Biological Journal of the Linnean Sociey (19891, 36: 281-294.
Genetic anomalies associated with Cerion
hybrid zones: the origin and maintenance of
new electromorphic variants called hybrizymes
DAVID S. WOODRUFF
Department of Biology and Center for Molecular Genetics, Uniuersity of Calzfornia San
Diego, La Jolla, California 92093 U.S.A.
Received 13 October 1987, accepted for publication 10 M a y 1988
Natural hybrid zones involving the West Indian pulmonate land snail Cerion are characterized by
the occurrence, in low to moderate frequencies, of allozymes that are unique to interspecific hybrid
zones. As such electrophoretically detected genetic anomalies havr also becn reported in hybrid
zones involving mammals, birds, reptiles, amphibians, and insects this appears to be a general
phenomenon. These unexpected allozymes are inappropriately called ‘rare alleles’ and the term
hybrizyme is introduced. The origins of hybrizymes are discussed in terms of suppressor-mutation
systems, transposon-induced hybrid dysgenesis and intragenic recombination, but available evidence
will not resolve this issue. Similarly, it is not clear whether the relatively high frequencies of
particular hybrizymes are due to selection or genetic drift or some combination of these agents.
Finally, the evolutionary significance of hybrizymes and the possible role of hybridization in
introducing new genetic variation into populations are discussed.
KEY WORDS:-Hybrid
zone - genetic variation - allozymes - mutation - hybrids
-
snails - Cerion.
CONTENTS
Introduction .
. . .
Terminology . . . .
The evidence .
. . .
Hybrizymes in Cerion .
Other cases . . .
Discussion.
. . . .
Nature of hybrizymes .
Origin of hybrizymes .
Frequency of hybrizymes
Conclusion .
. .
Acknowledgements
. .
References.
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281
282
282
282
285
287
287
287
290
29 1
292
292
INTRODUCTION
Natural hybrid zones are typically regions of genetic anomaly. In three
morphologically defined hydrid zones involving the West Indian land snail Cerion
I found that the populations in and around the hybrid zone were characterized by
the occurrence, in low to moderate frequency, of allelic electromorphs that are
either unique to the zone or of strictly localized occurrence in the homospecific
0024-4066/89/0302ai
+ 14 sos.oo/o
28 I
0 1989 The Linnean
Society of London
282
D. S. WOODRUFF
parental populations. As such electrophoretically detected genetic anomalies have
also been reported in hybrid zones involving rodents, bats, frogs, salamanders,
clams and insects this appears to be a general phenomenon. I n this report I
provide empirical data and discuss the possible etiology and significance of the
phenomenon.
1ERMINOLOGY
Previous reports of the genetic anomalies associated with hybrid zones have
referred to the unexpected alleles in the context of “the rare allele phenomenon”
(Sage & Selander, 1979; Woodruff & Gould, 1980; Barton & Hewitt, 198 1, 1985;
Harrison, 1986). As it is now clear that such alleles often reach frequencies of
above 0.10, and can occasionally become the commonest allele locally, the
adjective ‘rare’ is inappropriate. Furthermore, the term ‘rare allele’ has always
had a more self-evident use in the genetics literature meaning a truly rare, minor
or private variant (sensu Neel, 1973; Slatkin, 1985, 1987; Barton & Slatkin, 1986;
Takahata & Slatkin, 1986; and references therein). I am accordingly introducing
the term hybrizyme for these unexpected allelic electromorphs associated with
hybrid zones. This term avoids the etiological connotations of some alternatives,
seems less problematic than heterozyme or bastardizyme, and respects the prior
usage of ‘hybrid gene’ by developmental geneticists (Kelly & Darlington, 1985)
and others (Nei, 1987).
In the following accounts I have used conventional abbreviations for various
allozymic loci; the reader is referred to the original papers cited for Enzyme
Commission numbers and the specific details of enzymatic resolution.
THE EVIDENCE
HybriQmes in Cerion
The West Indian pulmonates of the genus Cerion promise to contribute much
to evolutionary biology; for a prolegomenon to these land snails and the
problems they present see Woodruff (1978) and Woodruff and Gould (1980).
