Resolving cryptic species complexes of major tephritid pests

Abstract An FAO/IAEA Co-ordinated Research Project (CRP) on “Resolution of Cryptic Species Complexes of Tephritid Pests to Overcome Constraints to SIT Application and International Trade” was conducted from 2010 to 2015. As captured in the CRP title, the objective was to undertake targeted research into the systematics and diagnostics of taxonomically challenging fruit fly groups of economic importance. The scientific output was the accurate alignment of biological species with taxonomic names; which led to the applied outcome of assisting FAO and IAEA Member States in overcoming technical constraints to the application of the Sterile Insect Technique (SIT) against pest fruit flies and the facilitation of international agricultural trade. Close to 50 researchers from over 20 countries participated in the CRP, using coordinated, multidisciplinary research to address, within an integrative taxonomic framework, cryptic species complexes of major tephritid pests. The following progress was made for the four complexes selected and studied: Anastrepha fraterculus complex – Eight morphotypes and their geographic and ecological distributions in Latin America were defined. The morphotypes can be considered as distinct biological species on the basis of differences in karyotype, sexual incompatibility, post-mating isolation, cuticular hydrocarbon, pheromone, and molecular analyses. Discriminative taxonomic tools using linear and geometric morphometrics of both adult and larval morphology were developed for this complex. Bactrocera dorsalis complex – Based on genetic, cytogenetic, pheromonal, morphometric, and behavioural data, which showed no or only minor variation between the Asian/African pest fruit flies Bactrocera dorsalis, Bactrocera papayae, Bactrocera philippinensis and Bactrocera invadens, the latter three species were synonymized with Bactrocera dorsalis. Of the five target pest taxa studied, only Bactrocera dorsalis and Bactrocera carambolae remain as scientifically valid names. Molecular and pheromone markers are now available to distinguish Bactrocera dorsalis from Bactrocera carambolae. Ceratitis FAR Complex (Ceratitis fasciventris, Ceratitis anonae, Ceratitis rosa) – Morphology, morphometry, genetic, genomic, pheromone, cuticular hydrocarbon, ecology, behaviour, and developmental physiology data provide evidence for the existence of five different entities within this fruit fly complex from the African region. These are currently recognised as Ceratitis anonae, Ceratitis fasciventris (F1 and F2), Ceratitis rosa and a new species related to Ceratitis rosa (R2). The biological limits within Ceratitis fasciventris (i.e. F1 and F2) are not fully resolved. Microsatellites markers and morphological identification tools for the adult males of the five different FAR entities were developed based on male leg structures. Zeugodacus cucurbitae (formerly Bactrocera (Zeugodacus) cucurbitae) – Genetic variability was studied among melon fly populations throughout its geographic range in Africa and the Asia/Pacific region and found to be limited. Cross-mating studies indicated no incompatibility or sexual isolation. Host preference and genetic studies showed no evidence for the existence of host races. It was concluded that the melon fly does not represent a cryptic species complex, neither with regard to geographic distribution nor to host range. Nevertheless, the higher taxonomic classification under which this species had been placed, by the time the CRP was started, was found to be paraphyletic; as a result the subgenus Zeugodacus was elevated to genus level.


Introduction
Tephritid fruit flies (Diptera: Tephritidae) are among the world's worst pests of agriculture, being of major economic importance in nearly all tropical, subtropical and temperate countries (Cavalloro 1983, White andElson-Harris 1994). By laying their eggs directly into fruit, where the maggots feed and develop, these pest species cause enormous devastation to both food production and international trade in spite of often intensive insecticide applications. They are among the primary causes of poverty, malnutrition and poor production and trade in fresh horticultural commodities in large areas of tropical developing countries, impeding the development of lucrative and labour-intensive fruit and vegetable-based agroindustries in rural areas (Waterhouse 1993, Allwood andLeblanc 1996).
