J. Moll. Stud. (2002), 68, 385–391 © The Malacological Society of London 2002
Unionid bivalves are one of the most imperiled groups of ani-
mals in the world, with 70% of the recognized species in North
America considered extinct, endangered, threatened or of
special concern (Williams, Warren, Cummings, Harris & Neves,
1993; Neves, Bogan, Williams, Ahlstedt & Hartfield, 1997; Master,
Flack & Stein, 1998). One of the centres of greatest diversity
in North America is the Tennessee-Cumberland basin of the
southeastern United States (Parmalee & Bogan, 1998). Historic-
ally, 111 unionid taxa were found in the two river systems,
including 35 endemics (Starnes & Bogan, 1988). The genus
Epioblasmawas once widely distributed across the Tennessee and
Cumberland systems, but 15 species and four subspecies are
now presumed extinct due to habitat destruction or modifica-
tion (Williams et al., 1993; Neves et al., 1997). Currently consist-
ing of 20 species and eight subspecies (Turgeon, Quinn, Bogan,
Coan, Hochberg, Lyons, Mikkelsen, Neves, Roper, Rosenberg,
Roth, Scheltema, Thompson, Vecchione & Williams, 1998), the
genus is noted for the presence of often extreme sexual dimor-
phism in shell shape, which is filled by a fleshy, soft, mantle pad
apparently used as a fish-host attractant. Little is known about
the life histories and habitat requirements of the remaining
extant Epioblasmataxa, and all but one (E. triquetra) are federally
listed as endangered. Epioblasma triquetra is considered im-
periled, but not federally listed.
Many taxonomic questions remain unanswered due to the
uncertainty of whether shell variation represents a response
to environmental conditions or genetically-based diagnostic
characteristics (e.g. Williams & Mulvey, 1994). The vast majority
of currently recognized unionid species are based on interpreta-
tions of how to partition qualitative shell differences, although
recent studies have coupled genetic data along with shell char-
acteristics (Mulvey, Lydeard, Pyer, Hicks, Brim-Box, Williams &
Butler, 1997; Hoeh, Bogan, Cummings & Guttman, 1998; King,
Eackles, Gjetvaj & Hoeh, 1999; Lydeard, Minton & Williams,
2000; Hoeh, Bogan & Heard, 2001). Whether species are deemed
valid or not alters our current views of conservation status, dis-
tribution and general biodiversity assessment (Lydeard et al.,
2000). This, in turn, may influence the management or active
recovery plan for federally or state listed species (Lydeard &
We conducted a molecular genetic study on populations of
two federally endangered freshwater Epioblasma species, the
tan riffleshell, E. florentina walkeri (Wilson & Clark, 1914), and
oyster mussel, E. capsaeformis(Lea, 1834). Epioblasma capsaeformis
(Lea, 1834) was formerly found throughout the Tennessee and
Cumberland River systems in Virginia, Tennessee, northern
Alabama and Kentucky. Currently, it persists at extremely low
numbers in three locales: (1) the Nolichucky River, Tennessee
(extremely rare, only three specimens found since 1980); (2)
the Clinch River (now rare in Virginia, but still relatively com-
mon in Tennessee), and (3) the Duck River, Tennessee. It is
listed as federally endangered [United States Fisheries and
Wildlife Service (USFWS), 1997], but has no approved recovery
plan. Epioblasma f. walkeri (Wilson & Clark, 1914) is considered
the Tennessee basin, headwater form of E. f. florentina, which is
now presumed extinct (Parmalee & Bogan, 1998). Epioblasma
f. walkeri has also been reported historically throughout the
Cumberland drainage, including the Stones, Red and Harpeth
rivers (USFWS, 1984). Four extant populations of Epioblasma
f. walkeri are known: (1) Big South Fork, Cumberland River,
Tennessee and Kentucky (reproducing populations); (2) Indian
Creek (tributary to upper Clinch River, Virginia); (3) Hiwassee
River, Tennessee (extremely rare, one live female found in
CONSERVATION GENETICS OF TWO ENDANGERED UNIONID
BIVALVE SPECIES, EPIOBLASMA FLORENTINA WALKERI AND
E. CAPSAEFORMIS (UNIONIDAE: LAMPSILINI)
JENNIFER E. BUHAY, JEANNE M. SERB, C. RENÉE DEAN, QSHEQUILLA PARHAM
AND CHARLES LYDEARD
Department of Biological Sciences, Biodiversity and Systematics, University of Alabama, Box 870345, Tuscaloosa, Alabama 35487, USA
(Received 1 November 2001; accepted 7 May 2002)
The genus Epioblasma consists of 20 species and eight subspecies, of which 15 species and four sub-
species are presumed or probably extinct. Remaining taxa are considered endangered or threatened
largely due to habitat destruction or modification. We conducted a molecular genetic study on all
known extant populations of two federally endangered freshwater Epioblasma species: the tan riffle-
shell, E. florentina walkeri(Wilson & Clark, 1914), and oyster mussel, E. capsaeformis(Lea, 1834), to deter-
mine the extent of genetic variation within and among populations of the two species. Mitochondrial
DNA sequence data from two protein-coding genes (COI and ND1) failed to differentiate E. capsae-
formis from E. f. walkeri. Molecular variation within and between the two species does not exceed limits
observed within Epioblasma brevidens, although substantial genetic differences are observed among
E. brevidens, E. triquetra and E. capsaeformis ? E. florentina walkeri. Although data do not support the
recognition of the two taxa as separate phylogenetic species, they still warrant endangered status due to
the fact that few reproducing populations are known. Life history and population biology studies have
already been conducted on the single known extant reproducing population of nominal E. f. walkeri.
