ORIGINAL ARTICLE

doi:10.1111/j.1558-5646.2007.00091.x

HAWTHORN-INFESTING POPULATIONS OFRHAGOLETIS POMONELLA IN MEXICO ANDSPECIATION MODE PLURALITYXianfa Xie,1 Juan Rull,2 Andrew P. Michel,1 Sebastian Velez,1,3 Andrew A. Forbes,1 Neil F. Lobo,1

Martin Aluja,2 and Jeffrey L. Feder1,4,5

1Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556-03692Instituto de Ecologıa, Asociacion Civil, Km 2.5 Antigua Carretera a Coatepec No 351, 91070 Xalapa, Veracruz, Mexico

5E-mail: Feder.2@nd.edu

Received July 13, 2006

Accepted January 11, 2007

Categorizing speciation into dichotomous allopatric versus nonallopatric modes may not always adequately describe the geographic

context of divergence for taxa. If some of the genetic changes generating inherent barriers to gene flow between populations

evolved in geographic isolation, whereas others arose in sympatry, then the mode of divergence would be mixed. The apple maggot

fly, Rhagoletis pomonella, has contributed to this emerging concept of a mixed speciation mode “plurality.” Genetic studies have

implied that a source of diapause life-history variation associated with inversions and contributing to sympatric host race formation

and speciation for R. pomonella in the United States may have introgressed from the Eje Volcanico Trans Mexicano (EVTM; a.k.a.

the Altiplano) in the past. A critical unresolved issue concerning the introgression hypothesis is how past gene flow occurred given

the current 1200-km disjunction in the ranges of hawthorn-infesting flies in the EVTM region of Mexico and the southern extreme

of the U.S. population in Texas. Here, we report the discovery of a hawthorn-infesting population of R. pomonella in the Sierra

Madre Oriental Mountains (SMO) of Mexico. Sequence data from 15 nuclear loci and mitochondrial DNA imply that the SMO flies

are related to, but still different from, U.S. and EVTM flies. The host affiliations, diapause characteristics, and phylogeography of

the SMO population are consistent with it having served as a conduit for gene flow between Mexico and the United States. We

also present evidence suggesting greater permeability of collinear versus rearranged regions of the genome to introgression, in

accord with recent models of chromosomal speciation. We discuss the implications of the results in the context of speciation mode

plurality. We do not argue for abandoning the terms sympatry or allopatry, but caution that categorizing divergence into either/or

geographic modes may not describe the genetic origins of all species. For R. pomonella in the United States, the proximate selection

pressures triggering race formation and speciation stem from sympatric host shifts. However, some of the phenological variation

contributing to host-related ecological adaptation and reproductive isolation in sympatry at the present time appears to have an

older history, having originated and become packaged into inversion polymorphism in allopatry.

KEY WORDS: Chromosomal inversions, differential introgression, host races, Sierra Madre Oriental, sympatric speciation.

One of the most contested issues in speciation theory has centered

on biogeography: Is the complete geographic isolation of popula-

tions a prerequisite for speciation or can divergence be initiated in

3Present address: Museum of Comparative Zoology, Harvard Uni-

versity, 26 Oxford St, Cambridge, Massachusetts 02138.4Corresponding author.

the face of gene flow? Recently, however, there has been a grow-

ing awareness that dichotomizing speciation into strictly allopatric

versus nonallopatric modes may not always adequately describe

the geographic context of divergence for taxa. As noted by Mallet

(2005), “If some genetic changes leading to reproductive isola-

tion occur in allopatry, and others in sympatry, then what is the

geographic mode of speciation? An obvious answer would be a

1091C© 2007 The Author(s). Journal compilation C© 2007 The Society for the Study of Evolution.Evolution 61-5: 1091–1105

XIE ET AL.

mixture.” In this regard, Coyne and Orr (2004) have proposed sev-

eral new terms describing situations of mixed geographic mode

including “allo-parapatric” speciation (e.g., speciation by rein-

forcement), “para-allopatric” speciation (when populations ini-

tially differentiate while still in partial contact, but accumulate

substantial reproductive isolation only after a subsequent period

of geographic isolation), and “allo-sympatric” speciation. These

terms are not without their practical difficulties. For example, if

populations underwent repeated episodes of isolation and contact

during divergence, with varying degrees of spatial overlap occur-

ring during different contact periods, then we could have specia-

tion modes such as allo-para-sym-para-allo-parapatric. Neverthe-

less, the endeavor heralds the development of a more pluralistic

view of speciation mode, advancing the field beyond the mindset

of strict allopatric versus nonallopatric exclusivity.

Distinguishing the signature of a mixed, pluralistic mode of

speciation from a single mode of divergence can be a difficult

task, however. Identifying key traits responsible for the reproduc-

tive isolation of taxa and establishing their involvement in the

speciation process is daunting enough. To assess a mixed mode

hypothesis one must further integrate these data with historical in-

formation to show that the genetic bases for key traits separating

taxa originated in different geographic contexts.

Despite the complexity of the undertaking, one might still

expect to see certain patterns of genetic differentiation charac-

teristic of a mixed speciation mode. For example, taxa experi-

encing episodes of population subdivision interspersed with pe-

riods of contact and differential gene flow may display a mosaic

genome structure, with genes associated with different isolating

barriers varying in their coalescence times in a manner reflect-

ing the chronology of their respective evolutionary origins. Thus,

coalescence times for certain genes underlying reproductive isola-

tion may be congruent with periods of past geographic separation

between taxa when the trait differences they encode arose. In con-

trast, coalescence times for other genes may converge on different

periods of ecological radiation or reinforcement when populations

were in contact. Moreover, genomic regions not associated with

reproductive isolation may show little or no divergence if pop-

ulations recently or currently overlap and hybridize (Rieseberg,

et al. 1999; Noor et al. 2001a, b; Machado et al. 2002; Feder

et al. 2003a, 2005; Stump et al. 2005; Turner et al. 2005; Rogers

and Bernatchez 2005; Payseur and Nachman 2005; Osada and

Wu 2005).

The apple maggot fly, Rhagoletis pomonella (Diptera:

Tephritidae), has contributed to the emerging concept of a mixed

speciation mode plurality in which the genetic seeds for reproduc-

tive isolation that germinate in speciation may be planted in differ-

ent geographic contexts. Rhagoletis pomonella has long served as

a model for sympatric speciation via host plant shifting and spe-

cialization for phytophagous insects (Bush 1966, 1969). Indeed,

Bush (1966, 1969) has argued that the four described and sev-

eral undescribed taxa comprising the R. pomonella sibling species

complex in North America, including the apple and hawthorn

races of R. pomonella, the snowberry maggot (R. zephyria), the

blueberry maggot (R. mendax), the shrubby dogwood fly (R. cor-

nivora), and the undescribed flowering dogwood (Cornus florida)

fly, all arose via sympatric host shifts. Molecular sequence data,

however, have added a pluralistic twist to the sympatric story for

Rhagoletis: a geographic source of genetic variation for diapause

life-history may have contributed to the adaptive radiation of the

R. pomonella species group (Feder et al. 2003a). Based on gene

trees constructed for three anonymous nuclear loci and mtDNA,

it was inferred that an ancestral, hawthorn-infesting fly popula-

tion became geographically subdivided into Mexican and U.S.

isolates about 1.57 million years ago (Feder et al. 2003a). During

this period of separation, inversions appeared to have arisen and

fixed in the isolated fly population located in the Eje Volcanico

Trans Mexicano (EVTM; a.k.a. Altiplano) for three different ge-

nomic regions on chromosomes 1, 2, and 3. These inversions may

have fixed in the EVTM isolate due to the capture of favorable

combinations of genes affecting diapause life history in accord

with recent theory supporting the local adaptive significance of

rearrangements (Kirkpatrick and Barton 2006). Following subse-

quent secondary contact, introgression from the EVTM fly pop-

ulation into the United States established inversion clines in the

United States for diapause traits adapting flies to latitudinal and

local variation in host phenology influenced by seasonal climatic

conditions. Sometime later, the prestanding diapause variation in

the latitudinal clines appears to have aided the U.S. population

in sympatrically shifting and adapting to a variety of new plants

with differing fruiting times, including introduced apple. There-

fore, a portion of the genetic variation contributing to the adaptive

radiation of the R. pomonella complex in the United States may

have originated in an earlier time and a different place than the

proximate behavioral and ecological factors triggering sympatric

host shifts and divergence in more recent times.

