Reproduction by worker-derived kings under natural conditions in the termite Reticulitermes speratus

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In many species, however, workers are not absolutely sterile but are instead functionally sterile—they retain reproductive potential that can be expressed under certain conditions. Why this potential persists despite the evolutionary expectation of trait loss remains an open question. In this study, we present the first field-based evidence that worker-derived male reproductives, previously known only from laboratory observations, can successfully reproduce under natural conditions in the subterranean termite Reticulitermes speratus . A survey of 702 field colonies identified one colony containing only worker-derived kings. Microsatellite genotyping confirmed that these individuals reproduced via mother–son inbreeding, and the caste fate of their offspring matched the expected pattern for matings between worker-derived kings and nymph-derived queens. Importantly, no male nymphs—the usual precursors of male reproductives—were present in the colony. Further investigation revealed that 40% of surveyed colonies (6 out of 15) entirely lacked male nymphs, indicating that such conditions may occur with non-negligible frequency in the wild. These findings suggest that worker-derived males gain rare but functional reproductive opportunities when typical male reproductives are absent, thereby maintaining their reproductive capacity over evolutionary timescales. This study provides important insight into the mechanisms that preserve reproductive totipotency in eusocial workers and underscores the value of examining reproductive roles in natural contexts to better understand the evolution of permanent sterility and true superorganismality. worker reproduction totipotency ergatiod termites social insects Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Eusociality is characterized by the presence of a reproductive caste (e.g., kings and queens) that monopolizes reproduction, and one or more sterile castes (e.g., workers and soldiers) that forgo reproduction for their entire lives and perform tasks such as foraging and colony defense under the presence of reproductives (Wilson 1971 ; Oster and Wilson 1978 ; Beshers and Fewell 2001 ; Hölldobler and Wilson 2009 ). In many species, the sterile castes—particularly workers—are not absolutely sterile but are instead functionally sterile; they retain the capacity to develop reproductive organs and produce offspring if the reproductive caste is removed (Boomsma 2009 ; Korb and Heinze 2016 ). Such workers are categorized as reproductively totipotent (or pluripotent) individuals (Revely et al. 2021 ), and this totipotency can give rise to reproductive conflict within colonies, as reproductively capable individuals may gain fitness through direct reproduction (Trivers and Hare 1976 ; Woyciechowski and Łomnicki 1987 ; Ratnieks 1988 ; Ratnieks et al. 2006 ). In contrast, in species where sterile castes have completely lost the ability to reproduce, reproduction is no longer governed at the level of individuals but at the colony level. These species are considered true superorganisms, with reproductive castes acting as the germline and sterile castes as the soma (Crespi and Yanega 1995 ; Boomsma 2007 , 2009 ; Hölldobler and Wilson 2009 ; Gardner and Grafen 2009 ; Korb and Heinze 2016 ; Bernadou et al. 2021 ). The evolution of such permanently sterile castes is thought to drive strong mutual dependence between reproductives and non-reproductives, along with increased behavioral and morphological specialization. This transition is regarded as one of the major evolutionary transitions (METs) associated with the rise in social complexity (Szathmáry and Smith 1995 ; Michod 1997 ; Bourke 2011 ; West et al. 2015 ). To understand the conditions that promote the evolution of complete sterility in workers, it is essential to investigate the mechanisms that maintain reproductive totipotency. A critical first step is to determine whether workers in reproductively totipotent species have actual opportunities to reproduce under natural conditions. This is because traits not used over time will be lost as a result of regressive evolution (Fong et al. 1995 ; Porter and Crandall 2003 ; Lahti et al. 2009 ). If workers in eusocial species never have access to reproductive opportunities, random mutation will eventually eliminate reproductive capacity. Reproductive totipotency in functionally sterile castes has been reported across diverse eusocial taxa, including lower termites (Thorne 1997 ), polistine wasps (Strassmann et al. 2002 ), bees (Michener 1974 ; Breed and Gamboa 1977 ), ants (Monnin and Ratnieks 2001 ), and naked mole-rats (Faulkes and Bennett 2001 ). Comparative studies across species that vary in the presence or absence of reproductive opportunities for workers shed light on how long reproductive totipotency persists following the loss of those opportunities. The subterranean termite Reticulitermes speratus is a lower termite species in which workers are reproductively totipotent. Colonies contain a median of approximately 25,000 workers and can reach up to 450,000 (Takata et al. 2023c ), inhabiting interconnected decayed logs via underground tunnels. The king and queen typically reside in a royal chamber within a log (Takata et al. 2023a ). Colonies are initially founded by a monogamous pair of primary reproductives, but over time, queens are replaced by multiple nymph-derived secondary queens produced via parthenogenesis by the primary queen (Matsuura et al. 2009 ). In most colonies, one primary king and several nymph-derived queens produce all other colony members, resulting in a simple family structure composed of a single reproductive pair and their offspring (Matsuura et al. 2009 ; Takata et al. 2023b ). Experimentally removing kings and/or queens induces the differentiation of both nymph-derived (nymphoid) and worker-derived (ergatoid) neotenic reproductives (Fig. 1 ) (Shimizu 1970 ; Miyata et al. 2004 ). However, to date, worker-derived male reproductives have not been found in natural colonies (Matsuura et al. 2018 ). If worker-derived male reproductives contribute to reproduction, their involvement can be detected through shifts in the caste differentiation patterns of their offspring. In this species, offspring undergo a bifurcated developmental pathway (Fig. 1 ), develop into either nymphal (fertile progenies) or apterous (functionally sterile worker) line by the third instar (Takematsu 1992 ; Roisin 2000 ; Roisin and Korb 2011 ). Caste fate is largely determined at oviposition and influenced by the phenotypes of both parents (Hayashi et al. 2007 ; Matsuura et al. 2018 ; Takata et al. 2023b ). For example, when both parents are nymph-derived reproductives, all offspring become workers, whereas if the father is worker-derived and the mother is nymph-derived, sons become workers while daughters become nymphs. Therefore, by analyzing caste fate and genotypes of offspring, it is possible to infer the phenotype of their parents. In this study, we conducted a large-scale field survey to investigate whether R. speratus workers have opportunities to reproduce under natural conditions. We examined the phenotypes of kings and queens (primary, nymph-derived, or worker-derived) in 702 field colonies. Upon discovering a colony containing worker-derived kings, we investigated why such individuals were able to differentiate and whether they had produced offspring by analyzing the colony’s caste composition and kin structure. Finally, to evaluate how commonly worker reproduction might occur, we examined the proportion of field colonies lacking male and/or female nymphs. Materials and Methods Field investigation of king and queen phenotypes in R. speratus The phenotypes of kings and queens were investigated in field colonies of R. speratus . Decayed logs containing king(s) and queens were collected from pine or Japanese cedar forests across Kyoto, Osaka, Nara, Shiga, Hyogo, Mie, Wakayama, Fukui, and Niigata, Japan, between 1998 and 2024 (see Dataset S1 for details). A total of 702 colonies were examined, with each colony processed individually. For colonies containing worker-derived kings, the entire logs in which the kings and queens were found were brought back to the laboratory. Within seven days of collection, all termites were extracted from the logs, and the number and phenotypes of kings and queens (primary, nymph-derived, or worker-derived reproductive) were recorded, with reproductive phenotypes classified according to developmental origin (see Fig. 1 ). Caste composition and kin structure of a colony with worker-derived kings Caste and sex composition The number of individuals in each caste and sex was counted and recorded for colonies with worker-derived kings. For workers (a mix of W4 and W5), a randomly selected set of 100 individuals was examined, while all individuals from other castes were recorded. Caste was identified based on the presence of wing buds, and sex was determined by sternite morphology (Weesner 1969 ; Zimet and Stuart 1982 ; Takata et al. 2020 ). Subsequently, these individuals were stored at − 20°C. Microsatellite genotyping If worker-derived kings successfully reproduces, inbreeding will occur, and its offspring can be identified based on their genotypes. Therefore, we first analyzed the genotypes of all 10 worker-derived kings present in the colony, as well as 10 randomly selected individuals from each of the other castes and sexes (nymph-derived queens, male and female workers, first-instar nymphs [N1], and first-instar workers [W1]). Total DNA was extracted using a modified Chelex protocol (Walsh et al. 1991 ). Heads or antennae were digested in 20 µL of Chelex solution (10% w/v; TE pH 8.0) and 0.2 µL of proteinase K at 55°C for 3 h, followed by incubation at 95°C for 15 min. Polymerase chain reaction (PCR) amplifications were performed in multiplex to analyze seven microsatellite loci: Rf24-2, Rf6-1, Rf21-1 (Vargo 2000 ), and Rs15, Rs10, Rs68, and Rs78 (Dronnet et al. 2004 ). Primers were labeled with fluorescent tags: Rf24-2 and Rs68 with NED, Rf6-1 and Rs10 with 6-FAM, Rf21-1 with VIC, and Rs15 and Rs78 with PET. The 10-µL PCR mixture contained 1 µL of template DNA, 0.20 µL of 10 mM dNTPs, 0.99 µL of 10× PCR buffer, 0.07 µL of 5 U/µL Taq DNA polymerase (New England Biolabs, Ipswich, MA, USA), 1.15 µL of 5 µM multiplex primers, and 6.59 µL of distilled water. Amplification conditions consisted of an initial denaturation at 95°C for 3 min, followed by 35 cycles of 95°C for 30 s, 60°C for 75 s, and 72°C for 2 min. PCR products were mixed with 10 µL of Hi-Di formamide and 0.3 µL of GeneScan 600 LIZ size standard and analyzed using an Applied BioSystems 3500 Genetic Analyzer. Raw data were processed with GeneMapper 5.0 software (Applied Biosystems, Foster City, CA, USA). Next, to determine the genetic origins of colony members, we reconstructed the genotypes of the primary queen and king of the colony. Nymph-derived queens are typically produced parthenogenetically by automixis with terminal fusion in primary queens (Matsuura et al. 2009 ), inheriting only the genes of the primary queen and resulting in homozygosity at all loci, except in cases where recombination occurs. To reconstruct the genotype of the primary queen, we first focused on nymph-derived queens that were homozygous at all microsatellite loci and listed all alleles present in these individuals. Next, using the genotypes of workers, we identified all alleles paired with those of the reconstructed primary queen, allowing us to determine the genotype of the primary king. Subsequently, we determined whether each individual was produced through outbreeding between the primary king and primary queen (i.e., carrying one allele from each parent at all loci) or through inbreeding within the colony (i.e., possessing at least one locus where only the primary king's or the primary queen's alleles were present). Nymph-derived queens produced via parthenogenesis were not classified as inbred individuals. Proportion of field colonies without male and/or female nymphs (dup: abstract ?) The proportion of colonies without nymphs (male and/or female) was examined in 15 colonies with a primary king out of the 702 colonies. After the extraction of all termites from the nests, the caste and sex were identified based on the presence of wing buds and sternite morphology, respectively. Then, presence of male and female nymph was recorded. Statistical analysis The proportion of inbred individuals across different developmental stages within the colony with worker-derived kings was compared using Fisher’s exact test with Bonferroni correction. The sex ratios of workers (W4 and W5) and young individuals (N1 and W1) in the same colony were tested for deviation from the expected 1:1 ratio using exact binomial tests. The difference in the proportion of colonies lacking male versus female nymphs in field colonies was assessed using Fisher’s exact test. A significance level of p < 0.05 was used for all statistical tests. For multiple comparisons, Bonferroni-adjusted p -values were applied. All analyses were conducted using R version 4.2.3 (R Core Team 2022 ). Results Phenotypic composition of kings and queens in R. speratus Among the 702 colonies examined, one was found to contain only worker-derived kings, which served as the sole male reproductives (Fig. 2 a). Regarding the phenotypes of kings, 93.4% (656 colonies) had only a primary king as the male reproductive. Colonies where the primary king had been replaced by nymph-derived kings accounted for 5.8% (41 colonies). In 0.6% (4 colonies), both a primary king and nymph-derived kings were present. Regarding the phenotypes of queens (Fig. 2 b), 2.0% (14 colonies) had only a primary queen, while 7.1% (50 colonies) contained both a primary queen and nymph-derived queens. The majority of colonies (90.5%, 635 colonies) had only nymph-derived queens. In 0.4% (3 colonies), worker-derived queens coexisted with nymph-derived queens. Caste composition and kin structure of the colony with worker-derived kings The colony consisted of 10 worker-derived kings, 57 nymph-derived queens, 4,296 workers, 822 nymphs, 4 male and 6 female soldiers, 27 male and 28 female pre-soldiers, and 50 L2 larvae. Both sexes were present among fourth- and fifth-instar workers, with a male-biased sex ratio (exact binomial test, 95% CI = 0.548–0.743, p = 0.004), but no male nymphs were found in the colony (Fig. 3 ). The caste fate of the larvae within the colony showed that all males differentiated into workers ( n = 55), while all females differentiated into nymphs ( n = 115). Their sex ratio was significantly skewed toward females (exact binomial test, 95% CI = 0.254–0.399, p < 0.001). Microsatellite genotyping revealed the presence of individuals produced through inbreeding. Among the analyzed individuals, 4 out of 10 worker-derived kings, 3 out of 10 nymph-derived queens, 2 out of 20 workers, and 18 out of 20 young individuals (N1 and W1) were identified as inbred (Fig. 4 ). The proportion of inbred individuals was significantly higher in young individuals compared to other older developmental stages (Fisher’s exact test with Bonferroni correction, p < 0.05). Proportion of field colonies without male and/or female nymphs Among the 15 colonies examined, 6 colonies (40.0%) lacked male nymphs (Fig. 5 ). Female nymphs were absent in 2 colonies (13.3%), both of which also lacked male nymphs. The difference in the proportion of colonies lacking male versus female nymphs was not statistically significant (Fisher’s exact test, 95% CI = 0.020–1.766, p = 0.215). Discussion The presence of reproductive opportunities for workers contributes to the maintenance of their