Glyptotermes nakajimai contains multiple asexual populations

To confirm the presence of asexual populations of G. nakajimai, we investigated the colony composition of 74 mature colonies from ten populations. Colonies contained multiple reproductives and up to 2300 non-reproductive members (soldiers, workers, nymphs, alates, and young instars; see Fig. 1). In lower termites, including the family Kalotermitidae, individuals (except young instars) usually exhibit sexual dimorphism in their external morphology, such as the female-specific character of having an elongated seventh sternite [23, 24] (but see [25]). Dissections revealed that our sexing of G. nakajimai individuals on the basis of the configuration of the caudal sternites was 100% accurate in reproductives (n = 30), soldiers (n = 30), and workers (n = 60) (Additional file 1: Figure S1), confirming that the sex of these castes of G. nakajimai can be easily determined from sternite morphology. In the Shikoku and Kyushu populations, all reproductives (n = 194), soldiers (n = 316), and workers (n = 3700) collected from 37 colonies were female (Fig. 2a and Table 1). In contrast, all 37 colonies collected from the other populations (Honshu, Amami-Oshima Island, Okinawa Island, and Ogasawara Islands) contained both male and female reproductives (females/[females + males] = 0.53 ± 0.02 SEM [n = 548]), soldiers (females/[females + males] = 0.42 ± 0.03 SEM [n = 713]), and workers (females/[females + males] = 0.49 ± 0.01 SEM [n = 3700]) (Fig. 2a and Table 2).

Fig. 2 Asexual and sexual populations of the termite Glyptotermes nakajimai. a Termite royals in mature colonies of asexual (top left) and sexual (top right) populations, and spermathecae of egg-laying queens without sperm in an asexual population (bottom left) and with sperm in a sexual population (bottom right). Spermathecae were stained by propidium iodide and observed under a confocal fluorescence microscope. Q, queen; K, king. Scale bars, 2 mm (top); 20 μm (bottom). b Geographical distribution of asexual and sexual populations. Each population may include more than one collection site if they are located less than 50 km apart. The number of colonies sampled in each population is shown in parentheses Full size image

Table 1 Compositions of mature field colonies in asexual populations Full size table

Table 2 Compositions of mature field colonies in sexual populations Full size table

To rule out the possibility that cryptic males exist in the all-female populations (Shikoku and Kyushu), we examined sperm storage among egg-laying queens by observing the spermathecae (sperm storage organs) stained by propidium iodide. Spermathecae of all examined queens (n = 12) in the all-female populations of G. nakajimai were empty, whereas those of all examined queens (n = 12) in the other mixed-sex populations were filled with sperm (P < 0.0001, Fisher’s exact probability test) (Fig. 2a). This is, to our knowledge, the first demonstration of queens in natural colonies that lack sperm in their spermathecae. In all the cases, we could find in the literature every termite queen in natural colonies that has been checked has had sperm in her spermatheca, regardless of the presence or absence of the kings (e.g., [26, 27]).

We also compared the hatching success of unfertilized eggs between all-female and mixed-sex populations. As expected, the all-female populations exhibited high rates of hatching success of unfertilized eggs (112 of 134 eggs [83.6%] hatched) (Fig. 3). Notably, some unfertilized eggs from the mixed-sex population also hatched (7 of 193 eggs [3.6%] hatched), indicating the presence of tychoparthenogenesis (occasional development of eggs without fertilization) [1, 28, 29] in sexual colonies of this species (Fig. 3). No significant difference in hatching success was observed between unfertilized eggs of the all-female population (112 of 134 eggs [83.6%] hatched) and fertilized eggs of the mixed-sex population (117 of 127 eggs [92.1%] hatched) (P = 0.12, Fisher’s exact probability test with Bonferroni correction) (Fig. 3). Overall, our results clearly indicate complete asexuality in the Shikoku and Kyushu populations, in contrast to the other normal sexual populations (Fig. 2b).