Four points should be emphasized concerning this mixed,

pluralistic model of divergence hypothesized for R. pomonella.

First, we are not arguing that allopatry was necessary at some

stage of divergence for R. pomonella flies to speciate or that host

race formation in the apple maggot could not have happened with-

out it. Rather, we are contending that some of the quantitative

genetic variation facilitating diapause life-history adaptation for

R. pomonella in the United States and its packaging into chro-

mosomal inversions had geographic roots predating host shift-

ing in the complex. Subsequently, the divergent ecological selec-

tion that transformed within-population diapause variation into

between-race and among-species differences in the United States

occurred in sympatry and was associated with shifts to new host

plants with differing fruiting times.

1092 EVOLUTION MAY 2007

MEXICAN RHAGOLETIS

Second, the inversions and their component genes do not

display fixed differences between the apple and hawthorn host

races or among the various R. pomonella group sibling species in

the United States, but rather differ in allele/haplotype frequency.

Nonetheless, host races and species differ greatly and even dis-

cretely in their eclosion phenotypes in a manner generating sub-

stantial reproductive isolation. Thus, it is possible to have quanti-

tative genetic differences between host races and species.

Third, the inversion polymorphisms are just one factor con-

tributing to race formation and speciation for R. pomonella flies.

Other, as yet uncharacterized, genes may also affect the dia-

pause differences between the taxa. In addition, host discrimi-

nation traits are also important for causing reproductive isolation

in R. pomonella (Feder et al. 1994; Linn et al. 2003). These other

genes and traits may have more recent, nonallopatric genetic his-

tories that do not involve inversions.

Finally, analysis of an additional set of 12 nuclear loci has im-

plied that differential introgression is occurring between Mexican

EVTM and U.S. populations (Feder et al. 2005). In particular,

the inverted regions of chromosomes 1–3 that introgressed in the

more distant past now appear to be less permeable to gene flow

than loci in putative collinear regions of the genome on other

chromosomes (Feder et al. 2005). Six of the 12 additional loci

analyzed in Feder et al. (2005) map to chromosomes other than

1–3, outside of the rearrangements. These six loci displayed less

divergence (shallower coalescence times) between Mexican and

U.S. populations than genes associated with the inversions (Feder

et al. 2005). The pattern is consistent with new models of chro-

mosomal speciation (Rieseberg 2001; Noor et al. 2001a; Navarro

and Barton 2003; Kirkpatrick and Barton 2006) that hypothesize

that reduced recombination associated with rearrangements facil-

itates the retention of linked blocks of genes conferring differ-

ential adaptation between hybridizing taxa. In contrast, collinear

portions of the genome tend to introgress because of uninhib-

ited recombination. Studies in sunflowers (Rieseberg et al. 1999),

the Drosophila pseudoobscura subgroup (Wang et al. 1997; Noor

et al. 2001a, b; Machado et al. 2002), and Anopheles mosquitoes

(Stump et al. 2005; Turner et al. 2005) have found evidence for

greater introgression in collinear segments of the genome. Simi-

larly, the molecular data for R. pomonella suggest repeated cycles

(n ≥ 2) of population isolation and contact between Mexico and

the United States, with differential gene flow occurring across the

genome (Feder et al. 2005).

There are alternative scenarios to differential introgression

of inversion polymorphism from the EVTM fly population into

the United States, however, that could potentially explain the pat-

tern of genetic differentiation in R. pomonella. For example, it is

always possible that the inversion polymorphism initially arose

in the absence of geographic isolation internally within the U.S.

fly population and formed primary clines, perhaps as a result of

adaptational stress during range expansion from a southern refuge

after a glaciation period about 1.57 million years ago. Under this

hypothesis, the simultaneity of chromosomal origins would be ex-

plained as a pervasive adaptive challenge occurring during range

spread rather than due to a combination of ecology and allopatry.

Later gene flow between the United States and Mexico of unin-

verted chromosomes would be responsible for the greater simi-

larity of these sequences. However, as enumerated in Feder et al.

(2003a), a primary cline scenario would appear less likely than

secondary contact. Even if selective challenges can be great dur-

ing range expansions, this does not insure that co-adapted sets

of alleles will necessarily be captured in an inversion or that

different inversions will arise congruently across the genome at

roughly the same time. Moreover, although genetic exchange is

reduced between rearranged chromosomal sequences, recombi-

nation and gene conversion often occur, albeit at a reduced level

(Charlesworth 1974). Given a 1.57 million years ago timeframe

for the origin and continued co-existence of the inversions in sym-

patry under the primary cline hypothesis, we may therefore expect

to see significant shuffling of sequences within loci on chromo-

somes 1–3 degrading haplotype differences, but we do not. Finally,

the EVTM still displays a mtDNA signature of past geographic

subdivision 1.57 million years ago. If gene flow of uninverted se-

quences occurred in the EVTM, then it would have had to have

been extensive and obliterated all traces of past subdivision that

would have been present in these nuclear haplotypes, as well,

which seems unlikely.

Several critical unresolved issues still remain, however, con-

cerning the differential introgression hypothesis for R. pomonella.

Perhaps the foremost question centers on the current ranges of

hawthorn-infesting flies in Mexico and the United States. Feder

et al. (2005) argued that relatively recent gene flow between the

Mexican population in the EVTM and the United States is re-

sponsible for the limited population structure displayed by the six

putative collinear loci. However, EVTM and U.S. populations are

currently separated by a distance of at least 1200 km, implying

the need for large range shifts in the relatively recent past to ac-

count for the hypothesized gene flow. Occasional long-distance

dispersal of flies could also be invoked to explain the data (e.g., by

hurricanes). But given the extensive gene flow for collinear regions

implied by the molecular data and the likely rarity of long-range

migration given the distances involved, this hypothesis seems un-

likely. Obviously, the current distribution of R. pomonella does not

reflect its past range, which certainly must have changed dramat-

ically along with its hawthorn hosts during cooling and warming

periods associated with Pleistocene glaciations. Despite the po-

tential for substantial fluctuations in fly distribution, however, it

still seems unlikely that the ranges of Mexican EVTM and U.S.

populations changed so drastically in the recent past to bridge the

1200-km gap.

EVOLUTION MAY 2007 1093

XIE ET AL.

The distribution of hawthorn-infesting flies in Mexico is not

fully resolved, however. Bush (1966) first documented the exis-

tence of R. pomonella in the central EVTM highlands of Mexico,

but he reported that the exact limits of the species’ distribution

were unknown. Based on four collecting sites, Bush (1966) con-

sidered the distribution of the fly to likely correspond to the range

of Crataegus mexicana, a native hawthorn host of R. pomonella

in Mexico. This distribution roughly matches the transvolcanic

biogeographic area of endemism in Mexico (Marshall and Lieb-

herr 2000). Moreover, initial molecular genetic studies of Mexican

flies by Feder et al. (2003a) were based on U.S. Department of

Agriculture intercepts of infested C. mexicana fruit from Mexico

City, located on the EVTM plateau.

DISTINCT HAWTHORN FLY POPULATIONS IN MEXICO?

