Download Distortion of symmetrical introgression in a hybrid zone

doi:10.1111/j.1420-9101.2005.01064.x
Distortion of symmetrical introgression in a hybrid zone: evidence
for locus-specific selection and uni-directional range expansion
J. JOHANNESEN,* B. JOHANNESEN,* E. M. GRIEBELER,* I. BARAN, M. R. TUNÇ,à
A. KIEFER* & M. VEITH*
*Institut für Zoologie, Abt. V Ökologie, Universität Mainz, Mainz, Germany
Department of Science Education, Buca Education Faculty, Dokuz Eylül University, Izmir, Turkey
àDepartment of Biology, Falculty of Science and Education, Akdeniz University, Antalya, Turkey
Keywords:
Abstract
allozymes;
anthropogenic disturbance;
cytonuclear disequilibrium;
hybridization;
Lyciasalamandra antalyana;
Lyciasalamandra billae;
mtDNA length polymorphism;
selection;
Turkey.
The fate of species integrity upon natural hybridization depends on the
interaction between selection and dispersal. The relative significance of these
processes may be studied in the initial phase of contact before selection and
gene flow reach equilibrium. Here we study a hybrid zone of two salamander
species, Lyciasalamandra antalyana and Lyciasalamandra billae, at the initial
phase of hybridization. We quantify the degree and mode of introgression
using nuclear and mtDNA markers. The hybrid zone can be characterized as an
abrupt transition zone, the central hybrid zone being only c. 400 m, but
introgressed genes were traced up to 3 km. Introgression was traced in both
sexes but gene flow may be slightly male-biased. Indirect evidence suggests
that hybrid males are less viable than females. Introgression occurred at two
levels: (1) locus-specific selection led to different allelic introgression patterns
independent of species, while (2) asymmetrical species-level introgression
occurred predominately from L. antalyana to L. billae due to range expansion of
the former. This indicates that foreign genes can be incorporated into novel
genomic environments, which in turn may contribute to the great diversity of
morphological variants in Lyciasalamandra.
Introduction
Hybrid zones offer rich possibilities to study genetic
distinctiveness of populations and the selective forces
that keep them different (Arnold, 1997). If hybrids are
less fit than the parent species, reproductive barriers may
evolve as the final step towards total separation.
Renewed gene flow may break specificity if hybrid fitness
is not reduced or, if fitness of one parental population is
lower than that of the other, hybridization may lead to
replacement of the less fit population. Alternatively,
hybridization may be a creative force shaping new hybrid
species. In the latter cases, genes of the failing species
may be absorbed into the successful one (Clarke et al.,
2002).
Correspondence: Jes Johannesen, Institut für Zoologie, Abt. V Ökologie,
Universität Mainz, Saarstrasse 21, D-55099 Mainz, Germany.
Tel.: +49 (0) 6131 3923946; fax: +49 (0) 6131 3923731;
e-mail: [email protected]
Different introgression rates of genetic markers permit
inferences about how selection and/or behaviour play a
role governing the admixture of the organisms (e.g. Della
Torre et al., 1997; Sweigart & Willis, 2003). The width of
a clinal hybrid zone is determined by a balance between
dispersal rates and fitness (Barton & Hewitt, 1985).
Characteristic of hybrid zones is the nonrandom association of alleles. When disequilibrium is strong, the central
region of a cline will be distinct from the tails and the
cline-width will depend on the ratio between recombination and selection (p116, Barton & Hewitt, 1985). If
selection acting on distinct characters differs, they may
introgress at different rates. Because mtDNA is assumed
selectively neutral, it is generally believed to have greater
rates of introgression than nuclear genes, but an equally
parsimonious explanation is positive selection that
sweeps the mitochondrial genome through the population (Dasmahapatra et al., 2002). On the other hand,
sex-specific gene flow will cause asymmetric rates of
introgression, particularly if one sex is less viable or
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J. JOHANNESEN ET AL.
fertile than the other. The less fit sex is nearly universally
the heterogametic sex (Coyne & Orr, 2004) and this is
known as Haldane’s rule (Haldane, 1922). In birds, male
biased gene flow (i.e. restricted mtDNA gene flow) across
species borders is, at least partly, due to reduced viability
of the heterogametic females (Helbig et al., 2001; Crochet
et al., 2003). However, mtDNA and nuclear polymorphisms often are coincident and concordant (Szymura &
Barton, 1986; Dasmahapatra et al., 2002).
Amphibians are well-known for their ability to hybridize naturally. The tendency to hybridize, even between
species with high sequence divergence (Szymura et al.,
2000) indicates genomic resilience and makes amphibians prone to build species-complexes (Tarkhnishvili et al.,
2001; Bogart, 2003). Hybridization occurs between
sympatric species (e.g. Szymura & Barton, 1986; Green
& Parent, 2003), as well as between introduced (allopatric) species (e.g. Riley et al., 2003). Several authors have
noted that hybridization in sympatric species can be
brought about by human disturbance of the natal habitat
(Riley et al., 2003 and references therein). Particularly
the possibility to hybridize between species from divergent mtDNA lineages raises the question whether resilient species require longer speciation times or,
alternatively, shorter time due to introgressive speciation. The former scenario requires strict allopatry while
the latter allows character divergence to build up in a
relationship between gene flow, selection and some
habitat divergence.
The Lycian salamanders of the genus Lyciasalamandra
Veith & Steinfartz (2004) (former Mertensiella luschani
Steindachner, 1891; see Veith & Steinfartz, 2004) consists
of nine morphologically distinct taxa, distributed allopatrically along the southern Turkish coast and adjacent
Greek and Turkish islands. The total range covers only
350 km. Weisrock et al. (2001) analyzed sequence
divergence in eight taxa and found highly divergent
lineages with 7.6–10.1% pair-wise sequence divergence,
rooted in a basal unresolved polytomy. Morphological
variants can even be recognized among populations of
the nine taxa (Veith & Steinfartz, 2004). The lineages
diverged between 5.9 and 7.9 MYBP, which corresponds
to vicariant speciation initiated during the rise of
Anatolia in the late Miocene (Weisrock et al., 2001;
Quennell, 1984). Habitat requirements include average
rainfall of >800–1000 mm, average temperature in
January above 0 and a soil structure with crevices
(Klewen, 1991; Steinfartz & Mutz, 1998; Veith et al.,
2001). The crevices are crucial for survival as refuges
during the dry and hot summers. These habitat requirements limit the distribution of Lyciasalamandra to island
populations associated with limestone outcrops.