The group is characterized by extreme interpopulation variation in shell size,
shape, colour and sculpture. This morphological variation has led to the naming
of over 600 species in Cuba and the Bahamas. With very few exceptions these
taxa are parapatric in their distributions but their ranges are typically connected
by very narrow hybrid zones. Woodruff (1981) described five such zones in
general terms and discussed their significance with respect to models of allopatric
and parapatric speciation. Two generalizations about the Cerion hybrid zones are
relevant to the present paper. First, the striking morphological differences in the
adult shell are apparently generated by relatively simple changes in shell
deposition rates (Gould, 1977, 1984; Galler & Gould, 1979). Cerion’s great
diversity may be the product of heterochronous evolution at relatively few
regulatory genes. Second, although the various species of Cerion show little
differentiation at electrophoretically detectable loci, hybrid zones separating
these taxa are areas of genetic anomaly. They are characterized by the
occurrence of unique electromorphs and the presence of typically rare
electromorphs at low to moderate frequences. Three Bahamian examples of this
phenomenon may now be described.
NOVEL GENES IN HYBRID ZONES
283
O n Long Island in the Bahamas, C. stevensoni and C.fernandina are restricted to
within 50 m of the east coast. Their ranges are separated by a hybrid zone less
than 300 m wide occupied by snails of intermediate conchological phenotypes.
Although the contrasting parental morphotypes are not clearly differentiated on
the basis of variation in anatomy or allozymes, the hybrid zone between them is
characterized by unexpected alleles at three loci: Es-2, Mdh-I and 6Pgd
(Woodruff, 1981). .Thirty-six samples involving over 900 snails were collected
along a 4175 m transect and were surveyed for variation at these three
polymorphic loci (Table 1). The data show that the genetic hybrid zone is
several times wider than the morphological transition and is asymmetrically
TABLE
1. Frequency of minor electromorphs in samples of land snails from a linear transect across
a zone of hybridization between Cerion stevensoni and C.fernandina on Long Island, Bahamas
Es-2
Sample*
42 1
612
614
420
616
615
617
Mdh-1
D
N
0
0
410
475
475
485
500
13
32
23
26
49
43
13
32
23
21
52
43
15
15
n
6-Pgdh-I
N
618
469
470
47 1
4728
477H
4788
473H
474H
621H
475H
476H
622H
459H
623
624
625
458
628
650
725
1100
1125
1195
1220
1235
1245
1295
1320
1345
1395
1395
1395
1475
1600
1750
1850
1850
27
17
35
29
20
11
15
28
25
32
21
36
32
6
55
31
27
30
32
27
24
20
19
19
9
15
18
19
32
18
19
32
6
48
31
23
24
32
626
627
378
377
375
542
540
539
538
537
2200
2200
2250
2425
2950
3350
3400
3500
3700
4175
21
43
1
6
4
30
12
4
42
30
21
43
I
15
4
30
12
4
42
30
n
-
~
-
-
~
~
N
q
13
32
24
0.06
~
~~
~
__
-
~
0.02
1
0.05
0.08
0.06
0.13
0.03
0.03
0.03
0.14
0.08
0.03
2
3
-
0.06
0.11
0.26
0.13
0.19
~
1
4
1
1
2
5
3
2
~
6
7**
12
5
10
4
-
-
n
44
37
13
0.05
0.01
0.04
27
0.17
0.02
-
28
4
1
1
~
25
35
29
20
11
15
28
28
32
27
37
32
6
48
30
20
18
32
22
39
1
6
4
30
12
4
42
30
9***
I
~
~
~
0.05
0.18
0.13
0.07
0.14
0.06
0.06
0.08
0.06
2
4
4
3
6
4**
3
5
2
-
-
0.04
0.35
0.05
0.19
0.28
0.02
0.03
4
15
2
5**
17**
I
2
-
.~
~
0.03
~
2
~
~
~
~
0.02
~
1
*Samples 421-617: C. stevensoni; 618-628: genetic hybrid zone; 626-537: C.fernandina. H, Sample of hybrid
morphology; D, Distance in m from north end of transect; N, No. of snails examined; q, Frequency of minor
electromorph(s); n, No. of snails with minor electromorph(s); **/*** 2 or 3 minor electromorphs detected.
