Research Article |
Corresponding author: Vicente Hernández-Ortiz ( vicente.hernandez@inecol.mx ) Academic editor: Teresa Vera
© 2015 Vicente Hernández-Ortiz, Nelson A. Canal, Juan O. Tigrero Salas, Freddy M. Ruíz-Hurtado, José F. Dzul-Cauich.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Hernández-Ortiz V, Canal NA, Tigrero Salas JO, Ruíz-Hurtado FM, Dzul-Cauich JF (2015) Taxonomy and phenotypic relationships of the Anastrepha fraterculus complex in the Mesoamerican and Pacific Neotropical dominions (Diptera, Tephritidae). In: De Meyer M, Clarke AR, Vera MT, Hendrichs J (Eds) Resolution of Cryptic Species Complexes of Tephritid Pests to Enhance SIT Application and Facilitate International Trade. ZooKeys 540: 95-124. https://doi.org/10.3897/zookeys.540.6027
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Previous morphometric studies based on linear measurements of female structures of the aculeus, mesonotum, and wing revealed the existence of seven morphotypes within the Anastrepha fraterculus cryptic species complex along the Neotropical Region. The current research followed linear and geometric morphometric approaches in 40 population samples of the nominal species A. fraterculus (Wiedemann) spread throughout the Meso-American and Pacific Neotropical dominions (including Mexico, Central America, Venezuela, Colombia, Ecuador, and Peru). The goals were to explore the phenotypic relationships of the morphotypes in these biogeographical areas; evaluate the reliability of procedures used for delimitation of morphotypes; and describe their current distribution. Findings determined that morphotypes previously recognized via the linear morphometrics were also supported by geometric morphometrics of the wing shape. In addition, we found an eighth morphotype inhabiting the highlands of Ecuador and Peru. Morphotypes are related into three natural phenotypic groups nominated as Mesoamerican-Caribbean lineage, Andean lineage, and Brazilian lineage. The hypothesis that lineages are not directly related to each other is discussed, supported by their large morphological divergence and endemicity in these three well-defined biogeographic areas. In addition, this hypothesis of the non-monophyly of the A. fraterculus complex is also supported by evidence from other authors based on molecular studies and the strong reproductive isolation between morphs from different lineages.
Cryptic species complex, linear morphometrics, geometric morphometrics, distribution
Phylogenetic relationships stated that infrageneric classification based on morphology of the genus Anastrepha Schiner recognizes nearly 21 species groups (
First evidence of the Af cryptic species complex appeared in the comprehensive taxonomic revision of the genus Anastrepha made by
Morphometric analyses have been a useful technique in detecting morphological differences among organisms to distinguish closely related species of fruit flies (
Despite all evidence gathered by different sources, it is still difficult to set out the taxonomic status of the morphotypes mainly due to two reasons. The first one is that other methodological approaches, such as DNA sequences or sexual compatibility have shown large interpopulation divergences, without allowing full identification of interspecific boundaries; and the second one is that information about the overall distribution of the cryptic species still remains uncertain. This is especially true for morphotypes occurring in the North and Central Andes, and for the Brazilian morphotypes.
According to
Another crucial issue for the resolution of this cryptic species complex is understanding the distributional patterns of their morphotypes.
Given this scenario, systematic studies that identify the incidence areas of the different Af morphotypes throughout the Neotropical region are needed. Increasing the number of samples from Colombia, Ecuador and Peru will confirm previous evidence that suggests that biogeographical and ecological factors in these countries, contribute to the understanding of the distributional patterns of the morphotypes. As such, the goals of this study were to explore phenotypic relationships among different morphs of the Af complex in the Mesoamerican and Pacific biogeographical dominions; to make comparisons of the usefulness of the linear morphometrics and geometric morphometry of the wing shape for delimitation of the morphotypes; and to describe their distributional patterns throughout the biogeographical provinces currently recognized.