Only two taxa, Lyciasalamandra billae and Lyciasalamandra antalyana, are known to have adjoining populations (Veith et al., 2001). The two taxa behave as discrete
species; they are morphologically different and show
7.8% mtDNA sequence divergence (Weisrock et al.,
2001). However, during a survey of the distribution of
Lyciasalamandra, Veith et al. (2001) reported several
morphologically intermediate individuals suggesting a
contact zone between the two species. The aim of
this paper is to delimit a putative contact zone between
L. billae and L. antalyana and to analyse the degree and
mode of introgression between the species. The species
are characterized by two diagnostic nuclear (allozyme)
loci and by a diagnostic mtDNA D-loop length polymorphism. We address four questions: (i) Is introgression
gradual or does hybridization follow a mosaic pattern?
(ii) Is gene flow sex-biased? (iii) Is introgression uniparental? and (iv) Is introgression locus-specific
(evidence for selection)? Gradual introgression is expected when the frequency of parental individuals gradually
decreases away from the pure populations, hybrids
increase towards the centre and hybrids have intermediate fitness. One expects deviation from HW proportions
and linkage disequilibria within the contact zone and
selection against hybrids. Because only two nuclear loci
(out of 31 loci) are polymorphic the expectation of
linkage build up across several loci in the centre of the
zone cannot be tested. Mosaic introgression is expected
when parental individuals mix within sites and hybrids
are rare (thus allowing parentals to be relatively common). Inference of sex-biased gene flow was gained by
comparing nuclear and maternal variance estimates
(Ennos, 1994; McCauley, 1994) and cytonuclear
disequilibria (Asmussen & Basten, 1996; Basten &
Asmussen, 1997). The former comparison is based on
equilibrium conditions assuming equal sex ratios, mating
success of the sexes, homoplasmy and unisexual transmission (Birky et al., 1983,1989); when maternal
(mtDNA) variance exceeds nuclear variance by four
times it indicates that females contribute more to gene
flow than males. Because of the assumptions, this
method should be considered with caution. Inference of
uni-parental introgression was gained from cytonuclear
disequilibrium (see section ‘Methods’ for details). Finally,
inference of locus-specific introgression (selection) was
gained from the frequency of heterozygotes at two
diagnostic loci. As we will show, the level of heterozygosity in hybrids differed at the two loci. There are two
possible explanations for this pattern. First, heterozygotes
at both loci may be lacking in hybrids, one more so than
the other. In this case, both loci are affected by negative
selection, one more than the other. In the second
outcome, a heterozygote at one locus may, despite being
lacking in the total gene pool, actually be overrepresented in hybrids relative to a situation with general selection
against hybrids. In other words, heterozygotes are less
lacking than expected in the total gene pool. In this case,
overrepresentation may be taken as evidence for positive
selection. To evaluate which of these possibilities
explained genotype-specific behaviour we simulated a
simple null-model assuming no selection and steppingstone admixture. The simulations further allowed testing
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Lyciasalamandra hybrid zone
for L. antalyana to move across the valley. Hence, the
opportunity for crossing at the village is considerably
older.
To delimit the contact zone we sampled populations in
the valley as well as putative parental populations found
in the heights on either side of the valley. Sampling was
done from 1996 to 1999 from 25 populations,
including 283 individuals. All animals, except from
populations 7 and 24, were individually marked and
genotyped at allozyme and mtDNA loci. Individuals from
populations 7 and 24 were sampled in 1996 when the
putative hybrid zone was discovered. The genetic estimates from these two populations are frequency estimates. To estimate the degree of introgression, we
sampled two transects on the L. billae side of the creek.
The first transects ascends a hill behind the population 23
while the second transect follows a road along the creek
and into the hills behind. All locations were positioned
with GPS.
genetic drift as a cause for different specific multi-locus
genotypes.
Method and materials
Species desciption
Lyciasalamandra antalyana and L. billae look very similar
in size and shape. However, they are well distinguished
in parotid and flank coloration. Lyciasalamandra antalyana
has bright yellow parotids, irrespective of the head’s
colour; in L. billae parotids are always coloured like the
head, either brown or incarnate. In hybrids, parotids
mostly resemble those of L. billae, although sometimes
with a whitish central spot. Flanks of L. antalyana are
yellowish; those of L. billae are white. Flanks in hybrids
are ivory-coloured.
Sample locations
The contact zone runs along a creek in a narrow valley
near the villages Gökdere and Hurma, 30 km southeast
of Antalya in southern Turkey (Fig. 1, Table 1). It seems
that L. antalyana has managed to cross the creek from the
north to the L. billae side where morphologically
intermediate individuals were identified (sample 23).
At first sight there are two potential centres of hybridization. The first site is the village Gökdere where a
bridge crosses the creek. The second potential centre is
2 km down-stream where permanently irrigated orange
groves, planted about 60 years ago, have made it possible
Allozyme polymorphism
All populations in the present study were screened for
25 presumptive allozyme enzymes and blood plasma
markers comprising 31 loci [investigated in all nine
species of the genus Lyciasalamandra M. Veith (in prep)].
The topotypical L. antalyana were monomorphic at all
31 loci. The topotypical L. billae were monomorphic at
30 loci and polymorphic at one locus, Pepidase-B. The
two species differed genetically by being fixed for
alternate Aat and Idh alleles (Table 2). The enzymes
52
Turkey
Hurma
51
1
Latitude 36°47–36°52
2
s
Bridge
50
11 12
8
10
Gökdere
49
3
4
48
13
20
14
15
Creek
6
17
16
ve
18
gro
nge
a
r
O
23
22
21
19
9
7
5
Fig. 1 Sampling locations of Lyciasalamandra
billae and Lyciasalamandra antalyana at a
contact zone in south Turkey. The subspecies
meet at a creek (thick line).
