MOLECULAR ECOLOGY AND EVOLUTION Comparative Study of Breeding Systems of Sympatric Subterranean Termites (Reticulitermes flavipes and R. hageni) in Central North Carolina Using Two Classes of Molecular Genetic Markers

1 EDWARD L. VARGO AND JOANNA R. CARLSON

Department of Entomology, Box 7613, North Carolina State University, Raleigh, NC 27695Ð7613

Environ. Entomol. 35(1): 173Ð187 (2006) ABSTRACT We used microsatellite and mtDNA markers to compare colony and population genetics of two sympatric species of subterranean termites, Reticulitermes flavipes (Kollar) (n ϭ 25 colonies) and R. hageni Banks (n ϭ 16 colonies), in two sites located 27 km apart in the Piedmont of central North Carolina. Colony breeding structure was inferred by examination of microsatellite genotypes of workers within colonies and by estimates of nestmate relatedness and hierarchical F-statistics. Similar to previous results on this species, nearly one-half of the R. flavipes colonies were simple families mainly headed by outbred primary (alate-derived) reproductives, about one-half were comprised of extended (inbred) families inferred to be headed by low numbers of neotenics (non alate-derived secondary reproductives) descended from the original primary pair, and two colonies contained the offspring of multiple reproductives. About two-thirds of the R. hageni colonies were comprised of simple families largely headed by related reproductives, and about one-third consisted of extended families headed by low numbers of neotenics. R. hageni differed from R. flavipes in having signiÞcant isolation by distance at one site as well as signiÞcant differentiation between sites at both the microsatellite and mtDNA markers. We conclude that dispersal in R. hageni is more limited resulting in higher levels of inbreeding within colonies and greater degrees of population genetic structure at small and large spatial scales than in sympatric populations of R. flavipes. These results indicate that closely related species of subterranean termites occurring in the same habitat can differ in their breeding systems with important consequences for higher level genetic structure.

KEY WORDS Reticulitermes flavipes, Reticulitermes hageni, Reticulitermes virginicus, microsatellites, mitochondrial DNA

TERMITES (ISOPTERA) ARE A LARGE and ecologically im- each other. Thus, the colony breeding system and portant group of social insects. Following the basic associated dispersal behaviors can strongly affect col- ground plan of highly cooperative insect societies, ony and population genetic structure: simple family termite colonies consist primarily of nonreproductive colonies headed by dispersive primary reproductives workers who labor in support of relatively few repro- promote gene ßow and outbreeding, whereas ex- ductively specialized individuals. Within this general tended family colonies headed by nondispersive neo- reproductive theme, however, termites exhibit con- tenics lead to inbreeding and increased genetic con- siderable variation in colony breeding structure, pri- trasts among colonies. There is considerable variation marily concerning the numbers of reproductives among termite taxa in their tendency to form neoten- within colonies and the degree of relatedness among ic-headed colonies, and this variation is related to them. Both of these variables largely depend on ecological conditions and behavioral traits. SpeciÞ- whether colonies are simple families headed by mo- cally, frequent production of neotenics seems to be an nogamous pairs of adult-form (primary) reproduc- ancestral trait associated with the formation of tem- tives or whether colonies are extended families con- porary nest sites located in or close to relatively un- taining numerous neotenic reproductives, nonwinged stable food sources (Shellman-Reeve 1997, Myles reproductive forms who develop within established 1999). colonies as precocial breeders from either nymphs or Subterranean termites (Rhinotermitidae), in par- workers (Shellman-Reeve 1997, Myles 1999). In gen- ticular, have exceptionally plastic breeding systems eral, primary reproductives disperse through mating and dispersal behavior, with a strong tendency to ßights to found new colonies, whereas neotenics re- produce neotenics and to form extended family col- main in their natal colony where they inbreed with onies. The Holarctic genus Reticulitermes is the best studied group of subterranean termites with respect to 1 Corresponding author, e-mail: [email protected]. breeding structure, and work on this genus has been