To better understand the biogeography of R. pomonella in Mexico,

Rull et al. (2006) initiated a survey of Rhagoletis on eight of the

13 endemic hawthorn species throughout the country. Three ma-

jor findings emerged from the study. First, the study expanded

the range of “R. pomonella-like” flies to cover much of the geo-

graphic distribution of Crataegus in Mexico, including the Sierra

Madre Oriental Mountain range (SMO) and the Sierra de las Altas

de Chiapas (Fig. 1). The two major exceptions were the apparent

absence of the fly from northwestern Mexico and perhaps parts of

the Sierra Madre del Sur Mountains. Second, the study enlarged

the host range of R. pomonella, documenting infestation for five

of the eight native hawthorn species surveyed. Third, the results

Figure 1. Collection sites for pomonella populations from Mexico

and the United States genetically analyzed in the current study.

See Table 1 for site information and designations. Sites MC and CJ

are located in the Eje Volcanico Trans Mexicano (EVTM) of Mexico,

sites SJ, CB, PL in the Sierra Madre Oriental Mountains (SMO) of

Mexico, site CP in the Sierra de las Altas de Chiapas, and sites NY,

MI, and TX in the United States.

revealed a significant difference in pupal body mass between flies

from the EVTM (mean = 11.17 ± 0.10 mg, n = 5 sites, 30 pu-

pae/site) and those from elsewhere in Mexico (mean = 7.86 ±0.45 mg, n = 6 sites; Fig. 2).

The difference in pupal body mass could either be due to pop-

ulation subdivision in Mexico or reflect environmental rather than

genetic variation. Flies from the EVTM primarily infest C. mex-

icana and C. rosei rosei (Rull et al. 2006). These two hawthorns

fruit late in the field season in the central EVTM highlands (infes-

tation period ranges from mid October to early January; Table 1).

In addition, C. mexicana bears large-sized hawthorn fruit, which

could help account for the larger pupal body size of EVTM flies.

In the SMO, C. rosei parrayana, C. gracilor, C. greggiana, and C.

cuprina are the predominant hosts (Rull et al. 2006). Infestation

occurs considerably earlier in the season in the SMO (late August

to early October; Table 1), suggesting possible allochronic isola-

tion between EVTM and SMO flies attacking different hawthorn

hosts (Rull et al. 2006). (Note: In the United States, R. pomonella

infests a variety of different hawthorn species mainly from mid

August to late October; Berlocher 2000.) However, in the transi-

tion zone between the EVTM and the SMO in the state of Veracruz

(Fig. 1), C. mexicana and C. rosei rosei co-occur with C. rosei

parrayana and C. gracilor, and all four hawthorns are infested by

Figure 2. Mean pupal mass for pomonella flies from Mexican

EVTM and SMO sites plotted against population altitude (n = 30

pupa measured per site) based on data from Rull et al. (2006). Re-

gression coefficient (r2) between altitude and pupal body mass =

0.16, P > 0.22, 10 df. Annual rainfall was also not significantly re-

lated to mean pupal mass (r2 = 0.01, P > 0.77, 10 df). The EVTM

sites in Mexico with increasing elevation from left to right along

the X-axis are Tancitaro, Michoacan (MC); Texcoco, state of Mexico;

Santa Marta, District Federal; Perote, Veracruz; and Coajomulco,

Morelos (CJ). The SMO sites in Mexico from left to right along the

X-axis are Piletas, Veracruz (PL); Los Pinos, Veracruz; San Cistobal,

Chiapas (CP); Casa Blanca, Veracruz (CB); Cerro el Potosı, Nuevo

Leon; and San Joaquin, Queretaro (SJ). Two letter abbreviations

given in parentheses for the sites above designate fly populations

genetically analyzed in the current study.

1094 EVOLUTION MAY 2007

MEXICAN RHAGOLETIS

Tab

le1.

Co

llect

ing

site

sfo

rR

.po

mo

nel

lafl

ies

gen

etic

ally

anal

yzed

inth

est

ud

y.Po

p.r

efer

sto

wh

eth

erth

esi

teg

enet

ical

lyb

elo

ng

sto

the

Eje

Vo

lcai

no

Tran

sM

exic

o(E

VTM

),Si

erra

Mad

reO

rien

tal

(SM

O),

or

U.S

.(U

S)fl

yp

op

ula

tio

n.

Site

abb

revi

atio

ns

(Ab

br.)

,la

titu

de

(deg

.N

),lo

ng

itu

de

(deg

.W

),al

titu

de

(met

ers)

,m

ean

ann

ual

pre

cip

itat

ion

(mm

),h

ost

tree

spec

ies,

infe

stat

ion

per

iod

,an

dco

llect

ing

dat

ear

eal

sog

iven

.

Pop.

Col

lect

ing

site

Abb

r.L

at.

Lon

g.A

lt.Pp

t.H

ostp

lant

(s)

Infe

stat

ion

peri

odD

ate

EV

TM

Tanc

itaro

,Mic

hoac

anM

C19

.20

102.

2220

8011

10C

.ros

eiro

sei

Ear

lyO

ctob

erto

late

Dec

embe

r15

Nov

embe

r20

02C

.mex

ican

aE

arly

Oct

ober

tola

teD

ecem

ber

15N

ovem

ber

2002

EV

TM

Coa

jom

ulco

,Mor

elos

CJ

19.0

399

.11

2670

1300

C.m

exic

ana

Mid

Oct

ober

tola

teD

ecem

ber

12N

ovem

ber

2002

SMO

San

Joaq

uin,

Que

reta

roSJ

20.5

599

.34

2450

1030

C.r

osei

parr

ayan

aL

ate

July

tola

teA

ugus

t24

Aug

ust2

002

SMO

Cas

aB

lanc

a,V

erac

ruz

CB

19.3

897

.06

2200

1220

C.m

exic

ana

Lat

eA

ugus

tto

earl

yO

ctob

er24

Sept

embe

r20

02SM

OPi

leta

s,V

erac

ruz

PL19

.35

96.5

615

2014

90C

.ros

eipa

rray

ana

Lat

eJu

lyto

late

Aug

ust

9A

ugus

t200

2SM

OSa

nC

rist

obal

,Chi

apas

CP

16.4

592

.38

2120

1105

C.m

exic

ana

Mid

Nov

embe

rto

earl

yJa

nuar

y15

Nov

embe

r20

02U

SG

enev

a,N

ewY

ork

NY

42.8

677

.00

150

810

C.m

olli

sL

ate

Aug

ustt

oea

rly

Nov

embe

r16

Sept

embe

r20

00U

SG

rant

,Mic

higa

nM

I43

.34

85.8

326

594

0M

alus

pum

ila

Lat

eJu

lyto

earl

ySe

ptem

ber

15A

ugus

t199

5C

.mol

lis

Lat

eA

ugus

tto

earl

yN

ovem

ber

15Se

ptem

ber

1995

US

Bra

zos

Ben

d,Te

xas

TX

29.2

295

.35

3211

15C

.mol

lis

Ear

lySe

ptem

ber

tom

idN

ovem

ber

6O

ctob

er20

00

flies (Rull et al. 2006). Here, the fruiting times of C. mexicana

and C. rosei rosei are earlier (infestation period late September to

early November) than they are on the EVTM, partly overlapping

with C. rosei parrayana and C. gracilor. Temporal and spatial

continuity are therefore possible among Mexican fly populations

(i.e., the observed differences in pupal mass could reflect host-

and altitude-related environmental variation). But in the Sierra de

las Altas de Chiapas (Fig. 1), an isolated fly population separated

by the lowland forests of the Isthmus of Tehuantepec was dis-

covered attacking the larger and later fruiting C. mexicana (mid

November to early January; Rull et al. 2006). The mean pupal

mass for these Chiapas flies was 7.98 mg (n = 30 pupae; Fig. 2),

equivalent to that seen at other sites in the SMO. Moreover, pu-

pal body mass among sites was not correlated with environmental

factors such as altitude or annual precipitation (Fig. 2). These

data are consistent with the trait being heritable, supporting the

hypothesis that Mexican flies are geographically and genetically

subdivided, with hawthorn host specificity being lax. The current

differences between Mexican flies highlight the potential for the

EVTM population to have served as a past source of variation for

diapause and possible other traits in sympatric host shifts in the

United States.

