Download HYBRIDIZATION BETWEEN AN INVASIVE AND A NATIVE

J OURNAL OF C RUSTACEAN B IOLOGY, 32(6), 962-971, 2012
HYBRIDIZATION BETWEEN AN INVASIVE AND A NATIVE SPECIES OF
THE CRAYFISH GENUS ORCONECTES IN NORTH-CENTRAL OHIO
Sierra T. Zuber 1 , Katherine Muller 2 , Roger H. Laushman 1 , and Angela J. Roles 1,∗
2 Department
1 Biology Department, Oberlin College, Oberlin, OH 44074, USA
of Plant Biology and Conservation, Northwestern University, Evanston, IL 60208, USA
ABSTRACT
Hybridization between native and invasive species is an important but little-studied factor in crayfish invasions, with few documented
natural cases. Here, we report genetic evidence of hybridization between invasive O. rusticus (Girard, 1852) and native O. sanbornii
(Girard, 1852) in the Huron River in north-central Ohio based on a combination of molecular markers: nuclear DNA, mitochondrial DNA,
and allozymes. Although we found no fixed differences between the species at nuclear DNA loci, fixed differences in mtDNA and allozyme
loci confirmed the presence of individuals of hybrid ancestry. We also found preliminary evidence of possible mitochondrial recombination
and biparental inheritance (though the existence of rare haplotypes cannot be excluded at the present time). We examined morphological
features of both species in sympatry and allopatry and confirmed species-diagnostic morphological features including gonopod traits and
shape of the annulus ventralis. This study is the second to detect hybridization between O. rusticus and a congener, which suggests that this
may be an important mechanism of invasion by O. rusticus when closely related species are present. Further study of this system, including
more extensive sampling and additional molecular markers, is necessary to understand the extent and implications of hybridization for the
native species.
K EY W ORDS: crayfish, hybridization, introgression, invasion, Orconectes
DOI: 10.1163/1937240X-00002091
I NTRODUCTION
A wide body of research has examined the ecological
impacts of invasive crayfish, including displacement of
native crayfishes (Capelli and Munjal, 1982; Hill and Lodge,
1999), reductions in prey taxa (Rosenthal et al., 2006; Pintor
and Sih, 2011), and other community changes (Hamr, 2002;
Roth et al., 2007; Bobeldyk and Lamberti, 2008). The
displacement of native crayfishes is a particularly important
consequence of invasion in North America, which holds the
majority of the world’s crayfish biodiversity (Master et al.,
1998; Perry et al., 2002; Crandall and Buhay, 2008).
While the ecological impacts of crayfish invasions are
well known, the evolutionary impacts have received little attention. In particular, hybridization between native and invader is an important evolutionary phenomenon in freshwater species invasions (Perry et al., 2002). Species that diverge
in allopatry do not experience selective pressure to develop
mating barriers, leading to the possibility of hybridization
after secondary contact (Fisher, 1930; Dobzhansky, 1940;
Kaneshiro, 1980; Dumont and Adriaens, 2009). Hybridization and reproductive interference can have a variety of impacts on an invasion, slowing or speeding the loss of a native
species depending on the viability and fertility of hybrids
and relative fitnesses of parental species and hybrid individuals (Ellstrand and Schierenbeck, 2000; Perry et al., 2001b).
Few examples of crayfish hybridization exist in the
literature. Induced hybridization has generally resulted in
non-viable or sterile offspring or reduced brood sizes of
∗ Corresponding
fertile offspring (Berrill, 1985; Lawrence and Morrissy,
2000; Reynolds, 2002; Lawrence, 2004). The relevance of
these studies to natural populations is, however, unknown.
Most reports of natural hybridization are supported solely
by morphological evidence (Crocker, 1957; Crocker and
Barr, 1968; Capelli and Capelli, 1980; Smith, 1981), which
is not conclusive. Genetic evidence of natural crayfish
hybridization has been found in two systems, both believed
to be the result secondary contact (Procambarus spp.:
Sbordoni et al., 1988; Cesaroni et al., 1992; Orconectes spp.:
Perry et al., 2001b).
While the studies of hybridization in Procambarus examined two native species, Perry et al. (2001b) studied hybridization between a native and an invasive species of Orconectes. Examining genetic markers (allozyme loci) and
morphology in a hybrid zone between invasive Orconectes
rusticus (Girard, 1852) and resident O. propinquus (Girard,
1852), Perry et al. (2001b) found evidence of introgression
between species. While an earlier study of hybridization
between these species suggested reproductive interference
favoring the invasion of O. rusticus (Berrill, 1985), Perry
et al. (2001b) demonstrated that hybrids outcompeted both
parental species within the hybrid zone, thus slowing the advance of the O. rusticus invasion front.
Orconectes rusticus is a notorious invader in North
American waterways. Within the last 50 years O. rusticus
has expanded its range from Indiana, Kentucky, and western
Ohio to much of the Midwest and Northeast United States,
author; e-mail: [email protected]
© The Crustacean Society, 2012. Published by Brill NV, Leiden
DOI:10.1163/1937240X-00002091
ZUBER ET AL.: HYBRIDIZATION BETWEEN SPECIES OF ORCONECTES
plus the western states of Utah and Oregon, as well as north
into Canada (Ontario), and south to Tennessee and North
Carolina (United States Geological Survey, 2012). Use of
O. rusticus as live bait is likely responsible for much of this
spread (Berrill, 1978).