500 m
24
47
29
30
31
32
33
Longitude 30°29–30°36
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35
36
708
J. JOHANNESEN ET AL.
Co-ordinates
Location
North
East
n
Altitude (m)
Slope
Side of creek
Population type
1
2
3
8
10
11
12
18
4
5
6
7
9
13
14
15
16
17
19
20
21
22
23
24
25
3651,10
3650,59
3648,90
3649,62
3649,33
3649,61
3649,47
3650,23
3648,25
3647,44
3648,34
3648,16
3648,47
3649,37
3648,89
3648,71
3649,53
3649,73
3649,31
3649,30
3649,64
3649,82
3649,96
3647,09
3641,08
3035,31
3030,11
3030,77
3032,56
3033,16
3032,96
3033,29
3034,60
3030,60
3030,17
3031,35
3032,00
3032,79
3033,39
3033,12
3032,63
3033,84
3033,75
3034,57
3034,41
3034,83
3034,86
3034,88
3034,03
3034,34
17
10
11
5
5
2
11
2
5
6
10
14
7
21
16
5
19
4
16
14
14
18
14
25
11
175
410
400
315
75
80
40
45
600
1000
450
230
270
35
275
150
35
25
140
370
260
150
35
15
35
SE
NE
SW
SE
E
NE
SSE
NE
N
NW
N
NE
N
NNW
N
NW
NW
SE
NW
N
NW
NW
N
E
SE
N (T)
N
N
N
N
N
N
N
S
S
S
S
S
S
S
S
S
S
S
S (T)
S
A
A
A
A
A
A
A
A
AB
AB
B
AB
B
AB
AB
B
AB
AB
B
B
AB
AB
AB
B
B
Table 1 Sampling locations of Lyciasalamandra antalyana and Lyciasalamandra billae
southwest of Antalya, Turkey; population
types: A, pure L. antalyana; B, pure L. billae;
AB, introgressed population.
Side of creek: T, terra typical.
Table 2 Multi-locus allozyme genotypes and D-loop length polymorphisms of parental Lyciasalamandra billae and Lyciasalamandra
antalyana.
Allozyme genotype
Species
Aat
Idh
Pep-B
D-loop
Lyciasalamandra antalyana
Lyciasalamandra billae
AA
BB
AA
BB
CC
C/D
1
2,3,4
Genotype designation based on all nine Lyciasalamandra taxa.
Pep-B (Leu-Gly-Gly) EC 3.4.11 or 13, Aat EC 2.6.1.1, Idh
EC 1.1.1.42 and the enzyme Pgd EC 1.1.1.44 (polymorphic in a single population) were analyzed by celluloseacetate electrophoresis. Running conditions for the
polymorphic enzymes in this study were as follows:
Pep-B, Idh and Pgd were analysed from blood; Aat from
muscle tissue. Pep-B was run in Tris–Glycine buffer pH ¼
8.5, 250 V; Idh in Tris–Maleic acid pH ¼ 7.0, 200 V; Pgd
in Tris–citrate acid pH ¼ 8.2, 200 V; Aat in Tris–Borate
pH ¼ 7.8, 200 V. All enzymes were run for 40 min.
Mitochondrial DNA
To characterize female gene flow and cytonuclear disequilibrium we amplified the mtDNA D-loop using the primers
L-Pro-ML and H12S1-ML (Steinfartz et al., 2000; DNA
extraction and amplification protocols are given therein).
We limited the mtDNA analyses to scoring a D-Loop length
polymorphism, which differs between the two species
(Johannesen, 2004). The L. antalyana D-loop is about
900 bp and that of L. billae about 1100 bp. However,
during the investigation we observed three length polymorphisms in L. billae (new length polymorphisms 1300
and 1500 bp). No additional length polymorphisms were
found in L. antalyana. To check for correct assignment of
length polymorphisms to parental species we sequenced
29 L. antalyana from eight populations (11, 17, 18, 10, 12,
23, 3, 8) and 20 L. billae from seven populations (16, 14, 22,
13, 19, 20, 21) for about 550 bp. Phylogenetic analysis
showed monophyly of L. antalyana haplotypes and of L.
billae length polymorphisms. The sequence data will be
published elsewhere (B. Johannesen & M. Veith in prep).
Because the present paper investigates introgression only,
we have treated the three L. billae length polymorphisms as
one haplotype.
Data analysis
Individuals were scored as introgressed if (i) they were
heterozygotic in any combination of the fixed parental
Aat and Idh alleles and (ii) for individuals with homozygote parental genotypes, those bearing an alternative
D-loop haplotype. We tested for nuclear linkage
disequilibrium and deviations from Hardy–Weinberg
(HW) proportions using G E N E P O P V . 3 . 2 (Raymond &
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Lyciasalamandra hybrid zone
Rousset, 2000). The observed multi-locus genotypes in
the hybrid zone were compared to a random mating
population. Genetic differentiation within species was
assessed for nuclear and mtDNA alleles with the program
G E N E P O P V . 3 . 2 . Cytonuclear disequilibrium was tested
with the program C N D M (Asmussen & Basten, 1996;
Basten & Asmussen, 1997). The cytonuclear disequilibrium has four estimators, D, D1, D2 and D3, where
positive D ¼ DA
M indicates a positive association between
nuclear alleles (A) and mtDNA (M) from the same
parental species. D1 ¼ DAA
M is the association between
parental mtDNA and its homozygote (AA), D2 ¼ DAB
M is
the association between parental mtDNA and the heterozygote (AB), and D3 ¼ DBB
M is the association between
mtDNA of one species and the homozygote of the other
species (BB). The estimator D2 is of special interest
because it estimates whether heterozygote (hybrid)
individuals are more likely to have one species’ mtDNA
than the other. A significant value implies fitness
differences relative to genetic backgrounds or mating
asymmetry between the species.