We used samples from forty populations obtained from different sources. Most of them were collected from nature directly on their hosts and afterwards reared to adult specimens in the laboratory. Others were collected in McPhail traps baited with hydrolyzed protein, and in few cases, we analyzed samples from laboratory strains established for long time at the Seibersdorf facilities of the FAO/IAEA Agriculture and Biotechnology Laboratories (Austria). Specific data of collection, country, location, and specimens examined are listed in Table
List of samples examined of the Anastrepha fraterculus along the Neotropics, showing data of location, georeferentiation, and source of sample
Sample-Key | Country | Locality | Altitude (m) | Coordinates | Source | N Linear | N Geometrics |
---|---|---|---|---|---|---|---|
MEX-Jica | Mexico | La Jicayana | 400 | 19°21'44"N, 96°39'23"W | McPhail trap | 10 | 10 |
MEX-Teoc | Mexico | Tejería | 980 | 19°23'14"N, 96°36'59"W | Psidium guineense | 10 | 10 |
MEX-Apaz | Mexico | Apazapan | 250 | 19°17'00"N, 96°39'23"W | McPhail trap | 10 | 10 |
MEX-Coat | Mexico | Coatepec | 1200 | 19°27'25"N, 96°57'29"W | Syzygium jambos | 10 | 10 |
MEX-Tuxt | Mexico | Los Tuxtlas | 160 | 18°35'06"N, 95°04'12"W | Psidium guajava | 10 | 10 |
MEX-Chis | Mexico | San Vicente | 1400 | 16°11'50"N, 92°02'57"W | Psidium guajava | 10 | 10 |
MEX-Tap | Mexico | Tapachula | 150 | ND | Seibersdorf Lab-strain | 6 | 6 |
MEX-QRoo | Mexico | Chunhuhub | 30 | 19°37'39"N, 88°38'56"W | McPhail trap | 10 | 10 |
GUA-City | Guatemala | Guatemala City | 1500 | 14°36'51"N, 90°32'22"W | Psidium guajava | 15 | 15 |
PAN-LCam | Panama | La Campana | 61 | 08°44'16"N, 79°51'29"W | Psidium guajava | 15 | 15 |
PAN-BCol | Panama | Barro Colorado Is. | 125 | 09°09'08"N, 79°50'47"W | Eugenia uniflora | 17 | 17 |
VEN-Corr | Venezuela | Corrales | 40 | 10°44'35"N, 71°21'10"W | McPhail trap | 15 | 15 |
VEN-LMit | Venezuela | Loma Mitimbís | 1570 | 09°16'57"N, 70°14'59"W | Rubus glaucus | 15 | 15 |
VEN-DDiaz | Venezuela | Diego Díaz | 1640 | ND | Eriobotrya japonica | 15 | 15 |
VEN-SDom | Venezuela | Santo Domingo | 2500 | 08°57'37” N, 71°02'54"W | Coffea arabica | 15 | 15 |
VEN-Tig | Venezuela | Tiguanín | 1900 | ND | Psidium caudatum | 15 | 15 |
COL-Cund | Colombia | La Mesa | 1350 | 04°38'09"N, 74°27'21"W | McPhail trap | 10 | 10 |
COL-Tol | Colombia | Vereda Gamboa | 1600 | 04°26'11"N, 75°11'29"W | Seibersdorf Lab-strain | 15 | 15 |
COL-Bar | Colombia | Barbosa | 1880 | 05°55'57"N, 73°37'16"W | Psidium guajava | 13 | 16 |
COL-Cach | Colombia | Tocarema alto | 1850 | 04°45'01"N, 74°23'01"W | Coffea arabica | 20 | 20 |
COL-Duit | Colombia | Duitama | 2569 | 05°49'29"N, 73°04'29"W | Acca sellowiana | 20 | 20 |
COL-Rold | Colombia | La Aguada | 1764 | 04°23'05"N, 76°13'20"W | Coffea arabica | 20 | 20 |
COL-Lun | Colombia | El Guabo | 1704 | 01°36'53"N, 77°07'53"W | Coffea arabica | 20 | 20 |
COL-Pen | Colombia | Pensilvania | 2091 | 05°22'03"N, 75°09'29"W | Acca sellowiana | 20 | 21 |
COL-Sev | Colombia | Sevilla | 1556 | 04°17'19"N, 75°54'23"W | Coffea arabica | 20 | 20 |
COL-Sibu | Colombia | Fatima | 2136 | 01°12'05"N, 76°54'48"W | Ps. acutangulum | 20 | 20 |
COL-Ibag | Colombia | Ibagué | 1433 | 04°24'53"N, 75°18'50"W | Lab colony-U Tolima | 20 | 20 |
ECU-Agro | Ecuador | Km39 via la Costa | 7 | 01°57'15"S, 79°55'17"W | McPhail trap | 17 | 20 |
ECU-Guay | Ecuador | Guayaquil | 80 | 02°12'13"S, 79°53'50"W | Ps. guajava | 14 | 15 |
ECU-Bab | Ecuador | Recinto Tauín | 91 | 01°45'29"S, 79°26'50"W | McPhail trap | 20 | 20 |
ECU-Chac | Ecuador | Chacras | 370 | 03°26'51"S, 79°49'53"W | McPhail trap | 20 | 20 |
ECU-Chot | Ecuador | Ambuquí | 1550 | 00°26'50"N, 78°00'18"W | McPhail trap | 20 | 20 |
ECU-Per | Ecuador | Perucho | 1861 | 00°06'48"N, 78°25'33"W | McPhail trap | 20 | 20 |
ECU-Pich | Ecuador | Guayllabamba | 2176 | 00°03'47"S, 78°20'56"W | McPhail trap | 20 | 20 |
ECU-Pat | Ecuador | Patate | 2034 | 01°19'04"S, 78°30'44"W | McPhail trap | 20 | 20 |
PER-Piu | Peru | Piura | 35 | 05°12'00"S, 80°37'00"W | Seibersdorf Lab-strain | 15 | 15 |
PER-LMol | Peru | La Molina | 300 | 12°05'21"S, 76°55'41"W | Seibersdorf Lab-strain | 15 | 15 |
PER-Chon | Peru | Chongona | 1502 | 12°45'49"S, 72°36'15"W | McPhail trap | 7 | 10 |
PER-Echa | Peru | Puente Echarate | 941 | 12°46'10"S, 72°34'37"W | McPhail trap | 11 | 14 |
PER-VSag | Peru | Valle Sagrado | 2859 | 13°19'00"S, 72°05'21"W | McPhail trap | 17 | 17 |
Permanent mounting slides were made prior to observations. Female aculeus was cleaned in a boiling solution, consisting of 10% sodium hydroxide, for approximately 15–20 min; in addition the right wing of each specimen was cut from its base. After that, structures were washed with distilled water and further dehydrated by gradual alcohol series (50, 70, 100% by holding them for 20 min at each step), placed in xylene 2–3 min, and immediately mounted with Canada balsam. Digital images of the mesonotum and wing were made with a digital camera (Olympus C5050) adapted to stereomicroscope (Olympus SZX7); and images of the aculeus were performed using an optical microscope (Olympus BX41) with objective 40X. Permanent slides and pinned voucher specimens of the studied samples were deposited at the Entomological Collections of the Instituto de Ecología AC (Xalapa, Mexico), Universidad del Tolima (Ibagué, Colombia), and the Universidad de las Fuerzas Armadas – ESPE (Quito, Ecuador).
