Tephritid Integrative Taxonomy: Where We Are Now, with a Focus on the Resolution of Three Tropical Fruit Fly Species Complexes

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1 ANNUAL REVIEWS Further Click here to view this article's online features: Download figures as PPT slides Navigate linked references Download citations Explore related articles Search keywords Annu. Rev. Entomol : First published online as a Review in Advance on November 2, 2016 The Annual Review of Entomology is online at ento.annualreviews.org This article s doi: /annurev-ento Copyright c 2017 by Annual Reviews. All rights reserved Corresponding author Tephritid Integrative Taxonomy: Where We Are Now, with a Focus on the Resolution of Three Tropical Fruit Fly Species Complexes Mark K. Schutze, 1, Massimiliano Virgilio, 2,3 Allen Norrbom, 4 and Anthony R. Clarke 1,5 1 School of Earth, Environmental, and Biological Sciences, Queensland University of Technology, Brisbane, 4001 Queensland, Australia; m.schutze@qut.edu.au 2 Department of Biology, Royal Museum for Central Africa, B3080 Tervuren, Belgium 3 Joint Experimental Molecular Unit, Royal Museum for Central Africa, B3080 Tervuren, Belgium; massimiliano.virgilio@africamuseum.be 4 Systematic Entomology Laboratory, United States Department of Agriculture, c/o National Museum of Natural History, Washington, DC 20560; allen.norrbom@ars.usda.gov 5 Plant Biosecurity Cooperative Research Centre, University of Canberra, Bruce, Australian Capital Territory 2617, Australia; a.clarke@qut.edu.au Keywords integrative taxonomy, iterative taxonomy, unified species concept, true fruit flies, horticultural pests, Diptera Abstract Accurate species delimitation underpins good taxonomy. Formalization of integrative taxonomy in the past decade has provided a framework for using multidisciplinary data to make species delimitation hypotheses more rigorous. We address the current state of integrative taxonomy by using as a case study an international project targeted at resolving three important tephritid species complexes: Bactrocera dorsalis complex, Anastrepha fraterculus complex, and Ceratitis FAR (C. fasciventris, C. anonae, C. rosa) complex. The integrative taxonomic approach has helped deliver significant advances in resolving these complexes: It has been used to identify some taxa as belonging to the same biological species as well as to confirm hidden cryptic diversity under a single taxonomic name. Nevertheless, the general application of integrative taxonomy has not been without issue, revealing challenges that must be considered when undertaking an integrative taxonomy project. Scrutiny of this international case study provides a unique opportunity to document lessons learned for the benefit of not only tephritid taxonomists, but also the wider taxonomic community. 147

2 INTRODUCTION Species exist in nature, yet their operational delimitation remains an ongoing challenge. A multidisciplinary approach to answer this challenge is integrative taxonomy, wherein independent lines of evidence, rather than evidence from a single discipline (e.g., morphology or molecular biology), delimit species boundaries. First coined in 2005 (17, 110) and operationally formalized in 2010 (78), integrative taxonomy has appeared in the titles of a steadily increasing number of papers (from 12 papers in 2010 to 44 in 2015, according to a Web of Science search). Today, the term is routinely applied to any systematic study in which more than one species delimitation tool is used. Given this uptake in usage, we review how successful the integrative taxonomic approach has been in dealing with complex taxonomic problems. We briefly cover integrative taxonomic theory but refer readers to other treatments for the approach s theoretical basis (36, 69, 70, 72, 112). We focus on a case-study and on how integrative taxonomy has been applied to a specific insect group, the subtropical and tropical tephritid fruit flies. More explicitly, we address how integrative taxonomy fared when implemented as part of a major international effort to resolve species complexes in three economically important tropical fruit fly groups. In doing so, we ask the following: Was the integrative taxonomic objective achieved? Did taxonomic resolution result? What lessons were learned, and what recommendations may be made for tephritid taxonomy? TEPHRITID FRUIT FLIES Tephritidae (true fruit flies) are a large and diverse family comprising over 4,900 species in more than 500 genera (67). Globally distributed, many are pests threatening food security and horticultural trade, especially in developing nations (57). Within the tropics and subtropics, three genera contain most of the economically important species: the predominantly African Ceratitis and Asian/Australasian Bactrocera (both of subfamily Dacinae) and the mainly Neotropical Anastrepha (subfamily Trypetinae) (1) (also see sidebar Tephritid Species Complexes). Though important, the temperate genus Rhagoletis is not included in our review. TEPHRITID SPECIES COMPLEXES The term species complex may mean subtly different things to different people, including tephritid workers. A key distinction is whether the group in question is a cryptic, sibling, or taxonomic species complex or a combination of any, or all, of these three kinds of complexes. The terms cryptic and sibling are often interchanged, yet they do not always describe the same thing: A sibling complex is a group of closely related species (i.e., a monophyletic group), whereas a cryptic complex is a group of virtually, or actually, morphologically identical species (108). Granted, most cryptic complexes consist of sibling species, and sibling species are more likely to be morphologically identical. Bactrocera carambolae of the B. dorsalis species complex, for example, may be confused with B. dorsalis (hence, it is cryptic), yet it is also a closely related sibling species (7). Nevertheless, a cryptic species complex need not be monophyletic and include only sibling species, and sibling species need not be cryptic. Certain lineages in the Anastrepha fraterculus species complex are virtually identical morphologically, yet may be more closely related to species outside the complex (56). Additionally, species complexes are sometimes proposed for convenience and without phylogenetic support (15). Such complexes may be neither cryptic nor monophyletic lineages. Bactrocera musae (Tryon), for instance, was taxonomically assigned to its own complex on the basis of morphology (musae species complex) (28), yet molecular phylogenetic evidence has revealed it may be more closely related to B. dorsalis than to some members of its complex [e.g., B. bancroftii (Tryon)] (50). 148 Schutze et al.

