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 (Norrbom et al. 1999, 2012). One of them is the “fraterculus species group” consisting of 34 species, with some widely distributed in the Neotropics (e.g., A. ludens (Loew), A. obliqua (Macquart), A. suspensa (Loew), and A. fraterculus). One of these nominal species, the “South American fruit fly” Anastrepha fraterculus (Wiedemann) occurs from south of the USA (Texas) through Mexico, Central America to Argentina and represents a cryptic species complex (hereafter denoted as the Af complex).

First evidence of the Af cryptic species complex appeared in the comprehensive taxonomic revision of the genus Anastrepha made by Stone (1942). Since then, other findings from distinct populations along its distributional range were reported supporting this hypothesis, such as differences of karyotypes (Mendes 1958, Bush 1962, Solferini and Morgante 1987, Selivon et al. 2005b); isozyme divergence (Morgante et al. 1980, Steck 1991); DNA sequences (Steck and Sheppard 1993, Smith-Caldas et al. 2001); or studies with multiple approaches including karyotype, isozymes, and morphology (Selivon and Perondini 1998, Selivon et al. 2004, 2005a). Moreover, differences in host range and pest status (Baker 1945, Aluja et al. 2003, Hernández-Ortiz and Morales-Valles 2004, Zucchi 2007); reproductive isolation and sexual incompatibilities (Selivon et al. 1999, 2005a, Vera et al. 2006, Cáceres et al. 2009, Rull et al. 2013, Devescovi et al. 2014); or data on pheromone composition and cuticular hydrocarbon profiles (Břízová et al. 2013, Vaníčková et al. 2015).

Morphometric analyses have been a useful technique in detecting morphological differences among organisms to distinguish closely related species of fruit flies (Adsavakulchai et al. 1999, Khamis et al. 2012, Schutze et al. 2012). Based on adult morphology of the Af complex, Hernández-Ortiz et al. (2004) developed a morphometric technique using linear measurements of the aculeus, wing, and mesonotum for the full recognition of the Mexican morphotype, separating it from other South American samples from Colombia, Brazil and Argentina. Further linear morphometric studies applied to 32 populations from Mexico, Central America, and South America (including Venezuela, Colombia, Ecuador, Peru, Brazil and Argentina) confirmed previous findings, and added the fact that seven morphotypes could be distinguished within the Af complex throughout the Neotropical region (Hernández-Ortiz et al. 2012).

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 Daly (1985), multivariate methods of morphometric analysis (e.g., DFA, PCA) can be widely applied in biology. However, two general kinds of problems may be encountered in canonical variate analysis of morphometric data: a) linear dependence when two or more variables are highly correlated; and b) heteroscedasticity of the covariance matrices (inequality of dispersion matrices). In this sense, ratios have been used for scaling morphometric variables to remove variation in general body size; to express shape by finding the proportion of one dimension of a structure to another; and to express growth in the size of some structure from one instar to the next. Additionally, because linear distance measurements usually are highly correlated with size, much effort was spent in developing methods for size correction, so that size-free shape variables could be extracted and patterns of shape variation elucidated (Bookstein et al. 1985, Sundberg 1989). The most widespread approach of the geometric morphometrics, is to represent each specimen by the relative positions of morphological landmarks, that can be located precisely and establish a one-to-one correspondence among all specimens included in the analysis (Klingenberg 2010). Shape is defined as all the geometric information about a configuration of landmarks and it is extracted by a procedure called Procrustes superimposition, which removes variation in size, position and orientation from the data on landmark coordinates, and which is at the core of geometric morphometrics (Goodall 1991, Dryden and Mardia 1998, Zelditch et al. 2012).

Another crucial issue for the resolution of this cryptic species complex is understanding the distributional patterns of their morphotypes. Morrone (2014) recently revised the biogeographic regionalization of the Neotropical region. The Mesoamerican dominion comprises lowlands of central and southern Mexico, and most of Central America (Guatemala, Belize, Honduras, El Salvador and northern Nicaragua). The Pacific dominion encompasses southern Central America (southeastern Nicaragua to Panama) and northwestern South America (including western Colombia, Ecuador, Peru, northwestern Venezuela, Trinidad and Tobago, and the Galapagos Islands). Contiguous to these dominions, the Mexican Transition Zone (MTZ) occupies an area where the Neotropical and Nearctic regions overlap, corresponding basically to the mountainous areas of central and southern Mexico and northern Central America; and the South American Transition Zone (SATZ) represented by highlands of the Andes between western Venezuela and northern Chile, and central western Argentina (sensuMorrone 2006). In this sense, correlating the occurrence of the different morphotypes to biogeography will add valuable information to delimit the distribution of the species involved.

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.

Results showed that the nominal species Anastrepha fraterculus (Wiedemann) includes several cryptic species in concordance with previous morphometric findings (Hernández-Ortiz et al. 2004, 2012). Specifically, the Af complex consists of eight morphotypes throughout the Neotropics, and in turn, these are related to each other within at least three phenotypic lineages. Major similarities were seen between morphotypes belonging to the same lineage; for example, closer distances were observed between the Mexican and Venezuelan morphotypes (Meso-Caribbean lineage), or between the Andean and the Ecuadorian morphotypes (Andean lineage). The phenotypic proximity between the Ecuadorian and the Andean morphotypes, together with the fact that individuals were partially classified within each other, means that they could have a partial and incomplete isolation.