In total, 612 female specimens were examined, considering 27 morphometric traits of structures such as mesonotum, aculeus, and wing. Variables as linear distances between two points, as ratios of two variables, and qualitative features of wing pattern were assessed following methods described by
Mesonotum (Figure
Aculeus (Figure
Wing (Figure
Additional variables of ratios between two measurements were assessed as follows: X1) aculeus length/mesonotum length (A1/M1); X2) aculeus length/wing length (A1/W1); X3) mesonotum length/wing length (M1/W1); X4) mesonotum length/mesonotum width (M1/M2); X5) width at beginning of serrated section/length of serrated section (A3/A5); X6) mesonotum width/wing length (M2/W1).
Eighteen homologous landmark coordinates were digitized on the wings. A total as 626 females belonging to 40 populations distributed from Mexico through Central America, Venezuela, Colombia, Ecuador and Peru were examined.
Landmarks were determined by the intersection or termination of wing veins as follows:
1) junction of humeral and costal veins; 2) subcostal break along costal vein; 3) apex of vein R1; 4) apex of vein R2+3; 5) apex of vein R4+5; 6) apex of vein M; 7) apex of vein CuA1 on posterior margin; 8) apex of vein CuA2 on posterior margin; 9) basal bifurcation of R2+3 and R4+5; 10) junction of R4+5 and cross vein r-m; 11) basal angle of cell bm; 12) junction of M and cross-vein dm-bm; 13) junction of M and cross vein r-m; 14) junction of M and cross-vein dm-cu; 15) junction of CuA1 and Cu2; 16) junction of CuA1 and cross vein bm-cu; 17) junction of CuA1 and dm-cu; 18) junction of A and Cu2 (= apex of cell bcu) (Figure
Linear measurements and the landmark coordinates were acquired from digitized images of wing, aculeus and mesonotum using the TPS DIG software package (
The wing shape information was extracted by the generalized Procrustes superimposition analysis, which is used to remove non-shape variation by scaling all specimens to unit size, translating to a common location and rotating them to their corresponding landmarks lined up as closely as possible (
The exploratory canonical variate analysis (CVA) of linear morphometrics, applied to 40 populations along the Mesoamerican and Pacific Neotropical dominions showed significant differences among them (F = 9.40; Wilk’s lambda < 0.0001; DF = 39/547; p < 0.0001). The tree of similarities computed from the Squared Mahalanobis distance matrix, supported the presence of six well-differentiated morphotype clusters: the Mexican, Venezuelan, Andean, and Peruvian (previously established by
The predictive model of linear morphometrics showed that centroid means for the Andean, Peruvian and Ecuadorian morphotypes were mainly differentiated by the CV-1 scores, which contributed with 61.5% of the differentiation. The CV-2 accounted for 19.6%, distinguishing the East-Peru sample. The CV-3 scores accounted for only 8.4% of the variation among groups (Table
Chi-Square tests with successive roots removed. Results of five significant variates produced by two morphometric models.
Model | Function | Eigenvalue | Canonical R | % Variance | % Cumulative |
---|---|---|---|---|---|
Linear morphometrics | 1 | 10.451 | 0.955 | 61.5 | 61.5 |
2 | 3.333 | 0.877 | 19.6 | 81.1 | |
3 | 1.442 | 0.768 | 8.4 | 89.5 | |
4 | 1.156 | 0.732 | 6.8 | 96.3 | |
5 | 0.624 | 0.620 | 3.7 | 100 | |
Geometric morphometrics | 1 | 2.996 | 0.866 | 43.5 | 43.5 |
2 | 2.171 | 0.827 | 31.5 | 75.0 | |
3 | 1.295 | 0.751 | 18.8 | 93.8 | |
4 | 0.309 | 0.486 | 4.5 | 98.3 | |
5 | 0.120 | 0.328 | 1.7 | 100.0 |
Means and Standard Deviations for all measurements of the morphotypes encountered. Linear measures are in mm, except qualitative traits (W5, W6, A7), and ratios (A9, A10, A11, W7, X1, X2, X3, X4, X5, X6). See methods for explanations.
