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Molecular Ecology (2008): The definitive version is available at www.blackwell-synergy.com doi: 10.1111/j.1365-294X.2008.03725.x
© 2008 The Author
Journal compilation © 2008 Blackwell Publishing Ltd
Blackwell Publishing Ltd
Good species despite massive hybridization: genetic
research on the contact zone between the watersnakes
Nerodia sipedon and N. fasciata in the Carolinas, USA
K. MEBERT
Department of Biological Sciences, Old Dominion University, Norfolk, VA 23529, USA
Abstract
Genomic markers generated with the amplified fragment length polymorphism method
revealed extensive, panmictic-like hybridization along the narrow contact zone between
the water snakes Nerodia sipedon and Nerodia fasciata in the Carolinas, USA. However,
asymmetric distributions of diagnostic markers between both species and low frequencies
of backcrossed hybrids with a high value of interspecific mixture infer selection against
certain genotypes. This is consistent with a pronounced genetic and morphological prepon-
derance of N. fasciata characters in the hybrid zone. Despite massive hybridization within
the contact zone, the existence of nearly fixed genetic markers and the potential inferiority of
certain hybrid genotypes support the species status of the two taxa and corroborate known,
but nondiagnostic differences in morphology and ecology. This study stretches the
applicability of species concepts to cases, where the genetic compatibility between two
closely related species is very high, yet, they still evolve and persist as independent entities.
Keywords: AFLP, hybridization, incomplete speciation, Nerodia fasciata, Nerodia sipedon, snake
Received 11 March 2007; revision received 8 January 2008; accepted 24 January 2008
Introduction
Scientists rely on studying the patterns of variation within
and among contemporaneous populations or between
different taxa in order to infer mechanisms of speciation
(Mayr 1963; Harrison 1993). Hybridization and introgression
are two such patterns of special interest that have received
increased attention over the last three decades, particularly
since the application of molecular techniques has allowed
identification of introgressive genes to investigate the extent
of hybridization (Hewitt 1988; Rieseberg & Wendel 1993;
Avise 2004).
Generally, hybridization refers to the interbreeding
between two taxa, usually closely related species. This implies
some type of interspecific barrier to keep the two taxa on
their diverging, evolutionary pathways (e.g. Harrison 1990;
Arnold 1997). Hybridization may continue over several
generations, through backcrossing between F1 hybrids
and their parental species or any other later-generation
hybrids. This generates a hybrid zone, a geographical area,
where hybridization results in steep character clines between
two differentiated populations that are themselves relatively
homogenous over large areas (Harrison 1993). In this
context, the dynamic aspect of hybridization events is
termed introgression, which refers to the movement of
genes mediated by backcrossing between two species or
genetically well-distinguished populations (Avise 2004).
The presence of a natural hybrid swarm, a hybrid zone
that includes abundant hybrids with a diverse array of
interspecific recombinant types, conflicts with the traditional
biological species concept (BSC, see Mayr 1963), under which
two species should be reproductively isolated from each
other and hybrids are uncommon and less fit or exhibit an
increased fitness only in an intermediate or altered (mostly
anthropogenic) environment uncharacteristic of either
parental species (Moore 1977; Arnold 1997). Particularly in
vertebrate groups that exhibit coitus to fertilize eggs, such
as mammals and snakes, hybrid zones are rare (best known
interspecific hybrid zones in snakes: Conant 1963; Thorpe
1984; Lawson et al. 1991). Nonetheless, hybrid zones
represent an evolutionary window by revealing aspects of
ecology, behaviour, genetics and geography that contribute
to speciation processes (Harrison 1990). There are two
principal models leading to the development of hybrid
Correspondence: Konrad Mebert, Alte Obfelderstrasse 44,
Affoltern am Albis, 8910, Switzerland.
E-mail: kmebe001@yahoo.com
2K. MEBERT
© 2008 The Author
Journal compilation © 2008 Blackwell Publishing Ltd
zones. First, the geographical-selection-gradient model
assumes that two closely related species are adapted to a
different environment in allopatry (rarely parapatry) and
produce a zone of secondary contact after expansion of their
ranges. A hybrid zone is then formed through exogenous
selection, involving adaptations to distinct environmental
parameters by the two taxa (Endler 1977; Moore & Price
1993). Hybrids are often either sterile, less viable, or less fit
in terms of healthy offspring produced as a consequence
of their minor adaptation to the environment. Position,
width, and maintenance of the hybrid zone may be deter-
mined by environmental parameters. In the second model,
a hybrid zone may arise through a secondary contact or in
parapatry, but its maintenance results solely from endo-
genous selection (internal genetic incompatibilities) and is
not associated with an environmental gradient. In this case,
the hybrid zone is called a tension zone because of the two
opposing forces, dispersal of the parental species towards
the hybrid zone and selection against hybrids (Key 1968;
Barton & Hewitt 1985). Another way of classifying hybrid
zones was proposed by Bogdanowicz & Harrison (1997).
In this classification, hybrid zones are either ‘unimodal’,
consisting largely of a hybrid swarm, ‘bimodal’, containing
mostly parental genotypes with a few hybrids, or an
intermediate, ‘flat’ type.
The watersnakes in the genus Nerodia contain 10 species,
ranging from Canada to Mexico. Interspecific relationships
within Nerodia have been studied repeatedly (e.g. Rossman
& Eberle 1977; Lawson 1987). However, the relationship
between the northern watersnake, N. sipedon, and the banded
watersnake, N. fasciata, has not been fully resolved, since
these two taxa were elevated to species status based on
small, but consistent differences in external characters
across most of their ranges (Conant 1963). This view has
been generally accepted up to a recent treatment of that
genus (Gibbons & Dorcas 2004) and by analysis of genetic
data (Alfaro & Arnold 2001). The two parapatric species
meet in contact areas roughly along the borders of the
Mississippi River Valley and east along the Lower Coastal
Plain to Georgia, and northeast to North Carolina (Fig. 1).
