-
Incipient speciation revealed in Anastrepha fraterculus(Diptera;
Tephritidae) by studies on matingcompatibility, sex pheromones,
hybridization,and cytology
CARLOS CCERES1, DIEGO F. SEGURA2, M. TERESA VERA3,VIWAT
WORNOAYPORN1, JORGE L. CLADERA2, PETER TEAL4,PANAGIOTIS
SAPOUNTZIS5, KOSTAS BOURTZIS5, ANTIGONE ZACHAROPOULOU1,6
and ALAN S. ROBINSON1*
1Joint FAO/IAEA Programme of Nuclear Techniques in Food and
Agriculture, International AtomicEnergy Agency, IAEA Laboratories,
A-2444 Seibersdorf, Austria2Laboratorio de Gentica de Insectos de
Importancia Econmica, IGEAF, INTA Castelar, Los Reserosy Las
Cabaas, Castelar (1712), Buenos Aires, Argentina3Estacin
Experimental Agroindustrial Obispo Colombres, William Cross 3150,
Las Talitas (4101),Tucumn, Argentina4Center for Medical,
Agricultural, and Veterinary Entomology, US Department of
AgricultureAgricultural Research Service, 16001700 SW 23rd Drive,
Gainesville, FL, USA5Department of Environmental and Natural
Resources Management, University of Ioannina, 2 SeferiStreet, 30100
Agrinio, Greece6Department of Biology, University of Patras, Patras
26500, Greece
Received 7 October 2008; accepted for publication 9 October
2008
It has long been proposed that the nominal species Anastrepha
fraterculus is a species complex and earlier studiesshowed high
levels of pre-zygotic isolation between two laboratory strains from
Argentina and Peru. Furtherexperiments were carried out on the same
populations and on their reciprocal hybrids, including pre-
andpost-zygotic isolation studies, pheromone analysis, and mitotic
and polytene chromosome analysis. A high level ofpre-zygotic
isolation had been maintained between the parental strains despite
3 years of laboratory rearing underidentical conditions. The level
of pre-zygotic isolation was reduced in matings with hybrids. There
were alsodifferences in other components of mating behaviour. There
were quantitative and qualitative differences in the sexpheromone
of the two strains with the hybrids producing a mixture. The
pre-zygotic isolation barriers werecomplemented by high levels of
post-zygotic inviability and sex ratio distortion, most likely not
due to Wolbachia,although there was evidence of some cytoplasmic
factor involved in sex ratio distortion. Analysis of
polytenechromosomes revealed a high level of asynapsis in the
hybrids, together with karyotypic differences between theparental
strains. The combined results of the present study indicate that
these two strains belong to differentbiological entities within the
proposed A. fraterculus complex. 2009 The Linnean Society of
London, BiologicalJournal of the Linnean Society, 2009, 97,
152165.
ADDITIONAL KEYWORDS: cryptic species hybrid incompatibility
pre-zygotic/post-zygotic isolation polytene chromosomes
speciation.
INTRODUCTION
Most biologists accept that speciation is a continuousprocess by
which genetic variation becomes se-
gregated between populations. Within Diptera, theTephritidae
family is an interesting field for evolu-tionary studies because
species complexes have beenidentified and cases of sympatric
speciation, hostshifts, and host race formation have been
documented*Corresponding author. E-mail: [email protected]
Biological Journal of the Linnean Society, 2009, 97, 152165.
With 7 figures
2009 The Linnean Society of London, Biological Journal of the
Linnean Society, 2009, 97, 152165152
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(Feder et al., 2003; Linn et al., 2003). Many of thesecases
involve species of great economic significance,providing an
important interface between basic andapplied research.
The South American fruit fly Anastrepha fraterculus(Wiedemann)
is a case in point. It is highly polypha-gous (Norrbom, 2004) with
a distribution from south-ern USA to Argentina (Steck, 1999). Early
studiesshowed population differences in host preference(Malavasi
& Morgante, 1983), karyotypes (Bush,1962), isozymes (Morgante,
Malavasi & Bush, 1980),and morphology (Stone, 1942) and
subsequent studieson hybridization (Santos, de Uramoto &
Matioli, 2001),egg morphology and embryonic development
(Selivon,Morgante & Perondini, 1997; Selivon &
Perondini,1998; Selivon, Vretos & Perondini, 2003),
mito-chondrial DNA (Smith-Caldas et al., 2001), highlyrepetitive
DNA (Rocha & Selivon, 2004), matingcompatibility (Vera et al.,
2006), and morphometrics(Hernandez-Ortiz et al., 2004) have
suggested that thenominal species A. fraterculus is a species
complex (fora revision, see Steck, 1999; for additional
discussion,see Hernandez-Ortiz et al., 2004, Selivon, Perondini
&Morgante, 2005; Goday et al., 2006). Studies on repro-ductive
isolation revealed both pre- and post-zygoticmechanisms (Selivon,
Perondini & Morgante, 1999,2005; Vera et al., 2006).
Sex pheromones play a key role in mate recognitionand mating in
Anastrepha (Nation, 1989) and theymay play a role in pre-zygotic
isolation. In the Bac-trocera dorsalis Hendel complex, there are
distinctdifferences between Bactrocera carambolae Drew andHancock
and Bactrocera papayae Drew and Hancockin the volatile components
of the male rectal gland(Perkins et al., 1990); however, this does
not preventhybridization in the field (Wee & Tan, 2005).
