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Good species despite massive hybridization: Genetic research on the contact zone between the watersnakes Nerodia sipedon and N. fasciata in the Carolinas, USA


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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 preponderance 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.
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Molecular Ecology (2008): The definitive version is available at 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
Department of Biological Sciences, Old Dominion University, Norfolk, VA 23529, USA
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
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.
© 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.
© 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.
© 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
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
© 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.
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: pure N. sipedon, pure N.
fasciata, N. sipedon missing an additional
genetic sipedon marker, possibly because o
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:
phenetic N. fasciata and phenetic N.
© 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
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.
© 2008 The Author
Journal compilation © 2008 Blackwell Publishing Ltd
Reliability and frequencies of genetic markers and
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
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
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
ackcross to Ns (five sipedon markers, 1–4 fasciata markers); later-generation hybrid (1–4
asciata markers, 1–4 sipedon markers). The lowest row shows the proportion of each phenetic
subgroup in the contact zone (rounded values).
© 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).
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
(Ss) among phenetic groups from the contact zone. The geneti
states in each individual are evaluated by scoring 10 loci
composed of five positive states (presence of markers of the firs
species) and five negative states (absence of markers of the secon
species). Groups: cf and cs, phenetic Nerodia fasciata and N. sipedon
respectively, from the contact zone; x, phenetic intermediat
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
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
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
© 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).
© 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
© 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.
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.
... In contrary, the expression of these 14 characters from two related and sympatric/syntopic species speaks strongly for F1-hybrids, and with a lower probability for an F2-or later-generation backcross (Mebert, 2008(Mebert, , 2010. This concludes that the expression of morphological traits follows the common Mendelian genetic system, be them alleles of dominant-recessive or co-dominant inheritance. ...
... Hence, morphological traits in a F1-hybrid show an expression of either (a) one of the parental species, or (b) an expression intermediate between those of the parents (Mebert et al., 2020). This is indeed possible herein, as illustrated in Figure 2. Furthermore, a study conducted across a large hybrid zone of related watersnakes, Nerodia sipedon and N. fasciata (Mebert, 2008(Mebert, , 2010 revealed more than 20% of snakes with a hybrid phenotype (based on 40 morphological characters), of which all individuals proved to exhibit also a hybrid genotype, hence, leading to a simple statement: "if it looks like a hybrid, it is a hybrid". In conclusion and with a clo-ser look, using a multitude of morphological variables, the intermediacy of dorsal pattern and the mix of bi-parental head characters from two Natrix species discussed above and exhibited in Figure 2 are strong indicators for F1-hybrids status. ...
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Bol. Asoc. Herpetol. Esp. (2021) 32(2):45-53. /// Natrix astreptophora and Natrix maura are known to sympatric over a wide area from southern France to almost the entire Iberian Peninsula, and from Tunisia to Morocco in North Africa. However, hybridization between these watersnakes is virtually unkown, possibly a consequence of their relatively great genetic distance. In this note, we describe illustrate three specimens from western Andalusia, southern Spain, which show strong evidence of hybridization with a mixture of characters, some intermediate and others typically found only in one or the other species. These specimens would represent the first possible and natural hybrids between the Iberian (or Red-eyed) Grass Snake and the Viperine Snake.
... Even though one may expect that snake genera delimit by different levels of cyt b divergence and might not be comparable to small mammals, various studies that include closely related snake species in sympatry, where species integrity is naturally tested (Harrison 1993), show cyt b divergence at an equivalent magnitude and range. For example, the Southern and Northern watersnakes (Nerodia fasciata and N. sipedon, respectively), diverge by 9% cyt b, but produce very wide hybrid zones (20-100 km) that align (are constrained) along environmental factors (Mebert 2008(Mebert , 2010; Western and Eastern grass snakes (Natrix helvetica and N. natrix, respectively), differ by 6.9% with limited unidirectional nuclear gene flow across a narrow contact zone between taxa (Kindler et al. 2017); a divergence of 5.2% exists between partly sympatric Mexican and Checkered gartersnakes (Thamnophis eques and T. marcianus, respectively), or 5.5% between Western Aquatic and Coast gartersnakes (T. couchii and T. elegans, respectively; de Queiroz and Lawson 1994); and sympatric North American brown snakes (Storeria spp.) differ by 8% (Alfaro and Arnold 2001). ...