N. fasciata is considered to be a lowland species, occupying
mainly lentic (quiet) water bodies in a subtropical climate,
whereas N. sipedon, replaces the former species towards
the more temperate climate, adding lotic and brackish
Fig. 1 Distribution of Nerodia sipedon and N. fasciata. Letters denote areas with evidence of hybridization at (A) Blaney & Blaney (1979),
Schwaner et al. (1980); (B) Schwaner & Mount (1976); (C) Seyle (1980); (D) Conant (1963: some sites of sympatry without morphological
evidence of hybrids); (E) Conant & Lazell (1973); (F) Gaul (1996); and sympatry-areas with no evidence of hybridization at (G) along the
Mississippi River Valley, Trauth et al. (2004). Figure is modified from Conant (1958) with permission from Houghton Mifflin Co., NY.
HYBRIDIZATION BETWEEN WATERSNAKES 3
© 2008 The Author
Journal compilation © 2008 Blackwell Publishing Ltd
water bodies to its ecological niche (e.g. Conant 1963;
Conant & Lazell 1973; Schwaner & Mount 1976). In the
southeast, the contact zone of the two species follows
mainly the physiographic fall zone, an area, where the flat
Coastal Plain meets the hilly Piedmont (see example for
North Carolina in Fig. 2).
Generally, Nerodia sipedon and N. fasciata appear to main-
tain their identity along their contact zone (Conant 1963).
However, a few studies have found morphological evidence
of substantial interbreeding at several sites of ecological
transition or environmental disturbance between Louisiana
and North Carolina (Fig. 1). For example, a zone of extensive
morphological mixing between N. sipedon and N. fasciata
along the Louisiana–Mississippi border is interpreted as an
intergradation between two subspecies (Blaney & Blaney
1979), but was refuted after the discovery that each putative
hybrid from that area possessed protein allelomorphs
(Schwaner et al. 1980) and morphological characters (Dundee
& Rossman 1989) of only one species. Wiley (1981) viewed
this population as a case representing residual geographical
variation (perhaps reinforced by local selection) of the
common ancestor of the two species. The various opinions
stated above raise the question of whether all currently
recognized contact zones characterized by intermediate
phenotypes actually are intergrades between subspecies,
hybrids between species, or whether many such inter-
mediates belong genetically to one or the other species.
Various molecular methods, e.g. allozymes (Lawson 1987),
microsatellites (Jansen 2001), restriction fragment length
polymorphisms (RFLP, Densmore et al. 1992), mitochondrial
sequence (Alfaro & Arnold 2001), and sequencing of a few
conservative nuclear genes (ZFY, ZNF6, and REP-genes
in Mebert 2003), have been applied for the last 20 years to
investigate the systematic relationships and population
dynamics within the genus Nerodia (Mebert 2003 and refer-
ences therein). However, none of the studies has clarified
the issues concerning the relationship in respect of the
hybridization between N. fasciata and N. sipedon. Amplified
fragment length polymorphism (AFLP) is a promising,
relatively novel method to study closely related taxa, but
has been neglected in the field of zoology (Bensch & Åkesson
2005), also reflected in the relatively few herpetological
studies applying AFLP (e.g. Olsson et al. 2005; Measey et al.
2007; Quinn & Georges 2007). AFLP generates mostly
selectively neutral fragments from genomic DNA alleles
(Avise 2004) with ≤5% being affected by selection (Wilding
et al. 2001; Campbell & Bernatchez 2004). These dominant
markers are inherited in a Mendelian pattern that can be
used to identify species-specific markers (Bensch & Åkesson
2005). Codominance does occur with a frequency of 4–15%
Fig. 2 Distribution of Nerodia sipedon and N. fasciata and hybrid zones in North Carolina, USA. Range limits are drawn from personal
observation (Mebert 2003) and data from Palmer & Braswell (1995). Cross-hatching shows the extent of hybrid zones and sympatry
currently documented with arrows pointing to sites of previous studies (Conant 1963; Conant & Lazell 1973; Gaul 1996). The white areas
represent regions from which records of either species are missing. The ‘Fall Zone’ extends approximately 30–50 km from the indicated
upper boundary towards the coast (adapted from Conant 1963; Clay et al. 1975). Numbered sites: (1) Richmond Co.; (2) city of Southern
Pines, Moore Co.; (3) city of Fayetteville to Cambro Pond in Cumberland and Harnett counties; (4) Holts Lake, Johnston Co.; (5) area around
city of Greenville in Pitt and Edgecombe counties; (6) lower Roanoke River near Oak City, Martin Co.; (7) Elizabeth City area in Pasquotank
and Camden counties; (8) Highway 264 through Alligator River National Wildlife Refuge in Dare and Hyde counties; (9) Tar River Reservoir
Lake and Falls Battle Park, Nash Co.; (10) Northampton Co.; (11) Hertford Co.; (12) Shackleford Bank, Carteret Co.; (13) Garner Creek,
Martin Co.; and (14) Lake Waccamaw, Columbus Co. Small map insert shows the hypothetical hybrid zone as presumed, if sufficient data
over the entire contact zone in North Carolina could be collected.
4K. MEBERT
© 2008 The Author
Journal compilation © 2008 Blackwell Publishing Ltd
(Mueller & Wolfenbarger 1999). The problem of homoplasy
is negligible, as O’Hanlon & Peakall (2000) found an
average of only 2.5% size homoplasy for AFLP fragments
among congeners, which could be counteracted by selecting
for long amplified fragments from gel areas of low fragment
density (Vekemans et al. 2002). Variation among individuals
and taxa may result from base substitutions within cleavage
sites or from corresponding additions or deletions of
DNA. AFLP has a great resolution, yields many diagnostic
markers, and is not affected by potentially anomalous
inheritance of the maternal lineage (Savelkoul et al. 1999).