The study of the biology and management of tephritids requires significant international attention to overcome transboundary hurdles and to assist the global community in developing and validating more environment-friendly fruit fly suppression systems to support viable fresh fruit and vegetable production and export industries. Such international attention has resulted in the successful development and validation of a Sterile Insect Technique (SIT) package for the Mediterranean fruit fly, Ceratitis capitata (Wiedemann, 1824) (Dyck et al. 2005). R&D support for this pest species is diminishing due to successful integration of the SIT into area-wide integrated pest management (AW-IPM) programmes to manage C. capitata populations (Enkerlin 2005). On the other hand there is increased demand from Africa, the Asia-Pacific and Latin America to address other major tephritid species or groups of economic importance. Some of these major pest fruit fly species occur within cryptic species complexes that include taxonomically described species that may actually be geographical variants of the same species. Conversely, some fruit fly populations grouped taxonomically within the same pest species display different biological and genetic traits, including reproductive isolation, which suggest that they are different species . This uncertain taxonomic status has important practical implications on the effective development and use of the SIT against such pest complexes where the species under mass-rearing is not the same as the population occurring in the target area. Uncertainty of taxonomic status can also result in the incorrect establishment of trade barriers for agricultural commodities that are hosts of pest tephritids.
The resolution of some of the taxonomic uncertainties that surround major cryptic species complexes is therefore critical both for integrated SIT application and for subtropical and tropical countries to overcome non-tariff trade barriers, enabling them to export their fresh fruit and vegetable commodities to international markets. In particular, it is essential that the sterile males from such species complexes produced in regional fruit fly rearing facilities and destined for release in different countries or regions are behaviourally fully compatible with the target native fruit fly pest populations in the various recipient regions (Cayol et al. 2002). If the taxomomic status of species complexes remained unresolved, it would be difficult or impossible to achieve this desirable goal.
To address these issues, a major international collaboration was initiated in 2010 under the auspices of the Joint Food and Agriculture Organization / International Atomic Energy Agency (FAO/IAEA) Programme on Nuclear Techniques in Food and Agriculture. This paper summarises the goals, achievements and results of this coordinated research project that are compiled in this special issue of Zookeys (2015, Special Issue 540).

Approach
During a Consultants' Meeting, held from the 6 th to 10 th of July 2009 in Vienna, Austria, the potential for conducting co-ordinated R&D in this area was assessed, and the major tephritid pest complexes were discussed and prioritised in terms of economic importance and potential for SIT application. Three complexes, the Anastrepha fraterculus complex (Latin America), the Bactrocera dorsalis complex (Asia and Pacific, Africa), and the Ceratitis FAR (= C. anonae Graham, 1908, C. fasciventris (Bezzi, 1920), C. rosa Karsch, 1887) complex (Africa) were confirmed to be of priority. The possibility that Bactrocera cucurbitae (Coquillett, 1899) (Asia and Pacific, Africa) also represents a species complex was evaluated and considered a lower, but still important, priority. In each of these groups (Figure 1), questions were raised concerning the validity of some of the described species, the capacity to diagnose described species, or the strong a priori evidence that unrecognised sibling taxa may occur.
A proposal for an FAO/IAEA Co-ordinated Research Project (CRP) on "Resolution of Cryptic Species Complexes of Tephritid Pests to Overcome Constraints to SIT Application and International Trade" was formulated and approved for the period 2010-2015. The specific objectives of this CRP were to define, using an integrative taxonomic approach (Schlick-Steiner et al. 2010), the species limits within the target complexes, and to develop robust species-specific diagnostic tools.
This international research network was operated under the IAEA Research Contract Programme and included 22 research teams. Other research teams also par- ticipated directly or indirectly and were fully funded by their institutions and governments. Overall close to 50 researchers from over 20 countries from all continents participated at one time or another during the six years of the CRP (2010)(2011)(2012)(2013)(2014)(2015).
Research networks were established to (1) encourage close collaboration among institutes from developed and developing countries, (2) provide a forum for information exchange between scientists, and (3) embrace a focused approach to the development, capacity building and technology transfer of environment-friendly technologies. A worldwide network of partners provided representative samples of the fruit fly populations in order to assess the genetic diversity throughout the distributional ranges of the members of each complex. A generic protocol for collection and shipment of live and dead insects for vouchering, rearing, morphological and morphometric studies, chemical ecology and molecular assays was developed at the start of the CRP and used for distribution of material between the participating research units. Whenever possible, colonies of populations were established at the FAO/IAEA Agriculture and Biotechnology Laboratories in Seibersdorf, Austria, to be able to carry out field cage cross-mating studies that would not have been acceptable at other locations due to quarantine regulations and the risk of pest establishment.
