Neotropical Entomology ISSN: 1519-566X journal homepage: www.scielo.br/ne

Systematics, Morphology and Physiology A Morphometric and Molecular Study of Anastrepha pickeli Lima (Diptera: ) ZV Bomfim1, KM Lima2, JG Silva2, MA Costa2, RA Zucchi1 1Depto de Entomologia e Acarologia, Escola Superior de Agricultura Luiz de Queiroz, Univ de São Paulo, Piracicaba, SP, Brasil 2Depto de Ciências Biológicas, Univ Estadual de Santa Cruz, Ilhéus, BA, Brasil

Keywords Abstract Fruit , geometric and traditional morphometry, molecular phylogeny, This study investigated the level of morphometric and genetic cytochrome oxidase I (COI), variability among populations of Anastrepha pickeli Lima from several localities in , one locality in and one in . Correspondence Traditional and geometric morphometric analyses were used, as Roberto A Zucchi, Departamento de well as sequencing of a fragment of the cytochrome oxidase gene Acarologia e Entomologia, Escola Superior (COI). Six variables were measured from the aculeus for traditional de Agricultura Luiz de Queiroz - ESALQ/USP, 13418-900, Piracicaba, SP, Brasil; razucchi@ morphometric analysis and 14 landmarks from the right wing were esalq.usp.br used for geometric analysis, using 10 specimes/population. The aculeus tip length, aculeus width at the end of the cloaca opening, Edited by Takumasa Kondo – CORPOICA and the serrate part length contributed with 62.7% for grouping. According to the results from traditional morphometry, there was Received 9 February 2011 and accepted 31 March 2011 no significant difference, but the multivariate tests showed that thephylogenetic canonical analysis variables indicated were statistically that the populations significant, clustered indicating into a differencethree clades in andthe revealedwing conformation a high level among of genetic populations. variation Molecular within A. pickeli populations from various geographic regions. Anastrepha pickeli used in this study, showing incongruence among the methods used. populations differed among them according to the methods Introduction morphometry) and on the shape (geometric morphometry) variationMorphometric of several methods structures based have on the been size used(traditional in the Anastrepha seriousSpecies problemsidentification for the is implementation highly problematic of quarantine in some landmarks (Strauss & Bookstein 1982, Sklorz 1992). In restrictions, integrated groups pest and misidentificationsmanagement, and can other pose Anastrephaidentification, morphometry of some has been species used, by for establishing example, to analyze populations of the Anastrepha fraterculus complex is an ongoing need to explore additional morphological (Hernández-Ortiz et al 2004, Selivon et al 2005). andcontrol molecular programs characters (McPheron so 2000).as to moreTherefore, accurately there Besides morphometric analysis, molecular methods have been used in Anastrepha systematics to complement morphological and molecular methodologies will also morphological and ecological data to address questions improveelucidate our species basic identification. knowledge of theUtilization relationships of combined among the species within the various Anastrepha species groups variation, structured genetic variation, and phylogeny et al 1999, Norrbom et al 1999, Smith-Caldas (Silvasuch as2000, species Silva identification,& Barr 2008). Sequencing speciation, of geographic fragments et al 2001). of mitochondrial and nuclear genes has been used to (McPheron Neotrop Entomol 40(5): 587-594 © 2011 Sociedade Entomológica do Brasil 587 A Morphometric and Molecular Study of Anastrepha pickeli Bomfim et al make inferences about relationships among specimens, morphometry, the wing for geometric morphometry, and populations, and species (Avise 1994). the thorax and legs for the molecular analysis). It is known that some of what were considered widespread species such as A. fraterculus are in fact Traditional morphometry cryptic species complexes and Anastrepha pickeli Lima Ten females from each locality were used. The aculeus of could be an example of such a complex based on the each female was dissected and treated with 10% sodium morphological study by Canal (1997) (Norrbom et al hydroxide for 24h and then mounted on a microscope 1999). Anastrepha pickeli belongs to the spatulata group slide with glycerine. Aculei were placed ventral side up (Norrbom et al 1999), and is reported from and carefully oriented in a consistent level position. The to and Tobago, to , Brazil, and aculei were photographed with a camera attached to a (Norrbom 2004). In Brazil, it is recorded in ® software was used for image analysis. Five variables: L1 (Zucchi 2007), where it has received attention as it infests (aculeusstereomicroscope tip length), and L2 (widththe Motic of the Images aculeus Advanced apex at base3.2 15 states comprising all five Brazilian geographic regions the fruit of cassava (Manihot esculenta, Euphorbiaceae), of serrate part), L3 (distance between the cloaca opening consuming the pulp and the , causing losses of up to and base of serrate part), L4 (aculeus length at the cloacal 30% in fruit production (Lozano et al et al opening), and L5 (serrate part length) were measured (Fig 1996). Although cassava is most important as a root crop, 1). The aculeus length (variable L6) was measured under a 1983, Morgante breeding programs (Farias & Bellotti 2006). the Ineffect this study, of we predationinvestigated negatively the morphometric affects cassava and genetic variation among A. pickeli populations using 4 traditional and geometric morphometry and sequencing 6 of a fragment of the COI of this cassava pest. We aimed at verifying if A. pickeli, a specialist species, shows gene cryptic to improve speciation identification based on specimens collected in Bolivia, Brazil and Paraguay. 1 2 5

