Evolutionary relationships within European Monochamus (Coleoptera:...

Evolutionary relationships within EuropeanMonochamus (Coleoptera: Cerambycidae) highlightthe role of altitude in species delineation

FOTINI A. KOUTROUMPA1†, DANIEL ROUGON1, CORALIE BERTHEAU1‡,FRANÇOIS LIEUTIER1 and GÉRALDINE ROUX-MORABITO1,2*

1Université d’Orléans, UPRES-EA-1207 (LBLGC), 45067 Orléans, France2INRA, UR0633 (URZF, Zoologie Forestière), F-45075 Orléans, France

Received 9 November 2012; revised 21 December 2012; accepted for publication 21 December 2012

Phylogenetic relationships within the European Monochamus (Coleoptera: Cerambycidae) remain understudieddespite their increasing importance in the Pine Wood Nematode spread in Europe. To clarify the delimitationand the evolutionary history of the two main European Monochamus species, Monochamus galloprovincialis andMonochamus sutor, as well as their sub-species, a comparative study using morphological, molecular, andbiogeographical criterions was conducted. Four morphological characters, including a newly-described morphologi-cal character on the male genitalia, separated the two species. Additionally, molecular data revealed twelve andtwo single nucleotide polymorphisms in cytochrome oxidase c subunit I and 28S, respectively, supporting speciessegregation. By contrast, incongruence between morphological and genetic results did not allow discriminating thesub-species of M. galloprovincialis and M. sutor, even though mitochondrial DNA revealed intraspecific differen-tiation, mostly consenting to a multiple refugia origin. Within-species variability was explained to a large extentby biogeography (i.e. altitude, climate). These different ecological adaptations within beetle species, together withpotential climate change impact, increase the risk of spreading the nematode across Europe to novel conifer hostsand challenge the European biosecurity. © 2013 The Linnean Society of London, Biological Journal of the LinneanSociety, 2013, 109, 354–376.

ADDITIONAL KEYWORDS: biogeography – genitalia – integrative taxonomy – morphology – mitochondrialDNA – nuclear DNA – PWN vector.

INTRODUCTION

The coexistence of closely-related taxa is an issue offundamental interest in evolutionary biology andaccurate taxonomy is crucial for such evolutionstudies, as well as biodiversity, ecology, and conser-vation studies (Cracraft, 2002; Agapow et al., 2004;

Mace, 2004). Nevertheless, much comtroversyappears to surround the species concept and lowertaxa delimitation (De Queiroz, 2007). Lacking stand-ardized operational criteria to delimit them, severalstudies have stressed the importance of integrativetaxonomy (i.e. a multidisciplinary approach to sepa-rate species) (De Queiroz, 2007; Roe & Sperling,2007b; Schlick-Steiner et al., 2010; Heethoff et al.,2011; Fujita et al., 2012). Even though monophylyusually supports species separation, discordancehas been observed not only between phylogeniesbased on morphological versus molecular markers(Wiens & Penkrot, 2002), but also between molecularmarkers (mitochondrial versus nuclear DNA) (Shaw,2002). The mitochondrial DNA and especially thecytochrome oxidase c subunit I (COI) gene, as

*Corresponding author.E-mail: [email protected]†Current address: Max Planck for Chemical Ecology,Department of Entomology, Beutenberg Campus,Hans-Knöll Strasse 8, 07745 Jena, Germany‡Current address: Department of Forest & Soil Sciences,Institute of Forest Entomology, Forest Pathology & ForestProtection, BOKU, University of Natural Resources & LifeSciences, Hasenauerstrasse 38, Vienna 1190, Austria

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Biological Journal of the Linnean Society, 2013, 109, 354–376. With 6 figures

© 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 109, 354–376354

proposed by ‘barcodes’ (Hebert et al., 2003a; Hebert,Ratnasingham & deWaard, 2003b), has a number ofadvantages for the distinction of species, althoughcollaboration with traditional taxonomy is necessary(Lipscomb, Platnick & Wheeler, 2003; Mallet &Willmott, 2003; Seberg et al., 2003; Will & Rubinoff,2004; DeSalle, Egan & Siddall, 2005; Taylor & Harris,2012). Thus, studies attempting to define speciesboundaries, particularly in cases of recent speciationevents or cryptic species, need the consensus ofnumerous independent criteria.

Monochamus (Coleoptera, Cerambycidae) is aworldwide distributed genus that has drawn atten-tion as a result of its association with the transmis-sion to conifers of the highly pathogenic pine woodnematode (PWN) Bursaphelenchus xylophilus Steiner& Buhrer, 1934 (Nickle, 1970). Five species of thegenus Monochamus have been described in Europe:Monochamus galloprovincialis (Olivier, 1795), Mono-chamus sutor (Linnaeus, 1758), Monochamus salt-uarius (Gebler, 1830), Monochamus sartor (Fabricius,1787), and Monochamus urussovi (Fischer, 1806); allattacking conifers and mainly Pinus and Piceaspecies. European Monochamus species are describedin the literature as closely-related species furtherseparated into sub-species (Cesari et al., 2005). Phy-logenetic relationships within the European Mono-chamus remain understudied despite the increasedimportance of such clarification subsequent to thediscovery of the invasive PWN in Portugal (Motaet al., 1999; Rodrigues, 2008; Fonseca et al., 2012)and more recently in Spain (Abelleira et al., 2011;Robertson et al., 2011). Of the five European Mono-chamus species, only M. galloprovincialis has been

proved to vector the nematode in Europe so far (Sousaet al., 2001). Monochamus sutor is absent from Por-tugal, whereas it is present in Spain and the rest ofEurope, at higher altitudes than M. galloprovincialis(Hellrigl, 1971; Villiers, 1978; Vives, 2000; Sama,2002, 2008). As a result of the progression of thenematode infestation and its recent discovery at theSpanish border with Portugal, M. sutor is the closestco-vectoring candidate in Europe (see geographicaldistribution; Fig. 1) and increases the undeniable riskof PWD expansion to the continent.

To date, the taxonomic status of Monochamusspecies is based on morphological features; however,some controversy remains as a result of the consistentvariability of these characters both within species andbetween sister-species. Taxonomic uncertainties stillremain, especially within the highly polymorphicM. galloprovincialis species, with the dark specimenseasily mistaken for its sister-species M. sutor or at thesub-species where the confusions are the most fre-quent. Hellrigl (1971), Sama (2002, 2008), Tomminen& Leppänen (1991), Villiers (1978) and Vives (2000)have morphologically and ecologically described thesetwo Monochamus species and their sub-taxa (Table 1).It is generally admitted that Monochamus gallopro-vincialis galloprovincialis is found in south-westEurope and North Africa, whereas Monochamus gal-loprovincialis pistor (Germar, 1818) occurs in north-ern, central, and eastern Europe. Monochamusg. pistor (Germar, 1818) is also present in southernEurope (central Spain, southern France, and theFrench Alps) but, in these cases, higher elevationscompensate the cooler conditions required by thissub-species (Vives, 2000). In his description of

Figure 1. Pine wood nematode (PWN) infested areas (stars) and Monochamus galloprovincialis versus Monochamussutor geographical distribution in Europe (lines). Combination map of the CABI Crop Protection Compendium data andobservations of Hellrigl (1971), Sama (2002) and Vives (2000). A, yellow line encircles the regions of M. galloprovincialisgeographical distribution. B, blue line encircles the regions of M. sutor geographical distribution.

EVOLUTIONARY RELATIONSHIPS WITHIN EUROPEAN MONOCHAMUS 355


Tab

le1.

Mon

och

amu

sga

llop

rovi

nci

alis

/Mon

och

amu

ssu

tor

taxo

nco

mpa

riso

nby

Hel

lrig

l(1

971)

,Sam

a(2

002)

,Tom

min

en&

Lep

pän

en(1

991)

,Vil

lier

s(1

978)

and

Viv

es(2

000)

Hos

tD

istr

ibu

tion

Ele

vati

onC

lim

atic

requ

irem

ents

Mor

phol

ogy

Mon

och

amu

sga

llop

rovi

nci

alis

gall

opro

vin

cial

is

Pin

us

spp.