We examined hybridization between introduced O. rusticus and native O. sanbornii (Faxon, 1884) in north-central
Ohio. The native range of O. sanbornii extends from central
Ohio to parts of Kentucky and West Virginia (Fitzpatrick,
1967; Thoma and Jezerinac, 2000). While O. sanbornii is
not classified as endangered or threatened, populations in
parts of Ohio and West Virginia are experiencing local decline with the introduction of O. rusticus through its use
as live bait (Thoma and Jezerinac, 2000). Several studies
have explored O. rusticus-O. sanbornii interactions, including one laboratory study demonstrating that O. sanbornii
males prefer to copulate with O. rusticus females rather than
with conspecifics (Butler and Stein, 1985). Butler and Stein
(1985) also found that O. rusticus exhibits faster juvenile
growth and lower susceptibility to predation than O. sanbornii, giving the invader additional competitive ecological
advantages. However, Butler and Stein (1985) did not observe the outcome of mating attempts, thus the possibility of
hybridization between these species in natural populations
remained speculation.
In the course of our field research on the Huron River,
where these two species exist in sympatry, species identification on the basis of morphology is not always clear.
We sought to identify species diagnostic genetic markers by
genotyping nuclear DNA loci, mitochondrial DNA loci, and
allozyme loci in allopatric populations, then genotyping the
sympatric population to determine whether hybridization is
occurring. We also explored morphological differences between the species in allopatry as we discovered that some
morphological traits frequently used to identify species in
the field exhibit more within-species variation than we had
expected.
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M ATERIALS AND M ETHODS
Sample Collection
Crayfish were collected in fall 2009 from the Portage River,
Vermilion River, and Huron River, and in summer 2010
from a second site on the Huron River (Fig. 1). The Portage
and Vermilion rivers were selected as allopatric sites for O.
rusticus and O. sanbornii, respectively, whereas the species
are sympatric in the Huron River. Crayfish were caught in
seine and hand nets, by eye and by disturbing rocks in riffle
zones. We collected 21 O. rusticus (9 male, 12 female) from
the Portage River, 24 O. sanbornii (12 male, 12 female)
from the Vermilion River, 36 sympatric crayfish (21 male,
15 female) from the Huron River Monroeville site (HRM)
and 12 sympatric crayfish (9 male, 3 female) from the
Huron River Lover’s Lane Bridge site (LLB). Crayfish were
transported back to the lab and maintained in 75.7 liter tanks
(20 gallon) until frozen and preserved whole at −80°C.
DNA Analysis
DNA Isolation.—DNA was isolated from pleopod tissue,
collected before we sacrificed crayfish, using the Qiagen
Puregene DNA Purification kit (Qiagen, Germantown, MD).
For each individual, one or more pleopods were removed for
DNA isolation and stored in 300 μl Cell Lysis Buffer (Qiagen). Tissue was disrupted with a mortar and pestle and
maintained at room temperature. Proteins were digested with
1.5 μl Proteinase K (20 mg/ml) at 55°C for 1 h, then precipitated with 100 μl Puregene Protein Precipitation Solution and pelleted by centrifugation; this step was repeated if
the supernatant was not clear. DNA was precipitated from
the supernatant with 300 μl 100% isopropanol and pelleted
by centrifugation, then washed with 300 μl 70% ethanol
and dried overnight at room temperature. DNA was rehydrated in 50 μl Puregene DNA Hydration Solution and incubated either for one hour at 65°C with occasional agitation
or overnight at room temperature. Purified DNA was stored
at −20°C.
Loci and Amplification.—We amplified four nuclear
DNA regions: glyceraldehyde-3-phosphate dehydrogenase
Fig. 1. Map of crayfish sampling sites in north-central Ohio on the Portage River (allopatric for Orconectes rusticus), Vermilion River (allopatric for O.
sanbornii), and Huron River (sympatric for both species), with GPS coordinates for each site. Crayfish were collected fall 2009 and summer 2010.
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JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 32, NO. 6, 2012
(GAPDH), 28S ribosomal RNA (28S), histone H3 (H3) and
phosphoenolpyruvate carboxykinase (PEPCK). We amplified two mitochondrial DNA regions: 16S ribosomal RNA
(16S) and cytochrome oxidase II (COII). Primer sequences,
amplification conditions, and sequencing details are reported
in the Supplementary Material in the online edition of this
journal, which can be accessed via http://booksandjournals.
brillonline.com/content/1937240x/32/6.
RFLP.—Restriction digests were performed on 16S and
COII amplified from allopatric samples to identify diagnostic differences in cut sites. For 16S, we used the restriction
enzyme AseI, which cuts in O. sanbornii but not in O. rusticus. For COII, we used PvuII, which cuts in O. rusticus
but not in O. sanbornii. Each 30 μl reaction contained 12 μl of undiluted PCR product, 3 μl of 10× restriction enzyme buffer (NEB), 20 units of restriction enzyme, and water to volume. Reactions were incubated at 37°C for 1-2 h
before being stopped with 1 μl EDTA (0.5 M). The resulting fragments were run on a 2% agarose gel and stained with
ethidium bromide. Species identity was assigned by visual
inspection of fragment number and length.
Allozyme Analysis
We extracted enzymes from frozen tail muscle tissue ground
with a mortar and pestle in liquid nitrogen and extracted
enzymes using three extraction buffers (recipes in Supplementary Material). Extracted enzymes were filtered through
Miracloth and absorbed onto nitrocellulose wicks (Whatman
3mm), then stored at −80°C. Enzymes were separated using horizontal electrophoresis on 11% or 12% starch gels
(StarchArt Lot #W670) for 6-8 h with various electrode/gel
buffer systems at 4°C (Table 1 and Supplementary Material).
We stained for 12 enzymes (Table 1) using stains given in
Murphy et al. (1990). Gels were examined by eye after staining and scored by hand relative to a standard run on each
gel (crayfish HL1; see Supplementary Material). For Idh, 18
allopatric O. rusticus and 24 allopatric O. sanbornii were
genotyped; for all other enzymes 12 allopatric individuals of
each species were genotyped to assess variability.