It was successfully applied to demonstrate hybridization
in plants (Chauhan et al. 2004; Guo et al. 2005; Lihova et al.
2007) and animals (Mavarez et al. 2006; Meyer et al. 2006;
Vallender et al. 2007).
The objectives of this study were to evaluate the relationship
between N. sipedon and N. fasciata by means of genotypically
characterizing their zone of sympatry and reveal the degree
of hybridization. The study of such hybrid zones provide
us with valuable insights into the mutual genetic com-
patibility between two closely related taxa, from which
inferences about their evolutionary paths can be drawn.
Hybrid zones might be active sites of evolutionary change,
either as sources of recombinant types, especially in plants
(Arnold 1997), or as areas, where selection against hybridiza-
tion leads to strong prezygotic barriers to gene exchange,
thus reinforcing characters of reproductive isolation and
promoting speciation (Hewitt 1988; Harrison 1993 and
references therein). The objectives included the search for
at least five diagnostic genetic markers per species, to reco rd
and map the distribution of distinct hybrid categories,
and thereby gaining insight into the extent and direction
of gene flow between the two taxa. The study of this hybrid
zones may extend the applicability of popular species con-
cepts to cases where massive interspecific genetic exchange
occurs.
Materials and methods
Sample and sites
From 1995 to 1999, approximately 330 specimens were
collected from eight anticipated sites of sympatry near the
fall zone between the states of Georgia and Virginia and
coastal areas of North Carolina, USA. Allopatric samples
were collected from nine sites at distances of more than
25 km from the border of any known area of sympatry in
the same states and in Florida. This distance was found to
be sufficient to keep the possibility of overlooked intro-
gression near the hybrid zone small. Sites of presumed
allopatry have been posteriorly confirmed by the genetic
data. All collection sites in North Carolina are indicated
in Fig. 2, whereas details of two major sampling sites from
the border area between Virginia and North Carolina, and
between South Carolina and Georgia, are shown in Fig. 3,
and in Gibbons & Dorcas (2004), respectively.
Morphological data were recorded from each individual,
which was subsequently assigned to one of five categories
according its geographical provenance (i.e. from within or
outside the contact zone) and their phenetic resemblance
to one of the species based on distinguishing features,
including diagnostic dorsal pattern and significant differ-
ences in other colour pattern characters and head shape/
proportions (Clay 1 938; Conant 1963). A complete morpholo-
gical analyis is given in Mebert (2003).
Abbreviations used in the text for these phenetic/geo-
graphical groups are
•s (allopatric sipedon): Nerodia sipedon from areas > 25 km
away from the contact zone;
•cs (contact zone sipedon): phenetic N. sipedon from the
contact zone;
•x (hybrids): phenetic intermediates (putative hybrids)
from the contact zone;
•cf (contact zone fasciata): phenetic Nerodia fasciata from
the contact zone;
•f (allopatric fasciata): N. fasciata from areas > 25 km away
from the contact zone.
A maximum of 1 mL of blood was extracted from the
subcaudal vein and stored in screw-cap tubes containing
9 mL lysis buffer (100 mm Tris-HCl, pH 8; 100 mm EDTA,
pH 8; 10 mm NaCl; and 1.0% SDS weight:volume). Most
snakes were photographed, scale-clipped with the individual
identification number, and later returned to their sites of
capture. Approximately 10% of snakes were preserved as
voucher specimens and deposited at the North Carolina
State Museum of Natural Sciences, Raleigh.
Genetic procedures
The DNA of most samples was purified using the GFX
Genomic Blood DNA Purification Kit (Amersham Pharmacia
Biotech), or slightly altered standard phenol–chloroform
procedures in Hillis et al. (1996). Subsequently, the DNA
samples were processed by the AFLP method (Vos et al. 1995),
with modifications by Brazeau et al. (2001). The amplified
fragments were separated by a 6% polyacrylamide gel
electrophoresis (PAGE). Urea was added to the gel solution
and formamide was added to the loading buffer to separate
the double strands. No doublets were observed. A current
of 60 watts was applied to the gel for 2–3 h. The bands were
visualized by silver staining according to modifications by
Promega Corporation. For permanent preservation, many
gels were scanned at 2000 dpi and photographed with 100
or 200 ISO film. Initially, species-specific markers were
identified for presence/absence with two different banding
patterns generated by two selective primer pairs; EcoRI
HYBRIDIZATION BETWEEN WATERSNAKES 5
© 2008 The Author
Journal compilation © 2008 Blackwell Publishing Ltd
primer with -ACC (bp) extension paired with either the
MseI primer with CTT-extension, or MseI primer with CTA-
extension (Mebert 2003). Potentially codominant marker
pairs, as for example when both species yielded a band of
nearly the same size and apparently differing by only a few
base pairs, were scored independently, since the sequences
remained unknown.
Markers
Overall, 286 specimens from all five phenetic groups contrib-
uted to the genetic study (s: n= 83; cs: n=37; x: n= 36; f:
n=50; and cf: n= 80). Ten genetic markers that exhibit high
diagnostic values were tested in the two allopatric (‘pure’
species) groups f and s. Those markers were labelled F1
Fig. 3 Distribution of geno- and phenotypes
in southeastern Virginia and northeastern
North Carolina. Individual genotypes are
based on 10 diagnostic genetic markers
and illustrated with circles, indicating the
proportional contribution of each species;
black from Nerodia fasciata and white from
N. sipedon: 䊊 pure N. sipedon, 䊉 pure N.
fasciata, 两 N. sipedon missing an additional
genetic sipedon marker, possibly because o
f
a low influence from N. fasciata (details in
Mebert 2003). Numbers in or next to large
circles near Elizabeth City represent the
sample size exhibiting that genotype.
