Reticulitermes Species Research Articles

A microsatellite-based test of the Reticulitermes speratus genetic caste determination model in Coptotermes lacteus

Coptotermes lacteus, a termite found in eastern Australia, is a central-site nester that constructs mounds up to 2 m in height. Mature colonies typically contain the primary king and queen, and up to a million or more worker and soldier offspring. Once a year, male and female nymphs are produced in approximately equal numbers and develop into new alates. Experimental removal of the queen in the field and subsequent mating of the king with a replacement queen, results in an unusual phenomenon whereby the production of female nymphs completely ceases, while the production of male nymphs, and workers of both sexes, continues. The proximate cause of this nymph male-bias is yet to be discovered. The production of male nymphs and workers of both sexes in de-queened C. lacteus colonies is equivalent to offspring production patterns in laboratory crosses of male nymphoid (nymph-derived) with female ergatoid (worker-derived) replacement reproductives in the related species Reticulitermes speratus. An X-linked genetically influenced caste determination (GCD) mechanism has been proposed to account for such offspring patterns in R. speratus. We examined microsatellite genotypes in C. lacteus to test a prediction arising from the R. speratus GCD model: that nymphs should result from reproduction by neotenics, rather than the primary pair. In five of six colonies examined, genotypes indicated that all workers and nymphs were derived from a single reproductive pair. In three of these cases, the primary queen was also located and examined; her genotype matched that inferred from worker and nymph genotypes. These results suggest that the GCD model proposed for R. speratus does not apply to C. lacteus, at least under field conditions. The male-nymph bias following queen removal therefore remains an unresolved issue in C. lacteus.

Evidence for genetically influenced caste determination in phylogenetically diverse species of the termite genusReticulitermes

A number of social insect species have recently been shown to have genetically influenced caste determination (GCD), challenging the conventional view that caste determination should be strictly environmental. To date, GCD has been found in phylogenetically isolated species; examples of GCD being present in multiple species of a genus are lacking. Through crossing experiments of neotenic (juvenile) reproductives, we have recently provided the first evidence for a royal versus worker GCD in the termite Reticulitermes speratus. To elucidate whether this system is more widespread, we performed crossing experiments using three additional Reticulitermes species. Offspring caste and sex ratios were found to be highly similar to those found previously in R. speratus, raising the possibility that GCD was present in an ancestral lineage of Reticulitermes, and subsequently maintained throughout several episodes of speciation.

Survey and Identification of Termites (Isoptera: Rhinotermitidae) in Indiana

Abstract In total, 289 termite samples were collected from 45 counties in Indiana during 2002– 2004. Approximately 89% of the collection sites were associated with artificial structures, and almost half of the samples were from inside homes. The rest of the samples were from forested areas. Termite samples were identified based on their morphological characteristics, molecular characteristics, or both. Five species from the genus Reticulitermes were identified, and the relative abundance (percentage of the total collections) of these five species was Reticulitermes flavipes (Kollar) (90.0), Reticulitermes virginicus (Banks) (7.6), Reticulitermes arenincola (Goellner) (1.0), Reticulitermes tibialis (Banks) (1.0), and Reticulitermes hageni Banks (0.3). Based on the distribution map, R. fiavipes was the dominant and the most widely distributed species in Indiana (44 counties); followed by R. virginicus (13 counties). The three other Reticulitermes species, R. arenincola, R. tibialis, and R. hageni, were encountered in only five counties. R. arenincola is considered a rare species and its distribution has been limited to sand dunes near Lake Michigan. However, in this study, two of the three R. arenincola samples were collected outside of its type location. R. tibialis was found in three counties, whereas R. hageni was only found in Evansville, IN. To complement the morphological identifications, a 389-bp region of the mitochondrial DNA (mtDNA) 16S rRNA gene was amplified and sequenced from all five Reticulitermes species. Based on species-specific polymorphisms exhibited in mtDNA sequences, a polymerase chain reaction-restriction fragment length polymorphism—based diagnostic tool was developed to identify samples lacking of diagnostic morphological characters.