Mexican | Venezuelan | Andean | Peruvian | Ecuadorian | East-Peru | |
---|---|---|---|---|---|---|
A1 | 1.773 ± 0.10 | 1.945 ± 0.05 | 1.801 ± 0.10 | 1.68 ± 0.07 | 1.905 ± 0.12 | 1.727 ± 0.06 |
A2 | 0.123 ± 0.01 | 0.131 ± 0.01 | 0.123 ± 0.01 | 0.120 ± 0.01 | 0.136 ± 0.01 | 0.116 ± 0.01 |
A3 | 0.087 ± 0.01 | 0.093 ± 0.01 | 0.080 ± 0.01 | 0.079 ± 0.01 | 0.083 ± 0.01 | 0.077 ± 0.01 |
A4 | 0.117 ± 0.01 | 0.142 ± 0.01 | 0.120 ± 0.01 | 0.114 ± 0.01 | 0.128 ± 0.01 | 0.122 ± 0.01 |
A5 | 0.161 ± 0.01 | 0.178 ± 0.01 | 0.126 ± 0.01 | 0.132 ± 0.01 | 0.132 ± 0.01 | 0.143 ± 0.01 |
A6 | 0.284 ± 0.02 | 0.328 ± 0.02 | 0.253 ± 0.02 | 0.252 ± 0.01 | 0.268 ± 0.02 | 0.271 ± 0.02 |
A7 | 11.83 ± 1.52 | 14.13 ± 0.77 | 10.97 ± 1.16 | 13.11 ± 1.12 | 10.80 ± 0.97 | 9.65 ± 0.63 |
A8 | 0.277 ± 0.02 | 0.32 ± 0.01 | 0.250 ± 0.08 | 0.246 ± 0.01 | 0.260 ± 0.02 | 0.265 ± 0.02 |
A9 | 0.730 ± 0.10 | 0.803 ± 0.06 | 0.954 ± 0.14 | 0.866 ± 0.10 | 0.974 ± 0.14 | 0.856 ± 0.11 |
A10 | 0.157 ± 0.01 | 0.165 ± 0.01 | 0.139 ± 0.04 | 0.146 ± 0.01 | 0.137 ± 0.01 | 0.153 ± 0.01 |
A11 | 0.420 ± 0.03 | 0.445 ± 0.02 | 0.485 ± 0.04 | 0.463 ± 0.03 | 0.491 ± 0.04 | 0.460 ± 0.03 |
W1 | 6.287 ± 0.51 | 7.033 ± 0.26 | 6.653 ± 0.50 | 6.383 ± 0.31 | 7.089 ± 0.35 | 7.521 ± 0.41 |
W2 | 2.681 ± 0.24 | 2.903 ± 0.12 | 2.837 ± 0.23 | 2.785 ± 0.17 | 3.002 ± 0.15 | 3.126 ± 0.20 |
W3 | 0.441 ± 0.04 | 0.411 ± 0.03 | 0.314 ± 0.04 | 0.366 ± 0.03 | 0.300 ± 0.03 | 0.454 ± 0.03 |
W4 | 1.401 ± 0.13 | 1.459 ± 0.10 | 1.317 ± 0.17 | 1.429 ± 0.18 | 1.749 ± 0.11 | 1.936 ± 0.13 |
W5 | 1.16 ± 0.37 | 1.93 ± 0.26 | 1.98 ± 0.12 | 2.00 ± 0.00 | 2.00 ± 0.00 | 1.59 ± 0.51 |
W6 | 1.00 ± 00 | 1.00 ± 0.00 | 1.70 ± 0.46 | 1.77 ± 0.42 | 1.62 ± 0.49 | 1.00 ± 0.00 |
W7 | 0.426 ± 0.01 | 0.413 ± 0.01 | 0.427 ± 0.02 | 0.436 ± 0.01 | 0.424 ± 0.01 | 0.415 ± 0.01 |
M1 | 2.884 ± 0.24 | 3.159 ± 0.12 | 2.879 ± 0.26 | 3.061 ± 0.17 | 3.083 ± 0.20 | 3.005 ± 0.22 |
M2 | 1.900 ± 0.16 | 2.103 ± 0.09 | 1.856 ± 0.19 | 1.987 ± 0.11 | 2.036 ± 0.13 | 1.984 ± 0.14 |
M3 | 1.815 ± 0.15 | 2.007 ± 0.08 | 1.792 ± 0.19 | 1.925 ± 0.12 | 1.992 ± 0.12 | 1.910 ± 0.15 |
X1 | 0.617 ± 0.04 | 0.616 ± 0.02 | 0.628 ± 0.04 | 0.550 ± 0.03 | 0.621 ± 0.06 | 0.576 ± 0.04 |
X2 | 0.283 ± 0.02 | 0.277 ± 0.01 | 0.271 ± 0.01 | 0.263 ± 0.01 | 0.269 ± 0.02 | 0.231 ± 0.02 |
X3 | 0.459 ± 0.02 | 0.449 ± 0.01 | 0.433 ± 0.02 | 0.479 ± 0.02 | 0.434 ± 0.02 | 0.399 ± 0.02 |
X4 | 1.520 ± 0.09 | 1.503 ± 0.04 | 1.555 ± 0.06 | 1.541 ± 0.06 | 1.518 ± 0.08 | 1.515 ± 0.05 |
X5 | 0.540 ± 0.04 | 0.523 ± 0.04 | 0.635 ± 0.05 | 0.598 ± 0.04 | 0.630 ± 0.07 | 0.537 ± 0.04 |
X6 | 0.303 ± 0.02 | 0.299 ± 0.01 | 0.279 ± 0.01 | 0.311 ± 0.01 | 0.287 ± 0.01 | 0.264 ± 0.01 |
Valid N | 123 | 15 | 258 | 101 | 98 | 17 |
MANOVA tests showed significant overall differences among morphotypes based on linear morphometry (F = 366.73; Wilk’s lambda = 0.0024; DF = 25/2237; p < 0.0001), and all Hotelling’s pairwise comparisons (post hoc) also led to significant differences (p < 0.0001). Furthermore, reliability of morphotypes based on the wing shape also proved to be statistically significant (F = 159.93; Wilk’s lambda = 0.0235; DF = 25/2289; p < 0.0001), as well as all Hotelling’s pairwise comparisons between each other (p < 0.0001).
