In Arkansas and Louisiana, subterranean termites cause millions of dollars of damage annually. Damage caused by subterranean termite activity probably exceeds $2.5 billion annually in the United States (Anonymous 2003) and $22 billion globally (Su 2002). Several structurally important species in the genus Reticulitermes are common throughout the southeastern U.S. (Weesner 1965). Messenger et al. (2002) completed a comprehensive survey of termites in Louisiana, providing information for the likely occurrences of Reticulitermes spp. in Arkansas. Previously, Snyder (1954) listed four species in Arkansas: Reticulitermes flavipes (Kollar), R. virginicus Banks, R. hageni Banks, and R. tibialis Banks. All four species have been documented in Louisiana, including R. tibialis, which was recently discovered (Messenger 2003). Information on the distribution of Reticulitermes spp. in Arkansas has become available through a current national termite survey, which confirms that Reticulitermes spp. are common throughout the State (Messenger 2003). Moreover, surveys from the neighboring States of Texas and Oklahoma have included valuable information on Reticulitermes distribution and genetic composition (Austin et al. 2004a, Austin et al. 2004b).

Previous genetic studies have primarily focused on phylogenetic relationships among Reticulitermes species from the eastern United States and Western Europe (Jenkins et al. 1998, Jenkins et al. 2001; Marini & Mantovani 2002; Uva et al. 2003; Ye et al. 2003). Recently, Austin et al. (2004a), Austin et al. (2004b) have conducted the first comprehensive genetic surveys of Reticulitermes sp. for Texas and Oklahoma using DNA sequencing of a portion of the mitochondrial DNA (mtDNA) 16S rRNA gene. We investigated the extent of genetic variation within and among Arkansas and Louisiana Reticulitermes relative to Texas and Oklahoma (Austin et al. 2004a, Austin et al. 2004b), evaluated these genetic markers for identifying species, and updated the geographical distribution of these taxa.

Materials and Methods

Termites were collected in Arkansas and Louisiana and preserved in 100% ethanol (Table 1). We solicited the assistance of Pest Management Professionals (PMPs) throughout both States to determine which species are most frequently recovered from infested structures. We provided collection kits, and PMPs returned samples to our laboratory for analysis. From various geographic zones throughout Arkansas and Louisiana (Table 1), 96 samples were used for molecular analysis.

When alates or soldiers were available, Reticulitermes sp. were morphologically identified to species by applying the keys of Krishna & Weesner (1969); Scheffrahn & Su (1994); Hostettler et al. (1995); and Donovan et al. (2000). In addition, all samples were identified to species with mtDNA 16S sequences (Szalanski et al. 2003). All of the morphological species identifications agreed with the molecular species identifications. Three additional taxa (Table 1) were included as outgroup taxa to corroborate relationships within the genus for phylogenetic analysis. Voucher specimens preserved in 100% ethanol are maintained at the Arthropod Museum, Department of Entomology, University of Arkansas, Fayetteville, AR.

DNA was extracted from alcohol-preserved specimens dried on filter paper according to Liu & Beckenbach (1992) and Jenkins et al. (1999) from individual worker termites with the Puregene DNA isolation kit D-5000A (Gentra, Minneapolis, MN). Extracted DNA was resuspended in 50 μl of Tris:EDTA and stored at -20°C. Polymerase chain reaction (PCR) was conducted with the primers LR-J-13007 (5′-TTACGCTGTTATCCCTAA-3′) (Kambhampati & Smith 1995) and LR-N-13398 (5′-CGCCTGTTTATCAAAAACAT-3′) (Simon et al., 1994). These PCR primers amplify an approximately 428-bp region of the mtDNA 16S rRNA gene. The PCR reactions were conducted with 1 l of the extracted DNA (Szalanski et al. 2000), having a profile consisting of 35 cycles of 94°C for 45 s, 46°C for 45 s and 72°C for 60 s. Amplified DNA from individual termites was purified and concentrated with minicolumns according to the manufacturer′s instructions (Wizard PCRpreps, Promega). Samples were sent to The University of Arkansas Medical School DNA Sequencing Facility (Little Rock, AR) for direct sequencing in both directions. GenBank accession numbers were AY603499 to AY603509 for termite DNA sequence haplotypes new to this study. Consensus sequences for each sample were obtained with Bioedit 5.09 (Hall 1999), and sequences were aligned by CLUSTAL W (Thompson et al. 1994). Mitochondrial DNA haplotypes were aligned by MacClade v4 (Sinauer Associates, Sunderland, MA). Haplotype distribution between populations, number of haplotypes, number of unique haplotypes, haplotype diversity (h), and nucleotide diversity (pi) were calculated with DNAsp v3.51 (Rozas & Rozas 1999).