reproductive totipotency and may prevent the evolution of true superorganisms, which are characterized by a complete reproductive division of labor (Crespi and Yanega 1995 ; Boomsma 2007 , 2009 ; Hölldobler and Wilson 2009 ; Gardner and Grafen 2009 ; Korb and Heinze 2016 ; Bernadou et al. 2021 ). Prior evidence of worker-derived kings in R. speratus had been limited to laboratory conditions (Shimizu 1970 ; Miyata et al. 2004 ; Matsuura et al. 2018 ), leaving open the question of whether such differentiation occurs in the field—and, crucially, whether these individuals are capable of producing fertile offspring. In this study, we present the first field-based evidence that worker-derived kings can attain reproductive opportunities in R. speratus . In the colony where worker-derived kings were discovered, no other male reproductive phenotypes were present, and their offspring were the result of mother–son inbreeding. Furthermore, the caste fate of these offspring—males developing into workers and most females into nymphs—matched the pattern observed when the father is a worker-derived king and the mother is a nymph-derived queen (Hayashi et al. 2007 ). Since sexually produced nymphs in this species develop into alates that go on to found new colonies (Matsuura et al. 2009 ; Wu et al. 2024 ), our findings strongly suggest that worker-derived kings are capable of producing the next generation under natural conditions. These results indicate that worker-derived kings are not merely a developmental vestige, but a viable alternative reproductive strategy when opportunities arise. The absence of male nymphs appears to be a key factor enabling the reproduction of worker-derived male reproductives. In the colony where worker-derived kings were found, no male nymphs were present. Colonies entirely lacking male nymphs were not uncommon in the field; in our survey, 40% of colonies had no male nymphs. This suggests that the absence of male nymphs occasionally creates reproductive opportunities for worker-derived males. Because nymphs differentiate into reproductives more rapidly than workers (Miyata et al. 2004 ), worker reproductive opportunities arising from the absence of nymphs are likely to be extremely rare or entirely lost in species that form large colonies where nymphs are continuously present or have life histories characterized by early nymph production. This could explain the discrepancy in frequency between colonies lacking male nymphs and those in which worker-derived kings were actually found. Although further research considering colony age is needed, it is possible that the nymph-less colonies were relatively young and therefore had lower primary king mortality, which could account for the gap. Our results highlight that, in addition to previously recognized factors such as increasing colony size and reproductive longevity—which reduce worker reproductive opportunities (Alexander et al. 1991 ; Bourke 1999 )—the timing of nymph production within a colony’s life history may also constrain worker reproduction in termite species where nymphs, rather than workers, serve as the main backup reproductive caste. Our findings also offer new insights into the phenomenon that occur during the transition from a primary king to secondary neotenics. In the colony with worker-derived kings, most young individuals (N1s and W1s) were the result of inbreeding, but a few inbred individuals were also found among the older castes, including workers, queens, and even some of the worker-derived kings themselves. This pattern suggests that inbreeding had already begun while the primary king was still alive. This inference is supported by our phenotypic survey of field colonies, which identified multiple colonies containing both a primary king and nymph-derived kings. Although the presence of a primary king typically suppresses the differentiation of additional male reproductives (Shimizu 1970 ; Miyata et al. 2004 ), our results imply that this suppression may weaken with age or declining fertility of the primary king. Consequently, there may be a window of reproductive opportunity for subordinate males even before the death of the reigning king. This study provides the first evidence that both male and female workers of R. speratus can attain reproductive opportunities under natural conditions. These findings contribute to our understanding of how reproductive totipotency is maintained in the workers of eusocial insects. Research on the evolutionary loss of traits suggests that even functionally neutral traits may persist for millions of years before being eliminated, and traits that occasionally confer fitness benefits can be retained over long evolutionary timescales (Hall and Colegrave 2008 ; Lahti et al. 2009 ; van der Kooi and Schwander 2014 ). This work highlights the importance of investigating reproductive totipotency and opportunities for reproduction in workers across diverse eusocial taxa. Such efforts will not only elucidate the timescales over which totipotency persists following the loss of reproductive opportunities, but also help identify the ecological and evolutionary factors that drive the transition toward permanently sterile castes and the evolution of true superorganisms. Declarations ACKNOWLEDGMENTS We thank Tadahide Fujita, Yao Wu, Takao Konishi, Shuya Nagai, Chihiro Tamaki, Shun Mizote, Hiroki Noda, Chen Jiaming, Kiyotaka Yabe, and Tomohiro Nakazono for assistance in collecting termites and helpful discussion. CONFLICT OF INTEREST STATEMENT The authors declare no conflicts of interest. ORCID Mamoru Takata https://orcid.org/0000-0002-8181-9987 Soshi Araki https://orcid.org/0009-0005-3518-6911 Michihiko Takahashi https://orcid.org/0009-0000-9572-4463 Kenji Matsuura https://orcid.org/0000-0002-9099-6694 Competing interests : The authors declare no competing interests. Funding :  This work was supported by JSPS KAKENHI Grant Numbers JP18H05268, JP20K20380, JP23H00332 to Kenji Matsuura, and JP21K14863 to Mamoru Takata. Ethics approval : Not applicable Consent to participate : Not applicable Consent for publication : Not applicable Availability of data and materials: All data generated or analyzed during this study are included in this published article. Data availability: Data is provided within the manuscript or supplementary information files. Code availability: All code used for graphing and statistical analyses in this study is included in this published article. Authors’ contributions : Ma.T. conceived the study, designed the methodology, performed validation and formal analysis, conducted the investigation, provided resources, wrote the original draft, reviewed and edited the manuscript, created visualizations, supervised the project, managed the project administration, and acquired funding. 