emphasizes the view that sufficient genetic divergence results in reproductive isolation between species (Wu & Hollocher, 1998). Indeed, several authors (e.g. Coyne & Orr, 1989; Sasa et al., 1998) have produced strong evidence of a correlation between genetic divergence and reproductive © 2002 The Linnean Society of London, Biological Journal of the Linnean Society, 2002, 75, 509–516 GENETIC DIVERGENCE FOR IDENTIFYING SPECIES isolation between species. This correlation appears to apply to both pre-mating and post-mating isolation (Gleason & Ritchie, 1998), at least in some taxa. The crucial unresolved question is whether the relationship between genetic distance and reproductive isolation is the consequence of cause-and-effect, or a product of correlation with other common genetic and evolutionary factors. Wu & Hollocher (1998) appear to favour the first alternative, while Templeton (1994) appears to favour the second. If this correlation resulted from cause-and-effect, we would expect postmating isolation between taxa with genetic divergence to be sufficient for consideration as separate species. However, numerous cases have been documented where this is not the case and where hybrids are fertile, e.g. for Drosophila (D = 0.212; Zouros, 1973, 1981), crickets (D = 0.22; Howard, 1983; Howard et al., 1993), and frogs (D = 0.4–1.11; Pyburn & Kennedy, 1961; Mecham, 1965). On the other hand, numerous cases exist where populations assigned to a single species appear to have pre-mating isolation, e.g. for Drosophila (Hollocher et al., 1997a; Hollocher, Ting & Wu, 1997b) and amphibia (D = 0.05–0.4; Kuramoto, 1984, and Tilley, Verrell & Arnold, 1990). Species with polyploid forms have post-mating isolation (White, 1978) and, by definition, little genetic distance. An increase in genetic distance into the range commonly observed between sibling species therefore does not necessarily lead to reproductive isolation. The converse applies too. SHOULD ONE RATHER USE GENETIC DIVERGENCE SUFFICIENT FOR PRE-MATING ISOLATION BETWEEN SPECIES? Within the context of genetic divergence, pre-mating isolation should be considered separately from postmating isolation. Wu, Johnson & Palopoli (1996) argued that post-mating isolation arises as a correlated effect of pre-mating isolation. If this were true, reproductive isolation between species, and the corresponding degree of genetic divergence, should be measured in terms of pre-mating isolation. Coyne & Orr (1989) investigated pre-mating and post-mating isolation in several Drosophila species and concluded that, among sympatric species pairs, pre-mating isolation arise more rapidly than post-mating isolation. However, this difference was not marked in allopatric species pairs. Gleason & Ritchie (1998) measured song characteristics, and pre-mating and post-mating isolation in the Drosophila willistoni species complex and found that pre-mating isolation increased much more rapidly with genetic divergence than post-mating isolation, while song characteristics did not relate to genetic distance in any way. These studies indicate 511 that, at least within Drosophila, pre-mating isolation may arise before post-mating isolation in many cases. This would appear to indicate that the degree of genetic divergence required for pre-mating isolation is often less than that required for post-mating isolation. However, the situation is apparently not so simple. Studies on salamanders by Tilley et al. (1990) and on the Tungara frog by Ryan, Rand & Weigt (1996) indicate that, after genetic divergence has been corrected for, a large proportion of pre-mating isolation is accounted for by geographical distance. In the salamander study, the correlation of 0.67 (P < 0.008) between geographical distance and pre-mating isolation is reduced to 0.46 (P < 0.027) by removing the effects of genetic divergence. In summary, these studies indicate that genetic divergence by itself is not sufficient to explain pre-mating isolation, but that several additional factors, e.g. degree of sympatry and geographical range, have a strong effect on the genetic distance measured. In addition, the detailed relationship between genetic divergence, pre-mating isolation and post-mating isolation is not clear. If pre-mating isolation proceeds faster than post-mating isolation, genetic divergence required for pre-mating isolation would need to be used for diagnosing species. But then the above studies suggest that, except at large degrees of divergence, genetic divergence is not a good predictor of degree of pre-mating isolation. Although there is a general trend for reproductive isolation to increase with genetic divergence, this relationship has very little predictive use for identifying new species, owing to the large number of violations of the trend. The relationship