During the implementation of the CRP, four Research Co-ordination Meetings (RCMs) were held to review research progress and to agree on future research directions and activities: the first RCM in Vienna, Austria from 2-6 August 2010, the second RCM in Brisbane, Australia from 31 January-3 February 2012, the third RCM in Tucumán, Argentina from 26-31 August 2013 and the fourth and final RCM in La Réunion, France from 1-5 June 2015.

Anastrepha fraterculus Complex Situation Analysis
The South American fruit fly, Anastrepha fraterculus (Wiedemann, 1830) s.l., is present in most countries of the Americas from the USA to Argentina (Hernández and Aluja 1993, Steck 1999, Zucchi 2007. Its centre of diversity is the South American subcontinent, where formerly it was thought to occurr in two, possibly unconnected bands: one along the western edge, including both highland and lowland areas of the Andean range, and the other along the east coast. However, recent data indicate its presence in parts of the Brazilian Amazon basin (Zucchi et al. 2011). It has been reported to infest about 110 host plants including major fruit crops (Norrbom and Kim 1988, Zucchi 2000, Norrbom 2004. The presence of this highly destructive pest results in quarantine restrictions for fruit export to many countries (Steck 1999).
The high levels of variability found among different populations throughout the geographical range of A. fraterculus led to the conclusion that it is a complex of cryptic species rather than a single biological entity (Stone 1942, Morgante et al. 1980, Malavasi and Morgante 1982, Solferini and Morgante 1987, Steck 1991, Steck and Sheppard 1993, Selivon 1996, Hernández-Ortiz et al. 2004. Differences have been reported based mainly on morphology, pest status and genetics (including karyotype, isozyme and molecular analyses); these are reviewed in Steck (1999) and some aspects discussed in subsequent studies (McPheron et al. 1999, Gomes Silva 2000, Smith-Caldas et al. 2001, Aluja et al. 2003, Hernández-Ortiz et al. 2004, Barr et al. 2005, Selivon et al. 2005, Goday et al. 2006, Selivon and Perondini 2007, Silva and Barr 2008, Prezotto 2008, Cáceres et al. 2009). However, in order to establish how much of this variation reflects population level variation, and how much reflects unrecognised cryptic species diversity, it is necessary to systematically correlate these genetic and morphological differences with the existence of reproductive isolation and other life history related traits (hosts, demography, etc).
Reproductive incompatibility has been reported both at pre-and post-zygotic levels between some A. fraterculus populations. At the pre-zygotic level, mating compatibility was evaluated among different populations from South America, involving lowland (Peru) and highland (Colombia) areas from the Andean region, and the south-eastern part of the continent (Brazil and Argentina). Most of the populations were shown to have some level of incompatibility with each other and thus appeared sexually isolated. Flies of different populations were often sexually active at different times of the day suggesting different sexual behaviour (Selivon 1996, Vera et al. 2006, Cáceres et al. 2009).
Post-zygotic studies between two populations from Brazil (Selivon et al. 1999) and between one Argentinean population and one Peruvian population (Cáceres et al. 2009) found partial hybrid inviability and sex ratio distortion confirming the existence of post-zygotic barriers. In the former case, cytological, isozyme and molecular studies revealed differences among groups (Malavasi and Morgante 1982, Selivon 1996, Selivon et al. 2005, Goday et al. 2006; while for the latter case, differences between groups were also found in terms of male sex pheromones and karyotypes (Cáceres et al. 2009 (Steck 1991).
Although previous studies provided strong evidence supporting the existence of several biological species, major knowledge gaps still existed in 2010. In particular, the described studies used different methodologies, did not use the same identified biological material and, most importantly, did not include all of the morphotypes. Therefore, in order to be able to formally describe and name these putative species, it was considered critical to apply a standardised, complete set of methodologies to all populations from the entire geographic distribution range in a comprehensive integrative taxonomy study. This would allow the characterization of each putative species and would provide sound diagnostic tools for addressing the related management and trade issues.