Post-zygotic isolation can limit gene flow betweenhybridizing
populations and this can lead to reducedfitness of the hybrids
(Burke & Arnold, 2001) andsex ratio distortion (Haldane, 1922),
which are bothconditions already demonstrated in A.
fraterculus(Selivon et al., 1999). Hybridization can also
revealphenotypes resulting from interactions between
dif-ferentiated regions of the nuclear genome and/orinteractions
between the nuclear genome and cyto-plasmic components (Burke &
Arnold, 2001). In somecases, symbiotic bacteria such as Wolbachia
are thesole determinant of hybrid sterility (Bourtzis &
Braig,1999). Wolbachia has been found in Brazilian popu-lations of
A. fraterculus and was suggested as a causeof reproductive
isolation (Selivon et al., 2002, 2005).Polytene chromosome analysis
can help to identifyspecies complexes (Coluzzi et al., 2002) and
previousobservations in A. fraterculus revealed
significantchromosomal polymorphism at the karyotypic level(Bush,
1962; Solferini & Morgante, 1987; Goday et al.,
2006). However, the polymorphism reported in Argen-tina
populations (Basso & Manso, 1998) was notassociated with
speciation because only one biologicalentity occurs (Alberti et
al., 2002).
The present study comprises a multi-disciplinaryapproach
involving studies on, pre- and post-zygoticisolation, male sexual
pheromones, cytology, and Wol-bachia in two laboratory strains of
A. fraterculus. Theresults obtained provide additional evidence
that fullysupports and strengthens earlier suggestions for
thisspecies being composed of an unknown number ofcryptic
species.
MATERIAL AND METHODSSTRAINS
The Argentina strain was derived from pupaesent from the Estacin
Experimental AgroindustrialObispo Colombres, Tucumn, Argentina and
the Perustrain from pupae sent from the La Molina facility,Lima,
Peru (for details of the history of the strains,see Vera et al.,
2006). Both strains were identified asA. fraterculus by Dr R.
Zucchi and Dr V. Hernandez-Ortiz. F1 hybrids were obtained from
Argentina malesmated with Peru females (HAP) and from the
recipro-cal cross (HPA). In addition, a hybrid inbred strain
wasestablished in 2005 by mating HPA females with HAPmales. Flies
from Piracicaba, Brazil, as well as from asingle population from
South America of unknownorigin, were also analysed for
Wolbachia.
PRE-ZYGOTIC ISOLATION STUDIES
Field cage experiments (FAO/IAEA/USDA, 2003) wereperformed in
Seibersdorf, Austria. In the bisexualtest, 25 virgin males from
each strain were releasedinto the cage within 1 h after sunrise,
and 15 minlater, 25 virgin females from each of the two strainswere
released. In the unisexual test, 25 femalesfrom one strain were
released in the cage together with25 males of two strains. Matings
were observed andthe type of male and female was identified,
matingduration was noted, as well as the time from therelease of
the females to the beginning of mating(i.e. latency).
Isolation was measured using the Index of SexualIsolation (Cayol
et al., 1999) and departures from zero(indicating nonrandom mating)
were evaluated usinga chi-square test of independence (bisexual
tests) andgoodness of fit (unisexual tests). Eight replicates
wererun for the parental tests and five for the hybrid
tests.Heterogeneity among replicates was assessed by achi-square
test (Zar, 1996). In bisexual tests, differ-ences in latency were
analysed using nonparametricanalysis of variance (ANOVA)
(KruskalWallis test)and differences in mating duration using a
one-wayANOVA. In unisexual tests, t-tests were used. For
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both variables, data from all cages within each testwere pooled.
Statistical analyses were performed withSTATISTICA (StatSoft,
2000).
POST-ZYGOTIC ISOLATION STUDIES
Reciprocal crosses were carried out in the laboratorybetween the
two parental strains with approximately50 virgin flies of each sex.
Eggs were placed on alarval diet and egg hatch, percent pupation,
percentadult emergence, and the sex ratio of the F1 adultswere
noted. F1 virgin hybrid males and females werebackcrossed to the
parental strains and also inbred.Eggs were placed on a larval diet
and egg hatch,percent pupation, percent adult emergence, and thesex
ratio of the F2 adults were noted.
CYTOLOGY
Mitotic metaphase spreads were from third-instarlarval
neuroblasts (Zacharopoulou, 1987) followed byC-banding (Selivon
& Perondini, 1997). Larvae fromthe two parental strains, F1
hybrids and the hybridstrain at generation 20, were analysed. For
eachstrain, more than 20 larvae were analysed. Polytenechromosome
preparations were from third-instarlarval salivary glands
(Zacharopoulou, 1987). Morethan 50 larvae were used from each
strain.Metaphase spreads and well spread polytene nucleiwere
photographed on negative film (100 ASA) at100 magnification using a
phase contrast Leitzmicroscope. Photographs were edited using
MicrosoftPicture Manager.
COLLECTION AND ANALYSIS OFVOLATILE PHEROMONES
Volatiles were collected between 05.30 h and 13.00 hunder
natural light conditions (Teal, Gomez-Simuta &Proveaux, 2000).