Morphological similarities through the Squared Mahalanobis Distance matrix (SMD) were assessed by pairwise comparisons among morphotypes. For example, closer distances were noted between morphs such as Ecuadorian vs. Andean (SMD = 15.2), and Peruvian vs. Andean (SMD = 19.7); or moderate distances between Peruvian vs. Ecuadorian (SMD = 37.9), and Mexican vs. Venezuelan (SMD = 37.7) (Table
Squared Mahalanobis Distances from linear morphometric data produced by pairwise comparisons among morphotypes from Mesoamerica and Pacific Neotropical dominions.
Mexican | Venezuelan | Andean | Peruvian | Ecuadorian | East-Peru | |
---|---|---|---|---|---|---|
Mexican | 0 | 37.7 | 62.8 | 54.6 | 87.0 | 84.3 |
Venezuelan | 0 | 50.8 | 48.1 | 67.0 | 100.7 | |
Andean | 0 | 19.7 | 15.2 | 81.2 | ||
Peruvian | 0 | 37.9 | 108.0 | |||
Ecuadorian | 0 | 80.1 | ||||
East-Peru | 0 |
Classification matrix of individuals by morphotypes according to tested models: Above line: Linear morphometrics. Below line: Geometric morphometrics. Rows: observed classifications; Columns: Predicted classifications.
% Correct | Mexican | Venezuelan | Andean | Peruvian | Ecuadorian | East-Peru | N | |
---|---|---|---|---|---|---|---|---|
Mexican | 93.5 | 115 | 7 | 1 | 0 | 0 | 0 | 123 |
Venezuelan | 100.0 | 0 | 15 | 0 | 0 | 0 | 0 | 15 |
Andean | 96.1 | 0 | 0 | 248 | 1 | 9 | 0 | 258 |
Peruvian | 97.0 | 0 | 0 | 3 | 98 | 0 | 0 | 101 |
Ecuadorian | 96.9 | 0 | 0 | 3 | 0 | 95 | 0 | 98 |
East-Peru | 100.0 | 0 | 0 | 0 | 0 | 0 | 17 | 17 |
Linear model | 96.1 | 115 | 22 | 255 | 99 | 104 | 17 | 612 |
Mexican | 87.8 | 108 | 9 | 0 | 4 | 0 | 2 | 123 |
Venezuelan | 100.0 | 0 | 15 | 0 | 0 | 0 | 0 | 15 |
Andean | 87.4 | 6 | 5 | 229 | 4 | 18 | 0 | 262 |
Peruvian | 95.2 | 0 | 0 | 1 | 100 | 4 | 0 | 105 |
Ecuadorian | 88.5 | 0 | 0 | 11 | 1 | 92 | 0 | 104 |
East-Peru | 100.0 | 0 | 0 | 0 | 0 | 0 | 17 | 17 |
Geometric model | 89.6 | 114 | 29 | 241 | 109 | 114 | 19 | 626 |
Moreover, the predictive model based on the CVA of the wing shape showed that 43.5% of the variability can be explained by the first canonical variable (CV-1), which recognizes the closely linked Andean and Ecuadorian morphotypes, and in turn, is clearly divergent from others. The second canonical variable (CV-2) described 31.5% of differences, recognizing the Mexican and Venezuelan morphotypes near each other, but differing from the Peruvian morphotype. The third canonical variable (CV-3) accounted for only 18.8% of the variability among groups. These wing shape variations are represented by the wireframes of morphotypes encountered, showing the change of the shape expected along the first two canonical variables (Table
Average scores for the first two canonical variates (CV1 and CV2) derived from CVA for the total variation of wing shape between morphotypes of the Af complex. Wireframes showing the shape changes (red lines) from the consensus configuration of landmarks (blue lines) to each extreme negative and positive of CV scores.