Fig. 3 Increased hatching success of unfertilized eggs in asexual populations. Comparison of the percentage of eggs hatched within 100 days after colony foundation among unfertilized eggs of an asexual population (n = 134), unfertilized eggs of a sexual population (n = 193), and fertilized eggs of a sexual population (n = 127). Different letters on the bars indicate significant differences (P < 0.0001, Fisher’s exact probability test with Bonferroni correction). For raw data, see Additional file 7 Full size image

A single origin of asexuality in G. nakajimai

To elucidate the evolutionary relationships among asexual and sexual populations of G. nakajimai, we conducted phylogenetic analyses of Glyptotermes termites based on two independently evolving markers: mitochondrial cytochrome c oxidase subunit II (COII) and nuclear internal transcribed spacer 2 (ITS2) sequences. The monophyly of G. nakajimai was unequivocally supported in each analysis (COII: Bayesian posterior probability [BPP] = 1.00; RAxML bootstrap support [RBS] = 100%, ITS2: BPP = 1.00; RBS = 100%) (Fig. 4, Additional file 2: Figure S2). All sequences of COII and ITS2 from six collection sites across the range of asexual populations were identical (COII: 100% identity, ITS2: 100% identity). Likewise, COII and ITS2 sequences from nine collection sites across the range of sexual populations were highly similar to each other (COII: 99%–100% identity, ITS2: 96%–100% identity), and the monophyly of all sexual populations was well-supported (COII: BPP = 1.00; RBS = 93%, ITS2: BPP = 0.97; RBS = 100%) (Fig. 4, Additional file 2: Figure S2). Given that all other known species of termites, including members of the genus Glyptotermes, are sexual, these results demonstrate a single origin of asexuality within G. nakajimai. The estimated divergence time between the two lineages was 14.1 million years ago (95% confidence interval = 8.1–22.4 million years ago [Mya]) (Fig. 5). Whether asexuality has persisted in G. nakajimai for this period of time is not known. It is possible that the most closely related sexual relatives of the asexual G. nakajimai were not represented among our samples (perhaps because they are extinct, or found in areas we did not collect from), and that asexuality evolved much more recently than 14.1 Mya. Further work is required to investigate this issue.

Fig. 4 Evolutionary relationships among asexual and sexual populations of Glyptotermes nakajimai. Phylogenetic trees were obtained by Bayesian analyses of mitochondrial COII (left) and nuclear ITS2 (right) sequences of G. nakajimai individuals representing each of the collection sites. The asexual lineage is highlighted in red, and the sexual lineage is highlighted in blue. Posterior probabilities (≥ 0.70) are shown at each node. The horizontal bar represents a distance of 0.1 substitutions per site. Multiple Glyptotermes spp. as well as Cryptotermes domesticus and Kalotermes flavicollis were used as outgroups. GenBank accession numbers are shown in parentheses. The topologies shown were very similar to those derived from maximum likelihood analyses, with some minor differences (see Additional file 2: Figure S2) Full size image

Fig. 5 Chronogram showing divergence times among Kalotermitidae, including the asexual and sexual lineages of Glyptotermes nakajimai. The tree was inferred based on an alignment of mitochondrial COII sequences, using the program BEAST v1.8.2. The asexual lineage is highlighted in red, and the sexual lineage is highlighted in blue. Branch lengths are drawn to a time scale given in millions of years. Bars represent 95% confidence intervals for estimates of node times. Four fossil termites provided minimum age constraints for calibration of the molecular clock at the nodes denoted by asterisks (see “Methods” for further details concerning these fossils) Full size image

Previous work has shown that the external morphology and cuticular hydrocarbon profiles of G. nakajimai representatives across its range are indistinguishable [21], including the asexual populations reported here. To further investigate potential fine-scale differences between the asexual and sexual lineages, we performed karyotyping. The sexual lineage consistently displayed 2n = 34 chromosomes (female: n = 12, male: n = 12), while the asexual lineage consistently harbored a complement of 2n = 35 (n = 18) and contained a trisomy, most probably of chromosome 16 (Fig. 6). To confirm our observations that the asexual lineage has an extra chromosome, we compared genome size (based on the C value or nuclear DNA mass) between the asexual and sexual lineages. As expected, the genome size of the asexual lineage was significantly higher than that of the sexual lineage (colony: F 4, 24 = 0.80, P = 0.54; lineage: F 1, 24 = 55.76, P < 0.0001; nested ANOVA with colonies nested within lineages [the asexual lineage and the sexual lineage]), although the ploidy levels were similar between the two lineages (Table 3). These results are consistent with the two lineages containing different chromosome profiles (Fig. 6), in addition to their molecular sequence profiles (Fig. 4, Additional file 2: Figure S2).

