DEFINICIÓN DE ESPECIES CRÍPTICAS EN … and...DEFINICIÓN DE ESPECIES CRÍPTICAS EN Anastrepha fraterculus Y A. obliqua (Dip., Tephritidae), EN COLOMBIA: ALTERNATIVAS DE MEJORAMIENTO

DEFINICIÓN DE ESPECIES CRÍPTICAS EN Anastrepha fraterculus Y A.

obliqua (Dip., Tephritidae), EN COLOMBIA: ALTERNATIVAS DE

MEJORAMIENTO DE LA TIE Y LOS SERVICIOS CUARENTENARI OS

(Resolution of cryptic species of Anastrepha fraterculus and A. obliqua (Dip.,

Tephritidae) in Colombia: Opportunities for improve the SIT and quarantine

services.)

PROGRESS REPORT 3

March/2012 – February/2013

Part of CRP: “Resolution of cryptic species complex es of Tephritid pests to

overcome constraints to SIT applications and intern ational trade.”

Research Contract No. 16069

Contracting Institute: UNIVERSIDAD DEL TOLIMA

Chief Scientific Investigator: NELSON A. CANAL

Main Scientific Staff:

Julio Cesar Carranza, Maria del Rosario Castañeda, Daniel Zabala (Universidad

del Tolima, Ibagué, Tol. Colombia)

Denise Selivon & Andre Luiz Perondini (Instituto de Biociencias, Universidade de

São Paulo, São Paulo, SP, Brazil)

Collaborator Staff (Students) :

Francy Edilma Gaitan, Elizabeth Tellez, Freddy Mauricio Ruiz, Yeniffer Campos.

Background

Species of the genus Anastrepha are the most important fruit flies in Latin America.

Seven species had been quarantined for international market, including A.

fraterculus and A. obliqua. The South American Fruit Fly is the common name of

Anastrepha fraterculus (Wiedemann, 1830), an important pest of fruits through

South America, from North of Mexico to North of Argentina and Chile. At least 14

synonymies had been referred for this species (Zucchi et al. 1996), but despite of

this fact, Stone since 1942 refer morphological variations of the populations and

suggests that could be a complex of species. Later studies had shown a cryptic

species complex in the taxon. Morphometrics (Hernandez-Ortiz et al., 2004, 2012),

cytogenetical (Goday et al, 2006, Caceres et al., 2009), biochemical (Morgante et

al., 1980; Steck, 1991), genetical (Ludeña et al., 2010; Selivon et al., 2005; Smith-

Caldas et al., 2001) and behavioral (Caceres et al., 2009; Vera et al., 2006) studies

had shown the presence of at least seven putative species in the Anastrepha

fraterculus complex.

The West Indian Fruit Fly A. obliqua belong to the fraterculus group and it is the

main pest in mango orchards (Aluja et al. 1996). Little is known about its population

and/or its host relation, only data of its kayotype by Selivon et al. (2005) and DNA

studies (Smith-Caldas et al. 2001, Ruiz-Arce 2012); data of DNA study suggest the

presence of six different clusters in the species.

Recently the fruits market become an important commodities among export

programs of the Colombian government, however, fruit flies are an important

barrier for development of the Colombian fruit-market (Miranda 2011).

The South American Fruit Fly is one of the most important pests in Colombian

fruits, especially referred to quarentenarian losses for international markets.

Despite of the importance for the country, just few specimens of Colombian

populations had been included in the morphometry works by Hernández-Ortiz et

al. (2004, 2012), behavior by Vera et al. (2006) and genetics by Smith-Caldas et al.

(2001), all of them coming from different places. Specimens from Colombia were

considered a different biological entity by Hernández-Ortiz et al. (2012), named as

Andean Morphotype together specimens from the high altitudinal places in

Venezuela. However, no comprehensive studies had been performed with

Colombian populations (or Andean morphotype) of A. fraterculus complex.

Mango is an important Colombian commodity for local and international market and

west indian fruit fly, A. obliqua (Macquart, 1835), appear to be an important pest for

this commodities, however, the knoledwege of the population structure of this

species in Colombia only include specimens studied by Ruiz-Arce et al. (2012).

The study of the population structure together dinamics, biological and ecological

data are the basement for the design of the more accuracy management systems

and/or to clear the quarentenarian status of these plagues.

Geographically, Colombia has three high mountain chains from South to North,

with two valleys among them and lowlands at West, North and Western.

Biogeography of the genus Anastrepha in Colombia is wide unknown, published

data support that apparently the South American Fruit Fly has altitudinal

distribution from 900 to 2,200 m and A. obliqua under 1,200 m (Castañeda et al.,

2010), making think, together biogeography of the insects and Colombian

geography, about isolation of Colombian populations of these species.