3 Included among these three genera are species complexes that have caused long-standing taxonomic uncertainty and disputes (41): the Bactrocera dorsalis complex, the Anastrepha fraterculus complex, and the Ceratitis FAR (C. fasciventris, C. anonae, C. rosa) complex (42), which are the focus of this review. Delineation among species within these complexes has largely centered on morphological characters, many of which are ambiguous (e.g., character-state definitions such as broad or narrow) (30) or sex specific (e.g., the female ovipositor) (92); alternatively, their withinspecies variation may be comparable to between-species variation (e.g., overlapping morphometric variation) (31). This lack of taxonomic certainty has hindered advances in evolutionary insight into these complexes, while also hampering agricultural trade, quarantine programs, and development of pest management programs (42). A BRIEF HISTORY OF INTEGRATIVE TAXONOMY Over 25 species concepts have been proposed during the past two centuries, resulting in a number of disciplines delimiting species boundaries using different, and often incompatible, theoretical frameworks (24). However, all species concepts agree in considering species as separately evolving metapopulation lineages (22, 23), and this primary defining property may be tested through a variety of secondary species criteria that may vary depending on the group and its evolutionary history (24). Integrative taxonomy originated from the operational difficulties in delimiting species through a single criterion. It uses multiple lines of evidence that may vary between different species pairs (12). Taxonomists have long appreciated the merits of using alternative lines of evidence to formulate evolutionary hypotheses and species delimitation (47, 88), including for tephritids (54, 109). Early integrative taxonomy papers (in particular, see 17) offered a set of guidelines to generate such hypotheses. Though some were criticized (96, 112), there was widespread agreement that multidisciplinary evidence increased the validity of species hypotheses (70), as revealed by the high failure rate for species delineation using a single discipline rather than two or three (78). From this understanding emerged a need to develop a unified approach for integrating data, particularly given species-concept advances that demanded an appreciation for multiple species criteria as part of an increasingly accepted (12) and unified species concept (24). In 2010, a more sophisticated set of guidelines for integrative taxonomy was proposed (78), identifying five study-system attributes for which the integrative approach was best suited: (a) long-standing taxonomic disputes, (b) ambiguous morphological delimitation, (c) pronounced variability, (d ) biodiversity hot spots, and (e) the importance of organisms to progress in other fields (arguably, all characteristics of tephritid complexes). A sequential approach using independent data from three disciplines (phenotype/morphology, nuclear DNA, and complementary data including other lines of evidence from, for example, behavioral, ecological, host, or mitochondrial DNA data) based on no prior species delimitation hypotheses (i.e., a discovery approach) was advocated. This core set of disciplines reflected earlier recommendations to use sequentially accrued information from DNA, morphology, reproduction, ecology, and/or geography to define independent lineages and to formally describe new species (25). In contrast to the discovery approach proposed by Schlick-Steiner et al. (78), a hypothesis-driven iterative taxonomic framework was proposed as the most defensible means of species delimitation (112), because a truly integrative approach (i.e., all data combined prior to rigorous statistical analysis) was considered beyond existing analytical capabilities. Thus, taxonomists were advised to test species hypotheses on the basis of an initial data source (equivalent to the null hypothesis; e.g., species-level lineages based on morphology, or a molecular phylogeny, alone) against which successive independent lines of evidence were to be evaluated (also see sidebar Definitions). Tephritid Integrative Taxonomy 149

4 DEFINITIONS Integrative taxonomy: term coined independently on three separate occasions to define practice as (a) a combination of independent data sets (37, p. 372); (b) an approach that uses a large number of characters including DNA and many other types of data to delimit, discover, and identify meaningful, natural species and taxa at all levels (110, p. 845); and (c) part of guidelines that delimit the units of life s diversity from multiple and complementary perspectives (phylogeography, comparative morphology, population genetics, ecology, development, behavior, etc.) (17, p. 407). Discovery approach: process formalized by Schlick-Steiner et al. (78) for evaluating three disciplines phenotype/morphology, nuclear DNA, and complementary (e.g., behavioral, ecological, mitochondrial DNA). Multiple lines of evidence are used without reference to an initial species hypothesis generated by a single discipline. This was deemed preferable because [a]n erroneous prior hypothesis might appear to be corroborated under the hypothesis-driven approach (78, p. 430). Iterative taxonomy: practice wherein species boundaries are hypothesized on the basis of an initial data set (e.g., morphology) against which subsequent disciplines are continually evaluated. Hypothesis-driven approach: process comprising iterative taxonomy and advocated as preferable because it was the most practical...and defensible, on the basis of classical scientific hypothesis testing (112, p. 214). Unified species concept: retains the general concept of species as separately evolving metapopulation lineages, which is treated as the only necessary property of species (24, p. 882). Competing species concepts are regarded as alternative perspectives of a common underlying principle for which no single line of evidence (or species criterion) is considered intrinsically superior to any other (e.g., morphological, phylogenetic, ecological). MOVING AWAY FROM A MORPHOLOGICAL FOCUS All good taxonomists consider species boundaries as hypotheses to be verified, and most would probably agree that the likelihood of a delimitation hypothesis increases when it is verified by new information from additional independent disciplines (71, 78). However, most insect species are still formally described and named on the basis of morphology, or morphology plus molecular data. Usually only when suspected problems arise (in pest taxa, for example) are other disciplines (e.g., biochemical, ecological, behavioral) used for a posteriori verification of species limits proposed by morphological taxonomy (sensu iterative taxonomy) (112). In contrast, extending and generalizing the integrative approach to tephritids would change the process of recognizing and accepting species from name first and verify second to one whereby the species delimitation hypothesis (i.e., new species name) originates only after independent evidence provided by three or more different disciplines has been compared. Granted, this approach is ideally suited to newly discovered and as-yet undescribed species; when it is applied to groups whose species have been named and need revision, the situation becomes more complex. Addressing this issue, Dayrat (17) suggests several guidelines to reduce the overabundance of species names originating from poor interdisciplinary feedback to morphological taxonomy. However, by further suggesting that only specimens available to molecular taxonomy (i.e., preserved to allow DNA extraction and genotyping) should be designated as type specimens (guideline VII), Dayrat (17) privileges molecular taxonomy in the integrative taxonomic framework. Thus, no new species names should be proposed until such specimens have been analyzed (guideline VI). These guidelines, as well as others, received strong criticism for their lack of operational feasibility (96). Not 150 Schutze et al.

5 labeling species until three or more separate lines of evidence are available also poses additional operational challenges (revisited in the section Recommendations and Concluding Remarks). THE SCHLICK-STEINER APPROACH The integrative approach opens up complex scenarios as it implies the need to resolve conflicts among disciplines and to weigh evidence from different characters. For example, compared with other morphological characters (e.g., thoracic color pattern), tephritid feeding preferences may be more ecologically relevant because they may more accurately reflect phylogenetic relationships (34, 106) and promote speciation (35, 83). In their protocol for decision making, Schlick-Steiner et al. (78) propose a multistep procedure with an initial exploratory phase relying on a prior-stated species concept, delimitation criteria, and analytical protocols. Their procedure also includes a primary and, possibly, sequential exploration phase (the latter in case two or more disciplines are inconclusive). For an integrative taxonomic study, the authors (78) suggest three independent delimitation tools, two of which are always recommended morphology (for compatibility with current taxonomic practice) and genetics [because species are genetic and not morphological entities (p. 429)]. Hypotheses generated by various methods are then compared, and possible disagreements are interpreted in evolutionary terms. Straightforward species delimitation is possible when the hypotheses are plausible and all disciplines are conclusive and in agreement. Conversely, data from inconclusive disciplines (i.e., disciplines that fail to delimit putative species), or from conclusive disciplines supporting implausible hypotheses, should be reprocessed in a hypothesis-driven iterative framework. Perhaps unsurprisingly, disagreement among disciplines is common (78). INTEGRATIVE TAXONOMY TAKES OFF, OR SO IT SEEMS Even though numerous multidisciplinary taxonomic studies existed prior to the entry of integrative taxonomy into the scientific vernacular, the literature has burgeoned with such studies since the term was introduced in the first years of the twenty-first century, mainly as a result of the rapid development of molecular technologies. In a review of nearly 500 species delimitation studies conducted between 2006 and 2013, including those that made formal taxonomic decisions, 65% used more than one line of evidence to evaluate species boundaries (72). The number of studies using more than two disciplines increased steadily each year, from 10 in 2006 to 39 in 2010 and 77 in (72). The remaining studies delimited species using only a single discipline, but their numbers also increased over the same time period, albeit at a lower rate, from 9 to 31 and 41, respectively. However, multiple authors inappropriately applied the term integrative taxonomy, for example, by referring to the integration of multiple analyses of the same data set. Several researchers also considered their work to be integrative because more than one discipline was used. However, as the majority of these studies (47%) applied only two forms of independent data (72), they did not conform to the three-discipline approach advocated by Schlick-Steiner et al. (78). Further, to what degree these studies followed an iterative (112), rather than integrative, framework remains a question. Circa 2005, momentum had built toward understanding several tropical fruit fly pest complexes. Evidence suggested that, for some complexes, several described species represented the same biological species and that in others, single species names were applied to different biological species. Owing to the economic and food-security importance of resolving these fundamental questions, the international community under the auspices of the Food and Agriculture Tephritid Integrative Taxonomy 151

6 Organization/International Atomic Energy Agency (FAO/IAEA) launched a multidisciplinary project to address this (42). RESOLVING THE TROPICAL TEPHRITID COMPLEXES: AN INTERNATIONAL INTEGRATIVE EFFORT A major international multidisciplinary effort to resolve the species boundaries within the most important tropical tephritid pest complexes began in More than 30 scientists met for the first of four research meetings in Vienna under a Joint FAO/IAEA coordinated research project (CRP). This project led to the revision of pest species within the B. dorsalis complex (32, 81) and significant advances in resolving the Ceratitis FAR (19) and A. fraterculus complexes (44, 102). Over 50 scientists, including all authors of this review, representing more than 20 countries participated in the five-year CRP (42). The complexity, size, and duration of the project offer a unique opportunity to review the integrative taxonomic paradigm when operationally applied. Bactrocera dorsalis Complex The B. dorsalis complex contains at least 85 species (29, 30, 32, 52), with diversity centered in the Asian and Australasian regions. Although most species are of little to no economic concern, some are of major importance owing to their wide geographic distributions and host ranges, which include valuable horticultural commodities (14). Pests of the B. dorsalis complex represent significant challenges to species delimitation because morphological variation can be very high. As a result, species identification is often impossible (52), which is problematic because most surveillance, quarantine, and monitoring programs use morphology as their primary means of diagnosis. Especially problematic were the most pestiferous species B. dorsalis (Hendel) sensu stricto; B. papayae Drew & Hancock; B. philippinensis Drew & Hancock; B. carambolae Drew & Hancock; and B. invadens Drew, Tsuruta & White. Such difficulties led to ongoing efforts to evaluate discriminatory characters among these taxa (14). As part of an iterative approach, researchers screened potential species delimiters using morphology, chemical ecology, allozymes, morphometrics, DNA barcoding, and phylogenetic tools (see reviews in 14, 41, 42). Some researchers (e.g., 94) challenged prevailing morphological taxonomy, yet many accepted the taxonomic descriptions and names as accurately reflecting underlying species boundaries, considering the absence of reliable diagnostic markers to be a case of not looking in the right place. Thus, there was unresolved disagreement among disciplines, compounded by a lack of coordination among researchers (41). Whereas initial CRP studies were based on morphological species hypotheses (e.g., 79), later work embraced a discovery paradigm (sensu 78). That is, multidisciplinary delimitation approaches were applied to specimens collected from across the native and introduced ranges of the taxa, without grouping according to morphological taxonomy (e.g., 49). Though existing species hypotheses were not used as a basis to group individuals prior to subsequent analyses, specific hypotheses were nevertheless proposed to test established taxonomy (see section below Truly Independent Tests: Can We Fully Escape Iterative Taxonomy? for B. dorsalis and B. papayae). Further, colonies of all target taxa (confirmed by the taxonomic authority) were established in centralized FAO/IAEA laboratories from which independent lines of inquiry on collection material, available equally to all working under the CRP, were undertaken by specialized researchers evaluating pheromones, cytogenetics, sexual compatibility, and phylogenetic relationships (see 81 and references therein). Building on prior work, this coordinated collegial approach resulted in formal taxonomic revision: Evidence from multiple independent disciplines was used to synonymize B. papayae and 152 Schutze et al.