Here, we report on a genetic analysis of Mexican hawthorn-

infesting flies to resolve issues concerning population structure,

geographic range, and host affiliation critical to determining

whether phenology traits that originated in the EVTM could have

set the stage for sympatric host shifts in the United States. We also

conduct rearing experiments to quantify eclosion time variation

among Mexican fly populations to assess its current potential as

an ecological barrier to gene flow. The results help clarify how

gene flow could have occurred in the distant and recent past be-

tween Mexican and U.S. populations along a corridor of hawthorn-

infesting flies in the SMO. The physical bridging of Mexican and

U.S. populations is significant because it provides a plausible route

for how phenological variation in EVTM flies could have intro-

gressed and served as a source of diapause variation contributing

to sympatric host shifts in the United States.

MethodsSITES SAMPLED

Hawthorn flies from nine different sites were genetically analyzed

in the study, six from Mexico and three from the United States

(Fig. 1). The three R. pomonella sites in the United States from

Texas (TX), Michigan (MI), and New York (NY) encompass much

of the range of the fly in the eastern United States. Two of the

Mexican fly sites (Tancitaro, in the state of Michoacan [MC] and

Coajomulco, in Morelos [CJ]) reside in the EVTM plateau. The

remaining four sites lie outside this region. Of these four sites, one

resides in the transition zone between the SMO and the EVTM

(Casa Blanca, Veracruz [CB]) and another represents an isolated

EVOLUTION MAY 2007 1095

XIE ET AL.

population in the Sierra de las Altas de Chiapas (San Cristobal

[CP]). The other two sites (Piletas, Veracruz [PL] and San Joaquin,

Queretaro [SJ]) are part of the SMO proper.

HOST PLANTS

Flies were collected as larvae in infested hawthorn fruit at all nine

sites and either immediately dissected from the fruit and frozen

for later genetic analysis or reared to adulthood in the laboratory.

At the Grant, MI, site, flies were collected from both sympatric

apple and hawthorn trees. Hawthorn host species and collecting

dates are given in Table 1. Note that the transitional CB popula-

tion at Casa Blanca, Veracruz, was sampled from C. mexicana, the

primary host for flies in the EVTM. The isolated CP population

from Chiapas was also collected from C. mexicana, a plant that is

not endemic to the region but was likely introduced by Tlaxcal-

tecan Indians accompanying Spanish conquistadors in the 1600s

(Standley and Steyermark 1946).

GENES SEQUENCED

Sequence data were generated for 15 nuclear loci and a 946 bp

fragment of the mitochondrial genome containing the 3′ portion

of COI, leucine tRNA, and the COII genes. The sequences are av

ailable in GenBank (accession numbers AY152477-AY152526,

AY930466-AY931013, and DQ812553-DQ812885). These nu-

clear and mtDNA loci formed the basis for the previous genetic

studies of EVTM and U.S. flies (Feder et al. 2003a, 2005). Nine

Table 2. Loci sequenced in the study. Given are chromosome map

positions (Chr.), probability level (P) for conformation to a molecu-

lar clock, the ML substitution model (Model) determined by Mod-

elTest using the Akaike information criterion (Posada and Cran-

dall, 1998), and the minimum number of recombination events

(Rec.) estimated by the method of Hudson and Kaplan (1985) for

each locus.

Locus Chr. P Model Rec.

P181 1 1.0000 HKY 0P220 1 0.9841 TIM 3P3072 1 0.9488 TIM+G 4P2473 2 0.9920 TrN+I 1P2956 2 0.9810 HKY+G 4P667 2 0.9992 TVM+G 5P8 2 1.0000 TVM 1P22 3 0.8646 TVM+I 0P7 3 0.7385 HKY 3P2963 4 0.9825 TIM+G 3P661 4 0.3171 TVM+G 5P1700 5 0.9999 HKY 4P2620 5 1.0000 K81uf 0P309 5 1.0000 K81uf+I 3P3060 – 1.0000 HKY 2mtDNA – 0.9991 TrN+I 0

of the loci (P220, P181, P3072, P2956, P667, P8, P2473, P7, and

P22) map to chromosomes 1–3 and are subsumed by chromoso-

mal inversions (Roethele et al. 2001; Feder et al. 2003b; Table 2)

(haploid n = 6 for R. pomonella). Allozyme markers within these

three inverted regions of the genome display significant allele fre-

quency differences between the sympatric apple and hawthorn

host races across the northeastern and midwestern United States

(Feder et al. 1988, 1990; McPheron et al. 1988; Feder and Bush

1989; Berlocher 2000). All three of the rearranged regions on

chromosomes 1–3 have been shown to correlate with diapause-

related traits, accounting for up to 30% of the phenotypic variation

for eclosion time within the hawthorn race at the Grant, MI, site

(Feder et al. 1993). The variation in diapause differentially adapts

the fly races to a three- to four-week difference in the peak fruiting

times of apples versus hawthorns, generating allochronic premat-

ing, as well as postzygotic ecological isolation between apple and

hawthorn flies (Feder et al. 1993, 1997a, b; Filchak et al. 2000).

Markers in these regions also display latitudinal allele frequency

clines within the U.S. host races (Feder and Bush 1989; Feder et al.

1990; Berlocher 2000). The clines match geographic variation in

host fruiting time; alleles associated with later adult eclosion time

and reduced propensity for nondiapause development are found in

higher frequencies in southern populations (Feder and Bush 1989;

Feder et al. 1990; Berlocher 2000).

Five other loci analyzed in the study (P661, P2963, P1700,

P2620, and P309) map to chromosomes 4 and 5 in the R. pomonella

genome (Table 2). The exact map position of P3060 is not known

but it is not located on chromosomes 1–3 (Feder, unpubl. data).

Allozymes on chromosomes 4 and 5 do not differ in allele fre-

quency between the host races, do not vary clinally, do not cor-

relate with the timing of eclosion, and do not display high lev-

els of linkage disequilibrium in nature (Feder et al. 1988, 1990,

1993, 2003b). The genetic data imply that P661, P2963, P1700,

P2620, P309, and P3060 are not associated with chromosomal

rearrangements (Feder et al. 2003b). In addition, these six loci ap-

pear to have introgressed more readily in the recent past between

EVTM and U.S. populations, possibly due to uninhibited recom-

bination in combination with a lack of differential selection (Feder

et al. 2005).

DNA CLONING AND SEQUENCING

Genomic DNA were isolated from individual flies and PCR am-

plified for 35 cycles (94◦C, 30 sec.; 52◦C, 1 min., 72◦C, 1.5

min.) using locus-specific primers for the 15 nuclear and mtDNA

fragment as described in Roethele et al. (2001). Products were

TA cloned into pCR II vectors (Invitrogen Corp., Carlsbad, CA).

PCR amplification products were initially cloned separately for

a minimum of two flies from each study site, with four to six

clones sequenced per locus per fly in both the 5′ and 3′ direc-

tions on an ABI 3700 sequencer using the ABI Prism�

BigDyeTM

1096 EVOLUTION MAY 2007

MEXICAN RHAGOLETIS

Terminator (Applied Biosystems Corp., Foster City, CA) version

3.0 system. To increase sample sizes for certain sites, we also sepa-

rately amplified genomic DNA for four to eight flies from the site,

and TA cloned the pooled amplification products for sequencing.

To avoid analysis of identical alleles from the same individual,

sequences generated from the pooled library were not included

unless they differed from each other.

