SUPPLEMENTARY MATERIAL
Description of host plants
The four host plants surveyed in this study (western mayhaw, blueberry hawthorn,
southern red hawthorn, and green hawthorn) differ from one another in leaf and fruit
morphology, as well as fruiting phenology and fruit volatiles.
The western mayhaw, C. opaca, is morphologically distinguished by its largely
unserrated, and cuneate leaves, and large, globose, and pale-red to pink fruit (see Fig 1 of text).
Its range extends from east Texas to western Mississippi with a northern range limit just above
the Louisiana/Arkansas border (Fig. 2 of text). Western mayhaw is the earliest fruiting hawthorn
species in North America, with fruit typically abscising from trees in late April to mid-May (Fig.
1; Table 1). The fruit of western mayhaw is commercially prized for making jelly in the southern
U.S. The western mayhaw blend differs from other R. pomonella host fruit volatiles in having a
very high concentration of 3-methylbutan-1-ol and possessing the compound pentyl acetate
(Table S1) (Cha et al. 2011a). Among other hawthorns only the green hawthorn and downy
hawthorn fruit blends contain 3-methylbutan-1-ol at levels within an order of magnitude as
western mayhaw fruit (Table S1). The compound 3-methylbutan-1-ol is a shared volatile found
in all hawthorn fruit, and is absent from apple (Table S1). Western mayhaw grows almost
exclusively in swamps and river bottoms, often in standing water.
The blueberry hawthorn, C. brachyacantha, is distinguished by its characteristic dark
blue and subglobose fruit, its small and waxy crenulate-serrated leaves, and its small curved
thorns (Fig. 1) (Sargent, 1933). Its native range is the “piney woods” region of eastern Texas
through much of Louisiana, extending into adjacent counties of Arkansas and Mississippi (Fig.
2). Blueberry hawthorn fruit typically ripens from late August through September (Fig. 1). The
behaviorally active fruit blend for blueberry hawthorns differ from those of other R. pomonella
hosts in having a high concentration of the volatile butyl acetate, a compound that only the
southern red hawthorn blend also contains, but in modest amount (Table S1) (Cha et al. 2011b).
Blueberry hawthorn is found in moist soil on the edge of forests bordering low-lying prairies
(Correll and Johnston, 1970). Phylogenetically, blueberry hawthorn is basal to remainder of the
genus Crataegus (Lo et al. 2010).
The southern red hawthorn, C. mollis var. texana, is a taxonomically distinct variety of
the downy hawthorn, C. mollis (Lance 2011). Southern red hawthorn has larger, more heavily
serrated leaves than the other southern host species and its fruit is relatively large, obovoid, and
bright red (Fig. 1). The fruit of the southern red hawthorn typically ripens in mid-October (Fig.
1). Southern red hawthorn differs from other hawthorns and apple in having butyl propionate and
isoamyl butanoate as active behavioral components in its fruit blend, and shares the compound
butyl acetate with blueberry hawthorn fruit (Table S1) (Cha et al. 2012). Southern red hawthorn
is primarily restricted to the coastal plain of Texas and the bottomland of rivers in east and
central Texas (Fig. 2) (Correll & Johnston, 1970).
The green hawthorn, C. viridis, has the broadest geographic range of any of the southern
hawthorns. It is found from southern Illinois southward to the coastal plain of Texas, eastward to
the Florida panhandle and as far north as the Virginia piedmont (Fig. 2). Green hawthorn is
sparsely distributed through much of its range, only occurring in high densities west of the
Mississippi River (Sargent, 1890). Green hawthorn is comprised of a complex of both sexual and
apomictic individuals (Talent and Dickinson, 2005), and is distinguished from other hawthorns
by having dark green, acute, and ovate leaves together with small, red-orange fruit (Fig. 1)
(Vines, 1977). The green hawthorn fruit blend has moderately high concentrations of 3methylbutan-1-ol and high concentrations of 4,8-dimethyl-1,3(E),7-nonatriene (DMNT) (Table
S1) (Cha et al. 2011b). Only downy hawthorn fruit also possess DMNT, but at trace levels. The
green hawthorn has the latest phenology of southern hawthorns, with fruit ripening in late
October through early December (Fig. 1).
Field collection of R. pomonella
Insects were collected as larvae in infested fruit, using standard R. pomonella collection
procedures. Collections were made when fruit was naturally abscising from trees (which is
accelerated by Rhagoletis infestation). In all cases, fruit was collected directly from the trees and
from the ground. When feasible, fruit was shaken from trees onto a ground cloth, rather than
picked directly.
