SYSTEMATICS Complexity in Dioryctria zimmermani ...SYSTEMATICS Complexity in Dioryctria zimmermani Species Group: Incongruence Between Species Limits and Molecular Diversity AMANDA

SYSTEMATICS

Complexity in Dioryctria zimmermani Species Group: IncongruenceBetween Species Limits and Molecular Diversity

AMANDA D. ROE,1,2 DANIEL R. MILLER,3 AND SUSAN J. WELLER2

Ann. Entomol. Soc. Am. 104(6): 1207Ð1220 (2011); DOI: http://dx.doi.org/10.1603/AN11051

ABSTRACT Dioryctria(Zeller 1846) (Lepidoptera: Pyralidae: Phycitinae) moths, commonly knownas coneworms, are a group of important coniferous pests. InterspeciÞc overlap of molecular, mor-phological, and behavioral traits has made identiÞcation and delimitation of these species problematic,impeding their management and control. In particular, delimitation of members of the Dioryctriazimmermani species group, a diverse group of Nearctic species, is notoriously difÞcult. To clarify thespecies boundaries in this species group we examined two independent molecular markers (cyto-chrome c oxidase I and II and elongation factor 1�), larval host plant association, geographicdistribution, and pheromone attraction in an integrated taxonomic framework. Congruence betweenthese diagnostic traits and established species limits in the zimmermani group was variable. Somespecies showed well-supported congruence between established taxonomic limits and mitochondrialDNA gene tree topology, whereas other species showed little phylogenetic resolution, little corre-spondence with diagnostic traits, and incongruence with previously described species limits. GenetreeÐspecies tree discordance may be caused by several evolutionary processes, such as imperfecttaxonomy, incomplete lineage sorting, or introgression. Additional information, such as highly variablemolecular markers, morphometrics, and larval host information, is needed to effectively evaluate anddifferentiate among these alternative hypotheses and fully resolve the species limits among D.zimmermani species group members.

KEY WORDS mitochondrial DNA, integrated taxonomy, Dioryctria, coneworm, species delimita-tion

Insects are known for their biological diversity, eco-logical importance, and economic impact (Scudder2009), making accurate and timely taxonomic classi-Þcation of insect species imperative (Wheeler 2009).Ideally, an integrative approach that incorporatesmany sources of evidence should be used to achievethis taxonomic goal. Integrative taxonomy combinesmorphological, molecular, behavioral, and ecologicaldata to improve identiÞcation, discover new species,delimit species boundaries, and reconstruct phyloge-netic relationships (Dayrat 2005, Will et al. 2005, Sper-ling and Roe 2009, Padial et al. 2010, Schlick-Steiner etal. 2010). Use of diverse data sources is invaluable inall aspects of insect taxonomy but is particularly im-portant when examining closely related species (Roeand Sperling 2007, Sperling and Roe 2009, Roe et al.2010). Evolutionary processes such as introgressionand incomplete lineage sorting lead to fuzzy speciesboundaries, particularly between closely related spe-cies where insufÞcient evolutionary time has passedfor diagnostic characters to become fully Þxed. Assuch, incongruence may exist between species limits

anddiagnostic traits,whichcouldbeundetectedwhena single character set is examined (Rubinoff et al. 2006,Elias et al. 2007, Roe and Sperling 2007, Twewick 2007,Roe et al. 2010).Dioryctria (Zeller 1846) (Lepidoptera: Pyralidae) is

a large, distinct genus of phycitine moths that requiresthe use of integrative taxonomy for accurate speciesdelimitation (Roe and Sperling 2007). Currently,there are 79 recognizedDioryctria species (Nuss et al.2010) and at least several undescribed species (Du etal. 2005, Knolke 2007, Powell and Opler 2009). Al-though 12 species groups were erected to help clar-ify morphological variation within Dioryctria (Mu-tuura and Munroe 1972, 1974; Wang and Sung 1982;Neunzig 2003; Knolke 2007), accurate identiÞcationof species is still problematic (Roe and Sperling2007, Roux-Morabito et al. 2008). Many speciesshow interspeciÞc overlap of molecular, morpho-logical, or behavioral traits, thereby impeding spe-cies delimitation and identiÞcation (Roe et al. 2006,Roe and Sperling 2007, Roux-Morabito et al. 2008).Larvae of all Dioryctria species feed on conifers,many on or in the cones of economically importantspecies (Pinaceae and Cupressaceae) (Neunzig2003, Roux-Morabito et al. 2008, Whitehouse et al.2011). As such, several Dioryctria species are con-sidered economically important pests and require

1 Corresponding author: 112 Denwood Dr., Sault Ste Marie, ON,Canada P6A 6T3 (e-mail: [email protected]).

2 219 Hodson Hall, 1980 Folwell Ave., St. Paul, MN 55108.3 USDA Forest Service, Southern Research Station, 320 Green St.,

Athens GA 30602.

targeted management (Whitehouse et al. 2011, andreferences therein), necessitating accurate speciesidentiÞcation.

DifÞculties with species delimitation are commonamong members of the zimmermani species group andtypify the taxonomic difÞculties commonly foundwithin Dioryctria. The zimmermani species group isone of the largest groups of Dioryctria, containing 18described species (Table 1), all of which are exclu-sively Nearctic (Mutuura et al. 1969; Mutuura andMunroe 1979; Neunzig 1990, 2003). Species are char-acterized by distinctive genitalic structures and prom-

inent forewing scale ridges (Fig. 1), and recent phy-logenetic analyses support the monophyly of thisgroup (Du et al. 2005, Roe et al. 2006, Knolke 2007).Although the majority of species have darkly coloredforewings, several distinctive pale colored species oc-cur in the western United States (Fig. 2; Table 1). Themajority of species in the zimmermani group feedalmost exclusively on Pinus (Munroe 1959, Neunzig2003, Roe et al. 2006). Larvae feed internally on cam-bium, shoots, cones, wounds, and rust cankers, causingextensive economic damage, particularly in commer-cial pine seed orchards (Whitehouse et al. 2011), and

Table 1. Members of the D. zimmermani species group

SpeciesWingcolor

Larval hostPheromonea Referenceb

Species Tissue

D. albovittella (Hulst)* L P. monophylla Torrey &Fremont, P.cembroides Zuccarini�P. edulis Engelmann�c

Cones, shoots Heinrich (1956), Cibrian-Tovar et al. (1986)

