Molecular Ecology (2014)
doi: 10.1111/mec.12674
Human disturbance causes the formation of a hybrid
swarm between two naturally sympatric fish species
DANIEL J. HASSELMAN,* EMILY E. ARGO,* MEGHAN C. MCBRIDE,† PAUL BENTZEN,†
T H O M A S F . S C H U L T Z , ‡ A N N A A . P E R E Z - U M P H R E Y ‡ and E R I C P . P A L K O V A C S *
*Department of Ecology and Evolutionary Biology, University of California, Santa Cruz, CA 95060, USA, †Marine Gene Probe
Laboratory, Biology Department, Dalhousie University, Halifax, NS B3H 4R2, Canada, ‡Marine Conservation Molecular
Facility, Duke University Marine Laboratory, Beaufort, NC 28516, USA
Abstract
Most evidence for hybrid swarm formation stemming from anthropogenic habitat disturbance comes from the breakdown of reproductive isolation between incipient species, or introgression between allopatric species following secondary contact. Human
impacts on hybridization between divergent species that naturally occur in sympatry
have received considerably less attention. Theory predicts that reinforcement should
act to preserve reproductive isolation under such circumstances, potentially making
reproductive barriers resistant to human habitat alteration. Using 15 microsatellites, we
examined hybridization between sympatric populations of alewife (Alosa pseudoharengus) and blueback herring (A. aestivalis) to test whether the frequency of hybridization and pattern of introgression have been impacted by the construction of a dam that
isolated formerly anadromous populations of both species in a landlocked freshwater
reservoir. The frequency of hybridization and pattern of introgression differed markedly between anadromous and landlocked populations. The rangewide frequency of
hybridization among anadromous populations was generally 0–8%, whereas all landlocked individuals were hybrids. Although neutral introgression was observed among
anadromous hybrids, directional introgression leading to increased prevalence of alewife genotypes was detected among landlocked hybrids. We demonstrate that habitat
alteration can lead to hybrid swarm formation between divergent species that naturally
occur sympatrically, and provide empirical evidence that reinforcement does not
always sustain reproductive isolation under such circumstances.
Keywords: Alosa, anadromous, hybrid swarm, hybridization, introgression, landlocked
Received 30 October 2013; revision received 13 January 2014; accepted 17 January 2014
Introduction
Much of the world’s biodiversity is of recent evolutionary origin and is sustained by divergent adaptation in
heterogeneous environments (Rundle & Nosil 2005;
Seehausen et al. 2008). However, rugged adaptive landscapes that promote and maintain diversification in nature can be smoothed by human activities, hindering
adaptive radiation (Hendry et al. 2006; De Le
on et al.
2011) and contributing to the loss of evolutionary
Correspondence: Daniel J. Hasselman, Fax: (831) 459 3833;
E-mail: [email protected]
© 2014 John Wiley & Sons Ltd
lineages through ‘reverse speciation’ (Seehausen 2006;
Taylor et al. 2006; Seehausen et al. 2008).
Most evidence for reverse speciation comes from
studies of introgressive hybridization between incipient
fish species that have recently (i.e. postglacially)
diverged in sympatry through disruptive selection in
heterogeneous environments (e.g. threespine stickleback
benthic–limnetic pairs (Taylor et al. 2006); Laurentian
Great Lakes ciscoes (Seehausen et al. 2008); European
whitefish (Vonlanthen et al. 2012); African cichlids
(Seehausen et al. 1997)). Although reproductive isolation
between incipient species may be incomplete, divergence can be reinforced through prezygotic (e.g. assortative mating) and postzygotic (e.g. reduced hybrid
2 D. J. HASSELMAN ET AL.
fitness) isolating mechanisms when gene flow is low
(Gow et al. 2006). However, the loss of environmental
heterogeneity can relax disruptive selection and eliminate reproductive isolating mechanisms, facilitating
introgressive hybridization and the collapse of incipient
species into a hybrid swarm (Seehausen et al. 2008).
Indeed, several studies have shown how habitat
homogenization through human activities (e.g. species
introductions, eutrophication) can weaken assortative
mating and facilitate reverse speciation for multiple
incipient species (Todd & Stedman 1989; Seehausen
et al. 1997; Behm et al. 2010).
Introgressive hybridization stemming from anthropogenic habitat disturbance also threatens the integrity of
species with deeper evolutionary histories (Wiegand
1935; Anderson 1948; Rhymer & Simberloff 1996). For
example, introgression between grey wolf (Canis lupus)
and coyote (C. latrans), species that diverged 1.4–
2.1 million years ago, occurs across a broad range
where forests have been converted to agricultural land
(Lehman et al. 1991). Similarly, despite a 2.6-millionyear evolutionary history of ecologically maintained
parapatry, hybridization between forest elephant
(Loxodonta cyclotis) and savannah elephant (L. africana)
threatens species integrity in regions of ongoing deforestation in Africa (Roca et al. 2005). However, the formation of a hybrid swarm resulting from the
breakdown of reproductive isolation between divergent
species that naturally occur in sympatry is not well
established (Seehausen 2006). Indeed, under these
conditions, theory predicts that reinforcement should
sustain reproductive isolation, potentially making reproductive barriers resistant to habitat disturbance.
The classic definition of reinforcement implies selection against hybrids through a variety of intrinsic (e.g.
hybrid inviability, sterility, behavioural dysfunctions) or
extrinsic (e.g. lowered fitness associated with a specific
set of ecological conditions) mechanisms (see Rundle &
Schluter 1998). However, this restrictive definition does
not take into account processes that reflect classic criteria for reinforcement (see Servedio & Noor 2003). Here,
we refer to reinforcement in the ‘broad sense’ (sensu
Servedio & Noor 2003), where reinforcement constitutes
‘an increase in prezygotic isolation between hybridizing
[species] in response to any type of selection against
interspecific matings, regardless of whether hybrids
themselves [exhibit reduced fitness]’. Under this definition, reinforcement could manifest as reproductive isolation via spatiotemporal differences in breeding for
divergent species in sympatry.