However, similar comparative studies should be conducted on the different mantle-pad morphs of
nominal E. capsaeformis populations to provide valuable data for more effective management of the
recovery of the phylogenetic species and to further test our hypothesis.
Correspondence: C. Lydeard; e-mail: firstname.lastname@example.org.
1999); and (4) Middle Fork Holston River, Virginia (extremely
rare, one live male found in 1997). One other relatively recent
population of E. f. walkeri from the Clinch River below Indian
Creek, was extirpated in 1999 from a chemical spill. Epioblasma
florentina curtisi is presumably a member of the same species,
but is distributed allopatrically in the Spring River system of
Arkansas and Missouri. It is listed federally as endangered and,
although a recovery plan has been developed for the taxon
(USFWS, 1986), it is now probably extinct (S. Ahlstedt, personal
The objective of this study was to determine the degree of
genetic differentiation among extant populations of Epioblasma
florentina walkeri and E. capsaeformis. We chose to focus our
efforts on mitochondrial DNA (mtDNA) sequence data because
mtDNA haplotypes have a smaller effective population size and,
therefore, coalesce four times more rapidly than nuclear mark-
ers (Moore, 1995; Wiens & Penkrot, 2002). In addition, we were
interested in testing the validity of the two recognized morpho-
species using the Phylogenetic Species Concept (sensu Mishler
& Theriot, 2000). A phylogenetic species is the smallest mono-
phyletic group deemed worthy of formal recognition, because
of the amount of support for monophyly and/or because of its
importance in biological processes operating on the lineage in
question (Mishler & Theriot, 2000).
MATERIALS AND METHODS
Specimens and vouchers
Permission was obtained to collect a limited number of
Epioblasma capsaeformis (n ? 4), E. f. walkeri (n ? 3), E. brevidens
(n?4) and E. triquetra (n?1) specimens or remove tissue clips
from the edge of the mantle without killing the animal from
extant populations, under the auspices of federal collecting
permits SA96-31 to Paul Hartfield and C. Lydeard, and SA00-14
to C. Lydeard. Given the rarity or endangered status of the
study taxa, sample sizes were extremely limited. Specimens of
Lampsilis siliquoidea, Villosa taeniata and V. villosa were included
as out-group lampsiline taxa (Lydeard, Mulvey & Davis, 1996;
Graf & ÓFoighil, 1999). Table 1 lists the taxa, localities and
tissue sources included in the present study. Voucher material
of Epioblasma specimens are deposited at the University of
Alabama Unionid Collection (UAUC).
DNA processing sequence procurement, alignment and
Whole genomic DNA was extracted from mantle tissue from
specimens using standard phenol/chloroform extraction
methods followed by ethanol precipitation as described in Roe
& Lydeard (1998). Initially, a 650-base pair (bp) region of the
cytochrome oxidase C subunit I (COI) gene was amplified using
primers LCO1490 and HCO2198 (Folmer, Hoeh, Black &
Vrijenhoek, 1994), and sequenced using standard amplification
parameters described in Roe & Lydeard (1998) and Lydeard et
al. (2000). Our pilot study examining one Epioblasma capsae-
formis, two E. f. walkeri, one E. brevidens, one E. triquetra and two
Lampsilis siliquoidea specimens revealed little variation within a
449-bp aligned data matrix of COI (see results below), therefore
a 700-bp region of the 5?-end of the first subunit of the NADH
dehydrogenase (ND1) gene was amplified using primers Leu-
uurF (5?-TGGCAGAAAAGTGCATCAGATTAAAGC-3?) and NIJ-
12073 (5?-TCGGAATTCTCCTTCTGCAAAGTC-3?). ND1 and
ND1-flanking primers were designed based on examination
of the complete mitochondrial genome sequence of Lampsilis
ornata (J. M. Serb & C. Lydeard, unpublished data). Leu-uurF
was designed from an alignment of Leu-tRNA, which included
sequence of Lampsilis ornata, Drosophila melanogaster and various
molluscan mt genomes available on GenBank (Benson, Karsch-
Mizrachi, Lipman, Ostell, Rapp & Wheeler, 2000). NIJ-12073
was modified from N1-N-12051 (Simon, Frati, Beckenbach,
Crespi, Liu & Flook, 1994). Reactions were amplified for 34
cycles at 94?C for 40 s, 50?C for 60 s and 68?C for 90 s. Sequencing
of all specimens was conducted using Big Dye (Perkin Elmer)
terminator cycle sequencing, and the products were visualized
using an ABI373 or ABI3100 automated sequencer.