7 B. invadens with B. dorsalis (81) following the synonymy of B. philippinensis under B. papayae (32). There was broad congruence among disciplines, yielding the clearest result (78) from which subsequent studies (e.g., 4, 13, 40) corroborated widespread acceptance of taxonomic changes. Yet, resistance to aspects of the revision remains, principally from morphological taxonomists who consider B. papayae distinct from B. dorsalis and those who argue that B. invadens does not belong in the B. dorsalis species complex (32, 39). Nevertheless, the synonymy has been widely accepted by the scientific community and is endorsed by national and international governments as well as nongovernmental organizations. Ceratitis FAR Complex The Ceratitis FAR complex is a small group of morphologically similar species: C. fasciventris (Bezzi), C. anonae Graham, and C. rosakarsch. None of the commonly considered species concepts (24) unambiguously resolved the limits of these species: In particular, (a) the phenetic species concept (63, 89) is not fully appropriate because females of C. rosa and C. fasciventris cannot be separated on the basis of morphological characters (18, 20); (b) the ecological species concept (3, 98) does not support the nomenclature, as the three species have largely overlapping ecological niches with sympatric and parapatric distributions (19); and (c) C. rosa and C. fasciventris might not even comply to the biological species concept (BSC) (61) as the production of fertile hybrids, under laboratory conditions, seems to suggest (33). Consequently, integrative taxonomy was used to define the species limits of the FAR complex (19). Preliminary phylogenetic analyses failed to resolve the three species as separate clades (105); thus, the three morphospecies represented a single taxonomic unit according to the phylogenetic species concept (27, 76). Further, larval morphology was inconclusive, yet it suggested C. fasciventris might be separated from the other two species (91). Microsatellites revealed the three morphospecies were composed of five genotypic clusters corresponding to C. anonae (cluster A), C. fasciventris (clusters F1 and F2), and C. rosa (clusters R1 and R2) (107). Interestingly, patterns of differentiation among clusters were not hierarchically organized. As such, following the genotypic cluster species concept (58), they should be considered as five separate taxonomic units. Iterative a posteriori morphological analysis of adult specimens (sensu hypothesis driven) (78) further supported subdivision into five groups, as consistent morphological differences (i.e., midtibial shape and coloration) were found between clusters R1 and R2 and, to a lesser extent, between clusters F1 and F2 (19). Sequential integrative research was implemented to resolve the conflicting hypotheses generated by different approaches. The same set of specimens already morphologically identified and genotyped (105) was subjected to wing morphometric analyses, which resolved the three morphospecies as separate entities and provided an indication of differentiation across genotypic clusters (97). Additional and hypothesis-driven evidence was obtained from analysis of (a) distributions of C. rosa R1 and R2 at different altitudes (64); (b) developmental physiology of colonies of C. rosa R1andR2(95);(c) cuticular hydrocarbons of C. anonae, C. rosa R1 and R2, and C. fasciventris F2 (100, 103); and (d ) pheromones of C. anonae, C. rosa R2, and C. fasciventris F2 (9). Considering and weighting a priori and a posteriori species hypotheses supported by different disciplines resulted in informal acceptance of two different C. rosa types (R1/lowland/hot rosa and R2/highland/cold rosa) (19) and the subsequent description of C. rosa R2 as C. quilicii (21). Further evidence is needed for the possible taxonomical revision of C. fasciventris (including the two genotypic clusters F1 and F2), as populations of this species have not been examined with all the delimitation approaches used for R1 and R2. Thus, the integrative approach, as applied to the FAR complex, has resulted in the confirmation of a new species, C. quilicii,whichis robustly supported by multiple lines of evidence. Further, what was learned from the R1/R2 system Tephritid Integrative Taxonomy 153

8 may form a model to apply to other members of the FAR complex and the genus Ceratitis as a whole. Anastrepha fraterculus Complex The A. fraterculus complex occurs from Mexico to northern Argentina (43, 44, 92). This taxon has been considered unusually variable in morphology and behavior (5, 90, 92), and crucially for horticulture, it is a severe pest in only some areas of its range (2, 5, 45, 114). As early as the 1940s, A. fraterculus (Wiedemann) was considered likely to comprise a cryptic species complex (5). Its taxonomic history also differs from the above-mentioned complexes: Although ten nominal species have been proposed in addition to A. fraterculus (eight from Brazil and two from Peru), they have all been considered synonyms of A. fraterculus (8, 48, 55, 68, 92, 113). Historically, various methods have been used to study the A. fraterculus complex, all generally supporting the multiple species hypothesis. However, most studies used different samples and were geographically restricted until recent CRP efforts; thus, results have been difficult to compare and evaluate (42). Hypotheses based on isozymes recognized at least four entities (90), whereas morphometric studies of adults recognized seven morphotypes (43). Differences in adult morphology, egg morphology, karyotype, and isozymes among three putative cryptic species from Brazil were also reported (84 87; for a review, also see 102). The CRP and other studies made significant progress to resolve further the A. fraterculus complex via a predominantly iterative approach using a range of methods to test the initial species hypotheses (42). Eight morphotypes grouped into three clusters based on adult female morphometric analysis have been recognized (43, 44). Strong supportive evidence also comes from mating studies (e.g., 26, 46, 77, 104), differences in male pheromones (10, 101), cuticular hydrocarbons (99), DNA sequences (93), and isozymes (90), suggesting that at least four of these entities are distinct biological species. Despite this progress, significant questions remain. Most nonmorphometric studies lacked sufficient samples to determine if all morphotypes represent distinct species. For example, the lowland Venezuela morphotype (currently difficult to sample due to political instability) has been distinguished only by adult morphometrics, and no significant differences in ITS1 sequences between Mesoamerican and lowland Venezuelan morphotypes, or between Andean and highland Ecuadorian morphotypes, were found (93). Thus, conclusive concordance is lacking among disciplines within an integrative taxonomic context. The question of how much these differences comprise population-level variation versus differences between truly cryptic species also remains unresolved for some putative taxa, and additional disciplines need to be sequentially investigated. The highland Ecuadorian morphotype, which was recently recognized, has been differentiated by only adult and larval morphometrics and karyotype (11, 44). The Brazilian-2 and Brazilian- 3 morphotypes have been compared with only the Brazilian-1 morphotype (75, 84 87, 111). Further, though morphotypes of the Andean region were once thought to be allopatric, at least three different ITS1 types occur in Colombia and three in Peru, with two in common, and their ranges are nearly contiguous (93). Yet, how ITS1 types correlate with morphotypes remains to be fully determined, as does whether they consistently occupy different ecological regions or if any zones of sympatry exist. This situation reiterates the need to apply alternative lines of evidence to the same specimens as part of an integrative approach. Ongoing efforts to apply consistent molecular approaches to populations across the entire geographic range continue and will shed further light on the A. fraterculus complex (e.g., 51, 59). However, morphometric results must be fully integrated with molecular and other data to determine the status and limits of each morphotype and to determine the full distribution and host range of each species. 154 Schutze et al.