Sterile Insect Technique had been improved for A. ludens and has been performed

for species like A. fraterculus and A. obliqua. The South American Fruit Fly (A.

fraterculus) is a complex of cryptic species with so different economic and

quarantine importance. In order to improve the SIT for Anastrepha species, to

implement new biorational pest management and to remove some quarantine

barriers, the taxonomic status of the complex has to be defined and local

population structure has to be study.

RESEARCH CARRIED OUT

METHODOLOGY

General methodology was included in the second progress report, in this report

only new data will be referred.

At this time we had included in our studies three new populations of A. fraterculus

and one of A. obliqua. The populations of A. fraterculus were from the South region

of the country, near to Ecuador (Sibundoy, Putumayo and La Union, Nariño) and

other one from the West, in the way to Venezuela (Tab. 1, fig. 1). The population of

A. obliqua included was from the Valle del Cauca; recently we obtained two new

populations of A. obliqua from the Colombian Caribean Coast and from Mexico, but

studies of these material are in progress.

Table 1. Collection data of populations of Anastrepha obliqua and A. fraterculus

from Colombia.

N W

A. obliqua Mangifera

indica Cundinamarca Anolaima El limon Arturo Orjuela 04⁰ 43' 23,6" 74⁰ 25' 4,3'' 972

A. obliqua Spondias

purpurea Tolima Guamo Luisa

A. obliqua Spondias

mombin Tolima Coello Arenal 04⁰ 16' 55,0'' 74⁰54' 16,6' 309

A. obliqua Spondia sp. Quindio

Calarca vìa

La Tebaida Carretera Carretera 04⁰ 29' 42,9'' 75⁰ 41' 36,6'' 1409


indica Tolima Espinal Corpoica Nataima 320


indica Valle del Cauca Zarzal Fogansevalle 04⁰ 25' 33,9'' 76⁰ 03' 43,1'' 916


indica Magdalena Cienaga 11⁰ 58' 92,4'' 74⁰ 12' 18,3''

A. obliqua MEXICO

A.

fraterculus

Acca

sellowiana Caldas Pensilvania La Estrella

Fernando

Patiño 05⁰ 22' 3,5'' 75⁰ 9' 29,6'' 2091

A.

fraterculus

Coffea

arabica Cundinamarca Anolaima

Tocarema

alto Chay Raul Romero 04⁰ 45' 1,6'' 74⁰ 23' 1,3'' 1850

A.

fraterculus

Coffea

arabica Tolima Ibaguè El placer Jesus Antonio 04⁰ 24' 53,5'' 75⁰ 18' 50,6'' 1433

A.

fraterculus

Coffea

arabica Valle del Cauca Roldanillo La Aguada San Luis

Norbey

Almeciga 04⁰ 23' 05,8'' 76⁰ 13' 20,36'' 1764

A.

fraterculus

Acca

sellowiana Boyacà Duitama La trinidad

Jorge Hernana

Camargo 05⁰ 49' 29,9'' 73⁰ 04' 29,7'' 2569

A.

fraterculus

Coffea

arabica Valle del Cauca Sevilla 04⁰ 17' 19,8'' 75⁰ 54' 23,3'' 1556

A.

fraterculus

Coffea

arabica Nariño La Unión El Guabo Granja INPEC 01⁰ 36' 53,9'' 77⁰ 07' 53,6'' 1704

A.

fraterculus

Psidium

acutangulum Putumayo Sibundoy Fatima Corpoamazonia 01⁰ 12' 05,33'' 76⁰ 54' 48,9'' 2136

A.

fraterculus

Coffea

arabica Santander

Florida

Blanca Vericute

Centro

Experimental CENICAFE 07⁰ 06' 07,6'' 73⁰ 04' 13,4'' 1558

ALTITUDESPECIECOORDENADAS

HOSPEDERO DEPARTAMENTO MUNICIPIO VEREDA FINCA PROPIETARIO

Figure 1 . Places of collections of Colombian populations of Anastrepha fraterculus

and A. obliqua. Red colour show populations of A. fraterculus and yellow colour of

A. obliqua. Number refer populations of the table 1.

Studies of mitochondrial genes CO I and CO II, karyotypes, adult morphometry,

larval morphometry and post-zygotic reproductive isolation were performed.

Studies of adult morphometry were performed following Hernández-Ortiz et al.

(2012); cytogenetical data were obtained following Selivon & Perondini (1997).

The study if larval morphology was performed in order to test a new tool for

taxonomic diagnostic. The first test were performed with Colombian populations,

however, we expected to perform it including Latinoamerican populations

according with the work of G. Steck. 11 morphological character, especially from

the cephalofaryngeal skeleton and eight different ratios were selected (fig. 2). A

multivariate analyse were used to study data.

Figure 2 . Morphometric variables of the cephalofaryngeal skeleton of larve of

Anastrepha fraterculus and A. obliqua.