Sequences were initially entered into the software program
XESEE (Ver. 3.0, Cabot & Beckenbach, 1989). A visual align-
ment was constructed by eye. The aligned data matrices are avail-
able electronically from the authors, and individual sequences
have been submitted to GenBank (see Table 1 for accession
codes). Parsimony analysis was performed by using version
4.0b5 of PAUP* (Swofford, 2001) with ACCTRAN, MULPARS
and TBR options. Branch-and-Bound searches were conducted
for both the COI and ND1 sequence data separately. Characters
were treated as unordered and equal weight for the phylogenet-
ic analyses due to the presumably close phylogenetic affinity of
the in-group taxa (Lydeard et al., 1996). Bootstrap values (1000
replicates) using the FAST step-wise addition option of PAUP*
(Felsenstein, 1985) and decay indices/Bremer support values
(Bremer, 1988, 1994) using the Decay Index option of Mac-
Clade 4.0 (Maddison & Maddison, 2000) in conjunction with
PAUP* were calculated to assess support for the individual
nodes of the resulting phylogenetic hypotheses.
Alignment of the COI sequences resulted in a data matrix of 449
bp. The two Epioblasma f. walkeri DNA sequences were identical
with the exception of an ambiguously scored (n) nucleotide for
one of the sequences. One of the two (UAUC 1717) E. f. walkeri
sequences differed from the E. capsaeformis by a single nucleo-
tide (the other E. f. walkeri was ambiguous at this site). In con-
trast, interspecific genetic distances (uncorrected p-distances)
ranged from 6.95 to 7.15% for E. capsaeformis/f. walkeri versus
E. brevidens; 4.26–4.46% for capsaeformis/f. walkeri and E. tri-
quetra; and 5.79% for E. brevidens and E. triquetra. The in-group
Epioblasma taxa differed from the Lampsilis siliquoidea out-group
specimens (n ? 2) from 7.35 to 8.49%. The aligned data matrix
including all taxa yielded 19 variable/parsimony-uninformative
characters and 37 parsimony informative characters.
Alignment of the mitochondrial ND1 sequences resulted in a
data matrix of 610 bp. Intraspecific genetic distances ranged
from 0 to 0.984% for E. capsaeformis, 0.492% for E. f. walkeri, and
0–0.164% for E. brevidens. Interspecific differences between
E. capsaeformisand E. f. walkeriare within the range of intraspecific
values observed for E. brevidens (0.492–0.984%). Interspecific
uncorrected p-distance values ranged from 6.557 to 7.541%
for E. capsaeformis/f. walkeri versus E. brevidens; 6.23– 7.213% for
E. capsaeformis/walkeri versus E. triquetra and 6.885–7.049% for
E. triquetra versus E. brevidens. The out-group lampsiline taxa
differed from the Epioblasma from 10.328 to 14.562%. The
aligned data matrix including all taxa yielded 79 variable/parsi-
mony-uninformative characters and 77 parsimony informative
sites. The number of parsimony informative sites for each codon
position of the mtDNA ND1 gene is 1st ?12, 2nd ? 3, 3rd ?62,
which follows patterns described for other mitochondrial pro-
tein coding genes in molluscs (Roe & Lydeard, 1998).
Maximum-parsimony analysis of the COI and ND1 genes
was conducted by treating each character transformation as
J. E. BUHAY ET AL.
unordered and of equal weight due to the presumably close
relationship among the study taxa (Lydeard et al., 1996). Parsi-
mony analysis of the COI data resulted in three equally parsi-
monious trees [consistency index (CI) ? 0.953; homoplasy
index (HI) ? 0.0465; retention index (RI) ? 0.9636; total
length ? 65]. The strict consensus tree is shown in Fig. 1. All
three parsimonious trees support the monophyly of Epioblasma,
with E. capsaeformisand E. f.walkeriforming an unresolved clade.
Epioblasmabrevidensis the most basal species. Epioblasma triquetra
is the sister taxon to an E. capsaeformis/f. walkericlade.