D. amatella (Hulst)* D P. palustris Miller, Pinusspp.

Cones, shoots cambium,ßowers, rust cankers

Z11Ð16:Ac � C25-p Heinrich (1956), Coulsonand Franklin (1970),Hedlin et al. (1980),Meyer et al. (1986),Miller et al. (2010)

D. banksiella Mutuura,Munroe & Ross

D P. banksiana Lambert Rust cankers Mutuura (1982), Mutuuraet al (1969)

D. cambiicola (Dyar)* D P. ponderosa Douglas exC. Lawson, P. contortaDouglas ex Loudon, P.coulteri D. Don

Cambium, rust cankers,shoots, cones

Heinrich (1956),Mutuura et al. (1969),Mutuura (1982)

D. contortella Mutuura,Munroe & Ross*

D P. contorta Cambium, rust cankers Mutuura et al. (1969)

D. cuitecensis Neunzig D UnknownD. delectella (Hulst) D UnknownD. fordi Donahue &

Neunzig*L �P. sabiniana Douglas ex

D. Don�cDonahue and Neunzig

(2002)D. merkeli Mutuura &

Munroe*D P. elliottii Engelmann, P.

palustrisFlowers, shoots, cones Z9Ð14:Ac � E9Ð14:Ac Mutuura and Munroe

(1979), Hanula et al.(1984), Meyer et al.(1984), 1986(??),Miller et al. (2010)

D. monticolellaMutuura, Munroe &Ross

D P. monticola Douglas exD. Don

Cambium Mutuura et al. (1969)

D. mutuurai Neunzig L UnknownD. resinosella Mutuura* D P. resinosa Aiton Shoots, cones Z9Ð14:Ac � E9 14:Ac

� Z9Ð14:OH �Z9Ð12:Ac

Mutuura (1982), Grant etal. (1993)

D. taedae Schaber &Wood

D P. taeda L., P. echinataMiller

Cones, shoots Schaber and Wood(1971)

D. taedivorella Neunzig& Leidy*

D P. taeda Cones Neunzig and Leidy(1989)

D. tumicolella Mutuura,Munroe & Ross*

D P. ponderosa Rust cankers Z9Ð16:Ac Mutuura et al. (1969); G.Grant, unpublished

D. westerlandi Donahue& Neunzig

L �P. jeffreyi Balfour�c Donahue and Neunzig(2002)

D. yatesi Mutuura &Munroe*

D P. pungens Lambert Cones

D. zimmermani(Grote)*

D P. strobus L., P. resinosa,P. sylvestris L., P.mugo Turra, P. nigraJ. F. Arnold, Pinusspp.

Cambium, shoots Z11Ð16:Ac Heinrich (1956); Munroe(1959); Mutuura(1982); G. Grant,unpublished

Species examined in this study are indicated by an asterisk (*). Host plant information is summarized from Neunzig (2003) and Whitehouseet al. (2011), with additional host plant and pheromone references included.a Z11Ð16:Ac, (Z)-11-hexadecenyl acetate; C25-p, (3Z,6Z,9Z,12Z,15Z)-pentacospentaene; Z9Ð14:Ac, (Z)-9-tetradecenyl acetate; E9Ð14:Ac,

(E)-9-tetradecenyl acetate; Z9Ð14:OH, (Z)-9-tetradecen-1-ol; Z9Ð16:Ac, (Z)-9-hexedecenyl acetate; Z11Ð16:Ac, (Z)-11-hexedecenyl acetate.b Includes both pheromone and larval host literature.cHypothesized.

1208 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 104, no. 6

host plant may be an important diagnostic character(Neunzig 2003). Pheromone lures, designed to im-prove management and control of Dioryctria pests,show distinct species differences (Table 1) (Meyer etal. 1986, Grant et al. 1993, Miller et al. 2010), althoughcross-species attraction does occur (Hanula et al.1984).

Despite pheromone and host plant differencesamong species (Table 1), accurate species identiÞca-tion remains elusive and species limits in this group

need further examination. IdentiÞcation of speciesrelies primarily on minor forewing differences, geo-graphic distribution, and larval host plant associations(Neunzig 2003), although these traits show consider-able interspeciÞc overlap (Sopow et al. 1996, Roe et al.2006), complicating species diagnostics. Furthermore,previous molecular work on Dioryctria has found lowlevels of molecular variation separating members ofthe zimmermani group, particularly among dark-scaled species (Richmond and Page 1995, Du et al.2005).

The objectives of this study were to examine thegenetic diversity found within species in the zimmer-mani species group and relate molecular variation tolarval host plant association, geographic distribution,and pheromone attraction. Using this integrated tax-onomic approach, we hope to clarify species bound-aries within this difÞcult group.

Materials and Methods

SpecimenCollection.Adult and larval specimens inthe D. zimmermani group were sampled from sitesacross North America by using a variety of methods,

Fig. 1. D.merkelihabitus showing raised scale ridges thatare found on all members of the zimmermani species group.(Online Þgure in color.)

Fig. 2. Members of the D. zimmermani species group included in the study (missing D. yatesi). (A) D. albovittella, CO:12 miles NW Fort Collins VIII-1971 R. Stevens reared Pinus edulis cones. (B)D. amatella, GA: Belleville 30-IX-1969 G. DeBarrreared Pinus elliottii second-year cones. (C) D. cambiicola, BC: Summerland 9-VIII-1967 reared Pinus ponderosa fresh pitchmass. (D) D. contortella, BC: Barriere 3-VII-1967 reared Pinus contorta blister rust, paratype. (E) D. fordi, CA: 3 miles NEDiablo 2,100 feet 4-X-1966 rearedPinus sabiniana. (F)D.merkeli, GA: Belleville rearedP. elliottii second-year cones G. DeBarr,paratype. (G) D. resinosella, ME: TWP 30 Wash. Co. 2-VII-1980 G. S. Patterson, type series. (H) D. tumicolella, BC:Summerland 31-VII-1967 rearedP.ponderosaold pitch mass. (I)D.zimmermani, ON: Gormley, Lk. Simcoe 10-VIII-1961 rearedPinus sylvestris. (Online Þgure in color.)