Nor

thA

fric

aS

outh

-wes

tan

dC

entr

alE

uro

pe

<80

0m

Med

iter

ran

ean

War

m,

dry

Bra

nch

es

(a)

•V

form

scu

tell

um

•R

oun

dap

exof

the

med

ian

lobe

(mal

ege

nit

alia

)(b

)•

Leg

san

dan

ten

nae

brow

n–r

ed•

Ely

tra

cove

red

grey

orre

d–w

hit

ebr

istl

e•

Lar

gebo

dysi

ze

Mon

och

amu

sga

llop

rovi

nci

alis

pist

or

Pin

us

sylv

estr

isP

inu

sn

igra

Pic

ea(o

ccas

ion

ally

)

Nor

thE

uro

peC

entr

alan

dE

ast

Eu

rope

Sou

thE

uro

pe(o

ccas

ion

ally

)R

uss

iaN

orth

Kaz

akh

stan

Cau

sasi

aTr

ansc

auca

sia

Arm

enia

Nor

thTu

rkey

>80

0m

Col

d,h

um

idB

ran

ches

Sam

eas

(a)

(c)

•L

egs

and

ante

nn

aebl

ack

•E

lytr

ara

reye

llow

–wh

ite

bris

tle

•S

mal

lbo

dysi

ze

Mon

och

amu

ssu

tor

suto

rP

icea

abie

sP

icea

exce

lsa

Pin

us

spp.

Mid

dle

and

east

Eu

rope

Th

eP

yren

ees,

Alp

sR

uss

iaN

orth

Asi

au

pto

Japa

n

Col

d,h

um

idTr

un

k(d

)•

IIfo

rmsc

ute

llu

m•

Fla

tten

edap

exof

the

med

ian

lobe

(mal

ege

nit

alia

)(e

)•

Leg

san

dan

ten

nae

blac

k•

Ely

tra

blac

kw

ith

few

wh

ite–

yell

owbr

istl

e•

Lar

gebo

dysi

ze

Mon

och

amu

ssu

tor

pell

ioP

icea

abie

sP

icea

exce

lsa

Pin

us

spp.

Eas

tE

uro

peS

iber

iaC

old,

hu

mid

Tru

nk

Sam

eas

(d)

(f)

•L

egs

and

ante

nn

aebl

ack

•E

lytr

abl

ack

wit

hou

tbr

istl

e•

Lar

gebo

dysi

ze

356 F. A. KOUTROUMPA ET AL.


M. g. galloprovincialis specimens from North Africa,Sama (2008) mentions four specimens identical to theM. g. pistor holotype from Slovenia. Similarly, withinM. sutor, it is unclear whether Monochamus sutorsutor and Monochamus sutor pellio represent morpho-logically different forms or sub-species with distinctgeographical distribution. Moreover, further clarifica-tion is needed on their geographical distributionbecause the limits of their sympatry or syntopy arenot clearly defined (Sama, 2002, 2008) (Fig. 1).

We consider the clarification of the phylogeneticrelationships of M. galloprovincialis and M. sutor fun-damental for future investigation on integrating pestmanagement programmes. Although the Europeanspecies of the genus Monochamus, and mainly M. gal-loprovincialis, have been subject of numerous studieson their biology and association with PWN (Sousaet al., 2001, 2002; Naves, Sousa & Quartau, 2006a,2006b; Naves, 2007; Naves et al., 2007; Akbulut et al.,2008; Koutroumpa et al., 2008b, 2009b; Akbulut,2009; Akbulut & Stamps, 2012), literature reports ontaxonomy and geographical distribution of PalearcticMonochamus species are scarce (Hellrigl, 1971;Villiers, 1978; Tomminen & Leppänen, 1991; Vives,2000; Sama, 2002, 2008). With the exception of onemolecular phylogenetic study on the European Mono-chamus (Cesari et al., 2005), the genetic variability atthe intra- and interspecific level is unknown inEurope.

The present study focused on M. galloprovincialis–M. sutor as the most expanded European Monocha-mus species, and investigates their relationshipswith the sub-species M. g. pistor and M. s. pellio.Under the assumption of a morphological delimita-tion, we use molecular characters to examine thelevel of differentiation between and within the twosister species: M. galloprovincialis and M. sutor. Weapplied a multi-marker approach on samples mainlycollected from their southern European distribution,which represents the area under direct risk ofnatural invasion by PWN (Robinet et al., 2011) andwhere the delimitation of the different sub-species iseven more complex (Vives, 2000). France is a keyarea and thus occupies a core position in this studyas a result of the occurrence of the two Monochamusspecies and their sub-species, as well as its strategicposition for the natural dissemination of the nema-tode from Portugal and Spain to the rest of Europe.A broad range of elevations and potential host treeswas considered because they offer potential environ-mental factors hiding cryptic taxa within thesespecies. The results obtained highlight new morpho-logical characters in Monochamus male genitalia andprovide essential knowledge on European Monocha-mus genetic delimitation in combination with theirecological requirements.

MATERIAL AND METHODSMONOCHAMUS SAMPLING

From June to September 2003 and 2004, specimens ofMonochamus were sampled either by cross van traps(Ibeas et al., 2007; Koutroumpa, 2007; Koutroumpaet al., 2008a) or by field collection. A total of 150specimens of M. galloprovincialis were collected at 32locations in natural pine stands in seven Europeancountries plus Morocco (Table 2). In addition, 50specimens of M. sutor were sampled from 17 locationsoriginating from natural populations of Pinus speciesand Picea abies (L. H. Karst, 1881) in four countriesin Europe. A sampling effort was conducted in theFrench Mountains, following an elevation gradient(up to 1700 m) suspected to house cryptic forms of thestudied species. Insects were stored at -80 °C or inabsolute ethanol.

MORPHOLOGICAL FEATURES

Seventeen morphological features in total, sevenbinary and ten multi-states, including eight externalcharacters and seven internal characters on malegenitalia, were analyzed (see Supporting information,Appendix S1). Seven were chosen based on previoussystematic and morphological studies on Monochamusspecies and other Coleoptera (Hellrigl, 1971; Villiers,1978; Tomminen & Leppänen, 1991; Bense, 1995;Vives, 2000; Sanmartin & Martin-Piera, 2003; Ahrens,2005; Takami & Suzuki, 2005; Sama, 2008). Within themain characters usually used to differentiate M. gal-loprovincialis from M. sutor, one can underline theform of the scutellum and the colour of the pubescenceon the elytra (see Supporting information, Appen-dix S1, Figs S1, S2) (Table 1). Tomminen & Leppänen(1991) reported a difference in the shape of the medianlobe of the male genitalia of the two species (Table 1;Fig. S3). In M. galloprovincialis lower taxa, M. g. gal-loprovincialis has brown-red legs and antennae,whereas M. g. pistor has black ones (Table 1). Accord-ing to Vives (2000) and Villiers (1978), M. g. pistor issmaller than M. g. galloprovincialis and has lesspubescence on the elytra of yellow–white colour(Table 1). Based on these generally accepted charac-ters and considering the distribution of M. g. pistor athigher altitude, latitudes, and longitudes in Europe(north and central-east european distribution sensuHellrigl, 1971, Sama 2002, 2008; Tomminen &Leppänen, 1991), we consider individuals from ninepopulations in our sampling (Table 2) as potentiallybelonging to the M. g. pistor sub-species. Monochamussutor individuals from Austria were recognized asM. s. pellio by their collectors (Table 2). Furthermore,nine new morphological features were included in ouranalysis (see Supporting information Appendix S1).



Tab

le2.

Abb

reva

tion

sof

Mon

och

amu

sga

llop

rovi

nci

alis

and

Mon

och

amu

ssu

tor

popu

lati

ons,

sam

plin

gsi

tes,

coll

ecto

rs’

nam

es,

hos

ttr

ees,

date

ofca

ptu

re,

alti

tude

,an

dge

ogra

phic

alco

ordi

nat

esar

eli

sted

Pop

ula

tion

code

(AM

OV

Age

ogra

phic

algr

oup)

Cou

ntr

yL

ocat

ion

Col

lect

orH

ost

spec

ies

Dat

eA

ltit

ude

(m)

Lat

itu

deL

ongi

tude

Mon

och

amu

sga

llop

rovi

nci

alis

gall

opro

vin

cial

isG

Pig

A(E

)F

ran

ceF

D3

Pig

non

sB

.G

erm

ain

Pin

us

pin

aste

r20

0376

48°2

4′N

02°3

3′E

GL

or(E

)F

ran

ceL

orri

sG

.R

oux

Pin

us

sylv

estr

is20

03,

2004

124

47°5

3′N

02°3

0′E

GO

le(D

)F

ran

ceS

tP

ierr

ed’

Olé

ron

R.