Those circles are enlarged to emphasize
their increased contribution, but are not
in proportion to the sample size. Many
genotypes from sites along the border
between Pasquotank and Camden counties
are shown slightly displaced for clarity.
Squares represent snakes evaluated solely
by distinct phenotypic features of colour
pattern, such as dorsal banding pattern,
postocular stripe, and ventral marking:
䊏 phenetic N. fasciata and ⵧ phenetic N.
sipedon.
6K. MEBERT
© 2008 The Author
Journal compilation © 2008 Blackwell Publishing Ltd
to F5 for N. fasciata and S1 to S5 for N. sipedon (see Table 1).
Criteria for the choice of genetic markers were visual clarity,
high fixation rate (fixed at ≥98% in one species, and
occurring at ≤4% in the other species), and reproducibility
(40 specimens were reprocessed with new reagents to
confirm the repeatability of the banding pattern), and reliably
scoring of > 250 individuals. Hence, each specimen was
scored for 10 loci composed of five positive states (presence
of markers of one species) and five negative states (absence
of markers of the other species).
To examine the extent of hybridization and introgression,
every individual was assigned to one of six categories
depending on its combination of species-specific genetic
markers (Lamb & Avise 1986; Miller 2000):
1Genotypic Nerodia fasciata, having all of the fasciata markers
but none of N. sipedon;
2Genotypic N. sipedon, having all of the sipedon markers
but none of N. fasciata;
3F1 hybrid, having all markers from N. sipedon and N. fasciata;
4N. fasciata backcross, having all five fasciata markers plus
one to four sipedon marker(s);
5N. sipedon backcross, having all five sipedon markers plus
one to four fasciata marker(s);
6Later-generation hybrid, having fewer than five markers
of either species, but otherwise a varying combination of
markers from both species, including no marker. These
specimens may represent F2 from F1 hybrid matings or
any other mating that involved a backcross.
No clear evidence for linkage between markers was
found after screening frequencies for all possible pairwise
combinations of species-specific loci in the presumptive
first-generation backcross to either species. Recombinant
genotypes were observed with relatively high frequencies
of 31% (between markers S2 and S3) to 69% (between markers
S2 and S5, as well as between F3 and F5). This suggests that
the species-specific markers probably are not tightly linked
and segregate according Mendelian inheritance. Only the
pairing of S3 and S4 yielded a low recombination frequency
of 15%, possibly indicating a linkage or a statistical type
I error. They could be in close proximity to each other,
increasing the probability of linked crossovers during
recombination. However, their simultaneous occurrence
in phenetic Nerodia fasciata from the contact zone was low
(< 4%), indicating their independence.
K, the probability of misclassifying a specimen into the
false category according to Mendelian segregations, is
related to the number of codominant, diagnostic markers
(n) per species via the equation k= (0.5)n, equivalent to a
situation with 10 dominant markers (Lamb & Avise 1986;
Miller 2000). In a dominant marker system, when n=5,
k= 0.031 (< 5%) for a first generation backcross showing
the same genotype as a pure specimen (= one out of 32
possible genotypic combinations after backcrossing an F1
hybrid with a pure parental specimen). Additional, rare
misclassification may accrue because of the incomplete
fixation of some genetic markers, with some markers
occurring up to 4% in the other species (Table 1). Overall,
potential misclassifications remain small, affecting one to
three individuals from the contact zone, and would mainly
increase the frequency of mixed genotypes. Numerous
calculations involving frequencies of species markers and
genotypes followed. In a first step, frequencies of markers
(including null markers = absent bands) were compared to
the Hardy–Weinberg equilibrium (Nei 1987). The frequency
of the null allele of a particular marker (bi-allelic) could
be derived simply from the square root of the observed
number of specimens missing that species marker. Second,
putative backcrosses of F1 hybrids to pure species (F1
hybrids ×parental genotype) were grouped according
their level of interspecific mixture of genetic markers. When
a backcrossed individual contains the complete set of
genetic markers from species A, then the term ‘introgressive
markers’ refers to the number of genetic markers it con-
tains from species B. This corresponds to a classification of
the degree of interspecific heterozygosity in a codominant
system with five diagnostic nuclear markers. A chi-squared
test of independence was applied to compare the fre-
quencies in those groups to Hardy–Weinberg expectations.
Corrections in the degrees of freedom were made for the
absence of two groups, those exhibiting a parental geno-
type (zero introgressive markers) and those with an F1
hybrid genotype (five introgressive markers).
Table 1 Proportion of AFLP genetic markers expressed among
phenetic groups
Phenetic
group
sipedon markers
NS1 S2 S3 S4 S5 Smean
s 83 100 100 99 99 99 99.4
cs 35 83 86 74 81 81 81.0
x 36445035385043.4
cf 76 30 12 13 5 18 15.6
f 50400221.6
fasciata markers
NF1 F2 F3 F4 F5 Fmean
s 83402142.2
cs 34 26 34 56 29 29 34.8
x 33837582796877.4
cf 74 95 84 97 95 92 92.6
f 50 100 100 100 98 100 99.6
N, mean number of snakes per group. Fn and Sn,markers
expressed by Nerodia fasciata and N. sipedon respectively. Phenetic
groups: cf and cs, phenetic N. fasciata and N. sipedon, respectively,
from the contact zone; f and s, allopatric N. fasciata and N. sipedon,
respectively, from an area > 25 km from the contact zone;
x, phenetic intermediates between f and s.