0046-225X/06/0173Ð0187$04.00/0 ᭧ 2006 Entomological Society of America 174 ENVIRONMENTAL ENTOMOLOGY Vol. 35, no. 1 accelerating, largely because of the availability of sen- sitive molecular markers (Cle´ment 1981, 1984, Reilly 1987, Jenkins et al. 1999, Bulmer et al. 2001, Cle´ment et al. 2001, Bulmer and Traniello 2002a, b, Vargo 2003a, b, DeHeer and Vargo 2004, Dronnet et al. 2004, 2005, DeHeer et al. 2005). These studies show variable breeding structures and suggest that local ecological conditions are important in shaping colony reproduc- tive structure. In the North American species, R. flavipes (Kollar), Bulmer et al. (2001) reported dif- ferences in breeding structure between two Massa- chusetts sites located 0.5 km apart, based on allozyme and mitochondrial DNA (mtDNA) markers. One site with rocky and poorly drained soil consisted of a mixture of extended families (60%) and simple fami- lies (40%), all with limited foraging ranges, whereas the other site containing more porous soil had equal numbers of spatially expansive extended families and mixed family colonies. Three recent studies of R. fla- vipes from central North Carolina using microsatellite markers give consistent results concerning colony breeding structure in this geographic region (Vargo 2003a, b, DeHeer and Vargo 2004): some 75% of the 126 colonies studied were simple families, 24% were neotenic-headed extended families descended from simple families, and a single colony contained genet- ically diverse individuals originating from the fusion of two distinct colonies. Thus, R. flavipes shows variation in breeding structure at both small and large spatial scales, presumably in response to ecological condi- tions. Although R. flavipes is only one of six recognized species of Reticulitermes in the United States, and there are probably several more unrecognized species (Austin et al. 2002, Page et al. 2002, Copren et al. 2005), almost nothing is known concerning the breeding sys- tem of the other species. Over most of its range in the eastern United States, R. flavipes is sympatric with two congeners, R. hageni Banks and R. virginicus (Banks) (Snyder 1954, Austin et al. 2002). Five colonies of the Fig. 1. Relative locations of R. flavipes, R. hageni, and latter species in North Carolina have been analyzed R. virginicus colonies sampled in the two transects. using microsatellite markers (Vargo 2003b, DeHeer and Vargo 2004); four of these were simple families Materials and Methods and one was an extended family. Only a single R. hageni colony, also from North Carolina, has been Sample Collection genetically characterized, and it was a simple family In May and June 2000, Reticulitermes spp. termites (DeHeer and Vargo 2004). were sampled from two undisturbed forest habitats: To better understand the causes and consequences Schenck Forest, a facility owned and managed by of variation in breeding system within and among North Carolina State University located in western Reticulitermes spp., we undertook a comparative study Wake County, NC, and Duke Forest, belonging to of colony and population genetic structure of R. fla- Duke University and located in central Durham vipes and R. hageni in two forests in central North County, NC. These sites, which consisted of mixed Carolina. Although R. flavipes is reasonably well stud- hardwoods and loblolly pine (Pinus taeda L.) Ͼ50 yr ied in this area (Vargo 2003a, b, DeHeer and Vargo old, are located Ϸ27 km apart. Samples of at least 50 2004), we included it in this study to control for vari- workers, some soldiers, and occasionally alates were ation in breeding structure caused by local ecological collected from natural wood debris along approxi- conditions or temporal differences. We used both mi- mately linear transects in each forest with a minimum crosatellite markers (Vargo 2000) and mtDNA mark- of 15 m between collection points (Fig. 1). The dis- ers to infer the breeding structure of colonies and to tance between collection points was measured and assess relative gene ßow within and among popula- recorded. Samples were taken from 20 collection tions. points in Schenck Forest and from 29 collection points February 2006 VARGO AND CARLSON:BREEDING SYSTEMS OF SUBTERRANEAN TERMITES 175 in Duke Forest. Originally, this project was meant to Fairfax, VA) to obtain individual genotypes. Mende- be a study of R. virginicus, a species whose worker and lian inheritance of all loci was shown previously for R. soldier body sizes are intermediate between the other flavipes by Vargo (2003a). two Reticulitermes spp. occurring locally, the larger The COII gene of mtDNA was sequenced using the and more common R. flavipes and the smaller R. hag- primers A-t Leu and B-t Lys (Liu and Beckenbach eni. When samples were encountered that appeared to 1992). In most cases, two individuals per collection be too large for R. virginicus, they were not collected. point were sequenced, but more individuals were se- Any samples that appeared too small to be R. flavipes quenced in the two collection points containing mixed were collected. However, on closer inspection, and family groups (see Results). Reaction volumes were especially after use of molecular markers, we deter- 50.0 ␮l, containing Ϸ20 ng (4 ␮l) of genomic DNA, 1ϫ mined that the samples consisted of a mixture of all PCR reaction buffer, 2 mM MgCl2, 0.25 mM dNTPs, three Reticulitermes spp., primarily R. flavipes and R. 0.06 U Biolase Taq polymerase (Bioline, Canton, MA), hageni. Thus, samples of the larger R. flavipes were and 1 pmol of each primer. PCR was performed on a biased toward colonies with small workers, whereas PTC-100 thermal cycler (MJ Research, Littletown, there was no obvious bias in the R. hageni samples. MA) using the following program: initial denaturation Immediately on collection, samples were placed at 94ЊC (2 min) followed by 35 cycles of 94 (1 min), into vials containing 95% ethanol. The day after col- 50 (1 min) and 70 (2 min), with a Þnal extension step lection, the ethanol was replaced, and any dirt or at 70ЊC (5 min). debris was removed from the vial. The vials were PCR products were either gel puriÞed or cleaned up stored at Ϫ20ЊC until DNA extraction. using the QIAquick PCR PuriÞcation kit (Qiagen) before ampliÞcation using the ABI Prism dRhodamine Terminator Cycle Sequencing Ready Reaction Kit Species Identification (Applied Biosystems, Foster City, CA). Both forward Soldiers in each sample were examined morpholog- and reverse reactions were performed using 1ϫ ABI ically for species identiÞcation using the keys of Schef- reaction mixture (ABI Prism DRhodamine Termina- frahn and Su (1994) and Hostettler et al. (1995). Ac- tor Cycle Sequencing Ready Reaction Kit; Applied cording to these keys, R. flavipes generally has a soldier Biosystems) and 0.3 pmol of primer A-t Leu or B-t Lys. pronotal width Ն0.85 mm and the point of the left The dye terminators were removed from the sequenc- mandible curves inward at 70Ð90Њ, that of R. virginicus ing reaction mixture by passing the solution through usually measures 0.71Ð0.80 mm with the point of the CentriSep columns (Princeton Separations, Adelphia, left mandible curves inward at 70Ð90Њ, whereas that of NJ) according to the manufacturerÕs instructions. R. hageni is generally Ͻ0.70 mm and the point of the Products were dried on a speed-vac and submitted to left mandible curves inward at 45Њ. From one to three the North Carolina State University Sequencing Fa- soldiers per sample were examined for both pronotal cility, where the samples were run on an ABI 377 width and mandible curvature. We determined that automated sequencer. Each template was sequenced identiÞcation based on morphological measurements in both directions. Sequence editing and alignment proved unreliable; many specimens that keyed to R. were performed using the software program Vector virginicus by these criteria were later reclassiÞed NTI Suite (Informax, Frederick, MD). based on sequence data from the cytochrome oxidase II (COII) gene as either R. flavipes or R. hageni (Sza- Genetic Data Analysis lanski et al. 2003). Voucher specimens have been de- posited in the North Carolina State University Insect Tests for Hardy-Weinberg Equilibrium and Link- Collection. age Disequilibrium. Deviations from Hardy-Wein- berg equilibrium (HWE) and linkage disequilibrium (LD) were analyzed by means of exact tests using the Genotyping program Genetic Data Analysis v.1.1 (Lewis and Individual whole bodies of termite workers were Zaykin 2000) with 3,200 iterations. Because genotypes pulverized in liquid nitrogen, and genomic DNA was within colonies were not independent due to the close extracted using the DNeasy Tissue Kit (Qiagen, Va- family relationships among colony mates (see Re- lencia, CA). At least 20 workers from each collection sults), only a single individual per colony was used for point were genotyped at eight trinucloetide microsat- these tests. We used a resampling procedure in which ellite loci following the methods of Vargo (2000), in one individual from each colony was selected at ran- which the polymerase chain reaction (PCR) products dom for a total of 20 replications. are labeled by the addition of a ßuorescent labeled Colony Affiliations. Colony associations for the ter- primer to the reaction. The PCR products were sep- mites from each collection site were made based on arated in 6.5% polyacrylamide sequencing gels run on microsatellite genotypes and mtDNA haplotypes. For a Li-Cor 4000 or 4200 automated DNA sequencer. each of the two sites, genotypes of workers from each Locus Rf 21–1 was ampliÞed separately, but the other collection point were compared with those of all other seven primer pairs were multiplexed in the following collection points of the same species. First, we com- combinations: (Rf 1–3 and Rf 5–10), (Rf 6–1, Rf 11–2 pared the number of private alleles, i.e., alleles unique and Rf 15–2), (Rf 11–1 and Rf 24–2). Gel images were to one group of workers, and the genotypes present at scored using GeneImagIR v. 3.56 (Scanalytics, Inc., all microsatellite loci. Collection points were consid- 176 ENVIRONMENTAL ENTOMOLOGY Vol. 35, no. 1 ered part of the same colony if they had all the same notation of Thorne et al. (1999) and Bulmer et al. alleles present and a compatible set of genotypes at all (2001) in which genetic variation is partitioned among microsatellite loci, and they had identical mtDNA the individual (I), colony (C), and total (T) compo- haplotypes. In addition, we performed pairwise FSTs nents. Using this notation, FIT is equivalent to the among all conspeciÞc pairs of collection points within standard inbreeding coefÞcient FIS in the absence of each transect using the program FSTAT (Goudet differentiation between transects, FCT represents ge- 2001). The signiÞcance of the FSTs was assessed by a netic differentiation among colonies and is similar to permutation test performed by FSTAT using 1,000 FST, and FIC is the colony inbreeding coefÞcient. This iterations and the Bonferroni correction applied. Non- last term has no analog in solitary organisms; it is signiÞcant FSTs between workers from different col- especially sensitive to the numbers of reproductives lection points were taken as additional evidence that and their mating patterns within social groups and is collection points were part of the same colony. In therefore highly informative in inferring colony practice, the high variability of the markers and the breeding structure (Thorne et al. 1999). In addition, close family structure of colonies (see Results) made we estimated FST-values among transects within sites colony designations unambiguous. as well as the overall FST among sites using Genetic Classification of Colonies. Following the terminol- Data Analysis. SEs of the F-values obtained using ogy of Vargo (2003a, b), Vargo et al. (2003), and FSTAT were generated by jackkniÞng over loci. Sig- DeHeer and Vargo (2004), colonies were classiÞed as niÞcance of the values was determined by means of a simple families, extended families, or mixed families. one-sample t-test; a two-sample approximate t-test as- Simple family colonies were those in which worker suming unequal variances (Sokal and Rohlf 1981) was genotypes at all microsatellite loci were consistent used to determine signiÞcance when comparing F- with being the offspring of monogamous pairs of re- values of two groups. F-statistics were estimated for all productives, and the observed frequencies of the ge- colonies together, as well as for simple family colonies notypes did not differ signiÞcantly from the expected and extended family colonies separately. For R. hag- Mendelian ratios. Deviation of genotype frequencies eni, analyses of colony-level F-statistics were done was assessed by means of a G-test in which the locus separately for the two transects to eliminate effects speciÞc G-values were summed to obtain an overall caused by higher level structure between transects G-value for each colony. Colonies were considered (see Results). These values were compared with val- extended families if they contained no more than four ues generated by the methods of Thorne et al. (1999) alleles per locus and had genotypes compatible with a in computer simulations of possible breeding systems single pair of reproductives at one or more loci (e.g., of Reticulitermes species. Genotypes of the reproduc- Þve or more genotypic classes or three classes of ho- tives in each simple family colony were inferred from mozygotes), or they had genotypes that were com- worker genotypes. F-statistics and the average relat- patible with simple families but their frequencies de- edness between the reproductives in these colonies viated signiÞcantly from expected (P Ͻ 0.05, G-test). were estimated from the reconstructed genotypes. In Extended family colonies presumably were headed by addition, we used the program Kinship v. 1.3.1 (Good- multiple neotenic reproductives by themselves or pos- night and Queller 1999) to determine the probability sibly together with one or both primary reproductives. that the nestmate reproductives within each simple Mixed families had more than four alleles at a locus family were close relatives. This program uses maxi- indicating the presence of multiple same-sex repro- mum likelihood methods to determine whether pairs ductives originating from outside the colony. of individuals share a speciÞed pedigree relationship. Colony and Population Genetic Structure. Colony We tested whether the two reproductives within each and population genetic structure were assessed at sev- simple family were putative full siblings by testing eral levels. To determine kin structure within colonies, each pair against the average coefÞcient of relatedness we estimated the average relatedness for workers from among workers in each population and using zero as the microsatellite genotype frequencies using the pro- the null hypothesis. The statistical signiÞcance of the gram Relatedness v.5.0.8 (Queller and Goodnight results was based on 10,000 permutations. 1989) with colonies weighted equally. SEMs were To ensure that the COII gene was not under strong obtained by jackkniÞng over loci. For R. hageni, with- selection, sequences were tested for conformance to in-colony relatedness was corrected for genetic struc- the neutral mutation hypothesis by means of the D ture between sites using the Demes option, because statistic of Tajima (1989) as implemented in the pro- there was signiÞcant differentiation between the two gram DnaSP (Rozas and Rozas 1999, available at sites in this species (see Results). The signiÞcance of http://www.ub.es/dnasp/). We also calculated nucle- the coefÞcients was determined by comparing them to otide diversity (␲), deÞned as the average number of ␪ ϭ zero with a t-test. A two-sample approximate t-test pairwise nucleotide differences per site, and ( 2Nfu, assuming unequal variances (Sokal and Rohlf 1981) where Nf is the effective population size of females was used to determine whether two coefÞcients were and u is the mutation rate) for each species and each signiÞcantly different. population using this program. Population genetic Structure at the level of the colony, the transect, and structure derived from the mtDNA sequence data site was assessed simultaneously by estimating F-sta- were assessed at the level of the transect by estimating tistics using the method of Weir and Cockerham ⌽-statistics using the analysis of molecular variance (1984) as implemented in FSTAT. We followed the method of ExcofÞer et al. (1992) as implemented in February 2006 VARGO AND CARLSON:BREEDING SYSTEMS OF SUBTERRANEAN TERMITES 177 the program ARLEQUIN (Raymond and Rousset 1995). This analysis was performed on the Euclidian square distances between pairs of haplotypes ac- cording to the method of Tajima and Nei (1984), and the signiÞcance probabilities for the ⌽-statistics were generated using permutation analysis on 10,000 ran- domly permuted distance matrices. The program Mo- lecular Evolutionary Genetics Analysis v.3.1 (Kumar et al. 2004) was used to construct a neighbor-joining tree depicting the genetic relationships of the haplo- types from the pairwise Euclidian distances, and con- Þdence levels for the nodes were obtained by boot- strapping 1,000 times. In addition, a minimum spanning tree was constructed using ARLEQUIN. Isolation by Distance Analysis. Isolation by distance was assessed from the microsatellite genotypes by estimating the FCT between workers from one colony and those in another colony for all pairs of conspeciÞc colonies within each transect. The Pearson product correlation coefÞcient was computed for the pairwise

