Allan Wilson Centre Summer Studentship Report 2012-2013
Stick Insect Phylogenetics
Megan Iminitoff
Supervisor: Thomas Buckley
Introduction
Stick insects are surprisingly common in New Zealand, belonging to the insect order
Phasmatodea. There are 23 recognised species of stick insect in New Zealand that fall into 10 genera. This project focussed on two of these genera, Asteliaphasma and
Spinotectarchus . Individuals belonging to these genera can be found in the upper half of the North Island, as well as on some offshore islands. Other similarities between the genera include short spines covering their eggs and that they are both most likely to be found on climbing rata. Spinotectarchus contains only one species
Spinotectarchus acornutus , whereas there are two species belonging to the
Asteliaphasma genus, Asteliaphasma naomi and A. jucundum . The aim of this project was to add to the growing stick insect genetic dataset. The project involved DNA extraction, PCR amplification, DNA sequencing and some phylogenetic analysis of two mitochondrial genes: COI and COII .
Methods
DNA Extraction
DNA was extracted from 180 stick insect specimens, of which 84 belonged to the genus Asteliaphasma and 96 to the Spinotectarchus genus. A leg was removed from each specimen, chopped up and combined with a mix of 420µl of tissue digest DXT and 4.2µl of digest enzyme, proteinase K. The samples were incubated overnight at
56°C. DNA was then extracted using the robot with the X-tractor Gene™ protocol.
Extracted samples were stored at 4°C.
Asteliaphasma
Amplification of the COI and COII genes was done by PCR amplification of the extracted DNA. The primers utilised were; C1-J-2195 and TL2-N-3014 for COI and
TL2-J-3034 and TK-N-3785 for COII . PCR amplifications were initially run with an annealing temperature set at 55°C, which was later raised to 56°C to give a cleaner product. To determine the success of the PCR reaction, the PCR products were run on a 1.5% w/v agarose gel in TAE buffer, stained with GelRed. All PCR products from the Asteliaphasma genus appeared as bright and/or thick bands for both genes.
Successful PCR products were then sequenced using BigDye
®
Terminator v3.1. The sequencing reactions were then purified using the BigDye
®
XTerminator™ Purification
Kit and loaded onto the sequencing machine. A total of 73 out of the 84 sequences were successful for COII, and 80 out of 84 were successful for COI. Attempts to correct messy or failed sequences included raising the annealing temperature to 58
°C and gel extraction; none of which improved the sequence quality. Sequences were analysed and edited using Geneious Pro 5.6.5. This program was also used to create two simple neighbour-joining trees, one for COI and one for COII .
Spinotectarchus
The genes COI and COII were also PCR amplified for the Spinotectarchus samples.
Initially, primers C1-J-2195 and TL2-N-3014 were used for amplification of COI and

TL2-J-3034 and TK-N-3785 were used for COII . After running the PCR products on a gel, a secondary band became apparent in the COI products. This band interfered with the COI sequencing causing the majority reads to be messy. To try and remedy this, PCR reactions were repeated on a small set of samples using BSA in the reaction mix and dropping the number of amplification cycles from 40 to 37. When these PCR products were run on a gel the desired band seemed to be more defined, yet there was still a faint secondary band and the sequencing traces remained unreadable. In order to try and eliminate the additional band, gradient PCR was used in attempt to ascertain a more optimal annealing temperature. BSA was included again and four annealing temperatures: 56°C (as a control), 58°C, 60°C, and 62°C, were trialled. The desired band was brightest at 56°C, as was expected. Also as expected, the secondary band diminished both at 58°C and 60°C, a 62°C annealing temperature resulted in the desired product bands being too faint. Bands resulting from the 58°C and 62°C reactions, where the secondary bands were the faintest, were sequenced. However, this still proved unsuccessful. Finally, a different forward primer, C1-J-2183 was tried. The secondary band was still present in these samples, but at a weaker intensity.
Therefore clean sequencing traces were able to be obtained. PCR amplification of
COI was repeated for all of the Spinotectarchus extractions using the 2183 and 3014 primers. COII had no such issues and sequenced cleanly with the original primers.
Sequencing for Spinotectarchus was done using the same methods as previously mentioned for the Asteliaphasma genus. Geneious was likewise used to analyse and edit the Spinotectarchus sequences. A total of 90 out of the 96 samples were sequenced successfully for COII , however only 69 out of 96 were successful for COI .
These sequences were used to create two neighbour-joining trees, one for each of COI and COII .
Discussion
Phylogenetic analysis of the datasets showed some distinct trends across the two genera. Two neighbour-joining trees (one for COI and one for COII ) created from an alignment of all of the consensus sequences showed the clear distinction between genera, with Asteliaphasma and Spinotectarchus falling into separate groups. The single genus trees for Asteliaphasma indicated strong groupings for populations from
Northland. The clades for Northland localities, such as Warawara Forest, Ngaiotonga
Scenic Reserve and Tangihua Forest, were very range specific, encompassing only individuals from a particular area. Clades containing individuals from areas further south showed a range expansion and comprised specimens from more spread out regions, for example individuals collected from Pureora Forest and from Bridal Veil
Falls were grouped together. These trends were supported by both the COI and COII trees. Similar patterns were seen in the Spinotectarchus genus. The trees for both genes showed separate clades specific to Northland areas, such as Te Paki and
Tangihua Forest. Also apparent were clades that had individuals from more southern regions, which extended over larger areas such as from Pureora Lodge to Tapu-
Coroglen Track.