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ORIGINAL PAPER
A molecular phylogenetic analysis of Speyeria and its implications
for the management of the threatened Speyeria zerene hippolyta
Anne McHugh •Paulette Bierzychudek •
Christina Greever •Tessa Marzulla •
Richard Van Buskirk •Greta Binford
Received: 16 May 2013 / Accepted: 9 October 2013
Springer Science+Business Media Dordrecht 2013
Abstract The genetic structure of lineages can provide
important information for delineating ‘‘evolutionarily sig-
nificant units’’ (ESUs) for conservation, and for planning
actions to protect and restore taxa threatened with extinction.
Speyeria zerene hippolyta, the Oregon silverspot butterfly, is
a U.S.A. federally threatened subspecies that is the focus of
considerable conservation effort, but whose evolutionary
relationships with other Speyeria taxa are not well-under-
stood. We conducted a genetic analysis of nine Speyeria
species and 25 subspecies from western U.S.A., using both
mitochondrial and nuclear markers. Our goal was to deter-
mine whether such data supported (a) S. z. hippolyta’s
designation as an ESU, and (b) the current morphologically-
based taxonomy of Speyeria spp. Our data for S. z. hippolyta
were equivocal; while nuclear markers resolved all these
individuals into a single clade, mtDNA data suggested the
existence of two clades. Aside from S. cybele, which was
consistently supported as monophyletic, our data provided
little support for most of the species currently recognized for
western U.S. Speyeria, including S. zerene, and even less for
the many subspecies designations. These genetic findings
stand in contrast to the morphological differences recog-
nized by experts, and suggest a relatively recent origin for
many of these taxa. Two of 66 individuals screened for
Wolbachia infection tested positive for this symbiont. Our
results provide no persuasive evidence that S. z. hippolyta
should lose its status as an ESU, but they have important
implications for ongoing management actions such as pop-
ulation augmentation.
Keywords ESU Evolutionarily significant unit
Lepidoptera Oregon silverspot butterfly Species
delineation Wolbachia
Introduction
Developing effective strategies for protecting and restoring
sensitive taxa requires that we be able to define and identify
those units in need of protection. It is widely recognized
that protected groups should represent distinctive evolu-
tionary histories and potentials (Moritz 1994), i.e. that they
be ‘‘evolutionarily significant units’’ (ESUs) (Waples 1991;
Crandall et al. 2000). Knowledge of the genetic structure of
lineages is an important complement to behavioral, mor-
phological, and ecological information in determining the
distinctiveness of groups (DeSalle and Amato 2004), the
A. McHugh P. Bierzychudek (&)C. Greever
T. Marzulla G. Binford
Department of Biology, Lewis & Clark College, 0615 S.W.
Palatine Hill Road, Portland, OR 97219, USA
e-mail: bierzych@lclark.edu
C. Greever
e-mail: christina.greever@colorado.edu
Present Address:
A. McHugh
Department of Biology, University of Vermont, 109 Carrigan
Drive, Burlington, VT 05401, USA
Present Address:
C. Greever
University of Colorado, 3100 Marine Street, 563 UCB 35,
Boulder, CO 80303, USA
Present Address:
T. Marzulla
Oregon Health and Sciences University, 3181 SW Sam Jackson
Rd., Portland, OR 97239, USA
R. Van Buskirk
Department of Environmental Studies, Pacific University, 2043
College Way, Forest Grove, OR 97116, USA
123
J Insect Conserv
DOI 10.1007/s10841-013-9605-5
recognition of which can help guide management decisions
and set conservation priorities (Dayrat 2005).
The Oregon Silverspot Butterfly (Speyeria zerene hip-
polyta) provides one example of a group for which ambi-
tious conservation activities are underway even though
little is known about the population processes and histori-
cal biogeography that underlie its current distribution.
Listed in the U.S. as a threatened subspecies in 1980 (45
FR 44935–44939), S. z. hippolyta has been the focus of
considerable management efforts, including habitat
improvement as well as augmentation of low and/or
declining populations with captively reared larvae (Crone
et al. 2007). Some sites of extirpated populations are
undergoing habitat restoration in preparation for re-intro-
duction of individuals from other locations (U.S. Fish and
Wildlife Service 2001). But little is known about the
genetic relationships among the five extant populations of
S. z. hippolyta in Oregon and northern California, nor is
there much information about their genetic relationships to
other subspecies of S. zerene or other species of Speyeria.
Speyeria are a North American group of Nymphalid
butterflies. The 16 recognized species (Pelham 2008;
Dunford 2009) were defined on morphological grounds by
dos Passos and Grey (1947), and are mostly distributed in
western North America. The larvae feed exclusively on
Viola spp. (Brittnacher et al. 1978; Hammond 1981), and
the ranges of various taxa are limited, in part, by the dis-
tribution of these plants. The group is notoriously variable
in wing pattern and color (Pyle 2002; Dunford 2009), and
this variation has formed the basis of numerous subspecies
designations. Adding to the complexity is the parallel
nature of the morphological variation observed among
sympatric taxa. Within the same region, species tend to be
similar in color and size. Differences between subspecies
from different regions sometimes exceed differences
observed among sympatric species (Hovanitz 1943). Con-
vergence in form within a region may be influenced by
selection favoring an advantageous phenotype, develop-
mental response to a common environment, a more recent
shared ancestry than the species designations indicate, or to
hybridization. While Brittnacher et al. (1978) report that
naturally occurring hybrids are rare, most subspecies and
even many species have successfully interbred under lab-
oratory conditions (Paul Hammond and David McCorkle,
personal communication, Feb 25, 2012). No key to the
group has been published (but see keys in Hammond 1978
and Dunford 2007), and only experts are able to reliably
identify individuals (Pyle 2002), even then requiring
Fig. 1 Map of collection locations. Three-letter geographical codes correspond to those used in the trees and in ‘‘Appendix 1’’
J Insect Conserv
123
knowledge of the geographic region from which they were
collected. Though a preliminary investigation of mito-
chondrial DNA variation was conducted by Dunford
(2007), Brittnacher et al.’s 1978 allozyme study of Cali-
fornia Speyeria remains the only published investigation of
genetic relationships among western North American
Speyeria to date.
In addition to the threatened S. z. hippolyta,Speyeria
includes three U.S. ‘‘endangered’’ subspecies, S. z. behrensii,
S. z. myrtleae, and S. callippe callippe, and two other taxa
regarded as vulnerable, S. z. bremnerii and S. idalia (Xerces
Society 2012). The taxonomic confusion characterizing
Speyeria raises the question of whether these taxa are as
genetically distinct as their protected status implies, and
suggests that a genetic analysis of Speyeria could provide
important information to help guide conservation efforts.