The allometric variation of the wing shape assessed by multiple regression of log-centroid size vs. shape scores, revealed significant differences (p < 0.0001), proving that wing size predicted for only 2.26% of the total shape variation. However though this test proved to be significant it is considered relatively minor given the low percentage shown (Figure
In accordance with the results from previous cluster analysis of the 40 populations examined, morphotypes were linked at higher distance forming three different phenotypical groups herein called the Meso-Caribbean, Andean and Brazilian phenotypic lineages. Multivariate regression analysis (MANOVA) applied to scores obtained from the CVA, proved that accuracy of lineages was highly significant. The linear model showed highly significant differences between lineages (F = 1150.8; Wilk’s lambda = 0.0437; DF = 4/1216; p < 0.0001), and among all pairwise comparisons (Hotelling’s test p < 0.0001). The predictive model, using the geometric method, also demonstrated highly significant differentiation between lineages (F = 433.3; Wilk’s lambda = 0.1746; DF = 4/1244; p < 0.0001) and all paired comparisons among them as well (Hotteling’s test, p < 0.0001). Mahalanobis distances exhibited remarkable divergence when contrasting morphotypes from distinct lineages; for instance, pairwise comparisons between East-Peru (Brazilian lineage) with all other morphotypes (SMD = 80.1–108), or distances among samples from the Andean lineage vs. the Meso-Caribbean lineage (SMD = 48.1–87.0) (Table
Mesoamerican-Caribbean lineage (shortly named Meso-Caribbean). It clustered all samples from Mexico, Central America, and the Caribbean coast of Venezuela. This lineage consisted of the two vicariant Mexican and Venezuelan morphotypes (sensu
Scatter plots of individuals tested by CVA grouping samples by distributional areas: 9a–b Mesoamerican-Caribbean lineage represented by 12 populations from Mexico, Guatemala, and Panama, including the single Venezuela lowland for comparisons 10a–b Five populations from Venezuela 11a–b Eleven populations from Colombia a linear morphometrics b geometric morphometrics of wing-shape. Confidence ellipses 95%.
This lineage exhibited distinctive morphological features such as the aculeus length (A1 = 1.77–1.95 mm); wider aculeus tip at beginning of serrated section (A3 = 0.087–0.093 mm); longer serrated section (A5 = 0.161–0.178 mm); ratio of non-serrated section/aculeus tip (A11 = 0.420–0.445); and lowest ratio of width/length of serrated section (X5 = 0.523–0.540), like specimens of the Brazilian lineage. Remarkable qualitative features in the wing pattern were also recorded: the typical Costal, S- and V- bands are broad and heavily colored; the upper connection between arms of V- band (W6) in nearly 100% of specimens examined; and wider apical section of S- band (W3 = 0.411–0.441 mm). In the Mexican morphotype, aculeus tip constriction at beginning of serrated section is almost unnoticeable, and connection between S- and V- bands is always present; whereas in the Venezuelan morphotype S- and V- band connection is typically absent in most specimens, and the aculeus tip wider with numerous marginal teeth (A7 = 14.1 teeth per side) (Figures
Typical shape of the acuelus tip in morphotypes from the Meso-American and Pacific dominions. Mexican morphotype: 14 Mexico-Apazapan 15 Guatemala-City 16 Panama-La Campana. Venezuelan morphotype: 17 Venezuela-Corrales. Andean morphotype: 18 Venezuela-Loma Mitimbis 19 Colombia-Barbosa. Peruvian morphotype: 20 Ecuador-Agroficial 21 Peru-La Molina. Ecuadorian morphotype: 22 Ecuador-Chota 23 Peru-Echarate. Brazilian lineage: 24 Peru-Valle Sagrado 25 Argentina-Tucuman. Scale bar 0.05 mm.
Typical wing patterns in morphotypes from the Mesoamerican and Pacific dominions. Mexican morphotype: 26 Mexico-Apazapan 27 Guatemala-City 28 Panama-La Campana. Venezuelan morphotype: 29 Venezuela-Corrales. Andean morphotype: 30 Venezuela-Loma Mitimbis 31 Colombia-Barbosa. Peruvian morphotype: 32 Ecuador-Agroficial 33 Peru-La Molina. Ecuadorian morphotype: 34 Ecuador-Chota 35 Peru-Echarate. Brazilian lineage: 36 Peru-Valle Sagrado, 37 Argentina-Tucuman.
Andean lineage. It comprises three clusters of samples: a) the Andean morphotype grouped all 15 populations coming from high mountains of Venezuela and Colombia; b) the Peruvian morphotype clustered six lowland populations along the Pacific coast of Ecuador and Peru; and c) the Ecuadorian morphotype, here recognized by the first time, including six highland populations from Ecuador and Peru. We highlight some variables, which may distinguish the morphs of this lineage from others: the apical section of S- band extremely narrow (W3 = 0.300–0.366 mm); S- and V- band connection (W5) missing in near 97% of the specimens examined; V- band arms upper connection (W6) absent in nearly one half of the specimens; and higher ratio between width/length of serrated section (X5 = 0.598–0.635). The Peruvian morph exhibited higher average teeth on the aculeus tip (A7 = 13.1 teeth per side) when compared to Ecuadorian and Andean morphotypes (A7 = 10.8, 10.9 teeth per side, respectively). The Andean morph showed a strong narrowing of apical section of S- band, in addition to distal arm of V- band diffuse and reduced (Figures
Brazilian lineage. It was recognized by a single population from the high mountains of the East-Andean region in Peru, which showed a clear differentiation from all other samples studied, and a preliminary analysis placed it closely related to the Brazilian morphs (sensu
The dendrogram of morphometric similarities also provided evidence that more than one morphotype could occur in some South American countries located in the Pacific dominion. Therefore, further discriminant analyses were performed separately.