FCT-values and the physical distance for all colony pairs within each site. The signiÞcance of the corre- lation coefÞcients was assessed by means of a Mantel test with 1,000 replications as implemented in Gene- pop on the Web v. 3.1c (Raymond and Rousset 1995).

Results Species Identification Based on soldier pronotal width and mandible Fig. 2. Neighbor-joining tree of COII haplotypes: length shape, samples from 9 of the 49 total collection points ϭ 164; CI ϭ 0.67, RI ϭ 0.90. Sequences for each species from keyed to R. hageni (mean pronotal width Ϯ SD ϭ GenBank were included for comparison. 0.67 Ϯ 0.03, range ϭ 0.64Ð0.72, n ϭ 15), whereas 39 samples keyed to R. virginicus (mean ϭ 0.81 Ϯ 0.04, range ϭ 0.72Ð0.88, n ϭ 67); 1 sample did not contain present in R. flavipes, whereas the R. hageni population soldiers. However, in the course of examining se- was slightly less polymorphic with between 3 and 20 quence variation in the COII gene, we found that the alleles per locus present. Based on mtDNA haplotype samples separated into three groups, aligning them- and microsatellite genotypes, we determined that all selves with sequences from GenBank for R. flavipes, R. of the R. virginicus samples belonged to the same virginicus or R. hageni (Fig. 2). Szalanski et al. (2003) simple family colony. Because this species was repre- also found that the COII gene provides a reliable sented by a single colony, we excluded it from further means to distinguish among these three species. We analysis and conÞned our comparison to R. flavipes used this grouping as the basis for our species iden- and R. hageni. tiÞcations, yielding 26 samples of R. flavipes (10 from For R. flavipes, locus Rf 11–2 deviated signiÞcantly Schenck Forest and 16 from Duke Forest), 16 samples from HWE in 16 of the 20 resampled data sets, whereas of R. hageni (3 from Schenck Forest and 13 from Duke none of the other loci showed signiÞcant deviation in Forest), and 7 samples of R. virginicus, all from more than two cases. Locus Rf 11–2, which also was Schenck Forest and all with identical haplotypes. The found to deviate signiÞcantly from HWE in another mean number of base pair differences among species study of nearby populations of R. flavipes (Vargo was three to four times higher than within species 2003a), was excluded from subsequent analyses. The (Table 1). The mean pronotal widths for soldiers of mean number of alleles for the remaining seven loci Ϯ these three species in our sample were 0.82 Ϯ 0.03 for was 12.6 8.7. For R. hageni, Þve loci showed no R. flavipes (n ϭ 49), 0.78 Ϯ 0.02 for R. virginicus (n ϭ 12), and 0.71 Ϯ 0.06 for R. hageni (n ϭ 24). Table 1. Mean no. base pair substitutions in the COII gene bp) within and among Reticulitermes spp. from the 680 ؍ length) study area Basic Genetic Data A total of 1,140 individuals was genotyped at eight R. flavipes R. virginicus R. hageni microsatellite loci, including 680 R. flavipes workers, R. flavipes (n ϭ 19) 10.0 R. virginicus (n ϭ 1) 53.9 Ñ 320 R. hageni workers, and 140 R. virginicus workers. ϭ As shown in Table 2, there were 3Ð27 alleles per locus R. hageni (n 9) 42.1 34.1 14.8 178 ENVIRONMENTAL ENTOMOLOGY Vol. 35, no. 1