Computer simulations
Because not only selection but also genetic drift in small
populations may produce different locus-specific
introgression rates, we simulated a simple null-model
(Griebeler et al., in press) to test whether (1) specific multilocus genotypes were more or less common than expected
and whether (2) observed multi-locus genotype frequency
differences could be explained by genetic drift alone. The
simulation model of Griebeler et al. (submitted) studies
stepping-stone dispersal and population admixture. The
model was adapted to the current settings only by
changing parameter values and population numbers (see
below).
The model
We suppose in our individual-based model that two
neutral diallelic loci (Aat and ldh) represent the genetic
system of the two diploid admixing salamander species.
Crosses between species are fertile and no selection is
assumed. Maximum recombination between the two loci
is allowed. The individuals inhabit a linear array of demes
with dispersal between them. Each deme harbours a
constant number of individuals (n). These may be of the
two parental types or may be hybrids. Admixture of
species results from stepping-stone dispersal of individuals. Emigration of an individual occurs once within a
generation with a constant probability. The displacement
of each emigrant is possible in both directions. The two
adjacent demes of a source deme may be chosen by a
migrant with distinct probabilities to allow for symmetric
and asymmetric introgression. Dispersal is followed by
n random matings in each subpopulation. Parents for
offspring are selected randomly. The offspring produced
in each subpopulation constitutes the next generation.
709
Simulations
The simulated introgression zone consisted of 60 demes
aligned evenly along a 3 km single-dimensional transect.
This corresponds to sampling every 50 0m. These settings
were based on observed natural conditions: introgression
proceeded linearly and was traced 3 km. At the beginning of the simulated introgression process demes located
at one edge were fixed for the parental multi-locus
genotype AA/AA (L. antalyana) and the others for BB/BB
(L. billae), respectively. All demes were populated at size
50 or 100, respectively, except of the two subpopulations
at the transect ends. To avoid edge effects, we assumed
that each of these subpopulations had 10 000 individuals
of each parent species.
We simulated two extreme introgression scenarios. In
the first scenario dispersal was symmetric, while dispersal
in the second scenario was limited to one direction only.
The first simulation scenario was based on observed ratio
of L. antalyana to L. billae alleles 1 : 4. In the modelled
transect; 12 L. antalyana subpopulations were followed by
48 L. billae subpopulations (corresponds to the 1 : 4 ratio).
Individuals of both species and their hybrids were allowed
to migrate with an equal chance to the two neighbouring
subpopulations with a probability of 0.1 per generation.
The second scenario corresponds to the assumption that L.
antalyana has crossed the creek, is expanding and replacing
L. billae in its natal habitat. Here we assumed initially a total
population of 5% pure L. antalyana (three subpopulations)
and 95% pure L. billae (57 subpopulations). For the
introgression of L. antalyana into L. billae subpopulation we
assumed a dispersal rate of 0.1. Both scenarios for introgression were simulated with subpopulations of 50 and
100 individuals, respectively.
We performed 100 Monte–Carlo simulations for both
introgression scenarios. We simulated 250 generations
in each simulation run. After each 25th generation,
15 individuals were sampled randomly per subpopulation.
The final genotypic multi-locus distribution across the
zone of introgression was obtained from the total sample of
randomly drawn individuals, i.e. 900 (60 demes · 15
individuals sampled) parental and hybrid individuals.
Due to the expanding front of introgression, each
simulated generation will have different relative multilocus distributions. In order to know which generation
from the simulated data to use for our null-model, we
qualitatively compared frequencies of the parental multilocus genotypes obtained from the simulations to the
observed data. The best fitting generation was compared
to the observed data. It should be noted that for practical
reasons the simulations were performed for a single onedimensional zone of admixture. In reality, we observed
two one-dimensional clines. In the comparison of simulated and observed data we have pooled the observed
data from the two transects. This seems appropriate
because the two transects behaved identically (see
section ‘Results’).
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The level of polymorphism from 25 sample locations is
presented in Appendix A. All populations north of the
creek designated as L. antalyana (locations 1–3, 8, 10–12
and 18) were completely monomorphic, holding the
diagnostic genotypes and haplotype listed in Table 2. In
contrast, populations of L. billae showed signs of introgression from L. antalyana in lowland populations but
had only parental genotypes at higher elevation. Population 7 had a unique Pgd allele.
Of the 283 studied individuals, 39 (14%) were hybrids
(5 adult males, 15 adult females, 11 juveniles, 4 subadults
and 4 no information). Because hybrids were delimited
by only two nuclear loci and one mtDNA polymorphism
the number of hybrids should be considered a minimum
value. Multi-locus genotypes together with corresponding D-loop haplotypes show that introgression includes
F2 and/or backcross animals. Six double homozygote
individuals were all juveniles. Only one individual was
double heterozygote (Table 3). Both ‘pure’ parental types
were never found at the same location.
We observed two clines extending from sample 23,
along the creek (locations 13–14 and 16–17) and up the
hill (locations 21–22) (Fig. 2). We termed these locations
the ‘primary hybrid zone’. Transect sampling revealed an
abrupt transition zone between the two species in both
transects. The central zone of introgression is only about
400 m. The total cline width is about 3 km wide, based
on the road distance along the creek. The introgression
Primary
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
20
21
22
23
0
200
400
600
800
1000
1200
Distance (m)
Frequency
Polymorphisms
Frequency
Results
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
16
14
15
9
13
23
17
0
500
1000
1500
2000
2500
3000
3500
Distance (m)
Fig. 2 Frequencies of Lyciasalamandra billae specific alleles across
two transects, from location 23 to location 20 and 9, respectively.