HYBRIDIZATION BETWEEN WATERSNAKES 7
© 2008 The Author
Journal compilation © 2008 Blackwell Publishing Ltd
Results
Reliability and frequencies of genetic markers and
genotypes
Most genetic markers were fixed or nearly fixed (98–100%)
for their respective species (Table 1). In contrast, a few
potentially introgressed markers or AFLP-bands with the
same size were detected in four individuals (~2%) from
allopatric populations at distances of 40–300 km from the
hybrid zone. This proportion is small and the genetic
markers were considered reliable, as also the frequencies
of null alleles (recessive homozygous loci) in allopatric
populations were < 4%.
The abundance of genotypes with mixed ancestry cor-
roborates the detection of a hybrid zone in the Carolinas
based on the occurrence of morphologically intermediate
watersnakes (e.g. Conant 1963; Gaul 1996). However, the
genetic data often revealed a larger width of the hybrid
zone than expected by morphological data alone. The width
of the hybrid zone is narrowest along the coast (~5 km) and
reaches its greatest extension of 70 km along the Tar River
between Greenville and Rocky Mount, the northwestern
contact zone in North Carolina (area between sites 9, 10,
and 13 in Fig. 2). From there, the hybrid zone decreases in
width in southwesterly direction to approximately 20 km
at the Edgefield–Aiken countyline in South Carolina.
Approximately 10% of individuals in the contact zone
were ‘pure’ genotypes, all Nerodia fasciata (Table 2). No F1
hybrids were found. Backcrosses of F1 hybrids to a parental
species included about 35% of all specimens within the
contact zone, whereby backcrosses to N. fasciata were twice
as frequent as those to N. sipedon. The majority of snakes
(55%) were late-generation hybrids, which included crosses
among all hybrid genotypes and those between non-F1
hybrids and either parental genotype.
Within the contact zone, frequencies for fasciata markers
ranged from 70.6% to 86.2% and were in Hardy–Weinberg
equilibrium (75.0%), as expected for a dominant system
in a freely interbreeding population [χ2= 0.062 (F1)–1.835
(F3)]. Null alleles of the fasciata markers (snakes missing
the markers, and thus being recessive homozygous at
those loci) also met Hardy–Weinberg expectations, except
null-F3 (χ2= 5.51, P< 0.025). In contrast, all sipedon markers
revealed lower than expected frequencies, 29.4–50.0%,
while their corresponding null alleles had unexpectedly
high frequencies of 56.1–70.6% [χ2= 12.33 (S1)–30.26 (S4) for
markers, and 36.99 (null-S1)–90.82 (null-S4) for associated
null alleles; all with P< 0.0001]. Both, phenetic N. sipedon
(cs) and N. fasciata (cf) from the contact zones yielded lower
frequencies of their corresponding species markers com-
pared to specimens from their respective allopatric groups
(s and f; Fig. 4 and Table 1).
Distribution of genotypes among phenetic groups
(cs, cf, x) in the contact zone
After evaluating individuals with all 10 genetic markers
(n=109), no pure Nerodia sipedon genotype was found in the
contact zone and only approximately 15% of phenetic N.
fasciata were also genotypically pure N. fasciata. Hence, all
other phenetic N. fasciata contained a mixed genotype. They
contribute to a group of cryptic hybrids, as their hybrid-
ization status was not recognizable using traditional
colour pattern characteristics. Half of those cryptic hybrids
exhibited one out of 10 genetic sipedon states, whereas the
remainder possessed two or more sipedon states. Similar
was the situation with phenetic N. sipedon, whereby 45% of
Table 2 Proportions (percentage) of differen
t
categories of genotypes among phenetic
groups from the contact zone
Genotype category cs x cf Total genotype
Nerodia fasciata (Nf) 0 0.9 8.3 9.1
Nerodia sipedon (Ns) 0000
F1 hybrid between Nf and Ns 0 0 0 0
F1 hybrid backcross to Nf 0.9 8.3 14.7 23.9
F1 hybrid backcross to Ns 10.1 1.8 0.0 11.9
Later-generation hybrid 13.8 10.1 32.1 55.0
Total phenetic group 24.8% 21.1% 55.1%
n= 109 for which all 10 genetic markes could be scored. Phenetic groups: cf and cs, phenetic
Nerodia fasciata, and N. sipedon, respectively, from the contact zone; x, phenetic intermediates
b
etween N. fasciata and N. sipedon. Genotype categories: Nf, ‘pure’ genotypes of N. fasciata
(five fasciata markers, no sipedon markers); Ns, ‘pure’ genotypes of N. sipedon (five sipedon
markers, no fasciata markers); F1 hybrid between Nf and Ns (five sipedon and five fasciata
markers); F1 hybrid backcross to Nf (five fasciata markers, 1–4 sipedon markers); F1 hybrid
b
ackcross to Ns (five sipedon markers, 1–4 fasciata markers); later-generation hybrid (1–4
f
asciata markers, 1–4 sipedon markers). The lowest row shows the proportion of each phenetic
subgroup in the contact zone (rounded values).
8K. MEBERT
© 2008 The Author
Journal compilation © 2008 Blackwell Publishing Ltd
their cryptic hybrids expressed one fasciata state, and higher
proportions of fasciata states were far less common. One
phenetic N. sipedon even possessed six genetic fasciata states
and thus genetically resembled more N. fasciata. Generally,
the frequency of genes from N. fasciata in phenetic N. sipedon
(maximum of 56% for marker F3) was larger than the
frequency of sipedon genes in N. fasciata (max. 30% for marker
S1; Table 1).