Material and Methods 7 Anastrepha pickeli 3 traps in Brazil, in the states of Bahia, Espírito Santo, Rio Fig 1 Aculeus apex of Anastrepha pickeli Grande do Norte and females Amazonas, were and collected also in Boliviain McPhail and with the landmarks. 1 = aculeus apex; 2 = end of the cloaca opening; Paraguay and preserved in frozen ethanol (Table 1). 3-4 = base of the serrate part; 5 = line showingfrom Montes the baseClaros, of MGthe Voucher specimens were deposited at the insect collection serrate part; 6-7 = line showing the end of the cloaca opening. L1 = 1-2, aculeus apex length; L2 = 3-4, width of the aculeus apex at at the Escola Superior de Agricultura “Luiz de Queiroz”, base of serrate part; L3 = 2-5, distance between the cloaca opening The same specimens were used and base of serrate part; L4 = 6-7, aculeus apex width at the end of for both morphometric studies (the aculeus for traditional the cloaca opening and; L5 = 1-5, serrate part length. USP, Piracicaba, SP, Brazil. Table 1 Colletion data and information on Anastrepha species used in this study.

Codes Species Collection sites Locations Altitudes (m) N1 N2 (morphometry) A. pickeli Teixeira de Freitas, BA, Brazil 18º06’S, 39º53’W 186 13 10 APBA Linhares, ES, Brazil 19º23’S, 40º04’W 33 10 10 APES Natal, RN, Brazil 05º47’S, 35º12’W 30 10 10 APRN

Manaus, AM, Brazil 03º06’S, 60º01’W 92 10 10 APAM/APMA Cochabamba, Bolivia 17º38’S, 66º15’W 2574 15 10 APBO Asuncíon, Paraguai 25º26’S, 57º66’W 55 15 10 APPA A. serpentina Manaus, AM, Brazil 03º06’S, 60º01’W 92 02 00 - A. striata Manaus, AM, Brazil 03º06’S, 60º01’W 92 02 00 -