Phylogenetic analysis of the mitochondrial ND1 data resulted
in five equally parsimonious trees (CI ? 0.679; HI ? 0.321;
RI ? 0.754; total length ? 218). A strict consensus tree of the
five most parsimonious trees and a phylogram of one of the five
most parsimonious trees is shown in Figure 2A and B, respectively.
The topology is similar to that of the COI-based tree, with the
genus Epioblasma being monophyletic. In addition, support is
found for the monophyly of E. brevidens and E. capsaeformis/
E. f. walkeri; however, as in the COI-based tree, E. capsaeformis
and E. f. walkeri are not reciprocally monophyletic. Unlike the
COI-based tree, the strict consensus tree reveals an unresolved
trichotomy between the E. brevidens, E. triquetra and E. capsae-
Epioblasma f. walkeri and E. capsaeformis are medium-sized
species, rarely exceeding 60–70 mm in length, and each species
possesses a dull green periostracum and bluish-white nacre
(USFWS, 1984; Parmalee & Bogan, 1998). Isaac Lea described
nearly half of the currently recognized species of Epioblasma
(Turgeon et al., 1998). After examining two or three of his
own specimens, and an unknown number from Mr Cooper’s
cabinet, Lea described E. capsaeformis as a distinct species (Lea,
1837). Lea (1857) described Epioblasma florentina from which
a less inflated, medium-sized subspecies, E. f. walkeri, was
described (Wilson & Clark, 1914). Johnson (1978) reviewed
the genus Epioblasma noting that E. capsaeformis most closely
resembled E. florentina (he did not recognize E. f. walkeri as a
distinct species or subspecies), with the male E. capsaeformis
being longer, lower and less swollen than that of E. florentina. He
noted that female E. capsaeformishas a darker marsupial swelling
than the rest of the shell, while in E. florentina the periostracum
is a uniform honey yellow or yellowish brown.
The mtDNA sequence data reveals that E. f. walkeriand E. capsae-
formis are genetically indistinguishable from one another,
forming a single, non-exclusive clade. Failure to distinguish
CONSERVATION GENETICS OF EPIOBLASMA
Table 1.Taxa, locality, collector and GenBank accession number of material used in this study
Species UAUC # Locality, collector, and voucher materialGenBank sequences
509Kyles Ford, Clinch River, Tennessee
River drainage, Hancock Co., TN; J. Khym; mantle clip
Station Camp Creek, Big South Fork, Cumberland River drainage, Scott Co., TN; S. Ahlstedt;
no shell, whole tissue
Parch Corn Creek, Big South Fork, Cumberland River drainage, Scott Co., TN; S. Ahlstedt;
Frost Ford, Clinch River, Tennessee. River drainage, Hancock Co., TN; S. Ahlstedt; whole animal ND1 ?AY094377
1527Lillard Mill Dam, Duck River,
Tennessee River drainage, Marshall Co., TN; L. Koch; adductor muscle clip
Nolichucky River, Tennessee River, drainage, Hamblen Co., TN; S. Ahlstedt & S. Fraley;
Venable Spring, Duck River, Tennessee. River drainage, Marshall Co., TN; S. Ahlstedt &
C. Hobbs; whole animal
Frost Ford, Clinch River, Tennessee River drainage, Hancock Co., TN; S.Ahlstedt;
E. florentina walkeri
(Wilson & Clark, 1914)
1690 Cedar Bluff of Indian Creek, upstream of confluence with Clinch River, Tennessee
River drainage, Tazewell Co., VA; C. Kane & L. Koch; whole animal
Cedar Bluff of Indian Creek, downstream from railroad bridge, Tennessee River drainage,
Tazewell Co., VA; L. Koch; whole animal
Big South Fork below Parchcorn Creek, Cumberland River drainage, Scott Co., TN; S. Ahlstedt;
COI ?AY094374 1717
2777 ND1 ?AY094384
2779 Frost Ford, Clinch River, Tennessee. River drainage, Hancock Co., TN; S. Ahlstedt;
882South Fishtail Bay, Douglas Lake, Great Lakes drainage, Cheboygan Co., MI; A.G.A. Pinoswka;
2718Venable Spring, Duck River, Tennessee. River drainage, Marshall Co., TN; S.
Ahlstedt & C. Hobbs; whole animal
652Suwannee River at campground near Flanning Springs, Suwannee River drainage,
Dixie Co., FL; D. Ruessler; whole animal
E. capsaeformis and E. florentina walkeri as mutually exclusive or
monophyletic indicates the two taxa are not valid phylogenetic
species (de Quieroz & Donoghue 1988; Baum & Donoghue,
1995; Mishler & Theriot, 2000; Wiens & Penkrot, 2002).