284
D. S. WOODRUFF
distributed about it, The precise width of the genetic transition varies with
) a
different loci. There are five alleles segregating at Es-2; one of these ( E S - ? ' ~has
) , found only in the
frequency of 0.8--0.95 in this area, another ( E S - , ? ~ ~was
heterozygous state in three conchological hybrids. In the case of NAD-dependent
Mdh-1, the parental populations are fixed for Mdh-l'.". Two other alleles were
detected in the hybrids: Mdh-l'." was found in samples up to 1125 m apart and
rises to a frequency of 0.26; Mdh-1°.50was detected as a single heterozygote in an
animal with intermediate morphology. Variation at a third locus, 6Pgd was also
noted by Woodruff (1981); subsequent work has revealed a total of five alleles at
this locus in this area. Samples of both parental species from outside the hybrid
zone are characterized by high frequences of 6Pgdl.I' and moderate frequencies of
6Pgdl.". Three other electromorphs have now been found in samples from the
hybrid zone: 6Pgdag3is widespread and rises to a frequency of 0.35, 6Pgd1.05is rare
was found in a single
(found as heterozygotes in four samples), SP~O?.~~
heterozygote from one edge of the zone. T h e relative frequencies of these
unexpected electromorphs are shown in Table2; they are one or two orders of
magnitude more common in the hybrid zone than in homospecific populations
from elsewhere.
The second example involves two allopatric semispecies, C. abacoense and
C. bendalli, on Great Abaco Island. A zone of allopatric hybridization between
these taxa has been described in broad outline by Gould and Woodruff (1978)
and Woodruff (1981). As in the previous case the zone is narrow; the
morphological transition is about 500m wide in one area and the genetic
transition is several times wider and asymetrically distributed around the
morphological zone. Based on a survey of 20 loci in 1798 snails from 58 localities it
was found that the parental taxa were weakly differentiated (Nei's (1978)
D = 0.04) and that the hybrids were characterized by higher frequencies of unique
or normally rare alleles of nut-1, 6Pgd and Es-2 (Table 2). This observation,
coupled with the finding of higher levels of mean genic heterozygosity, H, in
hybrid samples ( H = 0.083-0.1 15 vs. 0.053-0.070 for samples from away from the
interaction), define the hybrid zone as an area of marked genetic anomaly. I n the
case of 6Pgd, the parental populations are fixed for electromorph 6Pgd'.O0. The
hybrid zone is associated with the unexpected appearance of electromorph
6Pgd.""; this allele is very rare elsewhere on Great Abaco but rises to a frequency of
0.67 in one hybrid zone sample. Similarly, in the case of Es-2, where two alleles
are segregating in the most populations on Abaco, two additional alleles were
~ ~ of Gould & Woodruff, 1978; Woodruff, 1981)
found in the hybrids, E S - Z ' .(Es-26
occurs at fre uencies of up to 0.10 in the hybrid zone and is very rare elsewhere on
Abaco. Es-29'11 (formerly Es-25) was detected as a single heterozygote within the
hybrid zone and has not been found elsewhere on the island. The relative
frequencies of these minor alleles are set out in Table 2.
The third example involves C. glans and C. gubernatorium on New Providence
Island. A morphological hybrid zone less than 500 m wide is associated with the
presence of unexpected electromorphs (Woodruff, 1981). A survey of variation at
23 presumptive loci in 1086 snails from 34 sites across the island revealed that the
parental taxa are weakly differentiated (D=0.05) and that the zone of genetic
anomaly is 2-3 times as wide as the morphological hybrid zone (Gould &
Woodruff, 1986). Five unexpected alleles were detected in or near the hybrid
zone and were not found elsewhere (loc. cit., see Figs 23, 25-27, 31, 33). These
NOVEL GENES IN HYBRID ZONES
285
TABLE
2. Frequency of Cerion with unexpected electromorphs in homospecific (P)and hybrid (H)
samples from three regions in the Bahamas
Interaction
Locus
Type
S*
N
n/ 1000
P
H
H
P
P
H
17
19
19
19
17
19
394
509
401
435
381
500
0
6
0
I49
42
172
P
H
24
34
24
34
24
34
841
957
841
960
841
957
2
13
5
32
0
392
P
H
P
H
P
H
P
H
12
24
12
24
12
24
12
24
336
750
336
750
336
750
336
750
0
4
Cerion steuensoni-C. fernandina, Long Island
Es-2
Mdh-1
6Pgd
Cerion abacoense-C. bendalli, Great Abaco Island
Aat-1
H
Es-2
P
H
6Pgd
P
Cerion glans-C. gubernatorium, New Providence Island
Aat- I
Es-2
Gpi
I'gm-1
~~
~
1
79
0
128
0
44
~
*S, number of samples, N, no of snails; n, no. of variant snails.