Sampling coverage differed among sites due to reasonable constraints on workload and
collection capacity at host plant patches of different size. At small sites with a few host trees and
no others in the immediate vicinity, most of the available fruit was collected from all trees. At
medium sized patches with continuous host plant distribution (0.5-20 ha), fruit was collected
along multiple haphazard transects that covered the full breadth of the patch. These patches were
typically bounded either by changes in habitat type, bodies of water, or human development. At
sites too large for such thorough spatial sampling, such as the 25,000 ha Dewey Wills WMA,
fruit was collected along transects surrounding access points over a roughly 0.5km radius. A
previous multiyear study of apple and hawthorn flies found no evidence for significant
microgeographic (among trees within a site) genetic variation within hosts (Feder et al. 1990a).
Rhagoletis flies are highly vagile, capable of flying several kilometers in just 24 hrs, the flies
also disperse and forage for 7 to 10 post eclosion before they become receptive to host fruit cues
(Boller and Prokoy 1976).
Sampling of populations covered a range of four years. Previous detailed studies of apple
and hawthorn-infesting populations of R. pomonella have year to year variation in allele
frequencies within hosts, but these differences have never approached the level of differentiation
between these two hosts (Feder et al. 1990a; Feder et al. 1990b).
Mitochondrial sequence data.
A maximum parsimony mtDNA gene tree was constructed using PHYLIP 3.69
(Felsenstein 2005) to quantify the extent of matrilineal divergence among southern hawthorninfesting populations or R. pomonella. The data used in the gene tree were derived from both
newly-generated and previously published (Feder et al. 2003) sequences of an 864 bp fragment
covering a 3’ partial sequence of the cytochrome oxidase subunit I gene (COI), the entire tRNALeu gene, and a 5’ partial sequence of the cytocrhome oxidase subunit II (COII) gene (See table
B.1 in Appendix B) for details of mtDNA sequences). New mtDNA sequence data was
generated using the forward and reverse primers 5’-ATA CCT CGA CGT TAT TCA GA-3’ and
5’-GAG ACC ATT ACT TGC TTT CAG TCA TCT-3’ (Simon et al. 1993) for five western
mayhaw flies from four sites, five blueberry hawthorn flies from four sites, five green hawthorn
fives from four sites, four southern red hawthorn flies from three sites, and 2 downy hawthorn
flies from a site at the southern edge that host plant’s range. See Table S2 for sample details.
Sequences were aligned using Clustal X 2.1 (Larkin et al. 2007). Sequences from two outgroup
species R. electromorpha and R. pomonella nr., an undescribed partially intrinsically-isolated
species from the highlands of central Mexico (Rull et al. 2010), were used to root the network.
Microsatellite genotyping
Genomic DNA extraction was performed on whole fly bodies using Puregene extraction
kits (Qiagen). Purified DNA was transferred to 96-well plates for PCR amplification of 26
microsatellite loci. Microsatellite loci were previously developed by Velez et al. (2006) from a
GT-repeat enriched, restriction-based subgenome (Hamilton et al. 1999) using R. pomonella flies
from Grant, MI. These loci have been used effectively in previous studies covering the extent of
the latitudinal distribution of R. pomonella in the United States (Michel et al. 2010; Cha et al.
2012) as well as with more distantly diverged (~1.5 Mya; Xie et al. 2008) populations of R.
pomonella nr. in central Mexico (Michel et al. 2007). See Table S3 for a list of loci, including
GenBank accession numbers. PCR was conducted under the following conditions: initial
denaturing of 94 °C for 60 s; 35 cycles of 94 °C 20 s, 58 °C for 15 s, and 72 °C for 30 s;
followed by a final elongation incubation of 72 °C for 10 min. The annealing temperature of
some loci differed from the 58 °C used for most, specific annealing temperatures for these loci
can be found in the supporting documentation of Michel et al. (2010). Genotyping was
performed via capillary electrophoresis using a Beckman-Coulter CEQ8000. Fragment length
polymorphisms were scored using Beckman-Coulter’s Fragment Analysis software. Consistency
of allele-calling was maintained using a reference database established from previous Rhagoletis
microsatellite studies (Michel et al., 2007, 2010; Cha et al., 2012; Powell et al. 2013). As with
previous Rhagoletis microsatellite studies, many loci contained alleles with 1 basepair phase
shifts. This is due to the combined factors of biogeographic history (deeply divergent inversion
polymorphisms assorting in the population) and the complex nature of some of the repeat motifs
(Velez et al. 2006). These 1 bp alleles were unambiguously resolved using our genotyping
methods. From 179 individuals re-genotyped (including PCR-amplification and capillary
electrophoresis) the mean difference in estimated fragment size between runs was 0.09bp with a
standard deviation of 0.04bp, well within the 0.85 bp default bin-width of the genotyping
software.