Admittedly, one cannot prove the null hypothesis that genetic
differences between the putative taxa are absent; however,
management decisions should be based on the best available
scientific information (see Avise, 1994, 2000, for other similar
conservation genetic studies). The vast majority of unionid
species have been described based implicitly on a morpho-
species concept. Although considerable intraspecific shell varia-
tion within unionids and hyriids appears to be a response to the
hydro-dynamics of living in large versus small river or stream
environments (e.g. Walker, 1981; Watters, 1994), there was
a tendency for taxonomists of the late nineteenth and early
twentieth centuries to describe each morph as a unique species.
From a monograph by Simpson (1900, 1914) to a check list by
Turgeon et al. (1998), the taxonomy of Epioblasmahas exhibited
instability and subjective synonomy. Many biologists fail to
understand that our view of currently recognized unionid
species represents hypotheses that are subject to testing and
may not adequately reflect species that actually exist in nature.
For example, based on Johnson (1978) the Mobile River system
of Alabama, Tennessee, Georgia and Mississippi would have
only one Epioblasmaspecies, compared with three recognized by
Turgeon et al. (1998). Table 2 provides a comparison of the
three primary classification schemes and current conservation
status of Epioblasmataxa.
One intriguing feature that warrants further scrutiny, is the
significance and mechanism explaining population variation
in mantle pad colour. Ortmann (1924) reported geographic
variation in the mantle pad colour of nominal E. capsaeformis
with individuals from the Clinch, Powell and Nolichucky rivers
possessing an iridescent bluish-white, while individuals in the
Duck River possessing a slate-greyish almost black colour.
Although some specimens from the Duck have been observed to
have a small brown mottling along the outer edge of the mantle,
the remaining part of the mantle is always grey or almost black
in colour. In contrast, E. f. walkeri is mottled brownish/tan in
Indian Creek and Big South Fork of the Cumberland River (S.
J. E. BUHAY ET AL.
Figure 1. A strict consensus tree of three equally most-parsimonious trees
(TL ?65; CI ?0.953) based on a maximum-parsimony analysis of 449 bp of
the mitochondrial COIgene. Numbers above the nodes are bootstrap values
(1000 replicates) and numbers below the nodes are Bremer support values.
Specimen numbers ? UAUC identification numbers as shown in Table 1. E
cap ?Epioblasma capsaeformis, E wal ?E. florentina walkeri, E tri ?E. triquetra,
E bre ?E. brevidens and L sil ?Lampsilis siliquoidea.
Figure 2. A strict consensus tree (A) of five equally most-parsimonious trees
and (B) phylogram of one of the five equally most-parsimonious trees (TL ?
218; CI ? 0.679) based on an analysis of 610 bp of the mitochondrial ND1
gene. Numbers above the nodes are bootstrap values (1000 replicates) and
numbers below the nodes are Bremer support values. Specimen numbers ?
UAUC identification numbers as shown in Table 1. E cap ? Epioblasma cap-
saeformis, E wal ?E. florentina walkeri, E tri ?E. triquetra,E bre ?E. brevidens,
L sil ?Lampsilis siliquoidea; V tae ?Villosa taeniata; V vil ?V. villosa.
Ahlstedt & J. Jones, personal communication). Although the
two nominal E. capsaeformis specimens from the Duck River,
which possess black mantle pads, are monophyletic, the remain-
ing specimens did not form monophyletic groups associated
with mantle pad colour. It is possible that other more rapidly
evolving neutral genetic markers will delineate other phylo-
genetic species within the E. capsaeformis ? E. f. walkeri (e.g. the
Duck River black mantle pad morph) clade, which warrant
recognition. However, our data refute the recognition of the
currently recognized species boundaries based on shell mor-
phology. Alternatively, mantle pad colour may be a response to
some unknown environmental parameter or represent intra-
specific allelic variation.
Although there is a desire for some biologists to accept and
protect all currently recognized taxonomic units (e.g. Berg &
Berg, 2000), we believe it is more important to test and deter-
mine species boundaries using rigorous scientific methods and
principles, preferably prior to developing conservation recovery
plans, particularly when advocating augmentation or trans-
planting of populations (Mulvey & Lydeard, 2000). Admittedly,
sometimes decisions must be made on limited data due to the
imperiled status of many unionids, particularly when faced with
the complete loss of the putative species. We believe the Phylo-
genetic Species Concept, based on the recognition of minimal
monophyletic groups (Mishler & Donoghue, 1982; Mishler &
Brandon, 1987; de Queiroz & Donoghue, 1988, 1990), offers an
objective and rigorous approach to test the validity of problem-
atic unionid species complexes. This method has been applied
to other unionid taxa (Mulvey et al., 1997; Roe & Lydeard 1998;
King et al., 1999; Lydeard et al., 2000) and should eventually
result in a sound classification and understanding of unionid
biodiversity and evolution. Our interpretation of the mtDNA
sequence data, however, should be treated as an hypothesis sub-
ject to further testing.