N1 - number of specimens by locality N2 - number of specimens for morphometric analyses 588 Neotrop Entomol 40(5): 587-594 © 2011 Sociedade Entomológica do Brasil Bomfim et al A Morphometric and Molecular Study of Anastrepha pickeli stereomicroscope attached to a digital micrometric ocular. The average length (arithmetic mean) was calculated for fragment of 1,050 bp within the mitochondrial COI gene preserved specimens by Han & McPheron (1997). A followed by a principal component analysis to verify primers tRNA-cys2 (ACTCCTTTAGAATTGCAGTCTAAT) each population and used for the UPGMA cluster analysis, andwas amplifiedCOld-r (GGGCTCATACAATAAATCCTAAT) by polymerase chain reaction (PCR) (Ruiz- using among populations were assessed by the Wilk’s Lambda testwhich (Statistica variable most9.0®). contributed to clustering. Differences according to Gasparich et al genomicArce 2009). DNA. PCR The reactions cycle program were consistedcarried out of anin initial25 μl Geometric morphometry denaturation step of 3 min at (1995)94°C, followed using from by 39 3-5μl cycles of Ten females from each locality were used. The right wing of each female was dissected and mounted on a of 1 min at 94°C, 1 min at 50°C, 1 min at 72°C, with a final microscope slide with glycerine and photographed with extension step of 10 min at 72°C. The® amplified fragments a camera attached to a stereomicroscope and the analySIS phosphatase (SAP) (Fermentas ) and incubated in a were purified using exonuclease I and shrimp alkaline getIT® thermocycler for 1h at 37°C, followed by 15 min at 80°C. ® following DNA sequencing was carried out at the Centro de Estudos Rohlf (2009a). software Fourteenwas used. Ahomologous TPS file was landmarks created from were the manuallyimage file plottedin the software at the designated tpsUtil version vein 1.4 intersections do Genoma Humano at the Universidade de São Paulo (Fig 2) for each specimen using the software tpsDig (CEGH-USP) using the DYEnamic ET Dye Terminator version 2.12® following Rohlf (2009b), based on previous sequenced one specimen per location for each population Kit in a MegaBACE 1000 automated DNA sequencer. We morphometric studies on Anastrepha (Nascimento 2005, of A. pickeli and one specimen for each species used as Selivon et al 2005). The centroid size was calculated for all outgroups. Sequences were aligned using the Bioedit Sequence Alignment Editor 7.0.9® (Hall 1999) which ® as the canonical variables and matrix of relative warps, tool (Thompson usinganatomic the landmarksoftware tpsRelw configurations version for 1.46 each® (Rohlf wing, 2009c)as well et al 1994). uses the ClustalW Multiple Alignment and tpsRegr version 1.37® Phylogenetic analyses were conducted using

® test (centroid size) and Wilk’s (Rohlf Lambda 2009d). (relative Differences warps) software (Tamura et al maximum parsimony (MP) and neighbor-joining (NJ) (Statisticaamong populations 9.0®). We were used assessed Statistica by the 9.0 Mann-Whitney® to calculate methods using the MEGA 4.1 in2007). Kumar A consensus et al NJ tree was generated using the the Mahalanobis distance matrix and to carry out the Jukes-Cantor distance (chosen based upon criteria multivariateMolecular analyses analysis, MANOVA. species of the Anastrepha1993). Bootstrapping serpentina group, of the Anastrepha MP and serpentinaNJ analyses (Wiedemann) (1,000 replicates) and Anastrepha was performed. striata Schiner Two Total nucleic acid extractions from thorax and legs of were used as outgroups. individual specimens followed the protocol for alcohol-

Results 1 2 3 Traditional morphometric variables L1 (aculeus apex 11 length), L4 (aculeus width at the apex of the cloacal 8 9 4 10 12 13 differences among the analyzed populations (Wilk’s 5 Lambdaopening), test, and P L5 < 0.05). (serrate The part principal length) component showed significant analysis 14 showed that these three morphometric characters were 7 6 Santo, Bolivia and Rio Grande do Norte, Paraguay and Fig 2 Wing of Anastrepha pickeli from Natal, RN, with landmarks 1 significantly different among Amazonas and Espírito = intersection of the veins umeral and costal; 2 = intersection of the Rio Grande do Norte population pairs (Wilk’s Lambda test, P < 0.05), whereas Amazonas and Bolivia, and Rio veins R1 and costal; 3 = intersection of the veins R2+3 and costal; 4 = Grande do Norte and Espírito Santo populations were not intersection of veins R4+5 apical margin; 6 = intersection of vein CuA1 with apical margin; 7 = intersection of vein CuA andwith costal; posterior 5 = intersection margin; 8 of= intersection vein M with 2 In all characters analyzed by traditional morphometry, of vein CuA 1 thestatistically population different of A. pickeli (Wilk’s Lambda test, P > 0.05). = intersection of veins CuA1and bm-cu; 11 = intersection of the veins r-m and and R M; 9 = intersection of the veins M and bm-cu; 10 different from all other populations (Wilk’s Lambda 4+5 test, P < 0.0001). The variablesfrom L1, Bahia L4 andwas L5 significantly explained and CuA ; 12 = intersection of the veins r-m and M; 13 = 62.6% of the data variability. The dendrogram (Fig 3) intersection1. of the dm-cu and M; 14 = intersection of veins dm-cu Neotrop Entomol 40(5): 587-594 © 2011 Sociedade Entomológica do Brasil 589 A Morphometric and Molecular Study of Anastrepha pickeli Bomfim et al

