Introduction

The ant genus Pheidole is found all around the world, and is estimated to contain over 1000 species. This genus has presented challenges for taxonomists, not only because it is species-rich, but because the workers in this genus occur in two (or rarely three) forms. Major and minor workers in a single colony look vastly different even though they are sisters. Species identifications are usually based on the morphology of major workers, so when minor workers are collected unassociated with majors (e.g., in leaf-litter samples), it is not always possible to identify them to species. It has been proposed that DNA barcoding could be a very useful tool not only for identifying cryptic species within the genus Pheidole, but also for associating major and minor workers of the same species.

Biodiversity studies are an integral part of ecology and conservation, but one of the greatest challenges in this field is estimating species diversity efficiently and accurately. Leaf litter-dwelling ants, including Pheidole species, have been used in many studies across the world as a metric for biodiversity.

DNA barcoding is a method used for identifying species based on variation of mitochondrial (mt) COI DNA sequence. In general, the more similar the mtCOI sequences of two individuals, the more likely they belong to the same species. Because mitochondrial DNA has a relatively fast mutation rate, we can expect even recently diverged lineages to have genetic variation.

Questions

• Can DNA barcoding help to identify cryptic species within the ant genus Pheidole?
  
  
• Will DNA barcoding allow taxonomists to associate the major and minor worker specimens of the same species?

Methods

Specimens: Using a quantitative leaf-litter collecting protocol, approx 6500 Pheidole ants were collected from eight sites in Guyana and identified to species based on morphological characteristics.

Molecular Methods: The Pheidole specimens from the leaf-litter samples were subsampled in an unbiased manner. Three legs were removed from each voucher specimen. The genetic material was then extracted from the legs, amplified via PCR, and sequenced for the mtCOI barcode region. The sequence data were then analyzed using both parsimony and neighbor-joining methods. Putative Pheidole species identified by these methods were compared with those previously identified based on morphology; associations of major and minor workers were also noted.

Morphological Review: Voucher specimens of putative DNA barcode species were photographed using an extended focus Auto-Montage microphotography system that produces highly resolved images. These images serve to document both the morphology of cryptic species and the major-minor worker associations revealed by DNA barcoding.

Results and Conslusions

Can DNA barcoding help to identify cryptic species within the ant genus Pheidole?

Preliminary data suggest that there may be several cryptic species and synonymous morphospecies:

• 21 species were identified through morphology.
• 26 putative species were produced by the sequence data.

These results are preliminary and need to be corroborated with more extensive identifications, morphological study, and additional data from one or more nuclear genes.

Will DNA barcoding allow taxonomists to associate the major and minor worker specimens of the same species?

A major and minor worker from the species allarmata were successfully sequenced and were shown to be related not only from initial morphological identification, but from the genetic analysis as well, illustrating the value of DNA barcoding in associating worker castes.

Acknowledgments

I would like to thank Dr. Natasha Mehdiabadi and Dr. Ted Schultz for their guidance and support. Also Eugenia Okonski, Jeffery Sosa-Calvo, Molly Rightmyer and my fellow interns Tam Dang and Jessica Louton. Special thanks to Laura Florez whose work from last summer provided invaluable information for this research. This project was funded by Smithsonian Latino Initiatives.