The traps containing volatiles wereeluted with methylene chloride
containing 1 ng mL-1of n-tetradecane (internal standard) and
analysedchemically using a HP-5890 gas chromatograph(GC) equipped
with EC-1 and EC-5 columns (both30 m 0.25 mm inner diameter 0.25 mm
film thick-ness; Alltech Associates), cool-on-column injectorsand
flame ionization detectors (Teal, Gomez-Simuta &Meredith,
1999). The oven temperature was pro-grammed from 40 C (hold for 4
min) to 210 C (EC-1)or 200 C (EC-5) at 10 C min-1. The identities
ofcompounds were confirmed by both chemical (isobu-tane reagent
gas) and electron impact mass spectros-copy using a HP 5890 GC
interfaced to a 6890 massspectrometer (MS). The GC had a
cool-on-columninjector and a 30 m 0.25 mm inner diameterDB-1MS
capillary column (J&W Scientific Inc.) as theanalytical column
and using the same conditions
used for GC-flame ionization detector (FID) analyses.Authentic
synthetic samples including isomers,obtained from the CMAVE
chemical collection, wereused to calculate retention indexes for
FID and MSanalyses and for determination of MS
fragmentationpatterns (Teal et al., 1999).
WOLBACHIA ANALYSIS
DNA was extracted from whole insects (Nirgianakiet al., 2003)
and the wsp gene amplified using stan-dard primers (Zhou, Rousset
& ONeill, 1998). Poly-merase chain reaction (PCR) products were
analysedon 1% agarose gels, stained with ethidium bromide,digested
with AluI (Minotech) and the restrictionproducts separated on 2%
agarose gels. Purified wspgene PCR products were cloned into pGEM
T-easyvector and then transformed into Escherichia coliDH5a
competent cells. All sequencing reactions werecarried out in
Macrogen (http://www.macrogen.com)using T7 and SP6 universal
primers. Direct sequenc-ing was performed using the wsp-specific
primers(Zhou et al., 1998).
The phylogenetic relationship among Wolbachiastrains was
analysed using the wsp nucleotidesequences but excluding the
hypervariable regions(Baldo, Lo & Werren, 2005). The sequences
werealigned using the CLUSTALW multiple SequenceAlignment Program,
version 1.81 (Higgins, Thomp-son, Gibson, Thompson & Higgins et
al., 1994). Thephylogeny test was performed with bootstrap
analy-sis, whereas tree inference was determined using
theNeighbour-joining method. The substitution modelwas the
JukesCantor and all the sequences werenucleotide-coding. The tree
was constructed withMEGA, version 3.1.
RESULTSPRE-ZYGOTIC ISOLATION
There were high levels of pre-zygotic isolationbetween the
parental strains in the bisexual andunisexual tests as shown by the
index of sexual iso-lation (ISI) values (Table 1). Although the
mean ISIwas lower in the unisexual tests with Argentinafemales than
with Peru females, the difference wasnot significant (t = 1.911;
d.f. = 14; P = 0.078). ForF1 hybrids, the level of pre-zygotic
isolation wasreduced with random mating in three cases (Table
1).However, Argentina females still significantly pre-ferred
Argentina males to HAP males in two out ofthe five replicates (c2 =
3.83; P < 0.05), although thefive replicates were homogeneous
(c2 = 4.93; d.f. = 4;P > 0.05).
In bisexual tests, matings between Peru malesand females had
significantly longer latency periods
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(Table 2) (KruskalWallis test: H = 87.461; d.f. = 3;N = 252; P
< 0.001; and Dunns multiple comparisons;P < 0.001). In the
unisexual tests, the type of male didnot affect the latency, either
for Argentina (t = 1.486;d.f. = 145; P = 0.115) or Peru (t = 0.514;
d.f. = 120;P = 0.608). When data within tests were pooledwithout
considering the origin of the male, there weredifferences in
latency between Argentina and Perufemales (MannWhitney U-test =
1453, P < 0.001)with Peru females mating much later in the
day(Table 2).
For hybrids, there were no differences in latencybetween males
within tests, except for Peru femaleswith Peru and HPA males (Table
2) where homotypicmatings started earlier than heterotypic (t =
2.23;d.f. = 71; P = 0.029). Comparison of latency amongtests showed
significant differences (KruskalWallistest: H = 102.98; d.f. = 3; N
= 301; P < 0.001) evenbetween tests that involved females from
the samestrain. Tests with HPA males had shorter latency thanthose
with HAP males and significantly so for Perufemales (Dunns test, P
< 0.05). Tests involving
Table 1. Mating percentage (PM) and index of sexual isolation
(ISI) in bisexual and unisexual field cage tests for twostrains of
Anastrepha fraterculus from Peru and Argentina (Arg) and F1
hybrids
Test Males Females PM ISI c2 N
Bisexual Arg Peru Arg Peru 63.0 2.1 0.77 0.05 142.74***
8Unisexual Arg Arg Peru Arg 73.5 2.3 0.73 0.05 76.96*** 8Unisexual
Peru Arg Peru Peru 61.0 3.6 0.86 0.04 88.66*** 8Unisexual HAP Arg
Arg HAP Arg 63.2 5.4 0.30 0.12 6.70** 5Unisexual HPA Arg Arg HPA
Arg 55.2 4.3 0.15 0.11 1.17 5Unisexual HAP Peru Peru HAP Peru 58.4
6.9 0.10 0.10 0.67 5Unisexual HPA Peru Peru HPA Peru 64.0 8.0 0.13
0.09 1.80 5
HAP, F1 hybrid from matings between Argentina males and Peru
females.HPA, F1 hybrid from matings between Peru males and
Argentina females.Chi-square values are after pooling data from all
replicates in each test. **P < 0.01; ***P < 0.001.Replicates
within tests were homogeneous (c2 test of homogeneity; P <
0.05).