... Hence, one needs to compile more data from different lines of evidence (integrative approach) to show that there is a coherent pattern of distinct morphology, genetics, and geographic structure. Adequate sampling is the basis to compensate for the lack of required information with proximate populations or even contact zones between two taxa investigated for free gene flow today or in the past by molecular means (Mebert 2008(Mebert , 2010 Hillis 2019). We strive to improve our knowledge in locating these contact zones or proximate populations by molecular means, and if otherwise not possible, by consensus of color pattern variation. ...
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Turkey harbors a high diversity of viperid snakes, many with a high threat level on the International Union for Conservation of Nature (IUCN) Red List, yet perception about even basic topics, such as distributions and conservation statuses, remain poor. We initiated a multi-year project 7 y ago to compensate these shortcomings and present herein dramatically improved information on the status of mountain vipers of central-eastern Anatolia (Asian Turkey): Bolkar Viper (Montivipera b. bulgardaghica), Albizona Viper (M. b. albizona), Wagner's Viper (M. wagneri), and partly Ottoman Viper (M. xanthina). The data originate from our fieldwork and a comprehensive search of all records available, including information from literature, online resources, locals, and herpetological experts. This resulted in 51 new localities, complemented by 36 published records, which were refined with new information, including four corrected/removed records and two records that were combined with new records due to their proximity. We summarized all records with precise information in a supplemented list of 85 localities, which is compared to current literature and the range maps available on the IUCN Red List of Threatened Species, the global standard reference for consultation on range maps and conservation status of species. Consequently, we report on large range extension of > 100 km in all four mountain viper taxa, increase the extent of occurrence for each viper taxon 4-8 times, reduce the distribution gaps between all pairs of parapatric, related, and ecologically similar mountain vipers, and discuss taxa delimitation, putative contact zones and conservation aspects.
... In snakes, it has been shown that some valid species are hybridizing in the wild (e.g., Mebert, 2008;Mebert et al., 2011), but it was mostly observed for phylogenetically closely related ones. However, introgressive hybridization is still a rarely observed phenomenon in snakes (e.g., Sanders, Rasmussen and Guinea, 2014;Kindler et al., 2017;Schultze et al., 2019). ...
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Asp vipers ( Vipera aspis ) and adders ( Vipera berus ) coexist in the Loire-Atlantique department in France where the two species reach their respective range limits. This contact zone is of special interest since hybridization has been recently discovered there. We carried out extensive sampling to further investigate the hybrid status of morphologically typical individuals and to evaluate the proportion of genetically admixed individuals in this area. Using microsatellite markers, no hybrids with typical morphological traits of either asp vipers or adders were detected. All recently investigated individuals with intermediate morphological traits were shown to be hybrids. A rather low proportion of genetically admixed individuals (1.5-3%) and a very small number of supposed second-generation hybrids suggest reduced fertility of first-generation hybrids or low viability of their progeny. The investigation of mtDNA of newly sampled hybrids support the finding that hybridization only occurs between female V. aspis and male V. berus . Several possible explanations for the unidirectional hybridization are discussed and consequent future studies suggested.
... Considering the cloaca is the point of entrance for the hemipene (not just the anchoring point to the spines), this divergence in vaginal pouch widening could indicate a femaleevolved reproductive isolation mechanism to prevent interpopulation mating that is worth investigating in a future study. Current evidence suggests that hybridization of these two species in contact zones is common, but N. fasciata genes dominate in the hybrids (Mebert, 2008). ...
Genital evolution can be driven by diverse selective pressures. Across taxa we see evidence of covariation between males and females, as well as divergent genital morphologies between closely related species. Quantitative analyses of morphological changes in coevolving male and female genitalia have not yet been shown in vertebrates. This study uses 2D and 3D geometric morphometrics to quantitatively compare the complex shapes of vaginal pouches and hemipenes across three species of watersnakes (the sister taxa Nerodia fasciata, N. sipedon, and a close relative N. rhombifer) to address the relationship between genital morphology and divergence time in a system where sexual conflict may have driven sexually antagonistic coevolution of genital traits. Our pairwise comparisons of shape differences across species show that the sister species have male and female genitalia that are significantly different from each other, but more similar to each other than to N. rhombifer. We also determine that the main axes of shape variation are the same for males and females, with changes that relate to deeper bilobation of the vaginal pouch and hemipenes. In males, the protrusion of the region of spines at the base of the hemipene trades off with the degree of bilobation, suggesting amelioration of sexual conflict, perhaps driven by changes in the relative size of the entrance of the vaginal pouch that could have made spines less effective.