Male or female soldiers? An evaluation of several factors which may influence soldier sex ratio in lower termites

In termites, the soldiers’ sex ratio is often biased toward one sex. Unlike in the Hymenoptera, this bias cannot easily be explained by relatedness asymmetries because termites are diploid. Matsuura proposed that when large body size is adaptive for colony defence (e.g. in case of phragmotic defence) then the larger sex (given sexual size dimorphism exists) should be more likely to reach a threshold size and develop into soldiers. This would explain biased sex ratios. Matsuura validated his hypothesis for four Reticulitermes species. Here, we tested his hypothesis for two species of Cryptotermes with phragmotic defence. These drywood termites have a life type that is thought to be ancestral in termite’s evolution, thus giving us potential insights into the evolution of the soldier caste. In one of these species, the sex ratio of soldiers was highly female biased, but we could not support Matsuura’s hypothesis. Both species lacked sexual size dimorphism in all castes. Additionally, in both species, the sex ratio of helpers and sexuals did not deviate from a 1:1 ratio, and hence can also not account for the bias observed in soldiers. However, this study showed that there were behavioural differences between the sexes in both species, which could shed some light on biased sex ratio in soldiers. Our findings also indicate that the developmental pathway taken by individuals reflects a ‘decision’ at the colony level. The discovery of behavioural differences between sexes in termites should open the way to similar studies in other taxa with helpers/ workers of both sexes, as it might reveal more task partitioning in colonies than previously thought and it raises questions concerning the selective pressures that acted on caste evolution in termites.

Reticulitermes malletei (Isoptera: Rhinotermitidae): a valid Nearctic subterranean termite from Eastern North America

The taxonomic status of Reticulitermes Holmgren in North America has been in need of revision for years, but because of morphological ambiguity, traditional morphological identification of Reticulitermes species has always been difficult and unreliable. Analysis of termites, applying non-morphological genetic comparisons of mitochondrial DNA from numerous populations across North America, has implicated new species which are presently under investigation. Applying the 16S rRNA gene with biometric, cuticular hydrocarbons, and ethological data, a cryptic species of Nearctic Reticulitermes has been identified from Delaware, Georgia, Maryland, North Carolina, and South Carolina and determined to constitute a valid species with an apparently Atlantic distribution. Epicuticular hydrocarbon analysis showing the presence of rare triunsaturated alkenes, and a total absence of methyl branched alkanes also support this cryptic group as a distinct species. The name for this species is Reticulitermes malletei, previously described, but not generally accepted by termite experts in the United States. Comparisons from a 403 bp region of the mtDNA 16S rRNA gene was used to discriminate known Reticulitermes species from North America: The eastern subterranean termite R. flavipes (Kollar), dark southern subterranean termite R. virginicus (Banks), light southern subterranean termite R. hageni Banks, arid subterranean termite R. tibialis Banks, and western subterranean termite R. hesperus Banks. When compared to this new species, both maximum parsimony and maximum likelihood support their genetic isolation from sympatric populations of known species and eliminate either exotic Palearctic introductions or western Nearctic involvement.

Distribution and Phylogenetic Analysis of Termite Egg-Mimicking Fungi “Termite Balls” in <I>Reticulitermes</I> Termites

