A1 Bertrand

EXPLORING SHALLOW SPLITS IN COSTA RICAN BARCODED LEPIDOPTERA
Bertrand, C. (1), Taidi, S.(1), Janzen, D.H. (2), Hallwachs, W. (2), Hajibabaei, M. (1)

(1) Biodiversity Institute of Ontario, Guelph, Ontario, Canada
(2) University of Pennsylvania, Philadelphia, Pennsylvania, United States

Workflow A
MHARB180-05|CbDHJ02|COI-5P

A total on-going inventory of 10,000 species of Lepidoptera of the Area de Conservación Guanacaste
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(ACG) in northwestern Costa Rica has integrated DNA barcoding to assist in identification and in discovery • Table 1. summarizes the results of the cloning experiments for
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of new and cryptic species. Over 100,000 individuals from this inventory have been barcoded in this
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each species. 23
MHMX
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Q169-08|CbDHJ02|COI-5P

densely sampled DNA regional barcode campaign (Janzen et al. 2005). Barcoding has revealed numerous • All sequences with insertions, deletions or stop codons were
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cases where a morphologically-defined species contains two or more sympatric barcode clusters in a
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XAA057-04|CbDHJ02|COI-5P

removed as well as any sequences with more than 6 SNPs following 85

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neighbour-joining (NJ) tree. At least half of these (often shallow but consistent) cases are found to be traditional DNA barcoding guidelines.
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supported by morphological and/or ecological differences, reinforcing their status as different (usually
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• In both Target1 and Target2 species, each group of cloned 37

MHMX
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T648-08|CbDHJ02|COI-5P

undescribed) species (Burns et al. 2008). However, a significant number of these cases lack these sequences when aligned and compared with the original data from
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correlates and therefore could be cases where nuclear pseudogene copies of mitochondrial genes
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MHMXO045-08|CbDHJ02|COI-5P

BOLD (Barcode of Life Datasystems) clustered with the parent 63

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(NUMTs) have been amplified from some conspecific individuals, while the true barcode has been sequences and separate from their sister lineages, illustrated in Fig 3.
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amplified from other conspecifics, giving the result of two adjacent clusters in an NJ tree (Song et al.
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Workflow B
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2008). Most NUMTs show obvious signs of pseudogenes, such as stop codons and frame shift mutations, 66
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thereby allowing their rejection as barcodes. Deeper genetic exploration can bring clarity to where this
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•Massive 454 sequences of Target 2, Carathis byblis, were aligned
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rejection is not possible. We are currently cloning and Sanger sequencing the 658pb standard barcoding and compared with the original data from BOLD and the results
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MHMX
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O047-08|CbDHJ02|COI-5P

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region of cytochrome c oxidase (COI) and comparing the results with deep amplicon sequencing utilizing
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showed clustering with the parent sequences AND the secondary 69
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next generation 454 Pyrosequencing. In a later phase of this project we will use nuclear markers to sister lineage, corresponding to the original dichotomy seen.
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augment our current results.
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MHMXQ168-08|CbDHJ02|COI-5P
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TARGET TAXA
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Fig 3. NJ-tree of sample MHMXQ166-08 clustering with 27

•All samples analyzed were collected from the Area de Conservación Guanacaste in northwestern Costa
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92 successful clones and representative sequences in
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Rica.
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BOLD (outlined in red) and separately from the atypical 15
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•NJ trees were constructed of CO1 barcodes for the families Sphingidae and Arctiidae. branch.
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MHMXT649-08|CbDHJ01|COI-5P
BLPCG696-08|CbDHJ01|COI-5P

Target 1: Aellopos ceculus and Pachylia ficus were chosen from the Sphingidae family. Two specimens
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of each species from the atypical branch were analyzed. 0.001

NUMT? Target Species Number of Number of Screened clones Number of Successful
NUMT? Specimens/Specie Sequences/Specimen
s
Pachylia ficus
Aellopos ceculus 2 24 / specimen 16
Aellopos ceculus 10
Pychalia ficus 2 24 / specimen 17
10
Carathis byblis 8 100 / specimen 93
Target 2: Carathis byblis was chosen from the Arctiidae family. Eight specimens were chosen, five from
the representative and three from the atypical branch. One individual was used for preliminary 454 94
analysis. 95
NUMT? 92

Carathis byblis 87
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Table 1: Summaries the depth of screening and cloning success rate of target taxa sequences.