Snakes with an intermediate colour pattern (group x)
also exhibited mixed genotypes, being more intermediate
to those of cf and cs (Fig. 4). However, the general tendency
towards increased expression of fasciata markers in snakes
from the contact zone is repeated for x, which expressed
close to twice as many fasciata markers than sipedon markers
(Table 1 and Fig. 4). The phenetic hybrids produced the
widest range of genetic variation, from snakes with one
sipedon state and nine fasciata states to those with seven
sipedon states and three fasciata states (details in Mebert
2003). However, specimens with more than five fasciata
states predominated among the phenetic hybrids. No true
F1 hybrids, expressing all 10 genetic species markers, were
identified, but three snakes exhibited nine markers. One
phenetic hybrid exhibited a ‘pure’ fasciata genotype.
Distribution of introgressive markers in backcrosses of F1
hybrids to parental species
The distribution of introgressive species markers (com-
parable to the degree of interspecific heterozygosity in a
codominant system) within both genotypic categories of
presumptive F1 hybrids backcrossed to one of their parental
species deviated significantly from Hardy–Weinberg equ-
ilibrium (backcross to Nerodia fasciata: (χ2= 45.2, P< 0.0001;
backcross to N. sipedon: χ2= 26.4, P< 0.0001). For example,
there was a preponderance of individuals with only one
introgressive marker in both groups of backcrossed F1
hybrids (Table 3). Perfect Hardy–Weinberg expectations
would predict a proportion of 16.7% for individiduals from
such a category, but they were about twice as frequent as
all other categories combined (those containing two, three,
or four introgressive markers). Therefore, the backcrossed
hybrids that genetically resembled more the ‘parental’
genotype were abundant, whereas others were less frequent,
the more introgressive markers they contained (i.e. exhibiting
increased interspecific mixture).
Discussion
Efficacy and distribution of genetic markers
AFLP emerged as a highly efficient method for distin-
guishing the two closely related species, revealing structure
of their hybrid zone, and comparing phenotypic and
genotypic data sets. Although, the initial morphological
classification of individuals was fairly accurate, as fasciata
phenotypes generally reflected more fasciata-like genotypes
and sipedon phenotypes showed a similar but less prono-
unced pattern, it required genotypic data to detect the vast
majority of hybrids generated through backcrossing. The
occurrence of a few fasciata genes in ‘allopatric’ populations
of Nerodia sipedon at great distances up to 300 km from the
contact zone, and vice versa, affected the reliability of
such markers. However, it is unlikely that these ‘foreign’
markers are based on current introgression, as the distances
to potential source populations are far and both taxa yield
a low dispersal rate (e.g. Michot 1981; Tiebout & Cary 1987;
Prosser et al. 1999). Rather, these putative introgressive genes
may be the product of homoplasy (a different band of similar
size), a synplesiomorphic character, a procedural artefact,
Fig. 4 Proportions of genetic fasciata states (Fs) and sipedon state
s
(Ss) among phenetic groups from the contact zone. The geneti
c
states in each individual are evaluated by scoring 10 loci
,
composed of five positive states (presence of markers of the firs
t
species) and five negative states (absence of markers of the secon
d
species). Groups: cf and cs, phenetic Nerodia fasciata and N. sipedon
,
respectively, from the contact zone; x, phenetic intermediat
e
snakes.
Table 3 Proportions (percentage) of introgressive species markers
in individuals resulting from backcrosses between F1 hybrids to
one parental species (equivalent to heterozygotes in a codominant
system). Corrections in the degrees of freedom are made for the
absence of individuals with five- and zero-introgressive markers.
Thus, the proportion of introgressing markers was calculated for
30 different genotypic combinations rather than for 32, as predicted
b
y Mendelian segregations, because ~6% (2/32) of such backcrosses
would be indistinct from either a pure genotype or an F1 hybrid
Number of introgressive markers
1234
Nerodia fasciata backcross
Observed 65 15 12 8
Expected 17 33 33 17
Nerodia sipedon backcross
Observed 69 8 15 8
Expected 17 33 33 17
HYBRIDIZATION BETWEEN WATERSNAKES 9
© 2008 The Author
Journal compilation © 2008 Blackwell Publishing Ltd
a relict of an introgression in the past, or originate from
introduced specimens from the other species. Nonetheless,
the overall reliability of genetic markers remained high.
The general abundance of hybrids of various categories
initially suggested that the two species created a hybrid
swarm, a unimodal hybrid zone with freely interbreeding
members similar to a panmictic population (Bogdanowicz
& Harrison 1997; Bensch & Åkesson 2005). Although the
genetic influx from peripheral parental populations appears
to have been small, continued introgression could result
in a complete convergence of their genomes. Moreover,
bidirectional mating between both species has been con-
firmed with mitochondrial data (Gaul 1996) and under
controlled laboratory conditions during this study (N. Ford,
personal communication). Survival and fertility of second-
generation hybrids have also been demonstrated by
Riches (1976), corroborating the high fertility of wild-caught
hybrids collected in this study (Mebert 2003). However, an
asymmetrical distribution of genetic markers within the
contact zone in favour of Nerodia fasciata indicates the
presence of selective forces preventing the formation of
a single species. First, all genetic markers for N. sipedon
showed frequencies lower (< 46%) than expected at
Hardy–Weinberg equilibrium (~75%), whereas frequencies
of markers for N. fasciata were near equilibrium or higher,
indicating selection against genes of N. sipedon. Genetic
drift, assortative mating, migration, and mutation pressure
are potential mechanisms of selection. But they are unlikely
to act in this situation, in part because conditions in the
hybrid zone appear to be relaxed, as extensive natural
habitat is available, individuals are locally abundant, and
mating is not assortative. Second, the proportion of fasciata
genes in phenetic N. sipedon (group cs) is higher than the
reverse, sipedon genes in phenetic N. fasciata (group cf). This
suggests that genes of N. sipedon disrupt the phenotype of
N. fasciata less than the phenotype of N. sipedon is affected
by the introgression of N. fasciata. Third, genetic fasciata traits
dominate in the phenetic hybrids (group x). Finally, N. fasciata
was the only species contributing ‘pure’ genotypes into the
contact zone from adjacent parental populations. In con-
clusion, N. fasciata appeared to have an advantage over N.
sipedon within the contact zone during the period of sampling.
