DNA Sequencing as a Method for Larval Identification in Odonates Adeline Harris 121 North St Apt 3 Farmington, ME 04938 Adeline.harris@maine.edu Christopher Stevens 147 Main St Apt 1 Farmington, ME 04938 Christopher.stevens@maine.edu Faculty Sponsors Dr. J.A. Doty jeandoty@maine.edu Dr. R.G. Butler butler@maine.edu 1
Introduction Damselflies (Odonata: Zygoptera) are a diverse group of organisms ranging from the subarctic to the tropical that play an important role in lentic and lotic ecosystems (Artiss et al 2001). They are considered valuable biological indicators as they lay eggs in specific plant structures and sometimes on specific plant species (Butler & demaynadier 2008). Due to this fact, they are at an increased risk from near-shore development and disturbance which may damage near-shore vegetation required for reproduction (Butler & demaynadier 2008). Unlike the dragonflies (Anisoptera), the majority of the Zygoptera are weak flyers. Therefore any habitat disturbance may have large effect on their fitness. As a result, quantifying their diversity is an important aspect of their conservation. Unfortunately, abundance is often estimated by simply surveying adult populations (Fleck et al 2006; Rach et al 2007), which may underestimate the actual diversity within a given body of water (Fleck et al 2006). Monitoring the biodiversity of small, ecologically significant areasfor conservation depends on reliable identification of species. Larval odonate identification in particular presents a major challenge to scientists (Rach et al 2007). Identification of adults, especially males, is often quite easy and allows some understanding of their diversity. Larval identification, however, is very difficult and sometimes impossible for some species (Westfall & May 2006). Taxonomy and identification of zygopterans has previously been largely based on morphological characteristics (Fleck et al 2006). This may present an issue when determining phylogenies and identities of closely related species (Pilgrim et al 2002). This is especially true when looking at larval specimens. Methods such as DNA barcoding allow for consistent and reliable results, which complement traditional morphological identification (Rach et al 2007). Assessment using DNA as a taxonomic tool will allow a more accurate evaluation of species identity and diversity (Pilgrim et al 2002). 2
In addition to identification purposes, DNA sequencing is important in phylogenetic research. The 16S rrna and rdna regions of the mitochondrial genome have often been used to identify odonate species relationships (Misof et al 2000; Hasegawa & Kasuya 2006; Pilgrim & Dohlen 2008). In more recent studies, researchers have used multiple genomic sequences that allow them to examine different levels and patterns of variation for phylogenetic research (Chippindale et al 1999). Phylogenies can also be used to trace patterns of biodiversity in species of damselflies over time by looking at sequence divergences in DNA (Brown et al 2000). Local diversity is greatly influenced by speciation and extinction in addition to ecological factors, and studies have shown that climatic patterns have had large impacts on species radiation and diversification (Brown et al 2000; Turgeon et al 2005). The only way to reliably determine historical radiations is through the use of fossil evidence or DNA. However fossil evidence is often limited in terms of temporal scope and geographical scale, as climatic variability plays a large role in the fossilization process (Turgeon et al 2005). DNA sequencing allows a more robust phylogeny to be created that illustrates speciation events and helps to explain local and global species diversity. The phylogenetic relationship between the two species concerned in this study places them only one to three nodes apart on various parsimonious tree diagrams based upon both morphological and genomic data (Brown et al 2000). In particular, the morphological tree places them closer together than any of the genetic trees, suggesting that morphological similarity does not necessarily denote genetic similarity (Brown et al 2000). The goal of this project is to amplify a sequence of DNA that will allow reliable species determination for two congeneric damselflies (Enallagma ebrium and E. hageni), females of which are morphologically indistinguishable as larvae. 3