Structure analysis
The program STRUCTURE 2.3.4 (Pritchard et al. 2000) was used to examine patterns of
individual-level clustering between host associations. The strong clinal variation, which is a key
feature of the R. pomonella species complex, makes a poor fit for the discrete HW model
implemented in STRUCTURE. In order to limit the potential confounding effects of clines, we
focused these analyses on the five paired populations at local sites described in the text. For the
sake of comparison, we also included analyses of two apple and hawthorn sympatric populations
from Grant, MI and Urbana, IL (original data from Michel et al. 2010) and one paired sympatric
population of downy hawthorn-race R. pomonella and the flowering dogwood fly from
Dowagiac, MI (data from Powell et al. 2013).
Structure analyses involved using a burn-in length of 500,000 followed by 750,000
MCMC repetitions for five replicates each of K =1-2 under the admixture and correlated allele
frequencies model. Because the analyses did not extend beyond K=2, the often used method of
Evanno et al. (2005) for evaluating the successive decrease in log likelihood for increasing
values of K was inapplicable. However, the results here were unambiguous such that no
additional analysis was needed. Higher likelihoods for K =1 were found in all cases except that
of the downy hawthorn vs. flowering dogwood fly sibling-species comparison (Table S12).
The southern hawthorn-infesting populations of R. pomonella, like the classic apple and
downy hawthorn races, are distinguished by significant allele frequency differences at multiple
loci. However, these STRUCUTRE results indicate that the magnitude of this genetic
differentiation is insufficient to distinguish separate genetic clusters. This is not surprising for
two reasons. First, while the allele frequency differences in question are strong relative to
expectations of neutral migration/drift dynamics, they are not strong enough to make a particular
genotype statistically unlikely to occur in both populations. Second, this type of discrete
individual-level clustering of sympatric populations may not emerge until effective migration
rates are low enough to approach sibling-species levels of divergence. Gene flow estimates
between the flowering dogwood fly and hawthorn race of R. pomonella have been estimated to
be ~1%, which is near the threshold for sibling species vs. races proposed by Drès and Mallet
(2002)
Supplementary References
Boller EF, Prokopy RJ (1976) Bionomics and management of Rhagoletis. Annual Review of
Entomology. 21, 223-249.
Cha DH, Powell THQ, Feder JL, Linn CE (2011a) Identification of Host Fruit Volatiles from Three
Mayhaw Species (Crataegus Series Aestivales) Attractive to Mayhaw-Origin
Rhagoletis pomonella Flies in the Southern United States. Journal of Chemical Ecology, 37,
961–973.
Cha DH, Powell THQ, Feder JL, Linn CE (2011b) Identification of Fruit Volatiles from Green
Hawthorn (Crataegus Viridis) and Blueberry Hawthorn (Crataegus Brachyacantha) Host
Plants Attractive to Different Phenotypes of Rhagoletis Pomonella Flies in the Southern United
States. Journal of Chemical Ecology, 37, 974–983.
Cha DH, Powell THQ, Feder JL, Linn CE (2012) Geographic variation in fruit volatiles emitted by
the hawthorn Crataegus mollis and its consequences for host race formation in the apple maggot
fly, Rhagoletis pomonella. Entomologia Experimentalis Et Applicata, 143, 254–268.
Correll DS, and Johnston, MC 1970. Manual of the vascular plants of Texas. Vol. 6 Texas Research
Foundation.
Evanno GS, Regnaut S, Goudet J (2005) Detecting the number of clusters of individuals using the
software STRUCTURE: a simulation study. Molecular Ecology, 14, 2611-2620.
Feder JL, Chicote CH, Bush GL (1990a) Regional, local, and microgeographic allele frequency
variation between apple and hawthorn populations of Rhagoletis pomonella in western Michigan.
Evolution. 44, 595-608.
Feder JL, Chicote CH, Bush GL (1990b) The geographic pattern of genetic differentiation between
host associated populations of Rhagoletis pomonella (Diptera: Tephridtidae) in the eastern
United States and Canada. Evolution. 44, 570-594.
Feder JL, Roethele FB, Filchak K, Niedbalski J, Romero-Severson J (2003) Evidence for inversion
polymorphism related to sympatric host race formation in the apple maggot fly, Rhagoletis
pomonella. Genetics, 163, 939–953.
Lance RW (2011) New alignments in North American Crataegus (Rosaceae). Phytoneuron 3, 1–8.
Larkin M A, Blackshields G, Brown NP et al. (2007) Clustal W and clustal X version 2.0.
Bioinformatics 23,2947–2948.
Lo EYY, Stefanovic S, Dickinson TA (2010) Reconstructing Reticulation History in a Phylogenetic
Framework and the Potential of Allopatric Speciation Driven by Polyploidy in an Agamic
Complex in Crataegus (rosaceae). Evolution, 64, 3593–3608.
Michel AP, Rull J, Aluja M, Feder JL (2007) The genetic structure of hawthorn-infesting Rhagoletis
pomonella populations in Mexico: implications for sympatric host race formation Molecular
Ecology, 16, 2867–2878.