GENE AND GENETIC DISTANCE TREE CONSTRUCTION

Parsimony and maximum likelihood gene trees were constructed

using PAUP∗b10 (Swofford 2002). For the parsimony analysis,

gaps were treated as a fifth base pair, with indels of identical

length and sequence position recoded to count as single muta-

tional steps. Rhagoletis electromorpha, which belongs to the sis-

ter species group (R. tabellaria) to R. pomonella, was used as an

outgroup taxon to root trees. Parsimony and maximum likelihood

trees were very similar and so we report the results for only the

parsimony trees here. Intragenic recombination was statistically

tested using the methods of Hudson and Kaplan (1985). Putative

recombinant alleles and gene regions were identified and these

alleles were excluded from initial parsimony gene tree construc-

tion. Recombinant alleles were then added to the trees by hand to

generate allele networks. The molecular clock was tested for each

locus for R. pomonella and R. electromorpha sequences by com-

paring log-likelihood scores enforcing versus relaxing the clock

hypothesis for the best supported DNA substitution model iden-

tified using ModelTest (Posada and Crandall 1998). To quantify

gene tree topology and genetic divergence, relative node depths

(RNDs) were calculated between the major haplotype classes of

alleles in Mexico and the United States as in Feder et al. (2005) by

dividing the number of substitution differences between a given

pair of Mexican and U.S. alleles by the mean number of sub-

stitutions between each and the outgroup R. electromorpha se-

quence. Assuming a molecular clock (which none of the nuclear

loci or mtDNA violated; Table 2) the mean RND for all pairs of

Mexican and U.S. alleles between two haplotype classes estimates

the age of separation of the haplotypes relative to the divergence

time of R. electromorpha plus the coalescence time in the com-

mon ancestor.

Neighbor-joining trees (Saitou and Nei 1987) summarizing

the overall genetic relatedness of populations were constructed

using PHYLIP, version 3.66 (Felsenstein 1989). Trees were con-

structed separately for loci mapping to chromosomes 1–3 and

those residing elsewhere in the genome. To construct the neighbor-

joining trees, mean pairwise uncorrected genetic distances were

first computed separately for each locus between each of the nine

EVTM, SMO, and U.S. populations, as well as between these

populations and the outgroup R. electromorpha using Mega, ver-

sion 3.1 (Kumar et al. 2004). The pairwise distance between two

populations for a locus was then divided by the average distance

of all R. pomonella populations to R. electromorpha to standard-

ize for sequence length and substitution rate differences among

loci. The standardized distances were averaged across the nine

loci on chromosomes 1–3 and the six loci mapping elsewhere to

give overall pairwise distance measures used for tree construction.

For chromosome 1–3 loci, north (N) and south-north (SN) hap-

lotypes were considered separately in the calculations of genetic

distance for NY and MI sites, generating NYN, NYSN, MIN, and

MISN populations for the analysis.

ANALYSIS OF POPULATION DIFFERENTIATION AND

STRUCTURE

Hierarchical analysis of molecular variance (AMOVA) was per-

formed using Arlequin 2.0 (Schneider et al. 2000) to test for

genetic structuring among R. pomonella populations. For the

AMOVA analysis, the nine sites under study were initially divided

into three populations based on the results from the neighbor-

joining genetic distance trees that distinguished (1) EVTM (MC

and CJ sites), (2) SMO (SJ, CB, PL, and CP sites), and (3) the

United States (MI, NY, and TX sites). AMOVA was performed for

the nine chromosome 1–3 loci with N haplotypes both included

and excluded from the analyses. The analysis with N haplotypes

excluded was performed because N haplotypes tend to inflate

intrasite and among site variation within populations relative to

among-population divergence for the nine chromosome 1–3 loci

due to the latitudinal clines they display in the United States. Note

that although the CP site from the Sierra de las Altas de Chiapas

is geographically isolated from the SMO, genetically the Chiapas

flies were found to be very similar to SMO flies (Fig. 3) and so

they were designated as part of the general SMO population in the

statistical analysis of population structure.

ECLOSION EXPERIMENTS

We performed a series of controlled rearing experiments to quan-

tify eclosion time variation among Mexican fly populations. Fly

larvae were collected from infested hawthorn fruit at the six Mex-

ican sites in the fall of 2003 and allowed to pupate in an ambient

temperature room at the Instituto de Ecologıa, A.C., in Xalapa,

Veracruz. Three weeks after puparium formation, pupae were col-

lected and placed in moist vermiculite in petri dishes. The petri

dishes were held at 5◦C in a refrigerator for 14 weeks to simulate

winter. After removal from the cold, pupae were placed in a 26.5◦C

constant temperature chamber and eclosing adults collected on a

daily basis as they emerged.

ResultsNone of the sequenced genes deviated significantly from a molec-

ular clock (Table 2). Twelve of the 15 nuclear loci displayed ev-

idence for possible recombination, as implied by the method of

Hudson and Kaplan (Table 2). Inferred recombination was gener-

ally limited, however, to alleles within the same haplotype class

EVOLUTION MAY 2007 1097

XIE ET AL.

of inversion or geographic population (EVTM, SMO, or United

States; see online Supplementary Figs. S1–S15). There was no

evidence for recombination among mtDNA sequences (Table 2).

GENES ON CHROMOSOMES 1–3

Neighbor-joining genetic distance and parsimony gene trees for

the nine nuclear loci on chromosomes 1–3 implied the existence of

three genetically distinguishable hawthorn-infesting populations

of pomonella flies in North America: (1) EVTM, (2) SMO, and

(3) United States (see Fig. 3A for the neighbor-joining tree and

Fig. 4A–C for representative gene trees for the loci P220, P2956,

and P7; online Supplementary Figs. S1–S9 provide more exten-

sive gene trees of all nine loci sequenced from chromosomes 1–3).

Genetic subdivision was also evident in the hierarchical AMOVA

analysis. FCT values among EVTM, SMO, and U.S. populations

were significant for all nine chromosome 1–3 loci, regardless of

whether N haplotypes were included or excluded from the analysis

(Table 3). Even with N haplotypes included in the AMOVA, the

percentage of variation explained was substantially higher among

EVTM, SMO, and U.S. populations (mean = 43.4 ± 4.27%, SE,

n = 9) than among sites within these populations (mean = 12.4 ±2.80% SE, n = 9). Population structuring was even more pro-

nounced when N haplotypes were removed from the AMOVA

analysis, with the mean percentage of genetic variation explained

by among-population differences rising to 51.2 ± 7.71%. Seven of

the nine genes displayed synapomorphic substitutions or unique

combinations of derived mutations distinguishing EVTM, SMO,

and U.S. flies. As in Feder et al. (2003a, 2005), N haplotypes

were unique to northern U.S. sites (MI and NY), whereas variation

within the southern clade of “SN/M” (south-north and Mexico) al-

leles differentiated among EVTM, SMO, and Texas flies (Fig. 4A–

C; online Supplementary Figs. S1–S9).

The SMO fly population was more closely related, in an over-

all sense, to the U.S. than to EVTM population (Fig. 3A). The

mean genetic distance between SMO and U.S. populations for

SN haplotypes relative to R. electromorpha was less (0.245 ±0.031 SE; n = 8 loci; locus P8 possessed only N haplotypes in the

United States) than that between the SMO and EVTM (0.353 ±0.0591; n = 9 loci). However, the loci P3072, P2473, and P667

deviated from this general trend (see online Supplementary Figs.

S3, S4, and S6), implying that EVTM and SMO flies (or at least

a subset of alleles in these two populations) were most closely

related. In no case, however, were EVTM and U.S. SN alleles

genealogically more closely related than EVTM and SMO haplo-

types (mean relative genetic distance between EVTM and United

States = 0.368 ± 0.052, n = 9 loci; see Fig. 4A–C and online Sup-

plementary Figs. S1–S9). Thus, the SMO population appears to

comprise a composite set of alleles with ties to both the EVTM and

United States.

Figure 3. Neighbor-joining trees based on overall genetic dis-

tances among EVTM, SMO, and U.S. fly populations relative to

the outgroup R. electromorpha for (A) chromosome 1–3 loci, and

(B) loci not residing on chromosomes 1–3. Trees are scaled so that

the lengths of the branch from the R. electromorpha/R. pomonella

node to terminal populations are the same in (A) and (B). Relative

genetic distances are given on branches. For chromosome 1–3 loci,

the N and SN clades of haplotypes present at NY and MI were con-

sidered as separate populations (NYN, NYSN, MIN, and MISN in [A],

respectively).