APES

APRN

APPA

APAM

APBO

APBA Fig 3 Dendrogram of populations of Anastrepha pickeli Espírito Santo (APES), Rio Grande do Norte (APRN), from Bolivia Amazonas (APBO), (APAM), and Bahia Paraguay, (APBA), 20 30 40 50 60 70 80 90 100 110 generated from distances of aculeus by (Dlink/Dmax)*100 clustering analysis

(UPGMA). shows that the population from Bahia did not cluster with that was significantly different from all remaining the remaining populations analyzed, which formed two clusters, one with populations from Paraguay, Rio Grande do Norte and Espirito Santo, and a second one with populations (Mann-Whitney test, P < 0.05), and the populations from Bolivia and Amazonas. However, the population from Bolivia that was significantly different from thatParaguay of Rio Grandeand Bolivia do Norte indicate (Mann-Whitney that they harbor test, P individuals< 0.05). The significantwith larger differences wings when between compared populations to the MANOVA of the traditional morphometric data indicated remaining populations. thatCentroid populations size ranged were not between statistically 7.0 and different 8.0. Average (Wilk’s and A total of 24 relative warps was generated (k = 2n - 4), standardLambda test deviation = 0.33, Pvalues > 0.05). are graphically represented where k represents the number of relative warps and n in Fig 4. Centroid size analysis indicated that most populations were not statistically different among the number of anatomic landmarks. The MANOVA of the canonical variables of relative warps showed significant themselves at the 95% confidence level (Mann-Whitney populationsdifferences amongfrom Paraguay populations and (Wilk’s Espírito Lambda Santo (Wilk’s= 0.006; test, P > 0.05), except for the population from Paraguay P < 0.05). Significant differences were observed between 9.5

9.0 Lambda test, P < 0.05), but they were not significantly weredifferent significantly from populations different from from Bahia each andother Rio (Wilk’s Grande 8.5 Lambdado Norte test,(Wilk’s P < Lambda 0.05). Populations test, P > 0.05), of Awhich,. pickeli in fromturn, Bolivia and Amazonas can be readily distinguished from 8.0 each other and also from the other populations (Wilk’s Lambda test, P < 0.05) 7.5 explained, respectively, 42.7% and 24.5% of the data

C entroid variability (Fig 5). . The two first canonical variables 7.0 largest distances between population pairs from Bolivia 6.5 andThe Bahia Mahalanobis (35.9%), Amazonas distance and matrix Rio Grande indicated do Norte the (30.6%), and Amazonas and Bahia (29.3%) (Table 2). 6.0 Aligned sequences, including the outgroups, resulted in a data matrix containing 685 characters, of 5.5 APBA APES APRN APAM APBO APPA which 129 were variable and 59 sites were informative State for parsimony analysis. Sequences were deposited Mean Mean ± SE Mean ± 1.96*SD Fig 4 Graph comparing average values and standard-error of the centroid size of the wing of Anastrepha pickeli females. in GenBankThree most under parsimonious accession trees numbers were recovered JN002428 from - JN002435. 590 Neotrop Entomol 40(5): 587-594 © 2011 Sociedade Entomológica do Brasil Bomfim et al A Morphometric and Molecular Study of Anastrepha pickeli

VC 2: upper end 6 1 2 3 5 11 4 9 4 8 13 10 12 5 3 14 2 6 1 7 VC 2: lower end 0 C 2: 24.53% V -1 APBA -2 APES -3 APRN -4 APMA -5 APBO -6 -4 -2 0 2 4 6 VC 1: 42.67% APPA VC 1: lower end VC 1: upper end

Fig 5 Dispersion graph of Anastrepha pickeli females from Bahia (APBA), Espírito Santo (APES), Rio Grande do Norte (APRN), Amazonas indicate the presumable wing conformations for individuals in the superior and inferior ends of the canonical variables. Deformation (APMA), Bolivia (APBO), and Paraguay (APPA) in the bidimensional space of canonical variables VC1 and VC2. The deformation diagrams magnitudes were amplified 3x for visualization. Discussion