Table 2. Mean values of latency and mating duration for two
strains of Anastrepha fraterculus from Peru and Argentina(Arg) and
F1 hybrids
TestMating combination( ) Latency (min) Duration (min)
Bisexual Arg Arg 32.7 4.6 (147)*a 67.6 4.4 (147)*a
Peru Arg 48.5 36.2 (10)a 68.4 20.3 (10)a
Arg Peru 31.5 15.4 (19)a 65.1 7.0 (19)a
Peru Peru 216.8 15.9 (76)b 49.3 4.9 (76)a
Unisexual Arg Arg Arg 17.8 2.1 (127)a 67.0 2.6 (127)a
Arg Peru 25.6 5.9 (20)a 56.8 4.6 (20)a
Unisexual Peru Peru Arg 132.0 32.3 (9)a 28.0 2.4 (9)a
Peru Peru 150.3 10.5 (113)a 34.0 1.4 (113)a
Unisexual HAP Arg Arg Arg 112.6 6.5 (51)a 62.0 7.0 (35)a
Arg HAP 122.1 3.2 (28)a 68.8 4.4 (24)a
Unisexual HPA Arg Arg Arg 47.0 6.5 (39)a 57.7 7.0 (39)a
Arg HPA 40.4 5.1 (30)a 53.8 5.8 (30)a
Unisexual HAP Peru Peru Peru 132.8 4.5 (40)a 70.7 5.0 (30)a
Peru HAP 173.1 3.9 (33)b 68.0 3.3 (21)a
Unisexual HPA Peru Peru Peru 73.8 6.8 (46)a 41.1 9.5 (45)a
Peru HPA 74.8 5.3 (34)a 47.5 6.0 (34)a
HAP, F1 hybrid from matings between Argentina males and Peru
females.HPA, F1 hybrid from matings between Peru males and
Argentina females.Means followed by the same superscript letter
within each test are not statistically different (P >
0.05).*Figures in brackets refer to the number of matings.
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Argentina females had lower values than thoseinvolving Peru
females.
In bisexual tests, there were no differences inmating duration
(Table 2) (F = 2.417; d.f. = 248;P = 0.067). In unisexual tests,
there was no effectof the type of male on mating duration (t =
1.516;d.f. = 145; P = 0.132; and t = 1.248; d.f. = 120;P = 0.214,
for Argentina and Peru, respectively) andArgentina females mated
longer than Peru females(t = 13.094; d.f. = 267; P < 0.001). In
the unisexualtests with hybrid males, mating duration
involvingparental or hybrid males did not differ (t-test:P >
0.05) (Table 2) and there were no differencesbetween females.
However, there was an effect of thehybrid involved and the
interaction between thesetwo factors was also significant [F
(female) = 0.943;d.f. = 254; P > 0.05; F (type of test) =
20.983; d.f. =254; P < 0.001; F (interaction) = 5.118; d.f. =
254;P = 0.025]. Matings with HPA males were shorter thanmatings
with HAP males but only statistically so for
Peru females (69.6 5.2 min for HAP males versus43.8 3.1 min for
HPA males; Tukeys test, P < 0.001).
POST-ZYGOTIC ISOLATION
All crosses produced some viable progeny with ahigh adult
emergence (Table 3). In four crosses,Arg Peru, HPA HAP, Arg HAP,
and HPA Peru,there was a significant reduction in egg hatch
com-bined with a reduced larval viability, although thelatter was
not statistically significant. The reducedegg hatch is restricted
to specific crosses where thefemales were either from Argentina or
HPA hybridsand the males were either from Peru or HAP hybrids(i.e.
males from a cross between Peru females andArgentina males),
suggesting a possible maternaleffect of Peru females. In addition,
one of thesecrosses, HPA HAP, showed a sex ratio distortion
infavour of females, as was also observed in the Hybridstrain
(Table 3). A similar sex ratio distortion was
Table 3. Mean SE (%) for egg hatch, larval survival, egg-pupal
survival, adult emergence, and sex ratios for matingsbetween two
strains of Anastrepha fraterculus from Peru and Argentina (Arg) and
their F1 hybrids
Number and cross( )
Egg hatch
Larvalsurvival
Egg-pupalsurvival
Adultemergence
Sex ratio(/)
Numberof eggs Hatch*
1 Peru Arg 4000 81 6b 87 7a 70 5a 98 1b 0.98 0.04c
2 Arg Peru 4000 27 3c 84 14a 22 3d 97 1c 1.02 0.32c
3 Arg Arg 6000 83 6b 87 5a 75 3a 96 2b 0.97 0.17c
4 Peru Peru 6000 80 3b 85 7a 72 4a 98 1b 0.99 0.12c
5 HPA HPA 3169 78 5b 87 4a 68 4a 95 7a 1.01 0.20c
6 HPA HAP 4448 47 6d 75 9a 36 6c 92 4a 1.64 0.20b
7 HAP HPA 3361 80 4b 80 15a 64 21a 84 12a 0.98 0.10c
8 HAP HAP 2531 72 9b 91 5a 65 9a 92 4a 0.74 0.10d
9 Arg HPA 3160 79 6b 85 15a 67 12a 97 1a 0.97 0.01c
10 Arg HAP 3154 55 6d 90 5a 50 7b,c 97 1a 0.88 0.01d
11 Peru HPA 3011 92 4a 87 12a 81 14a 93 8a 1.00 0.20c
12 Peru HAP 3747 85 3a,b 79 15a 67 21a 80 5a 1.03 0.10c
13 HPA Arg 4080 74 1b 68 24a 50 22b 86 12a 2.33 0.20a
14 HAP Arg 3043 89 2a,b 88 16a 78 15a 96 3a 0.93 0.01d
15 HPA Peru 5470 27 3e 76 15a 20 4d 93 2a 1.00 0.20c
16 HAP Peru 4618 70 5b 88 11a 62 9b 95 5a 0.86 0.10d
Hybrid 3000 34 9 57 12 19 2 91 5 2.81 0.38
F, female; M, male.HAP, F1 hybrid from matings between Argentina
males and Peru females.HPA, F1 hybrid from matings between Peru
males and Argentina females.Means in the same column followed by
the same superscript letter are not statistically different.*(F =
77.41, P = 0.00).(F = 0.82, P = 0.64).(F = 9.53, P = 0.00).(F =
2.02, P = 0.027.(F = 31.48, P = 0.00).