Fig. 6 Proposed karyotype of Glyptotermes nakajimai. Top: mitotic chromosomes of a female of the asexual lineage (left), a female of the sexual lineage (middle), and a male of the sexual lineage (right). MC, Mitotic chromosome. Bottom: mitotic karyotypes of a female of the asexual lineage (2n = 35) (left), a female of the sexual lineage (2n = 34) (middle), and a male of the sexual lineage (2n = 34) (right) Full size image

Table 3 Comparison of genome size (C value) between the asexual and sexual lineages Full size table

Greater uniformity of head size in all-female soldiers of asexual colonies

Phenotypic variations among individuals within a colony often provide opportunities for efficient task partitioning, such as sexual specialization in tasks in animal societies [13, 15, 30, 31]. However, such variations among individuals performing the same task may lower task-efficiency when individual variation reduces group performance. The most common defense mechanism in lower termites, including the genus Glyptotermes, is phragmosis, where soldiers with plug-like heads block tunnels connecting chambers, thus preventing enemies from invading the nest [32, 33]. Tunnel width within a nest is relatively uniform, and stabilizing selection acts on soldier head width for efficient phragmotic defense in these termites, because soldiers with narrower head widths would tend to allow enemies to pass through more easily, while those with greater head width would tend to clog tunnels within the nest [34, 35]. When sampling G. nakajimai in the field, we found that individuals of several ant species carried off struggling workers when logs infested with G. nakajimai were broken open. However, soldiers typically retreated to the small tunnels between chambers and plugged the tunnels with their heads to prevent the ants from invading intact parts of the nest (i.e., phragmotic defense) (Fig. 7a).

Fig. 7 Defensive advantage of colonies in the asexual lineage. a Cross section showing the structure of a Glyptotermes nakajimai nest. Small tunnels connecting chambers are indicated by arrows. (inset, right) A tunnel-blocking soldier (phragmotic defense). Scale bars, 2 mm. b Comparison of the within-colony coefficient of variation (CV) of soldier head width between the asexual and sexual lineages. Values are mean ± SEM (n = 5). Individual data points are represented by open circles. *, P < 0.05 (Mann–Whitney U test). For raw data, see Additional file 7. c Comparison of the proportion of soldiers to other individuals of mature field colonies between the asexual and sexual lineages. Values are mean ± SEM (n = 37). Individual data points are represented by open circles. ****, P < 0.0001 (GLM). For raw data, see Additional file 7 Full size image

In the sexual lineage of G. nakajimai, male soldiers were significantly smaller than female soldiers in head width (colony: F 4,191 = 5.16, P < 0.001, sex: F 1,191 = 6.86, P < 0.01, two-way ANOVA), as well as in head length (colony: F 4,191 = 10.67, P < 0.0001, sex: F 1,191 = 13.21, P < 0.001, two-way ANOVA) (Additional file 3: Figure S3). Stabilizing selection appears to act on soldier head width because the head width to length ratio differed significantly between the sexes (colony: F 4,191 = 12.46, P < 0.0001, sex: F 1,191 = 12.15, P < 0.001, two-way ANOVA), such that differences in head width between the sexes were reduced (Additional file 3: Figure S3).

To investigate whether asexuality leads to increased stabilization of soldier head width, we compared the within-colony coefficient of variation (CV) of soldier head width between the asexual and sexual lineages. The asexual lineage had significantly smaller within-colony CVs of soldier head width than those of the sexual lineage (P = 0.012, Mann–Whitney U test) (Fig. 7b), indicating reduced intracolonial variation in soldier head width in the asexual lineage. This is presumably due to a loss in sexual dimorphism in the asexual lineage, although the loss of genetic diversity resulting from parthenogenesis may also contribute. We also compared the proportion of soldiers to other individuals in mature field colonies between the asexual and sexual lineages. Mature field colonies of the asexual lineage had significantly lower proportions of soldiers to other individuals than those of the sexual lineage (P < 0.0001, GLM) (Fig. 7c), suggesting the number of soldiers required for colony defense in the asexual lineage appears to be lower than that in the sexual lineage. A reduction in the number of soldiers, which require sibling care because they are unable to feed themselves, is likely to allow investment elsewhere in the colony. Thus, our results hint at the possibility of increased defensive efficiencies arising from the greater morphological uniformity of soldiers in all-female asexual colonies. It is possible that such efficiencies contributed to the persistence and spread of the asexual lineage.