Definition of species limits and formal naming of these putative species will be relevant for plant protection authorities in determining which of them may or may not be quarantine pests. This would immediately allow some countries to gain access to international fresh fruit markets for those countries and commodities which can be determined to be outside the geographic and host range of correctly delimited A. fraterculus s.s. In addition, detailed studies on pest status, host range, economic impact and distribution would minimize any possible impact on trade between South American countries. Furthermore, knowing species boundaries and their levels of sexual compatibility within the complex would enable the implementation of the SIT.

Outputs on the Anastrepha fraterculus complex
Colonies from five A. fraterculus morphotypes (Mexican, Andean, Peruvian, Brazilian 1 and Brazilian 3) were established and used for behavioural, chemical, cytological, molecular and larval morphology studies. Linear and geometric morphometry were validated as tools for morphotype discrimination. Comprehensive morphometric studies supported the existence of eight morphotypes: the seven reported previously and a new Equadorian morphotype (Hernández-Ortiz et al. 2012.
Guidelines for performing mating compatibility field cage tests were developed. Reproductive compatibility studies were performed among the five morphotypes. Among the combinations studied, morphotypes were incompatible at the pre-and post-zygotic level (Rull et al. 2012, Devescovi et al. 2014. Male calling and courtship behaviour were recorded for four morphotypes. Sexual behaviour studies helped to identify behavioural characteristics that allowed distinct morphotype descriptions such as time of sexual activity, acoustical signals and sequence of courtship behaviour (Rull et al. 2012, Devescovi et al. 2014. One post-copulatory study revealed differences in sperm storage and remating propensity between the Peruvian and Brazilian 1 morphotypes (Abraham et al. 2014). Hybrid females tended to mate with hybrid males and as a result Segura et al. (2011) suggested that hybridization is a possible speciation mechanism. In all combinations analysed, post-zygotic isolation was found to be weaker than pre-zygotic (Devescovi et al. 2014). Wolbachia was detected in several morphotypes (Cáceres et al. 2009, Lima 2015 and more in-depth studies aiming to characterize these strains are in progress. The chemical profiles of the male pheromones and cuticular hydrocarbons of these five morphotypes were characterized as complex blends that were qualitatively and quantitatively unique for the different morphotypes (Břízová et al. 2013, Gonçalves et al. 2013,c,d, Milet-Pinheiro et al. 2015. The description of the mitotic karyotypes from the Mexican, Colombian and Equadorian morphotypes, from which information was absent or incomplete, allowed confirming that karyotypes are unique for each morphotype (Canal et al. unpublished data). The polytene chromosome map for one morphotype was constructed (Gariou-Papalexiou et al. unpublished data). Internal Transcriber Spacer 1 (ITS 1) was found to be a good molecular marker to identify different groups (Sutton et al. 2015). Microsatellites were developed ) and proved to be successful to discriminate among morphotypes (Lima et al. unpublished data, Manni et al. 2015). The phylogenetic relationships of Andean-Ecuadorian populations were determined with other molecular markers (Ludeña et al. 2011).
Based on all the collected evidence it is now possible to describe four morphotypes as new species with the exception of the three Brazilian morphotypes for which it is still necessary to solve problems with the unknown origin of the holotype male of Anastrepha fraterculus (Wiedemann) and the new Ecuadorian morphotype from which further studies are required for an integrative description . A manuscript with the description of the new species is in preparation (V. Hernández-Ortiz personal communication) and improved diagnostic tools are now available based on morphology, molecular markers, chemical profiles, cytology and sexual behaviour.
The knowledge and gaps identified at the start of the CRP, as well as the progress made by the end of the CRP in addressing the gaps identified for the A. fraterculus complex are summarized in Table 1.

Bactrocera dorsalis Complex Situation Analysis
Across Asia and the Pacific the fruit fly subfamily Dacinae contains some 47 recognised pest species (Drew and Romig 2013). Of these, eight were recognized within the Bactrocera dorsalis complex, with some being the most economically damaging of all pest species within the subfamily (Drew andHancock 1994, Clarke et al. 2005). Losses caused by B. dorsalis complex species include destruction of crops, restriction of international trade, and the establishment of a range of quarantine and regulatory activities carried out by various regional governments.