Post-zygotic isolation studies were performed following Selivon et al. (1999). Five

couples of each populations or each crosses were isolated in rearing cages

10x10x10 cm. Flies were provided with water and food following standard protocols

of our laboratory according with data referred in the second progress report. Every

three days fruits were offered for oviposition, guava feijoa for A. fraterculus and

mangoes for A. obliqua. Five replicates were used and the variables studied were

pupal viability and sex ratio. Eggs viability and larval viability cannot be studied

because many populations did not oviposit on artificial device. A non-parametric

statistical analyze were performed.

RESULTS

Anastrepha fraterculus

Mitochondrial genes. For the COI gene a band of 850 pb was obtained and a

fragment of 750 pb was sequenced; from 707 pb analyzed, 154 were variables and

56 were parsimony informative. The average nucleotide frequencies were 37.3%T,

15.4%C, 33.3%A and 14.0%G. The p values for the distances among Colombian

populations were average 0.001, lower than Latin-America´s previous studies

(Smith-Caldas et al. 2001). For the COII gen a fragment of 630 pb was obtained

and 602 were aligned, 94.35% of them belong to identities.

Phylogenetic trees performed with COI gene show two main cluster, the first with

the specimens collected in Sibundoy (Putumayo) (bootstrap 100); the second

branch is divided in two cluster (bootstrap 61), all specimens of Ibagué were put in

a same clade with specimens from others populations and the other clade include

specimens from all populations not Ibagué nor Sibundoy (fig. 3). These results

were supported by the gene COII analyze (fig. 4). Phylogenetic trees were

performed with data of populations from Latin America obtained from the

GENBANK (fig. 5). Populations from Colombia (not Sibundoy included) grouping

apart from other populations; this is a genetic support, not previous widely referred,

for the Andean Morphotype referred by Hernández-Ortiz et al. (2012); however, the

populations from Sibundoy (the southern place) group with specimens from

Ecuador and Brazil. Three haplotypes were identified in our populations, the

haplotype 6 is exclusive for Sibundoy; Ibagué and La Union have the haplotype 4

and the other populations 4 and 5.

The genetical divergence of the specimens from Sibundoy could mean that the

Peruvian Morphotype reach the South of Colombia or that other genetical entities

is present there. Other tools have to be analyzed ahead.

Figure 3 . Strict consensus tree from MP analysis of COI sequences (with

bootstrap in the branches) of Anastrepha fraterculus from Colombia.

Bdorsalis

Ccapitata

Rol1

Rol3

Rol5

Pen1

Pen2

Pen4

Pen5

Dui1

Dui2

Dui5

Cac1

Cac2

Cac4

Cac5

Rol2

Rol4

Pen3

Iba1

Iba2

Iba3

Iba4

Iba5

Dui3

Dui4

Cac3

COINa3

COINa4

61

100

COI put1

COIpu2

COIpu3

COIpu4

COIpu5

100

100

Figure 4. Strict consensus tree from MP analysis of COII sequences (with

bootstrap in the branches) of Anastrepha fraterculus from Colombia.


bootstrap in the branches) of Anastrepha fraterculus from Latin American (referred

by Smith-Caldas et al. 2001) and data of this study.

Adult Morphometry . Measurements of 21 variables were done and the values

found show high variability in the mean and standard deviation (no data included);

this variability was higher than that referred by Hernández-Ortiz et al. (2012).

However, differences found by discriminant analysis (manhattan distances) were

10

BdorsalisCcapitata

Rol1Rol2Rol3Rol4Rol5Pen1Pen2Pen3Pen4Pen5Iba1Iba2Iba3Iba4Iba5Dui1Dui2Dui3Dui4Dui5Cac1Cac2Cac3Cac4Cac5Nar3Nar4ColsevillaColmezamerida/Ven

99

Guat:PalinVencar

Put1Put2Put3Put4Put5EcuaBJanaubaBSto amaroB LinharesBChapeco

98

Mex ChiapaCos/Rica P

88

53

BvacariaB.Sao-joseB/MontealeBr CacondeArg Tucama

24

39

29

67

100

low (tab. 2, fig. 6). The distribution of the Andean Morphotype of the A. fraterculus

complex is the highlands mountain from Colombia and Venezuela (Hernández-

Ortiz et al. 2012). Colombian populations show some morphometric variability and

our data are representative for the Andean Morphotype due to the surveys cover a

wide range of its distribution and will be an informative data for taxonomic

comprehension of the morphotype. Morphometric analysis does not show any

difference between Sibundoy and other populations like variability found in the

genetical studies. Data from Hernández-Ortiz et al. (2012) show high morphometric

divergence between Andean and Peruvian morphotype, then, Sibundoy should

differ from the last, despite their genetical similarity. Both morphometric and

genetical data do not support the presence or not of other biological entities.

Table 2 . Manhattan distances of adults of nine populations of the Andean

Morphotype of the Anastrepha fraterculus complex collected in different places

from Colombia.