Venezuela. Samples from five locations were considered for the analyses and the results from both linear and geometric morphometry were almost identical. The single population examined of the Caribbean coast (Ven-Corrales) belonged to the Venezuelan morphotype (Meso-Caribbean lineage), and it was distinguished from a second group comprising all four populations coming from the highlands, identified as the Andean morphotype (Andean lineage) (Figure
Colombia. Linear morphometric analysis grouped all 11 Colombian populations under the Andean morphotype. Nevertheless, the wing shape analysis revealed three partially differentiated groups: one cluster with individuals from 9 populations, a second sluster with individuals from the laboratory strain of the Vienna facilities (Col-Tolima), and the other one from Ibagué (Col-Ibagué) (Figure
Ecuador. The linear morphometrics and wing shape analyses applied to eight populations from Ecuador yielded identical results, forming two distinct morphological clusters inhabiting this country. The four lowland samples were closely related to each other within the Peruvian morphotype (sensu
Peru. Both morphometric techniques applied to five populations analyzed of this country revealed the presence of three different morphological clusters. The first one comprised two lowland samples classified into the Peruvian morphotype (Per-Piura, Per-La Molina). The second cluster was represented by two samples from the highlands (Per-Echarate, Per-Chongona) and belonged to the Ecuadorian morphotype. The third morphological entity, consisting of a single population from the East-region of the Andes (Per-Valle Sagrado), proved to be distinct from all other samples examined, tentatively related to the Brazilian-1 morphotype within the Brazilian lineage (Figure
Distributional patterns based in the current classification of the Neotropical biogeographic provinces (sensu
Distribution of the morphotypes through biogeographical provinces of the Mesoamerican and Pacific dominions (sensu
Morphotype | Biogeographical Sub-region | Biogeographical Province | Country | Sample-Key |
---|---|---|---|---|
Mexican | Mesoamerica | Veracruzan | Mexico | MEX-Jica |
Mexico | MEX-Teoc | |||
Mexico | MEX-Apaz | |||
Mexico | MEX-Coat | |||
Mexico | MEX-Tuxt | |||
Pacific Lowlands | Mexico | MEX-Tap | ||
Yucatan Peninsula | Mexico | MEX-QRoo | ||
Mex Tran Zone | Chiapas Highlands | Mexico | MEX-Chis | |
Guatemala | GUA-City | |||
Pacific | Guatuso-Talamanca | Panama | PAN-Lcam | |
Panama | PAN-Bcol | |||
Venezuelan | Pacific | Guajira | Venezuela | VEN-Corr |
Andean | Pacific | Magdalena | Venezuela | VEN-Lmit |
Venezuela | VEN-DDiaz | |||
Venezuela | VEN-Sdom | |||
Venezuela | VEN-Tig | |||
Colombia | COL-Cund | |||
Colombia | COL-Tol | |||
Colombia | COL-Bar | |||
Colombia | COL-Cach | |||
Colombia | COL-Duit | |||
Colombia | COL-Pen | |||
Colombia | COL-Ibag | |||
Cauca (north) | Colombia | COL-Rold | ||
Colombia | COL-Lun | |||
Colombia | COL-Sev | |||
Colombia | COL-Sibu | |||
Ecuadorian | Pacific | Cauca (south) | Ecuador | ECU-Chot |
Ecuador | ECU-Per | |||
Ecuador | ECU-Pich | |||
Ecuador | ECU-Pat | |||
South Brazilian | Yungas | Peru | PER-Chon | |
Peru | PER-Echa | |||
Peruvian | Pacific | Western Ecuador | Ecuador | ECU-Guay |
Ecuador | ECU-Agro | |||
Ecuador | ECU-Baba | |||
Ecuador | ECU- Chac | |||
Ecuadorian | Peru | PER-Piu | ||
S-Am Tran Zone | Desert | Peru | PER-LMol | |
Brazilian complex | S-Am Tran Zone | Puna | Peru | PER-VSag |
The Andean morphotype only occurs in the Pacific dominion along the Magdalena province, occupying the highlands of Venezuela (from 1570–2500 m altitude) and Colombia (from 1350–2569 m); it was also found in several Colombian locations in the north of the Cauca province (Roldanillo, La Union, Sevilla, and Sibundoy). However, in the Colombian Pacific lowlands represented by the Chocó-Darién province, we did not record any sample of the Af complex so far.