River herrings (alewife, Alosa pseudoharengus, and
blueback herring, A. aestivalis) provide an opportunity
to empirically examine hybridization between divergent
lineages that naturally occur in sympatry, and in
response to anthropogenic habitat disturbance. Alewife
and blueback herring are anadromous (migratory searun) species native to the Atlantic coast of North America. Although they co-occur in rivers over much of their
historical range (alewife: Labrador to South Carolina;
blueback herring: Gulf of St. Lawrence to Florida),
reproductive isolation is maintained by differences in
spawning time (temperature dependent) and spawning
habitat (reviewed by Loesch 1987). In terms of timing,
alewife spawn at cooler temperatures (5–10 °C) than
blueback herring (10–15 °C) and typically begin spawning 3–4 weeks earlier. However, peak spawn timing
only differs by 2–3 weeks, resulting in considerable
temporal overlap in freshwater (Loesch 1987). In terms
of spawning habitat, alewife preferentially select lentic
(i.e. still-water) habitats for spawning, whereas blueback
herring prefer lotic (i.e. flowing-water) habitats (Loesch
1987). Prior molecular research revealed that these species diverged up to 1 million years ago (Faria et al.
2006), but also detected a shared mitochondrial DNA
haplotype, suggesting either incomplete lineage sorting
(Chapman et al. 1994) or introgressive hybridization
(Faria et al. 2006).
A dam constructed on the Roanoke River in 1953
created novel conditions for river herring in Kerr Reservoir (North Carolina/Virginia, USA). Alewife have
repeatedly established landlocked populations in lakes
(Palkovacs et al. 2008), and landlocked blueback herring
populations persist in several reservoirs following introduction (Prince & Barwick 1981; Guest & Drenner
1991). To our knowledge, Kerr Reservoir is the only
instance where these species have become landlocked
in sympatry. Further, the absence of fish passage has
prevented the immigration of anadromous alewife and
blueback herring for approximately 60 years. Thus, Kerr
Reservoir provides a unique opportunity to examine
the effects of anthropogenic habitat disturbance on the
frequency of hybridization and pattern of introgression
between divergent species that naturally occur in sympatry. Alewife and blueback herring populations have
declined dramatically in recent decades (Limburg &
Waldman 2009; Hall et al. 2012), making these species a
conservation concern (Atlantic States Marine Fisheries
Commission 2012; Palkovacs et al. 2013). Evidence for
human impacts on hybridization could hold important
implications in setting future conservation guidelines
(Allendorf et al. 2001) and for prioritizing river restoration projects through dam removal.
Using 15 microsatellite loci, we (i) examine the extent
and spatial distribution of hybridization between anadromous alewife and blueback herring across both species’
ranges, including Kerr Reservoir, (ii) determine whether
the frequency of hybridization and pattern of introgression differ between anadromous and landlocked
© 2014 John Wiley & Sons Ltd
HYBRIDIZATION AND INTROGRESSION IN ALOSINES 3
scenarios and (iii) resolve whether anthropogenic habitat
disturbance can result in the formation of a hybrid
swarm between divergent species that naturally occur in
sympatry.
Materials and methods
Sample collections
Sampling was conducted across the ranges of both species, and targeted a minimum of 50 individuals per collection (i.e. riveryear 1). Individuals were initially
assigned to species based on diagnostic peritoneal coloration (Jordan & Evermann 1896; Scott & Crossman
1973; Messieh 1977), but were ultimately identified as
alewife, blueback herring or hybrid using Bayesian clustering analyses (see below). Collections were replicated
for a subset of rivers over successive years and pooled
following an analysis of molecular variance (AMOVA) that
indicated nonsignificant (P > 0.05) genetic variation
among years within rivers (Palkovacs et al. 2013). Samples were obtained in freshwater from adult specimens
on their spawning run, with the exception of those
collected from Veazie Dam and Souadabscook Falls
(Penobscot River) that comprised young-of-the-year
specimens. Sampling effort from 2005 to 2012 provided
tissue for 5358 alewife and 1529 blueback herring
from 56 to 30 anadromous populations, respectively, as
well as 76 and 43 presumed alewife and blueback
herring, respectively, from Kerr Reservoir (Table 1;
Fig. 1). Tissue was preserved in 95% ethanol until DNA
extraction.
Laboratory protocols
DNA extraction and genotyping. Laboratory procedures
were conducted at separate institutions (i.e. Dalhousie
University and Duke University Marine Laboratory) as
parts of independent studies of river herring genetic
variation in the northeastern USA and Canada (McBride
2013) and the USA (Labbe 2012; Palkovacs et al. 2013).
Details regarding DNA isolation and genotyping protocols were reported previously (Palkovacs et al. 2013;
McBride 2013). We examined the variation at 21 polymorphic microsatellites: 15 loci developed for alewife
(Ap010, Ap033, Ap037, Ap038, Ap047, Ap058, Ap070,
Ap071) and blueback herring (Aa046, Aa070, Aa074,
Aa081, Aa082, Aa091, Aa093) (A’Hara et al. 2012) that
were common across studies and six additional loci
developed for alewife (Aps1, Aps2A; Bentzen & Paterson
2005), blueback herring (Aa039; A’Hara et al. 2012) and
American shad (A. sapidissima) [AsaD042, AsaC249
(Julian & Bartron 2007), Asa8 (Waters et al. 2000)] that
were examined by McBride (2013) alone. The data sets
© 2014 John Wiley & Sons Ltd
of McBride (2013) and Palkovacs et al. (2013) were
combined after a correction factor for allele scores had
been applied for each locus common to both studies.
Briefly, small (30 ll) aliquots of pure genomic DNA
for alewife (n = 46) and blueback herring (n = 44) of
known genotype originally scored at Dalhousie University were genotyped at the Duke University Marine
Laboratory following the protocol of Palkovacs et al.
(2013). These data were then used to establish a correction factor to adjust the allele scores of the McBride
(2013) data set.
Data analyses
Overview. Following conformance of the data to model
assumptions, we simulated data for purebred alewife
and blueback herring and various hybrid classes (F1, F2
and backcrosses) using confirmed purebred individuals
from the empirical data set (identified using Bayesian
methods; see below). We then analysed the simulated
data using two Bayesian methods to determine the
appropriate threshold (Tq) for designating individuals
as either purebred or admixed. Next, we applied Tq to
the empirical data set to identify purebred and admixed
individuals, examined the extent of hybridization across
the ranges of alewife and blueback herring and determined whether the frequency of hybridization differed
between anadromous and landlocked scenarios. We
then used the genomic clines method (Gompert &
Buerkle 2009) to identify whether the pattern of introgression differed among anadromous and landlocked
hybrids.
