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Geographic Differentiation of Colombian Neoleucinodes elegantalis (Lepidoptera:
Crambidae) haplotypes: evidence for Solanaceae host plant association and Holdridge life
zones for genetic differentiation
Jorge Mario Londoño¹, Ana Elizabeth Díaz2, Gerardo Gallego¹, Wilson Vasquez3, Harold Suárez¹ and Joe Tohme¹
Figure 1. Haplotype frequencies obtained for the evaluation of 272 samples, the most frequent haplotypes are H5
(light blue) and H1 (dark blue).
Figure 3. Genetic distances inferred using Neighbor-Joining (NJ). Classification follows the faunistic zones proposed by
Kattan et al (2004): yellow (Inter-Andean Slopes), blue (Cauca Valley watershed), violet (Magdalena Valley watershed),
red (Ecuador), deep blue (Honduras).
The composition and heterogeneity analysis of COI sequences allowed the establishment of variability at the inter and intra
specific levels. This variation could identify significative divergence between individuals of N. elegantalis.
Figure 3. Heterogeneity
(He) of the COI in N.
elegantalis.
Figure 2. a) Geographical distribution of most common N. elegantalis haplotypes in Colombia, b) Clasification of
the 5 sub-regions proposed by Kattan et al., (2004), also refered to by the author as faunistic zones. Cauca Valley,
Central Cordillera and Magdalena Valley are the three sub-regions where all Colombian individuals come from.
DIAZ A E., SOLIS M A 2007. A New species and species distribution records of Neoleucinodes (Lepidoptera:
Crambidae:Spilomelinae) from Colombia feeding on Solanum sp. Proceedings of the Entomological Society of Washington.
109 (4):897–908.
HOLDRIDGE L R 1947. Determination of World Plant Formations from Simple Climatic Data. Science Vol, 105 No. 2727:
367-368.
HEBERT P D N., CYWINSKA A., BALL S L., DE WAARD J R. 2003. Biological identifications through DNA barcodes. Proc.
R. Soc. B. 270, 313-321.
KATTAN G H., FRANCO P., ROJAS V., & MORALES G. Biological diversification in a complex region: a spatial analysis of
faunistic diversity and biogeography of the Andes of Colombia
DNA Barcoding was an accurate tool for the identification of haplotypes as well as discrimination of species (N. silvane) reported previously by Díaz & Solís (2007) using geometric morphology. The number of haplotypes obtained revealed a possible role
of biogeographic isolation between valleys and of possible human pressure through the use of pesticides inducing divergent selection in N. elegantalis. The NJ analysis shows a wide distribution of the species along the Magdalena valley watershed, this
sub-region is located between the Central and Eastern Cordilleras. The distribution of N. elegantalis in this region could explain the wide range of altitudinal adaptation of the species that could facilitate dispersal. With regard to the Cauca Valley, diversity
centers on the southwestern slope of the Central Cordillera and on the East of Western Cordillera. The classification of regions proposed by Kattan et al (2004), are highly correlated to the grouping of haplotypes recovered in the species.
The DNA Barcoding tool shows high sensitivity in N. elegantalis haplotype identification, and to correlation with the sub-regions of colombia which
suggest at least 3 different geographic groups, related to both biogeographic separation, and human intervention through the use of pesticides.
This is the first genetic analysis of N. elegantalis and the first attempt to obtain a molecular characterization of the species.
Genetic differentiation could mean that there is partial reproductive isolation in N. elegantalis; further research could center on resolving this issue.
Through this methodology we confirm the existance of a new species of Neoleucinodes genera (N. silvanie) previously proposed by Díaz & Solís (2007)
with the use of morphological characters.