Morphology Analysis
For sympatric samples from the Huron River, each crayfish was field identified to species based on coloration.
A reddish-brown lateral carapace “rusty spot” and orange
and black claw tips characterize O. rusticus; a mottled brown
tail and orange or orange and dark claw tips characterize O.
sanbornii. For the Huron River sites, we field-identified 23
O. rusticus, 10 O. sanbornii, and 3 putative hybrids at HRM
in 2009 and 12 O. rusticus at LLB in 2010.
Preserved allopatric (9 male, 12 female O. rusticus; 11
male, 7 female O. sanbornii) and sympatric (19 male, 18
female) crayfish were measured for 15 quantitative morphological characteristics (Fig. 2). Because overall size is highly
variable in crayfish and many traits covary strongly with
size, we analyzed 15 size-adjusted characters to account for
this source of variation, e.g., chela length divided by carapace length, plus carapace length itself. For each character, differences between the species in allopatry were analyzed by ANOVA using JMP (SAS Institute). Traits that
1) exhibited significant differences in the mean in allopatry,
and 2) had non-overlapping distributions were designated as
species diagnostic; these characters were then examined in
sympatric crayfish to observe similarities and differences between allopatric and sympatric populations. Rostral margins
and the annulus ventralis were photographed with a Canon
EOS Rebel digital camera mounted on a dissecting microscope (Westover Scientific, Mill Creek, WA, USA). Photographs of allopatric individuals were examined by eye for
qualitative differences in shape for these two traits. We then
assessed which species the photographs of sympatric individuals resembled most closely.
R ESULTS
DNA Analysis
Sequence Comparison of Allopatric Crayfish.—We obtained
sequences from both species for all nuclear and mitochondrial loci except the nuclear locus H3 (only one sequence,
from O. rusticus). The nuclear loci for which we had multiple sequences proved largely uninformative for species identification due to either lack of variation or lack of fixed dif-
Table 1. Allozymes extracted from Orconectes rusticus and O. sanbornii tail muscle tissue, separated by gel electrophoresis, and stained. R = O. rusticus,
S = O. sanbornii. * See Supplementary Material; + Multiple, resolution was not sufficient to determine exact number of alleles present.
Enzyme
Abbreviation
IUBMBNC number
Running buffer system∗
Alleles observed
Aspartate aminotransferase
Alkaline phosphatase
Esterase
Fructose-bisphosphatase
Aat
Alp
Est
Fbp
2.6.1.1
3.1.3.1
3.1.1.3.1.3.11
2a
7
10
5(1), 5(2), 5(3)
Glucose-6-phosphate dehydrogenase
Glutamate dehydrogenase
Isocitrate dehydrogenase
G6ph
Gdh
Idh
1.1.1.49
1.4.1.2
1.1.1.42
2a
7, 8(-)
5(1), 5(2), 5(3), P
Lactose dehydrogenase
Malate dehydrogenase
Malic enzyme
Phosphoglucoisomerase
Phosphoglucomutase
Ldh
Mdh
Me
Pgi
Pgm
1.1.1.27
1.1.1.37
1.1.1.40
5.3.1.9
5.4.2.2
MC 6.9
MC 6.9, 1a, 2a
MC 6.9
10
7, 8(-)
1
1
+
R−2
S−1
1
1
R−1
S−1
1
1
1
+
1
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ZUBER ET AL.: HYBRIDIZATION BETWEEN SPECIES OF ORCONECTES
Table 2. Summary of sequence variation within and between species.
R = Orconectes rusticus, S = O. sanbornii. GenBank Accession numbers
JQ397605-JQ397631.
Locus
GAPDH
28S
PEPCK
H3
16S
COII
No.
sequences
R
S
Base
pairs
aligned
1
3
3
1
3
3
3
3
1
0
3
3
763
698
544
–
543
556
No. shared
No. fixed
polymorphisms differences
3
0
2
–
1
3
1
0
0
–
17
28
two mitochondrial loci (HL17; Supplementary Material). Of
sympatric crayfish from the LLB site collected in 2010,
83% (10 of 12) had the O. rusticus mitochondrial haplotype
for both loci (Table 3). The other two individuals had
conflicting banding patterns for the two loci (Supplementary
Material). In total, three individuals were found to have
conflicting banding patterns at the two mitochondrial loci.
We repeated the amplification and digestion for all three
DNA samples and obtained the same result in all cases.
For two cases (HL17, SLL12) the individuals displayed the
pattern characteristic of O. sanbornii for the 16S locus and
the pattern of O. rusticus for the COII locus. The remaining
individual, SLL10, had the O. rusticus pattern for COII
but for 16S displayed three bands – with sizes matching
the two digested bands characteristic of O. sanbornii and a
single undigested band as in O. rusticus. We discuss possible
interpretations of these results below.
Allozyme Analysis
Fig. 2. Quantitative characters measured on preserved allopatric Orconectes rusticus and O. sanbornii, and sympatric crayfish of both species
and putative hybrids. Measurements were made to the nearest hundredth
of a millimeter with digital calipers (Mitutoyo Absolute Digimatic CD-6
CX, Mitutoyo America) and a dissecting microscope (Westover Scientific,
Mill Creek, WA, USA). Diagram modified with permission from Perry et al.