Locality 23 exhibited exclusively Lyciasalamandra antalyana mtDNA
but nuclear alleles from both species. mtDNA – open triangles, Aat –
solid diamond, Idh – solid squares. Numbers signify locality numbers.
likely proceeds from L. antalyana to L. billae because no L.
billae alleles or haplotypes were found on the L. antalyana
side of the creek, whereas pure L. antalyana were
Second
Genotypes
Haplotype 1
Haplotype 2,3,4
Unknown
haplotype*
Haplotype 1
Haplotype 2,3,4
Unknown
haplotype
AA/BB
AB/AA
AA/AB
AA/AA
AB/AB
BB/BB
BB/AB
AB/BB
BB/AA
0
0
2
8
1
1
0
1
1
3
0
1
1
0
60
14
1
1
–
1
2
2
–
6
–
–
–
0
1
2
1
0
0
0
0
0
1
0
0
0
0
3 (9) 0
0
2
–
–
–
–
–
12 –
3 –
Table 3 Multi-locus nuclear genotypes (Aat/
Idh) and D-loop length-polymorphisms
observed at two hybrid areas.
*DNA extraction was corrupted and amplification failed.
Allozyme data from population 7 is based on frequency data from 15 individuals that were
not individually assigned. The nine individuals scored for mtDNA in population 7 had
haplotypes 2 or 3. It is therefore likely that six BB/BB individuals have haplotype 2, hence the
designation (9).
The primary hybrid zone included the samples 23, 22, 21, 17, 13, 16 and 14. The second
hybrid area consisted of individuals from samples 4, 5 and 7. Lyciasalamandra antalyana, AA/
AA and haplotype 1; Lyciasalamandra billae, BB/BB and haplotypes 2,3,4.
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Lyciasalamandra hybrid zone
established on the L. billae side (location 23). The
frequency of L. antalyana alleles dropped continuously
away from location 23. Interestingly, the bridge in the
village Gökdere seems to play only a limited role, if at all,
in promoting species contact. Here, no pure L. antalyana
were established on the L. billae side as was observed at
location 23.
A second introgression area was found where two
mountain ranges converge south west of Gökdere (locations 4–5 and 7) but data are too sparse to make
generalizations about this area.
Mating patterns and gene flow
Tests for random association of nuclear alleles within the
primary hybrid zone [treating all sites (13–14, 16–17 and
21–23) as one sample] revealed significant linkage-disequilibrium for all locus combinations (Aat/Idh, Aat/Pep-B,
Idh/Pep-B, all P < 0.001) and deviation from HW proportions (Fishers exact test, v26 ¼ 106.2, P < 0.001). Each
locus deviated significantly from HW proportions
(P < 0.001). However, the two species-specific loci differed
in the amount of heterozygote deficit (expressed as
AA : AB : BB) Aat: 19 : 4 : 84, Idh: 14 : 20 : 72 (v22 ¼
13.3, P < 0.01). Deviations from HW proportions were not
significant within locations within the primary hybrid
zone. This may be caused by a combination of events and
was not entirely unexpected: locality-sample sizes were
small (location 17), hybrids few (particularly including Aat
– see below) and HW tests are conservative. However, a
pattern emerged where every sample had a deficit of Aat
heterozygotes (significant only for location 22, P < 0.05),
but which was significant across localities, P < 0.01. In
contrast, Idh had a heterozygote excess within localities in
all but one sample and did not deviate from HW proportions across samples (P ¼ 0.97). HW proportions calculated for 27 hybrids where mtDNA, Aat and Idh were scored
in all individuals showed significant lack of heterozygotes
for Aat (v21 ¼ 15.13, P < 0.001) but an excess of
heterozygotes for Idh, albeit only marginally significant
(v21 ¼ 3.33, P ¼ 0.068).
Cytonuclear disequilibria (Table 4) showed highly
significant positive associations between parental species’
mtDNA and alleles (D) and homozygotes (D1 and D3).
This was expected because several pure parental individuals were found within the hybrid area. More interesting
were the associations between heterozygotes and mtDNA
(D2) of either parental species. Despite low sample sizes,
L. billae had a significantly negative association between
mtDNA and Aat heterozygotes, P ¼ 0.036 (L. antalyana
significantly positive). In contrast, no association
between Idh heterozygotes and mtDNA was observed,
P ¼ 0.97. Applying the cytonuclear tests only to hybrid
individuals produced no significant associations (results
not shown).
Assessments of intra-specific gene flow were not
possible for L. antalyana because it was completely
monomorphic. For L. billae estimates of genetic differentiation, FST, based on the nuclear locus Pep-B and mtDNA
D-loop polymorphism were possible. Genetic differentiation based on 5 ‘pure’ L. billae locations (6, 15, 9, 20 and
19) were: FST(Pep)B) ¼ 0.11; FST(D-loop) ¼ 0.62. The distribution of the D-loop polymorphisms was particularly
subdivided; the easterly ‘pure’ locations 6, 9 and 15 (and
the hybrid locations not included in the estimate above)
exhibited three length polymorphisms (Fig. 3), whereas
the westerly locations 19–23 only had polymorphism 2
(Appendix A). The distance between location 16 and 19
was only c. 600 m by air.
Selection
As shown above, the level of heterozygosity differed
between Aat and Idh. Considering this pattern in more
detail on a L. billae background, the relationship of
heterozygotic Idh (BB/AB) to Aat (AB/BB) in the primary
hybrid zone is 14 : 2 (Table 3) and significantly different from equal proportions (v21 ¼ 5.24, P ¼ 0.02). Thus,
L. antalyana Idh but not Aat is introgressing into a L. billae
background. Interestingly, the same pattern is also
suggested on a L. antalyana background: the relationship
of heterozygous L. billae Idh (AA/AB) to Aat (AB/AA) is
Table 4 Cytonuclear disequilibria between Aat and mtDNA, and Idh and mtDNA from a hybrid zone of Lyciasalamandra antalyana and
Lyciasalamandra billae.