9 LESSONS LEARNED The CRP made significant scientific advances in our understanding of species boundaries in key tephritid pest groups. It also provided researchers with invaluable experience in the practicalities of applying an integrative taxonomic approach to complex operational, political, and scientific scenarios. This section identifies some of the major lessons learned, which should be considered when undertaking an integrative taxonomic project. Moving Beyond the Biological Species Concept Though infrequently discussed, Mayr s BSC was adopted by most CRP workers. According to this concept, species are defined on the basis of reproductive isolation (62); as such, the fruit fly community tends to follow the general pattern among biologists (66). However, acceptance of the BSC is contrary to the philosophical concept of integrative taxonomy: It implies that a final decision regarding species boundaries ultimately relies on only one criterion, reproductive isolation. Yet, even without the full reproductive isolation required by the BSC, breakdown in gene flow and subsequent population divergence via mechanisms other than those directly related to mating (e.g., spatial, temporal, or ecological vicariance) may provide strong support for the recognition of species boundaries. When done appropriately, compatibility trials can be highly informative in helping delimit tephritid taxa (46), even though forced mating and viable offspring between unequivocal species are possible (16, 73). Determining the validity of seminatural cage compatibility tests to delimit species caused tension among some members of the CRP, but the broader issue concerning mating trials as a unique definer of species was not raised (for a discussion, see 60, 65). We urge researchers involved in integrative taxonomy to move away from the BSC and to utilize more recent species theories that accommodate multiple lines of evidence. Truly Independent Tests: Can We Fully Escape Iterative Taxonomy? Ideally, integrative taxonomy uses independent delimitation tools from which species delimitation hypotheses are generated, but in practice, this may be very difficult to achieve. For example, when conducting mating compatibility studies using populations that are fully allopatric (e.g., crossing African B. invadens with Chinese B. dorsalis) (6), the geographic collection sites are sufficient to ensure that the two test groups are unambiguously aligned with established taxonomy. However, when taxa are sympatric, such alignment becomes a significant challenge. For instance, as part of the CRP, populations of B. dorsalis, B. carambolae, B. papayae, and B. philippinensis were established at the IAEA Insect Pest Control Laboratory in Seibersdorf, Austria. Cultures were subsequently used not only for mating compatibility trials (80) but also for cytogenetics (4) and parapheromone responses (40). Ensuring that each culture line represented its purported taxon was imperative. For B. philippinensis, B. carambolae, and B. dorsalis, alignment was achieved by collecting each species from a site where it is allopatric to the other target taxa (Philippines, Suriname, and central Thailand, respectively). For B. papayae, the culture was obtained from a region in which the species has been considered allopatric with B. dorsalis (southern Thailand), but despite this the taxonomic authority considered the culture as B. dorsalis. As we already had a B. dorsalis culture, we had to make a second collection from Peninsular Malaysia; this time on the basis of host-rearing data and morphological differences, the colony was considered by the taxonomic authority as B. papayae and distinct from B. dorsalis. Thus, in this situation, an iterative approach was necessary: Two species delimitation tools comprising classical taxonomy and host fruit origin informed subsequent disciplines. Tephritid Integrative Taxonomy 155

10 It is worth noting that for other complexes, such as A. fraterculus in particular, there is greater uncertainty about the geographical distribution limits among putative cryptic species. Hence, in such cases alignment between species (or populations) with geography continues to be problematic, and collections may be made without knowing whether a sample contains a single species from an allopatric distribution, or a mixed sample of two or more species from an area of sympatry. This is likely to be the case for any attempt to study a group for which cryptic species are suspected to exist. Thus the research approach should not assume that samples are always composed of single species. Using Independent Species Delimitation Tools to Inform Other Studies: Independence Is Not Always Good The experience of the CRP has shown that full independence in an integrative taxonomic project (particularly a large one) is not always ideal. For all three complexes targeted by the CRP (42), known or putative sibling taxa had a range of geographic distributions varying from fully sympatric to fully allopatric. Because activities had to be coordinated across laboratories and countries, some information had to be shared, which directed research down an iterative rather than integrative path. For the Ceratitis FAR complex, this was the recognition of genetic differences within C. rosa and C. fasciventris populations (107), which then informed the design of subsequent sampling and behavioral experiments (64, 95). For A. fraterculus, multivariate morphometric approaches identified populations (43, 44) to be targeted for mating compatibility (26, 77) and chemotaxonomic (100) studies. Such iterative taxonomy suffers from a lack of full independence