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 (Hernández et al. 2010); wing shape differences between the fruit flies Rhagoletis pomonella and R. zephyria, were hypothesized to have changed in relation to host associations (Yee et al. 2009). However, we consider this might not be the case within the Af complex for several reasons. Firstly, the nominal species A. fraterculus is highly polyphagous, and host usage, albeit not the same, is highly overlapping among morphotypes (Norrbom 2004, Hernández-Ortiz and Morales-Valles 2004, Zucchi 2007). For example, Psidium guajava L. is a host widespread along its geographic distribution and altitudinal range in Mexico, Central America, the Andean countries, Brazil and Argentina. Secondly, the morphometric analysis performed on A. fraterculus in Mexico distinguished a single Mexican morphotype, even though eight populations from a wide altitudinal range, and belonging to three distinct host species were examined (Hernandez-Ortiz et al. 2004). Thirdly, in the Brazilian territory there are proofs of the occurrence of three morphotypes (Hernández-Ortiz et al. 2012), and evidence of karyotype differentiation and reproductive isolation supporting the existence of distinct species (Selivon et al. 2004, 2005a, 2005b); however all of them feed on guava, among other hosts.

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 (Shaw 1998). In the biological species concept defined as “groups of interbreeding natural populations that are reproductively isolated from other such groups” (sensuMayr 1969), it is not clear that in all sexually reproducing species, reproductive ties such as gene flow between demes provide the major cohesive force. The concept of interbreeding is a rather complex idea, because hybridization can be discussed in terms of reproductive modes but also in terms of speciation, that is, hybridization as “prima facie” evidence for incomplete speciation (Wiley 1981).

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” (sensuWiley 1978, modified from Simpson 1961), all terminal lineages are evolutionary species or descendant of higher taxa represented by their ancestral evolutionary species. Each branch therefore is the result of a speciation event; however, the concept does not preclude a particular ancestral species from surviving a speciation event. In this regard, De Queiroz (1998) noted that the term lineage is used for a single line of direct ancestry and descent, while a clade is a unit consisting of an ancestral species and its descendants, and are monophyletic in terms of their component species, however, lineages can be paraphyletic or even polyphyletic in terms of their lower level components.

In the broad sense, the monophyly of the fraterculus species group has been accepted based on morphology (Norrbom et al. 1999, Norrbom et al. 2012). In several papers it is assumed that reproductive isolation between cryptic species of the Af complex has recently evolved, leaving implicit the idea that those morphospecies had direct relationships, and at some time of its evolutionary history there was an interpopulation divergence among them (Cáceres et al. 2009, Segura et al. 2011, Rull et al. 2013, Devescovi et al. 2014). This is probably true among morphospecies related within each phenotypic lineage that could have a common origin and most likely a more recent evolution. However, from a theoretical perspective, the monophyly of the Af complex has never been tested so far. Conversely, other studies from various methodological sources stated that this species complex is not monophyletic. This assumption is supported by extreme allele differences found between highland and lowland Venezuelan samples (presumably from distinct lineages), being the largest genetic divergence found among samples studied by Steck (1991). The phylogenetic relationships inferred from mtDNA sequences of COI supported the presence of multiple gene pools and the non-monophyly among samples of the nominal species A. fraterculus (Smith-Caldas et al. 2001). In the same way, a phylogenetic relationship analysis based on the nuclear gene period of Anastrepha (Barr et al. 2005) with samples from Venezuela (Mérida and Caracas), Mexico, and Brazil (Sao Paulo) found them to be related in different clades.

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 (Cáceres et al. 2009, Segura et al. 2011) which belong to the Brazilian and Andean lineages, respectively. In addition, pre-zygotic reproductive isolation resulted in strong assortative mating to gene flow among the Mexican morphotype and other populations classified in the Brazilian-1 and Peruvian morphotypes (Rull et al. 2013), all of them belonging to three distinct phenotypic lineages described herein. Moreover, there is strong pre-zygotic isolation through temporal partitioning of mating activity of a Colombian population (Andean morphotype) compared with four other morphotypes spanning from Mexico to Argentina (Devescovi et al. 2014).

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 Hoorn et al. (2010) plate subduction along the Pacific margin caused uplift in the Central Andes (Peruvian and Bolivian Andes) during the Paleogene (65 to 34 Ma). The posterior plate breakup in the Pacific, and subsequent collision with the South American and Caribbean plates, resulted in intensified mountain building in the Northern Andes (Venezuelan, Colombian and Ecuadorian Andes) by the late Oligocene to early Miocene (~ 23 Ma); while plate reorganization ultimately resulted in closing of the Panama Isthmus during the Pliocene (at ~ 3.5 Ma). These data sustain that the origin of the Northern and Central Andes, and their current connection with Mesoamerica, occurred in remarkable different times. This would mean prolonged periods of isolation between morphotypes inhabiting those geographical areas. In this sense, Drew (2004) stated that high levels of endemism in an area would indicate that speciation has occurred in relative isolation over a considerable time.

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 (Shubin and Marshall 2000). This idea is particularly relevant when large numbers of sibling species occur. Therefore, from a practical point of view, it is necessary to understand the mechanisms of reproductive isolation between morphotypes, also as an essential precondition for applying control methods such as the sterile insect technique (SIT). However, from an evolutionary perspective, implications of the non-monophyly of the Af complex prevent making direct inferences about mechanisms of genetic or reproductive divergence among populations, since morphotypes belonging to distinct phenotypic lineages might have evolved independently in different clades.

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).