To understand the evolution of parasitism, a fundamental question is what insuences the distribution of globally distributed parasites among different hosts. Reticulitermes termites often harbor brown fungal called balls, alongside their eggs. The termite ball was identiÞed as the sclerotium of a parasitic corticioid fungus, an undescribed species of genus Fibularhizoctonia, which mimics termite eggs physically and chemically so as to be protected in termite nests. This phenomenon has been found for Reticulitermes species in Japan and the United States, but not all the Reticulitermes species in these countries have termite balls. To identify factors insuencing the distribution of termite balls among their host species, we conducted wide-range sampling in the temperate zone and in subtropical zones in Japan and Taiwan. We also conducted phylogenetic analyses of termite ball fungi and their host termites based on molecular data. In Amami-Oshima Island, R. amamianus Morimoto colonies had termite balls only in temperate highland regions, but no termite balls were found in its subtropical lowland region. The introduced termite R. kanmonensis Takematsu had termite balls as well as sympatrically occurringR.speratus (Kolbe). The termite ball fungi isolated from these two species showed no signiÞcant molecular difference, suggesting no host race formation. Phylogenetic analysis showed thatR.kanmonensis in Japan did not diverge fromR.flaviceps (Oshima), which has no termite in Taiwan. Analyses of termite ball distribution among nine Reticulitermes species suggested that climate is the most important factor restricting the distribution of the termite ball fungi.

FIRST RECORD OF RETICULITERMES FLAVIPES AND RETICULITERMES HAGENI IN OREGON (ISOPTERA: RHINOTERMITIDAE)

The majority of pestiferous subterranean termites in North America belong to the endemic genus Reticulitermes (Isoptera: Rhinotermitidae). Reticulitermes flavipes (Kollar), the eastern subterranean termite, is the most economically important (Su 1993) and widespread termite (Austin et al. 2005a) in the United States. Existing taxonomic studies provide information on only 1 Reticulitermes spp. in Oregon, R. hesperus (Banks), the western subterranean termite, which is the most common termite pest species found from southern British Columbia to central California (Snyder 1954; Weesner 1965). Distribution studies on Pacific Northwest species of Reticulitermes have been addressed by Castle (1928) and Light & Pickens (1934). We report herein findings of two unreported Reticulitermes spp. (R. flavipes and R. hageni) from Oregon. Soldiers, if available, and worker termites were collected from a total of 79 different colonies from 34 locations in Oregon located in the following counties: Clackamas, Coos, Jackson, Klamath, Lane, Linn, Marion, Multnomah, Polk, Umatilla, Washington, and Yamhill by our own collection efforts, by Pest Management Professionals (PMPs), and through the 2002 national termite survey. A 428-bp region of the mt-DNA 16s rRNA gene was amplified by PCR from 79 samples consisting of 1 worker from each colony and subjected to DNA sequencing per Szalanski et al. (2003). Two R. flavipes soldier specimens collected from a colony at a collection site in Keizer, OR were identified morphologically, applying keys of Scheffrahn & Su (1994) and by evaluating soldier labra (Hostettler et al. 1995), and confirmed genetically via sequence data from a worker specimen (Szalanski et al. 2003). A worker specimen from a collection site in Salem, OR was identified genetically from sequence data as R. hageni. Soldiers were not collected from this colony and morphological identification was not performed. Both Keizer and Salem are located within 50 km from Portland, OR. Reticulitermes flavipes is the most widely distributed Reticulitermes, and is found in the entire eastern region of North America as far as Ontario, Canada, and south to Florida (Snyder 1954; Weesner 1965). The known western distribution of the species extends through the central plains to the Rocky Mountains and down to Monterrey, Mexico (Banks & Snyder 1920; Snyder 1954; Weesner 1965; Messenger 2003). Austin et al. (2005a) reported the first occurrence ofR. flavipes in California and Nevada, extending its distribution westward. The presence of R. flavipes in western states has subsequently been independently verified (Su et al. 2006; Tripodi et al. 2006). The R. flavipes 16S rRNA haplotype was FF (GenBank Accession D2001958), which is predominately found in the eastern United States (Austin et al. 2005a). Because this is outside of the previou ly known distributions of both R. flavipes and R. hageni (Banks & Snyder 1920; Snyder 1954; Weesner 1965; Messenger 2003; Austin 2005a), eastern introductions to Oregon from anthropogenic sources are implicated. Because R. flavipes is a primary pest of structures in the United States (Austin et al. 2005a) and around the world (Scheffrahn et al. 1999; Austin et al. 2005b; Su et al. 2006), assessment of this pest should be carefully evaluated to determine whether its establishment in western Ore-