as promoted by integrative taxonomy. Yet, if this information had not been shared, then confusion would have resulted. Problems of Analyses Requiring Groups Determined A Priori Though a very technical point, the following illustrates the difference between iterative and integrative taxonomy and identifies a useful lesson. Geometric morphometric shape analysis, now widely used in integrative taxonomy (53, 74), generates multivariate data. Such data may be analyzed in an exploratory fashion [e.g., through principal components analysis (PCA)] or used for hypothesis testing via multivariate analysis of variance and linear discriminant function analysis (38). In an approach to variation in the wing shape of B. dorsalis complex species using previously identified groups, canonical variate analysis accurately placed most specimens in their predefined groups (79). However, PCA generally failed to differentiate B. dorsalis complex species on the basis of wing shape (49, 82). Thus, the same data type could be used to support (32) or deny (81) the validity of species within the complex. The difference lies in what analyses are meant to do: Hypothesis-testing approaches determine whether samples fit into predefined groups, whereas exploratory PCA determines if the groups exist. In terms of independent analysis for integrative taxonomy, PCA is a far better approach. However, for iterative taxonomy, or to identify possible diagnostic features once species limits have been established (97), hypothesis-testing analysis should be used. Sovereign Rights and the Growing Challenge of Exchanging Samples For some CRP researchers, exchanging samples presented considerable difficulties owing to legal barriers, which have generally increased since the Nagoya Protocol came into effect. Many countries, in the interest of protecting biodiversity and patrimony, have passed laws that restrict 156 Schutze et al.

11 international research collaboration. Obtaining permits to collect and/or export biological material is often complicated and time consuming to the point that many scientists give up or simply do not start international research programs. In Peru, for example, separate permits are required to conduct research, export specimens, and extract and sequence DNA; approval of the latter took two years for the Steck group (based in the United States) and Peruvian collaborators to secure. Though restrictions have somewhat eased in Brazil (it is now possible to exchange samples via institutional agreements), much confusion remains, and a climate of fear of breaking the law persists, limiting the sharing of specimens for collaborative research. Indeed, if the CRP had started today instead of in 2010, it would be almost impossible to conduct the same research. In Kenya, for instance, local permits and institutional agreements were previously sufficient to exchange larvae between international researchers (e.g., between the International Centre of Insect Physiology and Ecology and laboratories in Europe and the United States). Now, additional prior informed consent and mutually agreed terms must be negotiated with a Kenyan national clearing house body, with agreements to be made with individual countries. Further, individual agreements with each country in which populations of the FAR complex exist would need to be secured to conduct a single comprehensive microsatellite study. If a colleague from another institution or country requests the same samples for subsequent integrative research, negotiations with each of the original countries must restart unless the sharing of material had been negotiated in the original mutually agreed terms. Thus, despite best intentions among researchers to collaborate and address central and, in some cases economically critical, taxonomic questions (as evidenced by the success of the FAO/IAEA CRP), these imposed restrictions will hinder future international integrative taxonomic research. How Long Is a Piece of String: When To Stop? The final lesson learned from the CRP experience is that, prior to beginning an integrative taxonomic project, researchers must decide when to stop; this is related to issues of hypothesis testing.in the synonymyofb. invadens and B. papayae with B. dorsalis, 14 species delimitation tools were used to varying degrees, many of which were applied to multiple populations (81). Similarly, A. fraterculus workers applied a wide range of delimitation tools to their system (see, for example, papers reviewed in 19). Though, arguably, each time a new independent test verifies a hypothesis, the degree of confidence in the conclusion increases, such an approach also means that a final endpoint may seldom be reached. For similar reasons, it is also important to decide the number of populations to which these delimitation measures are applied. Critically, in the absence of an agreed-upon suite of delimitation measures (and associated hypotheses linked to these measures), the opinion that researchers should continue looking may persist. RECOMMENDATIONS AND CONCLUDING REMARKS Fruit flies may never again be submitted to such intensive international integrative (and iterative) taxonomic treatment as was carried out under the auspices of the FAO/IAEA CRP. It is therefore crucial that we distill the essence of what was learned to provide a taxonomy road map for tephritids and other organisms. We couch these recommendations in light of previous guidelines, including those of earlier integrative taxonomy advocates. We emphasize the importance of guidelines II and III of Reference 17. That is, as part of a taxonomic revision, within- and between-species variation must be exhaustively quantified and based on an examination of a consistent number of specimens. This should be done using a range of independent disciplines and not only using morphological characters that may be highly variable Tephritid Integrative Taxonomy 157