Given that so few reproducing populations of E. capsaeformis
and E. f. walkeri exist, combining the two into a single phyloge-
CONSERVATION GENETICS OF EPIOBLASMA
Table 2.Currently recognized species of Epioblasmaaccording to a popular identification manual by Burch (1975), a monographic review by
Johnson (1978) and a compiled checklist by Turgeon et al. (1998). The conservation status of each species is provided in the Turgeon et al.
(1998) column as follows: X ?presumed extinct; X* ?probably extinct (based on recent survey data from S. Ahlstedt, W. Haag, P. Hartfield,
P. Johnson, J. Williams); E ?federally endangered ?date listed; I ?imperiled, not federally listed.
(17 species; 2 subspecies)
Turgeon et al. 1998
(20 species; 8 subspecies)
florentina curtisii(Frierson &
florentina walkeri(Wilson & Clark, 1914) E,1977
obliquata obliquata(Rafinesque, 1820)
obliquata peroblique(Conrad, 1836)
torulosa gubernaculumc(Reeve, 1865)
torulosa rangianae(Lea, 1865) E,1993
othcaloogensis(Lea, 1857) E,1993/X*
torulosa torulosa(Rafinesque, 1820)
torulosa propinqua(Lea, 1857)
aBurch (1975) largely followed the first monographic treatment of Epioblasma(?Truncilla) by Simpson (1900) with minor exceptions (e.g. Burch
synonymized E.compactaand E. othcaloogensiswith E. metastriata.
bBurch (1975) hypothesized that E. biemarginatawas a large river form of E. turgidula.
cBurch (1975) considered E. torulosa gubernaculumas an upper Tennessee River form of E. torulosa torulosa.
dBurch (1975) hypothesized that E. sampsoniimight be a Wabash River form of E. torulosa torulosa.
eBogan (1997) and Parmalee & Bogan (1998) replaced E. torulosa rangianawith E. biloba(Rafinesque 1831), because the name had priority over
netic species does not alter their conservation status. Life history
and population biology studies have already been conducted on
the single known extant reproducing population of E. f. walkeri
(Rogers, Watson & Neves, 2001). However, similar comparative
studies should be conducted on remaining nominal E. capsae-
formis populations with the intent of further testing our hypo-
thesis that the two taxa are one phylogenetic species and to
provide valuable data to manage more effectively the recovery of
This study was supported by the United States Fish & Wildlife
Service, North Carolina Department of Transportation, the
Howard Hughes Medical Institution (HHMI) and the National
Science Foundation (multi-user equipment grant). C. Renée
Dean was a NSF undergraduate research participant and Qshe-
quilla Parham was a HHMI undergraduate-sponsored student.
Thanks to Steve Ahlstedt, Steve Fraley, Jess Jones, Leroy Koch
and Jason Khym for providing material for this study. Thanks to
Leroy Koch, Steve Ahlstedt and anonymous reviewers for their
helpful comments on the manuscript.
AVISE, J.C. 1994. Molecular markers, natural history, and evolution. Chap-
man and Hall, New York.
AVISE, J.C. 2000. Phylogeography: the history and formation of species. Har-
vard University Press, Cambridge, Massachusetts.
BAUM, D.A. & DONOGHUE, M.J. 1995. Choosing among alternative
‘phylogenetic’ species concepts. Systematic Botany, 20: 560–573.
BENSON, D.A., KARSCH-MIZRACHI, I., LIPMAN, D.J., OSTELL, J.,
RAPP, B.A. & WHEELER, D.L. 2000. GenBank. Nucleic Acids
Research, 28: 15–18.
BERG, D.J. & BERG, P.H. 2000. Conservation genetics of freshwater
mussels: comments on Mulvey et al. Conservation Biology, 14: 1920–
BREMER, K. 1988. The limits of amino acid sequence data in angio-
sperm phylogenetic reconstruction. Evolution, 42: 795–803.
BREMER, K. 1994. Branch support and tree stability. Cladistics, 10: 295–
BURCH, J.B. 1975. Freshwater Unionacean clams (Mollusca: Pelecypoda) of
North America, revised edn. Malacological Publications, Hamburg.
CABOT, E.L. & BECKENBACK, A.T. 1989. Simultaneous editing of
multiple nucleic acid and protein sequences with ESEE. Cabios, 5:
DE QUEIROZ, K. & DONOGHUE, M.J. 1988. Phylogenetic systematics
and the species problem. Cladistics, 4: 317–338.
DE QUEIROZ, K. & DONOGHUE, M.J. 1990. Phylogenetic systematics
and species revisited. Cladistics, 6: 83–90.
FELSENSTEIN, J.F. 1985. Confidence limits on phylogenies: an approach
using the bootstrap. Evolution, 39: 783–791.
FOLMER, O., HOEH, W.R., BLACK, M.B., & VRIJENHOEK, R.L. 1994.
