Allan Wilson Centre for Molecular Ecology and Evolution
Summer Studentship 2013/2014 Report
Student: Emily Roycroft
Project: Molecular phylogenetics of stick insects
Supervisor: Dr Thomas Buckley
With the advent of molecular genetic techniques for resolving evolutionary relationships, there has been much recent revision of historically determined phylogeny and species classifications (Blair and Murphy 2010).For New Zealand species of stick insects (Phasmatodea) belonging to the clade Lanceocercata, misclassification and undescribed species have contributed largely to an incomplete understanding of the evolution, taxonomy and biogeographic history of the group (Buckley et al. 2010). With nine described endemic genera of stick insects within New Zealand, consisting of 23 species (Salmon 1991; Jewell and Brock 2002), it is important that this phylogenetic information is resolved to aid our understanding of the biogeographical history and evolutionary relationships of species in the Australasian region.
Until recently, there had been limited formal phylogenetic analysis performed on New Zealand genera of stick insects, however work by Buckley et al. (2009; 2010) has began to attempt to resolve these issues. The geographical distribution of the clade Lanceocercatahas been found to range from Mascarene Islands, Australia, New Guinea, New Caledonia and the Pacific Islands, in addition to New Zealand (Brader 2001; Brader 2009; Whiting et al. 2003; Buckley 2009).
Buckley et al. (2010) performed molecular phylogenetic analysis using two mitochondrial genes (COI and COII) and two nuclear genes (28S and Histon3)from Lanceocercata taxa across all major landmasses included within their geographical range. Their results indicated that the New Zealand genera of Lanceocercataare contained within a larger radiation of New Caledonian species and that the New Zealand genera do not form a monophyletic group (Buckley et al. 2010). Further studies by Dunning et al. (2013) using 19 genes for 29 Lanceocercata stick insect taxa found that the New Zealand radiation are monophyletic. This disparity between the results of these two papers may be due to poor taxon sampling by Dunning et al. (2013), compared to the ~80 Lanceocercata taxa sampling by Buckley et al. (2010).
In order to investigate the relationships between groups within Lanceocercata and their historical and current biogeography, further studies with more robust taxon sampling for more genetic regions is required. This project aimed to acquire new nuclear DNA sequences from 88 individuals across the geographic range of Lanceocercata.
Methods Overview and Results
Nuclear genetic markers were designed using a whole genome sequence of one New Zealand stick insect individual. Six sets of genetic markers were tested in the course of this research project, with the initial two primer pairs tested corresponding to region EF1a, and further testing performed on regions Dhsa, T179 and Pyg.
EF1a Amplification and Sequencing
Polymerase chain reaction (PCR) amplification was performed on pre-extracted samples for all 88 available individuals. To amplify the region EF1a, primer pairEF1a-18F/1323Rwas used for the majority of samples as this appeared most effective in initial primer testing on a sample panel of representative individuals.Primer pair EF1a-153F/1453R was only used for samples that proved difficult to amplify with the former set.
PCR cycling conditions were set at the following standard conditions: Initial denaturation at 95°C for 4 minutes, followed by 35 cycles of 1) denaturation at 94°C for 30 seconds, 2) annealing at 54°C for 60 seconds, 3) extension at 72°C for 90 seconds. Final extension was set at 72°C for 10 minutes and samples were subsequently held at 10°C.
For samples which failed to amplify under the above conditions, the annealing temperature was lowered to 48°C. Successful amplification was determined by running PCR products on a 1.5% w/v agarose gel with TAE buffer, visualised using GelRed stain under UV light. Successful PCR product was purified using the BigDyeXTerminator® Purification Kit and sequenced with Sanger methods using BigDye®Terminator v3.1. Sequences were then edited and aligned in GeneiousR7 (Biomatters). Samples that showed messy or failed sequences were repeated under varied PCR conditions to attempt to improve sequence quality. A number of individuals displayed a length variable region, and internal primers were designed within the EF1a region (611F and 685R) in order to resolve sequence ambiguity. These were aligned and edited alongside the original EF1a-18F/1323R sequences where required.
In total, clean and unambiguous sequences for EF1awere obtained for 51 out of the total 88 available samples.
T179 and Pyg Primer Testing
Amplification success rate during initial primer testing for primer pairs across a sample panel of representation individuals for T179-F/R and Pyg-F/R suggested that these could be viable markers for further sequencing. PCR amplification, gel electrophoresis and sequencingwere performed as per the above standard conditions. Taking only samples with successful amplification under standard PCR conditions (as described above), the success rate for clean sequence during an initial sequencing run for region Pyg and T179a were 29% and 13% respectively. Due to time constraints, further troubleshooting was not performed and will be required in order to obtain a higher success rate for each of these regions.
A further two primer pairs;Dhsa-F/R and If5a-F/R also underwent initial primer testing on a sample panel of representative individuals. If5a displayed multiple bands within each lane for successful individuals and thus was excluded from further testing. Gel band visualisation of PCR amplification for Dhsa under standard PCR conditions had a success rate of approximately 44%. Further amplification and sequencing using this primer could prove successful in future studies.
A preliminary phylogenetic tree for region EF1a only (Figure 1) was constructed using the Tamura-Nei Neighbour-Joining method in Geneious R7 (Biomatters). Major geographic regions have been colour coded. Broad biogeographic groupings can be seen, with New Zealand samples forming a clade within a larger New Caledonian radiation, supporting findings of Buckley et al. (2010). The placement of an individual from the Mascarene Islands (MAM3) and from New Zealand (MIH5) can be considered anomalous. Similarly, it is unusual that individuals from New Zealand (ARG1, SPA1), Australia (LAN10, CEB1)seem more distantly related to other groups. DIM2 represented a more distantly relatedindividual from Papua New Guinea, so it’s appearance as a more independent lineage was expected. On the whole, these data seem to fit with biogeographic expectations of Lanceocercata phylogeny, however there exist some key discrepancies which warrant further testing of the genetic markers discussed above, as well as design and investigation of novel markers in the future. Further testing may help to explain some of the anomalous groupings observed in the preliminary results discussed in this report.
This project has contributed new genetic information which can be built upon into the future, and in the long term this research will help to further our understanding of the evolutionary history, biogeography and phylogeny of New Zealand stick insects.
I would like to thank Dr Thomas Buckley for the opportunity to work on this project over summer, and for providing invaluable assistance and expertise. Julia Allwood provided exceptional laboratory training and was always happy to provide help where required, along with all other members of the research group. Chen Wu provided excellent assistance with primer design. Finally, great thanks go to the Allan Wilson Centre for Molecular Ecology and Evolution for providing the funding for this summer studentship.
Figure 1: Tamura-Nei Neighbour-Joining Tree for region EF1a generated in Geneious R7 (Biomatters).
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Geneious version R7 created by Biomatters. Available from
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