Madison Lallatin, Seth Bybee, Biology Department
Vanuatu is a string of active volcanic islands in Micronesia near Fiji and Australia. The islands undergo regular submersion and emersion from the ocean, and the most recent emergence was 2 million years ago (Hamilton et al. 2010). As a result, all organisms on the islands of Vanuatu have migrated there, likely from other islands, within the past 2 million years. Fiji is one likely candidate for contributing species, being geographically close to Vanuatu and exhibiting similar species. Fourteen of us went to Vanuatu for 6 weeks on a study abroad led by Dr. Seth Bybee to collect and observe several orders of insects, most notably Odonata (damselflies and dragonflies), Coleoptera (beetles), and Lepidoptera (butterflies and moths). The insects of Vanuatu are still very understudied, making this a unique opportunity for student research. I was the leader of the Odonata group and our goal was to collect an endemic genus of damselfly called Vanuatubasis and record relevant information on where the genus was found, as well as ecological data, biogeographical distribution, and naiad association (matching juveniles with adults). We visited 6 islands in all, and we were successful in finding the target genus, as well as numerous new species. For my project within the Odonata group, the specimens were used to reconstruct a phylogenetic tree to help illustrate how Vanuatubasis and related genera, specifically the Fijian genus Nesobasis, have distributed and speciated across the islands of the South Pacific.
Methods and Materials
6 weeks were spent sampling rivers on the islands of Tanna, Efate, Erromango, Malekula, Espiritu Santo, and Gaua. Target genus specimens were found on Malekula, Espiritu Santo, and Efate. Odonates were stored in small vials with ethanol as a preservative. Latitude and longitude were recorded for each site, and correct labels were assigned to the specimens. Upon returning from Vanuatu, specimens were stored in a freezer to preserve the DNA. For the DNA extraction process, we used a Qiagen kit. Careful incisions were made on the ventral surface of the thorax of specimens, and some wing muscle was removed to use for the extraction. Wing muscle is the most abundant source of DNA for these insects, but other parts, including legs, were used as well. The wing muscle was then put into a centrifuge tube, and several buffers and lysing agents were added in a specific sequence to isolate the DNA. A NanoDrop machine was used to verify that enough DNA has been extracted for the PCR process which amplified the DNA to a usable level. PCR procedure was taken from Beatty et al. (2017). With the PCR products, DNA was sequenced and used to reconstruct a phylogenetic tree using standard software.
I referred to the paper by Ober and Staniczek (2009) which describes the only 3 species of Vanuatubasis described so far: V. bidens, V. malekulana, and V. santoensis. I have grouped the specimens by site and general appearance, and I have looked at representatives from each group under a microscope to find similarities to the known species. The identifying features from the paper were the anal appendages and the pronotum.
When a phylogenetic estimate using only COI we generated, Nesobasis was recovered as non-monophyletic, with Vanuatubasis within the Nesobasis clade (Figure 5). However, in the phylogenetic reconstruction using three loci (COI, 12S and ITS), the phylogenetic relationship between Nesobasis and Vanuatubasis showed both to be monophyletic (Figure 4). Within Vanuatubasis itself, species from Espiritu Santo and Malekula do not form monophyletic groups. Furthermore, a species from Efate sits within the Malekula species clade. This is interesting as Espiritu Santo and Malekula are close together geographically and distant from Efate (Figure 1). Further research is required to determine which evolutionary history of the group is accurate.
As for the ecology of the species, we found that Vanuatubasis usually occurs in rivers that are of a higher pH, usually 8 or higher. The target genus could be found on both sunny and cloudy days, but more often on sunny days, and often in the shade among vertical foliage along the bank. It is more commonly found between the hours of 10am and 2pm, and outside these hours it is generally found high up among the trees, out of reach. We found Vanuatubasis more often on smaller rivers with lots of foliage, and less often on larger and more open rivers.
Discussion and Conclusion
If Vanuatubasis sits within Nesobasis, then Nesobasis is non-monophyletic and would need to be reexamined. It was difficult to obtain usable DNA sequences from the extraction and PCR steps due to DNA fragmentation from temperature change. With such a limited amount of time, it is difficult to know which phylogeny is the most accurate, and more research is required. One thing to look for is whether Vanuatubasis is monophyletic or not. This will help us understand if there were multiple invasions of Vanuatu by Nesobasis in the history of the genus. More research and time will be required to determine the relationship between the genera. I presented my findings in the form of a poster at the Entomological Society of America meeting in Vancouver in November of 2018.
Christopher D. Beatty, Melissa Sánchez Herrera, Jeffrey H. Skevington, Arash Rashed, Hans Van Gossum, Scott Kelso, and Thomas N. Sherratt (2017). “Biogeography and systematics of endemic island damselflies: The Nesobasis and Melanesobasis (Odonata: Zygoptera) of Fiji.” Ecology and Evolution, Sep; 7(17): 7117–7129.
Stefan V. Ober and Arnold H. Staniczek (2009). “A new genus and species of coenagrionid damselflies (Insecta, Odonata, Zygoptera, Coenagrionidae) from Vanuatu.” Zoosystema, 31 (3): 485-497.