... The utility of such photos is limited with respect to natural coloration and often geographic location, but they allow experts to focus on critical (diagnostic) characters and gathering these, especially of holotypes, would certainly have value. We also did not focus on gathering photos that could be identified only to the genus or higher taxonomic level, photos of snakes in captivity (including captive-bred color morphs), or photos of captive-bred or naturally-occurring hybrids (LeClere et al., 2012;Mebert, 2008). Aberrant coloration and patterning occurs at low frequencies in wild snakes (Borteiro et al., 2021), and individual, regional, ontogenetic, and coloration and patterning are common (Bechtel, 1978;Farooq and Uetz, 2020), so continuing to collect photos even of common species has value. ...
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The secretive behavior and life history of snakes makes studying their biology, distribution, and the epidemiology of venomous snakebite challenging. One of the most useful, most versatile, and easiest to collect types of biological data are photographs, particularly those that are connected with geographic location and date-time metadata. Photos verify occurrence records, provide data on phenotypes and ecology, and are often used to illustrate new species descriptions, field guides and identification keys, as well as in training humans and computer vision algorithms to identify snakes. We scoured eleven online and two offline sources of snake photos in an attempt to collect as many photos of as many snake species as possible, and attempt to explain some of the inter-species variation in photograph quantity among global regions and taxonomic groups, and with regard to medical importance, human population density, and range size. We collected a total of 725,565 photos—between 1 and 48,696 photos of 3098 of the world's 3879 snake species (79.9%), leaving 781 “most wanted” species with no photos (20.1% of all currently-described species as of the December 2020 release of The Reptile Database). We provide a list of most wanted species sortable by family, continent, authority, and medical importance, and encourage snake photographers worldwide to submit photos and associated metadata, particularly of “missing” species, to the most permanent and useful online archives: The Reptile Database, iNaturalist, and HerpMapper.
... We chose individuals representative of the full range of size for each species (Table 1). Because a few of the species pairs occasionally hybridize (Lawson et al., 1991;Mebert, 2008), we were careful to choose specimens showing no signs of morphological intermediacy. Because of the possibility of sexual dimorphism in eye morphology, we sexed those adults of each species whose sex could easily be determined by the presence or absence of hemipenes. ...
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Visual acuity and sensitivity positively correlate to eye size in vertebrates, and eye size relates to the ecology of colubrid snakes. We investigated whether eye morphology of North American colubrids of the genus Nerodia correlates with ecology as well. Although all members of the genus utilize aquatic habits, they differ widely in the proportion of anurans they eat. We specifically tested whether eye size and placement is associated with the proportion of frogs in the diet to determine whether these two aspects of eye morphology relate to feeding ecology. Using phylogenetic comparative methods, we found a significantly positive association between eye size and the proportion of anurans eaten by Nerodia species. Although the evidence is equivocal, the anterior placement of relatively small eyes in one species may also enhance anurophagy. Although eye size may improve a snake’s ability to feed on frogs, eye size must compete with other selective forces on head shape in trade-offs that may also influence eye size.
... At least four North American watersnake (Nerodia spp.) lineages are known to occur in high-salinity environments (Rasmussen et al. 2011). Watersnakes are also widespread, abundant, and many lineages are sympatricproviding ample opportunity for gene flow, including inferred hybridization, and enabling the investigation of the formation, maintenance, and/or degradation of species boundaries (Conant 1963;Lawson et al. 1991;Mebert 2008). The Nerodia fasciata-clarkii complex, in particular, is differentiated by only a few morphological characters and presumed ecological differences, with N. fasciata (Southern Watersnake) predominantly occupying-but not limited to-freshwater habitats and N. clarkii (Salt Marsh Snake) occupying coastal, mostly saltwater habitats (Lawson et al. 1991; see Gibbons and Dorcas [2004] for full distributions of each taxon; USFWS [2008]). ...