Pel

loqu

int

Pin

us

pin

aste

r20

03,

2004

745

°58′

N01

°19′

WG

Her

(D)

Fra

nce

Her

m(L

éon

)J-

L.

Hau

tclo

cqP

inu

spi

nas

ter

2004

6543

°51′

N01

°14′

WG

Pis

(D)

Fra

nce

Pis

sos

Pin

us

Nin

osqu

eP

inu

spi

nas

ter

2004

4744

°18′

N00

°46′

WG

Far

(D)

Fra

nce

Far

gues

R.

Del

pon

tP

inu

spi

nas

ter

2004

123

44°1

1′N

00°1

1′E

GH

ou(D

)F

ran

ceH

ourt

inJ-

Pin

us

Cos

teP

inu

spi

nas

ter

2004

,20

0518

45°1

2′N

01°0

4′W

GS

arI

(D)

Fra

nce

Sar

e(S

tIg

nac

e)C

.V

an-M

eer

Pin

us

nig

ra20

0417

243

°20′

N01

°36′

WG

Sar

L(D

)F

ran

ceS

are

(Liz

arri

eta)

C.

Van

-Mee

rP

inu

sn

igra

2004

485

43°1

6′N

01°3

7′W

GA

ud

(C)

Fra

nce

Pey

riac

deM

erC

.V

an-M

eer

Pin

us

hal

epen

sis

2004

226

43°1

7′N

01°4

8′E

GC

ou(C

)F

ran

ceC

oust

ouge

s(6

6)H

.B

rust

elP

inu

sn

igra

2003

841

42°2

2′N

02°3

8′E

GL

ar(B

)F

ran

ceL

arza

cJ-

Pin

us

An

son

nau

dP

inu

sn

igra

2005

800

43°5

8′N

03°1

1′E

GC

or(B

)F

ran

ceS

tJe

ande

Cor

nie

sP

inu

sG

irar

dP

inu

sh

alep

ensi

s20

03,

2004

7343

°44′

N04

°00′

EG

Mey

(B)

Fra

nce

Mey

rarg

ues

J.H

intz

yP

inu

sh

alep

ensi

s20

03,

2004

320

43°2

8′N

05°3

8′E

GE

yg(A

)F

ran

ceF

DE

ygu

esR

osan

sP

inu

sF

eeP

inu

sn

igra

2004

1058

44°2

4′N

05°2

8′E

GA

drF

ran

ceS

tA

ndr

éle

sA

lpes

L.

Mic

asP

inu

ssy

lves

tris

2004

1226

43°5

9′N

06°3

0′E

GD

igF

ran

ceD

ign

e(L

esB

ain

s)L

.M

icas

Pin

us

sylv

estr

is20

0512

1644

°03′

N06

°15′

EG

PC

(I)

Por

tuga

lC

ompo

rta

Pin

us

Nav

esP

inu

spi

nas

ter

2003

2438

°22′

N08

°45′

WG

PL

(I)

Por

tuga

lL

eiri

aG

.R

oux

Pin

us

pin

aste

r20

0533

39°4

0′N

08°4

8′W

GE

spM

(H)

Spa

inM

urc

iaD

.G

alle

goP

inu

spi

nas

ter

2004

173

37°5

9′N

01°0

7′W

GM

arA

(G)

Mor

occo

Atl

asm

oun

tain

D.

Gh

aiou

leP

inu

spi

nas

ter

2003

475

32°2

1′N

06°2

4′W

GG

rA(F

)G

reec

eA

fete

(Mag

nes

ia)

A.

Kou

trou

mpa

sP

inu

sbr

uti

a20

04,

2005

539

°22′

N22

°56′

EG

GrE

r(F

)G

reec

eE

ryth

res

A.

Kou

trou

mpa

sP

inu

sbr

uti

a20

0539

738

°13′

N23

°19′

EG

GrB

(F)

Gre

ece

Bil

iaA

.K

outr

oum

pas

Pin

us

bru

tia

2005

625

38°1

0′N

23°2

0′E

GG

mo

(K)

Ital

yM

onte

falc

one-

Pis

aC

esar

iet

al.,

2005

––

101

43°4

3′N

10°4

4′N



Mon

och

amu

sga

llop

rovi

nci

alis

pist

orG

pFau

(A)

Fra

nce

Sey

ne

(Le

Fau

t)D

.R

ougo

nP

inu

ssy

lves

tris

2004

1364

44°1

9′N

06°2

4′E

GpA

dr(A

)F

ran

ceS

tA

ndr

éle

sA

lpes

D.

Rou

gon

Pin

us

sylv

estr

is20

0512

2643

°59′

N06

°30′

EG

pVer

(A)

Fra

nce

Ver

dach

esD

.R

ougo

nP

inu

ssy

lves

tris

2005

1444

44°1

5′N

06°2

0′E

GL

G(B

)F

ran

ceP

uec

hag

ut

leV

igan

C.

Ru

llie

reP

inu

sn

igra

2003

,20

0410

1444

°01′

N03

°34′

EG

GrF

(F)

Gre

ece

Fra

kto,

Mt

Rod

opi

A.

Kou

trou

mpa

sP

icea

abie

s19

9712

0041

°15′

N25

°30′

EG

Pm

u(J

)It

aly

Mu

les-

Bol

zan

oC

esar

iet

al.,

2005

––

946

46°5

1′N

11°3

1′E

GP

vs(J

)It

aly

Vil

laS

anti

na-

Udi

ne

Ces

ari

etal

.,20

05–

–37

446

°25′

N12

°55′

EG

pAu

Ca

(J)

Au

stri

aC

arin

thia

(Au

stri

a)U

.To

mit

czek

Pin

us

sylv

estr

is20

0677

546

°43′

N14

°10′

EG

pAu

So

(J)

Au

stri

aS

olle

nau

(Au

stri

a)U

.To

mit

czek

Pin

us

sylv

estr

is20

0626

947

°53′

N16

°15′

EM

onoc

ham

us

suto

rS

uS

amF

ran

ceS

amoe

ns

A.

Dru

mon

t–

1992

1122

46°0

5′N

06°4

4′E

Su

Ch

aF

ran

ceC

hat

el(P

rès

laJo

ux)

M.

Sim

onP

icea

abie

s20

0416

2746

°14′

N06

°48′

ES

uIs

eF

ran

ceM

eau

dre

Iser

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A total of 60 specimens of M. galloprovincialisand 46 of M. sutor were analyzed for the majority ofthe morphological features. The other samples (e.g.larvae or seriously damaged specimens) could not beconsidered as a result of a lack of (or destroyed)morphological characters. Because of no obvious vari-ability, none of the characters studied on the femalegenitalia are mentioned in the present study. Malegenitalia were prepared using a modified version ofthe protocol of Chatzimanolis (2005); see also Jeannel(1955) and Dupuis (2005). Images of all of the mor-phological characters studied were analyzed usingIMAGEJ 1.32j (http://rsbweb.nih.gov/ij/). Because itwas impossible to show all characters for all individu-als studied, a representative sample is shown in theSupporting information (Figs S1, S2, S3, S4, S5).

MITOCHONDRIAL AND NUCLEAR DNA SEQUENCES

DNA was extracted from one or two legs to allowsubsequent morphological observations at the sametime as reducing contamination from organisms suchas nematodes. Tissue from the last abdominalsegment was used for larval DNA extraction. Bodyparts and DNA extracts were kept as vouchers inthe entomological collection at the URZF at INRA-Orléans in France. Extraction and isolation ofgenomic DNA was performed using the GenEluteMammalian Genomic DNA miniprep kit (Sigma-Aldrich) at a final elution of 100 mL.

Polymerase chain reactions (PCRs) were conductedusing the Sigma Red Taq package. The primers usedwere C1-J-2183a and TL2-N-3014 (Simon et al., 1994)for the COI gene and D1-F and D3-R (Lopez-Vaamonde et al., 2001) for the D2 region of the 28SrDNA gene. The annealing temperatures were 48 °Cfor the COI gene, and 57 °C for the 28S rDNA. Ampli-fication of 35 cycles was completed and PCR productswere then purified using GenElute PCR Clean-Up kit(Sigma-Aldrich).

