Incongruence between molecules and morphology: A seven-gene phylogeny of Dacini fruit flies paves the way for reclassification (Diptera: Tephritidae)
Graphical abstract
Introduction
Despite the advent of molecular data, the phylogenetic relationships among some groups remain disputed even with the inclusion of substantial molecular data sets (Barley et al., 2010, Losos and Ricklefs, 2009). One explanation for the incongruence between molecules and morphology is prevalent convergent evolution of morphological characters, which confounds relationships (Moore and Willmer, 1997, Willmann, 1998). Convergent evolution has been documented across broadly divergent groups, with classical examples including Hawaiian song birds (Fleischer et al., 2008), Heliconius butterflies (Endler, 1981) and cichlid fishes (Kocher et al., 1993). Taxonomic classifications based on a small number of characters are often vulnerable to continued revision until a robust molecular framework is developed, even more so in groups with convergent evolution (Endler, 1981, Fleischer et al., 2008). In invertebrates, convergent morphological evolution is likely more common than previously thought (Bickford et al., 2007, Burns et al., 2008), further highlighting the need to study morphology within a robust molecular phylogenetic framework (Breinholt et al., 2012, Moore and Willmer, 1997).
Tephritidae, or “true fruit flies”, are one of the most species-rich families in Diptera (Aluja and Norrbom, 1999, White and Elson-Harris, 1992). The tribe Dacini holds 936 species, among which about 85 are recognized as pests on commercial fruits (Vargas et al., 2015). Dacini are native to the Old-World tropics and subtropics, and some invasive pest species have spread beyond their native ranges to other parts of the world (Fig. 1), although the vast majority of species are not considered pests and are often specialists, associated with Old-World tropical and sub-tropical fruit trees (Clarke, 2016, Drew, 2004, Novotny et al., 2005). The pest species together infest a broad range of tropical and subtropical fruits and cucurbit crops and are often more generalists (Vargas et al., 2015). Adult females oviposit on fleshy fruits and vegetables, where the larvae subsequently feed and develop. Economic losses come from the loss of trade between countries or regions to prevent the spread of these pests, management and eradication programs that include costs for surveillance and treatments, and significant crop losses where pests are present. Accurate identifications are therefore of key importance for quarantine, which, in turn, relies on a stable systematic classification with a clear concept of the phylogenetic and diagnostic relevance of morphological characters of the species and higher groups. However, in contrast to most other tephritids, which usually have colorful and intricate wing patterns, and mating dances displaying various characters, species in Dacini have largely clear wings and overall few morphological characters to distinguish them, posing a challenge for taxonomy and agricultural diagnostics alike (Fig. 2). The taxonomic system for Dacini has seen large shifts throughout its history regarding generic and subgeneric classifications (see Text Box 1 for an overview). At present, there is a discordance between relationships suggested by morphological character data and molecular results, and contemporary authors have diverging interpretations for species delimitation and classification (e.g., De Meyer et al., 2015, Drew and Romig, 2016, Freidberg et al., 2017, Hancock and Drew, 2015, Virgilio et al., 2015). Given the overall sparsity of informative characters and intraspecific variation of characters, in addition to the assumed mimicry of wasps, the morphological convergence of unrelated species is likely (Drew and Hancock, 1999).
