Abstract

Using specimens from an ant leaf-litter biodiversity survey from Guyana, the usefulness of DNA barcoding was tested for identifying Pheidole nominal species and morphospecies, and for suggesting possible cryptic species.

The results revealed correspondence between morphologically identified ant species and mitochondrial lineages identified by DNA barcoding, and also revealed the existence of several distinct mitochondrial sublineages (i.e., possible cryptic species) within the Pheidole ruida specimens in our data set. In addition, DNA barcoding proved to be an efficient tool for associating major (soldiers) and minor (workers) castes within the same species, an important advance because the taxonomy of this genus is based largely on the major caste.

Applied to the genus Pheidole, DNA barcoding has shown consistency as a species identification method. However, it is important to keep in mind its purpose as a tool for species identification rather than description, and the need to integrate it with ecological, morphological and behavioral data in order to draw valid conclusions about taxonomy or evolution.

Introduction

Ants have major ecological importance. They dominate terrestrial ecosystems, play significant roles in seed dispersal and nutrient cycling, and constitute a good indicator for conservation issues. However, there are many aspects related to diversity and community structure of ants that are still not well understood.

DNA barcoding is a technique that uses a short mitochondrial DNA sequence (cytochrome c oxidase I, or COI) for species-level identification. It is intended to be a standardized, rapid and effective method. This new technique has been proposed as a useful tool for overcoming the difficulties of species-level identification in ants, and could also facilitate the association among castes and reveal potential cryptic species.

Ants of the genera Pheidole (primarily) and Crematogaster were subsampled from a leaf-litter biodiversity survey carried out in Guyana during 2002. Following collection, the species were identified using morphology (LaPolla et al. 2007).

Materials and Methods

Subsampling: 883 Pheidole and 182 Crematogaster specimens preserved in 95% ethanol were subsampled.

DNA extraction: The leg samples were transferred to 96-well extraction plates and submitted to an automated DNA phenol-chloroform extraction machine.

Amplification and Sequencing of COI: Amplification of a 658bp fragment of the mitochondrial cytochrome oxidase I (COI) gene was done using LCO and HCO standard primers. PCR products were sequenced in both directions. Contigs were assembled and edited, and alignment was done by eye based on conserved amino acid residues.

Data analyses: Results were analyzed with a neighbor-joining algorithm. Parsimony and maximum likelihood methods were also applied and bootstrap values calculated for the three methods.

Results and Discussion

Pheidole and Crematogaster Neighbor-joining Tree

Pheidole

MORPHOLOGICAL AND MOLECULAR CORRESPONDENCE
Clustering of DNA barcoding mitochondrial lineages consistently corresponds to the Pheidole species determined previously with morphological methods. Relationships among the Pheidole species were not resolved robustly under any method, highlighting the usefulness of DNA barcoding for species identification, rather than for establishing phylogenetic relationships.

CRYPTIC SPECIES
Within P. ruida, there are five distinct mitochondrial lineages.These five lineages are separated by sequence divergences (between 5.992 and 14.590%) much greater than thresholds applied in previous studies on ants (2 and 3%) (Smith et al. 2005). However, whether these represent true species or mitochondrial polymorphisms within a single species requires further study (e.g., including nuclear genes) and increased sampling.

MAJOR-MINOR ASSOCIATION
The morphological grouping of majors and minors in P. ruida and P. allarmata is congruent with the results obtained using DNA barcoding. Thus, the effectiveness of this method as a tool for caste association is confirmed.

Crematogaster

The COI data clearly distinguish Pheidole and Crematogaster as separate lineages. However, within Crematogaster there is poor correspondence between the previously identified morphospecies and the taxonomic units suggested by DNA barcoding. Although there is only weak support for the first of these lineages, these results suggest that the morphospecies identifications are in error.

Conclusions

DNA barcoding is a useful method for identifying nominal species and morphospecies of the genus Pheidole. It is also helpful for recognizing possible cryptic species. It did not prove useful for reconstructing phylogenetic relationships among species.

Association of minor and major castes is a valuable use of DNA barcoding in ants, providing a solution to the inability to identify many Pheidole species using the minor caste and to the resulting miscounting of species present in biodiversity samples.

It is important to integrate DNA barcoding with other molecular approaches, as well as with ecological, behavioral and morphological data, in order to make valid inferences and to draw valid conclusions about species boundaries and phylogenetic relationships.

Acknowledgements

I thank Dr. Seàn Brady and Dr. Ted Schultz for their invaluable guidance and interest. To Faridah Dahlan, Katrina Pagenkopp, Eugenia Okonski, Jeffrey Sosa-Calvo, John Lapolla, Andrea Ormos, Kathleen Ketchum, the Ant Lab in NMNH and L.A.B. staff at the MSC, my most sincere gratitute for their help and unparalleled support. I am grateful to Santiago Herrera, Laura Lagomarsino and the 2007 RTP class for their exceptional advice and support. This project was funded by the Bill and Jean Lane Internship Endowment.