Sequencing was performed using the BigDye Ter-minator sequencing kit, version 3.1 (Applied Biosys-tems) on both strands. Sequencing reactions werepurified using Sephadex G-50 (Amersham; Sigma-Aldrich) and detected with an ABI 3100 automaticsequencer. The sequences obtained for each gene werealigned using CLUSTAL W, version 1.4 (Thompson,Higgins & Gibson, 1994) as implemented inBIOEDIT, version 4.7.8 (Hall, 1999).

A fragment of 792 bp of the mitochondrial COIgene was sequenced for all 199 collected individuals,one to 11 individuals per population. This fragmentcorresponds to base pair positions 441–1319 ofthe Drosophila yakuba reference sequence (Simonet al., 1994). Owing to co-amplification of nuclearparalogues (i.e. numts) (Koutroumpa, Lieutier &

Roux-Morabito, 2009a), 55 M. galloprovincialis COIsequences with ambiguities were withdrawn from theanalysis. Three sequences of M. galloprovincialis andone of M. sutor from Italy, published by Cesari et al.(2005) were added to the dataset (GenBank ID:AY260835-37 and AY260843, respectively). Finally,76 M. galloprovincialis specimens originating from 35European and one Moroccan populations, as well asall 50 M. sutor individuals, were used for the intra-and interspecific mitochondrial (mt)DNA analysis(Tables 2, 3). Most of them were sampled in Francewith 21 locations (53 specimens) sampled for M. gal-loprovincialis and 13 locations for M. sutor (32 speci-mens). The sampling sites, host tree species,altitudes, and date of capture of each Monochamusspecimen are summarized in Table 2.

In addition, 878 bp of the D2 domain of the 28SrDNA were sequenced for 21 M. galloprovincialis and11 M. sutor individuals, including their two sub-species (Tables 2, 3). The individuals were chosenaccording to ambiguous morphological featuresdetected previously within M. galloprovincialis orrelated to the two mtDNA sequence clusters observedin M. sutor.

DATA ANALYSIS

Phylogenetic analysisPhylogenetic analyses were performed withPAUP*4b10 (Swofford, 2000) for each gene independ-ently (COI and the D2 region of 28S rDNA). COIsequences were used for the phylogenetic analysisand subsequently compared with the morphologicalcharacters and the 28S sequences. The congenerspecies Monochamus alternatus (Hope, 1842) wasused as outgroup. Trees were reconstructed usingboth maximum parsimony (MP) and maximum like-lihood (ML). MODELTEST, version 3.7 (Posada &Crandall, 1998) was used to select the substitutionmodel that best describes the data. Following thelikelihood ratio test (Felsenstein, 1988), the mostappropriate model of nucleotide substitution wasdetermined, as well as the proportion of invariantsites and g-shape parameter. The hypothesis of amolecular clock was also tested. For MP trees, weused a heuristic search with a simple stepwise addi-tion of sequences and tree bisection–reconnectionbranch-swapping option as implemented inPAUP*4b10. Support values for MP trees were esti-mated with 1000 bootstrap replicates. Uncorrected ‘p’genetic distances were computed using PAUP*4b10.

The incongruence length difference test was usedto test congruence between molecular (COI) andmorphological data sets and was completed for 1000iterations. D2 was not included in this analysisbecause of a lack of variability and therefore low



Table 3. Number of Monochamus galloprovincialis and Monochamus sutor individuals (all adults except seven larvaemarked with L) studied in the mitochondrial cytochrome oxidase c subunit I (COI) and morphological analyses, haplotypesand sequence types found in each population

Code

Number of individuals

Haplotype codes (number ofsequences when more than 1)COICOI 28S

Morphology

� �

Monochamus galloprovincialis galloprovincialisGPigA 1 1 – 1 GI 8GLor 1 2 – 1 GI 8GOle 3 – 1 2 GI 6, GI 8 (2)GHer 2 1 1 1 GI 8 (2)GPis 2 1 1 1 GI 8 (2)GFar 2 – 1 1 GI 8 (2)GHou 3 – 1 2 GI 8 (3)GSarI 5 – 3 1 GI 6, GI 8 (4)GSarL 3 – – 2 GI 6, GI 8 (2)GAud 1 1 1 – GI 4GCou 1 1 1 – GI 9GLar 1 – 1 – GI 9GCor 6 1 2 3 GI 2, GI 6, GI 5, GI 8 (3)GMey 2 1 1 1 GI 8, GI 9GEyg 1 1 – 1 GI 2GAdr 0 1 – – –GDig 0 1 – – –GPC 3 1 – – GI 3 (3)GPL 1 0 1 – GI 3 (1)GEspM 3 2 2 1 GI 3 (3)GMarA 2 – 1 1 GI 1 (2)GGrEr 4L – – – GI 2 (4)GGrB 3L – – – GI 2 (3)GGrA 4 2 3 1 GI 2 (2), GI 7 (2)GGmo 1 0 0 0 GI 2

Monochamus galloprovincialis pistorGpFau 2 1 2 – GI 2 (2)GpVer 5 – 3 2 GI 9 (5)GpAdr 5 0 2 3 GI 9 (5)GLG 3 1 2 1 GI 2, GI 9 (2)GGrF 1 1 1 – GI 8GPmu 1 0 0 0 GI 2GPvs 1 0 0 0 GI 2GpAuCa 1 1 1 – GI 4GpAuSo 2 – 1 1 GI 8 (2)

Monochamus sutor sutorSuSor 1 1 1 0 SI 15SuDig 1 0 1 0 SI 15SuCha 2 1 1 1 SI 10, SI 11SuSam 2 – – – SI 4, SI 14SuAdr 2 0 1 1 SI 7, SI 15SuLG 1 1 1 0 SI 9SuMon 1 1 0 1 SI 9SuIse 1 1 0 1 SI 8SuMed 2 1 0 2 SI 7(2)SuSeyF 14 2 10 4 SI 5, SI 7 (6), SI 15 (7)SuSeyM 7 0 2 3 SI 5, SI 7 (2), SI 15 (4)SuSeyC 1 0 1 0 SI 6SuPra 1 1 1 0 SI 4SuGrE 6 1 5 0 SI 1, SI 2, SI 3 (4)SuGrX 5 0 4 1 SI 3 (5)SUvg 1 0 0 0 SI 16

Monochamus sutor pellioSupAuNa 2 1 2 0 SI 12, SI 13

Monochamus alternatusAJapF 1 1 1 0



phylogenetic resolution. This method, as developed byFarris et al. (1994) and implemented within PAUP* asa partition homogeneity test, determines whether com-bining data sets is appropriate for further phylogeneticanalyses. A MP tree was also reconstructed using the15 morphological characters and a bootstrap procedureof 1000 iterations was completed using PAUP*. Weused MACCLADE, version 4.06 (Madison & Madison,2000) under constraints of species or species lower taxamonophyly to optimize morphological characters onmolecular topologies. We tested the significance oflikelihood differences among constrained versusunconstrained topologies using the Shimodaira–Hasegawa test (Shimodaira & Hasegawa, 1999) asimplemented in PAUP* (1000 replicates).

Population genetic analysismtDNA haplotype network was performed using TCS,version 1.21 (Clement, Posada & Crandall, 2000)for both species, M. galloprovincialis and M. sutor,independently.

Analysis of molecular variance (AMOVA; Excoffier,Laval & Schneider, 2005) was used to partitionmolecular variance into different hierarchical levelsin M. galloprovincialis populations in France usingARLEQUIN, version 3.11 (Excoffier et al., 2005).Specimens were grouped (Table 2) either according to:(1) the elevation > 800 m or < 800 m [800 m proposedby the literature as the highest elevation whereM. g. galloprovincialis occurs (Hellrigl, 1971; Vives,2000)]; (2) the geographical origin, based on geo-graphical distances and climatic conditions and (3)the host tree species (Table 2).