Data conformance to model assumptions. Genotyping artefacts were assessed using MICROCHECKER version 2.2.3
(Van Oosterhout et al. 2004). Departures from Hardy–
Weinberg equilibrium (HWE) and linkage disequilibrium (LD) were assessed within rivers (where n > 20)
for each species using GENEPOP version 4.0.6 (Rousset
2007) with default parameters for all tests. Sequential
Bonferroni adjustments were used to judge significance
levels for all simultaneous tests (Holm 1979; Rice 1989).
Selective neutrality of the microsatellite markers was
confirmed previously (Palkovacs et al. 2013).
Simulations and hybrid identification. Hybrid identification depends on the genetic markers used and the
degree of differentiation between parental species
(Anderson & Thompson 2002; V€
ah€
a & Primmer 2006).
To assess the utility of each microsatellite locus to discriminate alewife and blueback herring, we identified
purebred individuals using Bayesian clustering methods
(see below) and estimated the Kullback–Leibler divergence (Kullback & Leibler 1951) following Anderson &
4 D. J. HASSELMAN ET AL.
Table 1 Sampling locations and sample sizes (N) for alewife and blueback herring collected from across the species’ ranges from
2005 to 2012. Specimens were initially classified to species at the time of collection using peritoneal coloration and were later confirmed (or adjusted) based on results of Bayesian clustering analysis of microsatellite data
Watershed
Code
Latitude
Alewife
Miramichi River
Richibucto River
Tidnish River
River Phillip
River John
Hillsborough River
Tracadie Bay
Margaree River
Bras d’Or Lakes
West River
Sullivan Pond Outlet
Sackville River
LaHave River
Medway River
Mersey River
Tusket River
Gaspereau River
Shubenacadie River
Petitcodiac River
Saint John River
St. Croix River
Dennys River
East Machias River
Narraguagus River
West Bay Pond
Mt. Desert Island
Union River
Penobscot River
St. George River
Damariscotta River
Sheepscot River
Kennebec River
Androscoggin River
Presumpscot River
Saco River
Piscataqua River
Mystic River
Monument River
Town Brook
Taunton River
Gilbert-Stuart
Thames River
Bride Brook
Connecticut River
Quinnipiac River
Housatonic River
Pequonnock River
Mianus River
Hudson River
Delaware River
Nanticoke River
Rappahannock River
James River
MIR
RIC
TID
RPH
RJO
HIL
TRA
MAR
BRA
WES
SUL
SAK
LHV
MED
MER
TUS
GAS
SHU
PET
SJR
STC
DEN
EMA
NAR
WBP
MDI
UNI
PEN
STG
DAM
SHE
KEN
AND
PRE
SAC
PIS
MYS
MON
TOW
TAU
GIL
THA
BRI
CON
QUI
HOU
PEQ
MIA
HUD
DEL
NAN
RAP
JAM
47.1419
46.6914
45.9900
45.7928
45.7539
46.2403
46.3828
46.4300
45.8536
44.9269
44.9269
44.7292
44.2928
44.1339
44.0419
43.8756
45.0894
45.2603
45.9053
45.2594
45.1911
44.9089
44.5419
44.4906
44.3583
44.5011
44.4408
44.4408
43.9592
44.0294
44.0219
43.7519
43.9147
43.7153
43.4669
43.0664
42.3439
41.7244
41.9625
41.6094
41.4472
41.3256
41.3006
41.2828
41.2722
41.1708
41.1736
41.0156
40.6958
39.1122
38.1689
37.4933
36.9686
2005
2008
2009
2010
2011
2012
Unknown
54
63
63
57
117
60
60
51
156
51
52
71
56
51
57
155
58
51
49
50
111
59
213
50
60
44
323
59
25
30
59
30
30
29
28
232
132
85
28
321
86
28
29
47
20
49
230
51
53
311
51
74
49
46
49
89
44
1
13
36
34
7
25
25
25
25
13
30
27
26
35
32
48
45
58
62
1
Total N
54
63
63
57
117
60
60
51
156
51
52
71
56
51
57
155
58
51
49
50
136
30
59
30
30
29
136
675
233
198
28
999
196
28
29
121
69
46
49
89
44
66
61
33
61
38
25
57
61
45
58
62
1
© 2014 John Wiley & Sons Ltd
HYBRIDIZATION AND INTROGRESSION IN ALOSINES 5
Table 1 Continued
Watershed
Chowan River
Roanoke River
Kerr Lake
Alligator River
Code
Latitude
CHO
ROA
KER
ALL
36.0811
35.9228
36.5702
35.9125
Watershed
Code
Latitude
Blueback Herring
Miramichi River
River John
Margaree River
Petitcodiac River
Saint John River
East Machias River
Penobscot River
St. George River
Kennebec River
Exeter River
Mystic River
Monument River
Gilbert-Stuart River
Connecticut River
Quinnipiac River
Hudson River
Delaware River
Nanticoke River
Rappahannock River
James River
Chowan River
Roanoke River
Kerr Lake
Neuse River
Cape Fear River
Santee-Cooper River
Savannah River
Altamaha River
St .John’s River
MIR
RJO
MAR
PET
SJR
EMA
PEN
STG
KEN
EXE
MYS
MON
GIL
CON
QUI
HUD
DEL
NAN
RAP
JAM
CHO
ROA
KER
NEU
CFE
SAN
SAV
ALT
STJ
47.1419
45.7539
46.4300
45.9053
45.2594
44.5419
44.4408
43.9592
43.7519
43.0664
42.3439
41.7244
41.4472
41.2828
41.2722
40.6958
39.1122
38.1689
37.4933
36.9686
36.0811
35.9228
36.5702
35.0764
33.9286
33.1833
32.0481
31.3147
30.4081
2005
2008
2010
2011
2012
Unknown
55
50
76
49
2008
2009
2010
2011
2012
Unknown
58
36
46
6
2
8
41
10
34
30
58
51
38
46
62
25
48
31
12
51
24
58
97
59
50
44
65
57
62
52
53
37
Total N
55
50
76
49
1
8
47
48
50
Thompson (2002) and as implemented in the ‘flexmix’
package in R (R Development Core Team 2010). We also
estimated a multilocus global measure of FST (h; Weir &
Cockerham 1984) between the species using FSTAT version 2.9.3.2 (Goudet 2001).