November 2011 ROE ET AL.: INCONGRUENCE IN D. zimmermani SPECIES GROUP 1209

including light trapping, pheromone lures, and larvalrearing (Table 2). Pheromone trapping was con-ducted in the southeastern United States as describedby Miller et al. (2010). IdentiÞcation of specimens wasbased on forewing morphology, host association, andgeographic range, based on species descriptions inNeunzig (2003). All specimens are deposited in theStrickland Museum frozen tissue collection at the Uni-versity of Alberta.Molecular Methods. Total genomic DNA was ex-

tracted using a Qiagen DNeasy Blood & Tissue kit(QIAGEN, Valencia, CA) using manufacturerÕs in-structions. Two independent molecular markers weresequenced from all samples. Mitochondrial (mtDNA)from the cytochrome c oxidase I and II gene regions(COI-COII) was obtained (Table 2) using primersdescribed in Roe et al. (2006). For a subset of speci-mens, a 534-bp elongation factor 1� (EF1a) fragmentwas obtained using two overlapping sets of primers:E15f (5� CGGACACGTCGACTCCGG 3�) to rcM44.9(5� CTTCATCAAATCYCTGTGTCC 3�) and M44Ð1(5� GCTGAGCGYGARCGTATCAC 3�) to E600rc (5�TCCTTACGCTCAACATTCC 3�) (Cho et al. 1995,Reed and Sperling 1999). For specimens with DNAvoucher numbers from AR28 to AR332, mtDNA poly-merase chain reaction (PCR) ampliÞcation, puriÞca-tion, and cycle sequencing protocols are as in Roe etal. (2006). Protocols for all EF1a ampliÞcation and theremaining mtDNA sequences were as follows. PCRampliÞcation was performed in 50 �l reactions usingTakara Taq and supplied reagents (R001T, Takara,Otsu, Shiga, Japan). The reaction mix contained 0.25�l of Takara Taq (5 U/�l), 5 �l of 10� PCR buffer, 4�l of dNTP mixture (2.5 mM each), and 2 �l of ex-tracted genomic DNA, 2 �l per primer (5 �M each).PCR products were puriÞed with EXO-SAP (exonu-clease I and shrimp alkaline phosphatase, 70073Z and70092Y, USB Corp., Cleveland OH) according to man-ufacturerÕs instructions. Bidirectional sequencing ofpuriÞed PCR products with ABI BigDye Terminatorversion 3.1 on an ABI 3730xl (Applied Biosystems,Foster City, CA) was performed at the DNA Sequenc-ing and Analysis Facility in the University of Minne-sota Biomedical Genomics Center. Sequence datawere analyzed with Sequencher version 4.8 (GeneCodes Corp., Ann Arbor, MI). All sequence data weresubmitted to GenBank as follows: mtDNA, JN162706ÐJN162761; and EF1a, JN162704, JN162705.Phylogenetic Analyses. Previously published Dio-ryctria sequences also were included in this study (Duet al. 2005, Roe et al. 2006):D. cambiicola (DQ295183,DQ296169, DQ296170), D. fordi (DQ295184,DQ296173, DQ296174), D. taedivorella (DQ247731),D. tumicolella (DQ247729), and D. zimmermani(DQ247730) (Table 2). All sequences were initiallyaligned in Sequencher version 4.8, followed withmanual adjustments made by eye. Sequence frag-ment lengths were not equal and treated as missingdata. Alignments of mtDNA and EF1a data sets weredeposited in TreeBase (http://purl.org/phylo/treebase/phylows/study/TB2:S11682).

Parsimony haplotype networks for mtDNA andEF1a data sets were calculated using TCS 1.21 (Clem-ent et al. 2000). Haplotype networks were inferredusing a statistical parsimony framework (Templeton1998), with gaps treated as missing data and a con-nection limit of 95%. During network inference iden-tical sequences were collapsed, leaving a unique hap-lotype set (Table 2).

Given the low EF1a variability, genetic diversityindices (nucleotide and haplotype diversity), uncor-rected pairwise distances, and a maximum likelihood(ML) tree were calculated for only the mtDNA dataset. Haplotype diversity and nucleotide diversity (Nei1987) were calculated in DNAsp version 5.10.00 (Ro-zas et al. 2003). Uncorrected pairwise distances wereestimated with PAUP* version 4.0b10. ML trees werecalculated using only unique haplotypes under a max-imum likelihood framework implemented in RaxMLversion 7.0.4 (Stamatakis 2006) by using the CIPRESportal version 1.0 (Cyberinfrastructure for Phy-logenetic Research, http://www.phylo.org/portal/Home.do). Before ML analysis, two additional specieswere included as outgroup taxa: D. okanaganellaMu-tuura, Munroe & Ross (DQ295178, in theD. pondero-sae group) and D. penticronella Mutuura, Munroe &Ross (DQ295180, in theD. baumhoferi group) (Roe etal. 2006). These species represent the two additionalspecies groups characterized by raised forewing scaleswhich have been shown to form a “raised scale” cladewith the zimmermani group (Whitehouse et al. 2011).

Results

In total, 56 specimens were collected for this studythrough rearing, light, or pheromone trapping. Whencombined with previously published data, the totaldata set includes 66 specimens from 11 species (Table2). Specimens were collected from across Canada andthe United States, and represent half of the describedspecies in the zimmermani group (Table 1). IdentiÞ-cations were based on previously published descrip-tions of forewing morphology, host plant associations,pheromone attraction, and geographic location(Neunzig 2003, and references therein).

Phylogenetic relationships and genetic diversity ofthe zimmermani group species were assessed with twoindependent loci, COI-COII (mtDNA) and EF1a (nu-clear) (Figs. 3 and 4). mtDNA sequence length rangedfrom 450 bp of COI to the full 2.3 kb of COI-COII(Table 2). The zimmermani group formed a mono-phyletic clade, although the bootstrap support for thisclade was low (Fig. 4). Morphologically, the zimmer-mani group can be circumscribed into two groups ofspecies: dark-scaled and light-scaled species. The pres-ence of a “dark-scaled” group is further supported inthe ML tree, where it forms a well-supported mono-phyletic clade, whereas a “light-scaled” group wasparaphyletic with respect to the “dark-scaled” clade(Fig. 4).

mtDNA gene tree topologies within the dark- andlight-scaled groups contrasted sharply. For the light-scaled species,D. fordiandD.albovittella,mtDNA was


Tab

le2

.Sp

ecim

enco

llect

ion

data

and

hapl

otyp

ein

form

atio

n

Speci

es

Loca

lity

Lat

itude

Lon

git

ude

Dat

eC

ollect

or

Collect

ing

info

rmat

iona

mtD

NA

EF

1aD

NA

no.

Gen

Ban

k

Dioryctriazimmermanigr.