between genetic divergence and reproductive isolation is probably the result of correlated responses and not a cause-and-effect relationship. DO REPRODUCTIVE ISOLATION AND GENETIC DISTANCE HAVE THE SAME GENETIC BASIS? The reason why genetic divergence is proposed to result in reproductive isolation, is because it reflects the accumulation of genetic differences at many loci over a long period of time. Wu & Hollocher (1998) argued that large numbers of genes are responsible for reproductive isolation between taxa and that, because of gradual change at all these loci over time, there should be a correlation between genetic divergence and reproductive isolation. The proposal that reproductive isolation is caused by the action of many loci needs to be considered because there is a direct relationship between the number of loci involved and the genetic distance calculated from a random sample of loci, used to infer taxonomic status. Wu & Palopoli (1994) and Perez & Wu (1995) argued that a large number of factors cause post-mating isolation in fruit flies. In terms of pre-mating isolation, Ehrman (1961) © 2002 The Linnean Society of London, Biological Journal of the Linnean Society, 2002, 75, 509–516 512 J. W. H. FERGUSON argued that all the chromosomes contribute to mate preference behaviour in Drosophila paulistorum. Marin (1996)) estimated that up to a quarter of 60 sampled factors per chromosome are involved in male sterility in D. koepferae ¥ D. buzzatii crosses, thus supporting much of the ‘generalized polygenic model’ of Palopoli & Wu (1994). In contrast, Carvajal, gandarela & Naveira (1996), investigating post-mating isolation in the D. koepferae ¥ D. buzzatii combination, concluded that three marker areas contributed towards this phenomenon. Several studies have shown that smaller numbers of loci are involved in reproductive isolation. Zouros (1981) suggested that assortative mating in D. arizonae is affected heavily by the sex chromosomes, a phenomenon encountered in many studies and which is especially pronounced in the Lepidoptera (Prowell, 1998). Coyne, Simeonidisa & Rooneya (1998) found that 4 out of 117 chromosomal regions contribute to hybrid conditional inviability in D. melanogaster ¥ D. simulans crosses. Coyne, Crittenden & Mah (1994) indicated that chromosome 3 contributes strongly to the pheromonal constituents involved in male/female signalling. Similar results were obtained by Noor & Coyne (1996). At the other extreme, Henry (1994) suggested that three or four loci were involved in his studies on lacewing (Neuroptera) communication. Coyne (1993) suggested that, in male D. simulans, three major genes shape the male genitalia that, in turn, are involved in interspecific breakdown of mating with D. mauritiana. In a similar study, Liu et al. (1996), using QTL techniques, found that fewer than ten genes contribute to shape of genitalia in these flies. Sixteen out of twenty five studies surveyed by Ritchie & Phillips (1998) indicated major gene effects on pre-mating isolation, while the remaining nine studies did not suggest such a relationship. Overall, it appears that studies on the genetic basis of pre-mating isolation show many cases of major gene effects, whereas similar studies on post-mating isolation frequently suggest a more complex genetic basis. What does this mean in terms of genetic distance as a measure for diagnosing species? Firstly, species with strong pre-mating isolation and weak post-mating isolation are likely to have little genetic divergence because pre-mating isolation could be brought about by fewer than ten loci. Secondly, the long time periods required for sufficient genetic divergence of two taxa in order to be termed separate species are probably frequently not required for strong pre-mating isolation. Thirdly, the genetic basis of reproductive isolation varies so much between taxa that a predictive rule about degree of genetic divergence required for the recognition of separate species is not possible. For pre-mating isolation the results are in favour of a relatively simple genetic structure. For post-mating isolation the results do not show a clear trend. Although it is generally accepted that genetic distance is brought about by the slow accumulation of point mutations and small genetic changes between taxa (Johns & Avise, 1998), the same is not necessarily true for reproductive isolation. In fact, genetic divergence and reproductive isolation may have different genetic bases. THE TIME DURATION REQUIRED FOR SPECIATION As genetic divergence between two isolated populations takes place gradually, one would expect that, on an ecological timescale, speciation according to the polygenic model (Palapoli & Wu, 1994) would take a long time. Indeed, Klicka & Zink (1997), Avise & Walker (1998) and Avise, Walker & Johns, (1998) used DNA sequence data to estimate the mean duration of speciation in the vertebrate orders and compared genetic divergence between, on the one hand, withinspecies populations which have been separated for long times and, on the other, within-genus sister taxa. Their results revealed that a modal sequence divergence of some 13% was observed between sister species, whereas smaller degrees of divergence tended to characterize major within-species phylogroups. This led them to conclude that a large number of speciation events take in the order of 4 Myr and that the majority of speciation events may have occurred before the pleistocene. This evidence could be taken to support the gradual, multilocus approach outlined above. However, palaeontological data indicate that speciation in many cases occurs during much shorter periods. Firstly, the isthmus of Panama arose some 3 Myr (Holcombe & Moore, 1977) and separated the Pacific and Atlantic oceans. Extensive speciation events in many taxa, including shrimps (23 pacific species, 22 Caribbean species; Chace, 1972), gastropods (22 pairs of sister taxa; Vermeij, 1978) and sea urchins (Lessios, 1998) have been described around the isthmus. Secondly, many of the subfamilies of the Bovidae (e.g. Alcelaphinae and Hippotraginae) in Africa arose some 5 Myr (Gentry, 1978; Vrba, 1979, 1985). A radiation in the Bovidae has taken place over the last 3 Myr, resulting in 120 extant species (Vrba, 1985). Analysis of cyt b sequences of the Alcelaphinae (hartebeest clade) yielded sequence divergences between 2% and 10% (Matthee & Robinson, 1999), suggesting fairly recent origins for some of the lineages. Thirdly, the lakes of the African Rift Valley are rather young; Lake Victoria and Lake Malawi were formed between 1 and 2 Myr (Doornkamp & Temple, 1966). Lake Malawi presently has some 450 endemic cichlid fish species (Ribbink et al., 1983). Lake Victoria dried up some 15 000 years ago and was dry for several thousands of years (Johnson et al., 1996), yet it contains some 300 endemic fish species (Johnson © 2002 The Linnean Society of London, Biological Journal of the Linnean Society, 2002, 75, 509–516 GENETIC DIVERGENCE FOR IDENTIFYING SPECIES et al., 1996). In all the above cases, speciation events must have been considerably shorter than the 4-Myr yardstick posed by Klicka & Zink (1997) and Avise et al. (1998). I acknowledge that these last authors did not intend to show that most speciation events require more than 4 Myr: they intended to show that the majority of speciation events must have taken place before the Pleistocene. If the 450 extant cichlid taxa in Lake Malawi are taken as terminal taxa of a binary tree, some 21 consecutive bursts of speciation would be required (on average about 50 000 years per speciation event). If the same manipulation is performed on the Lake Victoria fish, there would be 17 bursts of speciation, each on average taking some 1500 years. In fact, McCune (1997) and McCune & Lovejoy (1998) report on genetic divergence of cyt b between species pairs of fish in the African lakes and commonly found values between 0% and 1%, implying times for divergence of less than 1 my. Taken together, these results suggest that the 4-Myr yardstick of Klicka & Zink (1997) and Avise et al. (1998) is not universal. Either there are geographical differences in speciation rates (their data were biased towards North America while my examples above come mostly from Africa) or the phylogroups that these authors analysed were much older than a large number of sister species, resulting in an overestimate. Another line of evidence suggesting speciation may take less time than the gradual genetic divergence implied above, is the rapid molecular diversification in marine invertebrate gametic recognition proteins reported by Swanson & Vacquier (1995), Palumbi (1996) and Metz, Robles-Sikisaka & Vacquier (1998). These authors found significantly higher non-synonymous mutation rates than synonymous mutation rates at the 5¢ end of the bindin protein, with some 17% amino acid substitutions among closely related sea urchin species (Metz & Palumbi, 1996). Overall genetic divergence in these organisms would result in a different temporal perspective, compared with the molecular analysis of the bindin gene that contributes largely to reproductive isolation (Palumbi, 1992). In terms of using genetic divergence as a yardstick for identifying species, this means that long periods are not always required for speciation and that the steady build-up of genetic differences between isolated taxa cannot be used as a predictive yardstick for identifying species. The most important conceptual problem about using genetic divergence for identifying species is that genetic distance is an indication of the duration for which two taxa being