Background research on these flies has generated data on diagnostics, field surveillance, quarantine strategies, field pest control, and market access protocols (e.g. Tan and Nishida 1996, 1998, Muraji and Nakahara 2002, Naeole and Haymer 2003, Smith et al. 2003, Armstrong and Ball 2005. But the key knowledge gap of the B. dorsalis complex was a lack of consensus on species limits of the major pest species in the complex, particularly B. dorsalis s.s. (Hendel, 1912), B. papayae (Drew & Hancock, 1994), B. philippinensis (Drew & Hancock, 1994), B. carambolae (Drew & Hancock, 1994) and B. invadens (Drew, Tsuruta & White, 2005) (Clarke et al. 2005, Drew et al. 2005, Wee and Tan 2005, Ebina and Ohto 2006, Drew et al. 2008. Failure to resolve the taxonomic status of the members of this complex prevented further development towards SIT integration into AW-IPM programmes against these pest insects and limited international horticultural trade. Background research on the taxonomy of the B. dorsalis complex has been unable to provide definitive identification of some species (Clarke et al. 2005). This has confounded collecting associated host plant records and defining geographic distributions. It was considered absolutely essential that the species be accurately identified to be able to apply AW-IPM field programmes that include a SIT component. Because the trade implications and response systems to detections and/or incursions are different for all members in the complex, "near-enough" identification is, unfortunately, not good-enough. Consequently countries have difficulty overcoming the phytosanitary barriers to export-trade to major importers such as Australia, Japan, Europe, New Zealand, South Africa, and the USA. Another severe problem would arise if one member of the complex is detected or becomes established in a country, but is unable to be differentiated from others in the complex. In this case that country would then be forced to admit that all members of the complex may in fact be present, which would result in extended trade embargoes. Therefore a comprehensive integrative taxonomy approach involving biological, morphological, chemo-ecological and molecular studies of the various members of the B. dorsalis complex were needed to: (1) resolve species limits by seeking a consensus result from different tests; (2) examine congruence between data from the different approaches to either support taxonomic revision or retain existing species status; and (3) develop robust diagnostic tools for the identified species. invadens; no evidence for hybrid incompatibility was detected (Bo et al. 2014).

Outputs on Five Priority Species in the Bactrocera dorsalis Complex
Chemical components and ratios of sex pheromone stored in male rectal gland and emitted during courtship after feeding on methyl eugenol (ME) were determined qualitatively and quantitatively for B. dorsalis s.s., B. invadens, B. papayae and B. philippinensis. The four ccomplex members had identical volatile emission profiles and rectal pheromonal components consisting of 2-allyl-3,4, dimethoxyphenol (DMP) and Econiferyl alcohol (E-CF) (Tan et al. 2011(Tan et al. , 2013, and the ratios of DMP: E-CF were not significantly different between the different members. Probit analysis showed that the responsiveness of these four members to ME was similar as their ED 50 values (= dose at which 50 % of the population responded) were not significantly different (Hee et al. 2015a). However, differences were found for B. carambolae.
Wing shape variation analysed through geometric morphometrics was used for the first time in the B. dorsalis complex (Schutze et al. 2012a,b, Krosch et al. 2013). Variation in wing shape proved to be extremely informative in interpreting variation within the B. dorsalis complex.