D.albovittella

USA

,N

V,W

hit

eP

ine

Co.,

Bak

er

39.0

13�

114.

123

14Ju

ne

2003

A.C

ogn

ato

Pmcone

33E

1A

R30

7JN

1627

06b,

JN16

2704

D.albovittella

USA

,A

Z,C

oco

nin

oC

o.,

Coco

nin

oN

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ear

Hap

py

Jack

34.7

43�

111.

407

25A

ug.2

007

S.Sh

ank

34A

R49

3JN

1627

07c

D.albovittella

USA

,A

Z,Y

avap

aiC

o.,

Min

gus

Mts

.34

.698

�11

2.12

211

Aug.2

007

S.Sh

ank

35A

R49

5JN

1627

08c

D.albovittella

USA

,A

Z,Y

avap

aiC

o.,

Min

gus

Mts

.34

.698

�11

2.12

211

Aug.2

007

S.Sh

ank

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R49

6JN

1627

09c

D.amatella

USA

,SC

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ele

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o,

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sM

ario

nN

tnl

Fore

st

33.1

67�

79.6

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Jun

e20

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oe

MV

-lig

ht

04A

R22

0JN

1627

22c

D.amatella

USA

,G

A,B

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o.,

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dgeville

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aldw

inse

ed

orc

har

d

32.9

99�

83.2

0928

Sept.

2006

D.M

ille

rZ

11Ð1

6:A

c04

AR

379

JN16

2723c

D.amatella

USA

,A

L,G

reen

eC

o.,

Fla

twood

seed

orc

har

d

33.1

25�

87.8

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1995

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05A

R33

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1627

24c

D.amatella

USA

,A

L,G

reen

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o.,

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har

d

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25�

87.8

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1995

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06E

2A

R33

2JN

1627

25b,

JN16

2705

D.amatella

USA

,G

A,B

aldw

inC

o.,

Mille

dgeville

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aldw

inse

ed

orc

har

d

32.9

99�

83.2

0928

Sept.

2006

D.M

ille

rZ

11Ð1

6:A

c�

C25

-p11

AR

380

JN16

2710c

D.amatella

USA

,G

A,B

aldw

inC

o.,

Mille

dgeville

,B

aldw

inse

ed

orc

har

d

32.9

99�

83.2

0928

Sept.

2006

D.M

ille

rZ

11Ð1

6:A

c�

C25

-p12

AR

381

JN16

2711c

D.amatella

USA

,G

A,B

aldw

inC

o.,

Mille

dgeville

,B

aldw

inse

ed

orc

har

d

32.9

99�

83.2

0928

Sept.

2006

D.M

ille

rZ

11Ð1

6:A

c13

E2

AR

382

JN16

2712b,

JN16

2705

D.amatella

USA

,G

A,B

aldw

inC

o.,

Mille

dgeville

,B

aldw

inse

ed

orc

har

d

32.9

99�

83.2

094

Jun

e20

07D

.M

ille

rZ

11Ð1

6:A

c�

C25

-p14

AR

474

JN16

2713c

D.amatella

USA

,G

A,B

aldw

inC

o.,

Mille

dgeville

,B

aldw

inse

ed

orc

har

d

32.9

99�

83.2

0917

Apri

l20

07D

.M

ille

rZ

11Ð1

6:A

c�

C25

-p14

AR

468

JN16

2714c

D.amatella

USA

,G

A,B

aldw

inC

o.,

Mille

dgeville

,B

aldw

inse

ed

orc

har

d

32.9

99�

83.2

094

Jun

e20

07D

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ille

rZ

11Ð1

6:A

c�

C25

-p19

AR

465

JN16

2715c

D.amatella

USA

,G

A,B

aldw

inC

o.,

Mille

dgeville

,B

aldw

inse

ed

orc

har

d

32.9

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83.2

0917

Apri

l20

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ille

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466

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D.amatella

USA

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Mille

dgeville

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inse

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Apri

l20

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c�

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-p21

AR

467

JN16

2717c


Tab

le2

.C

onti

nued

Speci

es

Loca

lity

Lat

itude

Lon

git

ude

Dat

eC

ollect

or

Collect

ing

info

rmat

iona

mtD

NA

EF

1aD

NA

no.

Gen

Ban

k

D.amatella

USA

,G

A,B

aldw

inC

o.,

Mille

dgeville

,B

aldw

inse

ed

orc

har

d

32.9

99�

83.2

094

Jun

e20

07D

.M

ille

rZ

11Ð1

6:A

c�

C25

-p22

AR

469

JN16

2718c

D.amatella

USA

,G

A,B

aldw

inC

o.,

Mille

dgeville

,B

aldw

inse

ed

orc

har

d

32.9

99�

83.2

094

Jun

e20

07D

.M

ille

rZ

11Ð1

6:A

c�

C25

-p23

AR

471

JN16

2719c

D.amatella

USA

,G

A,B

aldw

inC

o.,

Mille

dgeville

,B

aldw

inse

ed

orc

har

d

32.9

99�

83.2

094

Jun

e20

07D

.M

ille

rZ

11Ð1

6:A

c�

C25

-p24

AR

472

JN16

2720c

D.amatella

USA

,G

A,B

aldw

inC

o.,

Mille

dgeville

,B

aldw

inse

ed

orc

har

d

32.9

99�

83.2

094

Jun

e20

07D

.M

ille

rZ

11Ð1

6:A

c�

C25

-p25

AR

473

JN16

2721c

D.cambiicola

CA

N,B

C,P

rin

ceG

eorg

e53

.917

�12

2.75

019

July

2001

A.R

oe

UV

-lig

ht

01E

2A

R78

DQ

2951

83b,

JN16

2705

D.cambiicola

USA

,O

R,Ja

ckso

nC

o.,

Medfo

rd42

.327

�12

2.87

6C

.M

aste

rsPsmcambium

01A

R89

DQ

2961

69d

D.cambiicola

CA

N,B

C,C

L62

21Ju

ly20

05A

.R

oe

Ppcambium

01A

R39

0JN

1627

26c

D.cambiicola

CA

N,B

C,P

rin

ceG

eorg

e53

.917

�12

2.75

019

July

2001

A.R

oe

UV

-lig

ht

02A

R79

DQ

2961

70d

D.cambiicola

CA

N,B

C,P

rin

ceG

eorg

e53

.917

�12

2.75

019

July

2001

A.R

oe

UV

-lig

ht

03A

R20

4JN

1627

27d

D.cambiicola

CA

N,B

C,C

L62

21Ju

ly20

05A

.R

oe

Ppcambium

03A

R38

9JN

1627

28e

D.contortella

CA

N,B

C,P

rin

ceG

eorg

e53

.917

�12

2.75

019

July

2001

A.R

oe

Pccambium

01E

2A

R38

7JN

1627

29c,

XX

XX

XX

D.contortella

CA

N,B

C,P

ritc

har

d50

.599

�11

9.89

25

Aug.2

003

A.R

oe

MV

-lig

ht

02E

2A

R32

2JN

1627

30b,

JN16

2705

D.fordi

USA

,C

A,B

utt

eC

o.,

Ch

ico

39.7

28�

121.