Aat
Idh
Disequilibrium
D1
D2
D3
D
D1
D2
D3
D
Normalized, D
Standard error H0
Standard error H1
Test statistic
Probability
0.824
0.014
0.024
47.66
<0.001
)0.609
0.006
0.012
6.64
0.036
)0.661
0.013
0.023
38.23
<0.001
0.741
0.013
0.019
45.35
<0.001
0.794
0.017
0.021
23.58
<0.001
)0.031
0.014
0.015
0.07
0.97
)0.787
0.012
0.023
44.55
<0.001
0.791
0.013
0.017
40.21
<0.001
The estimates are shown for L. billae, i.e. allele B and haplotype 2. (The D estimates for L. antalyana are symmetrical with opposite sign.) Positive
D shows a positive association between nuclear alleles and mtDNA from the same parental species, D1 is the association between parental
mtDNA and its homozygote, D2 is the association between parental mtDNA and the heterozygote, and D3 is the association between mtDNA of
one species and the homozygote of the other species.
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Symmetric dispersal
6
15
17 16
100 BB/AB
100 AB/BB
0.045
50 BB/AB
7
50 AB/BB
0.040
100 AB/AA
0
1000
100 AA/AB
0.035
13 14
9
50 AB/AA
Frequency
Frequency
23
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
2000
3000
4000
5000
0.030
50 AA/AB
0.025
0.020
0.015
0.010
Distance (m)
0.005
Fig. 3 The distribution of D-loop length polymorphisms across the
creek transect. Lyciasalamandra antalyana: open circles – haplotype
H1; Lyciasalamandra billae: solid squares – H2, closed circles – H3,
open triangles – H4. Numbers signify locality numbers.
0.000
0
50
100
150
200
250
300
Generation
Asymmetric dispersal
100 BB/AB
100 AB/BB
0.035
50 BB/AB
50 AB/BB
0.030
100 AB/AA
100 AA/AB
50 AB/BB
Frequency
0.025
50 AA/AB
0.020
0.015
0.010
0.005
0.000
0
50
100
150
200
250
300
Generation
Frequency of parental genotypes
100 BB/BB
50 BB/BB
1.000
100 AA/AA
50 AA/AA
0.900
100 asym BB/BB
100 asym AA/AA
0.800
50 asym BB/BB
0.700
Frequency
5 : 1 (Table 3), although not significant due to small
sample size. The relative number of heterozygote Idh and
Aat in all hybrids was 20 : 3 and significantly different
from equal proportions (v21 ¼ 6.57, P ¼ 0.01). The
relationship of hybrid double homozygotes (AA/BB and
BB/AA) was about equal (3 : 2). Thus, different heterozygote behaviour lead to different introgression rates of
L. antalyana Aat and Idh alleles. The different behaviour
may either be due to under representation of (selection
against) Aat heterozygotes or over representation of
(selection for) Idh heterozygotes.
Computer simulations showed that the relative frequency of Idh and Aat heterozygotes were similar in all
generations and independent of simulation scenario
(Fig. 4a,b). Hence genetic drift could not explain the
skewed multi-locus heterozygote (BB/AB vs. AB/BB)
distribution. The maximal relative difference between
BB/AB and AB/BB found during simulations 8.4 : 6.8
(250th generation, symmetric gene flow) differed significantly from the observed 14 : 2 distribution (v21 ¼ 4.39,
P ¼ 0.04).
No single simulation produced the observed parental
genotype frequency (AA/AA & BB/BB) simultaneously
(Fig. 4c). In the symmetric dispersal scenario, the frequency of BB/BB was always more common than the
observed frequency, whereas AA/AA fitted the observed
frequency after 125 generations. At generation 125 the
simulated frequency of BB/AB, 3.0%, was far lower than
the observed frequency, 13.3%; the simulated frequency
of AA/AB, 2.7%, was also lower than observed frequency, 4.8%. In contrast, the simulated frequencies of
AB/BB, 2.7%, and AB/AA, 2.8%, were higher than the
observed frequencies, 1.9 and 1.0%, respectively
(Fig. 4a).
In the second scenario with skewed dispersal with 5%
L. antalyana and 95% L. billae, the observed frequency of
L. antalyana parental genotypes was reached after c. 75
generations and that of L. billae parental genotypes after
175 generations (Fig. 4c). The two skewed-dispersal
simulations behaved similarly at generations 75 and
50 asym AA/AA
0.600
0.500
0.400
0.300
0.200
0.100
0.000
0
50
100
150
200
250
300
Generation
Fig. 4 Simulated frequencies of multi-locus genotypes (Aat/Idh)
under symmetric and asymmetric dispersal at population sizes
n ¼ 50 and n ¼ 100. Heterozygote frequencies with symmetric
dispersal (a). Heterozygote frequencies with asymmetric dispersal
(4b). Parental frequencies (AA/AA and BB/BB) with symmetric and
asymmetric dispersal (c). The dotted lines in (c) represent the
observed frequencies of AA/AA (10.5%) and BB/BB (62.9%) in the
hybrid area.
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Lyciasalamandra hybrid zone
175 by showing a lack of genotype BB/AB, 1.6 and 2.6%,
respectively; and to lesser extent, a lack of AA/AB, 1.4
and 2.2%, respectively. The simulated frequency of
heterozygote Aat (AB/BB and AB/AA) was 1.6 and
1.5% in generation 75, and 2.0 and 2.2% in generation
175. Thus, both symmetric and asymmetric dispersal
scenarios suggest that Idh heterozygotes were highly
overrepresented in the natural (hybrid) sample while Aat
heterozygotes were found at frequencies slight under or
at those expected during random mating and dispersal.
Discussion
The two Lycian salamanders L. antalyana and L. billae
meet and reproduce in a narrow contact zone in
southern Turkey. The contact area was made up of a
small L. antalyana neighbourhood (sample 23) from
which two steep linear clines produced abrupt introgression into a L. billae back land (Fig. 2). Clinal variation of
nuclear alleles and mtDNA were coincident and concordant. The contact centre was situated south of the village
Hurma (sample 23) where orange groves within the last
60 years (c. 20–30 generations) have expanded the
habitat of L. antalyana to the hill area of L. billae. The
contact zones were about 400–500 m wide. An alternative contact centre, the village Gökdere, is considerably
older but here no parental L. antalyana were found on the
alternative side. Although hybridization may occur
where populations meet naturally, the present hybrid
zone was probably set off by human activity. The area
showed typical hybrid zone characteristics, including
nuclear–nuclear linkage disequilibria, cytonuclear disequilibria and deviations from HW proportions. We
further found introgression in an area southeast of the
primary hybrid zone. This area was unexpected and the
number of individuals analyzed from here are too few to
make generalizations about the level of introgression or
the cause of species contact.