The distance matrix option of PAUP* 4.0b10 (Swofford 2001) was used to calculate genetic distances according to the Kimura 2-parameter model of sequence evolution (Kimura 1980). Mitochondrial 16S sequences from the desert subterranean termite Heterotermes aureus (Snyder) (GenBank AY380299) and Formosan subterranean termite Coptotermes formosanus Shiraki (GenBank AY558910) were added to the Reticulitermes DNA sequences as outgroup taxa. Maximum parsimony analysis on the alignments were conducted with PAUP* 4.0b10 (Swofford 2001). Gaps were treated as missing data. The reliability of trees was tested with a bootstrap test (Felsenstein 1985). Parsimony bootstrap analysis included 1,000 resamplings and used the Branch and Bound algorithm of PAUP*. Because no previous accounts of the abundance of Reticulitermes in Arkansas have been published, we compiled all available data from existing sources and noted them on our distribution map (Fig. 1).

Results

The DNA sequencing of the 16S rDNA amplicon revealed an average size of 428 bp. The average base frequencies were A = 0.41, C = 0.23, G = 0.13, and T = 0.23. From 35 Arkansas counties 59 R. flavipes, 11 R. hageni, and seven R. virginicus were identified based on species diagnostic nucleotide sites from Szalanski et al. (2003) (Table 1). In Arkansas, 11 haplotypes were observed in R. flavipes, three in R. hageni and three in R. virginicus (Table 1, Fig. 1). Among the 12 Louisiana parishes sampled, 13 R. flavipes, three R. virginicus, and one R. tibalis were identified with six, three, and one haplotypes for each species, respectively (Fig. 2, Table 1).

Nine nucleotide sites were variable among the 11 R. flavipes haplotypes (Table 2), and Tajima-Nei distances (Tajima & Nei 1984) among the R. flavipes haplotypes ranged from 0.2 to 0.9% (Table 3). The most common haplotypes were F and G with 32 and 9 representatives, respectively. Within R. hageni, one nucleotide site was variable between the two observed haplotypes. Haplotype diversity for R. flavipes from Arkansas was 0.759, and 0.782 for Louisiana (Table 4). Both States had high levels of genetic diversity. Tajima’s test resulted in non-significant P values (P < 0.05) in both States leading to the acceptance of the null-hypothesis of neutrality for the mtDNA rRNA 16S gene (Table 4).

Bootstrap analysis of the aligned Reticulitermes species and the outgroup taxa resulted in a consensus tree with several distinct branches (Fig. 3). These distinct clades included R. flavipes, R. tibialis, R. hageni, and R. virginicus. No genetic relationship was observed among R. flavipes haplotypes.

Discussion

This study represents the first attempt to update the current geographic distribution of Reticulitermes spp. and genetically categorize the genus Reticulitermes in Arkansas. At the same time, this is the first molecular description of Reticulitermes spp. from Louisiana, albeit on a limited scale.