Figure 1: Neighbor-joining trees of the selected species showing shallow splits in their DNA barcode sequences. We examined
two individuals from atypical groups in Pachylia ficus and Aellopos ceculus. Carathis byblis underwent deeper analysis with five
individuals from the representative and three individuals from the atypical branch (shown with dashed rectangles).

MOLECULAR ANALYSIS
Workflow A: DNA was extracted using a standard silica-based approach. PCR amplification of COI Our initial analysis suggests, the shallow splits within these three species are not a result of
(658bp) using standard LepF/R primers (Hebert et al. 2003) was performed followed by gel purification. NUMTs and are true mitochondrial CO1 sequences that may represent cryptic species. However the 454
The purified products were cloned into TOPO vectors and transformed to competent E.coli cells. analysis detected two sequence types and we propose that deeper screening is required to reveal the
Colonies were directly amplified and sequenced through Sanger sequencing. This was performed for dichotomous branching patterns. Massive sequencing using 454 Pyrosequencing is the most effective
both Target 1 and Target 2 species. technology available.
Workflow B: Massive sequencing of one individual of Target 2 (Carathis byblis) was performed by high
throughput sequencing by synthesis (Pyrosequencing) utilizing an in house 454 FLX genome sequencer.
The 454 sequencing results were subjected to all quality filters. Sequences were compared with
Workflow A results to detect any variation in the barcoding region.
•We will extend this analysis to shallow splits in species of numerous families of Lepidoptera.
• We will continue our analysis of deep sequencing approach using 454 pyrosequencing to examine a
much higher number of sequences from each specimen in cloning.
•Our next workflow will compare nuclear markers with COI to explore any changes in clustering
patterns.
•RNA extraction from fresh samples followed by RT-PCR is a prospective analysis to solely reveal the
sequence of expressed copy COI.

Burns, J. M., D. H. Janzen, M. Hajibabaei, W. Hallwachs & P. D. N. Hebert (2008) DNA barcodes and cryptic species of skipper butterflies in the genus Perichares in Area de Conservacion Guanacaste, Costa Rica. Proceedings of the National
Academy of Sciences of the United States of America, 105, 6350-6355.

Hebert, P. D. N., S. Ratnasingham & J. R. deWaard (2003) Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. Proceedings of the Royal Society of London Series B-Biological Sciences, 270, S96-S99.

Cloning and Sanger sequencing Janzen, D. H., M. Hajibabaei, J. M. Burns, W. Hallwachs, E. Remigio & P. D. N. Hebert (2005) Wedding biodiversity inventory of a large and complex Lepidoptera fauna with DNA barcoding. Philosophical Transactions of the Royal Society B-
Biological Sciences, 360, 1835-1845.

Song, H., J. E. Buhay, M. F. Whiting & K. A. Crandall (2008) Many species in one: DNA barcoding overestimates the number of species when nuclear mitochondrial pseudogenes are coamplified. Proceedings of the National Academy of Sciences
of the United States of America, 105, 13486-13491.

We thank ACG parataxonomists for collecting Lepidoptera specimens and Tanya Dapkey for sorting, sub-sampling and shipping specimens for analysis.
We thank CCDB staff for barcode analysis and Shadi Shokralla for 454 pyrosequencing.
Fig 2. Experimental design showing samples going into the cloning and 454 Pyrosequencing workflows
Funding Provided By:

A1 Bertrand

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