Rebecca Clement and Faculty Mentor: Dr. Seth Bybee, Department of Biology
Anax dragonflies are found worldwide, with many species migrating across continents. One species of Anax, A. junius, makes annual migrations travelling thousands of miles each fall from Canada to Mexico (May 2013). Researchers from all over the world use Anax to learn about vision, insect musculature and insect migration. For example, A. junius is a favorite of many research projects in North America (Bybee et al. 2012), while A. imperator is often used by European collaborators (Sharkey et al. 2015), and A. parthenope by Asian collaborators (Futahashi et al. 2015). However, despite widespread interest in Anax, relationships among species remain murky. Specifically, the relationship between Anax and Hemianax is unclear. They were previously considered the sister to each other, but recently some have suggested collapsing the two groups to reduce paraphyly (Peters 2000; Schorr et al. 2007). It is still unclear if they should be combined or left as sister to each other, and some continue to distinguish the two groups as separate (Von Ellenrieder 2002). Because Anax is a highly mobile group, very few geographic structures limit populations. Therefore, it is interesting to investigate what genetic barriers or migratory events divided populations enough to limit gene flow and promote speciation. This can be then applied to other groups to better understand how geographic distribution relates to evolutionary relationships. In this study, we compared four genes from 22 species to answer these questions as well as clarify general evolutionary relationships between species in Anax.
We included 155 specimens of Anax and Hemianax, and 10 outgroup specimens, for a total of 165. These specimens were either donated from collaborators or museums, or collected by the Bybee lab, and come from 23 countries throughout the world. Two of these specimens, Anax congoliath and Gynacantha victoria, were obtained through field work funded by this ORCA (Fig 1). we also included data available on GenBank in our nalysis. These specimens included 22 of the 31 recognized species of Anax (Schorr et al. 2007). We extracted DNA from specimens using Qiagen DNAeasyTM extraction kit (Valencia, CA, USA) following standard protocol for insect extraction. We amplified portions of mitochondrial genes COI/COII and CYTB, and nuclear genes ITS1/ITS2 and PRMT using polymerase chain reaction (PCR). We sequenced the regions of DNA at the BYU DNA sequencing center using 3730xl for sanger dideoxy sequencing. We analyzed the sequences using Geneious version 6.1.8 (http://www.geneious.com, Kearse et al. 2012) and aligned them with MAFFT. We generated a phylogeny using maximum likelihood in IQ-tree (Nguyen et al. 2015) with an edge-linked partitioning file and ultrafast bootstrap analysis. We visualized and rooted the tree to the outgroup dragonflies using FigTree version 1.4.2 software (Rambaut 2012).
The reconstructed phylogeny is well supported with 74% of the interspecies bootstrap values being greater than 70% (Fig 2). Thirteen species of Anax were recovered as monophyletic. Seven species are not monophyletic, and two species had only one individual so it is unknown if they are monophyletic. According to our tree, Hemianax ephippiger and Hemianax papuensis do not form a monophyletic group. Several clades show close relationships among Anax in the same area. Discussion and Conclusion Because Hemianax does not form a monophyletic group, we recommend following Peters’ suggestion and collapsing Hemianax into Anax (Peters 2000). This will reduce further confusion in the literature. A few geographic patterns are interesting in our phylogeny. First, there are two clades of American Anax. This may indicate that there were two separate migration events across the Atlantic Ocean. There is also a well-supported clade of African Anax. However, many African specimens are more closely related with Asian or Oceanic species. This suggests that there may have been multiple migrations back to Africa. Understanding the relationships in Anax will allow better comparisons among Anax species that are widely used in studies of vision, musculature, and migration. We can use the separated clades of American and African Anax to compare how a similar environment affects distantly related groups morphologically and behaviorally. Future work may compare migratory behavior and morphology between different groups to determine possible geographic barriers
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