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J Theor Biol 128:317–327. https://doi.org/10.1016/S0022-5193(87)80074-7 Wu Y, Fujita T, Namba Y et al (2024) Inter-clonal competition over queen succession imposes a cost of parthenogenesis on termite colonies. Proceedings of the Royal Society B: Biological Sciences 291:20232711. https://doi.org/10.1098/rspb.2023.2711 Zimet M, Stuart AM (1982) Sexual dimorphism in the immature stages of the termite, Reticulitermes flavipes (Isoptera: Rhinotermitidae). Sociobiology 7:1–7 Additional Declarations No competing interests reported. Supplementary Files DatasetS1.csv DatasetS2.csv DatasetS3.csv DatasetS4.csv ergatoidkingcolony.rmd Cite Share Download PDF Status: Published Journal Publication published 24 Apr, 2026 Read the published version in The Science of Nature → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6510951","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":459459360,"identity":"cc9876a4-d98c-4fad-a0a5-71eb37cf0575","order_by":0,"name":"Mamoru Takata","email":"data:image/png;base64,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","orcid":"","institution":"Kyoto University","correspondingAuthor":true,"prefix":"","firstName":"Mamoru","middleName":"","lastName":"Takata","suffix":""},{"id":459459361,"identity":"1fd31b30-3f09-4459-8ac5-71d139c62a55","order_by":1,"name":"Soshi Araki","email":"","orcid":"","institution":"Kyoto University","correspondingAuthor":false,"prefix":"","firstName":"Soshi","middleName":"","lastName":"Araki","suffix":""},{"id":459459362,"identity":"71cbe584-af56-4085-9844-7cc661194016","order_by":2,"name":"Michihiko Takahashi","email":"","orcid":"","institution":"Kyoto University","correspondingAuthor":false,"prefix":"","firstName":"Michihiko","middleName":"","lastName":"Takahashi","suffix":""},{"id":459459363,"identity":"79bdc83d-df1d-4408-b738-5e2b45ebe70a","order_by":3,"name":"Kenji Matsuura","email":"","orcid":"","institution":"Kyoto University","correspondingAuthor":false,"prefix":"","firstName":"Kenji","middleName":"","lastName":"Matsuura","suffix":""}],"badges":[],"createdAt":"2025-04-23 09:08:44","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6510951/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6510951/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00114-026-02105-3","type":"published","date":"2026-04-24T15:58:18+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":83281925,"identity":"b67d438e-c635-475e-8103-71d9f3670b44","added_by":"auto","created_at":"2025-05-22 10:29:27","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":146732,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic representation of caste developmental pathways in the termite \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eReticulitermes speratus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e After two larval instars (L1 and L2), individuals differentiate into either the nymphal line, which gives rise to fertile progeny, or the apterous line, which results in functionally sterile workers. Reproductives occur in three phenotypic forms: primary reproductives (alates that have undergone dealation), and neotenic reproductives derived from either the nymphal or worker line. E: egg; L1 and L2: first- and second-instar larvae; W1–W5: first- to fifth-instar workers; N1–N6: first- to sixth-instar nymphs. Solid arrows indicate molts; the dashed arrow indicates dealation. Males and females share the same developmental pathways.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6510951/v1/00dfbf3d4e61fab6f03a29d7.jpeg"},{"id":83282422,"identity":"dc8d998b-7c42-4211-8e71-007e1a4cc3b6","added_by":"auto","created_at":"2025-05-22 10:37:27","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":148054,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhenotypic composition of reproductives in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eReticulitermes speratus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e field colonies.\u003c/strong\u003e (\u003cstrong\u003ea\u003c/strong\u003e) Distribution of king phenotypes. Most colonies (93.4%) had a primary king as the sole male reproductive. In 5.8% of colonies, the primary king had been replaced by nymph-derived kings. In 0.6%, primary and nymph-derived kings coexisted. One colony (0.1%) contained only worker-derived kings. (\u003cstrong\u003eb\u003c/strong\u003e) Distribution of queen phenotypes. A small number of colonies (2.0%) had only primary queens, while 7.1% had both primary and nymph-derived queens. The majority of colonies (90.5%) contained only nymph-derived queens. In 0.4%, worker-derived queens coexisted with nymph-derived queens. \u003cem\u003en\u003c/em\u003e = 702 colonies.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6510951/v1/be858be535349ced10c99e74.jpeg"},{"id":83282419,"identity":"8087a7b7-a51c-4341-a4ce-dec42bf074b9","added_by":"auto","created_at":"2025-05-22 10:37:27","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":44501,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSex composition by caste in the colony with worker-derived kings.\u003c/strong\u003e The bar graph displays the sex ratio of individuals in the nymphs (N1–N6) and workers (W4 and W5). While the sex ratio among fourth- and fifth-instar workers was male-biased (exact binomial test, \u003cem\u003ep\u003c/em\u003e = 0.004), no male nymphs were found in the colony.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6510951/v1/aeb8ad3fb1ce83d58a8e72bc.jpeg"},{"id":83282421,"identity":"33df2b80-9c25-426c-8039-1acf660021c4","added_by":"auto","created_at":"2025-05-22 10:37:27","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":68526,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProportion of inbred individuals across castes in the colony with worker-derived kings.\u003c/strong\u003e The bars indicate the proportion of inbred individuals in each group: worker-derived kings (\u003cem\u003en\u003c/em\u003e = 10), nymph-derived queens (\u003cem\u003en\u003c/em\u003e= 10), workers (W4 and W5; \u003cem\u003en\u003c/em\u003e = 20), and young individuals (N1 and W1; \u003cem\u003en\u003c/em\u003e= 20). Inbreeding was most frequent among young individuals compared to those at later developmental stages. Different letters indicate significant differences (Fisher’s exact test with Bonferroni correction, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6510951/v1/2c65a6dbe08606855b07f2f0.jpeg"},{"id":83281932,"identity":"b5abf8f7-09a1-492f-90b4-60759215b26a","added_by":"auto","created_at":"2025-05-22 10:29:27","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":45833,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProportion of field colonies lacking male and/or female nymphs.\u003c/strong\u003e Bar graph showing the proportion of colonies with and without male or female nymphs among the 15 colonies examined. Black bars indicate colonies where nymphs were absent; white bars indicate presence. Male nymphs were absent in 40.0% of colonies, while female nymphs were absent in 13.3% of colonies.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6510951/v1/b1fff1535df5dc2181203d58.jpeg"},{"id":107928105,"identity":"9f19a2ea-98fa-45ac-b529-27af7f12f431","added_by":"auto","created_at":"2026-04-27 16:07:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":714195,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6510951/v1/c0de525b-29d1-424f-a17e-982092b513dd.pdf"},{"id":83281923,"identity":"2c5af0fd-eab8-4154-b888-2e39fa20afdb","added_by":"auto","created_at":"2025-05-22 10:29:27","extension":"csv","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":36878,"visible":true,"origin":"","legend":"","description":"","filename":"DatasetS1.csv","url":"https://assets-eu.researchsquare.com/files/rs-6510951/v1/49f91e98644c2e10252595de.csv"},{"id":83281924,"identity":"ac3b17e5-627a-4b46-ad4f-c7d4f4acc0f1","added_by":"auto","created_at":"2025-05-22 10:29:27","extension":"csv","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":184,"visible":true,"origin":"","legend":"","description":"","filename":"DatasetS2.csv","url":"https://assets-eu.researchsquare.com/files/rs-6510951/v1/4f5812abd44748facbe06d05.csv"},{"id":83282420,"identity":"94af465d-6b24-497e-8ddc-59ddb823f143","added_by":"auto","created_at":"2025-05-22 10:37:27","extension":"csv","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":4964,"visible":true,"origin":"","legend":"","description":"","filename":"DatasetS3.csv","url":"https://assets-eu.researchsquare.com/files/rs-6510951/v1/2838eb0f66b37ce781838708.csv"},{"id":83281929,"identity":"b80ce147-dd41-4a88-8e96-239cddfd5769","added_by":"auto","created_at":"2025-05-22 10:29:27","extension":"csv","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":387,"visible":true,"origin":"","legend":"","description":"","filename":"DatasetS4.csv","url":"https://assets-eu.researchsquare.com/files/rs-6510951/v1/833b3583da95eb66c8889b1c.csv"},{"id":83281928,"identity":"863c20c1-585d-4d13-926c-2a60fb4843e6","added_by":"auto","created_at":"2025-05-22 