Journal of Fish Biology (2008) 72, 418–434
doi:10.1111/j.1095-8649.2007.01732.x, available online at http://www.blackwell-synergy.com
History repeated: recent and historical mitochondrial
introgression between the current darter Etheostoma
uniporum and rainbow darter Etheostoma caeruleum
(Teleostei: Percidae)
J. M. R AY *, N. J. L ANG †, R. M. W OOD
AND
R. L. M AYDEN
Department of Biology, Saint Louis University, 3507 Laclede Avenue,
St Louis, MO 63103, U.S.A.
(Received 6 July 2007, Accepted 10 October 2007)
Incongruence between recognized taxonomy and phylogenetic relationships between two
species from a diverse clade (Percidae: Etheostomatinae) of stream fishes was found in
a mitochondrial (mt) DNA gene tree. Two darters in subgenus Oligocephalus, Etheostoma
uniporum current darter and Etheostoma caeruleum rainbow darter were sampled throughout
their sympatric distribution in the Ozark Highlands of the central United States. Sequences
from cytochrome (cyt) b and the first intron of the nuclear marker S7 were analysed separately
using maximum parsimony and Bayesian methods. Cyt b recovered both species as polyphyletic; E. uniporum haplotypes were interspersed within E. caeruleum. However, both species
were monophyletic and non-sister taxa based on S7. The cyt b gene tree pattern is caused by
introgressive hybridization resulting in the mtDNA replacement of E. uniporum haplotypes by
those of E. caeruleum. Some E. uniporum haplotypes are shared with geographically proximate
E. caeruleum, and this is consistent with recent hybridization, while other E. uniporum
haplotypes indicate historical sorting of introgressed lineages. The mechanism of introgression
is likely asymmetric sneaking behaviour by male E. uniporum, a mating tactic observed in related
species. MtDNA replacement may have occurred in E. uniporum due to drift fixation in
a historically small female effective population. Additional evidence for darter hybridization
will likely be discovered in future molecular genetic surveys of the nearly 200 species in eastern
# 2008 The Authors
North America.
Journal compilation # 2008 The Fisheries Society of the British Isles
Key words: darters; Etheostoma; hybridization; introgression; mitochondrial DNA replacement;
polyphyly.
INTRODUCTION
As the size and taxonomic breadth of molecular datasets grow, species level
polyphyly is an increasingly encountered pattern in mitochondrial (mt) DNA
gene trees. Distinguishing among alternative explanations for polyphyly can
*Author to whom correspondence should be addressed at present address: Department of Biological
Sciences, The University of Tennessee at Martin, 574 University Street, Martin, TN 38238, U.S.A.
Tel.: þ731 881 7181; fax: þ731 881 7187; email: [email protected]
†Present address: Department of Zoology, Oklahoma State University, 430 Life Sciences West,
Stillwater, OK 74078, U.S.A.
418
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#
INTROGRESSION IN ETHEOSTOMA DARTERS
419
be difficult since processes such as incomplete lineage sorting and hybridization
may produce nearly identical mtDNA patterns in closely related species (Funk
& Omland, 2003). Examining the geographical distribution of haplotypes may
be useful in detecting hybridization since the clearest signature of hybridization
is the sharing of haplotypes between sympatric species (Hare & Avise, 1998;
Masta et al., 2002). However, if introgression has occurred and haplotypes
have sorted over time (lineage sorting), this phenomenon will be difficult to
detect with mtDNA data alone (Moore, 1995; Funk & Omland, 2003). Comparisons with morphology or nuclear data may provide additional insights
on mtDNA gene tree polyphyly (Zink, 1994; McMillan & Palumbi, 1995;
Redenbach & Taylor, 2002). Since hybridization is not always detectable via
morphological examination (Rhymer & Simberloff, 1996; Sang & Zhong,
2000), molecular data may provide insights that might otherwise go undetected
(Avise, 2004).
Hybridizations between vertebrate species are most common between freshwater fishes with external fertilization and have been documented in a variety
of ecological settings (Hubbs, 1955; Schwartz, 1972). Hybridization often follows human-mediated introductions of non-native species and can reduce or
eliminate the genetic integrity of endemics (Allendorf & Leary, 1988; Echelle
& Connor, 1989). Hybridization may also occur naturally in altered environments (e.g. glaciated regions) or in areas that are seemingly undisturbed. In
areas affected by glaciation, introgression between currently allopatric species
may be due to mitochondrial replacement (Bernatchez et al., 1995; Wilson &
Bernatchez, 1998; Gerber et al., 2001; Lu et al., 2001; Redenbach & Taylor,
2002; Gum et al., 2005). Molecular studies of fishes have also documented
recent hybridization between sympatric species in a variety of other situations
(Avise, 2004).