Genetic, cytogenetic and molecular analyses of B. dorsalis complex specimens collected across the geographical range were carried out in participating laboratories in Asia, Australia and Europe: Cytogenetics: One of the objectives was to identify and evaluate cytogenetic tools that could help to resolve the taxonomic status of the five taxa under study, focusing on chromosomal rearrangements, especially inversions. For this purpose, mitotic and polytene chromosomes were analysed from colonized specimen representing B. dorsalis s. s., B. papayae, B. philippinensis, B. invadens and B. carambolae. Analysis of mitotic karyotypes could not detect any differences among these five taxa (Yesmin and Clyde 2012, Yesmin 2013, Augustinos et al. 2014, Augustinos et al. 2015, showing that all had the typical B. dorsalis s.s. karyotype as previously described by Hunwattanakul and Baimai (1994). Polytene chromosome maps were developed for the first time of a member of the B. dorsalis complex, i.e. B. dorsalis s.s. (Zacharopoulou et al. 2011a). Subsequent analysis showed that the five members of the complex do not present any chromosomal rearrangements that could be used as diagnostic characters and therefore these taxa can be regarded as homosequential (Augustinos et al. 2014(Augustinos et al. , 2015. Although B. carambolae presented the same mitotic and polytene chromosome karyotype as the other members of the complex, the presence of a high number of minor asynapses in F 1 hybrids of B. dorsalis s.s. × B. carambolae crosses may indicate the presence of small differences in the chromosomal organization among the parental entities. However, these observations cannot be regarded as diagnostic at the species level (Augustinos et al. 2014).
Microsatellites: Microsatellite DNA markers derived from B. dorsalis s.s. were tested on populations of B. dorsalis s.s. from Bangladesh, Cambodia, China (multiple populations), Hawaii (two populations), Laos, Malaysia, Taiwan, and Thailand (multiple populations) (Shi et al. 2012, Krosch et al. 2013, Aketarawong et al. 2011, 2014a. The same set of markers combined with microsatellite markers derived from B. papayae were used to compare populations of B. dorsalis and B. papayae from the Thai/Malay Peninsula (Krosch et al. 2013, Aketarawong et al. 2014b. No genetic isolation was found between the B. dorsalis and B. papaya populations, supporting the hypothesis that both are the same entity. On the other hand, microsatellite markers, which amplify for B. carambolae and B. dorsalis, showed different genetic clusters between these two species, although admixture populations were observed. Admixture is evidence that some gene flow (i.e. hybridisation) may occur in the field between these species (Aketarawong et al. 2015).
Haplotype analysis: CO1 haplotype networks showed that common haplotypes were shared between B. dorsalis, B. papayae, B. philippinensis and B. invadens, but not with B. carambolae (Schutze et al. 2012b(Schutze et al. , 2015a. This supports the hypothesis that the first four taxa are a single biological species, while B. carambolae is distinct. Phylogenetic analysis: A phylogenetic study using six neutral genetic markers found that B. carambolae could be resolved as a monophyletic clade from the other four target species, which were mixed together as an unresolved comb , Schutze et al. 2015a).
Based on genetic, cytogenetic, pheromonal, morphometric and behavioural data, which repeatedly showed no or only minor variation between B. dorsalis, B. invadens, B. papayae, and B. philippinensis, formal taxonomic name changes were made. B. philippinensis was made a junior synonym of B. papayae by Drew and Romig (2013). Subsequently, also B. papayae and B. invadens were synonymised with B. dorsalis (Schutze et al. 2015b, while the status of B. carambolae has not been altered. This means that only B. dorsalis and B. carambolae remain as scientifically valid names. The name changes have been widely accepted by national and regional plant protection organizations around the world, the Secretariat of the International Plant Protection Convention and the FAO (http://www.fao.org/news/story/en/item/262972/icode/).
In the works of Drew and Romig (2013), San  and Schutze et al. (2015b) new morphological descriptions of the target taxa are provided. However, the use of morphology alone is not sufficient for definitive diagnosis of B. carambolae and B. dorsalis, but molecular and pheromone markers are now available to distinguish them. Molecular protocols using neutral genetic markers to distinguish the two species from each other, and from other closely related taxa, are provided in Boykin et al. (2014). Microsatellite markers which amplify for both species and which are used in population genetic studies, are provided in Aketarawong et al. (2015). The Y-specific marker will also separate the two species. For adult flies, the presence of the ME metabolites DMP and E-CF in the male rectal gland following ME feeding can be used to discriminate B. dorsalis from B. carambolae (which produces only E-CF) (Tan and Nishida 1996.
The knowledge and gaps identified at the start of the CRP, as well as the progress by the end of the CRP in addressing the gaps identified for the Bactrocera dorsalis complex are summarized in Table 2.