Many recent attempts to use genetic data to identify
ESUs have focused on mitochondrial DNA (mtDNA)
markers, because the rapid rate at which variation accu-
mulates in the mitochondrial genome makes mtDNA useful
for assessing differences among closely related taxa. In
particular, much attention has been given to the potential of
a fragment of the mitochondrial cytochrome c oxidase
subunit one (COI) gene to serve as a universal ‘‘barcode’’
for identifying and delineating taxa (Hebert et al. 2003).
However, there are many reasons why variation within a
single locus, especially one from the mitochondrial genome,
might be a poor indicator of evolutionary patterns and pro-
cesses, both for organisms in general and for Lepidoptera in
particular (Wahlberg et al. 2003a,b; Gompert et al. 2006;
Forister et al. 2008; Wahlberg et al. 2009). For this reason our
genetic analysis employs both a mtDNA marker and several
nuclear markers. Because infection by the endosymbiont
Wolbachia has been known to complicate the interpretation
of patterns of mtDNA and to pose a threat to the persistence
of arthropod populations (Nice et al. 2009), we screened a
subset of our samples for evidence of Wolbachia infection.
Our goal is to develop a molecular phylogeny for the
Speyeria taxa in the western U.S., with a particular focus on
S. z. hippolyta and other subspecies of S. zerene, in order to
determine whether molecular phylogenetic data support S. z.
hippolyta’s designation as a distinctive evolutionary lineage.
This analysis also provides an opportunity to determine
whether the phylogenetic patterns we discover are coincident
with current taxonomy for other taxa in this group.
Methods
Taxon sampling
We attempted to include a representative sample of indi-
viduals of S. z. hippolyta, from both extant and extirpated
populations, a representative sample of most of the other
subspecies of S. zerene, and a sampling of other species of
Speyeria from across a wide geographic distribution. We
sampled a total of 121 Speyeria individuals from the
western United States (Fig. 1) and two outgroup speci-
mens, Brenthis daphne and Argynnis aglaja (‘‘Appendix
1’’). Our sampling structure for individuals and populations
of species and subspecies is summarized in Table 1. Eighty
of the Speyeria individuals were collected for or donated to
this project by Paul C. Hammond, David McCorkle and
Anne McHugh. Thirty-two specimens were obtained from
the Arthropod Collection at Oregon State University
(OSU). Nine S. zerene specimens were provided by the
McGuire Center for Lepidoptera and Biodiversity at the
Florida Museum of Natural History in Gainesville, Florida.
Table 1 Species, subspecies, and numbers of populations and indi-
viduals represented in the dataset
Speyeria
species
Subspecies Number of
populations
Number of
individuals
atlantis cornelia 13
dodgei 23
hesperis 12
nikias 12
sorocko 13
atlantis/hollandi
a
N/A 1 1
callippe elaine 13
semivirida 14
coronis snyderi 39*
cybele leto 3 15*
egleis egleis 14
macdunnoughi 16
hollandi hollandi 12
hydaspe hydaspe 13
purpurascens 16
sakuntala 14
mormonia artonis 29*
erinna 29*
zerene bremnerii 4 12*
conchyliatus 2 12*
gloriosa 3 16*
gunderi 3 11*
hippolyta 11 79*
picta 3 10*
platina 26
sinope 24
This table includes samples from both LC and RVB
* Includes multiple individuals possessing the same COII haplotype.
These are represented as single terminal taxa in our phylogenies. For
more information about haplotypes, contact RVB
a
Possible hybrid individual, according to Paul Hammond
J Insect Conserv
123
In addition to these 121 samples, mitochondrial sequence
data from 6 haplotypes representing 67 individuals of S.
zerene hippolyta were provided by Richard Van Buskirk
(RVB), either from individuals or tissue collected in
1995–1996 under USFWS permit number PRT-806058 or
from additional specimens from OSU. All collections of S.
z. hippolyta pre-dated the augmentations from other pop-
ulations currently taking place. RVB also provided mito-
chondrial sequence data from 45 additional haplotypes
representing 81 individuals of other Speyeria species and
subspecies. DNA vouchers of all specimens and DNA
samples (where available) have been archived at OSU.
DNA isolation
All genomic DNA isolated at Lewis & Clark College (LC)
was extracted from one leg using QIAgen’s DNEasy extrac-
tion kit, according to manufacturer’s instructions, except
eluted in 30 ll water. DNA was stored at -20 C. Genomic
DNA data provided by RVB came from wing tissue non-
destructively sampled from live individuals (for S. z. hippol-
yta), leg tissue (from museum specimens), or thorax muscle
(for all other live-caught specimens). This genomic DNA was
isolated using a proteinase digest followed by phenol–chlo-
roform extraction (for details see Van Buskirk 2000).
Gene selection
At LC, we amplified a single 1,410-base pair (bp) mtDNA
fragment that included two genes, cytochrome c oxidase
subunit I (COI) and cytochrome c oxidase subunit II (COII).
RVB amplified a 613 bp region of the COII subunit for some
individuals, and a 456 bpregion for others. To optimize taxon
inclusion while minimizing missing data, our phylogenetic
analysis used the 554 base pair region of COII that allowed
maximal overlap among these three datasets (see sequence
assembly and alignment section below). At LC we also
amplified four nuclear markers: glyceraldehyde 3-phosphate
dehydrogenase (GAPDH), ribosomal protein subunit 5
(RpS5), triosephosphate isomerase (TPI), and wingless.We
also amplified an 850 bp fragment of carbamoyl phosphate
synthetase 2, aspartate carbamyltransferase, dihydrorotase
(CAD) from a small but diverse subset of taxa, but did not
analyze this fragment because it lacked variability. All these
nuclear markers have proven to be informative at the species
level for Lepidoptera (Brower and DeSalle 1998;Beltra
´netal.
2002;Wahlbergetal.2003b;Regieretal.2008; Wah lberg and
Wheat 2008;Wahlbergetal.2009).
Molecular data acquisition
We used polymerase chain reaction (PCR) protocols and
primers from several sources (‘‘Appendix 2’’). We purified
post-PCR products using QIAgen PCR purification kits
(LC) or Millipore filtration tubes with double-distilled
water as a rinsing agent (RVB). We analyzed LC samples
with a Nanodrop 1000 Spectrophotometer for DNA con-
centration and sent them to the University of Arizona
Genetics Core for sequencing in two directions. RVB
samples were sequenced with an ABI377 Perkin-Elmer
automated sequencer.
Sequence assembly and alignment
We assembled the two strands for each fragment and
checked sequence quality using Sequencher 4.6. To confirm
amplification of the intended gene fragments, we subjected a
subset of assembled sequences to homology searches in
GenBank using BLASTn. We aligned sequences using the
online server for MUSCLE (http://www.ebi.ac.uk/Tools/
msa/muscle/; Edgar 2004) and used default alignments for
phylogenetic analyses. We viewed alignments, trimmed
ragged ends, and concatenated our multigene datasets (see
below) using Mesquite version 2.75 (Maddison and Madd-
ison 2011). The COI/COII fragments were trimmed after
alignment to the 554 nucleotides of COII that maximized
overlap between the regions amplified at LC and by RVB.
Preliminary analyses of the entire 1,410 base pair region of
COI/II amplified at LC resulted in nearly identical tree
topologies to those created using the shorter fragment;
minor discrepancies between the analyses do not affect our
conclusions. Sequences were deposited in GenBank
(Accession Numbers available from the authors).
Phylogenetic analyses
We reconstructed separate phylogenetic hypotheses (a) for
individual genes, (b) for a concatenated dataset of all
nuclear genes, and (c) for a concatenation of all genes,
nuclear and mitochondrial. Though our goal was to amplify
four nuclear genes, there were some taxa for which we
succeeded in amplifying only one (either wingless or RpS5,
‘‘ Appendix 1’’). Preliminary analyses of concatenated
datasets that included taxa with single gene representation
yielded trees that placed these taxa in unresolved basal
polytomies. Therefore, our concatenated analyses only
include taxa for which we had sequences for two or more
genes. Most of the excluded taxa were S. zerene specimens
from OSU (‘‘Appendix 1’’) that were represented only by
wingless sequences, which were nearly invariant (Table 2).
We used jModelTest version 0.1.1 with the Bayesian
Information Criterion (BIC) to determine the optimal
model of evolution for each gene dataset. All phylogenetic
models were constructed using Bayesian inference as
implemented in MrBayes version 3.1.2 (Huelsenbeck and
Ronquist 2001; Ronquist and Huelsenbeck 2003), with
J Insect Conserv
123
model parameters optimized from the results of BIC. For
concatenated analyses we used separate model parameters
for each gene partition. All analyses were done with
10,000,000 Markov Chain Monte Carlo generations, saving
every 100th tree, with two iterations of four chains for each
analysis. We used Tracer version 1.5 (Rambaut and
Drummond 2007) to determine the appropriate burn-in
value, and in all cases discarded the first 10 % of saved
trees as burn-in. We assessed confidence in particular
clades using posterior probabilities.
In addition, we analyzed the data using parsimony,
neighbor joining, and maximum likelihood methods. These
resulted in either similar tree topologies as Bayesian
analyses or reduced resolution; therefore, for simplicity, we
only report the Bayesian results.
Pairwise genetic distances
We calculated uncorrected p-distances (i.e. the proportion of
nucleotide sites at which two sequences differ, with no cor-
rection for multiple substitutions at the same site) within and
among resolved clades for the COII dataset and for the con-
catenated nuclear dataset using MEGA version 5.1 (Tamura
et al. 2011) with pairwise deletion of gaps and missing data.
Wolbachia screening
We screened a subset of the LC individuals for Wolbachia
infection by amplifying a Wolbachia-specific 16S gene
from genomic DNA isolated from Speyeria tissue
(‘‘Appendix 1’’). As a positive control for PCR amplifica-
tion, we used a genomic DNA template from a spider
previously determined by GJB to be infected with Wol-
bachia. This template yielded a positive band in every PCR
reaction we attempted with Speyeria gDNA. Positive bands
were subjected to the same protocols for molecular data
acquisition, sequence assembly and alignment as other LC
samples (‘‘Appendix 2’’). Sequences were identified using
homology searching with BLASTn.
Results
Data characteristics and model choice
Of the 121 Speyeria sampled at Lewis & Clark, we
obtained quality sequences from 90 individuals; the taxa
included, collection locations, and other information are
summarized in ‘‘Appendix 1’’. With the addition of RVB
sequence data (see ‘‘Methods’’ section and Table 1), our
dataset included data from nine of the 16 recognized spe-
cies of Speyeria in the western U.S. and from 25 of the 104
described subspecies of these nine species, including eight
Table 2 Data set characteristics and model parameters as estimated from Bayesian Information Criterion implemented in jModelTest 0.1.1
Gene Model A C G T ti/tv AC AG AT CG CT GT G I # char all # char
inf
COII HKY?I?G 0.35 0.15 0.11 0.38 11.98 1.10 0.70 554 53
wingless TrNef?I 0.26 0.23 0.27 0.23 1.0 1.42 1.0 1.0 10.9 1.0 0.81 403 10
GADPH TPM2?I?G 6.3 25.5 6.3 1.0 25.5 1.0 0.69 0.80 692 28
TPI F81?G 0.34 0.11 0.21 0.33 0.16 362 12
RpS5 K80?I 3.25 0.76 613 20
Total nuclear 2,070 70
Total 2,624 123
Numbers of characters are reported for both parsimony-informative (‘‘inf’’) and total characters (‘‘all’’) in the aligned dataset
J Insect Conserv
123
of the 15–16 described subspecies of S. zerene (Pelham
2008; Dunford 2009; Table 1). In addition to S. zerene, five
other species in our analysis (S. atlantis,S. callippe,S.
egleis,S. hydaspe, and S. mormonia) were represented by
more than one subspecies (Table 1).
DNA from different taxa and different markers ampli-
fied with varying success; our final datasets were most
complete for COII and wingless (‘‘Appendix 1’’). We had
particular difficulty amplifying genes from museum spec-
imens (Watts et al. 2007), which constituted most of our
samples of S. z. hippolyta; as a result, these museum
specimens are represented only in the wingless and COII
datasets. The numbers of bases in final alignments, and
model characteristics, are summarized in Table 2. Models
selected by jModelTest for all individual gene partitions
indicate that two-rate parameters provided the best models
of substitution patterns for both nuclear and mitochondrial
datasets. Of the 2,624 nucleotides in our full-concatenated
dataset, the mitochondrial gene constituted 21 % of the
dataset, and 43 % of the parsimony-informative sites
(Table 2).
Phylogenetic analyses
The degree to which relationships were resolved varied
among markers, with mitochondrial COII resolving a
higher proportion of nodes than any analyses of the nuclear
sequences. More nodes were resolved in the concatenated
nuclear gene analysis than in individual analyses of nuclear
genes; however, analyses of nuclear data resolved fewer
nodes, even when concatenated, than did analyses of
mitochondrial data (Fig. 2).
Support of monophyly of nominal species
Speyeria cybele was the only nominal species that was
monophyletic in all of our analyses (Figs. 2,3,4). S. hy-
daspe and S. mormonia were supported as monophyletic by
the full concatenated analysis, which contained the most
complete dataset (Fig. 4), as well as by either the COII (in
the case of S. hydaspe) or the nuclear (for S. mormonia)
analyses, but not by all of the analyses. Many species were
present in unresolved polytomies that included other taxa,
or were resolved into clades that were not monophyletic.
Some species were more solidly supported than others as
not monophyletic; most notable were S. atlantis and S.
zerene, each consistently supported as polyphyletic, or
included in multiple clades that were not resolved by our
analyses.
S. zerene is not supported as monophyletic
None of our analyses supported the monophyly of the focal
species, S. zerene. In the COII analyses, S. zerene was
strongly supported as polyphyletic, with two clades (M1
and M2) that were themselves paraphyletic (Fig. 2, left). In
clade M1 the paraphyly was caused by the inclusion of a
single haplotype of S. callippe. Clade M1 was contained in
another clade that also included S. hollandi,S. atlantis
sorocko,S. atlantis dodgei, other S. callippe individuals,
and S. egleis (Fig. 2, left). Another S. zerene (spp. hip-
polyta from Westport, WA) resolved with S. callippe. Most
of our S. zerene specimens were resolved in clade M2,
which contained two clades that we refer to as M3 and M4
(Fig. 2, left). In clade M3, a subset of S. zerene would be
monophyletic except for the inclusion of five individuals of
S. coronis snyderi. Clade M4 consisted primarily of
S. zerene but also included S. atlantis subspecies and S.
egleis.
The concatenated nuclear analysis (Fig. 2, right) had a
large polytomy that did not resolve all S. zerene taxa.
However, it did resolve two clades that were predominantly
S. zerene. One of these, N1, while weakly supported
(posterior probability =0.52), contained individuals of all
S. zerene subspecies in our dataset except S. z. hippolyta.
(S. z. bremnerii is not included because this taxon was
represented by only a single nuclear gene.) Clade N1 also
included one S. callippe (of three in the dataset) and two S.
atlantis hesperis individuals. The second clade, N2,
included all five S. z. hippolyta for which we amplified
more than one nuclear marker. However, this clade of
S. z. hippolyta was paraphyletic; it included a monophyletic
clade of seven S. cybele. Nine of the S. zerene in this
dataset were in neither clade N1 nor N2, but were in an
unresolved polytomy that contained clades N1 and N2
(Fig. 2, right).
In the full concatenated analysis (Fig. 4), relationships
among S. zerene individuals reflected the influence of
signals from both the nuclear and mitochondrial data
(Fig. 2). There was one strongly supported monophyletic
clade of S. zerene, A1 (Fig. 4), that included all taxa
resolved in clade M1 of the COII analysis (Fig. 2, left). A
second strongly supported clade, A2 (Fig. 4), included all
S. zerene taxa in clade M3 (Fig. 2, left), including a S.
coronis snyderi individual that rendered S. zerene para-
phyletic. A third clade, A3 (Fig. 4), contained taxa that
corresponded to clade M4 (Fig. 2, left) and was paraphy-
letic, including the two S. atlantis hesperis individuals in
the analysis.
J Insect Conserv
123
Fig. 2 Mirrored COII (left) and nuclear concatenated (right) 50 % majority rule consensus phylogenies from Bayesian analyses. Branch width is proportional to posterior probabilities of clades,
with the widest branches equivalent to probabilities[.95. Scale bar on tree represents numbers of nucleotide substitutions per site. S. zerene in blue;S. z. hippolyta in red. Photos display ventral
morphology on the left and dorsal morphology on the right. They are scaled for relative size; scale bars in photos represent 1 cm. (Color figure online)
J Insect Conserv
123
Mitochondrial and nuclear data differ in support
of monophyly of S. zerene hippolyta
There was a striking disparity between the mitochondrial
and nuclear analyses in their degree of support for the
monophyly of S. z. hippolyta (Fig. 2). In the COII analysis,
S. z. hippolyta was strongly supported as polyphyletic, with
some individuals falling into clade M1 (those from popu-
lations at Rock Creek, Bray Point, Lake Earl, and Boiler
Bay), and others falling into clade M3 (from populations at
Cascade Head, Mt. Hebo, Cape Meares, and other indi-
viduals from the Boiler Bay and Lake Earl populations). In
neither clade M1 nor M3 did S. z. hippolyta resolve as
monophyletic. Moreover, a single individual from West-
port, WA, fell outside of both clades. The average p-dis-
tances between this individual’s sequence and those of S. z.
hippolyta in clades M1 and M3 were 2.8 and 1.6 %,
respectively. In the COII analysis, this individual paired
with S. callippe; it was not represented in our nuclear
analysis.
In contrast, in the analysis of concatenated nuclear data
(Fig. 2, right), all S. z. hippolyta resolved into a single
clade (N2); this clade included S. cybele as well. We
analyzed the wingless dataset independently, because it
0.1
S. hollandi hollandi (Sil)
S. atlantis hesperis (Sil)
S. atlantis sorocko (Rio)
S. mormonia erinna (Och)
S. hollandi hollandi (Sil)
S. atlantis/hollandi (Sil)
S. atlantis hesperis (Sil)
S. mormonia erinna (Och)
S. mormonia erinna (Och)
S. egleis egleis (Sis)
S. zerene platina (Ban)
S. zerene picta (Dea)
S. mormonia artonis (Ste)
S. atlantis dodgei (Sis)
S. atlantis dodgei (Och)
S. atlantis dodgei (Sis)
S. mormonia artonis (Ste)
S. atlantis sorocko (Rio)
S. atlantis cornelia (Rio)
S. zerene hippolyta (Cas)
S. zerene hippolyta (Nor)
S. zerene hippolyta (Roc)
S. zerene hippolyta (Roc)
S. zerene hippolyta (Sad)
S. zerene hippolyta (Rey)
S. cybele leto (Och)
S. cybele leto (Och)
S. cybele leto (Och)
S. cybele leto (Och)
S. cybele leto (Och)
S. cybele leto (Och)
S. cybele leto (Och)
S. zerene hippolyta (Roc)
S. zerene hippolyta (MtH)
S. zerene hippolyta (MtH)
S. zerene hippolyta (Boi)
S. zerene hippolyta (LoB)
S. zerene hippolyta (Roc)
S. zerene bremnerii (BeC)
S. zerene bremnerii (Oly)
S. zerene bremnerii (Thu)
S. zerene picta (Och)
S. zerene platina (Ban)
S. zerene conchyliatus (Dog)
S. coronis snyderi (Ben)
S. zerene platina (Ban)
S. zerene gunderi (Ste)
S. coronis snyderi (Dea)
S. coronis snyderi (Dea)
S. coronis snyderi (Ben) S. zerene picta (Dea) S. zerene gloriosa (Sis)
S. zerene gloriosa (Sis)
S. callippe elaine (Sis)
S. hydaspe hydaspe (Sis)
S. callippe elaine (Sis)
S. egleis egleis (Sis)
S. egleis egleis (Sis)
S. egleis egleis (Sis)
S. callippe elaine (Sis)
S. zerene sinope (Gor)
S. zerene sinope (Lar)
S. zerene gunderi (Ste)
S. zerene picta (Och)
S. zerene platina (Uin)
S. zerene platina (Uin)
S. zerene sinope (Lar)
S. zerene gloriosa (Sis)
S. zerene platina (Ban)
S. zerene conchyliatus (Dog)
S. zerene sinope (Lar)
S. zerene gunderi (Ste)
S. zerene gunderi (Dog)
S. zerene picta (Dea)
S. zerene conchyliatus (Dog)
S. zerene gunderi (Dog)
S. zerene gunderi (Dog)
S. zerene picta (Och)
S. hydaspe sakuntala (Och)
S. hydaspe sakuntala (Och)
S. hydaspe hydaspe (Sis)
S. hydaspe sakuntala (Och)
S. hydaspe hydaspe (Sis)
S. hydaspe sakuntala (Och)
S. mormonia artonis (Ste)
Ar
gy
nnis a
g
la
j
a (Val)
S. atlantis sorocko (Rio)
S. atlantis cornelia (Rio)
Fig. 3 50 % majority rule consensus phylogeny from Bayesian
analysis of the wingless dataset. Branch width is proportional to
posterior probabilities of clades, with the widest branches equivalent
to probabilities [.95. Scale bar represents numbers of nucleotide
substitutions per site. S. zerene in blue;S. z. hippolyta in red. (Color
figure online)
J Insect Conserv
123
included the full set of taxa, including many museum
specimens of S. z. hippolyta. This analysis (Fig. 3) resolved
all the S. z. hippolyta into a single clade that also contained
S. cybele, as well as S. z. bremnerii.
Mean pairwise genetic distances for nuclear and
mitochondrial sequences among the S. z. hippolyta
clades M1 and M3 and S. cybele were consistent with
the phylogenetic relationships just described. Mito-
chondrial p-distances (Table 3a, c) suggest that clades
M1andM3ofS. z. hippolyta differ from one another
just as much as each does from S. cybele. However,
nuclear p-distances between clades M1 and M3 are half
as great as the distances between each clade and S.
cybele (Table 3b, c).
S. coronis snyderi (Dea)
S. coronis snyderi (Dea)
S. coronis snyderi (Ben)
S. hydaspe sakuntala (Och)
S. hydaspe sakuntala (Och)
S. hydaspe hydaspe (Sis)
S. hydaspe hydaspe (Sis)
S. hydaspe hydaspe (Sis)
S. hydaspe sakuntala (Och)
S. hydaspe sakuntala (Och)
S. cybele leto (Och)
S. cybele leto (Och)
S. cybele leto (Och)
S. cybele leto (Och)
S. cybele leto (Och)
S. cybele leto (Och)
S. cybele leto (Och)
S. mormonia artonis (Ste)
S. mormonia artonis (Ste)
S. mormonia artonis (Ste)
S. mormonia erinna (Och)
S. mormonia erinna (Och)
S. mormonia erinna (Och)
S. coronis snyderi (Ben)
S. zerene platina (Ban)
S. zerene picta (Och)
S. zerene picta (Och)
S. zerene picta (Och)
S. zerene picta (Uin)
S. zerene platina (Ban)
S. zerene gloriosa (Sis)
S. zerene gloriosa (Sis)
S. zerene gloriosa (Sis)
S. zerene picta (Dea)
S. zerene sinope (Lar)
S. zerene sinope (Lar)
S. zerene hippolyta (Cas)
S. zerene hippolyta (MtH)
S. zerene hippolyta (MtH)
S. atlantis hesperis (Sil)
S. atlantis hesperis (Sil)
S. atlantis sorocko (Rio)
S. atlantis sorocko (Rio)
S. atlantis sorocko (Rio)
S. atlantis cornelia (Rio)
S. atlantis cornelia (Rio)
S. atlantis cornelia (Rio)
S. zerene picta (Dea)
S. zerene picta (Dea)
S. zerene gunderi (Dog)
S. zerene gunderi (Dog)
S. zerene gunderi (Dog)
S. zerene gunderi (Ste)
S. zerene gunderi (Ste)
S. zerene gunderi (Ste)
S. zerene hippolyta (Roc)
S. zerene hippolyta (Roc)
S. zerene sinope (Gor)
S. zerene sinope (Lar)
S. egleis egleis (Sis)
S. egleis egleis (Sis)
S. callippe elaine (Sis)
S. callippe elaine (Sis)
S. atlantis dodgei (Och)
S. atlantis dodgei (Sis)
S. atlantis dodgei (Sis)
S. egleis egleis (Sis)
S. zerene conchyliatus (Dog)
S. zerene conchyliatus (Dog)
S. zerene conchyliatus (Dog)
S. hollandi hollandi (Sil)
S. hollandi hollandi (Sil)
S. callippe elaine (Sis)
S. egleis egleis (Sis)
0.3
S. zerene platina (Ban)
S. zerene platina (Ban)
S. zerene platina (Uin)
Brenthis daphne (PdN)
Argynnis aglaja (Val)
S. atlantis/hollandi (Sil)
A1
A3
A2
Fig. 4 50 % majority rule consensus phylogeny from Bayesian
analysis for the full concatenated dataset. Branch width is propor-
tional to posterior probabilities of clades, with the widest branches
equivalent to probabilities [.95. Scale bar represents numbers of
nucleotide substitutions per site. S. zerene in blue;S. z. hippolyta in
red. (Color figure online)
J Insect Conserv
123
Monophyly of other Speyeria subspecies
Few subspecies of any other Speyeria species were sup-
ported as monophyletic. However, few of these taxa were
sufficiently well-sampled to allow a meaningful test
(Table 1). Interestingly, despite a lack of broad sampling,
all S. zerene subspecies except S. z. conchyliatus are
polyphyletic in the COII analysis with individuals in clades
M1, M3, and/or M4. S. z. conchyliatus is paraphyletic with
respect to the inclusion of S. z. gunderi in a monophyletic
polytomy (Fig. 2, left). The conchyliatus ?gunderi group
was unresolved by the nuclear data analysis.
Subspecies of some other Speyeria species were con-
sistently paired into clades that may provide insight into
their taxonomic affinities. For example, S. atlantis sorocko
was never resolved with other S. atlantis, but they were
supported as a sister taxon to S.hollandi in the mito-
chondrial, the nuclear and the full concatenated analyses
(Figs. 2,4).
Evidence of Wolbachia in the Speyeria lineage
Of 66 individuals screened for the Wolbachia 16S gene, we
amplified two positive PCR products, one each from S. z.
gunderi and S. z. picta (‘‘Appendix 1’’). Both were sub-
mitted for sequencing, and one provided clean sequence
(contact authors for accession number). The top 100 best
matches from NCBI BLAST searches of this sequence had
98–99 % identity (e-values =0) with a strain of Wolba-
chia pipientis. All of the matches that were annotated were
isolated from insect hosts.
Discussion
Western North American species of Speyeria are notable
for their complex and often subtle morphological varia-
tion and for the difficulty they present for making accu-
rate determinations of species and subspecies (Pyle 2002;
Dunford 2009). Our analysis of patterns of mitochondrial
and nuclear DNA variation does not provide a tidy res-
olution of this complexity. Patterns suggested by an
analysis of the mitochondrial COII gene were rarely
confirmed by an examination of nuclear genes. Nuclear
genes that have proven to be useful markers in other
Lepidoptera provided little phylogenetically informative
variation, leaving relationships among many taxa unre-
solved. Different genes showed considerable variation in
their ease of amplification, causing different analyses to
contain different subsets of individuals. DNA from
museum specimens proved difficult to extract or amplify,
reducing the taxon sample for our target group, S. z.
hippolyta. Nevertheless, we are able to draw some useful
insights about S. z. hippolyta and the larger group to
which it belongs.
Aside from S. cybele (clearly supported as monophyletic
by our COII analysis), our analyses provide little support
for the nominal species currently recognized for Speyeria,
and even less support for the many subspecies designa-
tions. In many cases, the lack of pattern we have found
should be regarded as quite tentative, because some taxa
are represented by only a few individuals. However, even
the species for which we have the largest sample, S. zerene,
fails to emerge as a distinct group in any of our analyses.
Interestingly, both the nuclear and mtDNA analyses sug-
gest the possible existence of a previously unrecognized
monophyletic group composed of S. atlantis sorocko and S.
hollandi hollandi, including a putative hybrid between the
two. But as a general rule, there is little molecular support
for most of the nominal taxa in our sample.
These genetic findings stand in contrast to the subtle but
distinctive morphological differences recognized by
Speyeria experts and used to make consistent species and
subspecies identifications. This contrast suggests that the
evolutionary history of Speyeria in North America may be
quite recent, allowing little opportunity for fixed molecular
markers to diverge within lineages. Barriers to inter-
breeding in this group may be the consequence of mor-
phological, behavioral or ecological traits that are expected
Table 3 Uncorrected mean p-distances within (shaded) and among
(unshaded) groups of individuals that resolved in clades M1 and M3
of S. zerene hippolyta (a) for COII and (b) for the concatenated
nuclear dataset. (c) Mean p-distances within cybele (shaded) and
between cybele and hippolyta clades (unshaded) for COII and the
concatenated nuclear dataset
a
COII Clade M1 Clade M3
Clade M1 0.21
Clade M3 3.31 0.12
b
Nuc all Clade M1 Clade M3
Clade M1 0.44
Clade M3 0.42 0.30
c
cybele COII cybele
nuclear
Clade M1 4.00 0.89
Clade M3 2.91 0.73
cybele 0.36 0.05
J Insect Conserv
123
to evolve more rapidly than neutral traits because they are
driven by selection. In such cases we would expect neutral
genetic variation to display a pattern consistent with
incomplete lineage sorting, as observed here (Forister et al.
2008).
The existence of mtDNA patterns that are discordant
with patterns of nuclear DNA raises questions about the
origins of these discordances and their implications (Toews
and Brelsford 2012). At least four mechanisms could
contribute to discordances. First, as discussed above, they
could be the result of incomplete lineage sorting. Secondly,
they could be caused by introgression of mtDNA haplo-
types into populations through hybridization. Because
females are the heterogametic sex in Lepidoptera, it is
expected, according to Haldane’s Rule, that females that
result from interspecific hybridization will experience
reduced viability relative to males. For this reason it is
thought that Lepidoptera will be less prone to the intro-
gression of maternally inherited genetic material when
hybridization occurs (Sperling 2003). However, clear cases
of mitochondrial introgression have been reported among
Lepidoptera (Forister et al. 2008; Gompert et al. 2008;
Zakharov et al. 2009). Given that some Speyeria localities
have as many as eight sympatric species (Hammond 1974),
and that some species, such as S. zerene, are considered
relatively vagile (Hammond 1974), hybridization is a
potentially important process in this group. Hammond and
McCorkle (personal communication, Feb. 25, 2012) report
that approximately 1/1,000 Speyeria individuals observed
in the field appear to be hybrids on morphological grounds.
Models of hybridization (Chan and Levin 2005) have
shown that even occasional long-distance dispersal by a
single migrant can lead to introgression. S. z. gloriosa is
thought to be capable of migrating 80–160 km during its
flight season (Hammond and McCorkle personal commu-
nication, Feb. 25, 2012).
A third possible source of discordance between mtDNA
and nuclear variation patterns is Wolbachia infection. This
bacterial symbiont is increasingly being recognized as
posing a particular challenge to genetic studies of Lepi-
doptera and other arthropods (Nice et al. 2009). By con-
ferring cytoplasmic incompatibility (Werren et al. 2008),
Wolbachia infection can drive maternally inherited traits in
the mitochondrial genome to spread through populations,
causing patterns of mtDNA variation to depart from
expectations (Hurst and Jiggins 2005; Galtier et al. 2009).
Our data indicate that Wolbachia infection is present in at
least some populations of S. zerene.
Heteroplasmy, the possession of multiple mitochondrial
haplotypes by a single individual, represents a fourth
potential source of mtDNA and nuclear DNA discordance.
If PCR selectively amplifies only one of the possible
haplotypes present in an individual, mtDNA will be a poor
reflection of the true species tree. Nearly half of the bee
species surveyed by Magnacca and Brown (2010) exhibited
some degree of heteroplasmy for the COI barcoding gene.
Additional data, perhaps obtained through pyrosequencing
(White et al. 2005), are necessary to distinguish among
these possible sources of discordance between mtDNA and
nuclear DNA patterns.
As is true for the Karner blue butterfly (Gompert et al.
2006), our analysis suggests that COII does not accurately
represent species and subspecies-level genetic relationships
within Speyeria. Insofar as COII is closely linked to the
classic barcode region of COI, Speyeria joins a growing list
of taxa for which COI may not be a particularly useful
‘‘barcode’’ marker (Wahlberg et al. 2003a; Roe and Sper-
ling 2007; Forister et al. 2008).
Our primary motivation for this study was to obtain
clearer information about the phylogenetic status of the
threatened Speyeria zerene hippolyta, in order to assess the
appropriateness of its current classification as an ESU. Our
results for this group were particularly perplexing. Bayesian
analysis of the wingless gene resolves all of the S. z. hippo lyta
individuals and their morphologically-similar geographical
neighbor S. z. bremnerii into a single clade with posterior
probability =1.0. The concatenated nuclear data, whose
phylogenetic pattern reflects that of wingless, also resolves
the five S. z. hippolyta included in this analysis into a single
weakly-supported clade, N2 (posterior probability =0.55).
However, interpretation of the N2 clade is complicated by
the fact that it also includes seven individuals of S. cybele.
While S. z. hippolyta is a small-winged, sexually-mono-
morphic butterfly, S. cybele is one of the largest Speyeria
species and is sexually dimorphic in the western U.S.A.
(Hammond 1978). It is likely that S. cybele’s inclusion with
S. z. hippolyta in the N2 clade was driven by their shared
pattern for a single marker, wingless, which could be the
result of convergence in this relatively invariant gene. No
analyses of other individual genes, nuclear or mitochondrial,
supported a S. cybele–S. z. hippolyta clade.
Results for the COII tree are quite different. Here,
individuals of S. z. hippolyta are represented in two distinct
clades whose average sequences differ by more than 3 %,
more than each differs from some other nominal species.
Furthermore, neither clade consists solely of S. z. hippol-
yta; each also includes five other subspecies of S. zerene
and two other nominal Speyeria species. In addition, mean
p-distances of the concatenated nuclear genes are similar
within and among S. z. hippolyta individuals that are
resolved in the mitochondrial clades M1 and M3 (Table 2).
That these p-distances are relatively high is apparent from
the long branches of the nuclear concatenated tree (Fig. 2,
right). Combined, these data suggest that there is some
genetic variability within and among S. z. hippolyta
populations.
J Insect Conserv
123
However, the pattern created by these data is not strong
enough to override other evidence supporting S. z. hip-
polyta’s status as a distinct ESU. This group displays
specific morphological, developmental, and ecological
traits that McCorkle and Hammond (1988) described as
adaptations to the salt-spray meadows and windswept
headlands that characterize its coastal habitat. Unfortu-
nately, we were unable to draw any inferences about the
two other sensitive subspecies of S. zerene,S. z. behrensii
and S. z. myrtleae. Our samples of these taxa were com-
prised only of museum specimens, and we were unable to
amplify genes from any of them.
Though our results do not call into question’s S. z.
hippolyta’s status as an ESU, they do have significant
implications for current management practices for this
group. Currently, individuals from the large, stable popu-
lation at Mt. Hebo are being captively reared and released
at Cascade Head and Rock Creek/Bray Point to augment
the much smaller, declining populations there. Our study
detected differences between the mitochondrial DNA
haplotypes of the Mt. Hebo and Rock Creek populations,
differences that in the future could either be erased by these
augmentations or that might render the augmentations
ineffective, if the genetic differences are great enough to
provide barriers to interbreeding. Work in RVB’s labora-
tory is currently underway to determine what proportion of
the Rock Creek population retains its distinctive mtDNA
haplotype as opposed to having acquired the haplotype of
the captively-reared individuals used for augmentation.
Our provocative finding that some populations of S.
zerene are infected with Wolbachia raises additional con-
cerns about population augmentations. While none of our
S. z. hippolyta samples scored positively for Wolbachia,it
is possible that we failed to detect Wolbachia in some
infected individuals. Introducing Wolbachia-infected indi-
viduals into an uninfected population temporarily reduces
its effective population size (until the infection is fixed or
extinct), and thus could cause augmentation to have the
opposite of its intended effect (Nice et al. 2009). Our
screen was only preliminary and did not provide a com-
prehensive survey of Wolbachia infection. A more com-
prehensive screening of both extant and extinct populations
of S. z. hippolyta is currently underway (Amy Truitt per-
sonal communication, Aug. 15 2011).
In conclusion, the recent ancestry that seems to char-
acterize this group of butterflies creates challenges for
delineation of ESUs. For several taxa in this group,
knowledge of ESU boundaries has important implications
for management actions. It is possible that ESU determi-
nation in Speyeria might be aided by the application of an
integrative taxonomic approach (Crandall et al. 2000;
Dayrat 2005; Roe and Sperling 2007; Padial and De la Riva
2010; Schlick-Steiner et al. 2010), combining molecular
data with information about the morphological, ecological,
and geographic variation of these taxa. However, in a
group as evolutionarily dynamic as Speyeria appears to be,
even the combination of these approaches is unlikely to
produce a phylogeny characterized by reciprocally mono-
phyletic taxa, particularly at the subspecies level (Roe and
Sperling 2007). In such a situation, it may be best to err on
the side of caution when making conservation decisions for
the Oregon silverspot.
Acknowledgments We benefited from Paul Hammond’s and David
McCorkle’s many years of studying Speyeria in the field and labo-
ratory, and the generosity with which they have shared their time,
samples, knowledge, and insights. Other individuals and institutions
providing materials or other assistance include Andrew Warren and
the McGuire Center for Lepidoptera and Biodiversity; Chris Marshall,
Dana Ross and the Oregon State University Arthropod Collection;
Niklas Wahlberg; Gary Albright and the Tillamook County Pioneer
Museum; Mike Patterson; Anne Warner of United States Fish and
Wildlife Service; Debbie Pickering of The Nature Conservancy; and
Mary Jo Anderson and David Shepherdson at the Oregon Zoo. We are
grateful for the field assistance of Charlie Blackmar, Megan Siefert,
Ryan Essman, Amanda Delzer, Steven Levitte, Becca Salesky, Terry
Stratton, Marrissa Hirt, and Beka Feathers, and for the advice and
assistance of Pamela Zobel-Thropp, Elise Maxwell and Wendy
McLennan. Work at Lewis & Clark College was generously sup-
ported by Oregon Zoo’s Future for Wildlife Program, James Dunford,
and Lewis & Clark College. Data collection and initial analysis by
RVB was supported by the Center for Population Biology, University
of California, Davis.
Appendix 1
See Table 4.
Table 4 Nominal taxa whose DNA was isolated at LC, their provenance, accession numbers, and genes successfully amplified
Taxon Collection Location
(abbreviation used
in trees)
COII CAD GAPDH RpS5 TPI Wingless Wolbachia
testing
(4
denotes
positive result
Full Taxon
identifier
Argynnis aglaja Vallentuna, Stockholmsla
¨n (Val) •• • NW76-15
Brenthis daphne Pic de Nore (PdN) •• NW907080101
S. atlantis cornelia Rio Blanco Co., CO (Rio) •••• PHC-1
J Insect Conserv
123
Table 4 continued
Taxon Collection Location
(abbreviation used
in trees)
COII CAD GAPDH RpS5 TPI Wingless Wolbachia
testing
(4
denotes
positive result
Full Taxon
identifier
S. atlantis cornelia Rio Blanco Co., CO (Rio) •••• PHC-2
S. atlantis cornelia Rio Blanco Co., CO (Rio) •••• PHC-3
S. atlantis dodgei Ochoco Mountains, OR (Och) ••••• LC907180404
S. atlantis dodgei Siskiyou Mountains, OR (Sis) •• • • •• • LC908010205
S. atlantis dodgei Siskiyou Mountains, OR (Sis) •••••• LC908010505
S. atlantis hesperis Silver Lake, MT (Sil) ••• • • LC908010101
S. atlantis hesperis Silver Lake, MT (Sil) ••• • • LC908010102
S. atlantis sorocko Rio Blanco Co., CO (Rio) •••• PHS-1
S. atlantis sorocko Rio Blanco Co., CO (Rio) •••• PHS-2
S. atlantis sorocko Rio Blanco Co., CO (Rio) •••• PHS-3
S. atlantis/hollandi Silver Lake, MT (Sil) •••• LC908010103
S. callippe elaine Siskiyou Mountains, OR (Sis) ••• • •• • LC908010204
S. callippe elaine Siskiyou Mountains, OR (Sis) •••••• LC908010221
S. callippe elaine Siskiyou Mountains, OR (Sis) •••••LC908010226
S. coronis snyderi Bennett Hills, ID (Ben) ••••• LC907110101
S. coronis snyderi Bennett Hills, ID (Ben) ••••• LC907110102
S. coronis snyderi Dearborn River, MT (Dea) ••• • • LC907310102
S. coronis snyderi Dearborn River, MT (Dea) •••• LC907310104
S. cybele leto Ochoco Mountains, OR (Och) •••• LC907180201
S. cybele leto Ochoco Mountains, OR (Och) •••• LC907180203
S. cybele leto Ochoco Mountains, OR (Och) •••• LC907180204
S. cybele leto Ochoco Mountains, OR (Och) •••• LC907180205
S. cybele leto Ochoco Mountains, OR (Och) ••••• LC907180207
S. cybele leto Ochoco Mountains, OR (Och) ••• • •• LC907180208
S. cybele leto Ochoco Mountains, OR (Och) •• • LC907180209
S. cybele leto Ochoco Mountains, OR (Och) •LC907180217
S. cybele leto Ochoco Mountains, OR (Och) •LC907180219
S. egleis egleis Siskiyou Mountains, OR (Sis) ••••• LC908010206
S. egleis egleis Siskiyou Mountains, OR (Sis) •• • • LC908010217
S. egleis egleis Siskiyou Mountains, OR (Sis) •••••• LC908010404
S. egleis egleis Siskiyou Mountains, OR (Sis) ••••• LC908010515
S. hollandi hollandi Silver Lake, MT (Sil) ••••• LC908010105
S. hollandi hollandi Silver Lake, MT (Sil) ••• • • LC908010106
S. hydaspe hydaspe Siskiyou Mountains, OR (Sis) •••••• LC908010223
S. hydaspe hydaspe Siskiyou Mountains, OR (Sis) ••••• LC908010407
S. hydaspe hydaspe Siskiyou Mountains, OR (Sis) •••••• LC908010513
S. hydaspe sakuntala Ochoco Mountains, OR (Och) •••• LC907180101
S. hydaspe sakuntala Ochoco Mountains, OR (Och) ••• • •• LC907180401
S. hydaspe sakuntala Ochoco Mountains, OR (Och) ••••• LC907180402
S. hydaspe sakuntala Ochoco Mountains, OR (Och) •• • LC907180403
S. mormonia artonis Steens Mountain, OR (Ste) •••• LC908080101
S. mormonia artonis Steens Mountain, OR (Ste) ••••• LC908080306
S. mormonia artonis Steens Mountain, OR (Ste) ••••• LC908080401
S. mormonia erinna Ochoco Mountains, OR (Och) ••• • •• LC907180206
S. mormonia erinna Ochoco Mountains, OR (Och) •••• LC908090232
J Insect Conserv
123
Table 4 continued
Taxon Collection Location
(abbreviation used
in trees)
COII CAD GAPDH RpS5 TPI Wingless Wolbachia
testing
(4
denotes
positive result
Full Taxon
identifier
S. mormonia erinna Ochoco Mountains, OR (Och) •••• LC908090503
S. zerene bremnerii Benton County, OR (BeC) •• OSU121594
S. zerene bremnerii Thurston County, WA (Thu) •• OSU121600
S. zerene bremnerii Olympic Mountains, WA (Oly) •• OSU121615
S. zerene conchyliatus Dog Lake, OR (Dog) ••••• LC908030102
S. zerene conchyliatus Dog Lake, OR (Dog) ••••• LC908030105
S. zerene conchyliatus Dog Lake, OR (Dog) ••• • • LC908030106
S. zerene gloriosa Siskiyou Mountains, OR (Sis) •••••• LC908010111
S. zerene gloriosa Siskiyou Mountains, OR (Sis) •••••• LC908010401
S. zerene gloriosa Siskiyou Mountains, OR (Sis) ••• • • • LC908010514
S. zerene gunderi Dog Lake, OR (Dog) ••• • •• • LC908030101
S. zerene gunderi Dog Lake, OR (Dog) ••••• LC908030103
S. zerene gunderi Dog Lake, OR (Dog) ••• • •• 4LC908030104
S. zerene gunderi Steens Mountain, OR (Ste) ••••• LC908080104
S. zerene gunderi Steens Mountain, OR (Ste) ••• • •• LC908080207
S. zerene gunderi Steens Mountain, OR (Ste) ••• • • LC908080209
S. zerene hippolyta Cascade Head, OR (Cas) •• • • •• • LC208270101
S. zerene hippolyta Rock Creek, OR (Roc) ••• • •• • LC208280101
S. zerene hippolyta Rock Creek, OR (Roc) ••• • •• • LC208280102
S. zerene hippolyta Mt. Hebo, OR (MtH) ••••• LC309180101
S. zerene hippolyta Mt. Hebo, OR (MtH) •• • •• • LC509020102
S. zerene hippolyta Long Beach, WA (LoB) •• OSU140631
S. zerene hippolyta Del Rey Beach, OR (Rey) •• OSU140632
S. zerene hippolyta Saddle Mt., OR (Sad) •• OSU140636
S. zerene hippolyta Boiler Bay, OR (Boi) •• OSU140639
S. zerene hippolyta Rock Creek, OR (Roc) •• OSU140641
S. zerene hippolyta Rock Creek, OR (Roc) •• OSU140642
S. zerene hippolyta Del Norte, CA (Nor) •• OSU140643
S. zerene picta Dearborn River, MT (Dea) ••• • •• 4LC907310101
S. zerene picta Dearborn River, MT (Dea) ••••• LC907310105
S. zerene picta Dearborn River, MT (Dea) ••••• LC907310109
S. zerene picta Ochoco Mountains, OR (Och) •••••• LC908090505
S. zerene picta Ochoco Mountains, OR (Och) •••••• LC908090513
S. zerene picta Ochoco Mountains, OR (Och) ••• • • • LC908090603
S. zerene platina Uinta Mountains, OR (Uin) ••• •• • LC907130101
S. zerene platina Uinta Mountains, OR (Uin) ••• • •• • LC907130102
S. zerene platina East Bannock Pass, MT (Ban) ••• • • • LC907310102
S. zerene platina East Bannock Pass, MT (Ban) •••••• LC907310106
S. zerene platina East Bannock Pass, MT (Ban) •••••• LC908020101
S. zerene platina East Bannock Pass, MT (Ban) •••• LC908020105
S. zerene sinope Gore Pass, CO (Gor) ••••• LC907170101
S. zerene sinope Laramie Mountains, WY (Lar) •• • • •• LC907200101
S. zerene sinope Laramie Mountains, WY (Lar) ••••• LC907200102
S. zerene sinope Laramie Mountains, WY (Lar) ••••• LC907200103
Identifications by Paul Hammond and David McCorkle. Sequences provided by RVB not included
J Insect Conserv
123
Appendix 2
See Table 5.
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