Streams of eastern North America support one of the most diverse temperate
fish faunas in the world. Darters (Percidae: Etheostomatinae) represent approximately one-fifth of this diversity with 173 described species in 2000 (Page,
2000; Warren et al., 2000). Darters exhibit a range of behavioural and lifehistory adaptations, and although some are widespread and abundant, others are
rare and endemic to single rivers (Page, 1983). The relationships among many
darter species and subgenera are not well resolved, but hypothesized sister taxa
are frequently allopatric (Ceas & Page, 1997; Page et al., 2003). Overlapping
ranges of many non-sister species, however, provide the potential for interspecific hybridization. Surprisingly, darter hybridization in nature has not been
widely investigated with molecular methods (Scribner et al., 2000), despite
extensive laboratory studies of this phenomenon. Laboratory-produced hybrids
are often viable (Hubbs & Strawn, 1957; Hubbs, 1958, 1967; Linder, 1958),
even among species pairs exhibiting sexual isolation (Mendelson, 2003b). Additionally, darters exhibiting intermediate morphological characteristics (presumably, F1 individuals) have been found in nature (Hubbs & Laritz, 1961; Page,
1976; Mayden & Burr, 1980; Hubbs et al., 1988).
The current darter Etheostoma uniporum Distler and the rainbow darter
Etheostoma caeruleum Storer are both members of subgenus Oligocephalus
(Bailey & Etnier, 1988). Within the subgenus, E. uniporum is a member of
the Etheostoma spectabile (Agassiz) species group and is endemic to portions
# 2008 The Authors
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420
J. M. RAY ET AL.
of the Black River system (White River drainage–Mississippi River basin),
including the Current and Spring rivers in the Ozark Highlands of the central
United States (Distler, 1968; Ceas & Page, 1997; Fig. 1). Other members of the
E. spectabile species group occur in adjacent streams, and the distribution of
the entire E. spectabile species group largely overlaps with E. caeruleum in
the eastern United States. Etheostoma caeruleum is distributed in much of the
eastern North America and occurs in tributaries to the Great Lakes, Hudson Bay,
Mississippi and Potomac rivers [Page, 1983; Fig. 1 (inset)]. Both E. uniporum and
E. caeruleum are widespread in upland streams of the Black River system and
were present in 19 and 51% of 461 total collections, respectively, from this system (Missouri Department of Conservation, unpubl. data). These species may be
collected at the same locality, although E. uniporum more often occurs in headwater streams, while E. caeruleum is found in larger, permanent stream reaches
(Pflieger, 1997; pers. obs.). Populations of both species in this system are fragmented by intervening lowland and big river habitats (Pflieger, 1997). These
species overlap in several meristic counts, but are diagnosable by pigmentation
patterns and infraorbital canal characteristics (complete in E. caeruleum and
incomplete in E. uniporum), and are unambiguously identifiable (Page, 1983;
Ceas & Page, 1997; Pflieger, 1997).
While no comparisons of reproductive habits have been made between
E. uniporum and E. caeruleum, Winn (1958) found no difference in migration,
FIG. 1. Map showing sample localities (1–35) for Etheostoma species from streams in the Ozark Highlands
of the central United States. The shaded area represents the distribution of Etheostoma uniporum.
Sample localities correspond to Fig. 2 and Table I. Inset: Distribution of Etheostoma caeruleum
(grey) including area of sympatry with Etheostoma uniporum (
). , Etheostoma caeruleum;
, Etheostoma uniporum; , Etheostoma caeruleum and E. uniporum and , Etheostoma spectabile;
R., River.
Journal compilation
#
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2008 The Fisheries Society of the British Isles, Journal of Fish Biology 2008, 72, 418–434
INTROGRESSION IN ETHEOSTOMA DARTERS
421
territorial or spawning behaviour between E. caeruleum and E. spectabile. However, E. caeruleum and E. spectabile may segregate by water depth, velocity and
substrate (Winn, 1958; Page, 1983, 2000). Breeding males of both species
actively defend shifting territories in riffles against conspecifics and spawn when
a female enters their territory and buries her ventral half in the substrate. Eggs
are externally fertilized and left unguarded in the substrate (Winn, 1958).
Hybridization in nature has been reported between E. caeruleum and E. spectabile (Distler, 1968). Using allozymes, Martin & Richmond (1973) found that
presumed E. caeruleum E. spectabile hybrids did not have genotypes consistent with a hybrid origin and suggested that F1 hybrids must be rare in nature.
Spawning occurred in laboratory experiments, in which F1 hybrids were
produced in tanks containing only reciprocal sexes of E. caeruleum and E. spectabile (Distler, 1968). Hubbs (1967) found no differences in survival of F1 offspring between crosses of E. caeruleum and E. spectabile v. conspecific crosses.