Ceratitis FAR Complex Situation Analysis
The Afro-tropical fruit flies Ceratitis fasciventris, C. anonae and C. rosa (i.e. the Ceratitis FAR complex), together with C. capitata and C. cosyra, are considered major horticultural pests of that region (White and Elson-Harris 1994, De Meyer 200la). These species are of quarantine significance (EPPO/CABI 1997) as they are highly polyphagous and damage a wide range of unrelated wild and cultivated crops (De Meyer et al. 2002), resulting in enormous economic losses wherever they occur (Barnes 2000, De Meyer 2001b. They have different distribution patterns that partially overlap, resulting in sympatric occurrence in particular areas. Ceratitis rosa, C. fasciventris and C. anonae were considered the three members of the Ceratitis FAR species complex (Virgilio et al. 2007a(Virgilio et al. , 2007b. Taxonomically, C. fasciventris was initially considered a variety of C. rosa (Bezzi 1920) but has recently been recognized as a different entity with species status (De Meyer 2001a).
Unlike C. capitata, which has over the last century spread from its home range in East Africa and attained an almost world-wide distribution (Fletcher 1989, White  invadens. Species sensitivity to ME across the four species has not been compared. Probit analysis on the males' sensitivity to ME showed no significant differences in the ED also showed no differences in the protein electropherogrammes. This also included that of males exposed to ME.

Hee et al. 2015a this issue
Pheromone Components Phermononal components following ME consumption identical in B. dorsalis s.s. and B. papayae. Luc Leblanc et al. (unpubl.)

Outputs for Ceratitis FAR Complex
Development of molecular diagnostics, using microsatellites , revealed a more complex structure than the mere existence of three entities within the Ceratitis FAR complex. Five genotypic groups were identified ) and later confirmed by morphological differences of the males (De ).
Morphological diagnostics for male specimens of the five entities, called R1, R2 (C. rosa type 1 and 2), F1, F2 (C. fasciventris type 1 and 2) and A (C. anonae) were developed. Morphometric diagnostics using wing landmarks were developed for both sexes to a certain extent (Van Cann et al. 2015). Microsatellites allowed distinction between the five entities. Cytological studies were restricted to one representative (F2) acting as a reference dataset (Drosopoulou, unpublished data). Adult morphology and morphometry, pheromone, cuticular hydrocarbon and distributional data were collected that provide evidence for the specific status of all three formerly recognized taxonomic entities within the FAR complex (i.e. C. fasciventris, C. anonae, C. rosa) (Vaníčková et al. 2014, Van Cann et al. 2015. More detailed studies were conducted for the two C. rosa types (R1, R2) (adult morphology and morphometry, pheromone, cuticular hydrocarbon, developmental physiology, behavioural, and ecological data), which provided evidence that the two C. rosa types represent two separate species (Tanga et al. 2015, Van Cann et al. 2015, Vaníčková et al. 2015b, of which one (currently referred to as R2) will be formally described. An altitudinal transect in Tanzania, where R1 and R2 occur in sympatry, confirmed that R1 is more tolerant to higher temperatures and R2 better adapted to colder environments . For the two C. fasciventris types (F1, F2) these additional studies could not be conducted as laboratory colonies of one of the two types could not be established, preventing experiments on developmental physiology and mating compatibility. Larval morphology did not provide evidence with regard to the specific status, except for C. fasciventris (F2) to some extent (Steck and Ekesi 2015). Moreover, as a spin-off of this research it was shown that characters previously considered diagnostic for differentiation between species and even between the genera Ceratitis and Bactrocera, proved to be variable.
The knowledge and gaps identified at the start of the CRP, as well as the progress made by the end of the CRP in addressing the gaps identified for the Ceratitis FAR complex are summarized in Table 3.

Zeugodacus cucurbitae Situation Analysis
The melon fly, Zeugodacus cucurbitae (initially referred to as Bactrocera (Zeugodacus) cucurbitae), is a major pest of cucurbit crops that has spread from its area of origin (South East Asia) across Africa, Hawaii, the Indian Ocean, Papua New Guinea and the Solomon Islands (Severin et al. 1914, Dhillon et al. 2005. In particular, it causes severe losses in food crops and restrictions to trade for some cucurbit crops. Some populations were identified in Africa, islands in the Indian Ocean, Hawaii and South East Asia with different host use, which could indicate the existance of very closely related species. Although the SIT has been effectively applied against the melon fly in certain regions (Koyama et al. 2004), this issue needed to be resolved to enable the application of species-specific treatments such as the SIT against all populations in all regions.