RESULTSBETWEEN-SPECIES ANALYSIS

Morphological featuresOf the seventeen characters analyzed, includingexternal and internal features, only four were diag-nostic at the species level (Fig. 2; see also Supportinginformation, Appendix S1 and Table S1, diagnosticcharacters c, k, l, o, q). So far, these four charactershave never been used (or described) to separate thetwo species (Hellrigl, 1971; Villiers, 1978; Tomminen& Leppänen, 1991; Vives, 2000; Sama, 2002, 2008).More precisely, M. galloprovincialis individuals hadpatches of coloured bristle that formed one to threelarge stripes on their elytra, whereas, for M. sutorindividuals, bristle formed points all over their elytra(Appendix S1, c and Fig. S2), The sclerotinizationdegree of the lateral styli of the male genitalia wasstronger for M. sutor than for M. galloprovincialis(Appendix S1, k) and their colour was red for the firstone and dark brown for the second one (Appendix S1,

l). The most unambiguous character that can sepa-rate the two species was the copulatory piece in theaedeagus (Appendix S1, o, q). This very small struc-ture that is attached to the aedeagus has a verydifferent shape between the two species. We present itfor each species in Figure 2 (for complete photo-graphic library, see Supporting information, Figs S4,S5). We found no variability between M. galloprovin-cialis and M. sutor on the median lobe (Tomminen &Leppänen, 1991) besides the sclerotinization degree(see Supporting information, Appendix S1, Fig. S3),and therefore this character is not referred to further.The scutellum shape is more variable for M. gallopro-vincialis than for M. sutor. Monochamus sutor adultsalways have a nude line in the middle of the scutel-lum. Monochamus galloprovincialis usually has Vform scutellum, although many individuals have anude line in the middle of the scutellum, as inM. sutor adults (see Supporting information, Fig. S1).In addition to the overlap observed at the shape, thecolour of the scutellum bristle also overlaps betweenthe two species. A matrix of all the morphologicalcharacters used is presented in the Supporting infor-mation (Table S1).

Sequence analysisThe alignment of the 76 and 50 partial COI sequencesof M. galloprovincialis and M. sutor, respectively,revealed 48 single nucleotide polymorphisms (SNPs)(no deletions nor insertions), 12 of which appear asdiagnostic mutations separating the two species. Thealignment of the 32 nuclear gene sequences also sepa-rated M. galloprovincialis from M. sutor, by the pres-ence of one diagnostic transitions and the deletion oftwo nucleotides in M. sutor.

Genetic distancesUncorrected nucleotide pairwise distances betweenM. galloprovincialis and M. sutor ranged from 0.023to 0.037 (mean 0.031) for the COI gene and no overlapwas observed between intraspecific and interspecificpairwise distances (Fig. 3). The 28S genetic pairwisedistances between the two species corresponded to0.0023 and reached 0.007 when considering M gallo-provincialis or M. sutor versus the outgroup species,M. alternatus.

Phylogenetic analysisThe incongruence length difference test appliedbetween mtDNA and morphology data sets revealedsignificant conflict (P = 0.001). Consequently, COI andmorphology were analyzed separately (Fig. 4A, B).

MP analysis of COI resulted in 28 equally mostparsimonious trees with a length of 121 steps, con-sistency index (CI) 0.7851 and retention index(RI) 0.9795. The transversional distance model



(TVM + I + G) with gamma shape parameter 0.7868and proportion of invariable sites of 0.7336 wasselected as the best model explaining the data. BothMP and ML consensus phylogenetic reconstructionsshowed a monophyletic clade for each of the twospecies: M. galloprovincialis (clade A) and M. sutor(clade B) (only the MP tree is shown; Fig. 4B).Monochamus galloprovincialis monophyly was sup-ported by low bootstrap values compared toM. sutor, although the Shimodaira–Hasegawa test

was not significant when M. galloprovincialis andM. sutor were constrained to be monophyletic. TheD2 domain of 28S rDNA also supported the mono-phyly of the two species with bootstrap values com-parable to the COI analyses. The MP tree is shownin Figure 4C.

MP analysis of seventeen variable morphologicalcharacters yielded 100 most parsimonious trees witha length of 112 steps (CI = 0.277, RC = 0.233). Theresulting topology was poorly resolved, with no boot-

Figure 2. Differences on elytra and male genitalia of the two species Monochamus galloprovincialis (top) and Mono-chamus sutor (bottom): three stripes on elytra characterize M. galloprovincialis and points all over elytra characterizeM. sutor. The lateral styli are less sclerotinized (A) with brown setae (B) for M. galloprovincialis and more sclerotinizedwith red setae for M. sutor. The form of the sclerotinized part of the copulatory piece (C) has an elongated thin shape forM. galloprovincialis and is heart-shaped for M. sutor



strap values above 50 (Fig. 4A). However, M. sutorappeared monophyletic, whereas M. galloprovincialisappeared polyphyletic. The optimization of morpho-logical characters onto the COI MP topology identi-fied the four synapomorphies corresponding to thefour diagnostic morphological characters describedabove, each of them clearly separating M. gallopro-vincialis from M. sutor; one of the four morphologicalcharacters (i.e. the elytra bristle) is shown inFigure 4B).

WITHIN-SPECIES ANALYSIS

Morphological featuresThe morphological characters proposed in the litera-ture as being diagnostic at the intraspecific levelbetween M. g. galloprovincialis and M. g. pistor(Hellrigl, 1971; Villiers, 1978; Tomminen &Leppänen, 1991; Vives, 2000; Sama, 2002, 2008)appear confusing and ambiguous (Table 1; see alsoSupporting information, Figs S1–S3). Several indi-viduals showed intermediate features and none of thecharacters noted in the literature (i.e. legs, antennae,and elytra bristle colour) was shown to be diagnosticfor sub-taxa identification.

By contrast, no morphological variability wasobserved within M. sutor, as also reflected by theabsence of a description of its sub-species, M. s. sutorand M. s. pellio, in the literature (Hellrigl, 1971;Villiers, 1978; Tomminen & Leppänen, 1991; Vives,2000; Sama, 2002). To our knowledge, only the geo-

graphical distribution is mentioned as a differencebetween these sub-species.

Sequence analysisThe final alignment of the M. galloprovincialis COIsequences consisted of 792 bp, with a total of ten(1.26%) polymorphic nucleotides (all parsimonyinformative). Nine different haplotypes were identi-fied (Table 3) and have been submitted to GenBankunder accession numbers KC692719-27 (Table 5).

On the same final alignment of 792 bp, 29 (3.28%)polymorphic nucleotides (of which 18 are parsimoni-ously informative) have been detected in M. sutor COIsequences. Sixteen different haplotypes were identi-fied (Table 3) and have been submitted to GenBankunder accession numbers KC692728-42. Onesequence from M. alternatus COI has been obtainedand used as outgroup in our analysis (GenBank acces-sion number KC692743) (Table 5).

Both species M. galloprovincialis and M. sutorshowed no intraspecific variation in the D2 regionof 28S rDNA, with each species showing one allele(submitted to GenBank under accession numbersKC692744 and KC692745, respectively) (Table 5).

Genetic distancesMonochamus galloprovincialis uncorrected p geneticdistances ranged between 0 and 0.009. Interestingly,higher genetic distances were observed withinM. g. galloprovincialis specimens (0.009) (Iberian and

Figure 3. Frequency distribution of intraspecific and interspecific (congeneric) genetic divergence in Monochamus.Intraspecific distances correspond to Monochamus galloprovincialis cytochrome oxidase c subunit I (COI) sequences (A)and to Monochamus sutor COI sequences (B).

Figure 4. A, consensus maximum parsinomy (MP) tree for Monochamus galloprovincialis and Monochamus sutor speciesusing 15 morphological characters. No bootstrap values were higher than 50% and therefore these are not shown. B,consensus MP tree for M. galloprovincialis and M. sutor species using cytochrome oxidase c subunit I (COI) sequence data.Only bootstrap values higher than 50% are shown. In this example, the four different lines, attributed to each branch ofthe tree, indicate the four different states of the first morphological character (patches on the elytra) separatingM. galloprovincialis from M. sutor. Identical patterns were observed for three other morphological characters (characters9, 10, and 13 in the Supporting information, Appendix S1). C, consensus MP tree for M. galloprovincialis and M. sutorspecies using 28S sequence data. Population codes are provided in Table 2.

�



(A) (B)



Moroccan specimens versus the rest of the Europeanspecimens) than between M. g. galloprovincialis andM. g. pistor (0.005).

Within M. s. sutor, genetic distances rangedbetween 0 and 0.019. Specimens from Greece hadlower genetic distances (0.017) compared to the puta-tive M. s. pellio sub-species than to the rest of theM. s. sutor in our sampling (0.019).