12 (52). Until such widespread assessment of variation is undertaken, we endorse guideline IV, which stipulates that putative species are designated sp. until broad biological evidence is generated to support formal description (guideline V). This is especially important for putative taxa that are actually, or potentially, major pests. For these in particular, it is better to fail to delimit species than it is to falsely delimit entities that do not represent actual evolutionary lineages (12, p. 4369). Even though this failure has occurred for pest species of the B. dorsalis complex, the application of multiple tools across a range of specimens in the A. fraterculus and Ceratitis FAR complexes means that formal taxonomic revision may now proceed with a high degree of confidence, as is now happening (21). With respect to recommended tools (15), future tephritid workers dealing with suspected or known complexes should use a maximum of four delimitation measures: (a) pre- and postzygotic mate compatibility tests, (b) multilocus phylogenetics and associated species delimitation approaches, (c) pheromone analysis, and (d ) morphometrics and geometric morphometrics. All these measures cannot be used at all times for all species, as doing so would bring most species descriptions to a virtual halt thus hindering all tephritid taxonomy (nota bene, the majority of tephritids are not of economic concern). Therefore, although a gold standard, an integrative approach should be applied especially to taxa from pest complexes and putative taxa that closely resemble pest species: In such cases, taxonomy is not only academic; it may have serious economic and food security consequences if it fails to match biological reality. Ideally, these measures are applied in a discovery/hypothesis-driven approach. Accordingly, in the absence of species-boundary assumptions based on a single line of evidence (the discovery component), such an approach includes a biologically relevant question that will deliver an outcome directly supporting or refuting a single- or multispecies scenario (hypothesis component). We further stress the value of drawing morphological, molecular, and ecological data from the same specimens wherever possible so as to link independent lines of evidence to cross-referenced voucher specimens that are deposited in public institutions. As such, researchers may avoid ambiguity or potential disagreement based on discipline-specific data from disparate collections; this is especially the case for sympatric cryptic taxa. This integrative approach should be undertaken in the generalized species concept framework (sensu 24), wherein no single line of evidence is regarded as superior to any other. Evolutionary explanations should then be sought in cases of disagreement among disciplines (sensu 78). Finally, although color pattern is extremely useful for discriminating species in many tephritid genera, we urge caution in its use as a central character in species delimitation, especially in strictly defining taxon membership to artificially created groups or complexes in the absence of corroborative integrative data supporting their evolutionary association. Using color pattern is restrictive and limits the potential to resolve actual relationships. Artificially defined species could stifle future research into this remarkably varied group of insects and may result in truly global economic and food security consequences. DISCLOSURE STATEMENT The authors are not aware of any affiliations, memberships, funding, or financial holdings that might be perceived as affecting the objectivity of this review. ACKNOWLEDGMENTS The authors sincerely thank Marc De Meyer and Teresa Vera for providing comments on earlier drafts that greatly improved the manuscript. We extend our appreciation to colleagues past and 158 Schutze et al.

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19 Annual Review of Entomology Volume 62, 2017 Contents Following the Yellow Brick Road Charles H. Calisher 1 Behavioral Sabotage of Plant Defenses by Insect Folivores David E. Dussourd 15 Neuropeptides as Regulators of Behavior in Insects Liliane Schoofs, Arnold De Loof, and Matthias Boris Van Hiel 35 Learning in Insect Pollinators and Herbivores Patricia L. Jones and Anurag A. Agrawal 53 Insect Pathogenic Fungi: Genomics, Molecular Interactions, and Genetic Improvements Chengshu Wang and Sibao Wang 73 Habitat Management to Suppress Pest Populations: Progress and Prospects Geoff M. Gurr, Steve D. Wratten, Douglas A. Landis, and Minsheng You 91 MicroRNAs and the Evolution of Insect Metamorphosis Xavier Belles 111 The Impact of Trap Type and Design Features on Survey and Detection of Bark and Woodboring Beetles and Their Associates: A Review and Meta-Analysis Jeremy D. Allison and Richard A. Redak 127 Tephritid Integrative Taxonomy: Where We Are Now, with a Focus on the Resolution of Three Tropical Fruit Fly Species Complexes Mark K. Schutze, Massimiliano Virgilio, Allen Norrbom, and Anthony R. Clarke 147 Emerging Themes in Our Understanding of Species Displacements Yulin Gao and Stuart R. Reitz 165 Diversity of Cuticular Micro- and Nanostructures on Insects: Properties, Functions, and Potential Applications Gregory S. Watson, Jolanta A. Watson, and Bronwen W. Cribb 185 Impacts of Insect Herbivores on Plant Populations Judith H. Myers and Rana M. Sarfraz 207 viii