Populations of nearly all species, social or otherwise, exhibit at least some degree of genetic differentiation among geographic locales (Ehrlich & Raven 1969). One of the purposes of the research presented herein was to estimate the baseline genetic variation which occurs both within and among Reticulitermes spp. in Arkansas and Louisiana. By combining haplotype observations in the present study with the studies of Austin et al. (2004a) from Texas and Austin et al. (2004b) from Oklahoma, we had hoped to observe some type of spatial continuity which may not have been revealed otherwise. As with animal populations, additional genetic structure normally is to be expected over increasing spatial scales, where populations can show additional differentiation due to spatial habitat structure, isolation by distance, or other factors (Avise 1994). There was no apparent consistency of haplotype occurrence for Reticulitermes spp. in this study based on geography. However, we found genetic divergence values similar to those detected in our previous works (Austin et al. 2004a, Austin et al. 2004b).

Of the four species of Reticulitermes presented in this study, R. flavipes was the most common, followed by R. hageni and R. virginicus. Haplotype F was the most common haplotype of R. flavipes observed in Arkansas, and represented 26 of the 79 (44%) samples from Arkansas. This haplotype is also present in both Texas and Oklahoma but in smaller distributions: 12 and 13% for Oklahoma and Texas, respectively. In Oklahoma and Texas, the most abundant R. flavipes haplotypes are L (23%) and G (28%), respectively (Austin et al. 2004a, Austin et al. 2004b).

A haplotype or allele is defined by one unique form of the gene and differs from any other haplotype by at least one nucleotide. Haplotype diversity or gene diversity quantifies the number of haplotypes in relation to their relative frequency to each other, and is the probability that two sequences randomly selected from a population are different (Nei 1987). Haplotype diversity for R. flavipes from Arkansas and Louisiana was lower than Texas or Oklahoma (Austin et al. 2004a, Austin et al. 2004b).

Tajima’s D (Tajima 1989) is a standard test for the neutrality of a gene region. This is a useful measure because nucleotide diversity (Pi) calculations are dependant upon the infinite alleles model that assumes gene neutrality. Values of D indicate not only if natural selection is influencing gene frequencies but the type of selection pressure in operation. Negative values of D can indicate recent expansions in population size while positive values indicate recent population bottlenecks. Tajima’s D value was negative for both Arkansas and Louisiana, indicating that R. flavipes in these States may be expanding in population size. A similar result was observed from R. flavipes from Oklahoma, while Texas R. flavipes have a positive D value, indicating a recent population bottleneck. The null hypothesis of this test is that the gene region of interest is neutral. If the D value is significant (P < 0.05) then the null hypothesis of neutrality may be rejected. Tajima’s test resulted in non-significant p values (P > 0.05) for Arkansas and Louisiana. This was also observed in Texas and Oklahoma (Austin et al. 2004a, Austin et al. 2004b) leading to the acceptance of the null-hypothesis of neutrality for the mtDNA rRNA 16S gene.

The phylogenetic relationships of Reticulitermes evaluated in this study are consistent with those from neighboring states (Austin et al. 2004a, Austin et al. 2004b) and in other areas (Austin et al. 2002; Jenkins 1998, Jenkins 1999, Jenkins 2001). Extensive collecting in Louisiana has produced only one sample of R. tibialis from Sulphur, Calcasieu Parish, which borders Texas. Although Snyder (1954) lists R. tibialis as occurring in Arkansas, we have not yet recovered this species to date.

We speculate that because R. tibialis was generally not recovered in our collecting efforts, an eastern transition zone (from east to west) for the distribution of this species may exist in the proximity of Calcasieu Parish, Louisiana. This species is known to prefer more arid climates which can be more readily found in Texas and Oklahoma where R. tibialis has been more frequently recovered. More intensive collecting efforts should be performed to validate this hypothesis.

The lack of a geographical haplotype continuity of Reticulitermes suggests that (1) we require samples from larger geographic zones, (2) we need to increase the number of samples sequenced, or (3) the observed lack of spatial continuity from Reticulitermes may be attributed to anthropogenic origins. To evaluate the latter element, evaluation of haplotype frequency from undisturbed habitats (e.g., protected forests) should be compared with our current data, which largely reflects samples obtained from urban landscapes. Selected sites in undisturbed locations are being evaluated for intensive collecting efforts in future studies where more comprehensive statistical measures can be applied, and a better overall understanding of population dynamics may be addressed than in the current study.