One of the few attempts to produce a morphology-based phylogeny for Dacini was by White (1999), who conducted a parsimony analysis using a 38-character matrix from 51 pest species. The resulting tree was highly polytomous with only partly monophyletic subgenera, and Dacus was paraphyletic with respect to a monophyletic Bactrocera. The use of molecular markers has provided greater insight into the systematic relationships within Dacini. However, these studies included relatively few taxa and genes (Nakahara and Muraji, 2008, Smith et al., 2003). In an extensive phylogenetic study, Krosch et al. (2012) found that Dacus rendered the Bactrocera paraphyletic and strongly suggested that “Zeugodacus should, following revision of the Zeugodacus ‘concept’, be elevated to genus level”. More recently, Virgilio et al. (2015) elevated Zeugodacus to genus level and De Meyer et al. (2015) provided a checklist where they assigned species from the subgenus Zeugodacus and a number of associated subgenera to the newly formed genus Zeugodacus. The little-studied genus Ichneumonopsis Hardy (three spp.) was initially placed within Adramini and was transferred to Dacinae by Hancock (1986). Norrbom et al. (1999) moved it to Gastrozonini, however, Drew and Hancock (1999) continued to consider it Dacini. Most recently Ichneumonopsis was moved again to the tribe Gastrozonini by Freidberg et al. (2017). Its new placement fits with its bamboo feeding association whereas Dacini are frugivorous. Currently, Dacini contains four genera; one small genus Monacrostichus Bezzi (two spp.) and three much larger genera, Dacus (270 spp.), Bactrocera (467 spp.) and Zeugodacus (189 spp.) (De Meyer et al., 2015, Drew and Hancock, 1999, Krosch et al., 2012, Virgilio et al., 2015, White, 2006). The three large genera are further divided into subgenera, species groups and species complexes based on morphology, however, the validity and the evolutionary relationships between these groups has been questioned (Clarke et al., 2005, Drew, 2004, Drew and Romig, 2013, Drew and Romig, 2016, Krosch et al., 2012).
Most of the species complexes in Dacini have not been studied under a phylogenetic framework. The few previous studies primarily have focused on two complexes containing several economically important species, viz. the Z. tau and B. dorsalis complexes. Various approaches were used to understand the relationships between species in these groups, including geometric morphometrics (Kitthawee and Dujardin, 2010, Krosch et al., 2013, Schutze et al., 2015b) and phylogenetics (Boykin et al., 2014, Jamnongluk et al., 2003, San Jose et al., 2013, Smith et al., 2003). Such studies mostly concluded that some of the species could be discriminated using either method or a combination of both. Leblanc et al. (2015) used DNA sequence data, based on analysis of three genes and 50 species, to show that the B. dorsalis complex is a polyphyletic assemblage. Although the B. dorsalis complex with close to 90 species is the largest, there are other dacine species complexes that contain over ten members and include species of economic importance, e.g., the B. musae and Z. tau complexes, that require further study and may not be natural groups.
Given the agricultural importance of many Dacini species, knowledge on their ecology has evident practical implications with large economic consequences. Current knowledge is confounded by the lack of a clear understanding of the phylogenetic relationships among species and species groups. Tephritidae, in general, are known for their intricate sexual behaviors which play a part in mate selection and most likely speciation (Sivinski et al., 1999). In most Dacini, however, males are also attracted to plant kairomones which are ingested and incorporated into their male mating pheromones (Tan and Nishida, 2007). There are three known kairomones (methyl eugenol, raspberry ketone [cue-lure is the artificial formulation] and zingerone) that are highly attractive to different subsets of Dacini species (Drew and Hancock, 1999, Royer, 2015, Royer et al., 2017). These compounds are involved in sexual selection by increasing male mating success (Kumaran et al., 2013, Raghu, 2004, Shelly, 2010, Shelly and Dewire, 1994). The kairomones are also used as lures in traps to detect new invading species and to monitor the spread and the population density of established species (Vargas et al., 2010, Vargas et al., 2016). In agricultural settings, they may be used to reduce or eliminate male flies to reduce population size in the field and mitigate pest damage by disrupting mating (Vargas et al., 2009, Vargas et al., 2010). Ecological studies that focus on the behavior of species, host selection, distributional patterns or pollination associations are hindered when species are inaccurately identified.