Michel AP, Sim S, Powell THQ et al. (2010) Widespread genomic divergence during sympatric
speciation. Proceedings of the National Academy of Sciences of the United States of America,
107, 9724–9729.
Nojima S, Linn C, Morris B, Zhang AJ, Roelofs W (2003) Identification of host fruit volatiles from
hawthorn (Crataegus spp.) attractive to hawthorn-origin Rhagoletis pomonella flies. Journal of
Chemical Ecology, 29, 321–336.
Powell THQ, Cha DH, Linn CE, Feder JL (2012) On the Scent of Standing Variation for Speciation:
Behavioral Evidence for Native Sympatric Host Races of Rhagoletis Pomonella (diptera:
Tephritidae) in the Southern United States. Evolution, 66, 2739–2756.
Powell THQ, Hood GR, Murphy MO et al. (2013) Genetic differentiation across the speciation
continuum: the transition from host race to species in Rhagoletis. Evolution, 67, 2561-2576.
Pritchard JK, Stephens M, Donnelly P (2000) Inference of population structure using multilocus
genotype data. Genetics. 155, 945-959
Rull J, Aluja M, Feder JL (2010) Evolution of intrinsic reproductive isolation among four North
American populations of Rhagoletis pomonella (Diptera: Tephritidae). Biological Journal of the
Linnean Society, 100, 213–223.
Sargent, CS (1890) Silva of North America volume iv. (reprinted 1947) Peter Smith. New York, NY.
Sargent CS (1933) Manual of the trees of North America. Houghton Mifflin Co. Boston, MA
Talent N, Dickinson TA (2005) Polyploidy in Crataegus and Mespilus (Rosaceae, Maloideae):
evolutionary inferences from flow cytometry of nuclear DNA amounts. Canadian Journal of
Botany-Revue Canadienne De Botanique, 83, 1268–1304.
Velez S, Taylor MS, Noor M a. F, Lobo NF, Feder JL (2006) Isolation and characterization of
microsatellite loci from the apple maggot fly Rhagoletis pomonella (Diptera : Tephritidae).
Molecular Ecology Notes, 6, 90–92.
Vines, RA (1977). Trees of East Texas. University of Texas Press. Austin, TX.
Xie X, Michel AP, Schwarz D et al. (2008) Radiation and divergence in the Rhagoletis Pomonella
species complex: inferences from DNA sequence data. Journal of Evolutionary Biology, 21,
900–913.
Zhang AJ, Linn C, Wright S et al. (1999) Identification of a new blend of apple volatiles attractive to
the apple maggot, Rhagoletis pomonella. Journal of Chemical Ecology, 25, 1221–1232.
TABLE S1. Relative percentages of chemical compounds comprising the fruit volatile blends of
the four southern hawthorn species (WMH = western mayhaw, GH = green hawthorn, BB =
blueberry hawthorn, SR = southern red hawthorn) and the two northern R. pomonella hosts,
apple (AP) and downy hawthorn (DH). Blends were developed using GC-EAD analysis as
previously reported in Cha et al. (2011a,b,c) Nojima et al. (2003), and Zhang et al. (1999). Note:
DMNT indicates 4,8-dimethyl-1,3(e),7-nonatriene. Table adapted from Powell et al. 2012.
Chemicals
3-methylbutan-1-ol
Butyl acetate
Pentyl acetate
Butyl butanoate
Propyl hexanoate
Butyl hexanoate
Hexyl butanoate
Pentyl hexanoate
Isoamyl acetate
Isoamyl butanoate
Isoamyl hexanoate
Pentyl butanoate
Ethyl acetate
DMNT
Dihydro-β-ionone
Butyl propionate
1-octen-3-ol
WMH
44
6
6
6
26
12
GH
5
19.5
1.5
24
24
2.5
BB
0.6
50
3.5
9
16.8
16.8
SR
0.4
9
AP
45
0.3
20
14
0.6
10
4
37
44
5
DH
4
0.01
1.5
2.5
3
1.5
0.5
3
20.5
0.2
5.5
0.5
0.3
94.3
0.07
0.1
TABLE S2. Information for mitochondrial sequence data used in Fig. 3. Samples are listed in
the vertical order in which they appear in the Figure.
Species
R. pomonella nr.
R. pomonella
R. pomonella
R. pomonella
R. pomonella
R. pomonella
R. pomonella
R. pomonella
R. pomonella
R. pomonella
R. pomonella
R. pomonella
R. pomonella
R. pomonella
R. pomonella
R. pomonella
R. pomonella
R. electromorpha
Host
C. mexicana
BB
DH
DH
BB
GH
GH
SRH
BB
MH
BB
GH
DH
GH
MH
SRH
SRH
Silky dogwood
Location
Altiplano, Mexico
Site 9
Geneva, NY
East Lansing, MI
Site 3
Site 1
Site 5
Sites 1, 2
Site 10
Site 8
Site 9
Site 3
New Madrid, MO
Sites 4,6
Sites 4,7,9
Sites 1,2
Site 11
Urbana, IL
Source
Feder et al. 2003
New data
Feder et al. 2003
Smith & Bush 1997
New data
New data
New data
New data
New data
New data
New data
New data
Powell et al. 2013
New data
Powell et al. 2013
New data
Powell et al. 2013
Smith & Bush 1997
Genbank
Accesion no.