837

12Ju

ne

2001

A.R

oe

UV

-lig

ht

37E

1A

R15

7D

Q29

5184b,

JN16

2704

D.fordi

USA

,C

A,B

utt

eC

o.,

Ch

ico

39.7

28�

121.

837

4Ð6

Oct

.200

2A

.R

oe

MV

-lig

ht

38A

R28

5D

Q29

6173d

D.fordi

USA

,C

A,B

utt

eC

o.,

Ch

ico

39.7

28�

121.

837

4Ð6

Oct

.200

2A

.R

oe

MV

-lig

ht

38A

R28

8D

Q29

6173d

D.fordi

USA

,C

A,B

utt

eC

o.,

Ch

ico

39.7

28�

121.

837

4Ð6

Oct

.200

2A

.R

oe

MV

-lig

ht

39A

R28

9D

Q29

6174d

D.fordi

USA

,C

A,B

akers

Þeld

,K

ern

Can

yon

35.5

33�

118.

619

13Ju

ly20

07A

.R

oe

MV

-lig

ht

40A

R43

4JN

1627

31c

D.merkeli

USA

,G

A,B

aldw

inC

o.,

Mille

dgeville

,B

aldw

inse

ed

orc

har

d

32.9

99�

83.2

0928

Sept.

2006

D.M

ille

rZ

9Ð14

:Ac

�C

25-p

07E

2A

R37

3JN

1627

33b,

JN16

2705

D.merkeli

USA

,G

A,B

aldw

inC

o.,

Mille

dgeville

,B

aldw

inse

ed

orc

har

d

32.9

99�

83.2

0928

Sept.

2006

D.M

ille

rZ

9Ð14

:Ac

�C

25-p

08A

R37

4JN

1627

34c

D.merkeli

USA

,G

A,B

aldw

inC

o.,

Mille

dgeville

,B

aldw

inse

ed

orc

har

d

32.9

99�

83.2

0928

Sept.

2006

D.M

ille

rZ

9Ð14

:Ac

09E

2A

R37

5JN

1627

35c,

JN16

2705


Tab

le2

.C

onti

nued

Speci

es

Loca

lity

Lat

itude

Lon

git

ude

Dat

eC

ollect

or

Collect

ing

info

rmat

iona

mtD

NA

EF

1aD

NA

no.

Gen

Ban

k

D.merkeli

USA

,G

A,B

aldw

inC

o.,

Mille

dgeville

,B

aldw

inse

ed

orc

har

d

32.9

99�

83.2

0928

Sept.

2006

D.M

ille

rZ

9Ð14

:Ac

�C

25-p

10A

R37

7JN

1627

32c

D.resinosella

USA

,M

I,E

mm

et

Co.,

UM

BS

Stn

Str

eam

Lab

45.5

60�

84.6

7420

06B

.Sch

olt

en

sM

V-l

igh

t14

E2

AR

386

JN16

2736b,

JN16

2705

D.resinosella

USA

,M

I,C

heboygon

Co.,

Wildw

ood

Rd.

45.3

65�

84.6

5215

July

2006

B.Sch

olt

en

s14

AR

413

JN16

2737e

D.resinosella

USA

,M

N,It

asca

Co.,

Nort

hSta

rC

ampgr.

47.5

57�

93.6

541

Aug.2

007

A.R

oe

UV

-lig

ht

14A

R48

0JN

1627

38c

D.resinosella

USA

,M

N,It

asca

Co.,

Nort

hSta

rC

ampgr.

47.5

57�

93.6

541

Aug.2

007

A.R

oe

UV

-lig

ht

14A

R48

1JN

1627

39c

D.resinosella

USA

,M

N,It

asca

Co.,

Nort

hSta

rC

ampgr.

47.5

57�

93.6

541

Aug.2

007

A.R

oe

UV

-lig

ht

14A

R48

2JN

1627

40c

D.resinosella

USA

,M

N,It

asca

Co.,

Nort

hSta

rC

ampgr.

47.5

57�

93.6

541

Aug.2

007

A.R

oe

UV

-lig

ht

14A

R48

3JN

1627

41c

D.resinosella

USA

,M

N,It

asca

Co.,

Nort

hSta

rC

ampgr.

47.5

57�

93.6

541

Aug.2

007

A.R

oe

UV

-lig

ht

14A

R48

4JN

1627

42c

D.resinosella

USA

,M

N,It

asca

Co.,

Nort

hSta

rC

ampgr.

47.5

57�

93.6

541

Aug.2

007

A.R

oe

UV

-lig

ht

14A

R48

8JN

1627

43c

D.resinosella

USA

,M

N,It

asca

Co.,

Nort

hSta

rC

ampgr.

47.5

57�

93.6

541

Aug.2

007

A.R

oe

UV

-lig

ht

14A

R48

9JN

1627

44c

D.resinosella

USA

,M

N,C

han

has

sen

Co.,

MN

Lan

dsc

ape

Arb

ore

tum

44.8

63�

93.6

171

Jun

e20

07A

.R

oe

Pccambium

14A

R49

1JN

1627

45c

D.resinosella

USA

,M

N,It

asca

Co.,

Nort

hSta

rC

ampgr.

47.5

57�

93.6

541

Aug.2

007

A.R

oe

UV

-lig

ht

23A

R48

6JN

1627

46c

D.resinosella

USA

,M

N,It

asca

Co.,

Nort

hSta

rC

ampgr.

47.5

57�

93.6

541

Aug.2

007

A.R

oe

UV

-lig

ht

26A

R48

5JN

1627

47c

D.resinosella

USA

,M

N,It

asca

Co.,

Nort

hSta

rC

ampgr.