From both laboratory and field observations, Distler (1968) hypothesized that
the evolution of the E. spectabile species group (although not specifically of
E. uniporum) was influenced by hybridization with E. caeruleum or Etheostoma
whipplei (Girard).
In molecular systematic studies of the subgenus Oligocephalus, a discrete
inconsistency was discovered between recognized taxonomy and relationships
suggested by mtDNA gene trees (Lang & Mayden, 2007). This inconsistency
involved the recovery of E. uniporum (a recognized E. spectabile species group
member) with E. caeruleum. In this study, an in-depth investigation of potential
hybridization and introgression between E. uniporum and E. caeruleum from
throughout their sympatric range is reported. Sequences of the mitochondrial
cytochrome (cyt) b gene and first intron of the nuclear ribosomal protein S7
were used. The objectives of this study were to characterize the cyt b and S7
tree patterns of E. uniporum and E. caeruleum to test whether introgression
explains the incongruence between taxonomy and gene-based relationships
observed by Lang & Mayden (2007).
MATERIALS AND METHODS
STUDY SPECIES
Etheostoma uniporum and E. caeruleum were comprehensively sampled throughout
their sympatric distribution (Fig. 1 and Table I). Within the Black River system,
E. uniporum and E. caeruleum shared six collection localities. Etheostoma caeruleum
was recorded separately at 12 sites, and E. uniporum at seven sites. Etheostoma caeruleum were sampled at 13 additional localities outside the range of E. uniporum. Specimens of focal taxa were independently identified by two researchers using standard
meristic counts, pigmentation pattern, characteristics and completeness of infraorbital
canals (Page, 1983; Ceas & Page, 1997; Pflieger, 1997).
Allopatric members of the E. spectabile species group were sampled from four adjacent Ozark streams systems, including the upper Black River proper (Etheostoma
burri Ceas & Page), Strawberry River (Etheostoma fragi Distler), White River proper
(E. spectabile) and St Francis River (E. spectabile). Other species in subgenus Oligocephalus were sampled from outside the study area and included Etheostoma collettei
Birdsong & Knapp, Etheostoma radiosum (Hubbs & Black) and E. whipplei. Percina
caprodes (Rafinesque) was designated as the outgroup taxon. Specimen vouchers were
# 2008 The Authors
Journal compilation # 2008 The Fisheries Society of the British Isles, Journal of Fish Biology 2008, 72, 418–434
Etheostoma caeruleum
Meramec River (1)
St Francis River (2)
Upper Black River (4)
Upper Black River (5)
Cane Creek (7)
Barren Fork (10)
Jacks Fork (11)
Jacks Fork (12)
Little Shawnee Creek (13)
Rocky Creek (14)
Current River (15)
Current River (16)
Current River (17)
Little Black River (19)
Fourche Creek (20)
Eleven Point River (22)
Eleven Point River (23)
Frederick Creek (25)
Spring River (26)
S. Fork Spring River (27)
Spring River (28)
Spring River (29)
Strawberry River (33)
White River (34)
Stream (locality)
024, 025
020, 021
069
070
164
072
160
163
184
169, 170
161
185, 186
172, 174
165, 171
071, 173
157
168
166
167
162
006, 031
100, 101
098, 099
028, 029
Specimens
D
D
D
D
D
D
D
D
F
F
D
F
D, F
D
D
B
B
B
B
B
B
B
C
H
cyt b
Clades
Journal compilation
#
J
J
J
J
J
J
J
J
J
J
J
S7
STL
STL
STL
STL
STL
STL
STL
STL
STL
STL
STL
STL
STL
STL
STL
STL
STL
STL
STL
STL
STL
STL
STL
STL
8.12
1026.01
173.01
6.11
1060.01
1034.01
246.01
1059.01
1068.02
1065.01
1057.01
32.17
832.11
1061.01
79.04, 1066.01
247.01
1064.01
1062.01
1063.01
1058.01
1017.01
382.01
384.04
78.11
Museum accession
DQ465090–91/EU046652
DQ465086–87/EU046649
DQ465129/EU046655
DQ465130
DQ465210
DQ465132/EU046657
DQ465206
DQ465209
EU046707/EU046667
EU046704–05/EU046664–65
DQ465207
EU046702–03/EU046668–69
EU046706, DQ465217/EU046666
DQ465211, DQ465215
DQ465131, DQ465216/EU046656
DQ465203
DQ465214
DQ465212
DQ465213
DQ465208
DQ465076, DQ465096
DQ465156–57/EU046659
DQ465154–55/EU046658
DQ465094–95/EU046653
GenBank accession
number (cyt b/S7)
TABLE I. Specimen information for darters sampled in this study. Streams and localities correspond to those in Fig. 1; specimens and clades