AY152483
KF877734
AY152477
RPU535231
KF877735
KF877738
KF877739
KF877737
KF877740
KF877736
KF877742
KF877741
JX003700
KF877743
JX003698
KF877744
KF247219
EU53242
TABLE S3. List of 26 microsatellite markers used in this study.
Locus
P3
P4
P5
P7
P9
P11
P16
P17
P18
P23
P25
P27
P29
P37
P39
P40
P46
P50
P54
P60
P66
P70
P71
P73
P75
P80
Genbank no.
AY734887
AY734888
AY734889
AY734891
AY734893
AY734895
AY734900
AY734901
AY734902
AY734907
AY734909
AY734911
AY734913
AY734921
AY734923
AY734924
AY734930
AY734934
AY734938
AY734942
AY734950
AY734954
AY734955
AY734957
AY734959
AY734964
Primer1 (5’ - 3’)
Primer2 (5’ - 3’)
TCCACTCAAATACGGCAACA
GCAAGCGAGTCGTAATCACA
GAGCAGCAGAGGAAAAAGGA
CATTGGCAACGCTAGTTCAA
CGGCAGGTAAATGACCAAAA
ATGCAGCCATGACTGAGATG
CGCTTTAGATTTTCGCTACACA
TTCGAAACCGTTTGTTACCTCT
CCCAATGTCCCGTAAACTTC
AAACTGCCTTGCCTGTCATT
ATGACATTCGCTACGGGGTA
TTCTCACATTTTCGCGTTTG
TCCATGTGTGCCAGAACATT
CAACAGCGCGACTTAGTGAA
GCGAAAATGTGGTCGTAGGT
CGAGCAGGTGATGATAATGC
GCGCATTTCTCCATTCATTT
GTGCAACCAGTGAGCAGTGT
TGTGCTAAATTACCCAAAAGC
TACAACCTAGGCAGCCCAAC
GCAAACCATTTTCCACGAAT
CAGCCTGCCAACACCATT
CGCAAGCACTTTTTGAACTG
TTTTCTCGTCTACTCGTGTTAGTTAT
GCCGACTGTCGATTCTCTTG
GGACAGTTGTGGTTGCTGAA
GCAGCCGATCTTTTCGTCTA
CCCTCATCATTGTGGTCCTC
TGCACTGGTGTATTCCAAGG
GCGCTGAAACCATGAAAAAT
GCAATGACCGTTGGCTATTA
TGGAAAGTAATTTCACAAAGGCTA
ACGCAGTGCCAAATCTTCTT
CGCTATTGGAGGCAATGAAT
TTCACTCAATGCCCATTTCA
GCACTTTGTCGTTGATGCAC
TCTCGGAGAGTGGCAGTTTT
CTGGCCAATGCATAAATCCT
GACGTTATTTCGCTCGGTTG
TGGCTTCCACCTTTGTTTTT
ACAGTGCGGCTGACACATAG
CCGAAATTTGAGCCCCTTA
GCGGTAATTGTGCGTATGTG
TCTGACTGGCCCGTATTTGT
GCGTCATTCAGTCAACCAAA
GTCTGGTTTGGCGATCACTT
CGAAGCATGAATGCAACAAC
GCAACGCCTTCAAATTCATC
CTGCTGAATTGGCAGCATAA
AAAATGCACTTTGTAAATAGTCACTCA
GGCAGTGATGACGAGAAACA
TCCTTTGCAATGTTATGGTAATTG
TABLE S4. Alleles included in one of two groups generated by Monte Carlo allele pooling
method for each of 26 microsatellite loci across five chromosomes (Chr.).
Locus
P71
P37
P75
P4
P39
P3
P70
P54
P46
P73
P17
P7
P80
P16
P23
P66
P40
P11
P29
P25
P50
P60
P5
P18
P9
P27
Chr.