A Nondichotomous Key to Protist Species Identification of <I>Reticulitermes</I> (Isoptera: Rhinotermitidae)

A key was developed using morphological and behavioral characters to identify nine genera and 13 species of protists found in the hindgut of three Reticulitermes species— Reticulitermes flavipes (Kollar), Reticulitermes virginicus (Banks), and Reticulitermes hageni Banks—by using the online IDnature guides by Discover Life. There are seven characters and 13 taxa, each attached to species descriptions, digital stills, or movies to aid in protist species identification. We chose characters for protist species identification that were easy to observe with live samples and a light microscope at 400× magnification. All 11 protists from R. flavipes and nine each in R. virginicus and R. hageni were recognized using original and revised species descriptions. This was the first report of the protist genera Trichomitus from both R. virginicus and R. hageni.

A novel hypothesis for the origin of the sexual division of labor in termites: which sex should be soldiers?

Sexual specialization and skewed sex ratios of the altruistic castes, especially soldiers, are common in many termite taxa. However, no theoretical or empirical studies have explained the origin of the sexual division of labor in termites. In most termite species, female alates are larger than male alates, and mature queens are much larger than kings, with females under consistent selection for high fertility. Therefore, females usually have the potential to be larger than males. Here, I present a novel preadaptation hypothesis that potential sexual differences in the suitability for the caste give rise to the sexual division of labor, and I provide the first evidence in support of this hypothesis in termites. Defense in Reticulitermes is typically performed by soldiers via mandibular and phragmotic defense in which soldiers with pluglike heads block openings, thus preventing enemies from invading the nest. Phragmotic defense requires that soldiers have heads wide enough to plug nest openings. Therefore, a size threshold for workers that develop into soldiers is a likely adaptation for effective defense. I show that sexual size dimorphism (SSD) and a size threshold for soldiers promote skewed sex ratios. A female-biased soldier sex ratio was observed in species with SSD, whereas there was no bias in soldier sex ratio in species without SSD. Thus, SSD and soldier sex ratio data from several Reticulitermes species support the preadaptation hypothesis.

GENETIC EVIDENCE FOR A NEW SUBTERRANEAN TERMITE SPECIES (ISOPTERA: RHINOTERMITIDAE) FROM WESTERN UNITED STATES AND CANADA

Genetic evidence for a new subterranean termite species herein named Reticulitermes okanaganensis is provided based on DNA sequence analysis. Partial sequences of the mitochondrial DNA rRNA 16S gene were obtained from 27 samples of R. okanaganensis from British Columbia, Idaho, Oregon, Nevada and California. Five nucleotide sites were variable among the four observed haplotypes. One haplotype occurred only once, while the most common haplotype, O3, occurred in 37% of the samples. Molecular phylogenetic analysis of R. okanaganensis relative to five other North American Reticulitermes species has clarified its distinct position within the genus.

Juvenile hormone III, hydroprene and a juvenogen as soldier caste differentiation regulators in three Reticulitermes species: potential of juvenile hormone analogues in termite control

The efficacy of juvenile hormone III (JH III) and two JH-mimicking compounds was compared in laboratory experiments with Reticulitermes lucifugus (Rossi), R. santonensis Feytaud and R. virginicus (Banks). Induction of presoldier, soldier and/or soldier-worker intercaste differentiation was taken as positive response to the treatment. The novel juvenogen, a fatty acid ester of a juvenoid alcohol, induced greatest soldier differentiation in all species tested, followed by hydroprene. JH III was less effective. Representatives of three Reticulitermes species showed similar trends in soldier induction rates. Differences in mortality in treatments of termites of the same species from different colonies with the same compound were observed and evidently were caused by differences in the conditions of respective colonies.