GENES MAPPING OUTSIDE CHROMOSOMES 1–3

The six loci mapping outside chromosomes 1–3 also showed sig-

nificant genetic structuring among EVTM, SMO, and U.S. flies,

as indicated by hierarchical AMOVA analysis (Table 3). P661,

P2963, P1700, P2620, P309, and P3060 all displayed significant

FCT values (Table 3). Although a significant proportion of the

genetic variation present for P661, P2963, P1700, P2620, P309,

1098 EVOLUTION MAY 2007

MEXICAN RHAGOLETIS

Table 3. Hierarchical AMOVA analysis for EVTM, SMO, and U.S. populations. See Figure 1 legend for designation of sites within popula-

tions. Values when N haplotypes were excluded from the analysis appear above those in which they were included for chromosome 1–3

loci. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, as determined by permutation tests with 10,000 replicates.

Locus Percent variation explained Fixation indices

Chr. Among pops. Among sites in pops. Within sites FCT (pop./total) FSC (site/pop.) FST (site/total)

P181 1 59.35 0.01 40.65 0.593∗∗∗ 0.000 0.594∗∗∗

46.81 5.20 47.99 0.468∗ 0.098∗∗∗ 0.520∗∗∗

P220 1 73.11 7.75 19.14 0.731∗∗∗ 0.288∗∗∗ 0.809∗∗∗

62.56 6.71 30.73 0.626∗∗∗ 0.179∗∗∗ 0.693∗∗∗

P3072 1 48.33 18.36 33.31 0.483∗∗∗ 0.355∗∗∗ 0.667∗∗∗

42.38 21.73 35.89 0.424∗∗∗ 0.377∗∗∗ 0.641∗∗∗

P2473 2 27.46 15.37 57.18 0.275∗∗ 0.212∗∗∗ 0.428∗∗∗

34.70 14.99 50.31 0.347∗∗∗ 0.230∗∗∗ 0.497∗∗∗

P2956 2 63.36 −5.10 41.74 0.634∗∗ −0.139 0.583∗∗∗

53.05 2.16 44.79 0.530∗∗∗ 0.046∗∗∗ 0.552∗∗∗

P667 2 36.23 23.54 40.23 0.362∗∗∗ 0.369∗∗ 0.598∗∗∗

30.31 26.20 43.49 0.303∗∗ 0.376∗∗∗ 0.565∗∗∗

P8 2 87.36 4.59 8.05 0.873∗∗∗ 0.363∗∗∗ 0.919∗∗∗

58.27 7.05 34.68 0.583∗∗ 0.169∗∗∗ 0.653∗∗∗

P22 3 21.98 26.67 51.36 0.220∗ 0.342∗∗ 0.486∗∗∗

25.08 19.19 55.73 0.251∗ 0.256∗∗ 0.442∗∗∗

P7 3 43.97 5.31 50.73 0.440∗∗ 0.095∗∗∗ 0.493∗∗∗

37.52 8.21 54.27 0.375∗∗∗ 0.131∗∗∗ 0.457∗∗∗

P2963 4 50.50 2.31 47.20 0.505∗∗∗ 0.047∗∗∗ 0.528∗∗∗

P661 4 13.40 25.25 61.35 0.134∗∗ 0.292∗∗∗ 0.387∗∗∗

P1700 5 46.64 10.96 42.39 0.466∗∗∗ 0.205∗∗∗ 0.576∗∗∗

P2620 5 33.00 1.92 65.08 0.330∗∗∗ 0.028∗∗ 0.349∗∗∗

P309 5 40.61 5.21 54.18 0.406∗∗ 0.088∗∗∗ 0.458∗∗∗

P3060 – 16.20 5.96 77.84 0.162∗ 0.071∗∗∗ 0.222∗∗

mtDNA – 87.70 −1.13 13.43 0.877∗ 0.071∗∗∗ 0.866∗∗∗

and P3060 was partitioned among EVTM, SMO, and U.S. pop-

ulations, the percentage of genetic variation among populations

displayed by these six loci (mean = 34.9 ± 6.86 %) was less

than that for the nine loci residing on chromosomes 1–3 (51.2 ±7.71% with N haplotypes excluded). The most striking differ-

ence between the two sets of genes, however, was the relatively

deeper coalescence times observed for the nine chromosomes 1–

3 loci versus P661, P2963, P1700, P2620, P309, and P3060, as

revealed by comparisons of genetic distances in the neighbor-

joining trees (Fig. 3A, B) and RND values (Fig. 5). The larger

RND values for loci on chromosomes 1–3 were due, in part, to

the presence of N haplotypes in northern U.S. populations. The

pattern of interpopulation differentiation seen for P661, P2963,

P1700, P2620, P309, and P3060 among the EVTM, SMO, and

the United States therefore tended to be of more recent tempo-

ral origin than that observed for chromosome 1–3 genes. For loci

outside of chromosomes 1–3, EVTM, SMO, and U.S. populations

were separated from each other by similar levels of differentiation

(overall genetic distance of SMO to U.S. populations relative to

R. electromorpha = 0.171 ± 0.031, n = 6 loci; SMO to EVTM =

0.152 ± 0.018, n = 6 loci; EVTM to United States = 0.192 ±0.043, n = 6 loci).

mtDNA

MtDNA displayed a different pattern of geographic variation than

the nuclear loci. For mtDNA, SMO and U.S. populations were

highly differentiated from EVTM flies (Fig. 4 D). In contrast,

SMO and U.S. populations were very similar to each other, sharing

several mtDNA haplotypes in common (Fig. 4 D).

ECLOSION TIME

Three general features emerged from the eclosion data for Mex-

ican flies. First, with the exception of Chiapas flies, EVTM

and SMO populations had dramatically different and essentially

nonoverlapping eclosion curves (Fig. 6), implying the potential

for substantial allochronic isolation. The mean eclosion time for

the two EVTM populations was 151.3 ± 0.2 days (MC site =151.2 ± 5.5 days SE, n = 8; CJ = 151.5 ± 2.3 days, n = 41). In

contrast, the mean eclosion time for the three populations in the

SMO proper was 83.4 ± 5.9 days (SJ = 93.7 ± 1.1 days, n = 106;

EVOLUTION MAY 2007 1099

XIE ET AL.

Figure 4. Most parsimonious gene trees for (A) P220, (B) P7, (C) P2956, (D) mtDNA, (E) P661, (F) P309, (G) P3060, and (H) P2620. Site

designations are given in Table 1. Trees are scaled so that the longest distance from an allele to the outgroup R. electromorpha are

relatively the same across loci. Gene trees do not include all of the sequenced alleles for each locus, but subsets that encapsulate the

general topological structure for trees. Additional alleles and networks incorporating recombinant alleles are provided in the online

Supplementary Figures S1–S16. Chromosome position for loci, sequence lengths (bp), branch lengths (steps), and bootstrap support for

nodes (10,000 replicates) are given. Exact location for P3060 is not known, but locus does not map to chromosomes 1–3. For (A–C), N =

north United States (blue colored clade of alleles), SN = south/north United States (red), SMO = Mexican Sierra Madre Oriental (orange),

and EVTM = Mexican Eje Volcanico Trans Mexicano (green). For (D–H), all of the U.S. alleles are colored blue. Yellow-colored circles demark

the deepest node depth connecting alleles at a locus among EVTM, SMO, and U.S. flies. Comparisons of these nodes among (A–D) versus

(E–H) highlight the deeper coalescence times between chromosome 1–3 and mtDNA genes versus nonchromosome 1–3 loci. The outgroup

taxon R. electromorpha was collected from gray dogwood (Cornus racemosa) at Dowagiac, MI, on 12 September 1999.