Phylogenetic reconstruction and haplotypedemographic historyWithin M. galloprovincialis, phylogenetic structurewas weak and displayed very poor bootstrap values.However, as confirmed on the haplotype network(Fig. 5), two major haplotypes, GI 8 and GI 9, clus-tered together. Interestingly, individuals belongingto the GI 9 haplotype were all (except 1) sampled atelevations between 800 m to 1350 m at differentlocalities in the Alps (mostly individuals previouslycharacterized as the pistor sub-species), as well asin the Massif Central and the Pyrenees Orientales(Fig. 6). GI 8 was the most widespread haplotype,shared mainly between French lowland populationsand one population in Austria (GpAuCa), as well as

one on the Rodopi Mountain in north-eastern Greece(GGrF). Individuals from the Iberian Peninsula andMorocco revealed unique haplotypes, respectively.

To check for spatial structure (by region or altitude)and/or host effect on our data, M. galloprovincialispopulations collected in France were groupedadequately (for the different groups, see Table 2 andthe Material and methods) and were analyzed byAMOVA. The results on the COI sequences are pro-vided in Table 4. Interestingly, the AMOVA showedthat a significant partition of total genetic variationoccurred between the altitudinal groups (24%,P < 0.01). However, no significant variation wasobserved between the groups made by region inFrance or host species. In the three differentcluster-ings, the variances among and within populationswere significant.

Within M. sutor, the two major subclades observedin the MP tree, (B1 and B2 haplotype groups; Fig. 4)were distant by up to 12 mutational steps on thehaplotype network (Fig. 5). Subclade B1 was wellsupported (bootstrap value 82) and was representedby two dominant haplotypes, SI 3 and SI 7, whereasB2 was supported by lower bootstrap value (59) and

(C)

Figure 4. Continued



had one major haplotype, SI 15. A contact zonebetween these two divergent subclades was observedin south-western Alps populations (Fig. 6, populationsSuSeyM, SuSeyF, and SuAdr). Interestingly, the twohaplotypes, SI 7 and SI 15, are the largest of eachsubclade and were only found in this geographicalregion. Individuals identified as M. s. pellio (SI 12 andSI 13 from Austria) grouped in the B2 clade togetherwith the SI 15 haplotype from the north-east Alps andthe most northern French alpine haplotypes foundnear the Swiss border, namely SI 11, 10, and 14(Fig. 6). Therefore, the most northern haplotypes inthe present study cluster together. Most individualsfrom Greece shared the same dominant haplotype (SI3). All Greek individuals were grouped in the B1 cladetogether with haplotypes from populations sampled

in the most southern part of our sampling in France,the southern French Alps, and the Pyrenees; moreprecisely, the two haplotype SI 4 and SI 8 groupindividuals from the French Alps (SuSam) and MassifCentral (SuLG) with the Pyrenean individuals SuPraand SuMon, respectively. Both haplotypes were posi-tioned in subclade B1 with the previously notedsouthern-most haplotypes.

DISCUSSIONSPECIES DELINEATION: MULTIPLE LINES

OF EVIDENCE

Monochamus galloprovincialis–M. sutor speciescomplex comprises a taxonomically challenging

GI 1 (2)

GI 2 (14)GI 3 (7)

GI 4 (2)

GI 5 (1)GI 6 (4) GI 7 (2)

GI 8 (26)

GI 9 (15)

GMarA

GEspM

GPC

GPL

GLG

GLar

GCou

GAud

GCor

GMey

GEyg

GpVer

GpAdr

GpFau

GPig

GLor

GHer

GHou

GPis

GFar

GOle

GSarI

GSarL

GGm o

GPm u

GPvs

GpAuSo

GpAuCa

GGrF

GGrA

GGrB

GGrEr

)B()A(

SI 1 (1)

SI 2 (1)

SI 4 (2)

SI 6 (1)

SI 5 (2)

SI 7 (11)

SI 8 (1)

SI 9 (2)

SI 10 (1)SI 11 (1)

SI 12 (1)

SI 13 (1)

SI 14 (1)

SI 3 (9)

SI 15 (14)

Clade B1

Clade B2

SupAuNa

SuCha

SuSam

SUvg

SuSor

SuIse

SuSeyC

SuMed

SuSeyM

SuSeyF

SuAdr

SuDig

SuLG

SuMon

SuPra

SuGrX

SuGrE

Clade B2

Clade B1

Clades B1-B2sut ur zone

SI 16 (1)

Figure 5. Cytochrome oxidase c subunit I COI TCS network showing haplotype distribution. A, Monochamus gallopro-vincialis. B, Monochamus sutor. The colour code corresponds to different populations. Population codes are provided inTable 2 and circle size is proportionate to the number of individuals having each haplotype. Clades correspond to thoseof the maximum parsinomy tree (Fig. 4).



GpAuSo

GpAuCa

GGrF

GGrA

GGrEr

GGrB

GEspM

GMarA

GPmu

GPvs

GGmo

GOle

GHou GPis

GFarGHerGSarI GSarL

A)

SuGrX SuGrE

SuAuNa

SUvg

B)

GLor

GPigA

GI 9

GI 8

GI 6

GI 5

GI 7

GI 4

GI 2

GI 3

GI 1

GCou

GAud

GCor

GMey

GLar GLG

GpFau

GpVer

GEyg

GpAdr

SuDig

SuSam

SuLG

SuPraSuMon

SuAdr

SuIse

SuCha

SuSor SuMed

SuSeyCSuSeyM

SuSeyF

GPC

GPL

SuI 12

SuI 13

SuI 10

SuI 11

SuI 14

SuI 16

SuI 15

SuI 8

SuI 7

SuI 6

SuI 5

SuI 9

SuI 4

SuI 3

SuI 2

SuI 1

Clade B2

Clade B1

Sut ure zone



assemblage of recently diverged species with ongoingspeciation. These species are interesting models forstudies on biodiversity and species conservation con-cepts for two reasons. First, because of the specificenvironmental requirements found in our studywithin these species lineages (i.e. altitude) and,second, because of the difficulty in finding adequatescreening tools to clarify present taxonomic and phy-logenetic confusion within each species. An importantconfirmation from this nontidal data, showing highintraspecific variability, was the need to use inte-grated data from multiple independent sources. Usingthe integrative taxonomy approach, we gain accuracyin solving the taxonomic impediment at the sametime as revealing the evolutionary relationshipsbetween the different taxa (Schlick-Steiner et al.,2010; Heethoff et al., 2011; Fujita et al., 2012). In arecent review, Schlick-Steiner et al. (2010) claimedthat there is no silver-bullet discipline and thatseveral disciplines are needed to guard against single-discipline failure. In the present study, we attempteda more rigorous delimitation between and withinMonochamus that would provide crucial informationregarding the monitoring of PWN in Europe.

Morphological evidenceEven though numerous characters have been pro-posed to distinguish Monochamus species (Pershing& Linit, 1985), scutellum form and elytra colour haveso far comprised the main morphological charactersused for their identification (Hellrigl, 1971; Villiers,

1978; Tomminen & Leppänen, 1991; Vives, 2000;Sama, 2002, 2008). We showed in the present studythat these characters are extremely variable withinthe species. The polymorphism observed withinM. galloprovincialis in contrast to the monomorphicM. sutor is clearly visualized on the phylogeneticreconstruction using the morphological characters.Most of these characters do not support a mono-phyletic group for M. galloprovincialis specimens.They do not appear to have reached fixation and aredifficult to interpret, especially for old and badlypreserved samples. Nevertheless, four morphologicalcharacters, including genitalia, do allow an accuratedelimitation of these two species. By contrast tothe Monochamus male genitalia description fromTomminen & Leppänen (1991), the differencesreported in the present allow the unambiguous iden-tification of the two species. We describe a new malemorphological character, the copulatory piece, which,as a result of its position and difference in shape,must be an important character for mating. Thisfinding reinforces the hypothesis that the two specieswould not hybridize in sympatric area, even thoughno difference was found between the females. We haveobserved aggressive behavior (i.e. mutilation of anten-nae and legs) of M. galloprovincialis females towardsM. sutor males attempting to copulate (D. Rougon,pers. observ.), although this statement should be con-firmed by crossing experiments. Regarding ourresults, only four morphological characters appear tobe valid for confirming the species status in further

Figure 6. A, Monochamus galloprovincialis cytochrome oxidase c subunit I COI haplotype geographical distribution. B,as for (A) but for Monochamus sutor. Dotted delimitations correspond to suture zone and double line shows the Pyreneesbarrier. Maps on the left show haplotype distribution in south-east France. Population codes are provided in Table 2.Clades correspond to clades of the maximum parsinomy tree (Fig. 4).�

Table 4. Analysis of molecular variance of Monochamus galloprovincialis French populations based on cytochromeoxidase c subunit I sequences

Source of variationVariancecomponents

Percentageof variation

Grouping by altitude< 800/> 800

Among groups 0.00391Va 23.97**Among populations within groups 0.01857Vb 29.00*Within populations 0.00196Vc 47.03**

Grouping by region Among groups 0.16325Va 07.36NS

Among populations within groups 0.01075Vb 40.19*Within populations 0.00000Vc 52.45***

Grouping by host Among groups 0.08504Va 17.02NS

Among populations within groups 0.00000Vb 34.23***Within populations 0.00000Vc 48.75***

*P < 0.05, **P < 0.01, ***P < 0.001. NS, nonsignificant.



studies: the elytra bristle pattern (for a first andquick identification in the field) and the three char-acters on the male genitalia (structure of the copula-tory piece and overall sclerotinization and colour).