compared have been separated. Avise et al. (1998) showed that some separated populations (phylogroups) show more divergence than many between-species comparisons. This indicates substantial genetic differentiation over long time periods, but without any speciation events. Proof of 513 long-term genetic isolation between two populations is not sufficient for implying that they are separate species. In order to become separate species, behavioural and/or ecological changes also need to take place that would result in distinct gene pools for each species. This is reflected in Templeton’s (1994) plea that the genetic processes involved in speciation should be separated from the genetic characteristics that differ between two extant species. This is a fundamental distinction for systematists. The second conceptual problem relates to the argument that the use of genetic distance for inferring species is not strongly bound to any particular theory of speciation. Scientists attempting to understand the relationship between genetic distance and specieslevel distintness have almost exclusively used the Biological Species Concept (Dobzhansky, 1941; Mayr, 1963) as a point of departure. The construct of premating isolation and post-mating isolation is one of the central components of the Mayr’s (1963) exposition of this species concept. The discussion above argues that each of the steps indicated in Fig. 1 is based upon theoretical presuppositions, some of which may be false and all of which do not have universal applicability. The use of genetic distance to infer species is therefore explicitly theory-laden. CONCLUSION While acknowledging that very few scientific methodologies are completely free of theoretical assumptions (Chalmers, 1986), and keeping in mind the criticism against the Biological Species Concept (e.g. Mallett, 1995), systematists still need to aim for tools that are not tightly bound to any particular theory of species. An approach to such a tool comes from the logic that I assume underlies the use of genetic divergence for identifying species (Fig. 1). If this logic is realistic, it would be advantageous to use an approach which omits as many of the steps as possible towards reaching a conclusion about the species status of taxa in question. Steps that could be omitted without affecting the genetic conclusion are (A) to (C) in Figure 1. Lack of gene flow could be demonstrated in a genetic way, e.g. by measuring FST (Raymond & Rousset, 1995) or by analysis of molecular variance (Michalakis & Excoffier, 1996). Because this type of estimate has a wide standard error, it often does not allow precise estimations of gene flow (Whitlock & McCauley, 1999). However, it does enable the detection of very small levels of gene flow as compared to extensive gene flow. This would simplify the argument in Figure 1 considerably. Using genetic divergence to infer lack of gene flow is unnecessary. From a genetic point of view, a much more unambiguous statement about the species status of taxa can be made if one could demonstrate © 2002 The Linnean Society of London, Biological Journal of the Linnean Society, 2002, 75, 509–516 514 J. W. H. FERGUSON that populations differ in fixed genetic characteristics. This would imply genetic differentiation as well as a lack of gene flow between the taxa. This approach is used by many systematists (but many others continue to use genetic divergence). My suggestion above has three advantages. Firstly, a conclusion could be reached using the same protein-electrophoretic or DNA sequence data as those analysed for genetic divergence, but perhaps incorporating larger sample sizes from each population. Secondly, such an approach towards the genetic identification of species would be much more removed from theories of speciation. Thirdly, the data would be in a form much more compatible with operational species definitions such as the phylogenetic species (Cracraft, 1983) that relies on constant diagnosable differences for inferring species. An important caveat of the above argument is that many populations have been geographically separated for long times and have no gene flow, but presumably still comprise single species. From the genetic point of view, coalescence theory (Templeton, 1994; Avise & Wollenberg, 1997) may add some perspective to the problem, but ultimately behavioural and ecological information reflecting the mate recognition or proposed pre-mating isolation between two populations would be crucial. Genetic distance would not contribute towards elucidating the problem. The aim of this paper is to contribute towards operational molecular yardsticks for identifying separate species. It does not aim to disqualify genetic divergence as a useful tool in systematics. 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