MP analysis of the COI data and the strict consensus tree (Figis shown 7). The in Figaverage 6. The distance neighbor-joining among A. pickelitree is populationsvery similar wasin its 0.040placement ± 0.031. of the The various level populations of sequence to thedivergence MP tree The MANOVA indicated that the differences among the ranged from a minimum of 0.003 to a maximum distance populations were not statistically significant based on of 0.085 between populations. The lowest distance Anastrephatraditional morphometry,pickeli is a specialist as opposed species to and the therefore significant it observed within A. pickeli (0.003) was between the woulddifferences be expected observed to havewith athe more geometric uniform morphometry.morphology populations from Amazonas and Rio Grande do Norte among its populations. However, these morphological differences may be attributed to variation in both between those from Paraguay and Bahia. biotic (host plants) and abiotic factors (temperature, and the highest level of divergence (0.085) was verified pluviosity and humidity among others). For example, fraterculus Anastrepha pickeli group have been shown to vary along the geographic aculeusdistribution measurements range and foralso five among species specimens in the from the SantoTable (APES),2 Mahalanobis Rio Grande distances do Norte (APRN),of Bolivia (APBO), andpopulations Paraguay from (APPA) Amazonas based (APMA),on wing Bahiashape (APBA), components. Espírito analyzed the aculeus of two generalist species, Anastrepha APBA APES APRN APMA APBO APPA fraterculussame host (Araujo (Wiedemann) & Zucchi and 2006). Anastrepha Alencar-Souza zenildae (1998)Zucchi, APBA 0.00 and one specialist, A. pickeli, from Rio Grande do Norte A. APES 9.49 0.00 pickeli populations and concluded that there were two APRN 21.40 13.77 0.00 newand found species remarkable in the sample. intraspecific It is likely differences that in the among sample APMA 29.35 26.05 30.64 0.00 studied by Alencar-Souza (1998), besides specimens of A. APBO 35.93 23.13 22.17 18.63 0.00 pickeli, there were specimens of a species that is not yet formally described and has been denominated Anastrepha APPA 14.37 17.75 15.93 26.48 25.17 0.00 pickeli et al 2005).

Neotrop Entomol 40(5): 587-594 © 2011 Sociedade Entomológica do Brasilsp. aff. (Araujo 591 A Morphometric and Molecular Study of Anastrepha pickeli Bomfim et al

A. pickeli, Manaus, AM, BRA 74 A. pickeli, Cochabamba, BOL 98 A. pickeli, Natal, RN, BRA A. pickeli, Linhares, ES, BRA 100 82 A. pickeli, Teixeira de Freitas, BA, BRA A. pickeli, Asuncion, PRY A. striata, Manaus, AM, BRA

A. serpentina, Manaus, AM, BRA Fig 6 Phylogenetic relationships inferred from the strict consensus of three most parsimonius trees. Numbers in the branches indicate ). Statistics of each most parsimonious tree: length = 151; consistency index = 0.90; retention index = 0.84. bootstrap confidence limits higher than 50% (1,000 replications

In our study, samples were collected in various from Rio Grande do Norte and Espírito Santo (Fig 5). The geographic regions (Table 1), which can explain the specimens of A. pickeli analyzed by Nascimento (2005) A. pickeli populations. According to had wings widened at the base and narrowed apically and were distributed along the axis between the two todifferentiation the size of the structuresamong among the populations studied some researchers, the morphometric differenceset al 2004).related infrageneric groups. The deformation diagram shows that populationsends when compared from Amazonas to generalist and Bolivia species diverged in different from inare populations doubtful (Rohlf from &Bolivia Marcus and 1993, Paraguay Adams relative to the However, the significant difference in wing size observed remaining populations generated by the centroid size in the landmark 4 (apex of vein R4+5), which indicated a tendencythe remaining to narrow populations, the wing, showing and landmark differences 6 (apex mainly of as this measurement represents the geometric center of vein CuA1), which widened the wing. analysis, minimizes the influence of some external factors This is the most extensive molecular study of A. pickeli populations to date, both in number of included 1999).the wing Our as results well as agree the mass with thecenter centroid of a configuration, size reported samples and localities, even though we could not obtain bywhich Nascimento is paramount (2005), for size who definition analyzed (Monteiro a laboratory & Reis specimens from the entire range of distribution of this population of A. pickeli and other populations of generalist species in Brazil. Populations of A. pickeli were recovered Anastrepha species. However, further comparisons were as a monophyletic group with strong support in the prevented as that author did not specify the origin of the laboratory colony of A. pickeli studied. of three groups within the populations analyzed with The canonical variables analysis revealed that NJ tree. The COI data obtained indicated the existence populations of A. pickeli tended to form clusters; however, from Amazonas, Bolivia and Rio Grande do Norte, the most of them showed partial or total overlap. Populations secondreasonable from support. Bahia and The Espirito first group Santo, included and the populations third was from Amazonas and Bolivia showed a tendency to be represented by a single population from Paraguay, which located in the superior end of VC 1, as opposed to those is a strongly supported and highly divergent clade at the