To maximize the accuracy of delineating purebred
and hybrid individuals, we conducted admixture analyses using two Bayesian clustering methods implemented in STRUCTURE version 2.3.3 (Pritchard et al. 2000;
Falush et al. 2003) and NEWHYBRIDS version 1.0 (Anderson
& Thompson 2002). Each method probabilistically
assigns multilocus genotypes to clusters using a Markov
chain Monte Carlo (MCMC) simulation procedure that
provides estimates from the posterior distribution that
reflects the membership of each individual. For STRUCTURE, the posterior probability (q) describes the proportion of an individual genotype originating from each of
© 2014 John Wiley & Sons Ltd
2009
33
34
Total N
1
8
47
48
50
58
36
46
16
41
68
51
38
142
55
79
51
57
58
97
71
50
44
65
57
62
52
53
71
K clusters. For NEWHYBRIDS, q describes the probability
that an individual belongs to each of six different genotype frequency classes (i.e. parental purebreds, F1
hybrid, F2 hybrid and backcrosses). For both programs,
we ran 10 iterations of a parameter set that included a
burn-in of 50 000 steps followed by 250 000 replicates
of the MCMC simulation. For STRUCTURE, we set K = 2
(i.e. assuming two species contributed to the gene pool
of the sample) and employed the admixture model and
correlated allele frequencies. For NEWHYBRIDS, we used
uninformative priors for both the allele frequency and
admixture distributions.
An important consideration when using Bayesian
methods to identify admixed individuals is the choice of
optimal threshold value (Tq) for the q associated with
their assignment as purebred or hybrid (V€
ah€
a & Primmer
2006). For both methods, we initially used q ≥ 0.9
6 D. J. HASSELMAN ET AL.
Fig. 1 Map of the Atlantic coast of North
America displaying sampling locations
for alewife and blueback herring. River
names associated with each sampling
location code are provided in Table 1.
(Pritchard et al. 2000) to designate purebreds (i.e. criterion #3 for NEWHYBRIDS; Burgarella et al. 2009). We then
randomly chose 100 individuals identified as purebred
alewife and blueback herring from across their range,
and simulated five data sets of parental and various
hybrid classes using HYBRIDLAB version 1.0 (Nielsen et al.
2006) to determine Tq. For each of the five simulated data
sets, we generated profiles for 200 alewife and blueback
herring. From these 400 simulated parental genotypes,
we generated 100 F1 hybrids that were used to generate
100 F2 hybrids, and 100 F1 backcrossed alewife and blueback herring.
Simulated data sets were analysed using STRUCTURE
and NEWHYBRIDS (as described above), and results were
summarized across iterations using CLUMPP version 1.1.2
(Jakobsson & Rosenberg 2007) and visualized with DISTRUCT version 1.1 (Rosenberg 2004). These results were
used to determine Tq and to develop classification rules
for assigning individuals from the empirical data set
as purebred or hybrid. For STRUCTURE, we assessed the
q value of each K cluster for every simulated parental
and hybrid class. In NEWHYBRIDS, assignment to a specific
hybrid class may be uncertain, with q split among genotype frequency classes (Burgarella et al. 2009). Therefore, we summed q across hybrid genotype frequency
classes, because we were only concerned with the identification of hybrids, not distinguishing among hybrid
classes per se. We then assessed the q value for every
simulated parental and hybrid class.
To determine Tq, we used the simulated data set to
examine how the proportion of misassigned parental
and hybrid individuals using STRUCTURE and NEWHYBRIDS
changed in response to a shifting q (0.005 increments)
from 0.85 to 1.00. The resulting intersection between the
proportion of misassigned parental and hybrid individuals minimizes overall misassignments and was taken
as Tq. We then used this optimal threshold to determine
the error rate for parental and hybrid classification and
to establish confidence bounds on our assignments for
the empirical data set.
The empirical data set was then analysed using STRUCTURE and NEWHYBRIDS (as described above). Results were
summarized across iterations using CLUMPP (Jakobsson
& Rosenberg 2007) and visualized with DISTRUCT (Rosenberg 2004). Hybrids were identified based on the classification rules established through the determination of
Tq (see Results; Table S1, Supporting Information), and
the frequency and extent of hybridization across the
ranges of both species were examined. Factorial correspondence analysis (FCA) was used to visualize the
relative similarity of multilocus allele frequencies
among purebred alewife and blueback herring, and
anadromous and landlocked hybrids using GENETIX 4.05
(Belkhir et al. 2004).
Analysis of genomic clines. To examine whether patterns
of introgression differed among landlocked and anadromous hybrids, we used the genomic clines method
(Gompert & Buerkle 2009) implemented in the R package ‘introgress’ (Gompert & Buerkle 2010; R Development Core Team 2010). For this analysis, we only
selected specimens that were identified by both STRUCTURE and NEWHYBRIDS as hybrids. The proportion of blueback herring ancestry among hybrids was assessed
using a maximum-likelihood estimator of hybrid index
that calculates genome-wide admixture based on the
© 2014 John Wiley & Sons Ltd
HYBRIDIZATION AND INTROGRESSION IN ALOSINES 7
proportion of alleles inherited from each parental species (Gompert & Buerkle 2009). Parental species were
represented by the 100 purebred alewife and blueback
herring used in prior HYBRIDLAB simulations. Multiallelic
microsatellite data were reduced to biallelic classification, without the loss of information or distortion of the
relationship between the parental species, using ‘introgress’. Hybrids were then recognized as interallelic
class (interspecific) heterozygotes (Aa/Ab) or homozygotes (Aa/Aa, Ab/Ab) (i.e. both alleles from the same allelic class). The probabilities of observing each of these
possible allelic classes at each locus (i.e. genomic clines;
Gompert & Buerkle 2009) were then predicted using
multinomial regression of the observed genotypes on
hybrid index.
We identified loci that deviated from expectations of
neutral introgression by comparing the likelihoods of
the regression model to that of a neutral model, given
the observed data. Expected genomic clines under neutral introgression were generated using 10 000 runs of a
parametric procedure implemented in ‘introgress’.
Genomic clines for the landlocked and anadromous
hybrid zones were then fitted with a logistic regression
using the observed data to estimate probabilities of
observing homozygote and heterozygote interallelic classes as a function of hybrid index (Gompert & Buerkle
2009). Significant deviations from neutral expectations
for genomic clines were adjusted for multiple comparisons using the false discovery rate (FDR; Benjamini &
Hochberg 1995). We then summarized deviations from
neutrality on the basis of whether interallelic class homozygotes and heterozygotes were more or less common than expected under neutrality, and determined
whether locus-specific patterns of introgression differed
among landlocked and anadromous hybrids.