DNA primers for amplification of mitochondrial cytochrome C
oxidase subunit I from metazoan invertebrates. Molecular Marine
Biology and Biotechnology, 3: 294–299.
GRAF, D.L. & ÓFOIGHIL, D. 1999. The evolution of brooding charac-
ters among the freshwater pearly mussels (Bivalvia: Unionoidea) of
North America. Journal of Molluscan Studies, 66: 157–170.
HOEH, W.R., BOGAN, A.E., CUMMINGS, K.S. & GUTTMAN, S.E.
1998. Evolutionary relationships among the higher taxa of fresh-
water mussels (Bivalvia: Unionoida): inferences on phylogeny and
character evolution from analyses of DNA sequence data. Malaco-
logical Review, 31: 121–140.
HOEH, W.R., BOGAN, A.E. & HEARD, W.H. 2001. A phylogenetic
perspective on the evolution of morphological and reproductive
characteristics in the Unionoida. Ecology and evolutionary biology of
freshwater mussels [Superfamily Unionoidea] (G. Bauer & G. Wachtler,
eds), 257–280. Springer Verlag, Berlin.
JOHNSON, R. I. 1978. Systematics and zoogeography of Plagiola (?
Dysnomia ? Epioblasma), an almost extinct genus of freshwater
mussels (Bivalvia: Unionidae) from Middle North America. Bulletin
of the Museum of Comparative Zoology, 148: 239–320.
KING, T.L., EACKLES, M.S., GJETVAJ, B. & HOEH, W.R. 1999.
Intraspecific phylogeography of the freshwater bivalve Lasmigona
subviridis (Bivalvia: Unionidae): conservation implications of range
discontinuity. Molecular Ecology,8: S65–S78.
LEA, I. 1834. Observations on the naiades, and descriptions of new
species of that, and other families. Transactions of the American
Philosophical Society, New Series, 5: 23–119, pls 1–19.
LEA, I. 1857. Description of six new species of uniones from Alabama.
Proceedings of the Academy of Natural Sciences of Philadelphia, 9: 83.
LYDEARD, C., MINTON, R.L. & WILLIAMS, J.D. 2000. Prodigious
polyphyly in imperiled freshwater pearly-mussels (Bivalvia: Union-
idae): a phylogenetic test of species and generic designations. The
evolutionary biology of the Bivalvia (E. M. Harper, J. D. Taylor & J. A.
Crame, eds), 145–158, Special Publication 177. Geological Society,
LYDEARD, C., MULVEY, M. & DAVIS, G.M. 1996. Molecular system-
atics and evolution of reproductive traits of North American fresh-
water unionacean mussels (Mollusca: Bivalvia) as inferred from 16S
rRNA gene sequences. Philosophical Transactions of the Royal Society of
London, Series B, 351: 1593–1603.
LYDEARD, C. & ROE, K.J. 1998. Phylogenetic systematics: the missing
ingredient in the conservation of freshwater unionid bivalves.
Fisheries, 23: 16–17.
MADDISON, D.R. & MADDISON, W.P. 2000. MacClade 4: analysis of
phylogeny and character evolution, Version 4.0. Sinauer Associates,
MASTER, L.L., FLACK, S.R. & STEIN, B.A., eds. 1998. Rivers of life:
critical watersheds for protecting freshwater biodiversity. Nature Conserv-
MCKITRICK, M.C. & ZINK, R.M. 1988. Species concepts in ornitho-
logy. Condor, 90: 1–14.
MISHLER, B.D. & BRANDON, R.N. 1987. Individuality, pluralism, and
the phylogenetic species concept. Biology and Philosophy,2: 397–414.
MISHLER, B.D. & DONOGHUE, M.J. 1982. Species concepts: a case
for pluralism. Systematic Zoology, 31: 491–503.
MISHLER, B.D. & THERIOT, E.C. 2000. The phylogenetic species
concept (sensu Mishler and Theriot): monophyly, apomorphy, and
phylogenetic species concepts. Species concepts and phylogenetic theory:
a debate (Q. D. Wheeler & R. Meier, eds), 44–54. Columbia Uni-
versity Press, New York.
MOORE, W.S. 1995. Inferring phylogenies from mtDNA variation:
mitochondrial gene trees versus nuclear-gene trees. Evolution, 49:
MULVEY, M. & LYDEARD, C. 2000. Let’s not abandon science for
advocacy: reply to Berg and Berg. Conservation Biology, 14: 1924–
MULVEY, M., LYDEARD, C., PYER, D.L., HICKS, K.M., BRIM-BOX, J.,
WILLIAMS, J.D. & BUTLER, R.S. 1997. Conservation genetics of
North American freshwater mussels Amblema and Megalonaias. Con-
servation Biology, 11: 868–878.