Table 2. Allele numbers and frequency of most common allele types. In R. hageni, there were nine haplotypes overall, at eight microsatellite loci for the R. flavipes and R. hageni study two in Schenck Forest and seven in Duke Forest. populations There were no haplotypes shared in common between R. flavipes R. hageni locations. There were Þve unique haplotypes, and the Locus No. Freq. most No. Freq. most frequency of the most common haplotype was 0.25 alleles common allele alleles common allele (Table 3; Fig. 3). Rf 1–3 10 0.36 6 0.29 Rf 5–10 7 0.62 7 0.68 Colony Designations Rf 6–1 12 0.47 11 0.37 Rf 11–1 7 0.47 5 0.67 Figure 1 shows the relative locations of colonies of Rf 11–2 4 0.78 3 0.87 the different species in the two transects. R. flavipes Rf 15–2 3 0.67 6 0.3 Rf 21–1 22 0.29 20 0.13 was the most commonly collected species, accounting Rf 24–2 27 0.17 13 0.25 for 26 (53%) of the 49 samples collected and 25 (60%) Mean Ϯ SD 11.5 Ϯ 8.6 8.9 Ϯ 5.5 of the 42 colonies represented (Table 4). Based on the following evidence, all of the R. hageni collection points were considered distinct colonies. There was an signiÞcant deviation from HWE in any of the 20 re- average of 18.7 Ϯ 4.5 (SD) private alleles (range ϭ sampled data sets. Three loci deviated signiÞcantly in 10Ð30) between pairs of collection points and the some casesÑRf 5–10 in six cases, Rf 11–1 in Þve cases, mean FCTs for all pairs of collection points within a and Rf 6–1 in one caseÑbut we did not consider these transect was 0.36 Ϯ 0.09 (range ϭ 0.18Ð0.60), and all deviations sufÞcient to warrant exclusion of these loci. of these were signiÞcant at P Ͻ 0.01 (Bonferroni cor- There was no evidence of LD in either species, be- rection applied). Of all the R. flavipes samples, only cause none of the 560 tests performed for each species two collection points separated by 82.6 m had identical ϫ (28 pairs of loci 20 resampled data sets) were sig- alleles and genotypes present. The FCT between these niÞcant. Thus, all eight loci in R. hageni and seven in two points was not signiÞcantly different from zero R. flavipes were considered independently assorting (Ϫ0.01, P Ͼ 0.77), and the coefÞcient of relatedness markers suitable for colony and population genetic between workers in the two samples was high (r ϭ analysis. 0.425 Ϯ 0.15) and did not differ signiÞcantly from the Of the 680 bp in the COII gene, there were 103 coefÞcient of relatedness among workers within each (15.1%) variable positions with 82 (12.1% of total) of sample (r ϭ 0.495 and 0.560; both P Ն 0.27, approxi- these parsimony informative. In all cases, the individ- mate t-test). In addition, they had the same mtDNA uals sampled from the same colony had identical hap- haplotype. Therefore, these two collection points lotypes. In addition, we performed PCR-restriction were considered part of the same colony. In contrast, fragment-length polymorphism analysis of the AϩT- all other pairs of collection points within a transect rich region of mtDNA on 20 individuals per colony as were clearly different from each other, with an aver- described by Vargo (2003a), and in every case, only a age of 20.4 Ϯ 4.6 private alleles (range ϭ 10Ð36) and ϭ Ϯ ϭ single haplotype was present in each colony. R. fla- mean pairwise FCT 0.26 0.06 (range 0.12Ð0.40) vipes and R. hageni showed similar haplotype diversity signiÞcant at P Ͻ 0.001 (Bonferroni correction ap- (Table 3). The neighbor-joining tree of haplotypes is plied); these were therefore considered to be distinct shown in Fig. 2. The R. flavipes GenBank sequences colonies. were placed in the largest branch. Colonies on this branch were considered to be R. flavipes. The R. hag- Colony Genetic Structure eni samples formed two branches, one containing the Schenck Forest haplotypes and the other possessing The family structure of the R. flavipes and R. hageni the Duke Forest haplotypes. Within R. flavipes, there colonies is shown in Table 4. With two exceptions, R. were 18 haplotypes, 7 in Schenck Forest and 12 in flavipes colonies S2 and S6 from the Schenck Forest Duke Forest, of which only 1 was shared in common transect (Tables 5 and 6), all colonies had at most four (Table 3; Fig. 2). The frequency of the most common alleles at a locus, suggesting that they were comprised haplotype was 0.33, and there were 14 unique haplo- of the descendants of a single pair of founding repro-

Table 3. Haplotype and nucleotide diversity for the COII gene in R. flavipes and R. hageni

N No. haplotypes Haplotype diversity No. variable sites ␲␪SD (␪) R. flavipes 25 18 0.967 34 0.0147 0.0145 0.0055 Duke Forest 16 12 0.958 27 0.0134 0.0132 0.0055 Schenck Forest 9 7 0.917 21 0.0146 0.0126 0.0061 R. hageni 16 9 0.912 37 0.0217 0.0200 0.0088 Duke Forest 13 7 0.973 8 0.0048 0.0048 0.0026 Schenck Forest 3 2 0.667 1 0.0015 0.0015 0.0015

N is the no. colonies studied; only one haplotype was present in each colony. ␲ ␪ ϭ ␮ , nucleotide diversity as determined by the avg no. pairwise nucleotide differences per site; 2Nf , where Nf is the effective population size of females and ␮ is the mutation rate. February 2006 VARGO AND CARLSON:BREEDING SYSTEMS OF SUBTERRANEAN TERMITES 179

Fig. 3. Minimum spanning trees for COII gene sequences for the study colonies. Symbol sizes are proportional to the number of haplotypes depicted. Each dash represents one base pair difference; for many differences, the number of base pair differences is given. ductives. Simple families made up slightly less than t-tests). The F-statistics and relatedness values for the one half of all the R. flavipes colonies sampled, and this extended family colonies were not signiÞcantly dif- proportion was similar in the two transects. A little ferent from the values obtained for simple families (all Ͼ60% of the R. hageni colonies were simple families, P Ն 0.17, one-sample t-tests), and all of the F-statistics including all three of the Schenck Forest colonies. The were signiÞcantly lower than expected for any of the difference in the frequency of simple and extended simulated colony breeding systems involving three or family colonies in the two species was not signiÞcant more neotenic reproductives (all P Յ 0.01, one-sample (P ϭ 0.51, Fisher exact test). t-tests). The lower than expected level of inbreeding

Because there was signiÞcant differentiation be- in individuals relative to their colony (FIC) strongly ϭ tween sites in R. hageni (FST between transects 0.19; suggests that the effective number of reproductives in see Population Genetic Structure), analyses were per- these colonies is small, although there must have been formed on each population separately to avoid inßa- at least three reproductives present in each of these tion of FIT due to higher-level structure. Colonies of R. colonies as evidenced by the number of worker ge- flavipes from the two transects were pooled because notypes present. there was no signiÞcant differentiation at this level Close inspection of the genotypes present in the ϭ (FST 0.01; see Population Genetic Structure). Work- two mixed R. flavipes colonies revealed the presence ers of R. flavipes were not signiÞcantly inbred relative of different family groups. Three distinct family ϭ ϭ to the total population (FIT 0.04; P 0.19, one- groups comprised of 62, 28, and 4 individuals could be sample t-test) and were as related as full sibs (r ϭ 0.50; discerned in colony S2 (Table 5). All three family Table 7). The F-statistics and relatedness values for groups had genotypes consistent with simple families, workers in simple family colonies were very similar to but the ratios of the genotypes differed signiÞcantly those expected for colonies headed by monomagous from those expected (P Ͻ 0.05, G-test) only in the pairs of unrelated primary reproductives (Table 7, larger group (Table 5, Family 1). There was a fourth case A), and in no cases did the values differ signiÞ- group of six individuals that did not share any alleles cantly from the expected (all P Ն 0.07, one-sample in common with any of the three family groups at locus

Table 4. Family composition of colonies of R. flavipes and R. hageni in two North Carolina Piedmont sites

Schenck Forest Duke Forest Total No. No. No. No. No. No. No. No. No. Species No. simple extended mixed No. simple extended mixed No. simple extended mixed colonies family family family colonies family family family colonies family family family colonies colonies colonies colonies colonies colonies colonies colonies colonies R. flavipes 9 4 3 2 16 7 9 0 25 11 12 2 (44.4%) (33.3%) (22.2%) (43.8%) (56.3%) (44.0%) (48.0%) (8.0%) R. hageni 330013760161060 (100%) (53.8%) (46.2%) (62.5%) (37.5%) 180 ENVIRONMENTAL ENTOMOLOGY Vol. 35, no. 1