Columna1 COL-Bmg COL-Cach COL-Duit COL-Ibag COL-Lun COL-Pen COL-Rold COL-Sev COL-SibuCOL-Bmg 0COL-Cach 18.1158647 0COL-Duit 39.1156831 26.3121328 0COL-Ibag 39.9702832 23.8355295 14.928948 0COL-Lun 28.2135185 18.4993415 26.7630177 30.2763064 0COL-Pen 37.5569278 24.1870307 10.3872332 13.3048305 25.5090554 0COL-Rold 25.5066099 19.5052724 23.3609215 22.2556584 24.8534194 19.969055 0COL-Sev 17.2743079 16.9214117 36.6219038 36.4870559 23.1145162 33.4365079 28.390508 0COL-Sibu 24.7561408 17.2951063 16.6154323 19.8086298 14.8692577 16.7907691 18.148498 22.554979 0.00

Figure 6 . Discriminant analysis obtained with 21 variables of adults of nine

populations of Anastrepha fraterculus s.l. from Colombia.

The morphometric structure of the populations show specimens collected from

Bucaramanga (East of the country) separated from the other populations. The

eight remaining populations are grouped in two clusters, the first one including

mainly populations from the Magdalena basin (center of the country) except

Cachipay and the second branch with populations from the South (Sibundoy and

La Union in a cluster) and specimens from the Cauca basin (west of the country)

inn other branch (fig. 7).

Figure 7 . Cluster analysis produced with the Euclidea distances matrix of nine

populations of the Andean Morphotype of the Anastrepha fraterculus complex

collected in Colombia.

Larval Morphometry . Measurements of 19 variables were done only with six

populations of the nine studied, 20 specimens were studied for all group, and the

values found show high variability in the mean and standard deviation (no data

included); this variability was similar to those found in the morphometry of adults.

Differences found by discriminant analysis (manhattan distances) were low, similar

to those from adults (tab. 3, fig. 8), however, in the discriminant analyses the

population from Sibundoy was separated from the others and 100% of the

specimens were correctly classified. Results are similar of those obtained from

adults morphometry suggesting that this could be a tool for study populations of

this species. Like genetical data, specimens from Sibundoy show some variability

that should be studied deeper.

The population structure of larva has a branch with specimens from Sibundoy

(South of the country) and a cluster with two branches with populations from the

central region (fig. 9). Only six populations were studied and may be this fact is the

cause of the differences with the adult population structure.

Table 3 . Manhattan distances of larvae of six populations of the Andean

Morphotype of the Anastrepha fraterculus complex collected in different places

from Colombia.

Figure 8 . Discriminant analysis obtained with 19 variables of larvae of six

populations of Anastrepha fraterculus s.l. from Colombia.

Columna1 COL- Pen COL-Cach COL-Duit COL-Ibag COL-Rold COL-SibuCOL- Pen 0COL-Cach 25.5809374 0COL-Duit 17.2362225 23.9239526 0COL-Ibag 19.3826179 11.8334612 25.7061554 0COL-Rold 24.3291731 10.6911507 24.2555686 13.5849531 0COL-Sibu 27.5931551 20.9952728 30.1496905 26.1463499 19.6792486 0

Figure 9 . Cluster analysis produced with the Mahalanobis distances matrix of six

populations of larvae of the Andean Morphotype of the Anastrepha fraterculus

complex collected in Colombia.

Karyotypes. The karyotypes of the populations from Colombia are like nominal A.

fraterculus, with six pair of acrocentric chromosomes, one of them is a pair (XY) of

heterochromatic sex chromosomes. The sexual chromosomes have particular

morphology. A large X-chromosome with one apical C-band and a short Y-

chromosome with a big apical C-band. Populations studied in this year do not show

important differences with those referred in the second progress report. The Y-

chromosomes, due to its short size, show a more or less dot-shape and a bigger

C-band. The Y-chromosome is about 30% of the chromosome I and the C band is

bigger than a half of the cromossome; the X-chromossome is near 90% of the

autosome I, whit a C-band about 15% of the cromossome.

Pos-zygotic reproductive compatibility . Post-zygotic compatibility studies were

carried out in six matings with populations from the central region, and data were

analyzed whit kruskal-Wallis test (tables 4, 5). No statistical differences were

found, however, data are not conclusive because observations performed over a

single population were variable, may be, due to an incomplete adaptation to

laboratory of some populations.

Table 5 . Larval survival and female ratio of the F 1 for matings among Colombian

populations of Anastrepha fraterculus.

Larv

al s

urvi

val a

nd fe

mal

e ra

tio o

f the

mat

ings

bet

wee

n C

olo

mbi

an p

opul

atio

ns o

f A

nast

reph

a fr

ater

culu

s.

Mín

Máx

Hp

nM

edia

0.38

5237

690.

0113

6364

0.96

0526

324

0.45

8795

470.

1272

1085

0.60

1036

270.

9590

1639

50.

5064

5743

0.12

5129

870.

6650

4854

0.97

1428

575

0.43

8360

330.