1100 EVOLUTION MAY 2007

MEXICAN RHAGOLETIS

Figure 5. Relative node depths (RND) for nuclear loci residing

on chromosomes 1–3 (white bars) versus RND values for genes

on chromosomes other than 1–3 (black bars) for comparisons be-

tween (A) EVTM versus United States, (B) EVTM versus SMO, and

(C) SMO versus U.S. fly populations. P-values for difference in RNDs

between chromosome 1–3 versus nonchromosome 1–3 loci were

determined by Whitney-Mann U tests.

CB = 83.5 ± 0.9 days, n = 88; PL = 73.1 ± 2.7 days, n = 18),

about 68 days earlier than for EVTM flies.

Second, the CB population in the transition zone between

the SMO and the EVTM had an eclosion time similar to flies

from the SJ and PL sites in the SMO proper (Fig. 6). This was

true despite infested C. mexicana at the CB site having interme-

diate fruiting times between the phenologies normally seen for

infested hawthorns in the EVTM (late fruiting) and SMO (early

fruiting). Consequently, if increased temporal overlap occurs be-

tween EVTM and SMO flies at sympatric sites in the transition

zone, then it may largely be due to environmentally induced, rather

than genetically based, shifts in fly eclosion time associated with

intermediate hawthorn fruiting times.

Third, the geographically isolated CP population infesting

late fruiting C. mexicana had an eclosion curve intermediate be-

tween EVTM and SMO sites (Fig. 6). The result suggests that

SMO flies had or have evolved sufficient diapause genetic vari-

ation, in combination with phenotypic plasticity, to adapt to the

Figure 6. Eclosion curves for flies from Mexican EVTM (CJ) and

SMO (SJ, CB, and CP) populations.

later phenology of C. mexicana, assuming that the fly was also

introduced into the Sierra de las Altas de Chiapas.

DiscussionThe findings from the current study help clarify several points con-

cerning the differential introgression hypothesis for R. pomonella

and provide further insight into the concept of speciation mode

plurality. At least three different populations of hawthorn-infesting

pomonella flies appear to exist in North America. In addition to

the two previously known populations in the eastern United States

and EVTM of Mexico (Feder et al. 2003a, 2005), a third geneti-

cally and ecologically distinguishable population also resides in

EVOLUTION MAY 2007 1101

XIE ET AL.

the SMO of Mexico. The genetic differences observed between

flies from the EVTM and SMO corresponded to variation in pu-

pal body weight observed among sites (Rull et al. 2006; Fig. 1),

suggesting that morphological as well as genetic differences exist

between these two populations.

The two Mexican populations appear to be primarily differ-

entiated by geography, diapause traits related to hawthorn fruiting

time, and body size, rather than host specificity. This contrasts with

the pattern seen for fly populations in the United States, where

host discrimination and diapause-related adaptations to host phe-

nology have together generated a diversity of host-specific sibling

species and races in sympatry (Berlocher et al. 1993; Berlocher

2000). (Note, however, that the scale of comparison is not equiv-

alent between the countries. In the United States, the host plants

attacked by R. pomonella represent different genera and families,

whereas in Mexico the hosts are different Crataegus species. In the

northeast United States, there is no evidence for different races of

R. pomonella on different hawthorn species, although we cannot

rule out this possibility in the southern United States.) Eclosion ex-

periments conducted in the current study indicate that the diapause

differences between the EVTM and SMO populations correspond

to differences in the fruiting phenologies of their respective host

plants. It is interesting that at one transition zone site between the

EVTM and SMO, flies infesting C. mexicana, one of the two hosts

used in the EVTM, but not found in the SMO proper, genetically

represent the SMO population. The same is also true for the iso-

lated CP population in Chiapas, where C. mexicana and the fly

may have been introduced. These results support the hypothesis

that host specificity among different hawthorn species may not be

strong in Mexico.

The phenology and phylogeography of the SMO population

imply that it may have been a conduit for gene flow between

Mexican EVTM and U.S. flies, bridging the about 1200-km gap

between the two populations. Our current working model is that

Mexican and U.S. fly populations have undergone repeated cycles

(≥ 2) of geographic isolation, contact, and differential introgres-

sion. Consistent with the hypothesis of fragmentation and periodic

contact, the SMO and EVTM populations currently abut through

parts of the Mexican states of Veracruz, Puebla, and Hidalgo (Rull

et al. 2006). Here, C. mexicana and C. rosei rosei co-occur with

C. rosei parrayana. Hawthorns identified as C. rosei rosei have

also been reported through much of the SMO. However, as dis-

cussed in Rull et al. (2006), the plant in this area likely represents

a different subspecies from the C. rosei rosei found on the EVTM,

as they differ in several morphological characters, including fruit

color and size. C. mexicana and C. rosei rosei in the transition

zone fruit earlier than they do on the EVTM and are infested by

fly larvae from late September to early November, with C. ro-

sei parrayana being infested from September to early October

(Rull et al. 2006). Thus, in the EVTM/SMO transition zone, host

species and host fruiting time overlap, creating the potential for

gene flow.

The extent to which the SMO population currently contacts

U.S. flies is not known. However, hawthorns are present in iso-

lated patches in southeastern New Mexico, and possibly the Davis

Mountains of Texas. In north-central Coahuila, the hawthorn C.

greggiana is found concentrated on lower north-facing slopes,

along streamsides, and in other locally mesic spots. Moreover, C.

greggiana is infested by R. pomonella in Mexico (Rull et al. 2006).

Although R. pomonella‘s current distribution does not necessarily

reflect its past geographic distribution, which likely shifted in the

Pleistocene during glacial and interglacial periods, the existence

of hawthorn flies along the SMO provides a possible mechanism

for the bridging of EVTM and U.S. populations at various times

in the past, when hawthorns were pushed further south in their

distribution during periods of northern glaciation. Further sam-

pling is needed in the northern range of the SMO population to

assess the degree of current contact with U.S. flies to determine

if a discontinuous genetic break actually exists or whether U.S.

flies represent the northern extension of SMO flies. If the latter

proves true, a result not inconsistent with the mtDNA data, then the

observed nuclear sequence differences in the current study may

be more accurately interpreted as geographic variation within a

semicontinuous SMO/U.S. population rather than reflecting di-

vergence between differentiated SMO and U.S. fly taxa.

A geographically disjunct population of hawthorn-infesting

pomonella flies now exists in the Sierra de las Altas de Chiapas

(represented by collection site CP in the current study), isolated

from other Mexican flies by the lowland forests of the Isthmus

of Tehuantepec. This population is genetically very similar to

SMO flies found to the north (SJ, CB, and PL sites), but the CP

population from the highlands of Chiapas has a different phenol-

ogy from the other SMO flies. In Chiapas, R. pomonella infests

C. mexicana from mid November through December and has an

eclosion curve intermediate between those of EVTM and SMO

flies from elsewhere. Hawthorns are not native to the Sierra de

las Altas de Chiapas, however. C. mexicana was likely introduced

into the region by Tlaxcaltecan Indians accompanying Spanish

conquistadors in the 1600s (Standley and Steyermark 1946). The

genetic similarity of flies from the SMO sites and CP flies sug-

gests that introductions of the plant and fly may have occurred

simultaneously, perhaps from the transition region between the

EVTM plateau and SMO where both occur. If true, then follow-

ing the introduction, the fruiting time of C. mexicana may have

been shifted later in the season in response to local conditions,

becoming similar in phenology to the plant on the EVTM. The

fly subsequently adapted by evolving a later eclosion life history

to match. This implies that the ancestral Chiapas fly population

possessed adequate diapause variation to track the changing phe-

nology of its host. Interestingly, this life-history shift does not

1102 EVOLUTION MAY 2007

MEXICAN RHAGOLETIS

appear to be reflected in significant genetic differentiation for the

CP population from the highlands of Chiapas for any of the 15

nuclear loci or mtDNA genes analyzed in the current study. It is

always possible that R. pomonella from Chiapas do not represent

an introduction, but an endemic population of more distant origin

in Central America that has experienced recent population bot-

tlenecks. Although we consider this scenario unlikely, additional

sampling of sites are needed to define the southern boundaries

of hawthorn-infesting flies in Central America and discount the

demographic hypothesis for Chiapas.