(2001b). Body measurements: 1, carapace length; 2, areola length; 3, areola
width; 4, carapace width; 5, acumen length; 6, rostrum width. Chela measurements: 7, dactyl length; 8, chela length; 9, palm width; 10, palm length;
11, chela gap width; 12, number of carpus spines. Gonopod measurements
(males only): 13, gonopod total length; 14, central projection length; 15,
mesial projection length.
ferences (Table 2). The mitochondrial loci, 16S and COII,
amplified consistently and contained both shared and fixed
differences (Table 2). Thus, we were able to use differences
in restriction sites of mitochondrial loci to identify maternal
lineage in sympatric crayfish, but could not gain additional
information on the ancestry of sympatric crayfish from any
of the nuclear loci that we sequenced. GenBank Accession
numbers are reported in Table 2.
RFLP Analysis of Sympatric Crayfish.—Of the sympatric
crayfish from the HRM site collected in 2009, 28% (9 of 32)
had the O. sanbornii banding pattern and 69% (22 of 32)
had the O. rusticus banding pattern for both loci (Table 3).
One individual had conflicting banding patterns for the
Of the 12 enzymes screened in allopatric individuals, eight
were monomorphic across both species and four polymorphic (Table 1). Three of the polymorphic loci were not
species diagnostic. The last polymorphic marker, Idh, was
monomorphic within each species but variable between the
species, thus Idh displays species-specific variability and
was informative in sympatric individuals (Table 3). Following Perry et al. (2001a), we scored the O. rusticus allele as
110. The O. sanbornii allele moved only 85% as far from the
cathode as the O. rusticus allele, thus we scored it as 94.
We genotyped 43 sympatric crayfish (31 from HRM
and 12 from LLB) for the Idh allozyme locus. Of these,
69.8% were homozygous for the O. rusticus allele, 18.6%
were homozygous for the O. sanbornii allele and 11.6%
were heterozygotes (Table 3). Two individuals exhibited
disagreement between the allozyme banding pattern and
mtDNA identity, with one individual having the O. rusticus
genotype for Idh (HL16; on-line Supplementary Material)
and the other having the O. sanbornii genotype for Idh
(HL12; on-line Supplementary Material), suggesting the
presence of backcrossed individuals.
Morphological Analysis
Annulus ventralis shape was qualitatively different for the
two species in allopatry (Fig. 3; O. rusticus N = 12,
O. sanbornii N = 10). No sympatric crayfish showed an
intermediate morphology for the annulus ventralis; every
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JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 32, NO. 6, 2012
Table 3. Summary of genotypes, haplotypes, and qualitative morphological characters of crayfish sampled
from the sympatric Huron River. R = Orconectes rusticus; S = O. sanbornii. Shape of the female’s annulus
ventralis was identified as described in Fig. 3. Note that for the allozyme locus Idh, the O. rusticus allele is
scored as 110 (Perry et al., 2001a) and the O. sanbornii allele as 94 based on relative migration distances.
Site
Idh genotype
RR
RS
SS
N
16s haplotype
R
S
R/S
N
COII haplotype
R
S
N
Annulus ventralis shape
R
S
N
HRM
2009
LLB
2010
Overall
sympatric
Allopatric
O. rusticus
Allopatric
O. sanbornii
20
3
8
31
10
2
0
12
30
5
8
43
18
0
0
18
0
0
24
24
23
9
0
32
10
1
1
12
33
10
1
44
7
0
0
7
0
7
0
7
24
10
34
12
0
12
36
10
46
7
0
7
0
7
7
7
5
12
3
0
3
10
5
15
12
0
12
0
10
10
sympatric female crayfish (N = 15) was confidently
assigned to one species’ shape or the other without reference
to additional features or genetic data (Table 3; 10 O.
rusticus shape, 5 O. sanbornii shape). In all but one case,
annulus ventralis shape corresponded to mtDNA identity.
The exception was HL16, which had O. sanbornii mtDNA
and the O. rusticus Idh genotype: this putative hybrid
individual had the O. rusticus annulus ventralis shape (online Supplementary Material).
Rostral margins were variable within and between species,
contrary to our expectations of concave margins in O. rus-
ticus and straight margins in O. sanbornii (on-line Supplementary Material; Jezerinac et al., 1995). Both species exhibited approximately straight rostral margins in allopatry
(except for two allopatric O. rusticus with concave rostral
margins), thus rostral margin shape was not species diagnostic. All sympatric individuals photographed (N = 43)
except HL1 had straight rostral margins. Thus, while only O.
rusticus exhibited concave rostral margins, more often they
displayed straight margins, as in O. sanbornii.
For quantitative characters, species in allopatry exhibited
significantly different means for 10 size-corrected traits (Ta-
Fig. 3. Typical annulus ventralis shapes. A, Oroconectes rusticus (left); B, O. sanbornii (right). The annulus ventralis of O. rusticus females has a fairly
straight medial suture on a raised ridge in the caudal half of the annulus ventralis, with two protrusions in the rostral quadrants, which slightly overhang
the central region to create a small pocket. The annulus ventralis of O. sanbornii females is nearly flat, with a three-segment zig-zag medial suture that is
strongly asymmetric. We observed a bias in our sample as to which direction the first suture segment slanted: of the 10 allopatric O. sanbornii individuals
photographed, nine had a first suture slant from the animal’s rostral right to caudal left, one had a first suture slant from the animal’s rostral left to caudal
right.
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ZUBER ET AL.: HYBRIDIZATION BETWEEN SPECIES OF ORCONECTES
Table 4. Mean (s.e.) and range of ratios of body, chela, and gonopod measurements for allopatric Orconectes rusticus (N = 21 total; 9 males) and O.
sanbornii (N = 18 total; 11 males). A difference between the species means was tested by one-way ANOVA. L = length, W = width.