The preponderance of Nerodia fasciata in morphological
traits (Mebert 2003) and genetic characters within the contact
zone implies two possible scenarios for the interspecific
dynamics in the contact zone. First, the contact zone may
represent an area of current introgression of fasciata genes
into the former range of N. sipedon, whereby directional
selection results in the local predominance of N. fasciata,
increasingly displacing N. sipedon. Asymmetric introgression
probably will not proceed beyond the borders of the hybrid
zone, as each species’ advantages are linked to environ-
mental parameters that can be found up to the borders of
the hybrid zone (Mebert 2003). Second, the area may repre-
sent a stable hybrid zone with a heterogeneous, mosaic-like
habitat, thereby allowing both species to co-exist in their
preferred ecological niches and hybridization to occur along
patches of intermediate habitats. In both scenarios, an overall
local, environmentally related selective advantage in the
sampled areas accorded to N. fasciata and limited intrusion
from N. sipedon promotes the asymmetric distribution of
species-specific genes. However, as indicated in the following
paragraph, selection in the hybrid zone may not act on the
species as a whole, but rather on certain genotypes.
Distribution of hybrid genotypes
The lack of F1 hybrids likely is the result of the abundance
of successful backcrossing and low dispersal rate in relation
to the usually wide hybrid zone (20–70 km), which reduces
the probability of direct mating between pure Nerodia sipedon
and N. fasciata from populations adjacent to the contact
zone (Szymura 1993). Increasing the sampling effort along
the borders of the hybrid zone would eventually reveal some
F1 hybrids, especially along the narrowest hybrid zone
near the coast (approximately 5 km in width). Nonetheless,
the occurrence of first generation (F1) backcrosses (F1 hybrid ×
parental genotype) indicates some regular migration of
parental genotypes that enable the production of F1 hybrids.
Because of the low probability of misclassifications (< 5%),
it is unlikely that a significant portion of the F1 backcrosses
has been misclassified as later-generation hybrids.
The unequal distribution of the various categories of
pure and hybrid genotypes may indicate the presence of
additional selective forces. In a comparative study, Lamb &
Avise (1986) documented that at one pond, where substantial
hybridization between the treefrogs Hyla gratiosa and
H. cinerea was observed, close to 38.4% of the population
constituted F1 backcrosses to a parental species and later-
generation hybrids were rare (3.6%), indicating either
selective disadvantage against the latter group or genetic
swamping by parental genotypes. In my study, the frequency
of F1 backcrosses (35%) to either parental snake species was
very close to the treefrog study, but the high proportion of
later-generation hybrids suggests an almost unrestricted
genetic exchange between the two snake species over large
areas. However, the significant high frequency of individuals
with only one introgressive marker among F1 backcrosses
to a parental species (equivalent to single heterozygotes in
a codominant system) indicates a potential selection against
watersnakes with an increased state of interspecific mixture.
Similarly, Lamb & Avise (1986) found significant deviations
among heterozygotes of presumptive backcross progeny
in treefrog hybrids, albeit not as strong as among Nerodia
backcrosses. The selective disadvantage of hybrids with a
greater degree of interspecific mixture may be mediated
by epistasis between loci because of the disruption of co-
adapted gene complexes within each species (Arnold 1997).
10 K. MEBERT
© 2008 The Author
Journal compilation © 2008 Blackwell Publishing Ltd
Results of various studies on hybrids in animals and plants
corroborate the decreased level of fitness in such heterozy-
gote genotypes (e.g. Reed & Sites 1995; Arnold 1997; and
references therein). The pattern of a mixed selection for and
against certain genotypes in the hybrid zone resembles a
structure proposed with the evolutionary novelty model
by Arnold (1997).
Aside from genetic incompatibilities between the two
watersnake species, reduced fitness and selection against
hybrids are probably related to habitat parameters, similar
to that inferred from hybrid zones between the fire-bellied
toads Bombina variegata and B. bombina in Eastern Europe
(e.g. Szymura 1993; Yanchukov et al. 2006 and references
therein). Although some diverging environmental preferences
between the two Nerodia species, correlating to topography,
temperature, and salinity, have been found and were
discussed in Mebert (2003), no high levels of linkage dis-
equilibria comparable to the Bombina hybrid zone was
revealed. This might be due to a greater degree of inherent
genomic compatibility between the watersnakes, the neutral
character of most AFLP markers, as well as the ecotone
nature and the large size of the hybrid zones, enabling
both species to co-exist and produce sufficient backcrosse s.
Many examples from animals and plants demonstrate
that hybrid populations can thrive in such ecotones (refer-
ences in Harrison 1993).
Systematic implications and species concepts
Reproductive isolation is a precondition for two taxa to
evolve successfully as separate entities and is the pillar of
the BSC (Mayr 1963). The ease with which Nerodia fasciata
and N. sipedon establish hybrid zones has generated
uncertainty about their taxonomic status (e.g. Schwaner &
Mount 1976; Blaney & Blaney 1979; Lawson 1987). Reports
of bidirectional mating have further eroded support for
their specific status (Riches 1976; Gaul 1996; this study).