47.5

57�

93.6

541

Aug.2

007

A.R

oe

UV

-lig

ht

27A

R48

7JN

1627

48c

D.resinosella

USA

,M

N,It

asca

Co.,

Nort

hSta

rC

ampgr.

47.5

57�

93.6

541

Aug.2

007

A.R

oe

UV

-lig

ht

28A

R49

0JN

1627

49c

D.resinosella

USA

,M

N,C

han

has

sen

Co.,

MN

Lan

dsc

ape

Arb

ore

tum

44.8

63�

93.6

171

Jun

e20

07A

.R

oe

Pscambium

29A

R49

2JN

1627

50c

D.taedivorella

USA

,M

D,Q

ueen

An

neÕs

Co.,

Gra

son

ville

38.9

58�

76.2

1019

86D

.C.F

erg

eso

n32

Du11

9D

Q24

7731b

D.tumicolella

USA

,N

EA

ug.1

990

G.G

ran

tPp

01A

R28

JN16

2751d

D.tumicolella

USA

,K

S,C

raw

ford

Co.

37.5

17�

94.8

5020

02K

.O.B

ell

30D

u11

2D

Q24

7729b

D.yatesi

USA

,G

A,R

abun

Co.,

Ch

atta

hooch

ee

N.F

.n

r.C

layto

n

34.8

96�

83.3

621

Aug.2

004

J.H

anula

Pucone

07A

R45

1JN

1627

53c

D.yatesi

USA

,G

A,R

abun

Co.,

Ch

atta

hooch

ee

N.F

.n

r.C

layto

n

34.8

96�

83.3

621

Aug.2

004

J.H

anula

Pucone

07A

R45

4JN

1627

55c


Tab

le2

.C

onti

nued

Speci

es

Loca

lity

Lat

itude

Lon

git

ude

Dat

eC

ollect

or

Collect

ing

info

rmat

iona

mtD

NA

EF

1aD

NA

no.

Gen

Ban

k

D.yatesi

USA

,G

A,R

abun

Co.,

Ch

atta

hooch

ee

N.F

.n

r.C

layto

n

34.8

96�

83.3

621

Aug.2

004

J.H

anula

Pucone

07A

R45

5JN

1627

57c

D.yatesi

USA

,G

A,R

abun

Co.,

Ch

atta

hooch

ee

N.F

.n

r.C

layto

n

34.8

96�

83.3

621

Aug.2

004

J.H

anula

Pucone

07A

R45

6JN

1627

58c

D.yatesi

USA

,G

A,R

abun

Co.,

Ch

atta

hooch

ee

N.F

.n

r.C

layto

n

34.8

96�

83.3

621

Aug.2

004

J.H

anula

Pucone

07A

R45

7JN

1627

59c

D.yatesi

USA

,G

A,R

abun

Co.,

Ch

atta

hooch

ee

N.F

.n

r.C

layto

n

34.8

96�

83.3

621

Aug.2

004

J.H

anula

Pucone

07A

R45

9JN

1627

61c

D.yatesi

USA

,G

A,R

abun

Co.,

Ch

atta

hooch

ee

N.F

.n

r.C

layto

n

34.8

96�

83.3

621

Aug.2

004

J.H

anula

Pucone

15A

R45

0JN

1627

52c

D.yatesi

USA

,G

A,R

abun

Co.,

Ch

atta

hooch

ee

N.F

.n

r.C

layto

n

34.8

96�

83.3

621

Aug.2

004

J.H

anula

Pucone

16E

2A

R45

2JN

1627

54b,

JN16

2705

D.yatesi

USA

,G

A,R

abun

Co.,

Ch

atta

hooch

ee

N.F

.n

r.C

layto

n

34.8

96�

83.3

621

Aug.2

004

J.H

anula

Pucone

17A

R45

3JN

1627

56c

D.yatesi

USA

,G

A,R

abun

Co.,

Ch

atta

hooch

ee

N.F

.n

r.C

layto

n

34.8

96�

83.3

621

Aug.2

004

J.H

anula

Pucone

18A

R45

8JN

1627

60c

D.zimmermani

USA

,M

S,H

inds

Co.

32.2

92�

90.4

1719

94M

.E.P

osh

ore

31D

u11

8D

Q24

7730b

Outg

roup

Dioryctriaponderosaegr.

D.okanaganella

USA

,C

A,E

ldora

do

Co.

Pla

cerv

ille

38.7

30�

120.

799

13Ju

ne

2001

A.R

oe

AR

148

DQ

2951

78b

Dioryctriabaumhoferi

gr. D.pentictonella

USA

,C

A,B

utt

eC

o.,

Ch

ico

39.7

28�

121.

837

3A

ug.2

000

C.R

udolf

AR

15D

Q29

5180b

aP

hero

mon

eab

bre

via

tion

sas

inT

able

1;H

ost

pla

ntab

bre

via

tion

sas

follow

s:Pc,Pinuscontorta;Pm,Pinusmonophylla;Pp,Pinusponderosa;Ps,Pinusstrobus;Pt,Pinustaeda;Pu,Pinuspungens;Psm,Pseudotsuga

menziesii.

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CO

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CO

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OII

sequen

cefr

agm

en

t(C

1-J-

2183

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2-N

-301

3).

dP

arti

al45

0-bp

CO

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quen

cefr

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1-J-

2183

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congruent with previously described species limits(Figs. 3 and 4). These species had low levels of in-traspeciÞc variation and high, nonoverlapping levelsof interspeciÞc variation (Fig. 5), with no evidence ofshared mtDNA haplotypes (Figs. 3 and 4). Both spe-cies formed strongly supported, monophyletic cladesin the parsimony haplotype network and ML tree(Figs. 3 and 4). Haplotype diversity was also very highfor both species, despite low levels of nucleotide di-versity (Table 3).

In contrast to the light-scaled species, mtDNA di-versity in the dark-scaled group was not congruentwith previously described species limits, host plantassociation, pheromone attraction, or geographic lo-cation (Figs. 3A and 4). Often individuals from dif-ferent host plants or pheromone blends were moreclosely related than individuals with similar ecologicaltraits. All dark-scaled species had overlapping intra-and interspeciÞc variation (Fig. 5), and several hap-lotypes were shared among species (Figs. 3A and 4).Five of the 32 dark-scaled haplotypes were sharedbetween species, even when separated by large geo-graphic distances (Fig. 4, e.g., mtDNA haplotype 14).There was little phylogenetic structuring among spe-cies (Figs. 3A and 4), and relationships among hap-lotypes were characterized by short internal branches

with little to no bootstrap support (Fig. 4). For specieswith multiple individuals, nucleotide diversity waslow, ranging from 0.00121 (D. contortella) to 0.0484(D. amatella). Despite the low nucleotide diversityand shared haplotypes among species, overall haplo-type diversity within species was high, above 0.900 inseveral species (Table 3), indicating that nearly allmtDNA sequences were unique.