1
1
1
1
1
1
2
2
2
2
2
3
3
3
3
3
3
4
4
4
4
4
5
5
5
5
Alleles
182,185,186,188,194,196
191,192,196,199,200,202,203,207,208,209,217,218,219
211
158,159,160,164,167,171,179,187,197,201,202,211
185,187,191.196,197,198,203,204,205,211,212,219,224
144,145,157,166,168,169,170,173,175,177,181,190,196,197,200,205,226
166,173,174,175,178,180,185,186,189
180,210,212,213,215,217,218,221,225,227,228,232,233,236,237,239,242
202,204,205,212,213,215,227,234,246,247,256,258,262,268,270
119,126,128,136,142,156,162,164,168,170,175,182,186,222
243,247,251,253,255,258,261,265,266,269,274,275,276
211,226,231,232,233,234,236,247,248,250,251
186,189,199,200,205,207,213
290,304,305,307,311,314,319
192,198,199,201,208,211,212,215,216,218
192,194,199,203,204,206,207,210,212,213,226,229,232,233,238,248,256,260
89,91,121,126,136,138,141,146,154
260,272,281,286,287,292,294,300,306,308,312,326
179,189,193,197,204,208,215,219
214,218,219,226,228,230,231,235,238,239,244,246,253,255,256,257,264,274
144,147,152,154,159,160,162,165,167,168,169,172,177,178
146,158,164
183,193,206,224,227,228,230
281,283,287,289,291,295,296,298,301,303,311,315,321,323,333,335,339,341,345
153,154,159,161,163
154,158,164,168
TABLE S5. (uploaded to DRYAD) Microsatellite allele frequencies for the 26 loci analyzed in
study for 16 field sites, including 6 green hawthorn, 4 western mayhaw, 3 blueberry hawthorn,
and 3 southern red hawthorn populations. See Table 1 of text for site descriptions. Allele
numbers represent the size (in base pairs) of the PCR amplified DNA fragment (including the
repeat sequence) generated using locus specific primers for each microsatellite, as determined by
capillary electrophoresis on a Beckman-Coulter CEQ8000.
TABLE S6. Mean demic inbreeding coefficient (f) and standard deviation (σ) across all 26 loci
for each of the 16 populations and sampling regime (S.R.) determined by host plant patch size as
described above. E = exhaustive sampling of a few trees with no others found in the immediate
vicinity; H-M = haphazard sampling along multiple transects running the full breadth of medium
sized stands of host trees (often bounded by habitat type, water bodies, or human development);
H-L haphazard sampling along transects in continuous distributions of host plants too large cover
fully (>20 hectares).
σ
S.R.
0.079
0.185
H-L
Palmetto SP, Gonzalez Co., TX
0.091
0.134
H-M
3
L. Sam Rayburn, Angelina Co., TX
0.089
0.182
E
4
Dewey Wills WMA, La Salle Pr., LA
0.047
0.127
H-L
5
Fort Necessity, Caldwell Pr., LA
0.093
0.107
H-M
6
Rolling Fork, Sharkey Co., MS
0.063
0.148
E
7
HW Jackson Farm, Polk Co., TX
0.071
0.124
H-M
8
LSU Idlewild, E. Feliciana Pr, LA
0.088
0.142
H-M
4
Dewey Wills WMA, La Salle Pr., LA
0.107
0.124
H-L
9
SFA Exp. Forest, Nacogdoches Co., TX
0.103
0.154
H-M
Morris Ferris Park, Angelina Co., TX
0.077
0.108
H-M
L. Sam Rayburn, Nacogdoches Co., TX
-0.022
0.154
E
SFA Exp. Forest, Nacogdoches Co., TX
0.052
0.174
H-M
Hungerford, Wharton Co., TX
0.086
0.093
H-M
Brazos Bend Sp, Fort Bend Co., TX
0.058
0.185
H-L
Palmetto SP, Gonzalez Co., TX
0.093
0.134
H-M
Host plant origin
#
Green hawthorn
(C. viridis)
1
Brazos Bend SP, Fort Bend Co., TX
2
Mayhaw
(C. opaca)
Blueberry hawthorn 10
(C. brachyacantha)
3
9
S. red hawthorn
11
(C. mollis v. texana)
1
2
Location
f
TABLES S7-S11. The following tables present detailed results of generalized linear modeling
(GLM) analyses of microsatellite allele frequency as a quasibinomial variable as a function of
host plant (Host), latitude (Lat.), and Host x Lat. These analyses were done separately for four
different pairwise comparisons of co-occuring host-association: mayhaw and green hawthorn;
mayhaw and blueberry hawthorn, green hawthorn and blueberry hawthorn, and green hawthorn
and southern red hawthorn. Significance determined by F-test; * = p<0.05; ** = p<0.01; *** =
p<0.001; **** = p<0.0001. Tables are divided by chromosome.
TABLE S7. Results of GLM analyses of allele frequency for loci on chromosome 1.