10:29:27","extension":"rmd","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":14975,"visible":true,"origin":"","legend":"","description":"","filename":"ergatoidkingcolony.rmd","url":"https://assets-eu.researchsquare.com/files/rs-6510951/v1/586b6b106431b2485bfd159f.rmd"}],"financialInterests":"No competing interests reported.","formattedTitle":"Reproduction by worker-derived kings under natural conditions in the termite Reticulitermes speratus","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEusociality is characterized by the presence of a reproductive caste (e.g., kings and queens) that monopolizes reproduction, and one or more sterile castes (e.g., workers and soldiers) that forgo reproduction for their entire lives and perform tasks such as foraging and colony defense under the presence of reproductives (Wilson \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e1971\u003c/span\u003e; Oster and Wilson \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e1978\u003c/span\u003e; Beshers and Fewell \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; H\u0026ouml;lldobler and Wilson \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). In many species, the sterile castes\u0026mdash;particularly workers\u0026mdash;are not absolutely sterile but are instead functionally sterile; they retain the capacity to develop reproductive organs and produce offspring if the reproductive caste is removed (Boomsma \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Korb and Heinze \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Such workers are categorized as reproductively totipotent (or pluripotent) individuals (Revely et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and this totipotency can give rise to reproductive conflict within colonies, as reproductively capable individuals may gain fitness through direct reproduction (Trivers and Hare \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e1976\u003c/span\u003e; Woyciechowski and Łomnicki \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e1987\u003c/span\u003e; Ratnieks \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1988\u003c/span\u003e; Ratnieks et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). In contrast, in species where sterile castes have completely lost the ability to reproduce, reproduction is no longer governed at the level of individuals but at the colony level. These species are considered true superorganisms, with reproductive castes acting as the germline and sterile castes as the soma (Crespi and Yanega \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Boomsma \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2007\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; H\u0026ouml;lldobler and Wilson \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Gardner and Grafen \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Korb and Heinze \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Bernadou et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The evolution of such permanently sterile castes is thought to drive strong mutual dependence between reproductives and non-reproductives, along with increased behavioral and morphological specialization. This transition is regarded as one of the major evolutionary transitions (METs) associated with the rise in social complexity (Szathm\u0026aacute;ry and Smith \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Michod \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Bourke \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; West et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo understand the conditions that promote the evolution of complete sterility in workers, it is essential to investigate the mechanisms that maintain reproductive totipotency. A critical first step is to determine whether workers in reproductively totipotent species have actual opportunities to reproduce under natural conditions. This is because traits not used over time will be lost as a result of regressive evolution (Fong et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Porter and Crandall \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Lahti et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). If workers in eusocial species never have access to reproductive opportunities, random mutation will eventually eliminate reproductive capacity. Reproductive totipotency in functionally sterile castes has been reported across diverse eusocial taxa, including lower termites (Thorne \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1997\u003c/span\u003e), polistine wasps (Strassmann et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), bees (Michener \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1974\u003c/span\u003e; Breed and Gamboa \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1977\u003c/span\u003e), ants (Monnin and Ratnieks \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), and naked mole-rats (Faulkes and Bennett \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Comparative studies across species that vary in the presence or absence of reproductive opportunities for workers shed light on how long reproductive totipotency persists following the loss of those opportunities.\u003c/p\u003e \u003cp\u003eThe subterranean termite \u003cem\u003eReticulitermes speratus\u003c/em\u003e is a lower termite species in which workers are reproductively totipotent. Colonies contain a median of approximately 25,000 workers and can reach up to 450,000 (Takata et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2023c\u003c/span\u003e), inhabiting interconnected decayed logs via underground tunnels. The king and queen typically reside in a royal chamber within a log (Takata et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e). Colonies are initially founded by a monogamous pair of primary reproductives, but over time, queens are replaced by multiple nymph-derived secondary queens produced via parthenogenesis by the primary queen (Matsuura et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). In most colonies, one primary king and several nymph-derived queens produce all other colony members, resulting in a simple family structure composed of a single reproductive pair and their offspring (Matsuura et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Takata et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e). Experimentally removing kings and/or queens induces the differentiation of both nymph-derived (nymphoid) and worker-derived (ergatoid) neotenic reproductives (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) (Shimizu \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1970\u003c/span\u003e; Miyata et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). However, to date, worker-derived male reproductives have not been found in natural colonies (Matsuura et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). If worker-derived male reproductives contribute to reproduction, their involvement can be detected through shifts in the caste differentiation patterns of their offspring. In this species, offspring undergo a bifurcated developmental pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), develop into either nymphal (fertile progenies) or apterous (functionally sterile worker) line by the third instar (Takematsu \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e1992\u003c/span\u003e; Roisin \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Roisin and Korb \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Caste fate is largely determined at oviposition and influenced by the phenotypes of both parents (Hayashi et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Matsuura et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Takata et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e). For example, when both parents are nymph-derived reproductives, all offspring become workers, whereas if the father is worker-derived and the mother is nymph-derived, sons become workers while daughters become nymphs. Therefore, by analyzing caste fate and genotypes of offspring, it is possible to infer the phenotype of their parents.