1135

8399

0.72

8571

431

40.

5498

7212

0.19

0937

170.

5495

4955

15

0.49

6928

470.

0963

9873

0.62

9629

630.

8666

6667

50.

5451

2161

0.09

5309

770.

7643

0976

0.98

0392

165

0.52

2113

610.

2047

6935

0.5

14

0.49

1558

960.

2568

6688

0.23

8095

240.

8204

2254

50.

4492

6828

0.12

2497

990.

6419

7531

0.94

2857

145

0.42

6505

190.

1402

5542

0.58

9887

640.

9545

4545

50.

4477

1798

0.18

4646

590.

4191

6168

0.88

0952

385

0.45

4175

180.

1491

543

0.55

5555

560.

9375

50.

4704

7706

0.14

6538

160.

5188

6792

0.85

7142

865

0.52

3232

050.

2383

9604

0.41

9847

330.

8888

8889

50.

5597

2486

0.19

3182

140.

4930

5556

0.97

2972

975

0.44

4506

090.

1654

7072

0.58

50.

9338

843

50.

5106

1977

0.18

5278

230.

4844

2907

15

0.53

9413

590.

3310

9874

00.

8344

3709

40.

3847

3384

0.07

1190

910.

7379

3103

0.92

9687

55

0.49

6552

480.

0443

4466

0.77

60.

8907

1038

B5

0.39

5654

680.

0706

1102

0.64

1891

890.

8220

339

AB

50.

4878

6882

0.11

1504

550.

5294

1176

0.8

A4

0.48

7537

20.

1036

3978

0.53

1791

910.

7790

6977

A5

0.43

1014

67

6,52

0,08

9

% S

obre

vive

ncia

larv

alK

rusk

al -

Wal

lis

3,18

0,36

5

1,58

0,66

3

0,06

0,99

5

5,23

0,15

5

7,83

0,04

9

Anastrepha obliqua

Mitochondrial genes. Phylogenetic trees performed with COI gene show two

main cluster, the first with the specimens collected in La Tebaida (Quindio)

(bootstrap 100); the second branch (bootstrap 95) include all specimens of the

others five places (fig. 10). These results were supported by the gene COII analyze

(fig. 11). Phylogenetic trees were performed with data of populations from Latin

America obtained from the GENBANK (fig. 12). Populations from Colombia (not La

Tebaida) grouping with some specimens from Brasil (bootstrap 100); specimens

from La Tebaida belong to a separated branch with bootstrap 95 together some

specimens of others places from Brazil and belong to a sub-cluster of the main

cluster divided in three branches. Two haplotypes were identified in our

Cruzamiento % Sob. Larv. % Hembras

Ibg♀x Pns♂ 0.62820513 0.45918367

Ibg♀x Ibg♂ 0.75 0.51937984

Ibg♂x Pns♀ 0.88157895 0.53731343

Pns♂x Pns♀ 0.7484472 0.50207469

Duit♀x Pns♂ 0.6686747 0.45045045

Duit♂x Duit♀ 0.73770492 0.64444444

Duit♂x Pns♀ 0.57446809 0.7037037

Pns♂x Pns♀ 0.85185185 0.51086957

Cach♀x Pns♂ 0.69268293 0.53521127

Cach♀x Cach♂ 0.60240964 0.52

Cach♂x Pns♀ 0.82119205 0.49193548

Pns♂x Pns♀ 0.56047198 0.52105263

Duit♀x Ibg♂ 0.8729097 0.50191571

Ibg♀x Ibg♂ 0.75 0.51937984

Duit♂x Ibg♀ 0.57142857 0.4375

Duit♂x Duit♀ 0.73770492 0.64444444

Rld♀x Rld♂

Rld♀xPns ♂ 0.825 0.58333333

Pns♀x Rld♂ 0.93292683 0.48366013

Pns♀x Pns♂ 0.78763441 0.46416382

Cach♀x Ibg♂ 0.62116041 0.45054945

Ibg♀x Ibg♂ 0.51152074 0.44144144

Cach♂x Ibg♀ 0.76315789 0.65517241

Cach♂x Cach♀ 0.60240964 0.52

populations, the haplotype 6 is exclusive for La tebaida and haplotype 7 from the

other five populations.


bootstrap in the branches) of Anastrepha obliqua from Colombia.

Figure 11. Strict consensus tree from MP analysis of COII sequences (with

bootstrap in the branches) of Anastrepha obliqua from Colombia.


bootstrap in the branches) of Anastrepha fraterculus from Latin American (referred

by Smith-Caldas et al. 2001 and Boyker et al. 2010) and data of this study.

