Phylogenetic relationships within the leaf-mining flies (Diptera: Agromyzidae) inferred from sequence data from multiple genes
Introduction
The leaf-mining flies (Diptera: Agromyzidae) are small flies having phytophagous larvae that feed within leaves, stems, roots, flowers, or seeds. Agromyzids attack a broad diversity of plant hosts, including more than 140 plant families representing all major terrestrial plant groups, with the exception of mosses and most gymnosperms (Spencer, 1990, Benavent-Corai et al., 2005). These flies are distributed worldwide with their greatest diversity in northern temperate regions (Spencer, 1977). Despite the broad diet breadth exhibited by agromyzids as a group, most agromyzid species are remarkably specialized, feeding on only one or a few closely related plant species (Spencer, 1990, Scheffer and Wiegmann, 2000). Host-use evolution within the Agromyzidae is a subject of great interest because diversification in this group appears to be largely associated with host shifts and dietary specialization (Spencer, 1990, Kulp, 1968, Scheffer and Wiegmann, 2000). However, modern phylogenetic analyses necessary for elaborating the history of agromyzid/host associations have only recently been applied to studies of these flies (Scheffer and Wiegmann, 2000, Dempewolf, 2001).
Despite their near ubiquity and interesting larval habits, the systematics of agromyzids has remained rather poorly understood due to their small size and morphological homogeneity. Species identification generally requires dissection of the male genitalia, and it is difficult to identify some female specimens even to genus using only morphological characters. Currently, the Agromyzidae contains two subfamilies, the Agromyzinae and the Phytomyzinae, based on one larval and one adult character (Spencer and Steyskal, 1986) although these characters are not always consistent (von Tschirnhaus, 1971). Twenty-eight genera are recognized containing approximately 2860 described species (Table 1), although it is clear from the number of new species found during regional surveys and detailed study of species groups (e.g., Scheffer and Wiegmann, 2000, Zlobin, 1994a, Zlobin, 2002) that this represents only a fraction of agromyzid diversity (see also Spencer, 1977, Spencer and Steyskal, 1986). Nearly a third of the known genera have fewer than 10 species (Table 1), while many of the other genera are large, containing many groups of morphologically similar species that often feed on closely related host plants (Spencer, 1990; also e.g., Griffiths, 1974, Griffiths, 1976, Griffiths, 1977, Scheffer and Wiegmann, 2000, Zlobin, 2000).
Modern hypotheses of relationships among agromyzid genera come primarily from two sources: a verbal “phyletic lines” scheme by Spencer (1990) in his consummate treatise on agromyzids and their host plants (Table 2), and a quantitative morphological phylogeny based on 83 larval and adult characters (Fig. 1) in a dissertation by Dempewolf (2001). Spencer envisioned four phyletic lines within the family with the “Penetagromyza group” corresponding to the Agromyzinae, and the traditional Phytomyzinae broken into the “Phytobia group,” the “Phytoliriomyza group,” and the “Napomyza group.” Relationships among the latter three groups are unspecified. Most important in Spencer’s scheme is the assertion, first suggested by Nowakowski (1962), that Phytobia, a widespread genus of large, cambium-mining species, is of ancient origin and represents the “primitive” condition within the family. This continues a long-held belief among agromyzid workers that groups of small leaf-mining species are derived from larger stem-feeding species (Nowakowski, 1962, Spencer, 1990, von Tschirnhaus, 1991). In fact, within each of Spencer’s four phyletic lines the genera are arranged from a “primitive” stem-mining genus to “more advanced” leaf-mining groups.
Dempewolf, 2001, Dempewolf, 2005 phylogenetic analysis of 83 larval and adult characters resulted in 90 most parsimonious trees obtained by successive weighting using the consistency index. The strict consensus of these trees is shown in Fig. 1 (redrawn from Dempewolf, 2005). In this analysis, the two subfamilies are found to be monophyletic, although support values for these and all other relationships are not available. Relationships differ from those suggested by Spencer in several ways, most notably in finding Aulagromyza and Gymnophytomyza to be associated with the Napomyza group of Spencer. Additionally, Amauromyza is found to be sister to Cerodontha and well inside a clade also including Liriomyza, Galiomyza, Phytoliriomyza, Metopomyza, and Calycomyza. Most importantly, reconstruction of larval feeding mode suggests that the ancestral feeding mode within the Agromyzidae was leaf-mining rather than stem- or cambium-mining (Dempewolf, 2005).
Here, we present the first comprehensive molecular phylogenetic analysis of the entire family Agromyzidae. A major goal is to provide new evidence from nuclear and mitochondrial genes on higher-level relationships within the family. With these new phylogenetic results, we investigate patterns of diversification within leafminers, evaluate relative support from genes and morphology for currently recognised genera as well as for key areas of agromyzid classification, and establish an initial framework for future investigations of agromyzid relationships and host-use evolution.
Section snippets
Materials and methods
Agromyzid specimens representing 86 species in 21 genera were obtained from a variety of locations around the world (Appendix A). We attempted to include congeners from different subgenera or different species groups whenever possible, particularly for the larger genera. For outgroups, we chose two Fergusonina species (Fergusoninidae). Several authors have suggested that the Agromyzidae and the Fergusoninidae are closely related, and at one time fergusoninids were even placed within the
Results
Our final aligned concatenated data set for the three genes contains 2965 positions of which 1463 are variable and 1269 parsimony informative. The three gene partitions are 824 (28S), 710 (CAD), 1431 (mt COI) nucleotides in length, respectively. Uncorrected pairwise distances for all three genes are shown in Table 4, across the range of agromyzid divergences included in the current sample. Because of the high variability, and thus high potential for homoplasy, found in 3rd codon position sites
Discussion
A major means of assessing the utility of a character system for phylogenetics is to investigate whether phylogenetic analysis recovers groups that are strongly supported by other sources of data (Kluge, 1989, Miyamoto and Cracraft, 1991, Friedlander et al., 1994). The Agromyzidae has only recently been the subject of quantitative phylogenetic analysis, leaving us with little previous work on agromyzid relationships for strong comparisons with the results of this study. However, for this
Conclusion
This project is the first to use molecular data to investigate higher-level relationships within the Agromyzidae. Many of our findings are consistent with what is known concerning agromyzid morphology and taxonomy, although several areas requiring additional study have been revealed. Most importantly, there is no evidence that cambium-mining or stem-feeding is the ancestral condition within the family or the general precursor to leaf-mining. Further work on higher-level relationships as well as
Acknowledgments
We thank collectors listed in Appendix A for providing specimens used in the study. We thank Matt Lewis and Charles Godfray for assistance with collecting trips. Matt Lewis also provided much of the sequence data. Dug Miller, Ronald Ochoa, Kevin Omland, and Alessandra Rung provided helpful comments on the manuscript. This project was supported in part by National Science Foundation/Sloan Foundation Postdoctoral Research Fellowship in Molecular Evolution Grant No. BIR-9510795 to S.J.S. and
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