Timothy D. O’Connor and Dr. Keith A. Crandall Department of Integrated Biology

There are an estimated 338 taxa of freshwater crayfish in the United States, with approximately 48% considered to be endangered, threatened, or of special concern (Taylor et al. 1996). Life history information for the majority of the primary burrowing species is lacking because they dig deep burrows and venture out only during breeding periods. Burrowing members of the genera Procambarus and Fallicambarus are found in seasonally moist habitats across Arkansas, such as roadside ditches and open pastures. To date, no phylogenetic study has been conducted that establishes a phylogenetic framework for the classification of these elusive species. Furthermore, estimates of genetic diversity and migration are needed to assess the conservation needs of these imperiled species. The objective of this study is to determine the phylogenetic relationships among various burrowing species of Arkansas crayfish using 16s sequence data. Preliminary results showed that the groups defined as Procamabarus and Fallicambarus do not separate into monophyletic clades while some previously defined Procambarus species are actually multiple species. This creates a concern for the conservation status of these species. Many are rare and are in need of specific, appropriate management measures.

Approximately 70 samples of Fallicambarus genera in Louisiana and Arkansas were collected in June of 2004. This was done by digging up various chimney burrows created by the crayfish and hunting for them at night in wet fields. A Qiagen DNeasy Extraction kit was used to extract and purify the DNA from the collected samples. Using this DNA, we amplified the 16s region of the crayfish genome using a Polymerase Chain Reaction. This product was then sequenced using ABI Big-Dye Ready-Reaction kit and submitted to the BYU Sequencing Center to establish the sequence of nucleotides using a 3730 XL automated sequencer.

A Baysian analysis using the program Mr. Bayes (Huelsenbeck and Ronquist 2001) was done on the sequence data and formulated into a tree. This was done on the cluster computer at Brigham Young University. The Bayesian analysis was run with four chains for five million generations with a 6 parameter model and gamma rate heterogeneity. The runs were sampled every 1000th generation. Using the program Tracer (Rambaut, A., and A. J. Drummond. 2003) I found the burn to be 500 trees. The Bayesian analysis shows that the species from the Procambarus genus do not form a monophyletic group but actually form two separate groups. One monophyletic group composed of Procambarus liberorum, Procambarus reameri, Procambarus simulans and a new species of Procambarus. Procambarus liberorum clusters into a monophyletic group and appears to be a sister species of a new species, Procambarus sp. nov., collected in Grandview Prairie, Hempstead County, Arkansas. More work is needed to determine range for this new species as well as a formal species description based on the breeding males. In addition to this, Procambarus reimeri from Arkansas and Procambarus simulans collected by the Crandall Lab in Texas formed sister clusters as expected by morphology.

The second cluster is composed of Procambarus acutus, Procambarus ouachitae and various other species of Procambarus previously collected by the Crandall Lab. The Procambarus acutus collected in Arkansas may be a new species because it forms a distinct sister clade to Procambarus ouachitae and the other Procambarus acutus previously collected by the Crandall lab in Texas and Louisianna (Fig 1). One incongruity is that of the Procambarus tenuis appearing in the middle of the Fallicambarus monophyletic grouping. This could be due to a mistake in the identification but will require further inquiry of other populations in that area to verify this conclusion. All other samples seem to cluster correctly by species although the distinction between the two subspecies of Orconectes palmeri does not seem to be genetically distinct; they seem to have ample gene flow (Fig. 2).

Using the 16s data we see that there is a possibility of two new species of crayfish, Procambarus sp. nov., a sister species to Procambarus liberorum and a split in the species classification of Procambarus acutus. Using this information, these species can now be assessed for conservation status and further studies need to be done to discover their relation to other burrowing crayfish. Conservation efforts can then be specifically tailored to these new species of crayfish.