Table 5. Genotypes of different family groups within R. flavi- because of the small sample size in the Schenck Forest pes colony S2 at locus Rf 24–2 population, only the results of the Duke Forest pop- ulation is discussed below. Compared with their R. Family 1 Family 2 Family 3 Others Genotype (n ϭ 62) (n ϭ 28) (n ϭ 4) (n ϭ 6) flavipes counterparts, workers from the Duke Forest ϭ R. hageni colonies were signiÞcantly inbred (FIT 134/197 14 Ͻ 140/212 4 0.19; P 0.05, approximate t-test). In addition, the 143/197 16 Duke Forest R. hageni population had a signiÞcantly 143/212 15 higher value of FCT and relatedness than the R. flavipes 158/182 1 colonies (both P Յ 0.03, approximate t-test). Workers 170/197 16 170/212 15 in simple family colonies from the Duke Forest pop- 176/188 1 ulation of R. hageni were signiÞcantly more inbred ϭ Ͻ 182/194 1 relative to the total population (FIT 0.14; P 0.02, 191/194 1 one-sample t-test) than expected for colonies headed 194/194 1 194/212 1 by outbred primary reproductives (Table 7, case A). 197/197 14 In addition, the FCT and relatedness values were sig- niÞcantly higher than expected, suggesting that the reproductives in these colonies were close relatives (both P Յ 0.01, one-sample t-test). The R. hageni Rf 24–2. Thus, this group could not be placed in any simple families were more inbred than those of R. of the other families nor did it form an obvious family flavipes; F , F , and r values were signiÞcantly higher group by itself. The average degree of relatedness IT CT than the corresponding values in R. flavipes (all P Յ among workers within families was greater than that 0.05, approximate t-test.) for full sibs and was signiÞcantly greater than relat- In Duke Forest, workers in R. hageni extended fam- edness among workers in the entire colony without ily colonies were more inbred (F ϭ 0.24) and slightly respect to family (P Ͻ 0.03, t-test; Table 8) and sig- IT more related (r ϭ 0.63) than workers in simple family niÞcantly greater than relatedness between workers colonies, but these differences were not signiÞcant belonging to different families (P Ͻ 0.005, t-test). The (both P Ն 0.20, approximate t-test). The number of genotypes of workers in the two families present in genotypes present in the extended family colonies colony S6 are shown for locus Rf 24–2 in Table 6. indicates that there were more than two reproductives Family 1 was classiÞed as an extended family because present so that breeding structures with a single pair of the presence of more genotypes than is possible of neotenics (Table 7, cases B1 and B2) can be ruled with a single set of parents at Rf 1–3 and Rf 21–1, out as possibilities for these colonies. The strongly whereas a simple family structure could not be ruled negative value of the coefÞcient of inbreeding in in- out for family 2. The average relatedness within each dividuals relative to their colony (F ϭϪ0.26; P Ͻ of these families was only slightly higher than within IC 0.01, one-sample t-test) suggests low numbers of neo- the colonies as a whole (Table 8), no doubt because tenics that were inbred for very few generations (e.g., of the large disparity in the number of workers from Table 7, case B3). Although all of the inbreeding co- the two putative families (92 versus 8), and neither efÞcients were higher in the R. hageni extended family value differed signiÞcantly from r ϭ 0.50, the value colonies than in those of R. flavipes, these differences expected for full sibs (both P Ն 0.09, one-sample were not signiÞcant (all P Ͼ 0.10, approximate t-test). t-tests). Although the between family relatedness in Analysis of the reconstructed microsatellite geno- this colony was positive (r ϭ 0.19), it was not signif- types of reproductives gave results that were consis- icant (P Ͼ 0.2, one-sample t-test). Interestingly, indi- tent with those of the worker genotypes. The repro- viduals from all groups in both colonies S2 and S6 had ductives in R. flavipes were not inbred (F ϭϪ0.01; the same COII haplotype, which was shared in com- IT P Ͼ 0.5, randomization test in FSTAT), and the king mon with one other colony, making it the most com- and queen within simple families were not signiÞ- mon haplotype with a frequency of 0.33. cantly related on average (r ϭ 0.03, P ϭ 0.26, one- The F-statistics and relatedness values obtained for sample t-test). The situation was somewhat different both R. hageni populations are given in Table 7, but in R. hageni. Again, because of the small sample size from Schenck Forest, colonies from this population Table 6. Genotypes of two different family groups within R. were not included in the analyses, but they showed the flavipes colony S6 at locus Rf 24–2 same pattern as those from Duke Forest. Although the reproductives in the Duke Forest simple family col- Family 1 Family 2 onies were not signiÞcantly inbred (F ϭ 0.03; P Ͼ 0.3, Genotype ϭ ϭ IT (n 92) (n 8) randomization test in FSTAT), cohabiting reproduc- 128/158 23 tives were signiÞcantly related (r ϭ 0.28, P Ͻ 0.02, 128/188 20 one-sample t-test) and signiÞcantly more so than the 140/179 3 Ͻ 140/194 3 nestmate reproductives of R. flavipes (P 0.04, t-test). 143/179 1 The higher degree of relatedness among reproduc- 143/194 1 tives in R. hageni may have been caused by a slightly 158/188 22 greater frequency of matings between close relatives. 188/188 27 Results of analysis with kinship indicated that 2 of 11 February 2006 VARGO AND CARLSON:BREEDING SYSTEMS OF SUBTERRANEAN TERMITES 181

Table 7. F-statistics and relatedness coefficients (mean ؎ SE) for nestmate workers of R. flavipes and R. hageni from two study sites in central North Carolina

Species/Site FIT FCT FIC r R. hageni Shenck Forest All colonies (all simple families n ϭ 3) 0.223 (Ϯ0.197) 0.436 (Ϯ0.143) Ϫ0.376 (Ϯ0.038) 0.716 (Ϯ0.121) Duke Forest All colonies (n ϭ 13) 0.190 (Ϯ0.069) 0.365 (Ϯ0.045) Ϫ0.277 (Ϯ0.048) 0.614 (Ϯ0.046) Simple family colonies (n ϭ 7) 0.140 (Ϯ0.053) 0.332 (Ϯ0.024) Ϫ0.289 (Ϯ0.035) 0.595 (Ϯ0.024) Extended family colonies (n ϭ 6) 0.236 (Ϯ0.097) 0.394 (Ϯ0.079) Ϫ0.257 (Ϯ0.079) 0.599 (Ϯ0.092) R. flavipes All colonies (n ϭ 25) 0.037 (Ϯ0.040) 0.244 (Ϯ0.024) Ϫ0.274 (Ϯ0.026) 0.498 (Ϯ0.029) Simple family colonies (n ϭ 11) Ϫ0.002 (Ϯ0.045) 0.249 (Ϯ0.023) Ϫ0.336 (Ϯ0.023) 0.531 (Ϯ0.027) Extended family colonies (n ϭ 12) 0.056 (Ϯ0.092) 0.275 (Ϯ0.027) Ϫ0.302 (Ϯ0.040) 0.487 (Ϯ0.036) Mixed family colonies (n ϭ 2) 0.060 (Ϯ0.069) 0.198 (Ϯ0.038) Ϫ0.174 (Ϯ0.049) 0.396 (Ϯ0.055) Simulated breeding system Colonies headed by outbred reproductive pairs 0.00 0.25 Ϫ0.33 0.50 Colonies headed by inbreeding neotenics ϭ ϭ ϭ Ϫ (1) Nf Nm 1, X 1 0.33 0.42 0.14 0.62 ϭ ϭ ϭ Ϫ (2) Nf Nm 1, X 3 0.57 0.65 0.22 0.82 ϭ ϭ ϭ Ϫ (3) Nf 2, Nm 1, X 3 0.52 0.59 0.17 0.78 ϭ ϭ ϭ Ϫ (4) Nf Nm 10, X 1 0.33 0.34 0.01 0.51 ϭ ϭ ϭ Ϫ (5) Nf Nm 10, X 3 0.37 0.38 0.02 0.56 ϭ ϭ ϭ Ϫ (6) Nf 200, Nm 100, X 3 0.33 0.34 0.00 0.50