49 A. pickeli, Manaus, AM, BRA

92 A. pickeli, Cochabamba, BOL

74 A. pickeli, Natal, RN, BRA

A. pickeli, Linhares, ES, BRA

60 A. pickeli, Teixeira de Freitas, BA, BRA

A. pickeli, Asuncion, PRY

A. striata, Manaus, AM, BRA

100 A. serpentina, Manaus, AM, BRA

0.05 bootstrap values higher than 50% (1,000 replications). Fig 7 Phylogenetic relationships inferred from neighbor-joining tree based on Jukes-Cantor distances. Numbers on the branches indicate 592 Neotrop Entomol 40(5): 587-594 © 2011 Sociedade Entomológica do Brasil Bomfim et al A Morphometric and Molecular Study of Anastrepha pickeli base of the remaining A. pickeli populations in the trees. das-frutas (Diptera: Tephritidae) no Semi-Árido do Rio Grande the geographic proximity among samples from north Entomol 34: 889-894. do Norte: plantas hospedeiras e índices de infestação. Neotrop southwards.Interestingly, This the topology is consistent of both with MP the and fact NJ that trees northern reflects Amazonia is considered the place of domestication of Anastrepha do grupo fraterculus (Diptera: cassava (Nassar 1978), which is the main host of A. pickeli AraujoTephritidae). EL, Zucchi Neotrop RA (2006) Entomol Medidas 35: do329-337. acúleo na caracterização throughout its geographic range. de cinco espécies de Our data revealed a high level of genetic variation within A. pickeli populations from various geographic Avise JC (1994) Molecular markers, natural history, and evolution. 2ed. Sunderland, Massachusetts, Sinauer Associates, 684p. distance = 0.074-0.085) between the population from of nuclear gene period in genus Anastrepha (Tephritidae). Ann BarrEntomol NB, Liwang Soc Am C, 98:McPheron 173-180. BA (2005) Molecular systematics Paraguay,regions. There situated is at a thestrong extreme molecular south of divergencethe distribution (JC range, and the remaining populations. Additional studies, Farias ARN, Belloti AC (2006) Pragas e seu controle, p.591-671. In including sampling across the species distribution, are Aspectos socioeconômicos e agronômicos da mandioca. Cruz das warranted to explore the boundaries of this species, since Souza L da S, Farias ARN, Mattos PLP de, Fukuda WMG (eds) recent studies have revealed that what were believed to be widespread species are actually cryptic species complexes, Almas, Embrapa Mandioca e Fruticultura Tropical, 817p. such as A. fraterculus (see Norrbom et al 1999). Analysis of mitochondrial DNA and development of PCR-based Further morphometric and genetic studies of Gasparich GE, Sheppard WS, Han HY, McPheron BA, Steck GJ (1995) populations along the entire distribution range would (Ceratitis capitata) diagnostic molecular markers for Mediterranean fruit fly help to better evaluate variations in A. pickeli. In order Hall TA (1999) Bioedit: populations. a user-friendly Insect Mol biological Biol 1: 61-67. sequence to advance further with molecular approaches, a gene alignment editor and analysis program for windows 95/98/NT. or genes with an even higher level of divergence may be Nucl Acids Symp Ser 41: 95-98. required to actually track patterns of the evolutionary history of the spatulata group. Tephritidae (Insecta: Diptera) using partial sequences of the Han HY, McPheron BA (1997) Molecular phylogenetic 7: study 17-32. of

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