If the sexual lineage produced only female soldiers (or only male soldiers), intracolonial variation in soldier head width could also be reduced. Indeed, sexual specialization of soldiers is common in many termite taxa with sterile workers [13, 15]. However, such sexual specialization is extremely rare in dry-wood termites (Kalotermitidae) [13, 15]. This can be partly explained by their caste-developmental pathway, through which all colony members finally develop into alates, neotenic reproductives (only a small proportion of individuals), or sterile soldiers [13, 14] (Fig. 1). Sexual specialization of soldiers would therefore lead to skewed sex ratios of other colony members within colonies, most importantly alates. This would potentially lead to a failure of some alates from the colony to successfully breed.

Pre-adaptive conditions for the evolution of maleless societies

When the costs of asexuality are not high, a mutation causing asexual reproduction is expected to rapidly spread within a sexual population due to the twofold reproductive advantage of asexual reproduction. Under this scenario, asexual females produce only female offspring, while sexual females continue to produce both male and female offspring [36]. Assuming that approximately equal numbers of offspring are produced by both asexual and sexual colonies, asexual females will increase in frequency in the population and eventually replace sexual females entirely unless mutations that suppress asexuality arise.

We hypothesize the existence of a number of key traits in the ancestors of asexual G. nakajimai, which permitted them to overcome the barriers required for complete loss of males from their mixed-sex societies. Firstly, we hypothesize that the sexual ancestors of asexual G. nakajimai were likely to have been pre-adapted to overcome at least some developmental constraints associated with parthenogenesis. This is because parthenogenetic eggs have to be activated without sperm, and centrioles need to be inherited only from mothers during reproduction [5]. This hypothesis is supported by our finding of tychoparthenogenesis (occasional development of eggs without fertilization) in the sexual lineage, whereby 7 of 193 unfertilized eggs (3.6%) successfully developed into larvae (Fig. 3). Tychoparthenogenesis is thought to provide an important pathway to the evolution of parthenogenesis [29], but is also relatively common in many animal groups, regardless of the presence or absence of asexual lineages [1, 28]. Therefore, tychoparthenogenesis is unlikely to be the only characteristic of the sexual ancestors of G. nakajimai that facilitated the evolution of all-female colonies.

The second trait we propose to have facilitated the transition to asexuality in G. nakajimai is cooperative colony foundation by queens (i.e., pleometrosis). Following the departure of alates from termite colonies on their nuptial flight, most termite incipient colonies are founded by a monogamous pair of primary (alate-derived) reproductives (i.e., a king and a queen) [37]. Both asexual and sexual G. nakajimai lineages produce alates that leave their colonies to undertake nuptial flights [21, 22]. However, rather than finding a single queen in incipient field colonies, we found that one incipient field colony of the asexual G. nakajimai lineage contained two queens, and three other incipient asexual field colonies were founded by more than two queens (range = 4–25). We found that the number of larvae per colony was positively correlated with the number of founders (Table 4). Incipient termite colonies typically experience high mortality rates, owing to disease and other factors [38, 39]. Therefore, increased numbers of colony members through cooperative colony foundation by reproductives would be adaptive, especially in asexual populations, given the presumed genetic disadvantages of parthenogenesis [40]. In the present study, we were not able to find incipient field colonies of the sexual lineage of G. nakajimai. However, we found that 29 of 37 mature field colonies (78.4%) of the sexual lineage, as well as 21 of 37 mature field colonies (56.8%) of the asexual lineage, contained more than two alate-derived reproductives (Tables 1 and 2). This is suggestive of cooperative colony foundation by multiple kings and queens, although we cannot rule out the possibility of colony fusion. Based on these results, we hypothesize that the ancestors of the asexual lineage benefited from multi-queen colony foundation during the transition to male-free societies.

Table 4 Compositions of incipient field colonies of the asexual lineage Full size table

The presumed presence of multi-queen colony foundation in the ancestor of asexual G. nakajimai is also likely to have removed one potential barrier to the spread of the asexual phenotype: the expected failure of many colonies founded by single asexual queens [41]. Termites cannot clean their own bodies by self-grooming, with the exception of their antennae. Mutual grooming by reproductives during incipient colony foundation is therefore thought to play a key role in disease avoidance [41]. During the evolution of termite all-female asexual societies, females that founded colonies with other females (rather than alone) are likely to have been at a significant advantage, both from the benefits of grooming, as well as faster colony growth due to enhanced reproductive output.