Measurement
Body ratios
Carapace W/L
Areola L/Carapace L
Areola W/L
Areola W/Carapace L
Rostrum W/ Carapace L
Acumen L/Carapace L
Rostral W/Acumen L
Chela ratios
Dactyl L/Chela L
Dactyl L/Palm L
Palm W/Chela L
Palm L/Chela L
Palm W/Palm L
Chela gap W/Chela L
Gonopod ratios
Central projection L/Gonopod L
(Central projection L – Mesial projection L)/
Gonopod L
O. rusticus
O. sanbornii
F -ratio
P -value
Mean (s.d.)
Min-Max
Mean (s.d.)
Min-Max
0.49 (0.005)
0.34 (0.003)
0.15 (0.007)
0.05 (0.002)
0.10 (0.002)
0.09 (0.003)
1.16 (0.06)
0.44-0.55
0.32-0.37
0.09-0.22
0.03-0.08
0.09-0.13
0.05-0.12
0.92-1.88
0.47 (0.005)
0.33 (0.003)
0.18 (0.007)
0.06 (0.002)
0.10 (0.002)
0.09 (0.003)
1.09 (0.06)
0.44-0.51
0.31-0.36
0.14-0.22
0.05-0.07
0.07-0.12
0.05-0.12
0.76-2.01
F1,40
F1,40
F1,40
F1,40
F1,40
F1,39
F1,39
= 8.452
= 6.273
= 6.068
= 4.178
= 0.7035
= 0.4305
= 0.8496
0.006
0.016
0.018
0.048
0.407
0.516
0.362
0.61 (0.01)
1.66 (0.07)
0.39 (0.008)
0.38 (0.01)
1.06 (0.04)
0.04 (0.004)
0.52-0.69
1.20-3.17
0.28-0.47
0.21-0.46
0.81-1.82
0.01-0.08
0.56 (0.01)
1.35 (0.08)
0.40 (0.008)
0.42 (0.01)
0.95 (0.04)
0.04 (0.004)
0.53-0.61
1.08-1.62
0.33-0.46
0.33-0.52
0.80-1.12
0.02-0.06
F1,37
F1,37
F1,37
F1,37
F1,37
F1,37
= 16.05
= 8.220
= 0.0964
= 5.162
= 4.447
= 0.3273
0.0003
0.007
0.758
0.029
0.042
0.571
0.31 (0.02)
0.05 (0.006)
0.16-0.44
0.00-0.18
0.08 (0.02)
0.01 (0.006)
0.05-0.11
0.00-0.03
F1,38 = 105.9
F1,38 = 23.93
ble 4). While significant differences in the mean establish
an average difference between the species, only traits which
exhibit non-overlapping distributions are useful for species
identification. Of these 10 traits, only the ratio of gonopod
central projection length to total gonopod length displayed
both significantly different means and non-overlapping distributions for the two species in allopatry (Table 4; Fig. 4). In
all gonopod measurements and ratios, the range of values for
allopatric individuals was far greater for O. rusticus (N = 9)
than for O. sanbornii (N = 11). When these gonopod measurements were assessed in sympatric crayfish (N = 28),
values fell primarily above the range for allopatric O. sanbornii – though a few in each case fell below the range of
allopatric O. rusticus. In no case did a sympatric crayfish
have a value for a gonopod length ratio that fell above the
range of values for allopatric O. rusticus. Thus, the sympatric population displays morphology somewhat intermediate to the two species considered in allopatry for the species
diagnostic gonopod length ratio.
D ISCUSSION
Our observations of nine sympatric individuals that exhibit
mixed genotypes for nuclear and mitochondrial loci confirms that O. rusticus and O. sanbornii are hybridizing in
the Huron River (Table 3). We identified five individuals
heterozygous for the allozyme locus (Idh), two individuals
with the allozyme genotype of one species and the mitochondrial genotype of the other, and two individuals with differ-
<0.0001
<0.0001
ent haplotypes for the two mitochondrial loci. The existence
of individuals with different nuclear and mitochondrial haplotypes suggests that hybrids are fertile and are backcrossing with one or both of the parent species. The mixed maternal origins of mitochondrial haplotypes in individuals of
hybrid ancestry suggests that hybridization does not occur
exclusively through males of one species mating with females of the other; instead, males of each species must have
mated with females of the other or with hybrid individuals.
As we have small samples sizes and few loci, we cannot at
present speculate on directional trends in hybridization nor
upon rates of hybridization. In fact, while we can establish
a minimum of nine individuals of likely hybrid ancestry, our
limited set of molecular markers lacks the resolution to identify all individuals of hybrid ancestry.
We observed three individuals with conflicting data for
the two, mtDNA loci. These individuals could represent rare
haplotypes that exist in the allopatric populations but were
not present in our samples. Alternatively, these unusual haplotypes could represent biparental inheritance followed by
recombination in the mitochondrial genome, which is most
commonly observed in cases of hybridization (Rokas et al.,
2003; Barr et al., 2005) and is known to occur in animals
(Sutovsky et al., 2000). Hybridization events may be especially prone to exhibiting evidence of recombination due to
the breakdown of species-specific mechanisms that prevent
paternal mtDNA inheritance (Barr et al., 2005). Our results
are the first to report evidence of possible paternal leakage in
Fig. 4. Boxplots of ratios exhibiting statistically significant differences (see Table 1) between allopatric Orconectes rusticus (N = 21 total; 9 males) and
allopatric O. sanbornii (N = 18 total; 11 males). Sympatric individuals from the Huron River are included for comparison (N = 37 total; 19 males). Three
Form II individuals are included (1 O. sanbornii and 2 sympatric individuals) as their values do not differ from the Form I individuals. A, carapace length;
B, carapace width/length; C, areola length/carapace length; D, areola width/length; E, dactyl length/chela length; F, dactyl length/palm length; G, palm
length/chela length; H, palm width/length; I, central projection length/total gonopod length; J, central minus mesial projection length/total gonopod length.