INTRODUCTION
¹Agrobiodiversity and Biotechnology Proyect, Centro Internacional de Agricultura Tropical (CIAT), Palmira, Valle del Cauca - Colombia
²Corporación Colombiana de Investigación Agropecuario (CORPOICA), Centro de Investigación La Selva, Rionegro, Antioquía - Colombia 3Instituto Nacional Autonomo de Investigaciones Agropecuarias (INIAP, Ecuador)
METHODOLOGY
RESULTS
DISCUSSION
Nucleotide composition and haplotype frequencies of N. elegantalis
Amplification of the mitochondrial gene COI produced a 658 bp fragment from a total of 272 individuals with
average base (nucleotide) frequencies of A=34.06%, T=34.06%, C=15.94%, G=15.94%. The number of haplotypes
obtained was 9 in which the most frequent haplotype, with 87 sequences, was H5 representing 32.2% including 90%
of the Ecuadorian individuals. H5 was followed by H1 with 82 individuals and corresponds to 50% of Colombian
sequences. Moreover, H1 was found in the Magdalena valley watershed while H3 correspond to Inter-Andean slopes
and H2 to Cauca valley watershed. The number of substitutions at the nucleotide level varied between 9 and 24 in all
haplotypes observed.
CONCLUSIONS
ACKNOWLEDGMENTS
REFERENCES
Special thanks to Fondo Regional de Tecnología Agropecuaria FONTAGRO for financial support.
Neoleucinodes elegantalis, known as tomato fruit borer, is an insect of Neotropical origin, widely distributed in Central America and South America. This species is considered one of the most important pests for fruit production in the
Solanaceae. N. elegantalis is adapted to a wide altitudinal range (0 – 2600 m.a.s.l), a considerable diversity of natural enemies, and exhibits variability in terms of oviposition and pupation habit, besides a wide spectrum of host wild
Solanaceae, and a differential behavior to sexual pheromone Neoelegantol. Using the DNA Barcode tool, 272 samples were processed and analyzed through Cytocrhome oxidase I (COI) gene, which represents a standard segment of
the mitochondrial genome ( 650 base pairs). The geographical differentiation and population structure in relation to host-plant association and to Holdridge life zones were examined. The mains goals of this study are to evaluate the
utility of DNA barcoding in the identification of haplotypes in the populations of N. elegantalis from Colombia and Ecuador, and to establish a DNA barcode database for this species.
DNA extraction from 163 individuals collected in 14 departments of Colombia and 95 individuals from
Ecuador was performed using the GF – 1 Nucleic Acid Extraction Kit (GF1-100-Vivantis). Amplification was
carried out using universal primers that flank the COI region, with COI-Forward
(5’GGTCAACAAATCATAAAGATATTGG3’) and COI-Reverse (5’
TAAACTTCAGGGTGACCAAAAAATCA 3’).
Purification of the PCR product was performed using the PCR Clean up system (Promega). The sequences
were obtained using an automatic sequencer ABI 3730 (Perkin Elmer/Applied Biosystem) and the assembled
with Sequencher 4.6 (Gene codes corporation Ann Arbor; MI).
Different bioinformatic tools were used for alignment and verification of sequence quality, nucleotide
composition for each sample, genetic distances and heterogeneity among sequences. Additionally, the
geographic distribution for each sample was determined, using the software ArcGIS – ArcMap (ERSI 1999 –
2008).