A DUAL ROLE FOR THE INVERSIONS IN SPECIATION?

In addition to their roles in facilitating sympatric host shifts, the in-

versions may also be serving as seeds fostering ongoing allopatric

divergence between Mexican and U.S. flies. The pattern of dif-

ferentiation across the nuclear genome among EVTM, SMO, and

U.S. populations varies in a manner consistent with reduced gene

flow for loci residing in the inverted regions of chromosomes

1–3. These three regions of the genome correlate with diapause

traits differentially adapting R. pomonella in the United States

to geographic and host-related variation in apple and hawthorn

fruiting time (Feder and Bush 1989; Feder et al. 1993, 1997a, b,

2003b; Filchak et al. 2000). These genes display greater diver-

gence among EVTM, SMO, and U.S. populations than loci in

putative collinear regions of the genome on chromosomes other

than 1–3. The reduced differentiation for collinear loci is not due to

an inherently slower substitution rate for these genes. Nucleotide

substitution rates between R. pomonella flies and the outgroup

species R. electromorpha are as great as or greater for these loci

as they are for loci on chromosomes 1–3 (mean Tamura-Nei ge-

netic distance as calculated by Mega, version 3.1, for chromo-

somes 1–3 loci = 0.051 ± 0.007, n = 9, range 0.0212–0.0895;

for nonchromosome 1–3 loci = 0.056 ± 0.006, n = 6, range

0.0331–0.718). Moreover, no locus deviated significantly from

a molecular clock. Rather, the lack of population differentiation

suggests more extensive and recent gene flow for collinear versus

rearranged loci among EVTM, SMO, and U.S. flies. Under this

scenario, past genetic introgression between Mexican and U.S. fly

populations generated the adaptive latitudinal inversion clines for

chromosomes 1–3 seen in the United States. However, subsequent

genetic changes that accumulated within the inversions, as well as

possible additional rearrangements involving these regions, have

since reduced their potential to introgress between Mexico and the

United States, consistent with models of chromosomal speciation

proposed by Rieseberg (2001), Noor et al. (2001a), Navarro and

Barton (2003), and Kirkpatrick and Barton (2006). Thus, even

when considering just the SN class of haplotypes, mean overall

genetic distances still tended to be greater among EVTM, SMO,

and U.S. populations for chromosome 1–3 than nonchromosome

1–3 loci (EVTM to SMO = 0.353 ± 0.0591 vs. 0.152 ± 0.018;

EVTM to United States = 0.368 ± 0.0520 vs. 0.192 ± 0.043;

SMO to United States = 0.245 ± 0.031 vs. 0.171 ± 0.032). The

reduced recombination associated with the rearrangements may

therefore accentuate the isolating effects of the inversions above

the genic consequences of their component loci considered alone.

Moreover, in addition to their role in adaptively differentiating R.

pomonella complex flies in the United States, the inversions may

also be facilitating population divergence on a broader geographic

scale among EVTM, SMO, and U.S. flies.

MtDNA displays a different pattern of genetic differentiation

than either the putative collinear or inverted regions of the nuclear

genome. For mtDNA, the SMO and U.S. populations are very

similar and differ from the EVTM. The pattern implies that mi-

tochondrial introgression has been inhibited between the EVTM

and the other populations since the original isolation of EVTM

flies about 1.57 million years ago (Feder et al. 2003a). However,

mtDNA gene flow appears to be extensive between SMO and U.S.

flies, suggesting that the populations may have only recently sep-

arated or that gene flow is ongoing. The cause for the apparent

impermeability of mtDNA to gene flow between EVTM versus

SMO and U.S. flies remains to be determined, with possible ex-

planations including differential migration of males, cytonuclear

gene interactions, and cytoplasmic incompatibilities due to en-

dosymbionts, such as Wolbachia.

ConclusionsThe discovery of the SMO fly population in Mexico supports

an emerging view of speciation mode plurality. For R. pomonella,

and perhaps many other organisms, allopatric versus nonallopatric

speciation modes may not be as dichotomous and mutually exclu-

sive categories of population divergence as they seem. Rather,

our results suggest that speciation is simultaneously unfolding

across different spatial and host-related axes in R. pomonella,

interconnected by considerations of life-history adaptation, host

specificity, genetic architecture, and gene flow.

In Mexico, geography appears to have contributed to initial

population divergence in a similar manner that host-specific mat-

ing plays in the United States. However, the physical isolation

of Mexican fly populations has not always been complete. In the

past, a portion of the diapause variation that evolved in the EVTM

and was associated with inversion polymorphism appears to have

introgressed via the SMO fly population into what is now the U.S.

population. The inversion polymorphism subsequently played a

complimentary role, when it became coupled with host discrim-

ination traits and ecologically different plant niches, in fostering

shifts to novel hosts and sympatric race formation/speciation in the

United States. Moreover, since the period of initial introgression,

rearranged regions of the genome appear to have evolved addi-

tional changes making them less permeable to gene flow. As a

EVOLUTION MAY 2007 1103

XIE ET AL.

result, Mexican and U.S. fly populations display a mosaic pattern

of differentiation across the genome, with inverted regions on

chromosomes 1–3 showing higher levels of divergence than pu-

tative collinear loci mapping elsewhere. Under this scenario, the

inversions served as focal points for the accumulation and reten-

tion of host and nonhost-related traits contributing to isolation (“is-

lands of speciation”; Turner et al. 2005). Reduced rates of recombi-

nation for rearrangements decreased the permeability of the result-

ing linked blocks of interacting genes contained within them to in-

trogress. Hence, these barriers to gene flow are maintained despite

genetic exchange, forming a foundation for possible speciation.

Several questions still need to be resolved concerning the

transvolcanic EVTM, SMO, and U.S. hawthorn flies and the dif-

ferential introgression hypothesis. Reciprocal crosses are needed

to determine whether EVTM, SMO, and U.S. flies can currently

hybridize (an indicator of the potential for gene flow past and

present), whether any nonhost related barriers to gene flow now

exist among the flies, and whether these populations represent

different species. The pattern of genetic variation suggests that if

such nonhost reproductive barriers exist, then they are likely to

be most pronounced between EVTM versus SMO and U.S. flies.

It would be interesting to determine whether the genes respon-

sible for any observed prezygotic isolation or inherent genomic

incompatibilities disproportionately map within the inversions on

chromosomes 1–3, as predicted by chromosomal speciation mod-

els. The paleobiology of Mexico and the United States must also

be further investigated to determine whether the distributions of

co-occurring fauna and flora, as well as environmental conditions,

are consistent with the historical hypothesis for introgression pro-

posed for R. pomonella.

In conclusion, our study underscores the need for a fully

resolved biogeography to study speciation, even in cases when

the proximate causes of divergence appear to be sympatric and

ecological in nature. We are not arguing to abandon the terms

sympatry or allopatry. Rather, we are advocating thinking beyond a

simple sympatric versus allopatric dichotomy for speciation mode.

The factors contributing to divergence may arise and evolve under

a variety of different geographic circumstances that do not always

neatly fit into sympatric versus allopatric categories.

ACKNOWLEGMENTSThe authors would like to thank S. Berlocher, A. Birke, O. Brunel,H. Dambroski, K. Filchak, D. Garcio, L. Guillne, and Z. Wang for assis-tance and moral support, as well as two anonymous reviewers for construc-tive criticisms and insightful comments for improving the manuscript. Inaddition, we are grateful for M. Pale’s help in collecting flies from Mexico.This research was supported by grants to JLF from the National ScienceFoundation, the U.S. Department of Agriculture and the State of Indiana21 Century Fund and to MA and JR from the Mexican Campana Na-cional Contra Moscas de la Fruta and CONACyT convenio 1100/596/04c-837-04.

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