968
JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 32, NO. 6, 2012
ZUBER ET AL.: HYBRIDIZATION BETWEEN SPECIES OF ORCONECTES
crayfish. Inter-population crosses in one crayfish species detected no evidence of mitochondrial recombination (Nguyen
and Austin, 2004), however, if the mechanisms preventing
paternal transmission are species-specific, then recombination would not be expected in intraspecific crosses. Our
evidence stems from proposed interspecific crosses, where
species-specific mechanisms may fail. In addition, we report evidence for recombination between two mitochondrial
loci. As the frequency of crossing-over is positively correlated with distance, within-locus recombination is less likely
than between-locus recombination. We caution that our results are suggestive but inconclusive: further, more extensive, sampling is necessary to confirm or refute the possibility of biparental inheritance and recombination in the mitochondrial genomes of these crayfish.
We also lack the resolution in terms of sample sizes and
number of loci to determine the rate at which hybridization
may occur. However, we observe that the percentage of
putative hybrids in our sample was fairly consistent between
sites approximately eight miles of river distance apart
(3/12 = 25% for LLB, sampled in 2010 and 6/32 =
19% for HRM, sampled in 2009) with an estimate of 20%
overall, similar to the 23.4% reported for O. rusticus and
O. propinquus in Trout Lake (Perry et al., 2001b). However,
the addition of more molecular markers and larger sample
sizes is necessary to make a reliable estimate of the rates of
hybridization and backcrossing in the Huron River (Perry
et al. used two diagnostic nuclear loci and sampled 781
individuals).
The ability to identify hybrids by sight in the field is invaluable. We found many morphological traits that exhibit
significantly different means between the two species in allopatry, though few appear promising for field identification of the two pure species and the hybrid due to overlapping ranges (Table 4). For example, the two ratios used
by Perry et al. (2001b) to distinguish male O. rusticus, O.
propinquus, and O. virilis (areola width/carapace length,
(central-mesial projection length)/gonopod total length) had
significantly different means in our sample, but were not
truly diagnostic due to overlapping ranges. Only two, nonsize-corrected gonopod traits (central projection length and
mesial projection length; Supplementary Material), one sizecorrected gonopod trait (central projection length/gonopod
total length), and the shape of the annulus ventralis exhibited
non-overlapping distributions. These differences are unsurprising as characteristics of gonopods and the annulus ventralis are commonly used to distinguish and classify crayfish species (Fitzpatrick, 1987). However, the lack of difference in the size-corrected values is surprising because differences in size-corrected characters have been used to identify crayfish species. Unexpectedly, rostral margin shape did
not show fixed differences between species; concave rostral margins are a distinguishing characteristic of O. rusticus in dichotomous keys (Jezerinac et al., 1995). However, it is unknown whether O. rusticus in the Portage River
may have experienced hybridization with another species of
Orconectes in the past, leading to higher variance in these
morphological measures. Additional sampling of O. rusticus populations throughout their native range is necessary to
determine the generality of this result.
969
The lack of species-specific variation in the nuclear DNA
and allozyme loci we tested suggests very little phylogenetic
distance between O. rusticus and O. sanbornii, contrary to
their current classification in separate subgenera: Procericambarus and Crockerinus, respectively. While the current
classification scheme is based on morphology (Fitzpatrick,
1987), recent studies have addressed phylogenetic relationships within Orconectes using molecular markers (Crandall
and Fitzpatrick, 1996; Fetzner, 1996; Taylor and Hardman,
2002; Wetzel et al., 2004; Taylor and Knouft, 2006). Of
these studies, only Taylor and Knouft (2006) included both
O. rusticus and O. sanbornii. In their parsimony analysis of
COI sequences in most of the 82 species of Orconectes, O.
rusticus and O. sanbornii were quite closely related, in a
clade with only five other species, with O. rusticus in the
group sister to O. sanbornii, the most basal member of the
clade (Crockerinus and Procericambarus contain nearly 40
species in Fitzpatrick’s classification). Thus, the available
molecular data seems to support a closer relationship between O. rusticus and O. sanbornii than previously determined. It is noteworthy that while O. propinquus is not part
of the clade that includes O. rusticus and O. sanbornii, it
does share a common ancestor with them one node back
from the clade for O. sanbornii and O. rusticus; thus O. rusticus is known to hybridize with two relatively closely related species within the genus.
Our data confirm hybridization between O. rusticus and
O. sanbornii in the Huron River of north-central Ohio. The
genotypes of putative hybrids also indicate that backcrossing is occurring; thus hybrids are viable and fertile. We are
not presently able to estimate the rate at which F1 hybrids or
backcrosses are formed; additional nuclear loci must be developed to further understand the dynamics and importance
of hybridization in this invasion. More generally, assays for
hybridization in additional O. sanbornii rivers that have been
invaded by O. rusticus will allow us to establish whether this
is a common or important mechanism of invasion for O. rusticus in this region. We also report the first evidence in crayfish of possible mitochondrial recombination and paternal
leakage, resulting from interspecific mating; more extensive
sampling is necessary to confirm or reject this hypothesis
(alternatively, these results could represent rare haplotypes).
We suggest that the use of multiple mitochondrial loci may
increase the power to detect rare events of recombination
and paternal inheritance in mitochondrial genomes. Finally,
phylogenies based on additional markers would aid in understanding the true evolutionary relationships within the genus
as well as provide information on the potential for hybridization of the invasive O. rusticus with other members of the
genus.
ACKNOWLEDGEMENTS
The work described above comes from the undergraduate Honors Theses of
KM and STZ. We would like to thank the Oberlin Biology Department and
the Oberlin College Office of Sponsored Programs for funding this study.