Ecu
ad
or
NA
Y11
M
Ecuad
or
NA
Y1
1 H
1
Ecu
ado
r N
CH
101
0
Ecu
ado
r 41
0
Ecuado
r 18
0
Ecuador
20
Ecua
dor
43
Ecu
ador
NC
H 1
1 V
Ecuad
or
NG
512 M
000
0
Ecuador
09
Ecuador
27
Ecuador
60
Ecuador
17
Ecuador
NG
511 M
0000
0 Ecuador
TA
CH
101
Ecuador
NA
Y12 H
1
Ecuador
48
0
Ecuador
21
0
Ecuador
06
0
Ecu
ador
10
Ecu
ador
28
Ecu
ador
61
Ecu
ador 25
Ecu
ador N
CO
11 H
00
0
0
0
0
Ecu
ador 05
Ecu
ador N
CH
11 V
b
0
Ecu
ador 59
0
Ecu
ador
26
0 Ecu
ador 08
0
Ecu
ador
16
Ecu
ador
TAC
H 1
03
1 Ecu
ador
42
0 Ecu
ador
19
0 Ecu
ador
11
Ecu
ador
40
Ecu
ador
TACH 1
02
Ecu
ador
NCH11
V
Ecu
ador
NCO12
M
1
00
0
0
0
0
Ecuad
or 0
7
0
Ecuad
or 2
20
Ecuador 5
8
0
Ecuador N
CH 101 b
0
Ecuador N
AY12 M
2
Ecuador N
AY13 M
57
Ecuador 4
9
Ecuador 5
0
Ecuador NG512 H
Ecuador NCO11 M
58
52
Ecuador 14
Ecuador 68
60
Ecuador 01
Ecuador 02
Ecuador 03
Ecuador 12
Ecuador 13
Ecuador 15
Ecuador 23
Ecuador 24
Ecuador 29
Ecuador 30
Ecuador 31
Ecuador 32
Ecuador 33
Ecuador 34
Ecuador 36
Ecuador 37
Ecuador 38
Ecuador 39
Ecuador 45
Ecuador 46
Ecuador 47
Ecuador 52
Ecuador 53
Ecuador 55
Ecuador 62
Ecuador 63Ecuador 64Ecuador 65Ecuador 66Ecuador 67Ecuador 69Ecuador 70Ecuador 71
Ecuador 75Ecuador NRV31 HEcuador NRV31 MEcuador NRV11 H
Ecuador NRV11 MEcuador NRV12 H
Ecuador NRV21 MEcuador NRV21 H
Ecuador 44Ecuador 51
62
8
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
58
28
69
Col Mlena 48Col Mlena 49
Col NteStder 45
Col NteStder 41
Col NteStder 39
Col Stder 37
Col NteStder 35
Col NteStder 34
Col NteStder 31
Col Stder 29
Col Nd 28
Col Nd 27
Col Nar 18
9
Col Stder 26
Col M
lena 50
63
8
Col Stder 25
Col S
tder 79
Col S
tder 80
Col S
tder 73
32Col A
nt 94
Col C
auca 121
Col S
tder 76
Col C
un 22
Col B
oy 05
39
Col C
un 170
Col A
PLG
Tm
1058
-Ant
2C
ol A
PS
ITa897-A
nt
0
Col C
auca
130
0
Col C
auca
120
0
Col T
ol 1
07
0
Col T
ol 9
6
0
Col H
uila
92
0
Col A
nt 9
10
Col H
uila
90
0
Col H
uila
89
0
Col C
un 4
70
Col C
un 2
40
Col C
un 2
30
Col C
un 2
00
Col C
un 1
71
Col B
oy 0
329
Col B
oy 0
1
Col B
oy 0
2
Col B
oy 0
4
Col B
oy 0
6
Col B
oy 0
7
Co
l Boy 0
8
Col H
uila
88
Col H
uila
93
Col H
uila
95
Col H
uila
97
Co
l To
l 99
Co
l Tol 1
00
Col T
ol 1
05
Co
l Cau
ca 1
19
Col C
au
ca 1
22
CO
I ColC
au
ca 1
23
Col C
auca 1
24
Co
l Cauca 1
25
Col C
auca 1
26
Col C
auca 1
27
Col C
auca
128
Col C
auca 1
29
Col C
ME
RT
m313-C
al
Col T
ol 1
04
Col A
PLG
Tm
1989-A
nt
Col S
tder 7
4C
ol C
un 1
68
Col T
ol 1
10
Col T
ol 1
73
67 1
8
10
0
0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 2
61
49
50
38
11
31
95
Col V
cau 5
1
Col V
cau 5
2
Col V
cau 5
3
Col R
isa 5
5