The final traits we hypothesize that may have permitted the loss of males in G. nakajimai are those generally associated with the life histories of dry-wood termites. Dry-wood termites are single-site nesters, living in a single piece of dead wood that serves both as nest and as food [42]. This means that individual members do not need to forage outside the nest, reducing the risk of exposure to pathogens and parasites. Importantly, dry-wood nesters can easily disperse over water through wood-rafting. This is evidenced by the presence of many species, including G. nakajimai, in coastal areas and on remote islands [20, 21, 43, 44]. According to the Red Queen hypothesis, sexual reproduction is favored because it helps the population to co-evolve with specialist parasites and pathogens [45]. Long-distance dispersal events might have released sexually reproducing ancestors of asexual G. nakajimai from selection pressures favoring sexual reproduction. This is because expansion into new areas may allow founders to escape from parasites and pathogens [46, 47]. Moreover, following dispersal to new habitats, asexuality could be adaptive because it is an effective way of circumventing the challenges associated with low population densities, such as inbreeding depression and the inability to find mates [48]. Indeed, many asexual lineages of ants are unusually widespread geographically [9].

The mode of parthenogenesis in G. nakajimai

The transition from sexual to parthenogenetic reproduction may have significant negative consequences as a result of increased homozygosity and inbreeding depression [5]. Such increases in homozygosity may occur under two forms of automictic parthenogenesis: gamete duplication or terminal fusion of gametes produced during meiosis. On the other hand, heterozygosity may be maintained in offspring under alternative forms of automictic parthenogenesis: central fusion of gametes produced during meiosis, or apomixis (in which eggs cells are essentially clones produced via mitosis) [5]. Indeed, the mode of thelytokous parthenogenesis in asexual lineages of hymenopteran social insects is either automixis with central fusion or apomixis [8, 9]. The presence of an extra chromosome in all examined members of the G. nakajimai asexual lineage (2n = 35, vs 2n = 34 in members of the sexual lineage; Fig. 6) is suggestive of apomixis, as opposed to automixis. This is because an uneven number of chromosomes is likely to lead to pairing problems during the first stage of meiosis. Apomixis, on the other hand, would lead to a consistent number of chromosomes being passed to offspring [5]. We performed a preliminary investigation of the mode of parthenogenesis in G. nakajimai by genotyping individuals from both the asexual and sexual lineages (including offspring produced through tychoparthenogenesis from the sexual lineage; Fig. 3) at six polymorphic microsatellite markers developed for Glyptotermes termites (Additional file 4: Table S1). In the sexual lineage of G. nakajimai, offspring produced by tychoparthenogenesis were homozygous for a single maternal allele at two polymorphic loci (Gly08 and Gly18; Additional file 5: Table S2) (Additional file 6: Table S3). This pattern is suggestive of either (a) automixis with terminal fusion, where offspring are homozygous for a single maternal allele at all loci if no crossing-over takes place, as reported in other lower termites [16, 49], or (b) automixis with gamete duplication where offspring are completely homozygous for a single maternal allele at all loci, as suggested in a study of higher termites [16]. Unfortunately, the six markers we analyzed showed no polymorphism in the asexual lineage of G. nakajimai (Additional file 5: Table S2). Therefore, further work involving deeper sampling of the genomes of G. nakajimai representatives is required to test the hypothesis that the mode of parthenogenesis in the asexual lineage is apomixis, as suggested by its uneven number of chromosomes (Fig. 6). If our hypothesis is correct, such sampling of the asexual and sexual lineages could also determine whether heterozygous genotypes in the asexual lineage (if present) are derived from the conservation of initial allelic diversity present in the sexual ancestor. Under this scenario, the asexual lineage would have evolved through a transition from normal sexual reproduction directly to apomixis, as is known in Timema stick insects [6]. Alternatively, any heterozygous genotypes in the asexual lineage could be derived from new mutations, indicating that the asexual lineage evolved through an automictic step (as appears to be the case in tychoparthenogenetic G. nakajimai (Additional file 6: Table S3)).