Adult Morphometry . Measurements of 21 variables were done with specimens

from five populations and the values found show low variability in the mean and

standard deviation (no data included). However, differences found by discriminant

analysis (manhattan distances) were low (tab. 6 fig. 13). Colombian populations

show some morphometric variability, but analysis does not show significant

differences between La Tebaida and other specimens, although over 95% of the

specimens from La Tebaida and Espinal were properely separated from other

populations. Both morphometric and genetical data do not support the presence or

not of other biological entities. Actually, studies with this species are lacked, only a

wide study with mitochondrial genes were performed by Ruiz-Arce et al. (2012) but

their data were not conclusive and suggest to perform other studies.

Population structure show the specimens from La Tebaida away from populations

from the Magdalena Basin and Coello separated from the other in the same basin.

Table 6 . Manhattan distances of adults of five populations of Anastrepha obliqua

collected in different places from Colombia.

Columna1 COL-Anol COL-Coel COL-Esp COL-Gum COL-LtebCOL-Anol 0COL-Coel 24.9753631 0COL-Esp 34.1829562 32.2653151 0COL-Gum 22.9149136 24.8677032 19.1752709 0COL-Lteb 21.5077343 29.4157346 39.2902324 24.3747495 0

Figure 13 . Discriminant analysis obtained with 21 variables of adults of five

populations of Anastrepha obliqua from Colombia.

Figure 14 . Cluster analysis produced with the Euclidea distances matrix of five

populations of adults of Anastrepha obliqua collected in Colombia.

Larval Morphometry Measurements of 19 variables were done and the values

found show high variability in the mean and standard deviation (no data included).

Differences found by discriminant analysis (manhattan distances) were low (tab. 7,

fig. 15), similar to those from adults, including over 95% of the specimens from La

Tebaida correctly classified.

Table 7 . Manhattan distances of larvae of five populations of Anastrepha obliqua

collected in different places from Colombia.

Figure 15 . Discriminant analysis obtained with 19 variables of larvae of five

populations of Anastrepha obliqua from Colombia.

Karyotypes . The karyotypes of the populations from Colombia are like species in

the fraterculus group, with six pair of acrocentric chromosomes, one of them is a

pair (XY) of heterochromatic sex chromosomes. The X chromosome is equal or

shorter than Y; sexual chromosomes have an apical C-band (only one

heterochromatic block), bigger in the Y chromosome and smaller in the X (fig. 16).

Karyotypes of A. obliqua had been studied in Brazil and Mexico were always the X

Columna1 COL-Anol COL-Coel COL-Esp COL-Gum COL-LtebCOL-Anol 0COL-Coel 21.0341297 0COL-Esp 17.3040703 15.9649901 0COL-Gum 22.4767308 23.2842593 21.1192399 0COL-Lteb 18.875964 22.9842997 20.6803848 29.4555383 0

chromosome is larger than Y (Selivon et al. 2005, Ibañez-Palacios et al. 2010),

contrary to those from Colombia; karyotype of the Brazilian specimens has a C-

band of the X chromosome divided in two blocks. Differences between knowing

karyotypes of A. obliqua are not clear like differences between populations of the

A. fraterculus complex, however, there are differences that should be better

understand.

Figura 16 . Karyotypes of the males of Anastrepha obliqua collected in different

places from Colombia A. Coello; B. Espinal; C. Guamo; D. Anolaima; E. La

Tebaida.

Post-zygotic reproductive compatibility . Post-zygotic compatibility studies were

carried out with eight matings with populations from the central region, and data

were analyzed whit kruskal-Wallis test (tables 8, 9). No statistical differences were

found, however, data are not conclusive like tests with A. fraterculus, observations

performed over a single population were variable, may be, due to an incomplete

adaptation to laboratory of some populations. However, data of the F1 show some

important deviation although no statistical difference was found. Espinal ♀ x Coello

♂ has strongly reduction of the larval survival and deviant sexual ratio towards

males. The same happened with the cross mating in both directions of La Tebaida

x Anolaima.

Table 9 . Larval survival (%) and females ratio of the F1 from matings among

Colombian populations of Anastrepha obliqua.

. La

rval

sur

viva

l (%

) an

d fe

mal

es r

atio

of t

he m

atin

gs a

mon

g C

olom

bian

pop

ulat

ions

of A

nast

reph

a ob

liqua

.

Med

iaD

.E.

Mín

Máx

Hp

nM

edia

D.E

.0.

6143

942

0.07

4094

540.

5446

4286

0.71

1640

21B

50.

4851

3759

0.02

9306

290.

1416

2946

0.04

9209

170.

0909

0909

0.20

1680

67A

50.

5057

7122

0.08

7369

160.

6221

2473

0.23

3895

210.

3090

2111

0.91

8918

92B

50.

4895

5623

0.02

3652

450.

4276

0837

0.23

5626

410.

2139

9177

0.81

3218

39A

B5

0.52

2563

20.

0226

7534

0.93

3660

910.

0274

6699

0.90

5325

440.

9774

4361

50.

4919

5156

0.04

9695

570.

7126

9108

0.40

5115

240

0.96

5648

854

0.50

8513

280.

0718

944

0.85

1016

550.