The Peruvian morphotype was distributed throughout the Pacific Coastal lowlands from Ecuador (7–370 m) and Peru (35–300 m), into the Western-Ecuador and Ecuadorian provinces (Pacific dominion), and the Desert province of the South American Transition Zone. The Ecuadorian morphotype exhibited a distribution along the mountains of the south of Cauca province in the inter-Andean valleys from Ecuador (1550–2176 m), together with two other Peruvian highland samples (Per-Chongona, Per-Echarate) located at 941–1502 m, respectively, in the East-side of the Andes within the Yungas province (South Brazilian dominion). A single population sample was characterized as belonging to the Brazilian lineage, and it was collected in Cusco at the Inca region called Sacred Valley (2859 m), located in the East-side of the Andes into the Puna province of the South American Transition Zone.
Results showed that the nominal species Anastrepha fraterculus (Wiedemann) includes several cryptic species in concordance with previous morphometric findings (
Linear and geometric morphometric analyses showed similar results, both demonstrating to be useful for diagnosis and recognition of morphotypes presumably representing the cryptic species of the Af complex. However, we should also mention that some differences were noted. For instance, differences between samples reared from laboratory colonies, originally stemmed from the same area in Colombia (Col-Tolima, Col-Ibagué) proved to be divergent in wing shape between each other. This is probably due to laboratory strains facing phenotypic selection under artificial conditions over many generations. Therefore, it is advisable to use wild samples for identification of natural morphs, especially if geometric morphometrics is applied. Wing shape analysis also differentiated two Panamanian samples (Pan-La Campana, Pan-B Colorado) from other populations belonging to the Meso-Caribbean lineage, even though they belonged to field collections. This highlights the need to further investigate other samples from that region to assess natural variation.
It could be argued however, that other factors may have influenced the ultimate morphological phenotype of the wing shape of flies. In particular, altitude has been found to have an impact on the wing shape of the potato moth (
Species boundaries are related with the extent and limits of gene flow, the selection intensities on ecologically or reproductively functional phenotypes across the species range, and their genetic architecture, all indispensable pieces of information for predicting the course of early lineage divergence and the origins of new species (
By contrast, in the evolutionary species concept defined as “a single lineage of ancestor – descendant populations, which maintains its identity from other such lineages and which has its own evolutionary tendencies and historical fate” (sensu
In the broad sense, the monophyly of the fraterculus species group has been accepted based on morphology (
The occurrence of strong sexual incompatibility between distinct phenotypic lineages also supports the non-monophyly hypothesis. For instance, high levels of pre- and post-zygotic isolation, karyotypic and polytene chromosome differences, and qualitative and quantitative differences in male pheromones were found in two laboratory strains from Argentina and Peru (
In fact, the current study reveals that the Af complex is integrated by eight morphotypes, which are related into three phenotypic lineages that are virtually endemic, as they are restricted to certain regions, and there is no evidence of contact zones among them so far. The Meso-Caribbean lineage is restricted to the Mesoamerican dominion, to part of the Mexican Transition Zone, and also to the northern of Pacific dominion in Central America and the Caribbean coast of Venezuela. The Andean lineage essentially occupies most of provinces in the Pacific dominion and some parts of the South American Transition Zone; while the Brazilian lineage would be distributed along the Parana dominion in the eastern part of Brazil, and the Chacoan dominion in southern Brazil and northern Argentina.
In this regard, there are also historical processes associated to each biogeographical dominion that cannot be neglected, since they explain the own history of the biota they inhabit. According to
The relationship between morphological structure and genotype is complex and poorly understood for most characters, since we need to know if there is a relationship between the morphological characterizations and the real units of evolution (
In this research, the presence of eight morphotypes is established within the Anastrepha fraterculus (Wiedemann) complex, including the first characterization of the Ecuadorian morphotype with samples coming from the mountains of Ecuador and Peru. The morphotypes clustered into three phenotypic lineages we called Meso-Caribbean, Andean, and Brazilian. Based upon their morphological divergence and the current distributional areas, we suggest that these lineages would not have a direct connection with each other and might have evolved separately in these biogeographical regions. In terms of distributional areas or countries, the Mesoamerican dominion was only occupied by the Mexican morphotype. In other countries from the Pacific dominion such as Colombia and Venezuela, two morphotypes were encountered, the Venezuelan inhabiting the Caribbean lowlands of Venezuela, and the Andean in the highlands of both countries. In the territories from Ecuador and Peru, the Peruvian morphotype was found in the lowlands, and the Ecuadorian morphotype in the highlands. Furthermore, in the Eastern side of the Andes in Peru, another morphotype was detected that appears closely related to the morphotypes of the Brazilian lineage.
We are grateful to the International Atomic Energy Agency (IAEA) Vienna, Austria for financial support of this investigation [Research Contracts No. 16080 (VHO), and No. 16069 (NC)]. To Jorge Hendrichs (IAEA) for his support and encouragement along these investigations. NC acknowledges COLCIENCIAS (Ref: 445–2009) for partial support for this research. We thank the comments and suggestions of the two anonymous reviewers and the editor to the first version of the manuscript. We acknowledge David Salas (Agrocalidad, Ecuador) for technical assistance and support for obtaining samples from different locations in Ecuador; and thank Allen L. Norrbom (USDA) and Gary J. Steck (Florida State Collection of Arthropods) for providing three new samples from the high mountains in Peru. This is a contribution to the project “Biodiversity of Diptera for the Neotropical region” of the Instituto de Ecología (Ref: VHO-INECOL-100128).