R. flavipes colonies from the two sites were pooled because there was no signiÞcant differentiation between the sites. Also included are values expected for several possible breeding systems as derived from computer simulations by Thorne et al. (1999) and Bulmer et al. (2001). For the simulated breeding systems, X represents the no. of generations of production of neotenic reproductives within a colony; Nf and Nm represent the no. replacement females and males, respectively, produced per generation. pairs of R. flavipes reproductives (18%) were putative size in the Schenck Forest population (n ϭ 3), this full sibs (P Ͻ 0.05), whereas four of the seven R. hageni conclusion should be taken as tentative. Results of the simple families (57%) were likely headed by full sib- analysis of the mtDNA data showed greater degrees of lings (P Ͻ 0.05), although the difference in this fre- differentiation. This value was considerably higher in quency was not signiÞcant (P ϭ 0.07, Fisher exact R. hageni than in R. flavipes, with effective number of test). Alternatively, such colonies could have been migrants (Nm) in the former species below 0.1, headed by one primary reproductive and one replace- whereas Nm was above one in R. flavipes. ment neotenic. The data from the COII gene conformed to neu- trality, according to the test of selective neutrality of Tajima (1989), for both R. flavipes and R. hageni (both Population Genetic Structure P Ͼ 0.1). The R. flavipes sequences formed a single

The FST and Nm values between the Schenck and well-supported clade in the neighbor-joining tree, Duke populations for both the microsatellite data and within which were located R. flavipes haplotypes from the mtDNA data are shown in Table 9. In R. flavipes, GenBank (Fig. 2). Although the haplotypes from the there was no signiÞcant differentiation between the two populations were not completely separated into two locations according to the microsatellite geno- different clades, some segregation between them can ϭϪ ϭ types of either the workers (FST 0.00; 95% CI be seen in the neighbor-joining tree and the minimum Ϫ0.01Ð0.00) or the inferred genotypes of reproduc- spanning tree (Fig. 3). The R. hageni haplotypes ϭϪ ϭϪ tives (FST 0.01; 95% CI 0.03Ð0.01). In contrast, formed two well-supported clades with the two pop- there was signiÞcant differentiation between locations ulations clearly separated from each other. There was in R. hageni using both worker microsatellite geno- only a single COII sequence for R. hageni available ϭ ϭ types (FST 0.19; 95% CI 0.10Ð0.28) and the re- from GenBank, and this haplotype showed strong af- ϭ constructed genotypes of reproductives (FST 0.24; Þnity to the haplotypes from the Schenck Forest pop- 95% CI ϭ 0.14Ð0.37), but because of the small sample ulation (Fig. 2). The R. virginicus haplotype and the R.

Table 8. Relatedness (r) of workers in two mixed family colonies of R. flavipes

r between workers r between workers Colony No. families present r among all workers in the same family in different families S2 3a 0.346 0.600 0.154 (SE ϭ 0.0842) (SE ϭ 0.068) (SE ϭ 0.119) (95% CI ϭ 0.2059) (95% CI ϭ 0.1663) (95% CI ϭ 0.291) S6 2 0.439 0.481 0.185 (SE ϭ 0.056) (SE ϭ 0.078) (SE ϭ 0.133) (95% CI ϭ 0.2109) (95% CI ϭ 0.1919) (95% CI ϭ 0.325)

a There was also a group of 6 individuals that did not form an obvious family group. 182 ENVIRONMENTAL ENTOMOLOGY Vol. 35, no. 1 virginicus GenBank sequence formed a well-supported clade within the tree with strong afÞnity to the Duke Forest haplotypes. The minimum spanning trees showed similar relationships, with the R. virginicus haplotype embedded within the R. hageni clade (Fig. 3). We can gain some insights into the relative effective population sizes from analysis of mtDNA diversity. ␪, as shown in Table 3, is a function of the effective population size of females and the mutation rate. As- suming that the COII gene has the same mutation rate and has reached mutation-drift equilibrium in the dif- ferent populations and species, variation in the mag- nitude of ␪ should reßect differences in effective pop- ulation size. Of the three populations with sufÞcient numbers to analyze, the two R. flavipes populations were the largest (mean ␪ ϭ 0.013 Ϯ 0.006 for both Schenck Forest and Duke Forest). The Duke Forest population of R. hageni was considerably smaller (␪ ϭ 0.005 Ϯ 0.003), amounting to only about 35% of the R. flavipes population from the same site (P Ͻ 0.0005, t-test). Fig. 4. Relationship between genetic differentiation