Results
Data conformance to model assumptions
Evidence for null alleles resulted in the exclusion of loci
for both alewife (Aa082, Ap037, Ap047, Ap070) and blueback herring (Aa081, Ap058) prior to further analyses (Microchecker). Remaining loci were retained as evidence
for null alleles was sporadic among loci and rivers. Exact
tests revealed that genotypic frequencies were largely in
accordance with HWE for both alewife (P > 0.05; sequential Bonferroni correction for 55 comparisons) and blueback herring (P > 0.05; sequential Bonferroni correction
for 25 comparisons). HWE departures for alewife and
blueback herring remained for 25 and 11 locus–river
comparisons, respectively, and were due to heterozygote
deficiencies from sporadic null alleles. Exact tests of
LD revealed that loci were physically unlinked and
© 2014 John Wiley & Sons Ltd
statistically independent (P > 0.05; sequential Bonferroni
correction for 2762 and 1270 comparisons for alewife and
blueback herring, respectively).
Simulations and hybrid identification
Global FST between alewife and blueback herring was
0.352. Although no locus was diagnostic, the Kullback–
Leibler divergence revealed several loci that would be
informative in species discrimination when used
together (e.g. KL ≥ 6.0; Fig. S1, Supporting Information).
In Bayesian analyses, q-values for randomly chosen alewife and blueback herring used in simulations ranged
from 0.94–1.00 (STRUCTURE) and 0.99–1.00 (NEWHYBRIDS),
exceeding Tq (see below) and confirming that we had
correctly selected pure strain individuals for simulating
parental and hybrid classes. Further, allele frequency
distributions for the randomly chosen individuals were
representative of each species [P > 0.05; two-sample
Kolmogorov–Smirnov test; SYSTAT version 11 (SPSS, Inc.
2004)].
Across the five simulated data sets, the assignment of
purebred individuals to their correct parental class
using either Bayesian method was highly accurate
(Fig. 2). STRUCTURE unambiguously discriminated F1 and
F2 hybrids from purebreds (Fig. S2a, Supporting Information), but the increased variance (by an order of
magnitude) of F2 hybrids reduced assignment accuracy
using NEWHYBRIDS (Fig. S2b, Supporting Information).
Both Bayesian methods had difficulty with advanced
introgression and could not unambiguously discriminate all backcrossed alewife and blueback herring from
purebred individuals and F2 hybrids (Fig. S2, Supporting Information). Similar results have been observed
even using diagnostic markers (Gow et al. 2007).
Using STRUCTURE, the lowest proportion of misassigned purebreds (0.007) and hybrids (0.006) across the
five simulated data sets occurred when Tq = 0.935. For
NEWHYBRIDS, the lowest proportion of misassigned purebreds (0.004) and hybrids (0.004) occurred when
Tq = 0.870. Using these optimal thresholds, we established a set of classification rules for delineating purebreds and hybrids from the empirical data set (Table
S1, Supporting Information). Using these classification
rules, we detected hybrids from across the range of
river herring. Across the species’ ranges, 162 anadromous specimens (2.4%) were identified as hybrids, with
Bayesian methods producing consistent results for 90
specimens. Every specimen from Kerr Reservoir
(n = 119) was identified as a hybrid (Figs 3 and 4). Discrepancies between Bayesian methods occurred predominantly where individuals identified as purebreds
by NEWHYBRIDS were identified as hybrids by STRUCTURE
(n = 67), and more infrequently vice versa (n = 5). The
8 D. J. HASSELMAN ET AL.
Fig. 2 Results of Bayesian clustering
analyses for five simulated data sets (alewife, blueback herring, F1, F2, F1 backcrosses) using STRUCTURE (K = 2; number
of species) and NEWHYBRIDS (K = 6; number of genotype frequency classes). Individuals are represented by a thin vertical
line which is partitioned into K-coloured
segments representing an individual’s
estimated membership fractions from
each of the identified clusters. Black lines
separate individuals from different genotype frequency classes (labelled below).
proportion of hybrids in anadromous populations generally ranged from 0.00 to 0.08, but there were notable
exceptions (Fig. 4). The greatest proportions of anadromous hybrids were observed for the Petitcodiac River
(0.227, 0.216) and Margaree River (0.133, 0.112) using
STRUCTURE and NEWHYBRIDS, respectively (Table S2, Supporting Information). Both Bayesian methods identified
identical proportions of hybrids from the St. John’s
River (0.056), Altamaha River (0.019) and Savannah
River (0.019), despite these rivers being beyond the
southern range limit for alewife. No purebred alewives
were detected south of the range limit.
Two factors in FCA explained 85.6% of the genetic
variation among purebred and hybrid river herring and
demonstrated the clear separation of landlocked
hybrids (Fig. 5). While Axis 1 (44.35%) delineated purebred anadromous alewife and blueback herring and
revealed an intermediate genetic composition of anadromous hybrids, Axis 2 (41.25%) isolated landlocked
hybrids from anadromous hybrids and purebred river
herrings. Interestingly, landlocked hybrids did not exhibit Axis 1 scores similar to anadromous hybrids, but
values that exceeded those of purebred alewife (Fig. 5).
This prompted an examination of the distribution of
probability values (q) among genotype frequency classes
in NEWHYBRIDS for hybrids and revealed a significantly (P < 0.001; two-sample KS test) higher frequency
of F1 and F2 individuals among anadromous than
among landlocked hybrids. Landlocked hybrids were
more deeply introgressed with alewife, consistent with
FCA results (data not shown) and genomic clines analysis (see below).
Analysis of genomic clines
The genomic clines method revealed striking differences
in patterns of blueback herring ancestry and patterns of
introgression between anadromous and landlocked
hybrids (Fig. S3, Supporting Information). While the
hybrid index for anadromous hybrids ranged from 0.00
to 1.00, the fraction of the landlocked hybrid genome
that was derived from blueback herring never exceeded
0.78. Further, an approximately equal proportion of alewife and blueback herring ancestry was observed
among anadromous hybrids, but there was little blueback herring ancestry among landlocked hybrids; only
two individuals exhibited blueback herring ancestry
≥0.5 (Fig. S3b, Supporting Information). Cumulatively,
these results support neutral introgression of blueback
herring genotypes among anadromous hybrids, but
directional introgression leading to increased prevalence
of alewife genotypes in Kerr Reservoir.