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observed in the cross, HPA Arg, together with anegg-to-pupae
survival of only 50%. The progeny inthese last two crosses (as in
the hybrid strain) havetwo common characteristics: Argentina
cytoplasm andthe male sex chromosome complement is expected tobe
XAYA, XPYA (Table 4).
CYTOLOGY
Mitotic chromosomesThe karyotype of the nominal A. fraterculus
has sixpairs of acrocentric chromosomes, including one pairof
highly heterochromatic sex chromosomes with themale being XY, as
shown by Giemsa and C-banding(Fig. 1). However, the two strains can
be differenti-ated by size and morphology of sex chromosomes
afterGiemsa staining and C-banding. The Argentina strainhas a large
X-chromosome with two prominentC-bands located at the two tips, one
being larger thanthe other. The Y-chromosome is smaller than
theX-chromosome and also shows two C-bands, one onthe proximal tip
and the other in the sub-medianregion (Fig. 1A). In the Peru
strain, both X- andY-chromosomes are large and similar in size
(Fig. 1B).The X-chromosome, at least in early metaphase or inless
condensed chromosomes, exhibits a large gap(Fig 1D, F),
approximately two-thirds from one end,surrounded by two C-bands.
However, in more
condensed chromosomes, only one large C-bandwas observed. In
some metaphases, two types ofX-chromosome were observed: one with
one largeC-band and the second with a gap and, subsequently,two
C-bands, although smaller than the unique one.The Y-chromosome has
a large C-band at the proxi-mal end in both condensed and early
metaphasespreads. These differences between the two parentalstrains
can also be seen in the hybrids (Fig 1C, D, E).
In the hybrid strain, the expected sex chromosomecytotypes were
XAXP, XPXP, XAYA, XPYA plus XAXA
(Table 4) and some of these genotypes were observedbut not XPYA
and XPXP. However, XAXA female larvaeas well as larvae with 13
chromosomes, either XXXor XXY, were observed both with XA
chromosomes(Fig. 1F).
Polytene chromosomesThe polytene chromosomes show poor
banding,numerous weak points and inter- and intra-ectopicpairing.
The polytene complement consists of five longchromosomes, probably
corresponding to the fiveautosomes because sex chromosomes are
highly het-erochromatic, are not expected to form polytene
ele-ments as in other tephritids (Zhao et al., 1998).
The banding pattern between the two parentalstrains is very
similar, especially at the chromosomeends, making differentiation
difficult. Figure 2A
Table 4. Sex chromosome genotypes and cytotypes in offspring of
matings between Anastrepha fraterculus strains fromArgentina (Arg)
and Peru and F1 hybrids
No. and cross ( ) Parental genotypes Expected genotypes
Cytoplasm
1. Peru Arg XPXP XAYA XAXP, XPYA Peru2. Arg Peru XAXA XPYP XAXP,
XAYP Arg3. Arg Arg XAXA XAYA XAXA, XAYA Arg4. Peru Peru XPXP XPYP
XPXP, XPYP Peru5. HPA HPA XAXP XAYP XAXA, XAXP, XPYP, XAYP Arg6.
HPA HAP XAXP XPYA XAXP, XPXP, XAYA, XPYA Arg7. HAP HPA XAXP XAYP
XAXA, XAXP, XAYP, XPYP Peru8. HAP HAP XAXP XPYA XAXP, XPXP, XAYA,
XPYA Peru9. Arg HPA XAXA XAYP XAXA, XAYP Arg10. Arg HAP XAXA XPYA
XAXP, XAYA Arg11. Peru HPA XPXP XAYP XAXP, XPYP Peru12. Peru HAP
XPXP XPYA XPXP, XPXP, XPYA, XPYA Peru13. HPA Arg XAXP XAYA XAXA,
XAXP, XPYA, XAYA Arg14. HAP Arg XAXP XAYA XAXA, XAXP, XPYA, XAYA
Peru15. HPA Peru XAXP XPYP XAXP, XPXP, XAYP, XPYP Arg16. HAP Peru
XAXP XPYP XAXP, XPXP, XAYP, XPYP Peru17. Hybrid XAXP, XPXP, XAYA,
XPYA, XAXA Arg
HAP, F1 hybrid from matings between Argentina males and Peru
females.HPA, F1 hybrid from matings between Peru males and
Argentina females.Bold indicates crosses with reduced egg
hatch.Underlined indicates crosses with distorted sex ratio.Bold
and underlined indicates crosses with reduced egg hatch and
distorted sex ratio.Italics indicates genotypes not identified
cytologically.