Phylogeography of <I>Reticulitermes</I> Termites (Isoptera: Rhinotermitidae) in California Inferred from Mitochondrial DNA Sequences

Existing taxonomic studies of Reticulitermes spp. (Isoptera: Rhinotermitidae) from California provide information on only two described species: Reticulitermes hesperus Banks and Reticulitermes tibialis Banks. However, while conducting a genetic evaluation of the genus from North America, we Þnd evidence of species that cannot be identiÞed morphologically with existing infor- mation. We also update more current information about other species detected during our investi- gations, including the positive identiÞcation of R. flavipes from California. Therefore, we have conducted a molecular genetics study involving DNA sequencing of a portion of the mitochondrial DNA (mtDNA) 16S gene to determine the extent of genetic variation within Reticulitermes from California. We analyzed 94 samples. Twenty-Þve nucleotide sites were variable in R. hesperus, and 19 mtDNA haplotypes were observed in the 428-bp mtDNA sequence. Fourteen haplotypes (37%) occurred only once, whereas the most common haplotypes, HE4 and HE9, each occurred in 18% of the samples. Although some haplotypes were found to have a broad geographical range across the state, some were restricted to the southern region, as were all samples identiÞed as R. tibialis. Twelve haplotypes of an undescribed western species, R.n . sp. OR. okanaganensis,O were found, and its distribution throughout the state is discussed. Additionally, genetic evidence of two additional undescribed Reticulitermes species from southern California is presented.

Identification of Chilean <I>Reticulitermes</I> (Isoptera: Rhinotermitidae) Inferred from Three Mitochondrial Gene DNA Sequences and Soldier Morphology

Abstract Results of Bayesian, maximum parsimony (MP), and neighbor joining (NJ) analysis of mitochondrial COII, 16S, and 12S gene DNA sequences and soldier morphology identified Chilean samples of a Reticulitermes species as the eastern subterranean termite, Reticulitermes flavipes (Kollar). All Chilean R. flavipes samples had identical gene sequences for all loci examined, suggesting a single geographic introduction. The combined DNA sequences for Reticulitermes santonensis Feytaud collected from France were almost identical to those of R. flavipes samples from Florida, which agreed with previous studies suggesting that R. santonensis is a junior synonym of R. flavipes. One unexpected finding was a R. flavipes sample collected from California that closely resembled the Chilean R. flavipes in their combined mitochondrial DNA sequences. We hypothesize that the Chilean R. flavipes may have been introduced from California or vice versa or that both Chilean and Californian R. flavipes may have the same origin...

Niche heterogeneity determines bacterial community structure in the termite gut (Reticulitermes santonensis)

Differences in microenvironment and interactions of microorganisms within and across habitat boundaries should influence structure and diversity of the microbial communities within an ecosystem. We tested this hypothesis using the well characterized gut tract of the European subterranean termite Reticulitermes santonensis as a model. By cloning and sequencing analysis and molecular fingerprinting (terminal restriction fragment length polymorphism), we characterized the bacterial microbiota in the major intestinal habitats - the midgut, the wall of the hindgut paunch, the hindgut fluid and the intestinal protozoa. The bacterial community was very diverse (> 200 ribotypes) and comprised representatives of several phyla, including Firmicutes (mainly clostridia, streptococci and Mycoplasmatales-related clones), Bacteroidetes, Spirochaetes and a number of Proteobacteria, all of which were unevenly distributed among the four habitats. The largest group of clones fell into the so-called Termite group 1 (TG-1) phylum, which has no cultivated representatives. The majority of the TG-1 clones were associated with the protozoa and formed two phylogenetically distinct clusters, which consisted exclusively of clones previously retrieved from the gut of this and other Reticulitermes species. Also the other clones represented lineages of microorganisms that were exclusively recovered from the intestinal tract of termites. The termite specificity of these lineages was underscored by the finding that the closest relatives of the bacterial clones obtained from R. santonensis were usually derived also from the most closely related termites. Overall, differences in diversity between the different gut habitats and the uneven distribution of individual phylotypes support conclusively that niche heterogeneity is a strong determinant of the structure and spatial organization of the microbial community in the termite gut.