1061

4408

0.7

0.96

7289

725

0.50

4782

740.

1229

1564

0.89

7406

950.

0318

2586

0.86

875

0.94

6564

895

0.48

3480

110.

0232

6479

0.89

3008

520.

0531

057

0.83

8709

680.

9629

6296

50.

4748

7397

0.08

1149

960.

9228

6864

0.03

0614

210.

8789

8089

0.96

50.

5052

2195

0.08

0163

890.

9035

9201

0.07

0475

260.

7872

3404

0.97

3684

215

0.49

4942

410.

1106

8702

0.82

8125

850.

0823

7065

0.70

5128

210.

9280

8219

50.

4687

0801

0.05

2262

560.

9340

563

0.02

2008

60.

9035

0877

0.96

50.

5424

054

0.05

9248

910.

9236

345

0.03

9216

950.

8684

2105

0.96

1240

315

0.46

9910

050.

0723

3503

0.85

7311

830.

0711

4165

0.78

1512

610.

9479

1667

40.

5014

5673

0.06

3156

090.

8575

9267

0.14

4688

810.

6459

144

0.95

0819

674

0.46

6178

160.

0377

1972

0.89

6589

050.

0978

638

0.76

1904

761

50.

5124

0176

0.03

8014

960.

8415

3291

0.08

7779

380.

7023

8095

0.92

4242

425

0.42

7166

850.

1200

2117

0.88

9847

990.

0545

6192

0.81

8181

820.

9579

8319

50.

4828

2063

0.05

3293

990.

8965

8905

0.09

7863

80.

7619

0476

15

0.51

2401

760.

0380

1496

0.82

4663

220.

1071

0354

0.66

5263

160.

9567

1982

50.

4884

1437

0.04

0327

210.

9040

0264

0.07

1292

80.

7939

3939

0.98

2456

145

0.51

4415

090.

0421

2473

0.79

0420

180.

1556

5705

0.56

7328

920.

9554

4554

50.

5187

0236

0.01

3304

930.

7301

1118

0.40

9910

650

0.96

6292

134

0.49

1085

160.

0364

9599

0.81

5154

770.

1140

8892

0.63

7362

640.

9299

6109

A5

0.50

7010

490.

0302

3566

0.94

6912

480.

0269

1724

0.91

8604

650.

9807

6923

B5

0.47

4552

090.

0464

2272

0.87

6292

840.

0228

2876

0.84

1176

470.

9044

586

AB

50.

5104

5513

0.02

2409

230.

6098

7729

0.26

0206

720.

1532

8467

0.81

0699

59A

50.

4873

6517

0.05

3579

90.

9123

3952

0.05

2095

350.

8518

5185

0.96

6666

67B

50.

4621

6587

0.04

7986

560.

8833

5483

0.04

7357

350.

8134

9206

0.93

3333

33B

50.

4889

3204

0.03

6340

650.

5928

9459

0.26

8205

170.

1315

7895

0.82

1192

05A

50.

5274

0204

0.11

9783

130.

8801

5746

0.10

0830

680.

7228

9157

0.96

7532

47B

50.