The steepness and concertedness of the clines came
from inclusion of parent individuals on both sides of the
cline. In no sample did we observe both parent species.
This and the lack of F1 equivalents in the hybrid zone
points towards L. antalyana displacing L. billae and limited
reproductive interaction between parental individuals.
F1’s may be lacking either due to selection against F1
hybrids or because they are rarely formed. If the latter
explanation is correct, presence of F2 individuals implies
that F1’s may backcross readily. However, the backcross
probability might be influenced highly by genotypic
interactions. The magnitude of locus-specific introgression differed significantly between the two nuclear loci
Idh and Aat. Cytonuclear disequilibrium at Aat, but not at
Idh, and an intra-locality deficit of Aat heterozygotes, but
not of Idh, indicate different genotypic behaviours and
resulted in L. antalyana Idh introgressing further than Aat
into L. billae. Still, heterozygotes at both loci were lacking
in the total gene pool within the contact area, raising the
713
question arises whether Idh is more common than
expected or Aat less common than expected. The simulation results suggested that Idh heterozygotes are more
common than expected, rather than Aat are heterozygotes under-represented. Hence there may be positive
selection for Idh in backcrosses as well as selection against
heterozygote Aat (which were lacking in hybrids). The
simulations were hampered by not knowing the exact
initial parental frequencies upon contact and speciesspecific dispersal. However, the simulations showed that
varying initial parental frequencies and dispersal rates
changed only the magnitude of introgression but not the
skewed genotype distributions, which implies that selection is needed in future simulations to fit the observed
distribution. This corroborates the finding that the actual
genotype differences possibly have evolved far faster in
nature than indications gained by simulations (20–30
generations vs. 75–175 generations as fitted by the
parental genotype distribution).
The fact that alleles of the less common L. antalyana
move into L. billae populations and the observation of
negative cytonuclear Aat-D2 in L. billae imply further that
L. billae genes are not crossing the hybrid barrier to the
same extent as L. antalyana. Thus not only is L. antalyana
expanding its range but it may also be selectively
replacing L. billae aided by selection against cytonuclear
interactions on a L. billae background. As mentioned
above, the genotypic patterns suggest selection against
hybrid Aat genotypes but positive selection for Idh.
Differential selection should make the Idh cline wider.
It is not unlikely that the introgressed genomes will be
stable at least for Idh. Hybrid genotypes may in some
cases have higher fitness components than parental ones.
Once the F1 barrier is crossed, positive selection may
increase the frequency of L. antalyana Idh. In hybrids of
Bombina bombina and Bombina variegata, Nürnberger et al.
(1995) hypothesized that rapid metamorphosis – relative
to parental individuals – may be influenced by selection
for fast development in dry habitats. A combination of
hybrid characters could hereby become established.
Asmussen et al. (1987) gave hypotheses explaining the
origin of cytonuclear disequilibria in a hybrid population
(which were not definitive). Hypothesis 2 (D2 ¼ 0 and
D ¼ D1 ¼ )D3 „ 0) fits exactly the pattern for Idh and
suggests random mating and no directionality to mating.
In contrast, the pattern for Aat, where D „ 0, D1 „ 0,
D2 „ 0 and )D3 „ 0, corresponds to scenario HNR
which may indicate nonrandom mating, directionality
to mating and a fairly young system. The two complete
opposite explanations for the behaviour of Idh and Aat
may stem either from differential selection or sex-specific
mating behaviour. Genetic drift seems unlikely for
causing the different cytonuclear disequilibria (e.g. Latta
et al., 2001) because HW proportions support a greater
lack of Aat heterozygotes, F1 equivalents were missing
and, given fixed parental multilocus genotypes at species
contact, the expectation of equal frequencies of BB/AB
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J. JOHANNESEN ET AL.
and AB/BB (shown in simulations) was not observed. In
sex-specific gene flow, permanent nonzero cytonuclear
disequilibrium can be maintained with an epistatic
interaction between a single nuclear locus (e.g. Aat vs.
Idh) and cytoplasmic gene that involves specifically the
nuclear gene responsible in mating preference. However,
when the mating preference involves an epistatic interaction between cytotype and the multilocus nuclear
genotype characteristic of the pure parental species, all
cytonuclear disequilibria break down (Arnold et al.,
1988). Asmussen et al. (1989) analysed the cytonuclear
pattern of two species of Hyla tree frogs and suggested
that, due to nearly equal migration rates, significant D2
(i.e. heterozygote association) was caused by asymmetrical mating behaviour where Hyla cinerea females are
more attracted to conspecific males than are Hyla gratiosa
females.
Two findings challenge sex-specific gene flow as the
reason for cytonuclear disequilibrium in Lyciasalamandra.
The first is intra-specific differentiation estimates in L.
billae that indicate that males contribute more to gene
flow than females. The mtDNA differentiation estimate
was six times higher than the nuclear estimate, which,
under equilibrium conditions, indicates that male gene
flow predominates (Birky et al., 1983,1989). (An estimate was not possible for L. antalyana but own field
observations indicate that males move through the
habitat on humid nights in great numbers.) Thus, nuclear
introgression should proceed faster than mtDNA, but was
not the case. The second reason why sex-specific gene
flow does not explain cytonuclear disequilibrium is
because it broke down when analyzing only hybrids;
this is not expected if gene flow is sex-specific. For
example, Shoemaker et al. (1996) found cytonuclear
disequilibrium in fire ants because adult parental individuals continuously move into the hybrid zone. However, the cytonuclear disequilibrium also broke down
when only hybrids were considered. This was caused by a
marked reduction of parental-like hybrids in areas where
the similar parental species was common. This could owe
to reduced fitness of the hybrids caused by extrinsic
selection due to competition with parental species.