correspond to those in Fig. 2
422
J. M. RAY ET AL.
# 2008 The Authors
2008 The Fisheries Society of the British Isles, Journal of Fish Biology 2008, 72, 418–434
Spring River (30)
Frederick Creek (25)
Spring River (26)
435
005, 009
010, 016, 098
001, 002
179
007
018, 078
008
013
185
006, 011
Barren Fork (10)
Jacks Fork (11)
Little Shawnee Creek (13)
Little Black River (18)
Fourche Creek (20)
Tennessee Creek (21)
Eleven Point River (22)
Hurricane Creek (24)
058
179
017
045
132
134
153
155
51
014, 617
017, 175
057,
033,
016,
037,
131,
133,
152,
154,
Specimens
Little Red River (35)
Upper White River
Gasconade River
Otter Creek
Sandusky River
Tennessee River
Green River
Osage River
Total
E. uniporum
Cane Creek (8)
Pigeon Creek (9)
Stream (locality)
A
A
A, B
A
D
A
G
D
D
A
A, B
E
D
H
H
N. Ozarks
Upper Mississippi
Eastern U.S.A.
Eastern U.S.A.
Eastern U.S.A.
N. Ozarks
cyt b
Clades
I
I
I
I
I
I
I
I
I
I
I
J
J
J
J
J
J
J
J
S7
1034.02
246.02
1068.01
61.04
1066.02
833.04
247.02
1067.01
STL 383.04
STL 1062.01
STL 180.09, 1063.02
STL
STL
STL
STL
STL
STL
STL
STL
STL 831.04
STL 243.01
STL 1019.01
STL 1013.01
STL 64.04
JFBM 31430
STL 1051.01
STL 1052.01
STL 1020.01, 1024.01
STL 1055.01
Museum accession
TABLE I. Continued
EU046689, EU046700/EU046640
EU046692, EU046696/EU046643,
EU046646
EU046678–79/EU046632
EU046697/EU046647
EU046682/EU046634
EU046693–94/EU046644
EU046683/EU046635
EU046688/EU046639
EU046698
EU046681, EU046686/EU046633,
EU046638
EU046680, EU046684/EU046636
EU046685, EU046691, EU046695/
EU046637, EU046642 EU046645
EU046699
DQ465118–19/EU046654
DQ465098, DQ465222/EU046650
DQ465083–84/EU046648
DQ465101, DQ465109/EU046651
DQ465181–82/EU046660
DQ465183–84/EU046661
DQ465198–99/EU046662
DQ465200–01/EU046663
GenBank accession
number (cyt b/S7)
INTROGRESSION IN ETHEOSTOMA DARTERS
423
# 2008 The Authors
Journal compilation # 2008 The Fisheries Society of the British Isles, Journal of Fish Biology 2008, 72, 418–434
Journal compilation
#
STL 1069.01
STL 183.01
STL 1070.01
KU 29327
STL 174.01
STL 1069.02
STL 195.06
1
2
1
1
2
1
STL 835.02
Museum accession
2
I
S7
UAIC 11396.04
A
cyt b
1
015, 618
23
Specimens
EU046670/EU046624
DQ465069, EU046674/EU046626
DQ465068/EU046628
EU046673/EU046630
DQ465070, EU046675/EU046625
EU046672/EU046631
EU046676–77/EU046627
EU046671/EU046629
EU046690, EU046701/EU046641
GenBank accession
number (cyt b/S7)
JFBM, the Bell Museum of Natural History; KU, the University of Kansas Museum of Natural History; STL, Saint Louis University; UAIC, the University
of Alabama Ichthyological Collection.
Flat Creek (31)
Total
E. burri
Upper Black River (6)
E. collettei
Red River drainage
E. fragi
Strawberry River (32)
E. radiosum
Ouachita River drainage
E. spectabile
White River drainage
St Francis River (3)
E. whipplei
Red River drainage
Percina caprodes
Arkansas River drainage
Stream (locality)
Clades
TABLE I. Continued
424
J. M. RAY ET AL.
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INTROGRESSION IN ETHEOSTOMA DARTERS
425
deposited at Saint Louis University, the University of Kansas Museum of Natural
History, the University of Alabama Ichthyological Collection and the Bell Museum
of Natural History, and sequences were deposited in GenBank (Table I).