Our current study contributes a broader sampling of species and genes for the Dacini than most previous work, and thereby provides additional, important perspective on relationships at multiple levels. Because generic assignments are still in flux (Schutze et al., 2015a, Schutze et al., 2015b, Virgilio et al., 2015), and have important implications for quarantine, control, monitoring and invasion potential, additional data on the Dacini is vital to understanding this economically important and evolutionarily complex group of insects. Specifically, in the present study, we assess the robustness of current taxonomic relationships within the tribe by constructing a phylogeny using seven genes (one mitochondrial and six nuclear) and 167 Dacini species. We used the resulting molecular phylogenies to examine the monophyly of morphology-based genera, subgenera, subgeneric groups and species complexes within the tribe. We also apply our phylogenetic data to examine which, if any, synapomophic characters define monophyletic groups and might be useful for identification purposes. Testing the utility of such characters has important implications for rapid morphology-based identifications at quarantine. Finally, we anticipate that, in the future, the synergistic impact of comparing multiple datasets will provide a more robust framework to more firmly understand systematic relationships in this complex group and permit more rigorous comparative studies which aim to understand how morphology, climatic thresholds of pest species, and attractiveness of kairomones have evolved and possibly shaped the diversity of Dacini.
Section snippets
Taxon sampling
We included 167 species of Dacini and five outgroup Tephritidae from throughout the distribution range of Dacini. A list of taxa and specimens, including collecting locality, species names and voucher codes is in Supplementary materials (Table 1). Specimens were collected using male attracting kairomone-baited traps between 2010 and 2016 using the protocol described in Leblanc et al. (2015). In addition to the widely used lures methyl eugenol (ME) and cue-lure (CL), we used zingerone (ZN) which
Results
Sequence data for a total of 167 species from seven genes was used for phylogenetic analysis. Concatenation of sequence data produced an alignment of 4521 bp. We included 100 species of Bactrocera, 24 Dacus and 43 Zeugodacus, which represents 23%, 8.8% and 22% of the diversity in these genera respectively, and added five outgroup species: two species of Anastrepha and three species of Ceratitis. The results from the ML and BI phylogenetic analyses are presented in Fig. 3.
Phylogenies inferred
A robust phylogeny
Our phylogeny based on 167 species and seven genes represents the most comprehensive phylogeny produced for Dacini to date. It is missing the two species from Monacrostichus as well as a significant portion of the African fauna (primarily Dacus species) and many of the Bactrocera and Zeugodacus species endemic to the Pacific and Papua New Guinea. Despite these gaps in our taxon sampling, we believe this study provides significant insight into the evolutionary history and phylogenetic
Conclusions
Our study supports the recent elevation of Zeugodacus to genus level. However, most groups below the genus level in Dacini are not monophyletic and are often composed of genetically very different species. A more extensive sampling of multiple representatives of all subgenera and complexes is required to fully resolve the phylogeny of Dacini. Morphological characters can be used to some extent to differentiate the three genera from each other (abdominal terga fused vs. not fused, shape of
Conflict of interest
There are no competing interests for this research.
Funding sources
Funding for this project was provided by the United States Department of Agriculture Agricultural Research Service (USDA-ARS) and the USDA Animal and Plant Health Inspection Service (USDA-APHIS) Farm Bill Section 10007 projects “Diagnostic Resources to Support Fruit Fly Exclusion and Eradication, 2012–2014” and “Genomic approaches to fruit fly exclusion and pathway analysis, 2015–2016” to USDA-ARS, USDA-APHIS, and University of Hawai’i at Mānoa (projects 3.0251.02 and 3.01251.03 (FY 2014),
Acknowledgments
We are indebted to Eleida Flanglan, Rudolph Putoa, Kemo Badji and Sylvain Ouedraogo for providing specimens from the Philippines, French Polynesia and Africa. We also thank Will Haines, Jesse Eiben, Tony Wong, Emmett Easton and David Haymer for providing additional material from Asia and Hawaii. Saba Young, Ciera Taylor, Chris Guo and Dan Nitta helped with molecular work. We greatly appreciate the help of Chi-Yeh Chien (Thai Royal Project Foundation), Thongsavanh Taipangnavong (United Nations
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