The genomic clines method also revealed marked heterogeneity in locus-specific patterns of introgression for
anadromous and landlocked hybrids. For anadromous
hybrids, variation at four loci (Aa046, Aa070, Aa093,
Ap033) deviated from neutral introgression based on
genome-wide admixture and remained significant after
FDR correction (P < 0.015) (Fig. 6a). For three of these
markers (Aa046, Aa070, Aa093), the total probability for
the heterozygous genotype was increased relative to
neutral expectations (Fig. 6a). For landlocked hybrids,
eight of the nine loci (except Aa046) deviated from neutral introgression and remained significant after FDR
correction (P < 0.039) (Fig. 6b), with highly variable patterns of deviation from neutral introgression among
markers. Nonetheless, for several of the deviant loci (i.e.
Ap071, Ap010, Aa070), the total probability for heterozygous genotype was decreased relative to neutral expectations (Fig. 6b). We also observed significant
differences in the patterns of introgression between
anadromous and landlocked hybrids for two of five
markers that could be directly compared (i.e. Aa074,
Ap033).
© 2014 John Wiley & Sons Ltd
HYBRIDIZATION AND INTROGRESSION IN ALOSINES 9
Fig. 3 Results of Bayesian clustering analyses for rangewide empirical data for alewife and blueback herring using STRUCTURE (K = 2;
number of species) and NEWHYBRIDS (K = 6; number of genotype frequency classes). Individuals are represented by a thin vertical line
which is partitioned into K-coloured segments representing an individual’s estimated membership fractions from each of the identified clusters. Kerr Reservoir (KER) is denoted because it showed a substantially greater number of hybrids than other sampling locations.
Discussion
Fig. 4 Spatial distribution of the proportion of hybrids identified using two Bayesian clustering analyses. MAR, PET and
KER (see Table 1) are denoted because they exhibited a substantially greater proportion of hybrids than other sampling
locations.
Most evidence for the formation of hybrid swarms
resulting from anthropogenic habitat disturbance comes
from studies of either the breakdown of reproductive
isolation between incipient species that have recently
diverged in sympatry (e.g. Todd & Stedman 1989;
Seehausen et al. 1997; Behm et al. 2010; Vonlanthen et al.
2012), or introgression between allopatric species following secondary contact (e.g. Echelle & Connor 1989;
Walters et al. 2008; McDevitt et al. 2009). Our rangewide
study of hybridization between alewife and blueback
herring demonstrates that human activities can also
impact the formation of hybrid swarms between divergent species that naturally occur in sympatry, providing
empirical evidence that reinforcement does not always
sustain reproductive isolation under such circumstances.
Spatial extent of hybridization
Fig. 5 Factorial correspondence analysis revealed two factors
that explained 85.6% of the genetic variation among purebred
and hybrid river herring and demonstrated the clear separation
of landlocked hybrids in Kerr Reservoir from alewife, blueback
herring and anadromous hybrids.
© 2014 John Wiley & Sons Ltd
Despite varying frequencies of hybridization across
their range (Figs 3 and 4), our study reveals that species
integrity is maintained in drainages where anadromous
alewife and blueback herring occur sympatrically. This
is consistent with hybridization studies of European
alosines (A. fallax and A. alosa; Alexandrino et al. 2006;
Coscia et al. 2010; Jolly et al. 2011) with similar divergence times (Bentzen et al. 1993; Faria et al. 2006), and
with studies of other hybridizing species (e.g. Helianthus
sp.; Strasburg & Rieseberg 2008). The frequency of
hybridization among anadromous populations generally
ranged from 0 to 8% (Table S2, Supporting Information). Whether this approximates the expected frequency of hybridization under pristine environmental
conditions is uncertain. Many of the rivers examined
have dams that may impact the spatiotemporal distribution of spawning migrations through restricted
10 D . J . H A S S E L M A N E T A L .
(a)
Fig. 6 Genomic clines plots generated in
‘introgress’ for (a) anadromous (n = 90)
and (b) landlocked (Kerr Reservoir)
(n = 119) river herring hybrids for nine
microsatellite loci common to both data
sets. The name of each locus and P-value
for the test of departure from neutral
introgression is provided [P < 0.015 and
P < 0.039 indicate significance for anadromous and landlocked hybrids, respectively, after FDR correction]. Solid
coloured clines represent the 95% confidence intervals for hybrids with homozygous (Aa/Aa or Ab/Ab; dark green) and
heterozygous (Aa/Ab; light green) genomic clines given neutral introgression.
The solid and dashed lines give the estimated genomic cline based on the
observed homozygous and heterozygous
allelic classes, respectively. Circles indicate the raw allelic class data (Aa/Aa
along the top, Aa/Ab in the centre and
Ab/Ab along the bottom), with counts of
each allelic class along the right vertical
axis. The hybrid index quantifies the
fraction of alleles derived from blueback
herring across the nine microsatellite loci.
(b)
© 2014 John Wiley & Sons Ltd
H Y B R I D I Z A T I O N A N D I N T R O G R E S S I O N I N A L O S I N E S 11
habitat access (Hall et al. 2011) and altered spawning
cues (i.e. water temperature; Loesch 1987), and may
facilitate hybridization (Boisneau et al. 1992; Maitland &
Lyle 2005). Spawning alewife and blueback herring are
known to co-occur where upstream migration is
blocked by dams (Loesch 1987), and this may provide
opportunities for interspecific matings. However, the
Delaware River has no mainstem dams and exhibited
5–6% hybridization, suggesting that some level of
hybridization may be natural.
Although we do not know the extent to which preand postzygotic reproductive isolating mechanisms
maintain species integrity, the general absence of deeply
introgressed (i.e. backcrossed) individuals in anadromous populations may indicate an important role for
selection against hybrids under natural conditions.
In contrast to the maintenance of species integrity
among anadromous populations, our study reveals that a
hybrid swarm has become established in Kerr Reservoir
following dam construction that has prevented immigration of purebred alewife or blueback herring since 1953.