NEVES, R.J., BOGAN, A.E., WILLIAMS, J.D., AHLSTEDT, S.A. &
HARTFIELD, P.W. 1997. Status of aquatic mollusks in the southeast-
ern United States: a downward spiral of diversity. Aquatic fauna in
peril: the southeastern perspective (G. W. Benz & D. E. Collins, eds),
43–85, Special Publication 1. Southeast Aquatic Research Institute,
Lenz Design & Communications, Decatur.
ORTMANN, A.E. 1924. The naiad-fauna of Duck River in Tennessee.
American Midland Naturalist, 9: 18–62.
PARMALEE, P.W. & BOGAN, A.E. 1998. The freshwater mussels of
Tennessee. University of Tennessee Press, Knoxville.
ROE, K.J. & LYDEARD, C. 1998. Molecular systematics of the fresh-
water mussel genus Potamilus (Bivalvia: Unionidae). Malacologia, 39:
J. E. BUHAY ET AL.
ROGERS, S.O., WATSON, B T. & NEVES, R.J. 2001. Life history and
population biology of the endangered tan riffleshell (Epioblasma
florentina walkeri) (Bivalvia: Unionidae). Journal of the North American
Benthological Society, 20: 582–594.
SIMON, C., FRATI, F., BECKENBACH, A., CRESPI, B., LIU, H. &
FLOOK, P. 1994. Evolution, weighting, and phylogenetic utility of
mitochondrial gene sequences and a compilation of conserved poly-
merase reaction primers. Annals of the Entomological Society of America,
SIMPSON, C.T. 1900. Synopsis of the naiades, or pearly fresh-water
mussels. Proceedings of the United States National Museum, 22: 501–
SIMPSON, C.T. 1914. A descriptive catalogue of the naiades or pearly fresh-
water mussels, Parts I–III. Bryant Walker, Detroit.
STARNES, L.B. & BOGAN, A.E. 1988. The mussels (Mollusca: Bivalvia:
Unionidae) of Tennessee. American Malacological Bulletin, 6: 19–37.
SWOFFORD, D.L. 2001. PAUP*. Phylogenetic analysis using Parsimony
(*and other methods), Version 4.0b5. Sinauer Associates, Sunderland.
TURGEON, D.D., QUINN, J.F., JR, BOGAN, A.E., COAN, E.V.,
HOCHBERG, F.G., LYONS, W.G., MIKKELSEN, P.M., NEVES, R.J.,
ROPER, C.F.E., ROSENBERG, G., ROTH, B., SCHELTEMA, A.,
THOMPSON, F.G., VECCHIONE, M. & WILLIAMS, J.D. 1998.
Common and scientific names of aquatic invertebrates from the United States
and Canada: mollusks, 2nd edn. American Fisheries Society, Special
Publication 26. AFS, Bethesda.
UNITED STATES FISH & WILDLIFE SERVICE 1986. Curtis’ pearly
mussel recovery plan. U.S. Fish and Wildlife Service, Twin Cities,
UNITED STATES FISH & WILDLIFE SERVICE 1984. Tan riffle shell
pearly mussel recovery plan. U.S. Fish & Wildlife Service, Atlanta,
UNITED STATES FISH & WILDLIFE SERVICE 1997. Endangered
and threatened wildlife and plants; determination of Cumberland
elktoe (Alasmidonta atropurpurea), oyster mussel (Epioblasma capsae-
formis), Cumberlandian combshell (Epioblasma brevidens), purple
bean (Villosa perpurpurea), and rough rabbitsfoot (Quadrula cylindrica
strigillata) to be endangered species. Federal Register, 62: 1647–
WALKER, K.F. 1981. Ecology of freshwater mussels in the River Murray.
Australian Water Council Technical Paper No. 63.
WATTERS, G.T. 1994. Form and function of unionoidean shell sculp-
ture and shape. American Malacological Bulletin, 11: 1–20.
WIENS, J.J. & PENKROT, T.A. 2002. Delimiting species using DNA and
morphological variation and discordant species limits in spiny
lizards (Sceloporus). Systematic Biology, 51: 69–91.
WILLIAMS, J.D. & MULVEY, M. 1994. Recognition of freshwater
mussel taxa: a conservation challenge. Principles of conservation biology
(G. K. Meffe & C. R. Carroll, eds), 57–58. Sinauer Associates,
WILLIAMS, J.D., WARREN, M.L., JR, CUMMINGS, K.S., HARRIS, J.L.
& NEVES, R.J. 1993. Conservation status of freshwater mussels of the
United States and Canada. Fisheries, 18: 6–22.
WILSON, C.B. & CLARK, H.W. 1914. The mussels of the Cumberland
River and its tributaries. U.S. Bureau of Fisheries Document, 781: 1–63.
CONSERVATION GENETICS OF EPIOBLASMA