alleles reached moderate frequencies (Table 2). Pgm-lo.' was noted in ten of the
24 hybrid samples and has a frequency of 0.02-0.14 (mean=0.056) when
( 7 sites; freq. =0.01-0.11;
present. Analogous patterns were seen for E S - P . ~
mean = 0.037) and Es-Z'.'~ (16 sites; freq. = 0.01-0.1 1; mean = 0.049). Cgpio.6was
detected at low frequencies at four sites at the Blake Road contact (0.02---0.11)
and at high frequencies (0.18-0.44) at five sites at the Gambier village contact
3 km further west (loc. cit., Fig. 1). A ~ t - l ' was
. ~ the rarest, being found as single
heterozygotes at only two sites.
Before leaving the Cerion examples, it is worth noting the similarity in relative
mobility between hybrizymes on different islands. The reader may have been
struck by the inferred similarities between ES-L?~~
$Long Island), Es-T9' Abaco)
and E S - . ? ~ (New Providence), between Es-~'.' (Abaco) and Es-2'. (New
Providence), and between the slow Aat-1 allele on Abaco and New Providence.
These allelic designations were developed during the course of separate studies
and unfortunately we have not yet compared the mobility of the hybrizymes from
the different zones on the same gels. The slight differences may be real, or the
electromorphs may be homologous; regardless of their relationships other
evidence suggests they probably arose independently in each interaction.
1
Other cases
Barton and Hewitt (1985 : 135) found that "In most (19 out of 23) thorough
electrophoretic surveys of hybrid zones, an increased frequency of rare alleles has
286
D. S. WOODRUFF
been found". Well-documented cases involving more than 600 specimens include
the house mouse, Mus, in Denmark, and the leopard frog, Rana, in Texas. I n the
case of the M . musculus-M. domesticus interaction unexpected alleles were detected
in hybrid populations at four loci: Es-2, Es-3, Me-1 and M d h - 2 (Hunt & Selander,
1973; Selander, personal communication). Es-3, for example, has a frequency of
< 1/1000 in homospecific mice but up to 57/1000 in hybrid zone samples. Me-1,
on the other hand, was only ever seen in three heterozygous mice from one hybrid
zone sample. I n the R. berlandieri-R. utricularia case unique alleles were found a t 3
of 10 loci examined from across the 36 km wide hybrid zone (Sage & Selander,
1979; Kocher & Sage, 1986; Selander, personal communication). Aat-1, A d a and
Ldh-2 hybrizymes had a background frequency in homospecific populations of
< l/lOOO and a mean frequency in the hybrid zone of 40/1000.
In the first paper devoted exclusively to this phenomenon Barton, Halliday &
Hewitt (1983) described the hybridization of two chromosomal races of the
grasshopper Podisma pedestris at two sites 100 km apart in France. They studied
variation at 2 1 loci and found hybrizymes at Idh, GPgd, M d h - 1 and Mdh-2. T h e last
two showed five-fold increases in frequency in the center of the hybrid zone at
both sites. This example is particularly interesting, and similar to that involving
Cerion on Abaco, in that the parental taxa were almost indistinguishable at the loci
surveyed.
Another example involves the deer mice Peromyscus c. calzfrnicus and
P. c. insignis which hybridize in California. Smith (1979) found unexpected
electromorphs were 3-20 (mean = 12) times commoner in hybrid samples. The
approximate frequencies of variant deer mice may be estimated from the data
presented for homospecific/hybrid samples: Post-Alb 0.0 159/0.0488, M e - 1 0.0 l06/
0.2195, Pgm-1 0.0476/ > 0.3415, GPgd< 0.002/0.0488 and Es-30.0053/0.0244.
Greenbaum ( 1981 ) described the genetic interactions between hybridizing
cytotypes of the Central American tent-making bat Uroderma bilobatum. The
northern U. b. dauisi (2n=44) interacts with the southern U. 6. conuexum (2n= 38)
in a karyologically defined zone of allopatric hybridization about 400 km wide
(Baker, 1981). Reinterpreting the allozyme data presented by Greenbaum,
Hafrier (1982) concluded that the hybrid zone was an area of genetic anomaly
characterized by nine unexpected rare alleles: Aat-216*, Aat-p8, AlbgO, Hb1I3',
Ldh-T", Mdh-1140, h4dh-T'46, 6Pgd"' and Sdh-62. These unexpected electromorphs
occurred at every locality in the hybrid zone (Greenbaum's locs. 2-10, where a
total of 246 bats were sampled) which according to Hafner's reinterpretation
may be closer to 40 km wide. These hybrizymes occurred at average frequencies
of 8-45/1000 bats.