All individuals in Kerr Reservoir were identified as
hybrids (Table S2, Supporting Information; Fig. 4) and
were deeply introgressed – likely via multiple generations of backcrossing. This finding suggests that both
pre- and postzygotic reproductive isolating mechanisms
may have broken down and that there may not be selection against hybrids in this altered environment. In unaltered drainages, differential spawning habitat selection
exhibited by these species may limit opportunities for
interspecific matings (Loesch 1987). However, the lotic
spawning habitats preferred by blueback herring are not
available in Kerr Reservoir, potentially leading to
increased interspecific matings. Decreased spatial segregation may be accompanied by altered (temperaturedependent) migratory and spawning cues, possibly
increasing temporal spawning overlap within Kerr Reservoir as well. The exact mechanisms at play require further investigation. Nonetheless, our study demonstrates
that anthropogenic habitat alterations can breakdown
reproductive isolation between distinct evolutionary lineages that naturally occur in sympatry.
Although hybridization in temperate fishes as a consequence of habitat disturbance is not a new concept
(Hubbs 1955), prior examinations were largely limited
to young evolutionary lineages of freshwater species, or
intraspecific comparisons of anadromous and resident
salmonids (Utter 2001). While hybridization between
anadromous species placed in novel freshwater environments has been previously reported (Rosenfield et al.
2000), their collapse as a hybrid swarm has not been
documented. Our study reinforces the contention that
reproductive isolating mechanisms may be incomplete
during the first 2–5 million years after speciation
© 2014 John Wiley & Sons Ltd
(Coyne & Orr 2004) and that a substantial portion of
global biodiversity may be susceptible to anthropogenically induced hybridization (Rhymer & Simberloff 1996;
Seehausen et al. 2008).
Elevated frequencies of hybridization in the Petitcodiac River (~22%) and Margaree River (~12%) (Fig. 4)
were unexpected, but may be explained by a combination of migratory barriers, decreased abundances and
range-edge effects. A causeway with ineffective fish
passage was constructed near the head of tide in the
Petitcodiac River in 1968. This barrier completely
blocked the access of migratory fishes to upstream
spawning habitat and dramatically reduced the abundance of alewife and blueback herring in the drainage
(Locke et al. 2003). Increased spatiotemporal overlap of
spawning adults below the causeway coupled with
population declines may have increased the chances of
interspecific matings (Reyer 2008). Although dams are
not present on the mainstem Margaree River, river herring have been heavily exploited in this drainage (R.G.
Bradford, personal communication), and low population abundances may have increased opportunities for
hybridization. Further, this drainage approximates the
northern range edge of blueback herring where population density is low and heterospecific matings may be
more likely (Reyer 2008). However, we did not observe
elevated levels of hybridization for other northern
populations (i.e. Miramichi River, River John). Elevated
levels of hybridization reported for European alosines
(Jolly et al. 2011, 2012) have been attributed to similar
factors (Boisneau et al. 1992; Maitland & Lyle 2005),
but the role of shifting phenologies (Ellis & Vokoun
2009) on the frequency of hybridization requires
attention.
Hybrids detected in rivers from South Carolina to
Florida are beyond the southern range limit for alewife
and could be strays; our sampling of adult specimens
does not preclude their origin in more northerly drainages. However, there is a paucity of evidence for
increased straying rates of hybrid fishes, especially
anadromous species (Scribner et al. 2001; but see Gilk
et al. 2004). Alternatively, these hybrids could be the
progeny of stray purebred alewife that reproduced with
blueback herring in southern rivers. These remain
untested hypotheses that require attention.
Detecting hybrids
Accurate identification of hybrids using molecular
methods depends on the markers used and the degree
of differentiation between parental species (Anderson &
Thompson 2002; V€
ah€
a & Primmer 2006). Nonetheless,
studies continue to employ a default Tq (0.90) that was
originally derived from simulations (V€
ah€
a & Primmer
12 D . J . H A S S E L M A N E T A L .
2006); rarely has Tq been assessed on an individual
study basis. Employing this arbitrary threshold in our
study would have inflated our estimates of hybridization. Our simulations revealed that misclassifications
were minimized using a Tq of 0.87 (NEWHYBRIDS) and
0.935 (STRUCTURE). This value for STRUCTURE approximates
that used in hybridization studies of European alosines
(0.94; Jolly et al. 2012). We advocate a simulation
approach (as outlined herein) for setting the most
appropriate Tq for discriminating purebred and hybrid
specimens on an individual study basis.
Hybrid divergence in isolation
Although hybridization can threaten biodiversity (Rhymer
& Simberloff 1996), genetic admixture in novel and perturbed environments can generate diversity by increasing
the adaptive potential of admixed individuals with novel
genetic variation (e.g. Buerkle et al. 2000; Barton 2001;
Lexer et al. 2003). Our study demonstrates that landlocked
hybrids are genetically distinct from anadromous
hybrids, alewife and blueback herring (Fig. 5). Indeed,
landlocked hybrids are nearly as differentiated from alewife (FST = 0.30) as alewife are from blueback herring
(FST = 0.38). This may have resulted from an initial population bottleneck followed by hybridization, and the
effects of drift in isolation from anadromous congeners. A
number of landlocked European alosine populations have
become adapted to lacustrine habitats (Faria et al. 2006)
and are genetically distinct from their anadromous congeners (e.g. A. f. killarensis; Jolly et al. 2012). Our data
suggest that Kerr Reservoir constitutes an admixed population in a novel environment that is in the process of
divergence.
Contrasting patterns of introgression
The distribution of blueback herring ancestry observed
among anadromous hybrids (Fig. S3a, Supporting Information) is consistent with neutral introgression. This
contrasts with landlocked hybrids, where the decreased
prevalence of blueback herring ancestry (Fig. S3b, Supporting Information) suggests directional introgression
of alewife genotypes in Kerr Reservoir. Although the
proportion of the two species that became landlocked is
unknown, available evidence suggests that blueback
herring were more abundant in the Roanoke River than
alewife when the dam was constructed (Carnes 1965).
Thus, it is unlikely that our result simply reflects a
chance colonization event where alewife were numerically dominant and where the introgressed gene pool is
largely of alewife origin.