Mayhaw
vs. green
haw
Host
p3
df
9
F 194.951
p4
9
11.813
p37
9
0.0042
p71
9
0.0154
p75
9
0.0403
p39
9
2.7934
Lat.
p
F
****
90.872
*
105.919
0.7005
3.1158
1.5115
0.4107
p
Host:Lat. F
****
50.614
****
41.243
0.2589
0.5186
0.0301
0.46
p
***
***
-
-
-
-
df
F
6
32.675
6
1.0807
6
2.7125
6
0.0076
6
0.5574
6
150.5587
p
*
-
-
-
-
**
F
63.609
21.8775
0.5667
2.5952
0.0152
34.8164
p
Host:Lat. F
**
18.34
*
3.9471
2.0647
0.0731
2.4499
**
6.0369
p
*
-
-
-
-
-
df
F
8
8.1883
8
3.5591
8
0.746
8
0.001
8
1.1352
8
0.1258
p
*
-
-
-
-
-
35.3588 126.0536 0.7689
2.4081
0.9757
0.5494
**
28.4502
****
5.7492
0.5833
0.1065
3.6033
0.0058
**
-
-
-
-
-
8
73.458
8
0.7872
8
9.4552
8
0.0008
8
1.5565
Mayhaw Host
vs.
blueberry
haw
Lat.
Green
Host
haw vs.
blueberry
haw
Lat.
F
p
Host:Lat. F
p
Green
haw vs.
s. red
haw
Host
df
8
F 155.845
p
****
***
-
*
-
-
F
53.241
98.915
0.757
2.155
2.2028
0.4179
p
Host:Lat. F
***
22.261
***
32.594
0.7125
3.2946
1.8052
2.4002
**
**
-
-
-
-
Lat.
p
TABLE S8. Results of GLM analyses of allele frequency for loci on chromosome 2.
Mayhaw
vs. green
haw
Host
df
F
p46
9
39.174
p70
9
10.1908
p73
9
3.4095
p17
9
0.1968
Lat.
p
F
***
14.913
*
9.7145
86.2256
11.3687 20.1187
p
Host:Lat. F
**
560.811
*
0.3483
****
92.2829
*
2.7807
**
2.2464
p
****
-
****
-
-
df
F
6
142.552
6
19.3814
6
409.77
6
0.0005
6
3.4246
p
**
*
***
-
-
F
563.954
0.4954
164.13
4.3164
5.9224
p
Host:Lat. F
***
17.182
0.2452
**
188.31
3.2739
0.8415
p
*
-
***
-
-
df
F
8
23.0221
8
0.0948
8
158.55
8
0.0448
8
0.5842
p
**
-
****
-
-
F
74.7321
9.3044
88.833
3.5513
9.2716
p
Host:Lat. F
***
0.0969
*
0.0218
***
76.852
3.6476
*
0.3553
p
-
-
***
-
-
df
F
8
1.6757
8
3.1048
8
0.2938
p
-
-
-
**
*
F
43.1231
9.0963
125.3125
7.0174
14.7771
p
Host:Lat. F
**
147.1882
*
0.5592
****
73.2098
*
1.251
*
7.6232
***
-
***
-
*
Mayhaw Host
vs.
blueberry
haw
Lat.
Green
Host
haw vs.
blueberry
haw
Lat.
Green
haw vs.
s. red
haw
Host
Lat.
p
p54
9
1.836
8
8
17.0024 10.7162
TABLE S9. Results of GLM analyses of allele frequency for loci on chromosome 3.
Mayhaw
vs. green
haw
Host
df
F
p7
9
0.0315
p16
9
0.2427
p23
9
8.7091
Lat.
p
F
0.7932
14.2351
p
Host:Lat. F
0.669
p
p80
9
8.6176
p40
9
0.3431
*
16.8515 133.3119
*
0.4726
24.9541
**
2.094
**
2.9193
****
0.3769
14.3505
**
0.0014
-
-
-
-
**
-
df
F
6
1.5813
6
0.2192
6
21.726
6
3.2989
6
0.1926
6
83.6878
p
-
-
*
-
-
**
F
0.6097
0.0391
0.2522
8.5276
2.6921
129.2437
p
Host:Lat. F
0.2389
0.1635
4.067
6.2565
0.1517
**
4.1782
p
-
-
-
-
-
-
df
F
8
1.0488
8
3.139
8
0.4505
8
0.6103
8
0.6892
8
3.4339
p
-
-
-
-
-
-
F
1.2322
52.6219
19.2278
78.7293
1.1248
18.0631
p
Host:Lat. F
0.0384
***
0.2805
**
1.0005
***
5.4789
0.02
**
0.1018
p
-
-
-
-
-
-
df
F
8
0.0665
8
2.7757
8
6.748
8
7.0324
8
15.1584
8
0.0638
p
-
-
*
*
*
-
F
1.1075
42.0276
19.6643 138.7873
4.3226
18.2575
p
Host:Lat. F
0.798
**
6.5236
**
0.0096
****
64.7339
0.5687
**
0.1277
-
-
-
***
-
-
Mayhaw Host
vs.
blueberry
haw
Lat.