Col Q
dio
57
Col C
al 5
8
Col C
al 5
9
Col C
al 6
0
Col C
al 6
1
Col C
al 6
4
Col C
al 6
7
Col C
al 6
8
Col
Ris
a 70
Col
Ris
a 71
Col
Std
er 7
2
Col
Ris
a 75
Col
Vca
u 83
100
72
Col
ASDRTa
1046
-Ant
25
Col
Nte
Std
er 3
6
8
Col T
ol 1
06
Col
Nte
Stder
32
Col
Nte
Stder
40
31 27
28
Col C
MLE
Ta296
-Cal
Hondura
s 03
Honduras
149
Honduras
12 2
Honduras 0
9 1
Honduras 0
7 0
Honduras 0
6 1
Honduras 05 2
Honduras 01 9
Honduras 04 86
Honduras 08
Honduras 11
Honduras 02Honduras 13
7341
95
47
Honduras 10
73
Col ASDRTa113-Ant
Col ASDRTa858-Ant
Col ASDRTa903-Ant 100
45
Col Qdio 54Col DosQ 69
Col APSITa853-Ant
Col RSLALu346-RisaCol CMLETa304-CalCol Vcau 82
23
Col RSLLTm297-RisaCol RSLLTm316-RisaCol CMERTm337-Cal
63
Col AJCLu527-AntCol AJILu847-AntCol AJTTa1932-Atq 45
52
Col AJILu931-Ant
29
Col Nar 117Col Nar 118
Col Nar 116
64
Col Vcau 115
6
Col AJCLu601-Ant
0
Col RSLALu289-Risa
0
Col RSLLTm340-Risa
0
Col RSLLTm272-Risa
0
Col CMERTm350-Cal
0
Col CMERTm320-Cal
0
Col Vcau 81
0
Col Cal 63
2
Col Risa 56
48
Col RSLALu92-StaRosa
Col RSLALu318-Risa46
55
Col CMERTm300-Cal
Col AJCLu1875-Ant
Col Cal 290
64
Col AJCLu1400-Ant
21
Col AJCLu1654-Ant
20
Col APLGTm1763-Ant
3
Col AJTTa1666-Ant
1
Col AJILu737-Ant
0
Col AJCLu1726-Ant
0
Col AJCLu1026-Ant
0
Col AJCLu400-Ant
0
Col RSLGLu331-Risa
4
Col Cal 65
46
Col CMERTm273-Cal
Col AJCLu1492-Ant
Col AJTTa1467-Ant
Col-CMLETa59-Cal
64
Col Ant 85
Col Ant 86
Col Ant 87
Col AJC
Lu453-Ant
Col AJC
Lu911-Ant
Col AJC
Lu1307-Ant
Col A
JCLu1585-A
nt
Col A
JCLu1797-A
nt
Col A
JCLu1917-A
nt
Col A
JCLu1931-A
nt
Col A
PLG
Tm
1475-Ant
Col A
JTTa1829-A
nt
Col C
al 62
Col A
nt 109
Col A
JCLu256-A
tq
Col A
JCLu703-A
nt
Col A
JCLu
870-A
nt
Col A
JCLu971-A
nt
Col A
JCLu998-A
nt
Col A
JCLu1166-A
nt
Col A
JLLLu708-A
nt
65
25
1
0
00
0
00
00
01
21
24
42
44
34
6
19
19
56
35
48
Col R
SLA
Lu106-S
taR
osa
41
Col C
al 6
6
99
Col cu
n 4
3
Ecu
ador S
2R
V11 H
Ecu
ador S
2R
V12 M
Ecuador S
2R
V11 M
100
73
Col N
d 2
09
0.01
ECUADOR
HONDURAS
COLOMBIA –
Inter-Andean
Slopes
COLOMBIA –
Magdalena
Valley
watershed
COLOMBIA –
Cauca Valley
watershed
The genetic distances between sequences were calculated using Neighbor-Joining/UPGMA algorithm implemented in MEGA
5. These distances yield 5 groups, of which 2 belong to Ecuador and Honduras. Additionally, 14 individuals from Honduras,
which were used as the outgroup, formed a separate group; and 3 Colombian groups can be distinguished. The faunistic
classification proposed by Kattan et al (2004) in order to group the origins of the samples.
30.4
6.7
25.2
1.5
32.2
1.5
0.4 0.4 1.9
H1H2H3H4H5H6H7