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The migration-selection balance often governs the evolution of lineages, and speciation with gene flow is now considered common across the tree of life. Ecological speciation is a process that can facilitate divergence despite gene flow due to strong selective pressures caused by ecological differences; however, the exact traits under selection are often unknown. The transition from freshwater to saltwater habitats provides strong selection targeting traits with osmoregulatory function. Several lineages of North American watersnakes (Nerodia spp.) are known to occur in saltwater habitat and represent a useful system for studying speciation by providing an opportunity to investigate gene flow and evaluate how species boundaries are maintained or degraded. We use ddRADseq to characterize the migration-selection balance and test for evidence of ecological divergence within the N. fasciata-clarkii complex in Florida. We find evidence of high intraspecific gene flow with a pattern of isolation-by-distance underlying subspecific lineages. However, we identify genetic structure indicative of reduced gene flow between inland and coastal lineages suggesting divergence due to isolation-by-environment. This pattern is consistent with observed environmental differences where the amount of admixture decreases with increased salinity. Furthermore, we identify significantly enriched terms related to osmoregulatory function among a set of candidate loci, including several genes that have been previously implicated in adaptation to salinity-stress. Collectively, our results demonstrate that ecological differences, likely driven by salinity, cause strong divergent selection which promotes divergence in the N. fasciata-clarkii complex despite significant gene flow.
... Furthermore, neither the triangular dorsal blotches nor the color pat-tern of head and venter can be generated through crosses between sympatric Dipsas species. There is also no evidence of a hybrid zone among sympatric Dipsas species that would include deep backcrossing beyond F1-hybridization and their more complex admixed phenotypes (Mebert, 2008(Mebert, , 2010. ...
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We describe a new species of snail-eating snake from two localities within the Atlantic Forest of the states of Bahia and Minas Gerais, Brazil. The new species differs remarkably from all congeners by its dorsal pattern consisting of sharply bordered triangles resembling the pattern of sympatric pitvipers, more precisely Bothrops jararaca and B. pirajai. Parameters of external morphology of the new species resemble those of the Dipsas incerta species group. Its scalation, body/head shape, and color pattern are most similar to D. alternans, a species known to occur 360 km farther south, across the Rio Doce river. The new species differs from D. alternans by exhibiting triangular dorsal blotches and a higher number of pileus blotches, by the distribution of ventral spots and morphology of the supratemporal and premaxillae, as well as in hemipenial morphology. We also provide comparisons of the new species with all sympatric and/or parapatric congeners. The finding of a new snake species in the Atlantic rainforest of southern Bahia and adjacent Minas Gerais is particularly surprising as this region is easily accessible and represents a well-sampled area regarding herpetological surveys in the last decades.
... Hybridization is considered an important driver of adaptive evolution. 1,2 Hybridization and gene introgression have been reported in many animals, including mollusks, 3 insects, 4 fishes, 5 amphibians, 6 reptiles, 7 and mammals. 8 Aquatic fauna, with the absence of dispersal barriers preventing their contact, are especially prone to produce hybrids among closely related species. ...
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BACKGROUND Apple snails from the genus Pomacea have spread widely in paddy fields and other wetlands of southern China since their introduction in the 1980s. Pomacea spp. are commonly identified using mitochondrial COI sequences. However, sequencing the nuclear EF1α gene revealed genetic introgression between field populations of P. canaliculata and P. maculata , which produce surviving hybrids in laboratory crossbreeding experiments. RESULTS In this study, we sequenced 1054 EF1α clones to design specific primers and established a fast and accurate multiplex PCR method for genotyping EF1α. Combined with genotyping P. canaliculata and P. maculata based on COI sequences, we revealed the genetic introgression patterns of 30 Pomacea populations in China. Purebred and hybrid individuals of P. canaliculata were widely distributed, while pure maculata‐EF1α type was detected only in a few individuals identified as P. canaliculata based on COI sequences. Each egg clutch had one to three genetic patterns, indicating multiple paternity or segregation in the progeny of hybrids. The higher percentages of hybrids in both wild populations and progeny than the homozygotes indicated a potential heterosis in the apple snail populations. Additionally, egg size and clutch size of the apple snails became homogeneous among the nonnative populations exhibiting introgression hybridization. CONCLUSION Our findings emphasize the value of apple snails as a model to study the mechanisms and impacts of introgressive hybridization on fitness traits. This article is protected by copyright. All rights reserved.