These findings do not conflict with the BSC, if the hybrids
were to exhibit a good fitness only temporarily or restricted
to a small area of sympatry. However, the hybrid zone
between the watersnake species possibly have existed up
to 5 million years based on fossil records (Holman 2000),
other molecular studies (Lawson 1987; Densmore et al.
1992; Gaul 1996; Alfaro & Arnold 2001), and a lack of clear
glacial isolates between them (Mebert 2003 and references
therein). Although the hybrid zone is relatively narrow, its
entire length of approximately 2000 km from North Carolina
to Louisiana probably is large enough to encompass more
than 100 000 of backcrossed hybrids at any given time, a
truly massive number for vertebrates with coital fertilization.
The high frequency of hybrids is inferred from the avail-
ability of suitable habitats the fact that each contact
zone investigated in this study yielded hybrids, personal
observations and the reports of the general abundance
of both species and their intermediates (e.g. Gibbons &
Dorcas 2004; Fig. 1).
The genetic results presented here indicate that the two
taxa qualify as species under both the phylogenetic species
concept (PSC, Cracraft 1983) and the evolutionary species
concept (ESC, Simpson 1961; Frost & Hillis 1990). First, a
combined set of genetic species markers with nearly 100%
fixation rates produced with the AFLP method fulfil the
requirement of the PSC (Cracraft 1983) by demonstrating
diagnosable characters clearly distinguishing two species
and implying a parental pattern of ancestry and descent.
In this context, all Nerodia sipedon from localities outside
the contact zone were unified by at least five diagnostic
species markers, including individuals from localities
as distant as Pohick Bay Regional Park, Virginia, south to
Augusta, Georgia (approximative airdistance 570 km), or
the distance between Roanoke Island, North Carolina, west
to Grapefield, Virginia (~400 km). N. fasciata was also iden-
tified by at least five diagnosable genetic markers that
unify pure individuals from north of the Albemarle Sound,
North Carolina, to those from 950 km farther south near
Sarasota, Florida. In contrast, there is a full transition of
genetic markers from one species to the other across a
relati vely short distance from 5 to 70 km, depending on the
regional extent of the hybrid zone (Mebert 2003). Although
the distinct genetic markers might be only a byproduct
of their separate evolution without any biological function,
they reflect the cohesion within each taxon.
Second, despite the new genetic evidence supporting the
species status of Nerodia fasciata and N. sipedon, substantial
gene flow between those species in the Carolinas confirms
previously suspected introgression based on morphological
intermediacy (e.g. Conant 1963; Schwaner & Mount 1976;
Blaney & Blaney 1979). However, the unbalanced genotype
frequencies, indirect indication of hybrid inferiority,
significant morphological differences between pure speci-
mens, and distinctive habitat preferences, suggest that
N. sipedon and N. fasciata are on independent evolutionary
trajectories, despite massive hybridization. As such, the
two lineages conform to the ESC. Which then are the likely
consequences for these two species in their area of sym-
patry? Presumably, the fitness of parental and hybrid
genotypes are linked to environmental factors, which
may change geographically and temporarily. However, in
a period of relatively stable environmental conditions (e.g.
decades and centuries within an interglacial period), none
of the two species will competitively exclude the other, as
both exhibit the full suit of their selective advantages only
in their species-specific, allopatric habitats. During that period,
hybridization is restricted to geographically well-defined
natural ecotones (Mebert 2003). However, the hybrid zone
may move back and forth between 50 and 200 km in
respect to changing environmental conditions over periods
of 100s to 1000s years and during fluctuations of glacial
HYBRIDIZATION BETWEEN WATERSNAKES 11
© 2008 The Author
Journal compilation © 2008 Blackwell Publishing Ltd
cycles. This seesaw of environmental factors and their influ-
ences on certain parental and hybrid genotypes generates
some stability in this hybrid zone over the long-term. Such
stable hybrid zones represent intermediate stages along
the evolutionary route that must be taken during parapatric
speciation (Jiggins & Mallet 2000) and ultimately maintain
the species’ integrity along their zone of contact.
In conclusion, the application of a high-resolving genetic
method contributed valuable information regarding the
relationship between Nerodia fasciata and N. sipedon. Tradi-
tional diagnostic features of colour pattern serve well to
distinguish specimens of both taxa from allopatric popula-
tions, but they were inadequate to detect the extent of
introgression in the contact zone (Mebert 2003). This study
has extended our knowledge of natural hybridization in
snakes, a group with only a few known hybrid zones (e.g.
Thorpe 1984; Lawson et al. 1991). I have demonstrated the
utility of AFLP for the detection of diagnostic markers of
two closely related species, revealed extensive interspecific
introgression and asymmetry of gene exchange in favour
of fasciata traits and certain hybrid types, as well as a possible
selective disadvantage against N. sipedon and against
specimens exhibiting a state of increased interspecific
mixture. The simultaneous application of different data
sets (genetic in this script; morphological and ecological in
Mebert 2003) represents an integrated approach to under-
standing the pattern of selection in hybrid zones (Arnold
1997). The study of this species-pair has provided an excellent
opportunity to investigate the consequences of incomplete
speciation and its applicability to modern species concepts
under natural conditions.
Acknowledgements
Financial support: Roche Research Foundation, Theodore Roosevelt
Memorial Fund, Foundation Dr Joachim de Giacomi, Virginia
Academy of Sciences, North Carolina Herpetological Society,
Sigma Xi, and University of Virginia. My special thanks for editing
go to Alan Savitzky and Sylvain Ursenbacher, as well as two
anonymous reviewers for constructive suggestions. I am grateful
for molecular support to the laboratories of Denise Cooper, Dan
Brazeau, and ‘Ginger’ Clark.
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Konrad Mebert has research interests in evolution, population
genetics, biogeography, ecology, and conservation of reptiles and
other vertebrate groups.