The second locus, EF1a, was sequenced for a subsetof individuals (n � 11), representing eight of the 11species examined in this study (Table 2). In total, 584bp were obtained which represented two unique hap-lotypes. A parsimony network (Fig. 3B) shows thatthese two haplotypes (E1 and E2) differ by a singlemutation and coincide with the light-scaled and dark-scaled groups, which was congruent with the mtDNAresults.

Discussion

Species limits among the dark-scaled members ofthe zimmermani group have always been consideredproblematic. Previous work on a Dioryctria speciescomplex demonstrated that the examination of mul-tiple molecular markers (COI and EF1a) and densetaxon sampling successfully clariÞed species limits

Fig. 3. Parsimonyhaplotypenetworks for two independent lociof 11membersof theD.zimmermani speciesgroupShadedcircles represent individual haplotypes, with circle size proportional to the number of specimens. Lines connecting haplotypesrepresent single mutational differences, with missing haplotypes represented by black circles. Numbers within black circlesrepresent the number of missing haplotypes. (A) mDNA locus (COI-COII). (B) Nuclear locus (EF1a). (Online Þgure incolor.)


between two sympatric species (Roe and Sperling2007). By applying a similar technique, we sought toclarify species limits, estimate the genetic diversitywithin species, and clarify the phylogenetic rela-tionships among species within the zimmermanigroup.

Congruence of the mtDNA gene tree with estab-lished species limits in the zimmermani group wasvariable. Species limits and the mtDNA gene tree wereclearly congruent for the light-scaled species. The twolight-scaled species (D. albovittella andD. fordi) werecharacterized by high interspeciÞc pairwise variationand low intraspeciÞc variation (Fig. 5), and each spe-cies was well supported as monophyletic (Fig. 4).

Gene tree congruence in the light-scaled species con-trasts with the broad gene treeÑspecies tree incon-gruence in the dark-scaled clade of the zimmermanigroup. The nine dark-scaled species showed little phy-logenetic resolution (Fig. 4) and had overlapping in-terspeciÞc pairwise variation (Fig. 5). The nuclearlocus (EF1a) lacked species-level variation, despitediagnostic success in other Dioryctria species (Roeand Sperling 2007).

Discordance between molecular variation andspecies limits is not unusual. In a recent survey,Funk and Omland (2003) estimate that at �23% oftaxa (26.5% of arthropods) show some species-levelpolyphyly (considered broadly to represent non-

Fig. 4. Maximum likelihood phylogram (�ln �3380.800) of mtDNA (COI-COII) for the D. zimmermani species group.ML model information as follows: GTR�G; A � 0.299, C � 0.140, G � 0.135, T � 0.456; G � 0.0200; A-C � 0.0000170, A-G �11.904, A-T � 2.474, C-G � 0.0000170, C-T � 29.935, G-T � 1.000. Thickened branches indicate clade support �70%. For eachhaplotype, sample size, haplotype number, sampling locality, host plant, and pheromone association are shown. Host plantabbreviations are as given in Table 2. Pheromone abbreviations are as given in Table 1. (Online Þgure in color.)


monophyly). Several processes can lead to this phe-nomenon of gene treeÑspecies tree incongruence.

First, it is possible that the currently recognizedspecies limits are incorrect (i.e., imperfect taxonomy)and the mtDNA gene tree accurately represents thespecies tree. In the case of the dark-scaled species, theclade would be considered “overspilt” with all the taxabelonging to a single, widely distributed, highly poly-morphic species, rather than multiple distinct species.Historically, these taxa have been separated based onminor forewing variation and larval host plant associ-ations, although all authors acknowledge that complex

species problems continue to exist within the group(Mutuura et al. 1969, Schaber and Wood 1971, Mu-tuura and Munroe 1979, Hedlin et al. 1980, Mutuura1982, Sopow et al. 1996, Neunzig 2003). In fact, al-though Heinrich (1956) tentatively recognized D.cambiicola as a species, he postulated that it mightactually represent a western race of D. zimmermani,rather than a distinct species, a sentiment later sup-ported by Munroe (1959). Furthermore, many specieshave sympatric or parapatric distributions, as well asextensive overlap of diagnostic characters (Sopow etal. 1996), supporting the hypothesis of a single dark-scaled species.

Widely distributed, highly polymorphic species arenot unusual in Dioryctria. Dioryctria abietivorella(Grote), an important cone pest throughout NorthAmerica, has broad larval host associations and a trans-continental distribution. This level of ecological andgeographic variation would be comparable to the vari-ation exhibited among the dark-scaled members of thezimmermani group. Morphological variability, partic-ularly in forewing coloration, is also well known forother Dioryctria species (Roe et al. 2006, Roe andSperling 2007). For example, Dioryctria pentictonella(Mutuura, Munroe, & Ross), another raised scale spe-cies, has highly plastic forewing coloration, rangingfrom nearly black to red to white, which all occurwithin a single season at a single collection locality(Roe et al. 2006). Again, forewing variability amongthe dark-scaled species is within the intraspeciÞcrange of variability previously documented inD. pen-tictonella.

The second possibility is that the current specieslimits in the dark-scaled clade are accurate and thatthe mtDNA gene tree fails to accurately reßect theevolutionary relationships among these species. Al-though many species show interspeciÞc overlap oflarval host associations, other species do not (Table 1).As well, distinct pheromone sex attractants have beendescribed for several dark-scaled species, particularlyfor dark-scaled species in the southeastern UnitedStates (Miller et al. 2010). Although cross speciesattraction occurs (Hanula et al. 1984), recent work hasshown thatDioryctria pheromones are complex (Mil-lar et al. 2005, 2010) and pheromone races exist withinDioryctria species (Grant et al. 2009), although it isuncertain whether these races represent distinct spe-cies or show reduced inter-race gene ßow.