MOLECULAR METHODS
Genomic DNA was extracted from specimens with a QIAGEN DNeasy Tissue Kit
(QIAGEN, Inc., Valencia, CA, U.S.A.). Cyt b was polymerase chain reaction (PCR)
amplified with flanking primers L14724 and H15915 (Schmidt & Gold, 1993). PCR reactions included a total volume of 50 ml. Each reaction was conducted with 30 cycles of
96° C, 1 min; 48° C, 1 min and 72° C, 2 min. The S7 marker was PCR amplified with
flanking primers S7RPEX1F and S7RPEX2R (Chow & Hazama, 1998). PCR reactions
consisted of a total volume of 50 ml. Each reaction was conducted with 30 cycles of
96° C, 1 min; 53° C, 1 min; 72° C, 2 min. PCR products were purified with a QIAGEN’s
QIAquick Gel Extraction Kit (QIAGEN, Inc.). The light strand of cyt b was sequenced
for E. caeruleum in two segments with primer L14724 and an internal forward primer,
LECO1 (Ray et al., 2006). Other taxa were sequenced with the PCR primers for both
cyt b and S7. Sequencing reactions used a Beckman Coulter DTCS Kit (Beckman
Coulter, Inc., Fullerton, CA, U.S.A.) at a total volume of 10 ml for 30 cycles at 96° C,
20 s; 50° C (53° C for S7), 20 s and 60° C, 4 min. Sequences were recorded with a Beckman Coulter CEQ 8000 DNA Analysis System aligned in CLUSTALX 1.8 (Thompson
et al., 1997) and edited in MACCLADE 4.0 (Maddison & Maddison, 2000).
All individuals were sequenced for cyt b; a sub-set of E. uniporum and E. caeruleum
were sequenced for S7 based on the phylogenetic position of their cyt b haplotypes. All
cyt b clades were represented by one or more individual in the S7 dataset for each
species, and samples not recovered with conspecifics from the same locality in analysis
of cyt b were also sequenced for S7.
Heterozygosity in S7 sequences was detected with CEQ 8000 under the default heterozygote analysis parameters (Dobbs & Gee, 2002), and all individual heterozygous nucleotide sites were verified by eye. Heterozygous sites appeared in both E. uniporum and
E. caeruleum, although only eight of 52 positions were shared among 13 individuals. A
sub-set of individuals was cloned and sequenced for each S7 allele, but analyses of the
dataset containing individual alleles recovered clades identical to those containing only
heterozygous bases. Therefore, only the results of the analyses containing heterozygous
sites are presented (Domingues et al., 2005).
P H Y L O G E N E T I C A N A L Y SE S A N D G E N E T I C V A R I A T I O N
All phylogenetic analyses were performed separately on the cyt b and S7 datasets.
Maximum parsimony analysis was run in PAUP* 4.0b10 (Swofford, 2003) by
heuristic search with 1000 replicates of random addition sequence and tree-bisectionreconnection (TBR) branch swapping. Nodal support was calculated with 1000 bootstrap (Felsenstein, 1985) replicates in a full heuristic search with TBR branch swapping.
MRMODELTEST 2.2 (Nylander, 2004) was used to select the best-fit model of DNA
sequence evolution for each of the three codon positions in cyt b sequences; these models were used in subsequent Bayesian analysis. Bayesian analysis was conducted in
MRBAYES 3.1 (Huelsenbeck & Ronquist, 2001; Ronquist & Huelsenbeck, 2003). Four
independent runs were each conducted for six million generations with a variable rate
parameter, with other parameters set on default. Trees were sampled every 1000 generations, with posterior probability and branch length values saved to the output file. The
burn-in period was determined by graphing the log-likelihood scores against generation
time, and trees generated before the burn-in were discarded. Each Bayesian run was
examined to ensure that independent runs produced comparable topologies and posterior probabilities. Nodal support was calculated using mean posterior probability from
each run after discarding burn-in trees. From these analyses, a 50% majority-rule consensus phylogram was generated for cyt b and S7.
# 2008 The Authors
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426
J. M. RAY ET AL.
Per cent nucleotide sequence divergence between individuals was calculated using
uncorrected (p) distances in MEGA 3.0 (Kumar et al., 2004). Clades for the cyt b and S7
datasets were determined based on monophyletic groups recovered in all analyses and
geographic distribution of samples. Divergences between clades were corrected for
within-group variation in MEGA.
RESULTS
M I T O C HO N D R I A L D A T A
Complete cyt b sequences (1140 bp) were generated for 85 individuals of
Etheostoma and Percina: 51 E. caeruleum, 23 E. uniporum, two each of E. collettei,
E. radiosum, E. spectabile, E. whipplei, and one each of E. burri, E. fragi and
P. caprodes with 60 haplotypes recovered (Table I). A total of 750 (66%) nucleotide sites were constant, and 339 (30%) were parsimony informative; 90% of
parsimony-informative characters occurred in the third codon position. Maximum parsimony analysis produced 289 equally parsimonious trees with 1102
steps. The best-fit model of sequence evolution for each codon position was
first: HKYþ I þ G, second: GTRþ I, third: GTRþ I þ G based on the Akaike
information criteria (AIC). Bayesian analyses reached stationarity after
c. 100 000 generations; trees from the first 150 000 generations were discarded
as burn-in, leaving 5850 trees for phylogeny estimation. Both Bayesian and
parsimony analyses recovered the same monophyletic groups [clades A–H;
Fig. 2(a)], which were 14–62% divergent.