We would also like to thank Michael J. Moore for guidance with the lab
work and two anonymous reviewers for their insightful comments on the
manuscript. STZ thanks Katie A., Anthony B., Marta R. and Robin T. for
lab and field assistance, and Joshua T. G. for help in creating maps and
figures.
970
JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 32, NO. 6, 2012
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R ECEIVED: 14 February 2012.
ACCEPTED: 16 June 2012.
S UPPLEMENTARY M ATERIAL
Nuclear and Mitochondrial Loci.—Isolated DNA was amplified using PCR with a final reaction volume of 15 μl:
0.6 μl DNA (0.02× of stock; 0.5× for PEPCK), 0.15 μl
of dNTPs (10 mM), 1.5 μl of 10× Sigma RedTaq PCR reaction buffer, 0.6 μl (0.225 μl for 16S and COII) of forward
and reverse primers (10 μM), 0.6 μl RedTaq DNA polymerase (1 unit/μl; Sigma), and water to volume. Primer sequences, sources, and amplification conditions are reported
in Table S1.
PCR products were purified by combining 10 μl of PCR
product with 4 μl of ExoSAP-IT (USB) and incubating
at 37°C for 15 min to degrade remaining primers and
nucleotides. A second incubation at 80°C for 15 min
deactivated the enzyme. Samples were stored at −20°C until
shipped on dry ice for sequencing at SeqWright (Houston,
TX, USA) with an ABI Prism 3730xL DNA sequencer.
Sequence Analysis.—Sequences were edited and aligned
with either Sequencher 4.10.1 (Gene Codes) or Geneious
(Biomatters). For the nuclear loci GAPDH, 28S, H3 and
PEPCK, we examined the sequences for fixed differences
between the species. For the mitochondrial loci 16S and
971
COII, we identified species-specific differences in restriction
enzyme cut sites using Enzymex3.1 (Mekentosj).
Buffers used for allozyme extraction from crayfish muscle
tissue and gel electrophoresis.
Extraction buffers
Agerberg (1990): 21.40 g sucrose, 41.75 ml 0.1 M KH2 PO4 ,
20.75 ml 1.0 M K2 HPO4 , 3.75 ml phenoxyethanol, in
distilled water to 250 ml total volume
Fetzner (1996): Saturated pyridoxal 5 -phosphate in distilled
water
W. L. Perry (pers. commun. to RHL): 0.04 g EDTA, 0.01 g
NADP, 0.01 g NAD, 0.25 ml 2-mercaptoethanol, in
100 ml 0.1 M Tris-HCl buffer, pH 7.0
Running buffer systems
1a – Citric acid electrode buffer, 0.010 Histidine-HCl gel
buffer, pH 7.0
2a – Tris-citric acid electrode buffer, 0.067 × (electrode
buffer) gel buffer, pH 7.0
5(1) – Tris-citric acid electrode buffer, 0.7 × (electrode
buffer) gel buffer, pH 7.2
5(2) – Tris-citric acid electrode buffer, pH 7.2, 0.7 ×
(electrode buffer) gel buffer, pH 7.4
5(3) – Tris-citric acid electrode buffer, pH 7.2, 0.035 ×
(electrode buffer) gel buffer, pH 7.4
7 – Lithium hydroxide-boric acid electrode buffer, Triscitric acid gel buffer, pH 8.3
8(-) – Lithium hydroxide-boric acid electrode buffer,
pH 8.1, Tris-citric acid gel buffer, pH 8.5
10 – Tris-Borate-EDTA electrode buffer, 0.25 × (electrode buffer) gel buffer, pH 8.6
MC 6.9 – Morpholine-citric acid electrode buffer, 0.05 ×
(electrode buffer) gel buffer, pH 6.9
P – Tris-citric acid electrode buffer, pH 6.3, Tris-citric
acid gel buffer pH 6.7
Table S1. Primers and amplification cycles for mitochondrial and nuclear loci amplified in Orconectes rusticus and O. sanbornii. 1 Perry et al., 2001b;
2 Buhay et al., 2007; 3 Fetzner and Crandall, 2002; 4 Tsang et al., 2008.
Genome
mtDNA
Nuclear
Primer sequence (5 → 3 )
Amplification profile
f: AGATAGAAACCAACCTGG
r: CCTGTTTAACAAAAACAT
36 cycles of: 30 s at 94°C, 2 min at 50°C, 2 min at 72°C
COII 1
f: AGCGCCTCTCCTTTAATAGAACA
r: CCACAAATTTCTGAACATTGACCA
36 cycles of: 30 s at 94°C, 2 min at 50°C, 2 min at 72°C
GAPDH 2
f: TGACCCCTTCATTGCTCTTGACTA
r: ATTACACGGGTAGAATAGCCAAACTC
Initial 2 min at 95°C; 40 cycles of: 30 s at 95°C, 30 s at
56°C, 1 min at 72°C; final 10 min at 72°C
28S rDNA3
f: CCCSCGTAAYTTAAGCATAT
r: CCTTGGTCCGTGTTTCAAGAC
Initial 2 min at 94°C; 35 cycles of: 30 s at 94°C, 30 s at
50°C, 1 min at 72°C; final 5 min at 72°C
H34
f: ATGGCTCGTACCAAGCAGACVGC
r: ATATCCTTRGGCATRGTGAC
Initial 5 min at 94°C; 35 cycles of: 30 s at 94°C, 30 s at
40°C, 1 min at 72°C; final 5 min at 72°C
PEPCK 4
f: GCAAGACCAACCTGGCCATGATGAC
r: CGGCYCTCCATGCTSAGCCARTG
Initial 3 min at 94°C; 35 cycles of: 30 s at 94°C, 30 s at
60°C, 1.5 min at 72°C; final 10 min at 72°C
Locus
16S
rDNA1
S1
JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 32, NO. 6, 2012
Table S2. Sympatric Orconectes rusticus, O. sanbornii, and putative hybrid crayfish from two sites on the Huron River with allozyme (Idh), mtDNA
(16s and COII), and morphology data (Field ID, rostral margin, and annulus ventralis for females). The Field ID is based on coloration (see Materials and
Methods). R = O. rusticus, S = O. sanbornii, PH = putative hybrid (mix of O. rusticus and O. sanbornii coloration), – = no band or did not assay, ) ( =
concave, | | = approximately parallel, / \ = wedge-shaped. For Idh, the O. rusticus allele was scored as 110 and the O. sanbornii allele as 94. ∗ Putative
hybrid ancestry.