(FCT) based on microsatellite genotypes and physical dis- Isolation by Distance tance among pairs of conspeciÞc colonies within each transect. Regression lines are described by y ϭ 1.85x ϩ 0.229 There was no signiÞcant correlation between geo- for Schenck Forest R. flavipes,yϭϪ2.89x ϩ 0.277 for Duke ϭ graphic distance and FCT for R. flavipes (Fig. 4; r 0.06 Forest R. flavipes, and y ϭ 1.40x ϩ 0.320 for Duke Forest R. and Ϫ0.07 for Scheck and Duke transects, respec- hageni. tively; both P Ն 0.27). However, in R. hageni there was a signiÞcant correlation in the Duke Forest transect (Fig. 4; r ϭ 0.22, P Ͻ 0.03), but not in the Schenck these were located 55 m apart and had 31 private Forest transect (Fig. 4; r ϭ 0.97, P ϭ 0.48) where there alleles between them. In the Duke Forest R. flavipes were only three colonies. populations, there were two pairs of colonies and a set If colony reproduction by budding were common, of three colonies who shared the same haplotypes. colonies located near each other should have the same However, in only one case were colonies with the mtDNA haplotype, because female reproductives in same haplotype located adjacent to each other, and daughter colonies are expected to develop from even in this case the average relatedness between within the colony. However, only two R. flavipes col- workers in the two colonies was r ϭϪ0.10, indicating onies in Schenck Forest shared the same haplotype; that these two colonies were no more related to each other than they were to other colonies from the same population. Overall, the average relatedness between Table 9. Genetic differentiation (FST) and effective migration rates (Nm) between two populations each of R. flavipes and R. R. flavipes workers in colonies sharing the same hap- hageni using nuclear (microsatellite) and mtDNA markers lotype was no greater than it was between workers with different haplotypes (r ϭ 0.071 Ϯ 0.054 and FST Nm Ϫ0.020 Ϯ 0.117, for same and different haplotypes, ϭ Ͼ R. flavipes respectively; t118 0.97, P 0.3). Similarly, two of the Microsatellites R. hageni colonies in Schenck Forest had the same Worker genotypes Ϫ0.000 NA haplotype, but the workers in these colonies were (95% CIs) (Ϫ0.010 to 0.000) ϭϪ Genotypes of reproductives Ϫ0.010 NA unrelated to each other (r 0.147). In the Duke (95% CIs) (Ϫ0.030 to 0.010) Forest R. hageni population, there were two haplo- mtDNA COII 0.217 1.804 types shared by more than one colony (two colonies (P)(P Ͻ 0.010) in one case and three in the other), but same haplo- R. hageni Microsatellites type colonies were no more related to each other than Worker genotypes 0.190 1.066 they were to colonies with different haplotypes (r ϭ (95% CIs) (0.100Ð0.280) Ϫ0.009 Ϯ 0.087 and Ϫ0.001 Ϯ 0.151, for same and Genotypes of reproductives 0.240 0.792 different haploytpes, respectively; t ϭϪ1.25, P Ͼ (95% CIs) (0.140Ð0.370) 79 mtDNA COII 0.923 0.042 0.9). (P)(P Ͻ 0.002) Discussion For the microsatellite data, Nm is calculated from FST by the ϭ ϩ equation FST 1/(4Nm 1) after Wright (1931), whereas for the ϭ ϩ Results of this study, the Þrst detailed comparative mtDNA markers, Nm is calculated from FST 1/(2Nm 1) because of the lower effective population size associated with maternally study of colony and population genetic structure of inherited haploid genes (McCauley 1998). two sympatric subterranean termite species, reveal February 2006 VARGO AND CARLSON:BREEDING SYSTEMS OF SUBTERRANEAN TERMITES 183 important differences and similarities between the Vargo (2004) documented the fusion of two distinct breeding systems of R. flavipes and R. hageni in central colonies in Schenck Forest, the same location where North Carolina. Both species had high proportions of the two mixed-family colonies were found in this simple family colonies: some 44% in R. flavipes and 63% study. We collected our samples in this study during in R. hageni. Colonies of R. flavipes were generally a single point in time and are therefore unable to say founded by pairs of outbred primary reproductives, whether the mixed family colonies studied here arose whereas those of R. hageni appeared to be frequently through colony fusion. Nonetheless, the fact that founded by close relatives resulting in colonies that nearly all of the 100 individuals genotyped in each were signiÞcantly inbred. About one-half the R. fla- colony could be assigned to a distinct family with no vipes colonies in the study population, and almost 40% indication of interbreeding between the reproduc- of the R. hageni colonies, were extended families likely tives heading each family is consistent with a fusion headed by low numbers of neotenic reproductives, event involving two or more colonies. either by themselves or together with the one or both Surprisingly, we found that all the families present primaries. A small percentage of R. flavipes colonies within each of the mixed family colonies shared the (8%) was composed of a mixture of two or more same mtDNA haplotype. This may suggest that these different families, although the majority of individuals mixed family colonies are comprised of distantly re- in these colonies were from a single family. In contrast, lated kin, a conclusion suggested by the moderate we did not Þnd any mixed family colonies of R. hageni degree of relatedness among families within the same at either study site. colony (r ϭ 0.15 and 0.18), although these values were The proportion of simple family colonies of R. fla- not signiÞcantly different from zero. The probability vipes found in this study is lower than that reported in that the different families present in these colonies previous studies of this species from these same and would have the same mtDNA haplotype by chance are other nearby locations. In a similar transect study of 56 less than 1 in 10 in the case of colony S6 and less than colonies from Schenck Forest, Duke Forest, and an- 3 in 1,000 in the case of colony S2. However, mixed other central North Carolina wooded area, Vargo family colonies of R. flavipes are not always comprised (2003a) found that about three-quarters of the colo- of individuals with the same mtDNA haplotypes; in nies were simple families and no mixed family colonies fact Bulmer et al. (2001) and Jenkins et al. (1999) used were observed. DeHeer and Vargo (2004) studied 30 variation in mtDNA to detect the presence of multiple R. flavipes colonies from Schenck Forest and a nearby matrilines within R. flavipes colonies. The combined forest site and found that 70% were simple families, results of all these studies indicate that a small pro- 27% were extended families, and one colony was com- portion of colonies of R. flavipes in several populations prised of a mixture of different families. The reason for consist of a mixture of different families that may or the lower proportion of simple family colonies found may not share the same mtDNA haplotype and that in this study is not obvious, but may be caused by the the combined use of sensitive markers of both the sampling method used. In the previous two studies, all mitochondrial and nuclear genomes can be a powerful termites encountered were collected, whereas in this means to uncover the complexities of colony breeding study we attempted to focus on the slightly smaller structure in subterranean termites. species, R. virginicus, so worker groups that had con- The apparent lack of interbreeding among repro- spicuously large body sizes were not collected. If sim- ductives from different families in these colonies sug- ple family colonies of R. flavipes tend to have larger gests that the presence of multiple families within workers than extended family colonies at these study colonies may be a temporary phenomenon, with re- sites, the samples of this species studied here may have production eventually being monopolized by a single been biased toward extended families. Nevertheless, family. Indeed, ongoing studies of R. flavipes colonies the genetic structure of simple family colonies was that were observed to fuse in the Þeld (DeHeer and consistent among all three studies, as well as another Vargo 2004) indicate that the newly produced larvae study conducted in an urban habitat in central North present 1 yr after fusion are all the progeny of the Carolina (Vargo 2003b) and a study in Massachusetts reproductives from only one of the original colonies, (Bulmer et al. 2001), providing strong support to the and the proportion of workers in this original colony conclusion that established simple families are gener- has increased signiÞcantly (C. DeHeer and E. Vargo, ally headed by outbred primary reproductives. How- unpublished data). Similarly, a laboratory study in ever, as discussed below, recent evidence suggests that which two colonies of R. flavipes were observed to fuse sib pairings among colony founders may be fairly com- indicated that reproduction within fused colonies is mon in R. flavipes, but that they are selected against limited to individuals of only one of the original col- and relatively few survive in established colonies (De- onies (Fisher et al. 2004). Heer and Vargo 2005). There is very little previous information on the We found two mixed family colonies of R. flavipes, breeding system of R. hageni, and our results on colony both of which were located in Schenck Forest. Such genetic structure of this species are the Þrst of which colonies of this species have been found in other we are aware. Our results show that most colonies in studies (Jenkins et al. 1999, Bulmer et al. 2001, DeHeer the study population were each headed by a monoma- and Vargo 2004), where they also comprised a small gous pair of reproductives. The male and female re- fraction of the total colonies present. In a detailed productives in colonies tend to be closely related (r ϭ study of colony foraging areas over time, DeHeer and 0.28). There are two possible scenarios leading to a 184 ENVIRONMENTAL ENTOMOLOGY Vol. 35, no. 1 high frequency of consanguineous matings: (1) re- species experience low survivorship because of in- lated primary reproductives tend to pair during mating breeding depression. No comparable study of R. hag- ßights or (2) a sizable portion of colonies are headed eni tandem pairs exists, but our data suggest that nearly by neotenics, either a single pair or one neotenic 60% of the simple family colonies are headed by sibling together with one of the original primaries. Our data pairs. Thus, in the study population, either the cost of provide indirect evidence for the Þrst scenario. We inbreeding in R. hageni colonies is less than R. flavipes found signiÞcant isolation by distance in the Duke or the former species has fewer opportunities for out- Forest population of R. hageni using the microsatellite breeding. markers, and a strong suggestion of it in the Schenck Of the 25 R. flavipes colonies sampled in this study, Forest population of this species in which only three only a single colony spanned more than a single col- colonies were sampled. Such population viscosity is lection site. The occurrence of spatially localized col- consistent with short range mating ßights of primary onies of this species is consistent with the results of reproductives and/or frequent colony reproduction other studies from central North Carolina (Vargo by budding. This latter possibility can be excluded 2003a, b, DeHeer and Vargo 2004) and coastal South based on the distribution of the mtDNA haplotypes. Carolina (E. Vargo, C. DeHeer, and T. Juba, unpub- Frequent budding should produce groups of nearby lished data), where colonies generally had foraging colonies with the same haplotype, but there was no areas Ͻ30 linear m in diameter. However, in this study, evidence of such a pattern. Thus, the most likely ex- we found one colony that spanned 83 linear m, sur- planation for population viscosity is short-range dis- passing the previous records of 79 and 71 linear m for persal of primary reproductives, a phenomenon which this species (Grace et al. 1989, Su 1994). Interestingly, could also result in close relatives pairing during mat- the one R. virginicus colony sampled spanned at least ing ßights. In the few mating ßights that we have 120 linear m, which is a record for a colony of any observed in this species (unpublished data), alates Reticulitermes spp., rivaling the foraging distances of ßew at a height of no more than 3 m and quickly the Formosan subterranean termite, C. formosanus, descended near their point of emergence. Two other several colonies of which have been documented to Þndings were consistent with short-range dispersal of forage over 100 m (Su and Scheffrahn 1988, Su 1994, reproductives in R. hageni. First, the apparently lower Messenger and Su 2005, Vargo et al. 2005) up to a effective population size in the Duke population com- maximum of 185 m (Su 1994). Similarly expansive pared with sympatric R. flavipes suggests populations colonies of R. virginicus have been reported previously of R. hageni have greater genetic structure locally, as from central North Carolina (Vargo 2003b, DeHeer would be expected if dispersal were more limited. And and Vargo 2004), suggesting that this may be a com- second, the strong genetic differentiation between the mon trait of this species. All of the R. hageni colonies Schenck Forest and Duke Forest populations, leading in this study were sampled at only a single point, to an effective number of migrants (Nm) well below indicating they are relatively localized. This conclu- the value of 1.0 thought to be the threshold for pre- sion is supported by the results of recent studies of this venting evolutionary divergence by drift (Wright species in the Coastal Plain of North Carolina and 1931), suggests very limited gene ßow at spatial scales South Carolina (E. Vargo, C. DeHeer, T. Juba, un- of 30 km and possibly less. In fact, the actual value of published data), in which all 49 colonies sampled at Nm may be lower than this estimate. This is because 15-m intervals were present at only a single location.