5183

5419

0.05

0058

16

% d

e so

brev

iven

cia

larv

al%

de

emer

genc

ia d

e H

embr

as

0,18

9

3,04

0,38

6

Kru

skal

- W

allis

12,4

10,

006

3,21

0,35

9

4,79

3,00

0,39

1

13,4

50,

003

9,95

0,01

9

5,37

0,14

6

Cruce Cruce

Esp♀x Esp♂ 0.95294118 0.44444444 Gam♀x Gam♂ 0.85096154 0.55932203

Esp♀x Anl♂ 0.65137615 0.5 Gam♀x Anl♂ 0.83119266 0.50772627

Anl♀x Esp♂ 0.70283019 0.51006711 Anl♀x Gam♂ 0.75438596 0.47238372

Anl♀x Anl♂ 0.97058824 0.57575758 Anl♀x Anl♂ 0.625 0.5

Esp♀x Esp♂ 0.81229773 0.45418327 Ltb♀x Ltb♂ 0.82608696 0.42105263

Esp♀x Coel♂ 0.60550459 0.375 Ltb♀x Coel♂ 0.92372881 0.4587156

Coel♀x Esp♂ 0.83126551 0.53134328 Coel♀x Ltb♂ 0.88429752 0.48831776

Coel♀x Coel♂ 0.86666667 0.43269231 Coel♀x Coel♂ 0.86666667 0.43269231

Esp♀x Esp♂ 0.61806452 0.50521921 Ltb♀x Ltb♂ 0.7393617 0.48920863

Esp♀x Ltb♂ 0.88905547 0.53625632 Ltb♀x Anl♂ 0.67346939 0.4040404

Ltb♀x Esp♂ 0.9055794 0.51895735 Anl♀x Ltb♂ 0.58585859 0.55172414

Ltb♀x Ltb♂ 0.79166667 0.42105263 Anl♀x Anl♂ 0.97058824 0.57575758

Esp♀x Esp♂ 0.70871985 0.4973822 Gam♀x Gam♂ 0.87142857 0.47540984

Esp♀x Gam♂ 0.8880814 0.5106383 Gam♀x Coel♂ 0.95327103 0.47385621

Gam♀x Esp♂ 0.88640974 0.53318078 Coel♀x Gm♂ 0.57616893 0.53141361

Gam♀x Gam♂ 0.87162162 0.5374677 Coel♀x Coel♂ 0.77777778 0.43809524

Work supported or not referred

The mainly results obtained by our group had been referred previous, however, we

had supported other not included studies. We had supplied Colombian biological

material for cross mating studies at Seiberssdoff la

our group (Ph. D. student) was part of the worker team for these test, under

financial support of the IAEA.

complement Dr. V. Hernández

with Dra. Ruth R. do Nascimento and a

offered to her.

Studies with eggs had been performed under electronic microscopy (pictures

ahead belong to the eggs from different Colombian specimens), however, studies

are in progress at this time.

Publications

Canal N.A., Castañeda M.R., Osorio F. A. 2010. Variación morfológica de

obliqua (Diptera: Tephritidae) en el departamento del Tolima. Rumenes

Sociedad Colombiana de Entomología. Bogotá, 29 de junio

(Resumen)

Zabala D., Canal N.A., Ortiz P.A., Carranza J.C. Caracterización molecular y análisis

filogenético de poblaciones de

Work supported or not referred



material for cross mating studies at Seiberssdoff laboratories and a researcher of


financial support of the IAEA. Morphological data of adults were offered to

complement Dr. V. Hernández-Ortiz´s work. We are planning collaborative

with Dra. Ruth R. do Nascimento and a sample of male volatiles as a test was

s had been performed under electronic microscopy (pictures


in progress at this time.

Canal N.A., Castañeda M.R., Osorio F. A. 2010. Variación morfológica de

(Diptera: Tephritidae) en el departamento del Tolima. Rumenes

Sociedad Colombiana de Entomología. Bogotá, 29 de junio – 1 de julio de 2010. p 72.


filogenético de poblaciones de Anastrepha fraterculus y A. oblliqua en la región central de



boratories and a researcher of


Morphological data of adults were offered to

We are planning collaborative work

sample of male volatiles as a test was

s had been performed under electronic microscopy (pictures


Canal N.A., Castañeda M.R., Osorio F. A. 2010. Variación morfológica de Anastrepha

(Diptera: Tephritidae) en el departamento del Tolima. Rumenes 37 congreso

1 de julio de 2010. p 72.


en la región central de

Colombia. Resumenes 45 congreso Asociación Colombiana de Ciencias Biológicas. 2011.

(Resumen)

Zabala D., Canal N.A. Análisis del gen COII en poblaciones de Anastrepha fraterculus y A.

obliqua (Diptera: Tephritidae) en la región central de Colombia. Resumenes 38 congreso

Sociedad Colombiana de Entomología. 2011. p 56 (Resumenes)

Canal N.A., Carranza J.C., Zabala D., Castañeda M.R. El complejo Anastrepha fraterculus

(Diptera, Tephritidae) en Colombia. Memorias 39 congreso Sociedad Colombiana de

Entomología. CD-Room. (Ponencia en extenso).

Canal N.A., Castañeda M.R., Perondini A.L., Selivon D. Karyotypes of the Anastrepha

fraterculus complex populations from Colombia. 5th International Meeting on Taxonomy

and Natural History of Tephritoidea. Australia 6-10 febrero 2012. (Resumen)

Castañeda M.R., Canal N.A. Estudio del cariotipo de poblaciones colombianas de

Anastrepha obliqua (Mcquart). Proceedings 8th Meeting of the Tephritid Workers of the

Western Hemisphere. Panamá 30 julio – 3 agosto 2012. (Abstract – Poster)

Tellez E., Canal N.A. Descripción morfológica de larvas de tercer instar de seis especies de

Anastrepha (Diptera, Tephritidae). Proceedings 8th Meeting of the Tephritid Workers of

the Western Hemisphere. Panamá 30 julio – 3 agosto 2012. (Abstract – Poster)

Acknowledgments

We are very grateful with people who had collaborated with us, Dra. Denise

Selivon, Dr. Andre L. Perondini, Dr. Vicente Hernández-Ortiz and Dr. Jesus Reyes

and people who offer the fruits for us, referred in the table of collection data. The

IAEA and the Oficina de Invesstigaciones – Universidad del Tolima for the financial

support.

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Documents

DEFINICIÓN DE ESPECIES CRÍPTICAS EN … and...DEFINICIÓN DE ESPECIES CRÍPTICAS EN Anastrepha fraterculus Y A. obliqua (Dip., Tephritidae), EN COLOMBIA: ALTERNATIVAS DE MEJORAMIENTO