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
Introduction
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
Lanceocercata has 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 Lanceocercata are
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
Primer Design
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 pair
EF1a-18F/1323R was 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 BigDye
XTerminator® Purification Kit and sequenced with Sanger methods using
BigDye®Terminator v3.1. Sequences were then edited and aligned in Geneious R7
(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 EF1a were 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 sequencing were 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.
Phylogenetic analyses
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 related individual 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.
Acknowledgements:
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).
References:
Blair C, Murphy RW (2010) Recent Trends in Molecular Phylogenetic Analysis:
Where to Next?, Journal of Heredity, doi:10.1093/jhered/esq092, accessed at
http://jhered.oxfordjournals.org/content/early/2010/08/07/jhered.esq092.full,
13/03/14.
Bradler S (2001) The Australian stick insects, a monophyletic group within the
Phasmatodea?, Zoology 104 (Suppl. IV), 69.
Bradler S (2009) Phylogeny of the stick and leaf insects (Insecta: Phasmatodea).
Species, Phylogeny and Evolution 2, 3–139.
Buckley TR, Attanayake D, Bradler S (2009) Extreme convergence in stick insect
evolution: phylogenetic placement of the Lord Howe Island tree lobster, Proceedings
of the Royal Society of London Series B 276, 1055–1062.
Dunning LT, Dennis AB, Thomson G, Sinclar BJ, Newcomb RD, Buckley TR (2013)
Positive selection in glycolysis among Australasian stick insects, BMC Evolutionary
Biology 13:215, doi:10.1186/1471-2148-13-215.
Geneious
version
R7
http://www.geneious.com/
created
by
Biomatters.
Available
from
Jewell T, Brock PD (2002) A review of the New Zealand stick insects: new genera
and synonymy, keys, and a catalogue, Journal of Orthoptera Research 11, 189–197.
Salmon JT (1991) The Stick Insects of New Zealand, Reed, Auckland.
Whiting MF, Bradler S, Maxwell T (2003) Loss and recovery of wings in stick insects,
Nature 421, 264–267.