If individuals in the dark-scaled clade represent asingle species, we would expect to observe some phy-logeographic structuring among the mtDNA haplo-types. Instead, haplotypes are shared across broadgeographic ranges (e.g., mtDNA haplotype 14), withindividuals collected in the same location more closelyrelated to individuals from distant locations than toeach other (Figs. 3A and 4). The lack of phylogeo-graphic structuring and ecological variation amongspecies suggests that more complex evolutionary pro-cesses may be responsible for the observed incongru-ence (Schmidt and Sperling 2008).

Gene treeÐspecies tree discordance is a commonissue when seeking to delimit species boundaries and

Fig. 5. Intra- and interspeciÞc uncorrected pairwisemtDNA distances for 11 species in theD. zimmermani speciesgroup. Range (min.Ðmax) of pairwise distances are shownwith mean distance indicated by a black bar. Where specieswere represented by less than three sequences (D. taedi-vorella, D. tumicollela, D. zimmermani) only interspeciÞcpairwise distance is shown.

Table 3. Genetic diversity estimates for members of the D.zimmermani species group

Species n H Hd (SD) Pi (SD)

Dark scaledD. amatella 16 14 0.983 (0.0280) 0.0484 (0.000510)D. cambiicola 6 3 0.733 (0.155) 0.00323 (0.00133)D. contortella 2 2 1.000 (0.500) 0.00121 (0.000610)D. merkeli 4 4 1.000 (0.177) 0.00323 (0.000780)D. resinosella 15 6 0.571 (0.149) 0.00162 (0.000530)D. taedivorella 1 1 N.A.a N.A.D. tumicolella 2 2 1.000 (0.500) 0.00842 (0.00421)D. yatesi 10 5 0.778 (0.137) 0.00365 (0.000900)D. zimmermani 1 1 N.A. N.A.

Light scaledD. albovittella 4 4 1.000 (0.177) 0.00908 (0.00209)D. fordi 5 4 0.900 (0.161) 0.00295 (0.000670)

n, number of specimens; H, number of haplotypes; Hd, haplotypediversity; and Pi, nucleotide diversity.aN.A., not applicable.


can be caused by several evolutionary processes (e.g.,Maddison 1997, Funk and Omland 2003), such as in-complete lineage sorting or introgression. Incompletelineage sorting results when gene lineages of closelyrelated species have not had sufÞcient time to coalesceand achieve reciprocal monophyly. Generally,mtDNA is considered more robust to incomplete lin-eage sorting than nuclear genes (Hudson and Turelli2003) but has been shown to fail among rapidly radi-ating clades (Funk and Omland 2003), particularlyamong groups experiencing ecological race formation(Dres and Mallet 2002, Scheffer and Hawthorne2007). If dark-scaled Dioryctria species are undergo-ing rapid ecological divergence based on larval hostassociation and pheromone attraction, then the spe-cies barriers separating these recently diverged spe-cies may be maintained by a small region of the ge-nome (Matsubayashi et al. 2009), whereas otherregions of the genome (e.g., mtDNA) will not havehad sufÞcient time for purifying selection to producereciprocally monophyletic clades (Funk and Omland2003).

Conversely, interspeciÞc hybridization and subse-quent introgression is the movement of foreign ge-netic material into a conspeciÞc genome. This processleads to reticulate evolutionary relationships and genetreeÐspecies tree discordance, clouding genealogicalspecies boundaries (Maddison 1997). InterspeciÞc hy-bridization is surprisingly common (Mallet 2005),with hybridization rates ranging from 6 to 29% amongspecies of Lepidoptera (Sperling 1990, Mallet et al.2007). mtDNA introgression may occur without nu-clear introgression (Ballard and Whitlock 2004, PetitandExcofÞer2009),particularly ifmtDNAis impactedby direct or indirect selection (Ballard and Whitlock2004, Hurst and Jiggins 2005). For hybridization tooccur, species must be sympatric/parapatic, syn-chronic, and be capable of interbreeding (Schmidtand Sperling 2008). Dark-scaled zimmermani specieshave sympatric and parapatric distributions, overlap-ping ßight times, and have shown evidence for cross-species pheromone attraction (Hanula et al. 1984,Whitehouse et al. 2011), all conditions necessary forhybridization to occur.

As stated previously in many studies, we must ac-knowledge that difÞcult species problems continue toexist in the zimmermani species group. Despite ourdense taxon sampling and inclusion of multiple lines ofevidence, we were unable to fully resolve specieslimits among dark zimmermani species group mem-bers. Many of the dark-scaled taxa are considered“good” species, with extensive information availableon their behavioral and ecological differences, as wellas their economic impacts (Whitehouse et al. 2011).Although mtDNA has been used extensively as a di-agnostic marker in Lepidoptera (e.g., DNA barcoding;Hebert et al. 2003, 2004), and is successful in otherspecies ofDioryctria (Roe and Sperling 2007), includ-ing light-scaled members of the zimmermani group,studies have shown that a single marker is prone tofailure, particularly when differentiating closely re-lated species (Roe and Sperling 2007, Schmidt and

Sperling 2008, Roe et al. 2010). Given the economicimportance of these dark-scaled species and in theinterest of nomenclatural stability, we choose not torecommend any taxonomic changes to this groupbased on a single molecular marker.

Based on the currently available data, we are unableto differentiate among the alternative hypothesis forthe cause of the gene treeÐspecies tree discordancedetected among the dark-scaled zimmermani species.Effective evaluation of these hypotheses requires datafrom multiple regions of the genome (Maddison 1997)and analytical means for resolving gene tree discor-dance (Degnan and Rosenberg 2009). Highly variablemolecular markers, such as microsatellites or single-nucleotide polymorphisms from regions throughoutthe genome, in addition to behavioral, ecological, andmorphological characters will be required to provideclarity to the dark-scaled zimmermani species com-plex.

Acknowledgments

We acknowledge A. Cognato, G. Grant, C. Rudolf, B.Scholtens, and S. Shank for providing specimens for thisstudy. Without the assistance of these collectors, it would beimpossible to have performed this study. We also thank themembers of the Weller and Sperling laboratories for assis-tance with the collection of the molecular data, as well as helpduring the numerous Þeld trips needed to collect specimens.We thank the anonymous reviewer who provided commentsthat helped clarify this manuscript. Financial support wasprovided by the National Science FoundationÕs Assemblingthe Tree of Life program (ATOL 0531769 to C. Mitter) andATOL 0531639 to S.J.W.), AES Experiment Station Project(Min-17-022 to S.J.W.), and National Sciences and Engineer-ing Research Council of Canada Postgraduate fellowships toA.D.R.

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Received 13 March 2011; accepted 6 July 2011.


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