Molecular evidenceOur molecular data confirm the close relationship ofM. galloprovincialis and M. sutor, already consideredas sister species in the phylogenetic analyses of theMonochamus by Cesari et al. (2005). COI mitochon-drial gene and 28S nuclear genes allow an accuratedelimitation of the two species. However, the geneticdistances observed between M. galloprovincialis andM. sutor were quite low for both markers compared tothose separating other Coleopteran species (Farrell,2001; Kerdelhué et al., 2002; Monaghan et al., 2005).They were close to the 3% mtDNA sequence diver-gence, a threshold usually designed in species-levelidentification (Hebert et al., 2003b). This underliesthe problems in associating a standard thresholdvalue for species boundaries, suggesting that deline-ation of species using the COI gene barcode distances

is fairly subjective (DeSalle et al., 2005; Taylor &Harris, 2012). The interspecific divergence valuesfound in the data of the present study are usuallyobserved in intraspecific lineages. Similarly, nucleargenetic distances correspond mostly to intraspecificdistances (Lopez-Vaamonde et al., 2001; Duan et al.,2004; Auger-Rozenberg et al., 2006), indicating that28S has also evolved much slower in this group.However, when the mtDNA genetic distances areplotted on a histogram, they show an interspecificdistribution. Moreover, these results are comparablewith the genetic distances found so far for otherMonochamus species (Cesari et al., 2005; Kawai et al.,2006), as well as those observed in other intragenericanalyses in Coleoptera (Clark, Meinke & Foster,2001). In the present study, the COI diagnostic SNPswere not found in the barcoding region proposed forspecies delimitation (i.e. 600 bp at the 5′ end of thisgene) (Hebert et al., 2003b). Instead, its extension(C1-J-2183a and TL2-N-3014) at the 3′ end was sig-nificantly more variable and better reflected thesequence divergence within and between species, assuggested by Roe & Sperling (2007a). Introduction ofthe barcoding theory by Hebert et al. (2003b) wasmeant to give a universal solution to the need forstabilized taxonomic criteria, although recent studiesprove that it should be interpreted with caution andin combination with additional data sources such asmorphology and geography or, when these are notavailable, other gene regions and ecology should beincluded (DeSalle et al., 2005; Roux-Morabito et al.,2008; Taylor & Harris, 2012).

Considering the genetic distances observed betweenM. galloprovincialis and M. sutor, although we areaware of the lack of such estimations, we could datetheir separation to approximately 1.35–2 Mya, whichwould correspond to the beginning of the Pleistocene.This estimation was calculated according to themolecular clock for Coleopteran mitochondrial genes,calibrated from other Cerambycidae, the genusTetraopes, for which 1.5% genetic divergence isequivalent to 1 Mya (Farrell, 2001). Speciation ofthese two species is thus likely to be associated withpre-Quaternary events, during which the area of dis-tribution was repeatedly covered by ice or affected bylocal climate change (Zhang, Comes & Kadereit,2001). These episodes of expansion and contractioninto refugia would have likely patterned the geneticdistribution of the species. Divergence in their eco-logical adaptation may have fostered distinct verticaland horizontal inter- and intraspecific distribution.

SUB-TAXA DIFFERENTIATION

In the literature, separation of M. galloprovincialisand M. sutor into two sub-species is based on mor-

Table 5. Genbank accession numbers

Haplotypes

Gen BankAccessionnumbers

Monochmus_COI.sqn GI-1 KC692719Monochmus_COI.sqn GI-2 KC692720Monochmus_COI.sqn GI-3 KC692721Monochmus_COI.sqn GI-4 KC692722Monochmus_COI.sqn GI-5 KC692723Monochmus_COI.sqn GI-6 KC692724Monochmus_COI.sqn GI-7 KC692725Monochmus_COI.sqn GI-8 KC692726Monochmus_COI.sqn GI-9 KC692727Monochmus_COI.sqn SI-1 KC692728Monochmus_COI.sqn SI-2 KC692729Monochmus_COI.sqn SI-3 KC692730Monochmus_COI.sqn SI-4 KC692731Monochmus_COI.sqn SI-5 KC692732Monochmus_COI.sqn SI-6 KC692733Monochmus_COI.sqn SI-7 KC692734Monochmus_COI.sqn SI-8 KC692735Monochmus_COI.sqn SI-9 KC692736Monochmus_COI.sqn SI-10 KC692737Monochmus_COI.sqn SI-11 KC692738Monochmus_COI.sqn SI-12 KC692739Monochmus_COI.sqn SI-13 KC692740Monochmus_COI.sqn SI-14 KC692741Monochmus_COI.sqn SI-15 KC692742Monochmus_COI.sqn ALT_COI KC692743Monochmus_28S.sqn GGALLO_28S KC692744Monochmus_28S.sqn GSUTOR_28S KC692745



phological and eco-geographical criteria. In thepresent study, we aimed to compare these criteriawith molecular data.

Morphology versus geneticsM. galloprovincialis displayed extreme variability inthe morphological features which did not allow accu-rate differentiation of M. g. galloprovincialis andM. g. pistor. The colour of the legs and antennas, usedso far as the main characteristic to separate M. g.pistor from M. g. galloprovincialis (Hellrigl, 1971;Villiers, 1978; Tomminen & Leppänen, 1991; Vives,2000; Sama, 2002, 2008), is represented by a palleteof light red to dark brown and black colours. Suchphenotypic plasticity is observed in other insectspecies and could reflect adaptation to intra- andinter-specific signalling and/or environmental needssuch as camouflage and thermoregulation (Tuomaala,Kaitala & Rutowski, 2012). We could assume thatnorthern individuals would be selected for a highermelanization degree, as proposed for M. pistor, andsouthern ones would be selected against, as observedin other cases (Trullas, van Wyk & Spotila, 2007). Inthe study of Sama (2008), this character was confus-ing for Moroccan specimen characterization. Four ofthose specimens look like the M. g. pistor holotypefrom Slovenia, even though Morocco is out of thegeographical distribution range of this taxon. Simi-larly, characters such as body size and elytra bristledensity and colour appear to be very variable over thewhole geographical distribution range of the species(Sama, 2008). The combination of all these charactersmakes the data even more contradictory and confus-ing. The extreme inconsistency between the morpho-logical characters emphasizes the risk of error whenusing such characters to delineate M. g. pistor fromM. g. galloprovincialis.

This great variability in morphological features isfollowed by high genetic divergence, although no cor-relation could be found between the phenotype andthe haplotype structure within our sampling borders.Furthermore, none of the morphological characterstaken separately was consistent with the revealedgenetic haplotypes. Consequently, we cannot specu-late on the existence of the sub-species proposed inthe literature and we propose the use of the termsub-forms to designate specimens with intermediatemorphology.

ECOLOGICAL TRAIT SIGNIFICANCE VERSUS

MORPHOLOGY AND GENETICS

More than being associated with morphological fea-tures, the genetic structure of M. galloprovincialisappears to be shaped by ecological requirements.European Monochamus species are known to be eco-

logically and geographically subdivided, with mostspecies occurring at high latitudes and/or moderate toelevated altitudes (Hellrigl, 1971). Monochamus gal-loprovincialis is unique in that sense because it is theonly species with a Mediterranean origin that occursmainly in lowland areas to low elevations (Hellrigl,1971). In a previous evolutionary study on the genusMonochamus, Cesari et al. (2005) indicated a moreprimitive condition of the Eurasiatic sub-alpinespecies Monochamus saltuarius (Gebler, 1830) withrespect to the studied taxa. Furthermore, althoughM. sutor, as M. saltuarius, infests the trunk of weak-ened spruces and pines, M. galloprovincialis isrestricted to branches of pines (Hellrigl, 1971;Starzyk & Hilszczanski, 1997). This shift to brancheswould have prevented competition between the twosister species and suggests that M. galloprovincialisis a photophilic taxa with a higher tolerance todryness. It is tempting to assume that this particularecological requirement may have fostered M. gallo-provincialis to spread all over southern countries,with distinct differentiation patterns regarding theclimatic conditions of the colonized area and theoccurrence of putative host tree species.