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn this study, we conducted a large-scale field survey to investigate whether \u003cem\u003eR. speratus\u003c/em\u003e workers have opportunities to reproduce under natural conditions. We examined the phenotypes of kings and queens (primary, nymph-derived, or worker-derived) in 702 field colonies. Upon discovering a colony containing worker-derived kings, we investigated why such individuals were able to differentiate and whether they had produced offspring by analyzing the colony\u0026rsquo;s caste composition and kin structure. Finally, to evaluate how commonly worker reproduction might occur, we examined the proportion of field colonies lacking male and/or female nymphs.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e \u003cb\u003eField investigation of king and queen phenotypes in\u003c/b\u003e \u003cb\u003eR. speratus\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe phenotypes of kings and queens were investigated in field colonies of \u003cem\u003eR. speratus\u003c/em\u003e. Decayed logs containing king(s) and queens were collected from pine or Japanese cedar forests across Kyoto, Osaka, Nara, Shiga, Hyogo, Mie, Wakayama, Fukui, and Niigata, Japan, between 1998 and 2024 (see Dataset S1 for details). A total of 702 colonies were examined, with each colony processed individually. For colonies containing worker-derived kings, the entire logs in which the kings and queens were found were brought back to the laboratory. Within seven days of collection, all termites were extracted from the logs, and the number and phenotypes of kings and queens (primary, nymph-derived, or worker-derived reproductive) were recorded, with reproductive phenotypes classified according to developmental origin (see Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCaste composition and kin structure of a colony with worker-derived kings\u003c/h2\u003e \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003ch2\u003eCaste and sex composition\u003c/h2\u003e \u003cp\u003eThe number of individuals in each caste and sex was counted and recorded for colonies with worker-derived kings. For workers (a mix of W4 and W5), a randomly selected set of 100 individuals was examined, while all individuals from other castes were recorded. Caste was identified based on the presence of wing buds, and sex was determined by sternite morphology (Weesner \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e1969\u003c/span\u003e; Zimet and Stuart \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e1982\u003c/span\u003e; Takata et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Subsequently, these individuals were stored at \u0026minus;\u0026thinsp;20\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003eMicrosatellite genotyping\u003c/h3\u003e\n\u003cp\u003eIf worker-derived kings successfully reproduces, inbreeding will occur, and its offspring can be identified based on their genotypes. Therefore, we first analyzed the genotypes of all 10 worker-derived kings present in the colony, as well as 10 randomly selected individuals from each of the other castes and sexes (nymph-derived queens, male and female workers, first-instar nymphs [N1], and first-instar workers [W1]). Total DNA was extracted using a modified Chelex protocol (Walsh et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e1991\u003c/span\u003e). Heads or antennae were digested in 20 \u0026micro;L of Chelex solution (10% w/v; TE pH 8.0) and 0.2 \u0026micro;L of proteinase K at 55\u0026deg;C for 3 h, followed by incubation at 95\u0026deg;C for 15 min. Polymerase chain reaction (PCR) amplifications were performed in multiplex to analyze seven microsatellite loci: Rf24-2, Rf6-1, Rf21-1 (Vargo \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2000\u003c/span\u003e), and Rs15, Rs10, Rs68, and Rs78 (Dronnet et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Primers were labeled with fluorescent tags: Rf24-2 and Rs68 with NED, Rf6-1 and Rs10 with 6-FAM, Rf21-1 with VIC, and Rs15 and Rs78 with PET. The 10-\u0026micro;L PCR mixture contained 1 \u0026micro;L of template DNA, 0.20 \u0026micro;L of 10 mM dNTPs, 0.99 \u0026micro;L of 10\u0026times; PCR buffer, 0.07 \u0026micro;L of 5 U/\u0026micro;L Taq DNA polymerase (New England Biolabs, Ipswich, MA, USA), 1.15 \u0026micro;L of 5 \u0026micro;M multiplex primers, and 6.59 \u0026micro;L of distilled water. Amplification conditions consisted of an initial denaturation at 95\u0026deg;C for 3 min, followed by 35 cycles of 95\u0026deg;C for 30 s, 60\u0026deg;C for 75 s, and 72\u0026deg;C for 2 min. PCR products were mixed with 10 \u0026micro;L of Hi-Di formamide and 0.3 \u0026micro;L of GeneScan 600 LIZ size standard and analyzed using an Applied BioSystems 3500 Genetic Analyzer. Raw data were processed with GeneMapper 5.0 software (Applied Biosystems, Foster City, CA, USA).\u003c/p\u003e \u003cp\u003eNext, to determine the genetic origins of colony members, we reconstructed the genotypes of the primary queen and king of the colony. Nymph-derived queens are typically produced parthenogenetically by automixis with terminal fusion in primary queens (Matsuura et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), inheriting only the genes of the primary queen and resulting in homozygosity at all loci, except in cases where recombination occurs. To reconstruct the genotype of the primary queen, we first focused on nymph-derived queens that were homozygous at all microsatellite loci and listed all alleles present in these individuals. Next, using the genotypes of workers, we identified all alleles paired with those of the reconstructed primary queen, allowing us to determine the genotype of the primary king. Subsequently, we determined whether each individual was produced through outbreeding between the primary king and primary queen (i.e., carrying one allele from each parent at all loci) or through inbreeding within the colony (i.e., possessing at least one locus where only the primary king's or the primary queen's alleles were present). Nymph-derived queens produced via parthenogenesis were not classified as inbred individuals.\u003c/p\u003e\n\u003ch3\u003eProportion of field colonies without male and/or female nymphs (dup: abstract ?)\u003c/h3\u003e\n\u003cp\u003eThe proportion of colonies without nymphs (male and/or female) was examined in 15 colonies with a primary king out of the 702 colonies. After the extraction of all termites from the nests, the caste and sex were identified based on the presence of wing buds and sternite morphology, respectively. Then, presence of male and female nymph was recorded.\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe proportion of inbred individuals across different developmental stages within the colony with worker-derived kings was compared using Fisher\u0026rsquo;s exact test with Bonferroni correction. The sex ratios of workers (W4 and W5) and young individuals (N1 and W1) in the same colony were tested for deviation from the expected 1:1 ratio using exact binomial tests. The difference in the proportion of colonies lacking male versus female nymphs in field colonies was assessed using Fisher\u0026rsquo;s exact test. A significance level of \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was used for all statistical tests. For multiple comparisons, Bonferroni-adjusted \u003cem\u003ep\u003c/em\u003e-values were applied. All analyses were conducted using R version 4.2.3 (R Core Team \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003ePhenotypic composition of kings and queens in\u003c/b\u003e \u003cb\u003eR. speratus\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAmong the 702 colonies examined, one was found to contain only worker-derived kings, which served as the sole male reproductives (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). Regarding the phenotypes of kings, 93.4% (656 colonies) had only a primary king as the male reproductive. Colonies where the primary king had been replaced by nymph-derived kings accounted for 5.8% (41 colonies). In 0.6% (4 colonies), both a primary king and nymph-derived kings were present.