Research Methods Specimens were collected in Maine from June to August, 2007. These were previously identified and preserved in ethanol [(70%) various sources]. Male individuals of each species will be selected for DNA isolation and sequencing. For each individual, the legs will be removed and crushed using liquid nitrogen. DNA will be extracted using a standard DNA extraction kit. Two primer pairs will be used to amplify 28S rrna on the mitochondrial genome. An automated sequencer will be used to sequence the amplified fragments. In addition to sequencing done on the premises a sample will be sent to a professional lab for sequencing to assure the correct sequence is isolated. Once a useable sequence has been obtained, the procedure will be repeated with female specimens of E. ebrium and E. hageni, which cannot be differentiated between morphologically. Scholarship Funds and Project Outcomes This is a joint proposal for two separate Wilson Scholarships in which each individual is in charge of a separate aspect of the project. Adeline Harris will be in charge of DNA extraction, isolation, and sequencing regarding E. ebrium and Christopher Stevens will be in charge of DNA extraction, isolation, and sequencing regarding E. hageni as all aspects of the project will be extremely time consuming. Results for both species will be compiled and used to identify indistinguishable female specimens and a joint presentation will be given on symposium day detailing the results and implications of the study. In addition, if results are significant, an attempt will be made to publish them in a peer reviewed journal such as the Journal of Insect Conservation or Odonatologica. The additional funds for research materials will be required for both the scholarships as our needs for extraction and polymerase chain reaction (PCR) kits and other chemicals will likely reach $600 (see supplies). 4
Supplies Needed DNA isolation kit - ~$340 Primers - ~$22/primer Agarose gel - ~$70 Ethidium Bromide - ~$21/g PCR purification kit - $96 Micropipet loading tips for sequencer gel plate - ~ $50 Total ~$600 5
References Artiss, T., T.R. Shultz, D.A. Polhemus, and C. Simon. Molecular phylogenetic analysis of the dragonfly genera Libellula, Ladona, and Plathemis (Odonata: Libullulidae) based on mitochondrial cutochrome oxidase I and 16S rrna sequence data. 2001. Molecular Phylogenetics and Evolution, 18:3 pp348-361. Brown, J.M., M.A. McPeek, and M.L. May. A phylogenetic perspective on habitat shifts and diversity in the North American Enallagama damselflies. 2000. Systematic Biology, 49:4 pp679-712. Butler, R.G., P.G. demaynadier. The significance of littoral and shoreline habitat integrity to the conservation of lacustrine damselflies (Odonata). 2008. Journal of Insect Conservation, 12: pp23-36. Chippindale, P.T., V.K. Dave, D.H. Whitmore, and J.V. Robinson. Phylogenetic relationships of North American damselflies of the genus Ischnura (Odonata: Zygoptera: Coenagrionidae) based on sequences of three mitochondrial genes. 1999. Molecular Phylogenetics and Evolution, 11:1 pp110-121. Fleck, G., M. Brenk, and B. Misof. DNA taxonomy and the identification of immature insect stages: the true larva of Tauriphila argo (Hagen 1869) (Odonata: Anisoptera: Libellulidae) Hasegawa, E., and E. Kasuya. 2006. Phylogenetic analysis of the insect order Odonata using 28S and 16S rdna sequences: a comparison between data sets with different evolutionary rates. Entomological Science, 9:pp55-66. Misof, B., C.L. Anderson, and H. Hadrys. A phylogeny of the damselfly genus Calopteryx (Odonata) using mitochondrial 16S rdna markers. 2000. Molecular Phylogenetics and Evolution, 15:1 pp5-14. Pilgrim, E.M., S.A. Roush, and D.E. Krane. Combinging DNA sequences and morphology in systematics: testing the validity of the dragonfly species Cordulegaster bilineata. 2002. Heredity, 89: pp184-190. Pilgrim, E.M., C.D. Von Dohlen. Phylogeny of the Sympetrinae (Odonata: Libellulidae): further evidence of the homoplasious nature of wing venation. 2008. Systematic Entomology, 33:pp159-174. Rach, J., R. DeSalle, I.N. Sarkar, B. Schierwater, and H. Hadrys. Character-based DNA barcoding allows discrimination of genera, species, and populations in Odinata. 2008. The Royal Society, 275:pp237-247. Turgeon, J., R. Stoks, R.A. Thum, J.M. Brown, and M.A. McPeek. Simultaneous quaternary radiations of three damselfly clades across the Holarctic. 2005. The American Naturalist, 165:4:ppE76-107. Westfall, M.J., and M.L. May. 2006. Damselflies of North America. Scientific Publishers. Gainesville, Florida. 6