Outputs for Zeugodacus cucurbitae
In spite of earlier observations and indication of different host-use by B. cucurbitae in different geographic regions, genetic studies using mitochondrial and nuclear markers indicated very low intraspecific variability worldwide. Population genetic studies using microsatellites were able to distinguish five major groups worldwide: African mainland and Seychelles, Réunion and Mauritius, Central Asia, SE Asia, and Hawaii (Virgilio et al. 2010). However no phylogeographic patterns could be discerned using cytogenetics analyses (Zacharopoulou et al. 2011b(Zacharopoulou et al. , 2013 or mitochondrial and nuclear gene fragments (total of 2764 bp) (Virgilio, unpublished data). The invasion history for the species on the African mainland was also reconstructed (Delatte et al. unpublished data).
Cross-mating experiments were conducted at the start of the CRP between populations of Mauritius, Seychelles and a genetic sexing strain of Hawaii and these indicated no mating incompatibility or sexual isolation (Sookar et al. 2013). Given this fact and the genetic assessments, it was decided there was no need for additional cross-mating studies.
Further studies, including host preference and microsatellite markers, did not show any relation between genetic structure and host plants (Virgilio et al. 2010, Sookar et al. 2013. It was concluded that there is no evidence of the existence of host races or cryptic species within B. cucurbitae. However, as a spin-off of the conducted research, recent studies on the higher phylogeny of dacines have shown that the higher taxonomic classification under which B. cucurbitae is placed, is a paraphyletic grouping, requiring a taxonomic change in generic placement (Krosch et al. 2012, Virgilio et  No studies yet specifically with regard to Z. cucurbitae cuticular hydrocarbons have been conducted The composition of the cuticle of virgin males and females -ages 5, 15, 20, 30 after emergence -was analysed by GCxGCMS. The preliminary data demonstrate sex-and age-specific differences. Vaníčková (unpubl.) al. 2015). A nomenclatorial act has raised the subgenus Zeugodacus (as well as other subgenera belonging to the Zeugodacus group, sensu Drew 1989) to genus level. As a result, Bactrocera cucurbitae was put in a new generic combination: Zeugodacus cucurbitae, and should be referred to by this name from now onwards . The knowledge and gaps identified at the start of the CRP, as well as the progress made by the end of the CRP in addressing the gaps identified for Z. cucurbitae are discussed in De Meyer et al. (2015b) and summarized in Table 4.

Conclusion
Following an integrative taxonomic approach, the biology, cytogenetics, ecology, morphology, genetics, and physiology of major pest tephritid cryptic species complexes is now much better understood. This increased knowledge has resulted in formal decisions on the species status of some taxa within these complexes, thus facilitating international horticultural trade and simplifing SIT application against pest species of these complexes. In the case of Anastrepha fraterculus it was shown that it consists of a complex of a number of different species of no monophyletic origin, with distinct geographic and ecological distributions in Latin America. Also for the Ceratitis FAR complex evidence has been provided for the existence of five different entities within this complex from the African region, i.e. Ceratitis anonae, C. rosa (R1 and a new species referred to as R2), while for C. fasciventris the biological limits between F1 and F2 are not fully resolved. On the other hand the Asian/African pest fruit flies B. papayae, B. philippinensis and B. invadens were shown to represent populations of B. dorsalis, while only B. carambolae remains a valid species for which molecular and pheromone markers are now available to distinguish it from B. dorsalis. Finally studies among populations throughout the geographic range of Bactrocera cucurbitae in Africa and the Asia/Pacific region showed no evidence for the existence of host races. However, the higher taxonomic classification under which Bactrocera cucurbitae is placed was found to be a paraphyletic grouping, requiring the elevation of the subgenus Zeugodacus to genus level. As a result, Bactrocera cucurbitae was put in a new generic combination: Zeugodacus cucurbitae.