Green
Host
haw vs.
blueberry
haw
Lat.
Green
haw vs.
s. red
haw
Host
Lat.
p
p66
9
2.5449
TABLE S10. Results of GLM analyses of allele frequency for loci on chromosome 4.
Mayhaw
vs. green
haw
Host
df
F
p11
9
11.4556
Lat.
p
F
*
0.1895
150.1504
0.6236
30.2404
2.8539
p
Host:Lat. F
41.8211
****
64.1452
5.5246
**
26.5703
1.3236
p
***
****
-
**
-
df
F
6
62.8187
6
6.3259
6
0.0653
p
**
**
-
-
F
16.8689
23.2208
50.2625 10.4508
0.0006
p
Host:Lat. F
*
0.0205
*
1.5626
**
0.0258
*
0.048
10.8587
-
-
-
-
*
8
92.2433
8
4.8788
8
5.644
8
0.0003
Mayhaw Host
vs.
blueberry
haw
Lat.
p
Green
Host
haw vs.
blueberry
haw
Lat.
df
8
F 452.2407
p29
9
0.17
p50
9
0.7484
p60
6
0.7157
6
6
151.7173 74.1581
**
p
****
***
-
-
-
F
24.6521
122.2926
1.609
62.9367
4.1523
p
Host:Lat. F
**
5.0719
***
0.0087
0.1867
***
0.4487
0.474
-
-
-
-
-
8
68.675
8
4.6229
8
4.2496
8
0.0011
p
Green
haw vs.
s. red
haw
p25
9
1.6229
Host
Lat.
df
8
F 250.0982
p
***
***
-
-
-
F
7.4478
148.085
1.4073
67.8539
4.3275
*
181.6942
****
18.156
2.2636
***
11.511
0.7005
****
**
-
*
-
p
Host:Lat. F
p
TABLE S11. Results of GLM analyses of allele frequency for loci on chromosome 5.
Mayhaw
vs. green
haw
Host
df
F
p9
9
0.0967
p18
9
9.9617
p27
9
0.3373
p5
10
0.0322
Lat.
p
F
10.0465
*
16.3958
0.1075
7.5984
p
Host:Lat. F
*
0.9729
**
0.3065
1.3337
*
4.6845
p
-
-
-
-
df
F
6
0.1072
6
41.3361
6
5.5501
6
0.4205
p
-
**
-
-
F
0.0097
2.1957
2.9206
2.6221
p
Host:Lat. F
0.2026
3.007
0.0377
3.2669
p
-
-
-
-
df
F
8
0.2991
8
18.682
8
3.718
8
0.674
p
-
**
-
-
F
2.9747
13.556
0.4036
1.92
p
Host:Lat. F
0.3924
*
2.905
0.0927
7.295
p
-
-
-
*
df
F
8
0.0139
8
0
8
0.3393
8
11.5432
p
-
-
-
*
F
7.744
14.298
0.3561
5.8652
*
15.7328
*
51.712
1.1226
30.2587
*
***
-
**
Mayhaw Host
vs.
blueberry
haw
Lat.
Green
Host
haw vs.
blueberry
haw
Lat.
Green
haw vs.
s. red
haw
Host
Lat.
p
Host:Lat. F
p
TABLE S12. Mean estimated Ln likelihood and standard deviation across five replicates of
STRUCTURE analysis of paired local populations for K=1 and K=2, using a burn-in of 500,000 followed
by 750,000 MCMC repetitions under a correlated allele frequency with admixture model. “Δ Ln Lik”
reports the change in mean Ln likelihood between K=2 and K=1. MH = mayhaw; GH = green hawthorn;
SRH = southern red hawthorn; BB = blueberry hawthorn; AP = apple; DH = downy hawthorn; DW =
flowering dogwood . Sites refer to designations in Table 1 for southern comparisons. MI apple and downy
hawthorn site is in Grant, MI. IL apple and downy hawthorn site is in Urbana, IL. MI downy hawthorn
and flowering dogwood comparison is in Dowagiac, MI.
K=1
K=2
Ln Lik
σ
Ln Lik
σ
Δ Ln Lik
MH vs GH (4)
-2237.13
0.15
-2265.40
13.96
28.27
GH vs SRH (1)
-3654.57
0.06
-3566.00
5.63
5.63
GH vs SRH (2)
-3288.73
0.03
-3617.87
50.29
329.13
MH vs BB (9)
-3437.97
0.15
-3669.17
38.03
231.20
GH vs BB (3)
-1595.43
0.23
-1681.73
42.77
86.30
AP vs DH (MI)
-2030.10
0.00
-2125.67
30.91
95.57
AP vs DH (IL)
-1835.68
0.19
-1942.08
154.91
106.40
DH vs DW (MI)
-1648.20
0.61
-1528.40
0.35
-119.80
Comparison (site)