Nm is inversely related to FST, the magnitude of which In summary, available evidence shows both intra- and is dependent on the level of genetic variation, being interspeciÞc variation in the spatial expanse of Reticu- lower in highly variable markers such as microsatel- litermes spp. colonies, but the causes of this variation lites (Hedrick 2005). An alternative explanation for remain to be identiÞed. the high FST between the two R. hageni populations, Reticulitermes flavipes, R. hageni, and R. virginicus however, is that they contain different species or sub- occur sympatrically over much of their ranges in the species as discussed below. southeastern United States (Snyder 1954). This, to- Our results suggest that primary reproductives gether with the fact that all three species appear to heading established simple family colonies in R. hageni play similar ecological roles, suggests some degree of are on average more closely related than those of R. resource partitioning among them. The relative dis- flavipes and that this is most likely caused by a higher tribution and activity of these species in a given lo- frequency of sibling reproductive pairs in the former cation is most likely inßuenced by variation in soil species (60% versus 20%). The actual proportion of moisture and temperature, the two major factors de- probable consanguineous matings in the study popu- termining the local distribution of termite species lation of R. flavipes is probably lower; in a more ex- (Kofoid 1934). Evidence in support of such a role for tensive study of established simple family colonies temperature and moisture in resource partitioning from these and nearby populations, DeHeer and among sympatric Reticulitermes spp. comes from a Vargo (2005) found that only 11 of 243 (5%) were recent study of seasonal foraging activity of R. flavipes headed by probable sibling primary reproductives. and R. hageni in central Texas, in which R. flavipes was However, these authors also reported a signiÞcantly found to be most active during the cooler, wet months higher proportion of sibling pairs present among and R. hageni during the warmer, dry months (House- newly formed tandem pairs of R. flavipes during mat- man et al. 2001). In southern Mississippi, Howard et al. ing ßights (18 of 65; 28%), suggesting such pairs in this (1982) found colonies of R. virginicus more commonly February 2006 VARGO AND CARLSON:BREEDING SYSTEMS OF SUBTERRANEAN TERMITES 185 in lower, wetter areas than colonies of R. flavipes. No responding to R. flavipes. Page et al. (2002) suggested effort was made in this study to relate species distri- that these different phenotypes represent different bution to seasonal activity or to variation in soil mois- species or subspecies, although a phylogenetic study ture, but this is an area that certainly deserves atten- by Jenkins et al. (2000) did not support the separation tion in future studies. of different cuticular hydrocarbon phenotypes of R. There have been very few comparative studies of flavipes into distinct taxa. Cuticular hydrocarbon anal- colony and population genetic structure of closely ysis was not performed on the samples used in this related termite species. Thompson and Hebert study so we are unable to say whether the strong (1998a) studied the breeding system of Nasutitermes genetic differentiation between the Duke and nigriceps and N. costalis in Jamaica using allozymes and Schenck Forest populations of R. hageni correspond to restriction fragment-length polymorphism analysis of differences in cuticular hydrocarbon phenotypes. the AϩT-rich region of mtDNA. Nearly all (95%) However, a recent study using both mtDNA 16S rDNA colonies of N. nigriceps were simple families, whereas sequence data and morphological characters suggests the few remaining colonies appeared to be extended families descended from simple families. However, that R. hageni in the far eastern United States consists these authors could not draw conclusions concerning of two species (J. Austin, A. Szalanski, R. Scheffrahn, the breeding system of N. costalis in the study popu- and M. Messenger, unpublished data), and an analysis lations because there was no variation in either the of the 16S gene in samples from this study suggest that allozyme or mtDNA markers, possibly because of re- the Schenck Forest population is R. hageni sensu cent colonization of Jamaica by this species. Thomp- stricto, whereas the Duke Forest population is R. n. sp. son and Hebert (1998a) assumed that most colonies of (J. Austin and E. Vargo, unpublished data). However, N. costalis species were headed by multiple reproduc- because R. hageni has not yet been formally split, we tives because a study based on nest dissections of have treated it as a single species in this study. Because populations on other Caribbean islands (Roisin and the two populations were analyzed separately in our Pasteels 1986) found that all 14 colonies examined study, eventual splitting of the populations into dif- contained multiple queens. Cle´ment and coworkers ferent species will not signiÞcantly affect the conclu- (Cle´ment 1981, Cle´ment et al. 2001) report variation sions presented here regarding colony breeding struc- in the breeding systems among and within several ture and dispersal. Reticulitermes spp. in Europe based on variation at two In conclusion, these results reveal some important allozyme loci, but the small number of alleles present similarities and differences in the breeding systems of ϭ in most populations in these studies (mean 2.3 R. flavipes and R. hageni, two ecologically similar spe- ϭ alleles/locus, range 1Ð5) did not provide sufÞcient cies that are sympatric over much of the southeastern sensitivity for detailed analysis of colony genetic struc- United States. Colonies of both species appear to be ture. founded by pairs of primaries. A substantial portion of The lack of genetic differentiation between the two the colonies in both species is comprised of simple R. flavipes populations is consistent with results from families, presumably still headed by the original previous Þndings, in which no or very weak differen- tiation was detected at scales of 0.5Ð100 km across a founding pair, with the remainder of the colonies broad part of the geographic range of this species consisting mainly of extended families headed by low (Reilly 1986, 1987; Bulmer et al. 2001; Vargo 2003a). numbers of neotenics, and a small proportion of R. flavipes colonies comprised of mixed families most The strong genetic differentiation (FST for microsat- Ϸ ␪ Ϸ likely arising from colony fusion. One of the main ellites 0.2; ST for mtDNA 0.9) between the two R. hageni populations separated by only 27 km would differences between these species appears to be the appear to be unusual for termites. In a study of N. distance over which they disperse. The evidence sug- nigriceps, Thompson and Hebert (1998b) found mod- gests that R. hageni disperses over relatively short ϭ erate levels of differentiation (FST 0.1) among Ja- distances, resulting in (1) greater levels of inbreeding maican populations separated by distances of 100Ð200 in simple family colonies caused by the pairing of km. Goodisman and Crozier (2002) estimated FST val- relatives, (2) smaller effective population size, and (3) ues of 0.51 and 0.37 for the primitive termite Mastot- signiÞcant structuring of local populations. Such lim- ermes darwiniensis for spatial scales of 2Ð350 and ited dispersal may allow for greater degrees of adap- Ϸ1,500 km, respectively, using microsatellites. tation to local ecological conditions and a higher rate The presence of strong population structure in R. of speciation. hageni should provide greater opportunity for local evolution in this species. Accordingly, R. hageni shows much greater variability in cuticular hydrocarbon composition than other North American Reticuli- Acknowledgments termes species, including R. flavipes. In a study of We thank K. Patel and T. Juba for invaluable technical Reticulitermes spp. samples from a large portion of the assistance and C. DeHeer for helpful comments on the manu- United States, Page et al. (2002) identiÞed 26 distinct script. This work was supported by the Keck Center for cuticular hydrocarbon phenotypes, 11 (42%) of which Behavioral Biology and funds from the U.S. Department of were identiÞed as R. hageni based on soldier morphol- Agriculture National Research Initiative Competitive Grants ogy. In contrast, there were only Þve phenotypes cor- Program to ELV (00-35302Ð9377 and 02-35302Ð12490). 186 ENVIRONMENTAL ENTOMOLOGY Vol. 35, no. 1

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