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shows the same chromosome end for the two strainsand indicates
regions of homology and Fig. 2B showsanother chromosome arm. The
Argentina strainshows very little polymorphism; however, there
ispartial asynapsis (Fig. 2C) and many rearrange-ments, notably
inversions (Fig. 2D), in the Perustrain. F1 and F2 hybrids showed
extensive asynapsis(Fig. 3A, B, C), sometimes along the whole
chromo-some complement. Asynapsis is observed in almost
allchromosome ends in spite of significant similarities inbanding
pattern. Homologous chromosomes also differin size (Fig. 3D), as
well as in the presence of inver-sion loops (Fig. 3E, G) and
deletions (Fig. 3H), anddifferences in the puffing pattern in
asynaptic areas(Fig. 3B) indicate differences in transcription.
Therewas extensive asynapsis in the hybrid strain in the14th and
20th generations (Fig. 4), similar to thatobserved in the F1 and F2
hybrids. The deletionobserved in the F1 (Fig. 3H) was also observed
after20 generations of inbreeding (Fig. 4D).
PHEROMONE ANALYSIS
There were significant qualitative and quantitativedifferences
in the pheromone from Argentina andPeru males. Argentina males
produced small amountsof (E)-b-ocimene, (Z)-nonanal and larger
amounts of(Z)-3-nonen-1-ol, benzoic acid, suspensolide,
(Z,E)-a-
farnesene, (E,E)-a-farnesene, anastrephin and epi-anastrephin
(Fig. 5A), but no detectable amountsof (E)-a-bergamontene or
b-bisaboline. Peru malesproduced small amounts of limonene,
(Z)-nonanaland (Z)-3-nonen-1-ol along with relatively largeamounts
of (E)-b-ocimene, (E)-a-bergamotene, (E,E)-a-farnesene and
b-bisaboline along with suspensolide,anastrephin and epianastrephin
but no (Z,E)-a-farnesene (Fig. 5B). Nonanol and benzoic acid
arenovel compounds for Anastrepha species. Volatilesfrom the two
hybrid males were surprisingly similarin both the number and ratios
of compounds released(Fig. 6) and contained all of the compounds
identifiedin both parental strains. No statistical differences
inaverage amounts of compounds released per hour bythe parental
strains were found (t = 1.06, P > 0.05)and the ratios of
compounds released by males fromdifferent groups within each parent
strain were notdifferent. Thus, the ratio of pheromone
componentsreleased by each strain was unique and did not
varysignificantly, despite collection of samples from malesfrom
different dates.
WOLBACHIA
Seven individuals from the strain of unknown origin,12 from the
Piracicaba strain, and ten from theArgentina and Peru strains
carried Wolbachia based
Figure 1. Mitotic chromosome spreads from: Argentina (A); Peru
(B); HPA (C, D); HAP (E) and hybrid strain (F). Sexchromosomes, X
and Y, are indicated. Arrows in (D) and (F) show a gap.
158 C. CCERES ET AL.
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on the wsp-specific PCR assay (Zhou et al., 1998). Allflies
tested were positive. AluI-based restrictionfragment length
polymorphism (RFLP) analysis sug-gested that the strains from
Piracicaba, Argentinaand Peru were singly infected, whereas the
strain ofunknown origin was double infected (data not shown).
PCR products from the wsp gene from five malesand females from
the Piracicaba, Argentina, and Perustrains were directly sequenced,
as were cloned wspgene PCR products from two individuals from
thepopulation of unknown origin. The results confirmedthe PCR-RFLP
analysis with three strains beingsingly infected and one having a
double infection. Thesingly-infected populations carry Wolbachia
identicalto wSpt (A Wolbachia supergroup) naturally infect-ing
Drosophila septentriosaltans Magalhaes & Buck(Miller &
Riegler, 2006) (Fig. 7), with the only differ-ence being a
conserved substitution in position 658 inthe Argentina strain. The
doubly-infected strain isinfected with an A-supergroup Wolbachia
strainidentical to wAlbA naturally infecting the mosquitoAedes
albopictus (Skuse) (Zhou et al., 1998) and aB-supergroup strain
closely related to the wMa strainnaturally infecting Drosophila
mauritiana Tsacas &David (Zhou et al., 1998).
DISCUSSION
Major differences were demonstrated in behaviour,chemistry,
cytology, and genetics between two labora-tory strains of A.
fraterculus from Argentina andPeru. The differences are so
significant that the twostrains can be said to belong to different
biologicalentities. The present study strongly supports andextends
previous work which has provided an increas-ing body of evidence
that this nominal species is aspecies complex (Hernandez-Ortiz et
al., 2004; Selivonet al., 2004, 2005; Goday et al., 2006). Although
thiswork was conducteed using laboratory strains, it isunlikely
that these differences were caused by labo-ratory adaptation
because both laboratory popula-tions have been shown to be fully
compatible withtheir respective wild populations and the wild
popu-lations are incompatible with each other (Vera et
al.,2006).
The high level of pre-zygotic isolation found earlier(Vera et
al., 2006) was confirmed, and was unaffectedby 3 years of identical
laboratory rearing. A largecomponent of the pre-zygotic isolation
is due to dif-ferences in time of mating between the two
parentalstrains (Vera et al., 2006), which was again demon-
Figure 2. Polytene chromosome from: Argentina and Peru (A, B)
showing homologous regions at the chromosome ends;Peru (C, D)
showing asynapsis, (thin arrows) and chromosome inversion (thick
arrow).