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRegarding the phenotypes of queens (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), 2.0% (14 colonies) had only a primary queen, while 7.1% (50 colonies) contained both a primary queen and nymph-derived queens. The majority of colonies (90.5%, 635 colonies) had only nymph-derived queens. In 0.4% (3 colonies), worker-derived queens coexisted with nymph-derived queens.\u003c/p\u003e\n\u003ch3\u003eCaste composition and kin structure of the colony with worker-derived kings\u003c/h3\u003e\n\u003cp\u003eThe colony consisted of 10 worker-derived kings, 57 nymph-derived queens, 4,296 workers, 822 nymphs, 4 male and 6 female soldiers, 27 male and 28 female pre-soldiers, and 50 L2 larvae. Both sexes were present among fourth- and fifth-instar workers, with a male-biased sex ratio (exact binomial test, 95% CI\u0026thinsp;=\u0026thinsp;0.548\u0026ndash;0.743, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.004), but no male nymphs were found in the colony (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The caste fate of the larvae within the colony showed that all males differentiated into workers (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;55), while all females differentiated into nymphs (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;115). Their sex ratio was significantly skewed toward females (exact binomial test, 95% CI\u0026thinsp;=\u0026thinsp;0.254\u0026ndash;0.399, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMicrosatellite genotyping revealed the presence of individuals produced through inbreeding. Among the analyzed individuals, 4 out of 10 worker-derived kings, 3 out of 10 nymph-derived queens, 2 out of 20 workers, and 18 out of 20 young individuals (N1 and W1) were identified as inbred (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The proportion of inbred individuals was significantly higher in young individuals compared to other older developmental stages (Fisher\u0026rsquo;s exact test with Bonferroni correction, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eProportion of field colonies without male and/or female nymphs\u003c/h3\u003e\n\u003cp\u003eAmong the 15 colonies examined, 6 colonies (40.0%) lacked male nymphs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Female nymphs were absent in 2 colonies (13.3%), both of which also lacked male nymphs. The difference in the proportion of colonies lacking male versus female nymphs was not statistically significant (Fisher\u0026rsquo;s exact test, 95% CI\u0026thinsp;=\u0026thinsp;0.020\u0026ndash;1.766, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.215).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe presence of reproductive opportunities for workers contributes to the maintenance of their reproductive totipotency and may prevent the evolution of true superorganisms, which are characterized by a complete reproductive division of labor (Crespi and Yanega \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Boomsma \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2007\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; H\u0026ouml;lldobler and Wilson \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Gardner and Grafen \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Korb and Heinze \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Bernadou et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Prior evidence of worker-derived kings in \u003cem\u003eR. speratus\u003c/em\u003e had been limited to laboratory conditions (Shimizu \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1970\u003c/span\u003e; Miyata et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Matsuura et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), leaving open the question of whether such differentiation occurs in the field\u0026mdash;and, crucially, whether these individuals are capable of producing fertile offspring. In this study, we present the first field-based evidence that worker-derived kings can attain reproductive opportunities in \u003cem\u003eR. speratus\u003c/em\u003e. In the colony where worker-derived kings were discovered, no other male reproductive phenotypes were present, and their offspring were the result of mother\u0026ndash;son inbreeding. Furthermore, the caste fate of these offspring\u0026mdash;males developing into workers and most females into nymphs\u0026mdash;matched the pattern observed when the father is a worker-derived king and the mother is a nymph-derived queen (Hayashi et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Since sexually produced nymphs in this species develop into alates that go on to found new colonies (Matsuura et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Wu et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), our findings strongly suggest that worker-derived kings are capable of producing the next generation under natural conditions. These results indicate that worker-derived kings are not merely a developmental vestige, but a viable alternative reproductive strategy when opportunities arise.\u003c/p\u003e \u003cp\u003eThe absence of male nymphs appears to be a key factor enabling the reproduction of worker-derived male reproductives. In the colony where worker-derived kings were found, no male nymphs were present. Colonies entirely lacking male nymphs were not uncommon in the field; in our survey, 40% of colonies had no male nymphs. This suggests that the absence of male nymphs occasionally creates reproductive opportunities for worker-derived males. Because nymphs differentiate into reproductives more rapidly than workers (Miyata et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), worker reproductive opportunities arising from the absence of nymphs are likely to be extremely rare or entirely lost in species that form large colonies where nymphs are continuously present or have life histories characterized by early nymph production. This could explain the discrepancy in frequency between colonies lacking male nymphs and those in which worker-derived kings were actually found. Although further research considering colony age is needed, it is possible that the nymph-less colonies were relatively young and therefore had lower primary king mortality, which could account for the gap. Our results highlight that, in addition to previously recognized factors such as increasing colony size and reproductive longevity\u0026mdash;which reduce worker reproductive opportunities (Alexander et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1991\u003c/span\u003e; Bourke \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e1999\u003c/span\u003e)\u0026mdash;the timing of nymph production within a colony\u0026rsquo;s life history may also constrain worker reproduction in termite species where nymphs, rather than workers, serve as the main backup reproductive caste.