Phylogenetic analyses recovered both species as polyphyletic; E. uniporum
haplotypes were completely interspersed within E. caeruleum [Fig. 2(a)]. Seven
individuals of E. uniporum were recovered in clades B and D and were most
similar to E. caeruleum from the same stream system (0–088% cyt b sequence
divergence). The remaining 16 E. uniporum occurred at multiple positions
within the E. caeruleum branches (clades A, E and G) and had sequences
17–35% divergent from E. caeruleum from the Black River system. A single
E. caeruleum clade (clade F) from the Current River was basal to other
E. caeruleum from streams within the Black River system (29–35% divergent).
Etheostoma caeruleum from the upper White and Little Red rivers (clade H)
were sister to all other E. caeruleum plus E. uniporum. Allopatric members of
the E. spectabile species group were not recovered with E. uniporum and had
large corrected cyt b sequence divergence values from this species (13–16%).
NUCLEAR DATA
S7 sequences were generated for 46 individuals: 22 E. caeruleum, 16 E. uniporum, four other E. spectabile species group members, three additional species
of Oligocephalus and P. caprodes. Of 515 nucleotide sites, 398 (77%) were
invariant and 72 (14%) were parsimony informative. Heuristic searches with
unweighted characters recovered 4003 equally parsimonious trees with 199
steps. The best-fit model of sequence evolution was HKYþ I þ G based on
the AIC. The four Bayesian analyses reached stationarity after c. 50 000 generations; trees from the first 100 000 generations were discarded as burn-in,
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427
INTROGRESSION IN ETHEOSTOMA DARTERS
100
100
100
100
100
81
100
79
0·01 substitutions/site
CAP110* Percina caprodes
SPE141* Etheostoma spectabile
BUR006* [6] E. burri
SPE077* [3] E. spectabile
FRA100* [32] E. fragi
100
EW01, 02* E. whipplei
RAD01*, 02 E. radiosum
100
100
BBC01 E. collettei
U002 [10]
BBC02*
100
U001* [10] U007* [13]
100/100
U005 [25] U011* [24] CLADE A
100
widespread
U009* [25] U435 [30]
78
100
U010* U016* [26]
U185
[22]
100
U015* U618 [31]
U006* [24]
U098* [26]
EC006 [28]
CLADE B
100 EC031 [28] EC157 [22] Spring R.
100 98 EC100* [29] EC166 [25] system
EC101 [29] EC168 [23]
84
EC162 [27]
EC167 [26]
CLADE C
EC098* EC099 [33]
Strawberry R.
U008* [20] U013* [21]
100
EC020 [2]
100
100
100
EC021 [2]
84
97
EC024* [1]
EC025 [1]
CLADE D
EC069* [4] EC164 [7]
Current,
EC070 [5]
upper Black,
St. Francis,
EC071* [20]
Meramec
EC160 [11]
rivers
EC165 EC171 [19]
EC173 [20] U 175* [9]
100
87 U017* [9]
EC161 [15]
U179* [11] EC174 [17]
EC072* [10]
EC163 [12]
CLADE E
100
U014* U617 [8]
Cane Cr.
100
EC170* [14]
52
100
EC169* [14]
77
98
CLADE F
EC172* [17]
100
80
Current R.
EC186* [16]
97
EC184* [13]
EC185* [16]
CLADE G
U018 [18]
100
Little Black R.
100
U078* [18]
EC016
100
Northern
Ozarks
100
82
EC017*
56
100 EC037* EC045 Upper Mississippi R.
EC154*
100
Northern Ozarks
EC155
100
85
100
100
96
100
EC131* EC132
EC152 EC153*
EC133* EC134
EC028 [34]
EC029* [34]
EC033* EC179
EC057* EC058 [35]
Eastern
US streams
CLADE H
White, Little
Red rivers
FIG. 2. (a) Bayesian 50% majority-rule tree from 5850 trees retained after burn-in period based on
cytochrome b sequences. Asterisks indicate that the specimen was also sequenced for S7. (b) Bayesian
50% majority-rule tree from 5900 trees retained after burn-in period based on S7 sequences. EC,
Etheostoma caeruleum and U, Etheostoma uniporum. Values above branches indicate bootstrap support
from parsimony searches and values below branches indicate posterior probabilities. Numbers in
brackets that follow sample codes represent localities in Fig. 1 and Table I.
