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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
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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
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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
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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)
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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
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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
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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. It is useful
in many ways, e.g. in population-level analysis and
phylogeography, but on its own it is not useful for
identifying separate species. Systematists need tools
that are parsimonious, have well understood foundations, and that can be used consistently across a wide
range of taxa. In terms of the identification of new
species, genetic divergence fails on all three of these
criteria.
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