1. Email: b.garner@nhm.ac.uk
Testing the utility of museum specimens for DNA barcoding and phylogenetics of the Colorado potato beetle genus, Leptinotarsa (Coleoptera)
Beulah Garner1, Mindy Syfert2, Stephen Russell2, Sandra Knapp3, Diana Percy4
Department of Life Sciences, Natural History Museum, London, SW7 5BD, UK
Introduction
The beetle genus Leptinotarsa Stål, 1858, with 41 described species, has its centre of diversity in Mexico but has a geographic distribution in North, Central and South America (Chittenden, 1907). The Colorado potato beetle (CPB), L. decemlineata, is a serious pest of potato Solanum tuberosum L. and has spread to parts of Asia and Europe in association with this crop. Despite extensive literature available on pest control of CPB, (Maharijaya & Vosman, 2015; Huseth et al., 2015), little is known about Leptinotarsa and the evolutionary relationships between species or their natural history. We are currently testing the utility of using specimens in the collections of the Natural History Museum, London (NHM), for molecular analysis of Leptinotarsa, in the light of progress in successful DNA extraction of old museum specimens (e.g. Price et al., 2015). We use a ‘non-destructive’ protocol for DNA extraction (but see cautionary note below). Subsequent sequencing success will likely be dependent on factors such as specimen age, past environmental conditions and collection methods. We intend to synergise the genus phylogeny with plant host and geo-referenced data collated from NHM museum specimens.
Molecular protocol
The cytochrome B (cytB) region was targeted because it is commonly used for systematic studies, and there are standard primers targeting a short region (385 bp) making it suitable for potentially degraded DNA.
Using the QIAGEN Blood and Tissue kit, a non-destructive digestion and lysis was performed (lasting approximately 15hrs). After lysis the beetles were removed, washed in distilled water, dried, re-pinned or legs repatriated, and labelled to indicate DNA voucher. A 1ul aliquot of DNA was measured using a Nanodrop 8000 spectrophotometer. PCR cycling was adjusted to amplify low concentrations of DNA in some samples.
Results
It was expected that DNA yield may be inconsistent or poor due to factors including size of specimen, age, preservation, exposure to external environmental factors, physical damage, and how the specimen was originally captured and prepared for storage. We extracted measurable DNA from 57 specimens (either a whole specimen or a single leg). All specimens produced amplification, however sequence length and quality decreased rapidly in older specimens. Whole specimens produced greater quantities of DNA compared with single legs, which was expected (Watts et.al. 2007). Preliminary results suggest that the cytB region will be relatively straightforward to sequence and will provide sufficient variation to investigate both intra- and inter-specific variation in Leptinotarsa.
 As a cautionary note, some specimens amplified for human DNA, potentially due to the frequent handling of specimens during manipulation and examination. To reduce this risk in future, we propose to experiment with pre-digestion specimen washes, grinding tissue parts (e.g. legs), more specific primers, and fewer numbers of PCR cycles.
L. decemlineata . Germany. leg. Krell, BMNH(E)
Materials and Methods
Specimen selection
Leptinotarsa was recurated as part of the NHM Crops and Wild Relatives Initiative (CWRI). The NHM’s 880 specimens range from 184 to 5 years old, with the majority of specimens collected during the 1930s-1950s. Where possible, the most recently collected material was used for DNA extractions.
Selection criteria: age, geographic location (within species), robustness of specimen (whole specimen or leg), number of specimens (type material was excluded).
Digitisation, transcription and geo-referencing
To mobilise the data from specimen labels (notably host plant and geographic location) the Leptinotarsa collections were incorporated into the workflow of the Digital Collections Programme (DCP). Specimens were barcoded and allocated a unique specimen number (imbedded within the barcode). A Canon EOS 650 DSLR camera equipped with a 90mm macro lens was used for imaging (fig. 1). Images were loaded into a transcription interface written in SQL with a Microsoft Access back end and label information manually entered via a front end interface. Localities were interpreted from labels and translated into lat/long coordinates, providing a map of the geographic distribution of NHM specimens (fig. 2).
Fig. 2 Geo- referenced label data illustrating distribution of Leptinotarsa
 Discussion
Understanding the spatial and temporal spread of pest species and their non-pest relatives will help to inform others working on more applied pest control methods, including bio-control. Clarification of the taxonomy of Leptinotarsa will help in delimiting the species taxonomic and geographic boundaries, and improve our understanding of speciation processes and host-plant associations.
Future directions
1. Sequencing a second barcoding region to confirm sequence identification.
2. Combine cytB and CO1 for Leptinotarsa s.str. Focus on decemlineata and undecemlineata including synonymies to clarify species complexes relative to geographic distribution.
3. Use the specimen label transcription data to answer questions about geographic spread and speciation within and between continents.
4. Collaborate with other researchers on a more comprehensive phylogeny using multiple gene regions.
5. Inform further selection of specimens for molecular analysis by
investigating variation in elytral stripe formation and metallic colouration between species using confocal microscopy and SEM (fig. 3).
Fig. 1 Example of specimen digitisation.
L. decemlineata specimen used in sequencing. DNA extraction from middle left leg. A B
Fig. 3 Confocal microscopy images.
A, elytra of L. decemlineata
B, head of L. cacica
References: Chittenden (1907) U.S. Dept. of Agriculture; Huseth et al. (2015) PLOS ONE, 10(6); Maharijaya & Vosman (2015) EUPHYTICA, 204(3); Price et al. (2015) PLOS ONE, 10(4); Watts et.al. (2007) Journal of Insect Conservation, 11(2).
Funding for the molecular analyses was provided through the NHM Life Sciences Departmental Investment Fund for Systematics of the Colorado Potato Beetle Genus, Leptinotarsa (Chrysomelidae) to DMP.
We thank Roy Canty for confocal imaging and Peter Wing for notes on transcription.