leaving 5900 trees for phylogeny estimation. Bayesian analysis [Fig. 2(b)] was
consistent with parsimony analysis (not shown) and had greater resolution
within each focal species (clades I–J). In contrast to the cyt b trees, both E. uniporum
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428
J. M. RAY ET AL.
and E. caeruleum were monophyletic and non-sister taxa (2–3% divergent) in the
S7 tree, as E. uniporum was recovered with other E. spectabile species group members from the Ozark Highlands. Overall, greater interspecific sequence divergences appeared for cyt b (186–0%) than for S7 (99–0%). Intraspecific divergence
of E. uniporum and E. caeruleum was also greater for cyt b (54–0 and 75–0%,
respectively) than for S7 (23–0 and 44–0%, respectively), indicating a greater
substitution rate in cyt b than in S7.
DISCUSSION
While interspecific hybridization, inadequate phylogenetic information or
incomplete lineage sorting may each result in polyphyly (Funk & Omland,
2003), careful consideration of the data suggests that hybridization is the most
likely cause for mtDNA polyphyly and has resulted in the replacement of
E. uniporum cyt b haplotypes by those of E. caeruleum. Neither incomplete
lineage sorting nor inadequate phylogenetic information is a plausible alternative explanation for the observed pattern. The expectation for incomplete lineage sorting is a basal position of haplotypes, not their interspersion (Funk &
Omland, 2003). Further, incomplete lineage sorting of S7 sequences would also
be expected due to a lower substitution rate, but both species are monophyletic
and non-sister taxa based on nuclear data. The phylogenetic pattern of cyt
b is also not caused by inadequate phylogenetic information as this marker
differentiated E. uniporum and E. caeruleum from all other species sampled
and has proven useful in molecular systematic and phylogeographic studies
of darters (Kinziger et al., 2001; Switzer & Wood, 2002; Ray et al., 2006).
Hybridization, resulting in mtDNA introgression, is interpreted to have
caused the observed pattern for two primary reasons. First, all 23 E. uniporum
cyt b haplotypes were recovered within E. caeruleum and had sequence divergences from E. caeruleum (0–35%) within the range of intraspecific variation
in E. caeruleum clades (maximum corrected value 62%). Second, cyt b haplotypes from the Black River system were shared or nearly identical between
E. uniporum and E. caeruleum, and provide the clearest signature of hybridization. Two of six sites where both species were sampled showed signs of recent
hybridization. Seven E. uniporum were classified as recent hybrids based on
their position in clades B and D and their similarity in cyt b sequences
(0–088% divergence) to E. caeruleum from the same clade and stream. As lineages are more likely to be phylogenetically basal as the time since last gene
flow increases (Moore, 1995; Funk & Omland, 2003), the other 16 E. uniporum
possess introgressed haplotypes that have sorted over time, as evidenced
by their phylogenetic position and larger cyt b sequence divergence values
(20–35%) relative to E. caeruleum from the Black River system. The interspersion of E. uniporum haplotypes within E. caeruleum may have been caused both
by episodes of historical and by more recent introgressive hybridization, and
the E. uniporum mitochondrial genome has been completely eliminated and replaced by an E. caeruleum mitochondrial genome. Analyses further suggest that
mitochondrial introgression is not strictly unidirectional as six E. caeruleum
(clade F) were not recovered with conspecifics from the Current River system
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INTROGRESSION IN ETHEOSTOMA DARTERS
429
CAP110 Percina caprodes
RAD01 Etheostoma radiosum
EW02 E. whipplei
EC017
EC154
EC170 [14]
EC172 [17]
EC020 [2]
EC100 [29]
EC131
EC133
EC024 [1]
EC069 [4]
CLADE I
EC029 [34]
EC098 [33] E. caeruleum
EC184 [13]
EC057 [35]
EC185 [16]
EC071 [20]
EC033
EC072 [10]
EC169 [14]
EC186 [16]
EC037
EC153
BBC02 E. collettei
SPE141 E. spectabile
BUR006 [6] E. burri
SPE077 [3] E. spectabile
FRA100 [32] E. fragi
U001 [10]
U017 [9]
U175 [9]
U007 [13]
U179 [11]
U014 [8]
U016 [26]
U006 [24]
U010 [26]
U011 [24]
U098 [26]
U008 [20]
U013 [21]
U009 [25]
U015 [31]
U078 [18]
0·005 substitutions/site
CLADE J
E. uniporum
FIG. 2. Continued.