For hybrids, the extent of introgression at individual loci is a consequence of the fitness effects of
genotype combinations. Contrasts among markers permit the identification of loci that lower hybrid fitness
and contribute to reproductive isolation, or that
increase fitness and promote adaptive introgression
(Gompert & Buerkle 2010). Although our analyses
revealed some concordance in locus-specific patterns
of introgression between anadromous and landlocked
hybrids, we also observed loci that exhibited markedly different patterns of introgression and that may
be subject to varying selection in different settings
(i.e. environments and genetic backgrounds; Nolte
et al. 2009). Differential patterns of introgression have
been previously reported for sculpin (Cottus spp.)
hybrid zones, where discordance in locus-specific patterns has been attributed to differing extrinsic factors
(i.e. local ecological conditions may impose different
selection pressures on admixed genotypes), differing
underlying genetic architecture of reproductive isolation and adaptation and/or the influence of stochasticity and drift in (small) hybrid populations (Nolte
et al. 2009). One or more of these factors may contribute to the discordance in patterns of introgression
that we observe.
Patterns of non-neutral introgression can be categorized based on their correspondence with different
models of genotypic effects in hybrids (Nolte et al.
2009). For anadromous hybrids, loci that exhibited
increased probability for heterozygous (Aa/Ab) allelic
classes are consistent with overdominance, while
decreased probability for homozygous (Aa/Aa, Ab/Ab)
allelic classes is consistent with negative selection
(Fig. 6a). Conversely, for landlocked hybrids, loci that
exhibited decreased probability for heterozygous allelic
classes are consistent with positive selection, while
increased probability of homozygous allelic classes is
consistent with adaptive introgression (Fig. 6b).
Although different patterns of introgression are
apparent between anadromous and landlocked hybrids,
we hesitate to invoke any particular mode of selection
pending analyses of a larger suite of molecular markers.
The genomic clines method requires a sufficient number
of loci distributed broadly across the genome to ensure
that estimates of genome-wide admixture are representative of neutral introgression (Gompert & Buerkle
2009). Although the loci used in this study are not
linked, without a linkage map it will be unclear to what
extent loci demonstrating similar patterns of introgression are independent, and it will not be possible to
determine the proportion of the genome experiencing
different forms of selection (Gompert & Buerkle 2009).
The investigation of contrasting patterns of introgression between anadromous and landlocked hybrids
warrants further study using advanced genomic
approaches.
© 2014 John Wiley & Sons Ltd
H Y B R I D I Z A T I O N A N D I N T R O G R E S S I O N I N A L O S I N E S 13
Conclusions
Our study reveals that anthropogenic habitat changes
can breakdown reproductive isolation between divergent evolutionary lineages that naturally occur in sympatry. While sympatric populations of anadromous
alewife and blueback herring maintain species integrity
across their range, reproductive isolation has broken
down in Kerr Reservoir, leading to the formation of a
hybrid swarm. The construction of dams constitutes a
dramatic alteration to habitat and may disrupt processes that sustain reproductive isolation under natural
conditions. We posit that decreased opportunities for
spatiotemporal spawning segregation in Kerr Reservoir
have increased the likelihood of interspecific matings
and have lead to the breakdown of reproductive isolation. The detection of deeply introgressed hybrids in
Kerr Reservoir indicates that hybrids are not disfavoured in this altered environment. This contrasts with
a general absence of deeply introgressed hybrids in
anadromous populations, suggesting an important role
for selection against hybrids under natural conditions.
Our results show that reinforcement may be insufficient
to sustain reproductive isolation in the face of some
types of human disturbance. The benefits of dam
removal for the restoration of anadromous fishes have
been largely discussed in the context of replenishing
historic spawning runs (e.g. Hasselman & Limburg
2012). Our study provides evidence that dam removal
could also help maintain species integrity for alewife
and blueback herring.
Acknowledgements
We are grateful to all those who contributed sampling effort to
our ongoing coast-wide genetic studies of river herring ecology, evolution and conservation. B. Wynne (North Carolina
Wildlife Resources Commission), D. Michaelson (Virginia
Department of Game and Inland Fisheries) and Casey Seelig
(Dominion Environmental Biology) assisted with the sampling
of Kerr Reservoir. E. M. Labbe, S. Blienbry and L. Thornton
assisted with microsatellite data collection, and T. M. Apgar
assisted with Fig. 1. We also thank three anonymous reviewers
and the associate editor for constructive comments that
strengthened the quality of this paper. This research was
supported by an NSERC Discovery Grant to P. Bentzen, a
National Fish and Wildlife Foundation Grant to E.P. Palkovacs
and North Carolina SeaGrant funding to E.P. Palkovacs and
T.F. Schultz.
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D.J.H. designed the study with E.P.P., conducted the
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A.A.P.-U. collected data and contributed to manuscript
revisions. E.P.P., T.F.S. and P.B. funded the project and
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Data accessibility
Microsatellite data used in this manuscript: DRYAD
Digital Repository. doi:10.5061/dryad.8v0c3.
All necessary input files required to replicate our
genomic clines analysis using the R package ‘introgress’: DRYAD Digital Repository doi:10.5061/dryad.
ft48k.
Supporting information
Additional supporting information may be found in the online version of this article.
16 D . J . H A S S E L M A N E T A L .
Fig. S1 Allele frequency distributions for alewife ( ) and blueback herring ( ) for 15 loci examined in this study. Estimates
of the Kullback–Leibler divergence between the species for
each locus are located in the top-right of each panel.
Fig. S2 Mean (SD) of q-values for five simulated data sets of
six hybrid categories (ALE: alewife, BBH: blueback herring, F1,
F2 and F1 backcrosses) analysed using (a) STRUCTURE (K = 2;
number of species) and (b) NEWHYBRIDS (K = 6; number of genotype frequency classes). The horizontal dashed line indicates
q = 0.90.
Fig. S3 Ancestry plots generated in ‘introgress’ for (a) anadromous (n = 90) and (b) landlocked (Kerr Reservoir) (n = 119)
river herring hybrids for nine microsatellite loci common to
both data sets. Dark green blocks indicate hybrids that are
homozygous for alewife allelic classes (Aa/Aa), light green
blocks indicate hybrids that are homozygous for blueback herring allelic classes (Ab/Ab), and intermediate green blocks correspond to hybrids that are interclass heterozygotes (Aa/Ab).
White blocks indicate missing data. The plot to the right in
each panel indicates the proportion of each individual’s genome that has blueback herring ancestry; equivalent to the
hybrid index. Individuals are sorted, with those that have
genomic compositions resembling alewife at the bottom and
increasing similarity to blueback herring toward the top.
Table S1 Classification rules for delineating purebred and
hybrid specimens based on the optimal threshold (Tq) determined using Bayesian analyses of five simulated data sets.
Table S2 Proportion of hybrids detected by river using
and NEWHYBRIDS.
STRUC-
TURE
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