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Dermatophytic pathogens are a source of disturbance to the host microbiome, but the temporal progression of these disturbances is unclear. Here, we determined how Snake Fungal Disease, caused by Ophidiomyces ophidiicola, resulted in disturbance to the host microbiome. To assess disease effects on the microbiome, 22 Common Watersnakes (Nerodia sipedon) were collected and half were inoculated with O. ophidiicola. Epidermal swabs were collected weekly for use in microbiome and pathogen load characterization. For the inoculated treatment only, we found a significant effect of disease progression on microbial richness and Shannon diversity consistent with the intermediate disturbance hypothesis. When explicitly accounting for differences in assemblage richness, we found that β-diversity among snakes was significantly affected by the interaction of time and treatment group, with assemblages becoming more dissimilar across time in the inoculated, but not the control group. Also, differences between treatments in average microbiome composition became greater with time, but this interactive effect was not evident when accounting for assemblage richness. These results suggest that changes in composition of the host microbiome associated with disease largely occur due to changes in microbial richness related to disease progression.
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Michot, T. C. 1981. Thermal and spatial ecology of three species of water snakes (Nerodia) in a Louisiana swamp. Ph.D. Dissertation, Louisiana State Univ., Baton Rouge. 157 p. ABSTRACT Biotelemetry was used to study thermal and spatial relationships in Nerodia fasciata confluens- , N-. -c. c yclopion, and N-. -r. rhombifera. Twenty snakes were monitored between April 1978 and September 1979 in Ascension Parish, Louisiana. Laboratory-determined preferred temperature ranges were compared to environmental temperatures from air, soil, and water to predict thermally optimum microhabitats. Comparison of observed and predicted microhabitats for each observation showed that N-. fasciata was found more frequently in the thermally optimum microhabitat than were -N. cyclopion and N-. rhombifera. Nerodia cyclopion consistently showed thermal nonconformity by altering the relationship between body (BT) and ambient (AT) temperatures so that BT was closer to the preferred range when AT was high or low. All species showed the highest degree of thermoregulation in spring/fall; the lowest degree of thermoregulation was found in summer for N-. cyclopion, and in winter for the other two species. Snake movements showed a high degree of variation. Snakes typically stayed in a home area for about 20 days before making a major movement 0100 m). Nerodia fasciata moved significantly more than -N. rhombifera. The latter species stayed closer to land, spent more time underground, and, when in water, was found at greater depths than the other two species. The mean home range polygon for all observations was 5.96 ha, with values ranging from 0.03 to 15.39 ha. Home range size showed much variation and was not significantly correlated with species, sex, reproductive condition, weight, time of year, length of tracking period or interval between observations.
Individuals of the F5 and FM2 cytotypes of the Sceloporus grammicus complex form a narrow zone of parapatric hybridization near Tulancingo, Hidalgo, Mexico. Reproductive parameters were examined among chromosomally parental and hybrid females to assess the degree to which reduced clutch size is correlated with the level of chromosomal heterozygosity. Although clutch size in the two parental groups was highly correlated with female body size, this was not the case for females with intermediate karyotypes. These females displayed increased levels of infertility manifested as smaller clutches and as inviable embryos. F1 females produced the smallest average clutches and suffered the most precipitous fecundity loss (up to 75%). The number of heterozygous marker chromosomes and heterozygosity at chromosome 2 had significant effects on the number of viable embryos. Analysis of embryo karyotypes revealed the production of triploid offspring and an excess number of embryos heterozygous at chromosome 1. Differences in viability, among females heterozygous for the same number of chromosomes, suggest that genetic background of the female and/or sire may be an important factor in determining reproductive success.
Several chromosome races of the mesquite lizard, Sceloporus grammicus complex, hybridize at localities in central Mexico. In most cases, the hybridizing populations are delineated by centric fissions at one or more of the macrochromosomes. One notable exception is the Tulancingo hybrid zone between the F5 and FM2 cytotypes. In addition to fission and/or inversion differences at chromosomes 1, 3, 4, and 6, these races differ by a complex rearrangement of chromosome 2, which carries the nucleolus-organizer region in this species. The meiotic consequences of heterozygosity at this chromosome were examined in males to assess the potential for this chromosome to contribute to the dynamics of the hybrid zone. Chromosomal analysis revealed several putative F1 hybrids and confirmed the production of nonparental chromosomal morphologies through recombination. Pachytene analysis revealed meiotic pairing difficulties associated with chromosome 2 in males heterozygous for the parental chromosomal morphologies. Significant aneuploidy is expected because of random disjunction of the chromosome-2 elements. As a result, these males likely suffer reduced fertiliity and fitness. In contrast, males heterozygous for recombinant chromosomal morphologies displayed low levels of meiotic irregularities and presumably exhibit higher fertility than individuals heterozygous for parental morphologies. It is hypothesized that the recombinant phenotypes facilitate gene flow between the F5 and FM2 cytotypes.