Other cases include those involving white-footed mice, Peromyscus (Nelson,
Baker & Honeycutt, 1987), pocket gophers, Thomomys (Patton, Hafner, Hafner &
Smith, 1979; Hafner, Hafner, Patton & Smith, 1983), warblers, Dendroica
(Barrowclough, 1980), lizards, Anolis (Case & Williams, 1984), Sphaerodactylus
(Murphy, McCollum, Gorman & Thomas, 1984), amphibians, Bolitoglossa
(Wake, Yang & Pappenfuss, 1980), Bombina (Szymura & Farana, 1978;
Gollmann, 1986; Gollmann, Roth & Hodl, 1988), Bufo (Green, 1984), Ensatina
(Wake, Yanev & Brown, 1986), Plethodon (Duncan & Highton, 1979, Wynn,
1986), Pseudacris (Gartside, 1980), grasshoppers, Chorthippus (Butlin & Hewitt,
1985), Caledia (David Coates, Chris Moran, personal communication) and field
crickets, Gryllus (Harrison, 1986), and clams, Anodonta (Kat, 1986).
NOVEL GENES IN HYBRID ZONES
287
DISCUSSION
Nature of hybrizymes
T h e evidence for hybrizymes involves such a variety of organisms that it would
appear to be a general phenomenon. T h e unexpected electromorphs were
detected at loci for a wide range of proteins including variable substrate esterases,
regulatory enzymes like Me-1, and non-regulatory enzymes like Mdh. Most often
the hybrizymes were found a t loci which were also polymorphic in homospecific
populations, that is, at 50%, 86% and 7 1 yo of the variable loci surveyed in Cerion,
Mus and Rana, respectively. The instances of hybrizymes at loci which were
monomorphic in parental populations are thus exceptional. Only additional
comparative studies will show whether hybrizymes fall more consistently into
Lewontin’s (1985) category of highly polymorphic loci than into his other
categories. However, it must be remembered that single-gel electrophoretic
surveys will underestimate true heterozygosity and some misclassification may
have occurred.
T h e variant electromorphs under discussion here behave as typical allozymes.
They presented staining patterns that were typical of codominant or null alleles
for the enzyme and species-group. Although formal genetic analyses have not yet
been undertaken I am convinced that the Cerion hybrizymes are allelic and not
artifacts of one particular laboratory. I n both Mus and Cerion it is clear that the
various loci at which hybrizymes were found are not closely linked to one another.
I t is also clear that in the majority of samples where hybrizymes were detected at
moderate frequencies the various presumed alleles were found segregating in
accordance with Hardy-Weinberg expectations (Woodruff, in prep).
I n the preceding account I have noted the frequency of variant electromorphs
in each sample and found that they were often one or two orders of magnitude
more common in hybrid than in homospecific samples. There are several reasons
why it is impossible to calculate the rate a t which new alleles are appearing in
the hybrid zones from data of the type available. First, in the absence of
information about the genotypes of the parents of each individual sampled we
have no way of separating variants created de nouo from those inherited. Second,
in the cases of Cerion and Mus, the sampling was biased towards adults; if
juveniles bearing hybrizymes have reduced fitness then the frequency of such
alleles will be underestimated among samples of adults. Finally, we d o not know
the relatedness of the individuals sampled and the degree to which the sample
represents the local gene pool, Notwithstanding these difficulties, it is clear that
the hybrizymes must be generated by processes that reach frequencies of perhaps
as high as l o p 3 in the hybrid zones studied.
Origin of hybrizymes
The hybrizyme phenomenon has two separate parts-first, we must seek a
mechanism for the creation of the variant alleles, and second, we must explain
how in some localities thay rise to moderate frequencies. I can offer three
hypotheses concerning the origin of hybrizymes-post-translational modification,
mutation and intragenic recombination-but at the outset, it must be emphasized
that these are neither mutually exclusive nor the only possible mechanisms that
might be involved.
288
D. S. WOODRUFF
Post-translational mod$cation
The prime involvement of heritable or non-heritable second site o r dietinduced post-translational modification in the generation of hybrizymes seems
unlikely. First, the banding and activity patterns and phenotypic frequencies are
characteristic of monomeric and dimeric enzymes rather than secondary
isozymes. Second, I can think of no reason why post-translational modification
should occur at higher frequencies in hybrid zones or why its products should be
restricted to the area of hybridization. Third, most workers have been careful to
avoid situations where post-translational modification is known to be a problem.