MITOCHONDRIAL DNA VARIATION AND DISTRIBUTION OF THE SUBTERRANEAN TERMITE GENUS RETICULITERMES (ISOPTERA: RHINOTERMITIDAE) IN ARKANSAS AND LOUISIANA

Limited information exists on genetic variation and distribution of Reticulitermes from the south central United States. Focusing on molecular sequence data from the mitochondrial DNA 16S gene, this study records the distribution and genetic variation of Reticulitermes species in Arkansas and updates the current distribution in a neighboring State, Louisiana. Termite samples were collected from the field, subjected to DNA analysis with Polymerase Chain-Reaction (PCR), and sequenced. Reticulitermes sp. sequence data were aligned, genetic distances recorded, and their respective haplotypes were evaluated for possible geographic structure. From 35 Arkansas counties, 59 R. flavipes, 13 R. hageni, and seven R. virginicus were identified. In Arkansas, 11 mitochondrial haplotypes were observed in R. flavipes, three in R. hageni and three in R. virginicus. Among the 12 Louisiana parishes sampled, 13 R. flavipes, three R. virginicus, and one R. tibialis were identified with six, three, and one haplotypes for each species, respectively. Genetic variation among the R. flavipes haplotypes from both States ranged from 0.2 to 0.9%. Reticulitermes flavipes haplotype diversity observed in Arkansas and Louisiana was lower than observed in Texas and Oklahoma.

DNA-based identification of the eastern subterranean termite, Reticulitermes flavipes (Isoptera: Rhinotermitidae).

A DNA-based system for the identification of Reticulitermes flavipes (Koller) was established based on the following criteria: adequate molecular variation, comprehensive specimen sampling, explicit morphological species identification, and cladistic analysis. Termite soldiers were identified by labral shape, and these specimens and associated pseudergates were used in subsequent molecular analyses. Mitochondrial DNA from the AT-rich region was sequenced for 173 R. flavipes, Reticulitermes hageni Banks and Reticulitermes virginicus (Banks) soldiers and pseudergates collected from several widely dispersed Texan localities. Cladistic analysis was performed after exploration of an optimal sequence alignment inclusive of DNA sequences from Texas and published sequences from Georgia and Canada. Among the Texan individuals, 29, 5, and 1 mitochondrial DNA haplotypes were identified respectively with R. flavipes, R. hageni, and R. virginicus. One haplotype from El Paso likely represented a different Reticulitermes species. Species were monophyletic; however, individual relationships were unresolved in a strict consensus of 700 most parsimonious trees. Texas haplotypes were isolated by distance, and a correlation between genetic and geographic distance was observed among the Texas, Georgia, and Canada haplotypes. These results suggest that the methods outlined in this study will allow for the identification of R. flavipes from localities between Texas and Canada.

2002 Oklahoma Termite Survey (Isoptera)

More than 200 soil-surface and in-ground termite-monitoring devices were established across Oklahoma to collect termites. Termites were also collected from tree stumps and wooden buildings. Four species of Reticulitermes (Isoptera: Rhinotermitidae), R. flavipes, R. virginicus, R. tibialis, and R. hageni were recovered. From previous records, Gnathamitermes tubiformans (Buckley) is also indigenous to Oklahoma. Based on previous records, non-subterranean species including the dampwood termites Zootermopsis angusticollis (Hagen) and Zootermopsis nevadensis (Hagen), and the drywood termites Cryptotermes brevis (Walker) and Incisitermes minor (Hagen), are occasionally reported in lumber and furniture shipped to Oklahoma from California and Oregon, or from Gulf coast states. Known distributions of R. virginicus and G. tubiformans are extended. Reproductive flights of subterranean termites in Oklahoma peak from mid-March to mid-May. Observations of relative foraging densities across the state are also reported.