SPECIATION IN A. FRATERCULUS 159
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strated. This pre-zygotic isolation can also be attri-buted to
the response of the females towards thesexual pheromone of the
males because major differ-ences in its composition were found
between the twotypes male.
In the unisexual tests with HPA males, neitherArgentina nor Peru
females showed a mating prefer-ence. However, for HAP males, the
Argentina femalesrejected them in the presence of Argentina males
intwo of the five replicates, whereas Peru femalesshowed no
preference. This asymmetric response isdifficult to explain but may
be related to the phero-mone composition of the hybrid males
because thereare some quantitative differences between them(Fig.
6). However, both types of male produced all ofthe parental
compounds and there were no statisticaldifferences among them.
Given that females respondto volatiles released by their own males,
it is likelythat parental females would not freely mate with amale
from the other strain or with hybrid males
because of the observed differences in pheromonecomposition
(Figs 5, 6). However, the field cage datasupport the first
assumption, but not the secondbecause only in one case (and only in
two out of fivereplicates) was nonrandom mating (favouring
theparental males over the hybrid males) found. Itshould be noted,
however, that these were unisexualtests and did not include hybrid
females. It is likelythat female hybrids would respond equally well
tovolatiles fron either type of hybrid male because theblends are
virtually identical, but they would beunlikely to respond to
volatiles released by parentalmales. Thus, if hybridization were to
occur in nature,the hybrids would be pheromonally isolated fromboth
parental strains and could constitute a newreproductively-isolated
population (rapid one stepspeciation). Pre-zygotic isolation was
also found incrosses involving a strain from Brazil (Vera et
al.,2006) and, again, male sexual pheromone may play arole because
Lima, Howse & de Nascimento (2001)
Figure 3. Polytene chromosomes from: HAP (A) showing asynapsis
(arrows) and (B) difference in puffing pattern (arrow);HPA (C)
showing asynapsis (arrows) and (D) difference in chromosome length
(arrow); F2 from HPA HPA (E) showingasynapsis (thick arrow) and an
inversion loop (thin arrow); F2 from HAP HAP; (F) showing asynapsis
(arrows); F2 fromHAP HPA (G) showing an inversion loop (arrow) and
hybrid strain (H) showing a deletion (arrow).
160 C. CCERES ET AL.
2009 The Linnean Society of London, Biological Journal of the
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identified several compounds in that strain that werenot present
in the strains from Argentina and Peru.
Argentina flies generally mated earlier than Peruflies,
especially the Argentina females. The one excep-tion to this is
Peru females mating with Argentinamales in the bisexual test.
However, in this test, onlyten matings were found in the eight
replicates. In thebisexual test, the duration of mating was
unaffectedby the strain but matings involving Peru flies,
inparticular Peru females, were shorter and this wasconfirmed for
the unisexual tests. The differences inmating duration may be
attributed to the time atwhich mating started (Vera et al., 2003).
For hybrids,the influence of the females disappeared and the typeof
male became important. Surprisingly, when thefemales faced a hybrid
of the same maternal origin,they behave as with the parental males
but, whenthey faced a hybrid with a different maternal origin,the
latency changed markedly. Females facing hybridmales with the
maternal background of the otherstrain may detect critical
differences in behaviourbecause the pheromones released by the two
types ofhybrid are almost identical.
Two phenotypes of relevance to post-zygotic isola-tion and hence
potential speciation were found,namely reduced egg hatch and sex
ratio distortion.However, the two phenotypes were not always
observed together in the same cross, suggesting thatthey could
have a different genetic basis. Reduced egghatch was observed in
four crosses in which femaleswere either from Argentina or were
hybrids fromArgentina females (HPA), whereas males were eitherfrom
Peru or were hybrids from Peru females (HAP).The reciprocal crosses
did not show reduced egghatch, indicating asymmetrical post-zygotic
isolation.The observed decrease in egg hatch could be due toeither
unfertilized eggs or embryonic lethality but,because all these
matings were carried out with largenumbers of flies in laboratory
cages, it is unlikely thatmany females remained unmated. The gross
asynap-sis in the hybrids, probably indicative of majorgenetic
differences between the strains, is the mostlikely the cause of
embryonic lethality. The asymme-try of the phenotype and together
with the fact thatidentical genotypes did not show reduced hatch
sug-gests some type of nuclearcytoplasmic interaction ispresent,
although probably not due to Wolbachia.
Sex ratio distortion was observed in cross 6(HPA HAP), cross 13
(HPA Arg), and in the hybridstrain (Table 4) with the expected
reduction in theproportion of males (Haldane, 1922). The
expectedprogeny from these two crosses and in the hybridstrain have
two common characteristics; they all haveArgentina cytoplasm and
the males have identical sex
Figure 4. Polytene chromosomes from the hybrid stain analysed at
the 20th generation after its establishment showingasynapsis (A, B,
C) and a heterozygous deletion (D) (arrow).
SPECIATION IN A. FRATERCULUS 161
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chromosomes (i.e. XAYA and XPYA) (Table 4). There areother
crosses producing males with the same sexchromosome genotype (Table
4) but these did notshow sex ratio distortion. A low viability of
XPYA
males due to an interaction between nucleus and thecytoplasm
could explain this sex ratio distortion.Indeed, individuals of this
chromosomal type werenever observed in larvae from the hybrid
strain. Thisobservation may be related to hybrid breakdownobserved
in the F2 and subsequent generations ofinter-specific or
inter-subspecific crosses (Burke &Arnold, 2001). Whatever the
explanation for the sexratio distortion, it contributes to
post-zygotic isolationbetween the strains. Coyne & Orr (1989)
demon-strated that Haldanes rule often results in a patternof
speciation where males from reciprocal crossesbetween two taxa show
sterility or inviability beforeany effect is observed in females,
indicating thatcomplete sterility/inviability in hybrids is
almostalways preceded by the inviability/sterility in malesonly.