I n snails, for example, diet-induced modification of esterases derived from the
hepatopancreas (Oxford, 1978) was avoided by studying enzymes extracted from
the foot muscle. Similarly, in the case of certain dehydrogenases whose patterns
vary with the extent to which the enzyme is saturated with coenzyme, we have
carefully standardized our staining techniques to avoid this problem. Thus,
although post-translational modification of common electromorphs cannot be
ruled out in some cases it seems highly improbable as the prime cause of the
hybrizyme phenomenon.
Mutation
The involvement of mutation in the genesis of hybrizymes is much harder to
exclude. The mutation hypothesis does, however, have several weaknesses: it
does not explain why the normally rare alleles are restricted to the hybrid zones
or why polymorphic loci are more likely to be affected. It also fails to account for
the very high frequency of even the rarest variants. While overall per locus
mutation rates of 0.7 x 10b5-3.2x loF5per generation are typical for man (Harris,
Hopkinson & Robson, 1974; Nee1 & Rothman, 1981) and a variety of other
eucaryotes (Dobzhansky, 1970), hybrizymes are being detected at frequencies of
more than
in most hybrid samples. If mutations are the cause of hybrizymes
than a mechanism must be found to explain their frequency, geographic
distribution and association with the more polymorphic loci.
Such a mechanism, albeit one whose mode of operation is largely
hypothetical, has been proposed in another context-transposon-induced hybrid
dysgenesis (Bregliano & Kidwell, 1983). I t has long been known that artificial
hybrids may show a whole range of abnormal conditions involving chromosomal
behavior, development and morphology; collectively, this syndrome is termed
hybrid dysgenesis (Sved, 1979; Kidwell, 1982, 1983). I t is also well known that
hybridization can somehow stimulate the production of new mutants
(Sturtevant, 1937; Sved, 1979). R. C. Woodruff, Thompson & Lyman (1979)
observed a threefold increase in the mutation rate at certain loci following
interstrain hybridization in Drosojhila; Thompson and R. C. Woodruff (1978)
obtained a tenfold increase in other experiments. Mutator genes were supposed
to be responsible for this phenomenon; it was argued that different populations
have evolved different suppressors of mutability and that hybridization causes a
breakdown of suppression systems (R. C. Woodruff & Thompson, 1980, 1982).
The result is a release of mutator activity and a n explosive increase in genetic
variation (Mackay, 1984). As hybridization produces heterozygosity in
suppressor genes the effects of mutator genes are manifest in the first and later
generations.
If in fact hybridization does disrupt genetic suppressor systems and release
NOVEL GENES IN HYBRID ZONES
289
mutator activity then the increased frequency of mutants in the hybrid zones
might be understood in terms of mutation alone. Attractive as this hypothesis is,
the evidence in its favour is far from conclusive. We still know far too little about
suppressor-mutator systems in laboratory strains of eucaryotes to extrapolate to
the situations occurring in nature. In particular, it is not at all clear why mutators
are active in creating new electromorphs at only a few loci and not others; in the
cases described above less than 20% of the structural genes surveyed showed any
evidence of electrophoretically detectable mutations. Nor is it clear why mutator
activity would produce so few types of variant electromorphs-the
same
hybrizymes were detected in geographically distant parts of each hybrid zone.
Although it is now clear that transposons can induce mutations that result in
complex and intricate changes in genes near their site of insertion their
significance in the hybrizyme phenomen and in the differential introgression and
asymetry observed in hybrid zones has simple not yet been established (Syvanen,
1984).