The present study reveals a mitochondrial lineagethat is likely to be adapted to particular elevations inFrance (between 800 m and 1444 m). This lineage isrepresented by haplotype GI 9, occurring in the south-western Alps, as well as in other mountain systems(i.e. the western Massif Central and the Pyrenees inthe South). This altitudinal structure is also shown inthe AMOVA analyses. Furthermore, even thoughM. galloprovincialis has been reported to occurmainly in lowland or in regions of intermediate eleva-tion, we sampled some individuals at altitude of up to1444 m, as previously described in the Alps byRougon (1975). According to the literature, M. g. pis-tor is mainly located in Eastern Europe, althoughsome specimens have been reported at high elevationsin central Spain, southern France and the FrenchAlps (Vives, 2000). It is difficult to determine whetherthe altitudinal lineage found in the present studycould be associated with M. g. pistor because no mor-phological evidence could confirm this hypothesis,although the present study supports a primary effectof vicariance on the genetic structure of these twoecologically divergent lineages, probably as a result ofdistinct refugia and recolonization paths during andafter the glaciations. However, the existence of aM. galloprovincialis ecotype adapted at higher eleva-tion revealed a reduced gene flow between thelowland populations and this lineage. So far, onlyunfavourable climatic conditions appear to stop theproliferation and the symptoms caused by the nema-tode because it has been observed in Japan at theHokkaido and Honshu islands, even though the



insects and the pine-hosts are indigenous to this area(Mamiya, 1984; Shoda-Kagaya, 2007). To spreadbeyond Spain, the nematode would have to overcomea physical barrier: the Pyrenean Mountains. Altitu-dinal and temperature barriers in the Pyrenees couldbreak down the proliferation of PWN, as alreadyobserved at the Ohu Mountain in Tohoku in Japanwhere M. alternatus populations have been foundisolated at the two sides of this mountain(Shoda-Kagaya, 2007).

The European situation is quite different from theAsian one; in that sense, a vectoring relay could bepossible with M. sutor, which occurs in elevatedareas. The similarities between Monochamus speciesaround the world are remarkable (Koutroumpa et al.,2008b, 2009b; Akbulut & Stamps, 2012). They sharesimilar life histories, even though they are thousandsof miles apart, and have similar survivorship fromegg to adult and similar emergence dynamics. AllMonochamus species vectoring the PWN (for detailson PWN vectors, see Akbulut & Stamps, 2012) feedand oviposit on various conifer host species underadequate conditions (stressed trees) and, in thepresent study, no genetic variability within thespecies could be attributed to the host range. Further-more, we found M. sutor on the same host as M. gal-loprovincialis. Even though no study has recordedM. sutor as a PWN vector so far, sympatry andcommon hosts between the two species could be adeadly combination for conifers susceptible to PWN athigh altitude and proliferation of PWN beyond thePyrenean barrier.

Our molecular study revealed a north-east/south-west geographical pattern of M. sutor haplotypedistribution, which fits with the two sub-species pre-viously described in the literature. Our molecularanalysis revealed that M. sutor separates into twowell-supported distinct subclades on the COI MP treeand higher genetic distances separate these distinctlineages, which may be attributed to the two sub-species: M. s. sutor and M. s. pellio. However, no mor-phological features could be associated with thisvariability and the two lineages co-occur in south-western Alps. Therefore, populations originating fromthe western Alps include both forms (both majorhaplotypes) in sympatry; thus, hybridization shouldnot be excluded at this suture zone, even though thetwo haplotypes remain distant. The Austrian indi-viduals (M. s. pellio) belong to the B1 clade, includingthe northern Alps populations. We hypothesize,according to geographical distribution, that this cladebelongs to M. s. pellio (Hellrigl, 1971), whereas the B2clade likely corresponds to M. s. sutor. Maximumdiversity on the area of the French Alps is common toother insects such as the mountain caddisfly Drususdiscolor (Rambur, 1842) (Pauls, Lumbsch & Haase,

2006). Especially, the western part of the French Alpsis often considered as an important hybridizationzone for many species, as well as a suture zone fordifferent lineages (Taberlet et al., 1998; Flanaganet al., 1999; Hewitt, 1999; Deschamps-Cottin et al.,2000; Wojcik, Ratkiewicz & Searle, 2002; Godoy et al.,2004; Barilani et al., 2007). A similar distinct patternof differentiation between closely-related species hasalready been observed within the alpine Erebiamelampus/sudetica (Staudinger, 1861) speciescomplex (Haubrich & Schmitt, 2007). Pleistoceneclimate cycles have also acted on the diversification ofthe alpine Nebria species, as characterized by altitu-dinal zonation and habitat preferences (Schoville,Roderick & Kavanaugh, 2012).

CONCLUSIONS

By contrast to what is currently assumed, M. gallo-provincialis and M. sutor are not clearly subdividedinto the cited sub-species but rather into multipleintermediate morphological forms. The origin of thishigh morphological variability and important geneflow is found in the life traits of these species, as wellas movement as a result of human forestry activityand world widewood trade. The different lineages ofthese species are hardly delimitated with the markersused. A more finescale analysis of the dispersion withhighly polymorphic microsatellites markers has beeninitiated aiming to specify the genetic structure anddispersal patterns of these two sister species. Thesestudies will be applied over a larger sampling and athigher elevations, especially in the French Alps andPyrenees, which could represent crucial areas for thesurveillance of PWN infestation.

ACKNOWLEDGEMENTS

We thank the Ministère de l’Agriculture, del’Alimentation, de la Péche et de la Ruralité(MAAPR), the Foundation Korialenio (Greece) forfinancial support; the DSF (Département de la Santédes Forêts), Athanasios Koutroumpas, and UteTomitzek for their enthusiastic assistance with pro-viding specimens of Monochamus; Christiane Rougonfor her assistance with morphological study;Emmanuelle Magnoux for her assistance with DNAsequencing; Carlos Lopez Vaamonde, Carole Kerdel-hué, Astrid Groot, and Agnès Horn for valuable dis-cussion and help with the analyses; and the threeanonymous reviewers for their comments thatimproved the manuscript. The authors state thatthere are no conflicts of interest and ethics concerningthe present study. G.R. and F.L. designed the study.F.K. conducted the laboratory work. C.B. helped withthe laboratory work. D.R. helped with the taxonomic



task. F.K. and G.R. analyzed the data and, togetherwith C. B., drafted the manuscript. All authorscarried out the sampling, and read, discussed, andapproved the final version of the paper.

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SUPPORTING INFORMATION

Additional Supporting Information may be found in the online version of this article at the publisher’s web-site:

Appendix S1. Morphological characters list.Figure S1. Variability of the scutellum: top of the dotted line Monochamus galloprovincialis and under thedotted line Monochamus sutor. Photographs made by F. A. Koutroumpa.Figure S2. Variability of the elytra colour, bristle pattern and size: top of the dotted line Monochamusgalloprovincialis and under the dotted line Monochamus sutor. See also see Supporting information, Appen-dix S1 (characters 1–3). Photographs and measurements made by F. A. Koutroumpa.Figure S3. Variability of the median lobe: top of the dotted line Monochamus galloprovincialis and under thedotted line Monochamus sutor. Photographs and measurements made by F. A. Koutroumpa.Figure S4. Variability of the copulatory piece of Monochamus galloprovincialis. Left of the dotted line shapecoded (1) and on the right shape coded (0). See also see Supporting information (Appendix S1, characters 13–15).Photographs and measurements made by F. A. Koutroumpa.Figure S5. Variability of the copulatory piece of Monochamus sutor. Top of the dotted line shape coded (1) andunder the dotted line shape coded (0). See also see Supporting information (Appendix S1, characters 13–15).Photographs and measurements made by F. A. Koutroumpa.Table S1. Morphological character matrix.



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