(clade D). Clade F may represent a mitochondrial ‘recapture’ caused by hybridization between E. caeruleum males and E. uniporum females.
The monophyly of E. uniporum and E. caeruleum based on S7 was concordant with the morphological identification of all individuals and established
taxonomy (Distler, 1968; Ceas & Page, 1997), and places E. uniporum with
other E. spectabile species group members. This marker clearly establishes
incongruence among mtDNA and nuclear sequence data and confirms interspecific hybridization as the mostly likely cause for this discordance.
The mechanism for the mitochondrial introgression between E. caeruleum
and E. uniporum may be due to interspecific male sneaking behaviour. In
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430
J. M. RAY ET AL.
egg-burying darters like E. uniporum and E. caeruleum, subdominant males may
position themselves next to the spawning pair and sneak fertilization or sneak
spawning while dominant males are engaged in antagonistic interactions with
other males (Fisher, 1990; Page, 2000; Fuller, 2003). Interspecific sneaking
has been observed in laboratory studies of taxa from the same subgenus,
E. radiosum and Etheostoma spectabile pulchellum (Girard) (Mendelson,
2003a), which hybridize extensively in the Blue River, Oklahoma (Distler,
1968; Branson & Campbell, 1969; Echelle et al., 1974). This interspecific
sneaking is strongly asymmetric as E. s. pulchellum males and E. radiosum
females spawned more frequently than E. radiosum males and E. s. pulchellum
females. Etheostoma s. pulchellum males achieved this by quickly entering
spawning territory and mating with E. radiosum females, while E. radiosum
males were actively guarding against conspecifics (Mendelson, 2003a). A
similar pattern of behavioural asymmetry involving interspecific matings that
occur more frequently between E. uniporum males and E. caeruleum females is
revealed by the data in this study, but has not been previously suggested
based on morphology or laboratory observations.
Why has a mtDNA replacement occurred in E. uniporum? One potential
explanation is drift fixation, which in a small population, could have been
rapid and significant (Avise & Saunders, 1984; Wilson & Bernatchez, 1998).
Etheostoma uniporum may have had historically small female effective population sizes that resulted in the complete introgression of E. caeruleum mtDNA
haplotypes. Periodic isolation in upland streams separated by lowland or big
river habitats, during the Pleistocene and recent (Fenneman, 1938; Fisk,
1944; Pflieger, 1971), may have contributed to differential sorting of introgressed E. uniporum lineages [clades A, E and G; Fig. 2(a)]. These clades fit
the expected pattern of a more basal phylogenetic position, a product of lineage sorting since last gene flow. Fortunately, E. uniporum has not sorted out of
the E. caeruleum cyt b phylogeny and still shows a geographic association of
haplotypes. Otherwise, it may have been extremely difficult to conclusively
identify hybridization as the cause of the gene tree pattern.
Although the sampling of representatives of the E. spectabile species group
does not indicate that mtDNA introgression has occurred outside the Black
River system, Sloss et al. (2004) presented a mtDNA phylogeny for Percidae
in which a putative E. burri (GenBank AY374262) was the sister taxa to a single
E. caeruleum (GenBank AY374263). Comparison of this E. burri sequence to
two sequences of sympatric E. caeruleum from the present study (EC069 and
EC070) showed that these individuals were virtually identical (01–02%
sequence divergence). It is concluded that the E. burri specimen possesses
a mtDNA haplotype of E. caeruleum as previously suggested (Lang & Mayden,
2007). Interestingly, no E. caeruleum from outside the Black River system possessed a mitochondrial haplotype of sympatric E. spectabile group members
(Ray et al., 2006), suggesting that the pattern is strongly asymmetric throughout the area of sympatry.
Interspecific hybridizations between darters have been known for some time,
but its potential influence on systematics of darters have not previously been
intensively examined with molecular data. This in-depth study of sympatric
E. uniporum and E. caeruleum provides insights into interactions between these
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INTROGRESSION IN ETHEOSTOMA DARTERS
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species that would not have otherwise been detected. Since mtDNA gene trees
may aid in taxonomy and the design of comparative ecological studies, the
importance of accurate phylogenies cannot be over stated. With nearly 200 species confined to the streams of eastern North America, many of which occur in
sympatry, it seems likely that additional evidence for darter hybridization and
introgression will be discovered with molecular data in the near future.
We thank R. Blanton, A. Kinziger, A. Simons and J. Switzer for assistance with the
collection of specimens. K. Conway, C. Dillman and K. Tang made helpful suggestions
on earlier versions of the manuscript. Procedures used in this study have been approved
by the Animal Care Committee of Saint Louis University (STL). Fishes were collected
under state permits issued to the authors and individuals listed above. Laboratory work
was supported by grants from the Beaumont Fund of STL.
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