Individual
MONROEVILLE
HL01
HL02
HL03
HL04
HL05
HL06
HL07
HL08
HL09
HL10
HL11
HL12∗
HL13
HL14
HL15
HL16∗
HL17∗
HL18
HR01
HR02
HR03
HR04∗
HR05
HR06
HR07
HR08
HR09
HR10
HR11
HR12∗
HR13∗
HR14
HR15
HR16
HR17
HR18
Field ID
Idh
16S
COII
Rostral margin
Annulus ventralis
R
S
PH
R
R
R
R
R
S
R
S
S
R
S
S
PH
R
R
R
R
R
S
R
R
R
R
R
R
R
PH
R
R
S
R
S
S
RR
SS
RR
RR
RR
RR
RR
RR
SS
–
SS
SS
–
SS
SS
RR
RR
RR
RR
RR
RR
RS
RR
RR
RR
RR
RR
–
RR
RS
RS
RR
SS
–
–
SS
R
S
R
–
–
R
R
R
S
R
S
R
R
–
–
S
S
R
R
R
R
S
R
R
R
R
R
R
R
R
R
R
S
R
S
S
R
S
R
R
R
R
R
R
S
R
S
R
R
S
S
S
R
R
–
R
R
S
R
R
R
R
R
R
–
R
R
R
S
R
S
S
)(
||
||
||
||
||
/\
||
/\
–
||
||
–
||
||
||
||
||
||
||
||
/\
||
||
||
||
/\
–
||
||
||
/\
||
–
–
/\
R
RR
RR
RR
RR
RR
RR
RR
RS
RR
RS
RR
RR
R
R
R
R
R
R
R
R
R
R/S
R
S
R
R
R
R
R
R
R
R
R
R
R
R
||
||
||
||
||
||
/\
||
||
||
||
||
LOVER’S LANE BRIDGE
SLL01
R
SLL02
R
SLL03
R
SLL04
R
SLL05
R
SLL06
R
SLL07
R
SLL08∗
R
SLL09
R
SLL10∗
R
SLL11
R
R
SLL12∗
R
S
S
R
R
S
R
R
R
S
S
R
R
R
S2
ZUBER ET AL.: HYBRIDIZATION BETWEEN SPECIES OF ORCONECTES
Table S3. Mean (s.e.) and range of gonopod and body measurements taken on allopatric Orconectes rusticus and O. sanbornii. Measurements in mm except
number of carpus spines. L = length, W = width. Numbers in front of character name refer to Fig. 3. A difference between the species means was tested
with one-way ANOVA.
Character
Body measurements
1. Carapace L
2. Areola L
3. Areola W
4. Carapace W
5. Acumen L
6. Rostrum W
Chela measurements
7. Dactyl L
8. Chela L
9. Palm W
10. Palm L
11. Chela gap W
12. No. carpus spines
Gonopod measurements
13. Total L
14. Central projection L
15. Mesial projection L
O. rusticus
O. sanbornii
F -ratio
P -value
Mean (s.e.)
Min-Max
Mean (s.e.)
Min-Max
28.09 (1.16)
9.69 (0.44)
1.45 (0.08)
13.99 (0.64)
2.43 (0.11)
2.74 (0.12)
16.07-41.47
5.45-15.51
0.72-2.42
7.67-21.55
1.53-3.44
1.68-4.30
24.19 (1.16)
8.07 (0.44)
1.42 (0.08)
11.47 (0.64)
2.19 (0.11)
2.30 (0.12)
16.30-34.26
5.45-11.63
1.00-1.92
7.39-16.07
0.96-3.06
1.49-3.28
F1,40
F1,40
F1,40
F1,40
F1,39
F1,40
= 5.64
= 6.74
= 0.05
= 7.70
= 2.33
= 6.88
0.023
0.013
0.832
0.008
0.135
0.012
13.03 (0.87)
21.15 (1.33)
8.40 (0.57)
8.07 (0.57)
1.00 (0.13)
1.33 (0.11)
5.97-21.26
9.61-34.35
3.22-14.00
3.96-12.98
0.23-2.48
1-2
10.06 (0.94)
17.82 (1.44)
7.08 (0.61)
7.42 (0.62)
0.74 (0.13)
1.39 (0.12)
5.12-17.42
9.33-29.36
3.95-11.73
4.46-11.54
0.21-1.27
1-2
F1,37
F1,37
F1,37
F1,37
F1,37
F1,37
= 5.35
= 2.88
= 2.52
= 0.60
= 2.05
= 0.12
0.026
0.098
0.121
0.442
0.161
0.727
10.52 (0.41)
3.35 (0.28)
2.78 (0.30)
7.42-12.82
1.39-5.03
1.10-4.99
8.11 (0.37)
0.68 (0.25)
0.60 (0.27)
6.54-10.51
0.37-0.97
0.35-0.84
F1,18 = 18.89
F1,18 = 49.53
F1,18 = 29.55
0.0004
<0.0001
<0.0001