Based on the considerations above, it is more likely
that the Lyciasalamandra cytonuclear disequilibrium is
influenced by F1 incompatibility. Data from the primary hybrid zone showed that both parental mtDNA
types and nuclear loci introgress. Thus, hybrid formation involves females of both species. Interestingly,
none of the five adult hybrid males had heterozygotic
Aat while four of 15 females did. Also, calculating the
total number of pure individuals sampled from the
hybrid
areas,
the
relationship
between
female : male : juvenile : subadult is 61 : 48 : 47 : 19
(63 pure individuals and 4 hybrids were not sexed).
Considering the relationship relative to the number of
females, this becomes 1 : 0.79 : 0.77 : 0.31. In the
hybrid zone the relationship was 1 : 0.33 : 0.73 : 0.27.
Thus, only males were missing. Neither of these two
examples were significant (P > 0.20) but combined
they point towards selection against male hybrids.
Unfortunately we do not know which sex is functionally heterogametic in Lyciasalamandra as there are no
visible sex chromosomes. There is no rule about the
heterogametic sex in salamanders where both females
and males can be heterogametic, even within the same
genus (reviewed by Duellman & Trueb, 1994).
The findings from L. antalyana and L. billae provide
evidence for different forces working simultaneously at
species’ transitions. Locus-specific selection and unidirectional range expansion both distort symmetrical
introgression. Symmetrical introgression may further be
complicated by sex-specific hybrid mortality. The locusspecific asymmetry can have a positive effect of hybridization by increasing biological diversity because one
genome does not out-compete the other, but incorporates foreign genes into novel genomic environments and
forges new specific trajectories. This may explain the
great diversity of morphological variants in Lyciasalamandra. Teasing apart these processes, between species-pairs
and within species, will shed light on the jagged process
of diversification. The findings indirectly show how
habitat alterations (be they man-made or not) may
influence the fate of species. Particularly, a comparison
between the young hybridization studied here with what
seems an old ‘natural’ hybrid area in the mountain
ranges (locations 4 and 5) will help differentiate the
selection processes at work.
Acknowledgments
We thank Olaf Godmann, Sebastian Steinfartz and
Mehmet Öz for field assistance. All samples were collected with permission of the Turkish government. We
thank two anonymous reviewers for helpful suggestions
that greatly improved the manuscript.
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Received 12 April 2005; revised 28 July 2005; accepted 06 September
2005
ª 2005 THE AUTHORS 19 (2006) 705–716
JOURNAL COMPILATION ª 2005 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY
Proportion
hybrids
D-Loop
Pgd
Pep-B
Idh-1
Aat-1
A
B
A
B
C
D
A
B
N
1
2
3
4
N
2
3
1.00 1.00 1.00
0
0
0
1.00 1.00 1.00
0
0
0
1.00 1.00 1.00
0
0
0
1.00 1.00 1.00
0
0
0
17
10
11
1.00 1.00 1.00
0
0
0
0
0
0
0
0
0
13
5
9
0
0
0
Population 1
0.20
0.80
0.10
0.90
0.60
0.40
1.00
0
5
0.20
0.60
0.20
0
5
0.20
4
6
7
0.58 0
0.10
0.42 1.00 0.90
0.75 0
0
0.25 1.00 1.00
0.92 0.15 0.10
0.08 0.85 0.90
1.00 1.00 0.32
0
0
0.68
6
10
14
0.50 0
0
0
1.00 0.87
0.50 0
0.13
0
0
0
6
8
8
0.80 0
0.14
5
1.00
0
1.00
0
1.00
0
1.00
0
5
1.00
0
0
0
4
0
8
0
1.00
0
1.00
0.50
0.50
1.00
0
7
0
0.40
0.40
0.20
5
0
9
11
1.00 1.00
0
0
1.00 1.00
0
0
1.00 1.00
0
0
1.00 1.00
0
0
5
11
1.00 1.00
0
0
0
0
0
0
5
9
0
0
10
13
14
1.00 0
0
0
1.00 1.00
1.00 0.14 0.06
0
0.86 0.94
1.00 0.31 0.31
0
0.69 0.69
1.00 1.00 1.00
0
0
0
2
21
16
1.00 0.05 0
0
0.52 0.50
0
0.10 0.25
0
0.33 0.25
1
21
16
0
0.10 0.13
12
16
0
0.11
1.00 0.89
0
0.08
1.00 0.92
0.60 0.39
0.40 0.61
1.00 1.00
0
0
5
19
0
0.06
0.20 0.76
0.80 0.06
0
0.12
5
17
0
0.32
15
18
19
20
21
22
23
24
25
1.00 1.00 0
0
0
0.17 0.86 0
0
0
0
1.00 1.00 1.00 0.83 0.14 1.00 1.00
0.88 1.00 0
0
0.07 0.11 0.86 0
0
0.13 0
1.00 1.00 0.93 0.89 0.14 1.00 1.00
0.88 1.00 0.17 0.39 0.25 0.14 0.86 0.03 0.63
0.13 0
0.83 0.61 0.75 0.86 0.14 0.97 0.38
1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00
0
0
0
0
0
0
0
0
0
4
2
16
14
14
18
14
25
11
0.75 1.00 0
0
0
0
1.00 0
0
0.25 0
1.00 1.00 1.00 1.00 0
1.00 1.00
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
4
13
13
14
14
18
11
10
9
0.50 0
0
0
0.14 0.28 0.43 0
0
17
Appendix A. Allozyme allele frequencies and mtDNA haplotype frequencies of Lyciasalamandra billae and Lyciasalamandra antalyana from a hybrid zone southwest of Antalya, Turkey.
Sample locations are found in Fig. 1 and Table 1. The number of investigated individuals (n) differs slightly between the two genetic markers because one round of DNA extraction was
faulty and amplification failed in roughly half of these individuals. A and B alleles at Aat-1 and Idh-1 signify species-specific L. antalyana and L. billae alleles, respectively.
716
J. JOHANNESEN ET AL.
ª 2005 THE AUTHORS 19 (2006) 705–716
JOURNAL COMPILATION ª 2005 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY