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mtDNA introgression in Erynnis
Notes on Erynnis natural history and species delimitation
2
3
The genus Erynnis Schrank, 1801 is composed of a large number of species, totaling 17
4
taxa in the United States and Canada (Pelham, 2008). All Erynnis are medium-sized and dark
5
brown in color, usually with hoary grey scaling on the forewings, diffuse yellow spots on the
6
margin of the hindwings, and a number of small white or translucent marks on the forewings.
7
Apart from these markings, the wing patterns vary in slightly lighter or darker shades of grey.
8
(Layberry et al., 1998).
9
Differentiated forms of Erynnis that are morphologically most similar to one another are
10
always entirely or largely allopatric and collectively wide-ranging (Burns, 1964). In this study
11
we follow Erynnis classification given by Burns (1964), whose revision was based on
12
morphological, geographical, and ecological grounds and critical examination of ~ 11,000 adults
13
of thirty New World members of the genus Erynnis. Two species of particular interest in this
14
study (see below), E. propertius and E. horatius, are combined by Burns (1964) with five other
15
species (E. juvenalis, E. telemachus, E. meridianus, E. scudderi, E. tristis) to form the E.
16
juvenalis species group (Pelham, 2008).
17
Burns (1964) suggested that the nearest relative of E. propertius is E. meridianus, an
18
allopatric species of the New Mexican SW and Mexico. Burns (1964) further specified that E.
19
propertius is more closely related to E. juvenalis than it is to E. tristis and that the nearest
20
relative of E. tristis is the allopatric and mostly eastern species E. horatius.
21
E. propertius feed exclusively on Quercus and range from northern Baja California,
22
Mexico, through California, Oregon, and Washington in the United States, and find their
23
northern boundary in southeast British Columbia, Canada, including portions of Vancouver
24
Island and the neighbouring Gulf Islands. Fully grown larvae hibernate, and adults fly February
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mtDNA introgression in Erynnis
25
to July depending on latitude and elevation. The species generally has one generation per year
26
but is known to produce a partial second brood in middle to late summer in California (A.M.
27
Shapiro and S. Pelini, personal communication). This relatively small (wingspan = 4 cm) species
28
does not occur in deserts or hot central valleys and is restricted to open oak woodlands, forest
29
openings and edges, meadows and fields where Quercus co-occur with flowering plants (Scott
30
1986; Guppy and Sheppard, 2001).
31
E. horatius also feed on Quercus, including Q. phellos, Q. velutina, Q. nigra, Q. stellata
32
and Q. virginiana (Burns, 1964). Its distribution extends from Massachusetts west to eastern
33
South Dakota; south through most of the eastern United States to Florida, the Gulf Coast, and
34
South Texas; south in the west through southeastern Utah, Colorado, northeastern Arizona, and
35
New Mexico.
36
E. propertius and E. horatius are allopatric and have clear morphological differences,
37
including distinct structure of male genitalia (Burns, 1964). Although reproductive isolation of
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these taxa has not been tested experimentally, circumstantial evidence is more than convincing
39
that E. propertius and E. horatius cannot be treated as conspecific under any major species
40
concept.
41
Classification of the E. juvenalis group is reproduced from Burns (1964)
42
Erynnis Schrank, 1801
43
I.
44
45
46
47
Subgenus Erynnis
[4 Eurasian species and 2 New World species]
II.
Subgenus Erynnides
Species group “juvenalis” group Burns, 1964
(Superspecies Erynnis juvenalis Burns, 1964)
3
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mtDNA introgression in Erynnis
Erynnis juvenalis (Fabricius, 1793)
49
Erynnis juvenalis juvenalis (Fabricius, 1793)
50
Erynnis juvenalis clitus (W.H. Edwards, 1883)
51
Erynnis telemachus Burns, 1960
52
(Superspecies Erynnis propertius Burns, 1964)
53
Erynnis propertius (Scudder and Burgess, 1870)
54
Erynnis meridianus E. Bell, 1927
55
Erynnis meridianus meridianus E. Bell, 1927
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Erynnis meridianus fieldi Burns, 1964
57
Erynnis scudderi (Skinner, 1914)
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(Superspecies Erynnis tristis Burns, 1964)
59
Erynnis horatius (Scudder and Burgess, 1870)
60
Erynnis tristis (Boisduval, 1852)
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Erynnis tristis tristis (Boisduval, 1852)
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Erynnis tristis tatius (W.H. Edwards, 1883)
63
Erynnis tristis pattersoni Burns, 1964]
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65
Supplementary Materials and Methods
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67
The computer program MODELTEST version 3.7 (Nylander, 2004) in combination with
68
PAUP* 4.10b was used to select an appropriate substitution model for each of the molecular data
69
partitions (i.e., ND5 and wingless). The substitution model with the best fit to the data was
70
chosen based on Akaike Information Criterion (AIC) (Akaike, 1974). All parameters for the
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mtDNA introgression in Erynnis
71
nucleotide substitution model indicated by AIC were estimated as part of the subsequent analysis
72
in MrBayes version 3.1.2 (Huelsenbeck and Ronquist, 2001).
73
All Bayesian analyses included two separate concurrent MCMC runs, each composed of
74
four chains, three heated and one cold (see Huelsenbeck and Ronquist, 2001). Each Markov
75
chain was started from a random tree and run for up to 5  106 generations, sampling the chains
76
every 100th cycle. At the end of each run we considered the sampling of the posterior
77
distribution to be adequate if the average standard deviation of split frequencies was < 0.01. The
78
log-likelihood scores of sample points were plotted against generation time to determine when
79
the chain became stationary. After discarding the “burn-in” samples (Huelsenbeck and Bollback,
80
2001), MCMC runs were summarized and further investigated for convergence of all parameters,
81
using sump and sumt commands in MrBAYES and the computer program TRACER version 1.4
82
(Rambaut and Drummond, 2005). Data remaining after discarding burn-in samples were used to
83
generate a majority-rule consensus tree where the percentage of samples recovering any
84
particular clade of the consensus tree represented the clade’s posterior probability (Huelsenbeck
85
and Ronquist, 2001). Probabilities of 95% or higher were considered significant support. The
86
mean, variance, and 95% credibility intervals were calculated from the set of substitution
87
parameters.
88
To distinguish between incomplete lineage sorting and lack of resolution as the cause of
89
non-monophyly in E. horatius and E. tristis on the wingless phylogeny (see results), we used
90
Bayes factors to compare the posterior odds of the inferred tree topology to Bayesian trees that
91
forced the reciprocal monophyly of E. tristis and E. horatius. This method differs from
92
traditional hypothesis-testing because it does not offer a criterion for absolute rejection of a null
93
hypothesis, but instead it provides an evaluation of the evidence in favor of the null hypothesis
5
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mtDNA introgression in Erynnis
94
(Kass and Raftery, 1995). Analyses that constrained E. horatius and E. tristis to monophyly
95
were conducted in MrBAYES with an absolute prior (=1.00) using the command prset
96
topologypr = constraint and otherwise the same parameters as the ones used in the unconstrained
97
runs. Using the sump command in MrBAYES, we sampled the stationary (post-burnin) posterior
98
distribution to obtain the harmonic mean of tree likelihood values (following Nylander et al.,
99
2004 and Ronquist et al., 2005). The predictive value of the constrained harmonic mean
100
likelihood (H1) were then compared to the original, unconstrained likelihoods (H0) using a Bayes
101
factor comparison, B10 = Harmonic Mean Ln Likelihood H1 − Harmonic Mean Ln Likelihood H0
102
(Kass and Raftery, 1995).
103
104
Supplementary Results
105
106
Selection of substitution model
107
108
For the mtDNA dataset using AIC, MODELTEST identified a general time reversible
109
model of nucleotide substitution with gamma (γ) distributed rates and a proportion of invariable
110
sites (GTR + I + G). Estimates of substitution rates under this model are A→C, 0; A→G, 7.5216;
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A→T, 0.3211; C→G, 0.9138; C→T, 0.9473; G→T, 1. The proportion of invariant sites is
112
estimated as 0.474, and the γ distribution shape parameter is 0.941.
113
For the nuclear gene, wingless, MODELTEST indicated a transitional model (Rodríguez
114
et al., 1990) with a proportion of invariant sites and the γ distributed rates (TIM + I + G).
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Estimates of substitution rates under this model are A→C, 1; A→G, 2.2928; A→T, 0.4783;
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C→G, 0.4783; C→T, 6.0278; and G→T, 1. The proportion of invariant sites and the γ
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mtDNA introgression in Erynnis
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distribution shape parameter are estimated as 0.364 and 0.678, respectively. For Bayesian
118
analysis of the wingless dataset, we used GTR + I + G model of nucleotide substitution, given
119
that it is the model available in MrBAYES that best matches the TIM + I + G.
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121
Test of alternative topologies
122
123
When Bayesian analysis was run with enforced reciprocal monophyly of E. horatius and
124
E. tristis, the likelihood of the unconstrained topology (H0, Fig. 3B) was slightly smaller
125
(−1527.40) compared to the alternative constrained topology (−1527.75). Therefore, the
126
reciprocal monophyly of E. horatius and E. tristis was not considered as a possible alternative.
127
There was positive evidence in favour of the nonconstrained topology when only E. tristis was
128
forced to monophyly (lnL(H1) = -1530.08) and slightly positive support in favour of the
129
constraint topology when E. horatius was forced to monophyly (lnL(H1) = -1526.27) (Table
130
SOM-2).
131
132
Microsatellite genotyping
133
134
The primers developed for E. propertius amplified DNA fragments in other species of
135
Erynnis (Zakharov et al., 2007) and thus were used to amplify microsatellite repeats in sampled
136
individuals of E. horatius. Out of five specimens, however, only one had positive amplification
137
across all 14 analyzed loci. The other four individuals had missing data for 2, 3, 3 and 5 loci,
138
with a total of eight loci being affected by missing data in the E. horatius data subset.
139
Comparison of microsatellite data between E. propertius and E. horatius did not reveal any fixed
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mtDNA introgression in Erynnis
140
allelic differences between the two species although species-specific alleles were present in eight
141
loci of E. horatius.
142
Inclusion of data sampled for E. horatius in STRUCTURE analysis did not affect
143
individual clustering and average assignment probabilities for E. propertius. All five individuals
144
of E. horatius were assigned to a mainland cluster(s) of E. propertius with a tested number of
145
assumed populations K = 3 to 8. The likelihood estimate with increasing value of K reached
146
stationarity at K = 5. Specimens of E. horatius did not differentiate as a separate cluster and were
147
grouped with mainland populations of E. propertius under K = 5 which had the best likelihood
148
estimate of the data among the tested values of K = 2 to K = 8.
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mtDNA introgression in Erynnis
Table SOM-1. Evaluating convergence of MCMC runs in Bayesian analyses.
"Burn-In"
ND5
samples in
A.S.D.
each run
S.F.*
0
Wingless
Run 1
LnL
Run 2
S.D.
LnL
S.D.
Included
A.S.D.
Run 1
Run2
samples**
S.F.*
LnL
S.D.
LnL
S.D.
samples**
-
-3910.572
3.382
-3910.834
3.780
10,000,000
-
-1497.687
1.286
-1497.629
1.274
4,000,000
1,000
0.197108
-3909.629
2.451
-3909.918
2.920
9,998,000
0.162217
-1497.222
0.848
-1497.098
0.775
3,998,000
100,000
0.049097
-3907.238
0.479
-3906.999
0.406
9,800,000
0.035732
-1496.463
0.359
-1496.489
0.398
3,800,000
250,000
0.029195
-3907.175
0.492
-3907.016
0.421
9,500,000
0.017894
-1496.707
0.367
-1496.590
0.452
3,500,000
500,000
0.021460
-3907.112
0.606
-3907.102
0.416
9,000,000
0.013452
-1496.474
0.388
-1496.657
0.504
3,000,000
1,000,000
0.015214
-3907.273
0.622
-3907.076
0.441
8,000,000
0.009018
-1496.445
0.502
-1497.062
0.593
2,000,000
2,000,000
0.015803
-3907.293
0.762
-3906.580
0.488
6,000,000
0.007097
-
-
-
-
-
4,000,000
0.009783
-3907.017
0.835
-3906.507
0.810
2,000,000
-
-
-
-
-
-
5,000,000
0.006410
-
-
-
-
-
-
-
-
-
-
-
Note: values in bold indicate selected portion of "burn-in" data
* Average standard deviation of split frequencies (estimated for two independent runs) established at the end of "burn-in" portion of the data
** Number of combined data points used to calculate MCMC run statistics.
149
150
151
152
153
154
155
156
Included
9
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158
Table SOM-2. Summary of Bayes factor comparisons of phylogenetic hypotheses alternative to the topology inferred for the wingless dataset
(H0, Fig. 3B)
Tested constrained hypothesis, H1
E. tristis monophyly
E. horatius monophyly
E. tristis and E. horatius monophyly
159
mtDNA introgression in Erynnis
LnL: constrained (H1)
LnL: unconstrained (H0)
- 1530.08
- 1526.27
- 1527.75
- 1527.40
- 1527.40
- 1527.40
Bayes factor
[2loge(B10)]
- 5.36
2.26
- 0.70
Interpretation of evidence
(Kass and Raftery, 1995)
Positive for H0
Positive for H1
Not worth mentioning
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mtDNA introgression in Erynnis
Table SOM-3. MtDNA haplotypes inferred from same individuals using two or three mtDNA genes
DNA /voucher
sample number
ND5 haplotype
(Zakharov and
Hellmann, 2008)
COI
haplotype
COII
haplotype
Haplogroup
EP002
EP003
EP004
EP005
EP007
EP008
EP010
EP013
EP014
EP015
EP016
EP019
EP020
EP021
EP022
EP023
EP024
EP025
EP026
EP027
EP028
EP029
EP030
EP087
EP089
EP090
EP091
EP092
EP099
EP100
EP103
EP104
EP105
EP106
EP189
EP01
EP01
EP01
EP02
EP01
EP02
EP02
EP01
EP01
EP01
EP01
EP01
EP01
EP01
EP01
EP01
EP02
EP02
EP01
EP01
EP01
EP01
EP01
EP02
EP02
EP01
EP02
EP01
EP01
EP01
EP14
EP03
EP01
EP01
EP03
n/a
n/a
n/a
COI-01
COI-01
COI-01
n/a
n/a
n/a
n/a
n/a
COI-01
COI-01
COI-01
COI-01
COI-01
COI-01
COI-01
COI-01
COI-01
COI-01
n/a
n/a
n/a
COI-01
COI-01
COI-01
COI-01
n/a
n/a
n/a
n/a
n/a
n/a
n/a
COII-01
COII-01
COII-01
COII-01
COII-01
COII-01
COII-01
COII-01
COII-02
COII-01
COII-01
COII-01
COII-01
COII-01
COII-01
COII-01
COII-01
COII-01
COII-01
COII-01
COII-01
COII-01
COII-01
COII-03
COII-01
COII-01
COII-01
COII-01
COII-01
COII-01
COII-01
COII-01
COII-01
COII-01
COII-04
Erynnis propertius
- “” - “” - “” - “” - “” - “” - “” - “” - “” - “” - “” - “” - “” - “” - “” - “” - “” - “” - “” - “” - “” - “” - “” - “” - “” - “” - “” - “” - “” - “” - “” Erynnis propertius
- “” - “” -
11
Supplementary Online Materials
mtDNA introgression in Erynnis
DNA /voucher
sample number
ND5 haplotype
(Zakharov and
Hellmann, 2008)
COI
haplotype
COII
haplotype
Haplogroup
EP190
EP191
EP192
EP009
EP011
EP012
EP101
EP111
EP112
EP115
EP116
EP117
EP118
EP119
EP120
EP01
EP01
EP03
EPRP
EPRP
EPRP
EPRP
EPRP
EPRP
EPRP
EPRP
EPRP
EPRP
EPRP
EPRP
n/a
n/a
n/a
n/a
n/a
n/a
n/a
COI-02
COI-02
COI-02
COI-02
COI-02
COI-02
COI-02
COI-02
COII-05
COII-01
COII-01
COII-06
COII-06
COII-06
COII-06
COII-06
COII-06
COII-06
COII-06
COII-06
COII-06
COII-06
COII-06
- “” - “” - “” Erynnis horatius
- “” - “” - “” - “” - “” - “” - “” - “” - “” - “” - “” -
161
162
Table SOM-4. Average divergence among haplogroups of E. propertius and E. horatius
Fragment
length
sequenced, bp
Number of
specimens
compared
Number of
haplotypes
inferred
Average divergence
between
haplogroups, %
ND5
851
527*
28
5.07
COI
394
30
2
COII
639
49
6
mtDNA gene
163
164
* See (Zakharov and Hellmann, 2008)
5.56
4.87
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166
167
168
169
170
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mtDNA introgression in Erynnis
References
Akaike H (1974). A new look at the statistical model identification. IEEE Transactions on
Automatic Control 19: 716–723.
Burns JM (1964). Evolution in Skipper Butterflies of the Genus Erynnis. Univ California Publ
Entomol 37: 214pp.
171
Guppy CS, Sheppard JH (2001). Butterflies of British Columbia. UBC Press, Vancouver.
172
Huelsenbeck JP, Bollback JP (2001). Empirical and hierarchical Bayesian estimation of ancestral
173
174
175
176
177
178
179
180
181
182
183
184
185
186
187
states. Syst Biol 50: 351-366.
Huelsenbeck JP, Ronquist F (2001). MRBAYES: Bayesian inference of phylogeny.
Bioinformatics 17: 754-755.
Kass RE, Raftery AE (1995). Bayes factors. Journal of the American Statistical Association 90:
773-795.
Layberry R A, Hall PW, Lafontaine JD (1998). Butterflies of Canada. University of Toronto
Press, Toronto.
Nylander JAA (2004). MrModeltest v2. Program distributed by the author. Evolutionary Biology
Centre, Uppsala University.
Nylander JAA, Ronquist F, Huelsenbeck JP, Nieves-Aldre JL (2004). Bayesian phylogenetic
analysis of combined data. Syst Biol 53: 47–67
Pelham JP (2008). A catalogue of the Butterflies of the United States and Canada. J Res Lepid
40: xiv + 658 pp.
Rambaut A, Drummond A (2005). TRACER version 1.3: MCMC trace file analyser. Program
distributed by the authors. (http://evolve.zoo.ox.ac.uk/software.html).
Supplementary Online Materials
188
189
190
191
192
193
194
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Rodríguez FJ, Oliver JL, Marín A, Medina JR (1990). The general stochastic model of
nucleotide substitution. J Theor Biol 142: 485-501.
Ronquist F, Huelsenbeck JP, van der Mark P (2005). MrBayes 3.1 Manual, Draft 5/26/2005,
online at http://mrbayes.csit.fsu.edu/manual.php.
Scott JA (1986). The Butterflies of North America: a Natural History and Field Guide. Stanford
University Press, Stanford, California.
Zakharov EV, Hellmann JJ, Romero-Severson J (2007). Microsatellite loci in the Propertius
195
duskywing, Erynnis propertius (Lepidoptera: Hesperiidae), and related species. Mol Ecol
196
Notes 7: 266-268.
197
Zakharov EV, Hellmann JJ (2008). Genetic differentiation across a latitudinal gradient in two co-
198
occurring butterfly species: revealing population differences in a context of climate
199
change. Mol Ecol 17: 189-208.