Intragenic recombination
A third mechanism that may account for the high frequency of rare alleles in
hybrid zones is intragenic recombination. Intracistronic recombination differs
from intercistronic recombination (the only type of recombination considered
until a few years ago) in one very important respect. Although the latter generates
new combinations of existing alleles, the former, under some circumstances,
generates new allelic variants (Ohno, Stenius, Christian & Schipmann, 1969;
Lewontin, 1977). Watt (1972) demonstrated that a variety of reciprocal and nonreciprocal (gene conversion) intracistronic recombination mechanisms may
generate new alleles at rates several orders of magnitude higher than those
associated with standard mutational processes. Subsequently, it was
demonstrated that intragenic recombination probably plays a significant role in
determining the distribution of neutral alleles in many finite populations and
especially those associated with hybrids (Strobeck & Morgan, 1978; Morgan &
Strobeck, 1979; Golding & Strobeck, 1983). These theoretical conclusions are
supported by laboratory studies of Drosophila melanogaster where intragenic
recombination rates a t the rudimentary and rosy loci are 7.6 x lop4 and 1.2 x
respectively (Carlson, 1971; Chovnick, Ballantyne & Holm, 1971); and in a
captive stock of Japanese quail (Coturnix) where the rate a t the 6Pgd locus is at
(Ohno et al., 1969).
least of the order of magnitude of
This hypothesis would appear to have fewer problems than the previous one.
Existing theory predicts that the products of intragenic recombination will be
several orders of magnitude more frequent than the products of other mutations.
It also predicts that the effects of intragenic recombination will be more easily
detected in hybrid zones than elsewhere. It predicts that the rates of intragenic
recombination will be locus specific and that polymorphic loci are more likely to
be affected; “variation begets variation: the more alleles there are, the more new
alleles can come into being” (Futuyma, 1986 : 67). Finally, it may explain why
only a limited number of new alleles will be produced along the length of a hybrid
zone. These points are important as they suggest ways of distinguishing between
the results of intragenic recombination and mutation.
T h e observation that novel alleles are most likely to be found at polymorphic
loci might lead one to favour intragenic recombination over mutation as the
290
D. S. WOODRUFF
prime cause of the hybrizyme phenomenon. Unfortunately, it is not possible to
prove that either process was responsible for the generation of any particular
hybrizyme at this time. Both processes will give rise to the same phenotypic
result and both can be expected to occur. One would need to complete detailed
genetic analyses at the codon or amino acid sequence levels to discriminate
between the two hypotheses. (One such study, of Peromyscus hybrizymes, is
underway (Susan Hoffman, University of Michigan, personal communication).)
It is also possible that ultimately it will be shown that the dichotomy is false and
that the alternative mechanisms are intimately related.
We can only speculate as to why recombination apparently affects polymorphic
more than monomorphic loci. This is, of course, but part of a much larger
problem facing population geneticists-that of the ultimate determinants of locus
variability (Lewontin, 1985). I t may well be that the rates of intragenic
recombination are simply a function of a gene’s size and structure and the extent
to which it is fragmented along the chromosome. Minigenes more widely
separated by families of repeated sequences will be more likely to undergo
recombination resulting in the generation of novel sequences. This might account
for the multiple origin of electromorphically similar alleles in the three
interactions involving different species-pairs of Cerion. Dover ( 1982) argued that
such repeated sequences are in a dynamic state of turnover through the constant
amplification and deletion of sequences. Variants or mutants in such areas may
increase in frequency stochastically through the turnover processes underlying
concerted evolution (Dover & Flavell, 1982). Changes in such repeated
sequences, which are thought to act as ‘regulatory genes,’ do not appear to affect
the sequence of the structural genes. In hybrids between populations which have
different repeated sequence families, on the other hand, it is conceivable that the
protein coding genes themselves are affected. Such heterozygotes for repeated
sequences may be characterized by the novel electromorphs discussed here. The
results of this process would be superficially indistinguishable from those caused
by traditional cistronic mutations. Unfortunately, available data do not permit a
test of these ideas at this time.
Frequency of hyybrizymes
The second part of the hybrizyme problem involves accounting for the
frequency and geographic distribution of the novel alleles. Barton, Halliday &
Hewitt (1983) suggested that since they are rare they must be deleterious and
maintained in a balance by rates of production and selective elimination. The
present survey shows that this view may be too simple and although a discussion
of the factors controlling their spread is beyond the scope of this paper (see, for
example, Takahata & Slatkin, 1986), additional comment on the factors
controlling their abundance seems appropriate.