\u003c/p\u003e \u003cp\u003eOur findings also offer new insights into the phenomenon that occur during the transition from a primary king to secondary neotenics. In the colony with worker-derived kings, most young individuals (N1s and W1s) were the result of inbreeding, but a few inbred individuals were also found among the older castes, including workers, queens, and even some of the worker-derived kings themselves. This pattern suggests that inbreeding had already begun while the primary king was still alive. This inference is supported by our phenotypic survey of field colonies, which identified multiple colonies containing both a primary king and nymph-derived kings. Although the presence of a primary king typically suppresses the differentiation of additional male reproductives (Shimizu \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1970\u003c/span\u003e; Miyata et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), our results imply that this suppression may weaken with age or declining fertility of the primary king. Consequently, there may be a window of reproductive opportunity for subordinate males even before the death of the reigning king.\u003c/p\u003e \u003cp\u003eThis study provides the first evidence that both male and female workers of \u003cem\u003eR. speratus\u003c/em\u003e can attain reproductive opportunities under natural conditions. These findings contribute to our understanding of how reproductive totipotency is maintained in the workers of eusocial insects. Research on the evolutionary loss of traits suggests that even functionally neutral traits may persist for millions of years before being eliminated, and traits that occasionally confer fitness benefits can be retained over long evolutionary timescales (Hall and Colegrave \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Lahti et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; van der Kooi and Schwander \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). This work highlights the importance of investigating reproductive totipotency and opportunities for reproduction in workers across diverse eusocial taxa. Such efforts will not only elucidate the timescales over which totipotency persists following the loss of reproductive opportunities, but also help identify the ecological and evolutionary factors that drive the transition toward permanently sterile castes and the evolution of true superorganisms.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Tadahide Fujita, Yao Wu, Takao Konishi, Shuya Nagai, Chihiro Tamaki, Shun Mizote, Hiroki Noda, Chen Jiaming, Kiyotaka Yabe, and Tomohiro Nakazono for assistance in collecting termites and helpful discussion.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONFLICT OF INTEREST STATEMENT\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003eORCID\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMamoru Takata\u003c/em\u003e https://orcid.org/0000-0002-8181-9987\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSoshi Araki\u003c/em\u003e https://orcid.org/0009-0005-3518-6911\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMichihiko Takahashi\u003c/em\u003e https://orcid.org/0009-0000-9572-4463\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eKenji Matsuura\u003c/em\u003e https://orcid.org/0000-0002-9099-6694\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e:\u0026nbsp;The authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e:\u0026nbsp;\u0026nbsp;This work was supported by JSPS KAKENHI Grant Numbers JP18H05268, JP20K20380, JP23H00332 to Kenji Matsuura, and JP21K14863 to Mamoru Takata.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e: Not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e: Not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e: Not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability:\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eData is provided within the manuscript or supplementary information files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode availability:\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eAll code used for graphing and statistical analyses in this study is included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003eMa.T. conceived the study, designed the methodology, performed validation and formal analysis, conducted the investigation, provided resources, wrote the original draft, reviewed and edited the manuscript, created visualizations, supervised the project, managed the project administration, and acquired funding. S.A. conducted formal analysis and investigation, provided resources, created visualizations, and reviewed and edited the manuscript. Mi.T. contributed to methodology development, validation, formal analysis, investigation, and resources, and reviewed and edited the manuscript. K.M. contributed to conceptualization, investigation, and resources, supervised the project, acquired funding, and reviewed and edited the manuscript. All authors reviewed and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAlexander RD, Noonan KM, Crespi BJ (1991) The Evolution of Eusociality. In: Sherman JU, Jarvis JUM, Alexander RD (eds) The Biology of the Naked Mole-Rat. 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Sociobiology 7:1\u0026ndash;7\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"worker reproduction, totipotency, ergatiod, termites, social insects","lastPublishedDoi":"10.21203/rs.3.rs-6510951/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6510951/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA defining feature of eusociality is the presence of a lifelong sterile caste, such as workers and soldiers, that forgo reproduction entirely. In many species, however, workers are not absolutely sterile but are instead functionally sterile\u0026mdash;they retain reproductive potential that can be expressed under certain conditions. Why this potential persists despite the evolutionary expectation of trait loss remains an open question. In this study, we present the first field-based evidence that worker-derived male reproductives, previously known only from laboratory observations, can successfully reproduce under natural conditions in the subterranean termite \u003cem\u003eReticulitermes speratus\u003c/em\u003e. A survey of 702 field colonies identified one colony containing only worker-derived kings. Microsatellite genotyping confirmed that these individuals reproduced via mother\u0026ndash;son inbreeding, and the caste fate of their offspring matched the expected pattern for matings between worker-derived kings and nymph-derived queens. Importantly, no male nymphs\u0026mdash;the usual precursors of male reproductives\u0026mdash;were present in the colony. Further investigation revealed that 40% of surveyed colonies (6 out of 15) entirely lacked male nymphs, indicating that such conditions may occur with non-negligible frequency in the wild. These findings suggest that worker-derived males gain rare but functional reproductive opportunities when typical male reproductives are absent, thereby maintaining their reproductive capacity over evolutionary timescales. This study provides important insight into the mechanisms that preserve reproductive totipotency in eusocial workers and underscores the value of examining reproductive roles in natural contexts to better understand the evolution of permanent sterility and true superorganismality.\u003c/p\u003e","manuscriptTitle":"Reproduction by worker-derived kings under natural conditions in the termite Reticulitermes speratus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-22 10:29:22","doi":"10.21203/rs.3.rs-6510951/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f625f423-6e17-4b8b-99a2-2938e66b3e3c","owner":[],"postedDate":"May 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-27T16:05:38+00:00","versionOfRecord":{"articleIdentity":"rs-6510951","link":"https://doi.org/10.1007/s00114-026-02105-3","journal":{"identity":"the-science-of-nature","isVorOnly":false,"title":"The Science of Nature"},"publishedOn":"2026-04-24 15:58:18","publishedOnDateReadable":"April 24th, 2026"},"versionCreatedAt":"2025-05-22 10:29:22","video":"","vorDoi":"10.1007/s00114-026-02105-3","vorDoiUrl":"https://doi.org/10.1007/s00114-026-02105-3","workflowStages":[]},"version":"v1","identity":"rs-6510951","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6510951","identity":"rs-6510951","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}