Thus, according to Coyne & Orr (1989),Haldanes rule represents
an obligatory initial step inthe evolution of post-zygotic
isolation. Sex ratio dis-tortion was previously demonstrated by
Selivon et al.(1999) in crosses of between sp 2 females and sp
1males, with sp 1 males probably being the same asthe Argentina
strain used in the present study basedon karyotype analysis.
Selivon et al. (1999, 2005) werethe first to validate Haldanes rule
in A. fraterculus.
The two strains clearly belong to different biologicalentities
based on the mitotic karyotype and poly-tene chromosome analysis
because they (and theirhybrids) can easily be identified based both
on sizeand C-banding of sex chromosomes. The Argentinakaryotype has
previously been reported in naturalpopulations from Argentina
(Basso & Manso, 1998;Basso et al., 2003) and Brazil, referred
to as A. sp.1aff. fraterculus (Selivon et al., 2005; Goday et
al.,2006). The Peru strain has not been analysed previ-ously, but a
similar karyotype (based on C-banding)was reported in a sample from
Guayaquil, Ecuadorand referred to as A. sp.4 aff. fraterculus
(Selivonet al., 2004; Goday et al., 2006).
The extensive asynapsis in the polytene chromo-somes in hybrids
leaves no doubt that this resultsfrom inter-subspecific crosses as
shown in Drosophila(Madi-Ravazzi, Bicudo & Manzato, 1997;
Machado,Madi-Ravazzi & Tadei, 2006). It can be due to
minutechromosomal rearrangements, specific interactionsof genes
that determine chromosome synapsis(Dobzhansky & Tan, 1936) or
point mutations thatdisturb the identity of homologous loci
(Kerkis, 1936).In the present study, considerable asynapsis
wasobserved, even though the banding pattern of the twohomologues
was identical. The degree of asynapsisobserved, especially in F1
hybrids (Fig. 4), can be due
Figure 5. Comparisons of total ion chromatograms (elec-tron
ionization spectra) obtained from analysis of volatilescollected
from groups of five males of the Argentina strain(A) or Peru strain
(B). Compounds are: (1) limonene;(2) (E)-b-ocimene; (3)
(Z)-nonanal; (4) (Z)-3-nonen-1-ol;(5) benzoic acid (IS, internal
standard); (6) (E)-a-bergamontene; (7) suspensolide; (8)
(Z,E)-a-farnesene; (9)(E,E)-a-farnesene; (10) b-bisaboline; (11)
anastrephin; and(12) epianastrephin.
Figure 6. Comparisons of total ion chromatograms(electron
ionization spectra) obtained from analysis ofvolatiles collected
from groups of five hybrid HAP males (A)or hybrid HPA males (B).
Numbers indicate the compoundsdescribed in the legend to Fig.
5.
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2009 The Linnean Society of London, Biological Journal of the
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to significant genetic differentiation between the twostrains,
which is not restricted to chromosome struc-ture, but also includes
differences in gene activity, asindicated by differences in puffing
pattern of homolo-gous chromosomes in the asynaptic regions (Fig.
3B)(Zhimulev et al., 2004).
Introgression of genes between the two strains ispossible
because the hybrids are partially fertile. In ahybrid strain
created from a cross between homo-sequential D. mauritiana and
Drosophila simulansSturtevant, the proportion of fertile males
derivedfrom F1 females backcrossed to either parent gradu-ally
increased; reaching 91% within eight genera-tions, and this
proportion was stable at least for afurther ten generations (David
et al., 1976). Thebehaviour of the current hybrid strain is
different assignificant asynapsis and reduced egg hatch
wasmaintained for at least 20 generations of inbreeding.The
persistence of asynapsis is difficult to explain;however, the
consistently reduced egg hatch couldsuggest some form of balancing
selection is operatingwith heterozygous genotypes being favoured
overhomozygous genotypes. Genetic recombination mayalso be severely
reduced because the level of somaticpairing in polytene chromosomes
is correlated withmeiotic pairing of chromosomes necessary for
geneticrecombination (Evgenev, 1971).
The data presented here for two A. fraterculuslaboratory strains
clearly show high levels of pre- and
post-zygotic isolation, karyotypic and polytene chro-mosome
differences, and qualitative and quantitativedifferences in male
pheromones. Although each ofthese factors alone would be indicative
of incipientspeciation, taken together, they provide a very
strongcase for a taxonomic revision of this species complex,as
suggested previously (Selivon et al., 2005). Theimportance of this
species as a major quarantine pestof fruit crops in many countries
makes this revisionessential and urgent.
ACKNOWLEDGEMENTS
We are grateful to Mnica Alburqueque and RafaelGuilln (SENASA,
Peru) for providing the materialfrom Peru and Roberto Zucchi
(Universidade de SoPaulo, Brazil) and Vicente Hernandez-Ortiz
(Institutode Ecologa, A. C., Mexico) for the taxonomic
identi-fication of the specimens. We are grateful to DrJulio Walder
for providing the material from Brazil.We thank A. Islam, A. Sohel,
and T. Dammalage forexcellent technical support. The work on
Wolbachiawas supported in part by intramural funding of
theUniversity of Ioannina to K.B.
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