Neither mutation nor intragenic recombination appear strong enough to
account for the high frequency (i.e. > 0.20) of a few of the hybrizymes described
above. Selection, linkage and hitch-hiking, and/or drift must be invoked to
explain the observed levels of some hybrizymes. Hunt and Selander (1973)
argued that introgression modifies a gene pool in such a way that selective
barriers to the incorporation of new alleles are relaxed. New alleles may even be
favoured in the new genetic environment created by hybridization (Stebbins,
1971) or in the ecotone in which the hybrid zone lies. Selectively favoured
NOVEL GENES IN HYBRID ZONES
29 1
electromorphs would quickly rise above ‘danger levels’ and might be maintained
by frequency-dependent selection by predators, parasites and competitors
(Clarke, 1979). Selection need not, of course, be directed a t the hybrizyme itself
if it is linked to some other gene whose frequency is under such control. Despite
the obvious appeal of such a selection-based explanation one cannot exclude the
possibility that the higher frequencies of some hybrizymes are due to genetic
drift. Various features of the population biology of the animals studied indicate
that drift may be a significant agent of evolutionary change. I n Cerion and
several other cases, for example, dispersal is so limited that populations a few
metres apart are effectively isolated from one another and the opportunities for
drift must be appreciable (Woodruff, 1978, 1981). I n Mus it is well known that
effective population sizes are very small and inbreeding is quite high. The
situation in Rana and Bombina is less well known but the restriction of breeding to
a finite number of small ponds must again reduce the effective population size
(Szymura & Barton, 1986). In conclusion, both selectionist and neutralist
arguments must be advanced to explain why some hybrizymes reach moderate
frequencies in hybrid zones.
Conclusion
Regardless of their etiology and evolutionary fate, hybrizymes promise to be
useful in the on-going debates surrounding far broader questions about the
origin, genodynamics and significance of hybrid zones (Woodruff, 1973, 1979,
1981; Endler, 1977; Moore, 1977; Barton, 1986; Barton & Bengtsson, 1986;
Barton & Hewitt, 1981, 1985; Szymura & Barton, 1986). I t is obvious that
further use can be made of their occurrence in mapping semispecies boundaries.
Their presence, frequency and distribution may be related to the degree of
overall genetic differentiation between hybridizing taxa and this could, in turn,
be relevant to establishing the history of a hybrid zone, its future significance,
and the systematic status of the interacting populations. For example, the
elevated frequencies of rare alleles in Cerion populations on south Grand Bahama
Island and on the west side of South Caicos Island may reflect the former
existence of hybrid zones in these areas (Gould & Woodruff, 1978, 1987;
Woodruff & Gould, in prep). More significant, perhaps, is the question of
whether hybrid zones are sources of new alleles which subsequently become
rapidly and widely established in one or both parental species-as suggested by
R. C. Woodruff, Slatko & Thompson (1983). At this stage we simply lack the
necessary data on the relative fitness of hybrizymes or on their probability of
spreading to do more than speculate. It remains to be established whether any
presently widespread species-characterizing allozyme had its origin as a
hybrizyme, but unilateral introgression would clearly make hybrizymes positive
forces in evolutionary differentiation. Is it possible that Cerion’s extraordinary
phenotypic variability has its roots in the continued disruption of genetic
regulatory systems by hybridization and the spread of these effects far from the
narrow hybrid zones themselves? The hybrizyme phenomenon, like some data
on mtDNA (Avise, 1986; Ferris et al., 1983; McNeil & Strobeck, 1987; Nelson et
al., 1987), and rDNA (Arnold et al., 1987) also suggests that species boundaries
may be better viewed as semipermeable membranes and that simpler models of
hybrid zones as monotonic clines maintained by a balance between gene flow
and natural selection are no longer adequate (see Harrison, 1986, 1988). The
292
D. S. WOODRUFF
view of hybrid zones as barriers to introgression or as genetic sinks requires
reappraisal in the light of hybrizymes and the possible involvement of
transposons. I t is quite clear that the resolution of the problems presented by
hybrid zones calls for the full integration of molecular genetics and traditional
empirical natural history (Rose & Doolittle, 1983; Krieber & Rose, 1986;
MacIntyre, 1986; Szymura & Barton, 1986). Until this is brought about hybrid
zones will remain as taxonomist's nightmares and evolutionist's delights.
ACKNOWLEDGEMENTS
The Cerion hybrid zones were discovered in collaboration with Stephen Jay
Gould. I thank Sarah Burgess and Michael Goldman for assistance in the
laboratory and Nick Barton, Godfrey Hewitt, Mike Johnson, Robert K.
Selander, Jacek Szymura and Graham Wallis for useful discussions. I thank the
Ministry of Agriculture and Fisheries of the Bahamas for permission to study
Cerion. This report was completed while on sabbatical leave at the Department of
Zoology, University of Western Australia. This work was supported by the U.S.
National Science Foundation.
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