Identification of Reticulitermes spp. (Isoptera: Reticulitermatidae) from south central United States by PCR-RFLP.

Because of morphological ambiguity, traditional identification of Reticulitermes Holmgren termites has always been difficult and unreliable. A molecular diagnostic method is presented for differentiating Reticulitermes species occurring in the south central United States, which are economically important urban pests. A 379-bp region of the mtDNA COII gene and a 415-bp region of the mtDNA 16S rRNA gene were amplified using polymerase chain reaction (PCR) and sequenced from Reticulitermes flavipes (Kollar), Reticulitermes virginicus (Banks), Reticulitermes hageni Banks, and Reticulitermes tibialis (Banks). Applying DNA sequence data, the PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of two restriction enzymes each for the COII amplicon and the 16S amplicon, were diagnostic for all of the Reticulitermes species analyzed. Based on putative mutation rates, >87% and 97% of the samples should be successfully identified to species with PCR-RFLP of COII and 16S, respectively. To verify the accuracy of our predictions, we examined unclassified Reticultermes populations from Arkansas, Louisiana, Missouri, Oklahoma, Texas, and Virginia using PCR-RFLP. Applying PCR-RFLP, 97 samples were correctly classified to species. This technique allows the use of field-collected specimens preserved in alcohol and can identify termite specimens regardless of caste. PCR-RFLP, resolved with agarose or polyacrylamide gel electrophoresis, provided an efficient method for identification of Reticulitermes species from the south central United States for diagnostic purposes.

Phylogenetic relationships of nearctic Reticulitermes species (Isoptera: Rhinotermitidae) with particular reference to Reticulitermes arenincola Goellner

DNA sequence comparisons of the mitochondrial COII, 16S, and 12S rRNA genes were used to infer phylogenetic relationships among the six known US Reticulitermes species ( Reticulitermes flavipes, Reticulitermes arenincola, Reticulitermes tibialis, Reticulitermes hageni, Reticulitermes virginicus, and Reticulitermes hesperus) and the closely related European species Reticulitermes santonensis. The interspecific pairwise sequence divergence, based on uncorrected “ p” distance, varied up to 10% across the COII, 4.9% across the 16S, and 3% across the 12S fragments. Phylogenetic trees were constructed using maximum parsimony, likelihood, and distance methods. The combined results suggest several phylogenetic relationships including: (i) R. flavipes, R. arenincola, and European R. santonensis are possibly conspecific; (ii) R. virginicus and R. hageni are closely related species; and (iii) R. tibialis and R. hesperus are closely related species. Interestingly, while there is apparent synonymity between R. flavipes and R. arenincola by DNA sequence, there are clear morphological differences in the soldier caste. This finding suggests a combination of molecular and morphological approaches are necessary for accurate species identification. These data lend resolution to the complex problem of Reticulitermes systematics, and will assist future efforts directed toward characterizing species distribution and ecology.

Origin of a new Reticulitermes termite (Isoptera, Rhinotermitidae) inferred from mitochondrial and nuclear DNA data

The Holoarctic termite genus Reticulitermes is widely distributed in Europe. A new Reticulitermes species, R. sp. nov, was recently found in France and Italy. Its phylogenetic position was investigated using a 743-bp fragment of mitochondrial 16S rRNA-ND1 genes and 382-bp of the nuclear ITS2 region. Phylogenies for these sequences were estimated by neighbor-joining, maximum-parsimony and maximum-likelihood analysis. The results strongly supported a relationship between R. sp. nov. and the termite species from the eastern Mediterranean area including Reticulitermes balkanensis from the Balkans, Reticulitermes lucifugus from Turkey and Reticulitermes clypeatus from Israel. The hypothesis of a relationship between R. sp. nov. and the Japanese Reticulitermes speratus was rejected by parametric bootstrap. The current distribution of R. sp. nov. could be linked to postglacial colonization routes between Balkan refuge and northern regions.