Amphisbaenians facultatively oviposit in ant and termite nests

Amphisbaenians facultatively oviposit in ant and termite nests | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Amphisbaenians facultatively oviposit in ant and termite nests Henrique Bartolomeu Braz, Lívia Cristina Santos, Selma Maria Almeida-Santos This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8864491/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Oviposition-site selection is a key maternal decision in oviparous animals, involving trade-offs among incubation conditions, offspring performance, and maternal constraints. In amphisbaenians (worm lizards), a clade of highly specialized fossorial reptiles, oviposition has long been assumed to occur obligatorily in ant and termite nests, implying strong ecological specialization. Here, we re-evaluate this assumption using new field observations and a critical synthesis of published records. We describe a natural oviposition site of the smallhead worm lizard ( Leposternon microcephalum ) located in a soil cavity with no evidence of ants or termites and review 18 records from nine amphisbaenian species. Across species, eggs occur not only in ant and termite nests but also in subterranean cavities and decaying logs, indicating facultative rather than exclusive use of social-insect nests. Field inspections of 31 ant nests during the oviposition season yielded no eggs or amphisbaenians, further supporting non-obligate use. Additional evidence suggests some species excavate or modify underground chambers and that at least one species oviposits communally. These findings challenge the view of strict dependence on ant and termite nests and instead support oviposition-site choice in amphisbaenians as a flexible, context-dependent maternal behavior shaped by ecological trade-offs rather than rigid specialization. Standardized surveys across microhabitats and experimental tests of incubation environments are needed to clarify how availability, costs, and benefits interact to shape nesting decisions in fossorial reptiles. oviposition-site choice maternal decision-making behavioral plasticity fossorial reptiles Figures Figure 1 SIGNIFICANCE STATEMENT How animals choose where to lay their eggs can strongly influence offspring survival, yet this decision remains poorly understood in secretive, underground species. Worm lizards (amphisbaenians) have long been thought to depend on ant and termite nests for egg-laying, implying rigid ecological specialization. By combining new field observations with a critical reassessment of published records, this study shows that such behavioral decision is not obligatory. Instead, amphisbaenians use social-insect nests as part of a broader and flexible set of nesting options. Our findings highlight oviposition-site choice in these reptiles as a context-dependent maternal decision shaped by trade-offs among environmental conditions, risks, and constraints, rather than fixed dependence on a single nesting strategy. This work reframes amphisbaenian reproduction within a behavioral decision-making framework and underscores the importance of considering plasticity and detectability biases when interpreting nesting ecology in fossorial animals. INTRODUCTION “The reason for this association has not yet been determined. Brazilians hold the pious belief that ants take pity on the blind lizard and welcome it into their nests, even bringing it food!” ( Tschudi 1866: 159 ) For oviparous animals in which embryos develop outside the maternal body, oviposition-site choice is a major determinant of reproductive success. In many of these taxa, parental care is limited or absent, so oviposition-site choice becomes a major maternal effect through which mothers influence offspring survival and phenotype (Resetarits 1996 ; Refsnider and Janzen 2010 ). The physical and biological attributes of the chosen site influence embryonic development and hatching success by modulating temperature, humidity, gas exchange, microbial activity, and predation risk (Refsnider 2016 ; Buxton and Sperry 2017 ; Schütz and Füreder 2019 ; Fowler et al. 2025 ). The relevance of these factors, however, varies widely among taxa and depends on species-specific life-history traits, creating trade-offs among maternal survival, incubation conditions, and offspring performance (Refsnider and Janzen 2010 ). Thus, oviposition-site selection is best viewed as a maternal reproductive decision with direct fitness consequences. In reptiles, temperature and moisture at the oviposition site can influence embryonic growth, hatching success, and a range of offspring traits, sometimes with long-term effects. Site structure and local biota also affect egg survival by altering predation risk, microbial colonization, and exposure to flooding or desiccation (Refsnider 2016 ; Abayarathna and Webb 2022 ). Together, these pressures have produced a wide diversity of oviposition strategies that differ in the degree of site preparation, from the use of unmodified, pre-existing shelters (e.g., rock crevices, decaying logs, or colonies of social insects) to actively excavated burrows or constructed nesting structures (Packard and Packard 1988 ; Refsnider 2016 ; Murray et al. 2020 ). Variation also occurs in how sites are used. Females may oviposit alone (solitary oviposition) or share sites with other females (communal oviposition). Communal oviposition is often associated with limited availability of suitable sites or with shared benefits such as stable microclimates or reduced predation risk (Graves and Duvall 1995 ; Doody et al. 2009 ). Despite the relevance of these maternal decisions, current knowledge of reptile nesting ecology and behavior is strongly biased toward large, surface-active taxa (Doody et al. 2021 ; Doody and Refsnider 2022 ), leaving fossorial, limbless, and secretive lineages underrepresented in comparative studies. This bias is especially pronounced in amphisbaenians (worm lizards), a monophyletic and highly specialized group of ~ 200 species of fossorial, mostly limbless reptiles (except for the forelimbed Bipes ), distributed across tropical and subtropical regions of the Americas, Africa, and parts of the Mediterranean and Middle East (Pough et al. 2003 ; Uetz et al. 2025 ). Their subterranean lifestyle severely limits direct field observations; consequently, fundamental aspects of their nesting ecology and nesting-related behavior are poorly documented, hindering the identification of general patterns (Andrade et al. 2006 ). This lack of information can also generate detection bias. Conspicuous microhabitats are more likely to be searched and reported, which can inflate the apparent importance of particular oviposition sites. The same limitation can bias inferences about how sites are used. For instance, communal oviposition has not been documented in amphisbaenians; however, given how widespread this behavior is among other squamates (Graves and Duvall 1995 ; Doody et al. 2009 ), its apparent absence in amphisbaenians may reflect limited observations rather than a true lack of communal nesting. From a functional perspective, it has been assumed that limblessness constrains nest construction, such that limbless reptiles, including amphisbaenians, rely mainly on pre-existing structures (e.g., decaying logs, rock crevices, insect colonies) for oviposition rather than constructing nests (Packard and Packard 1988 ; Doody et al. 2009 ; but see Burger and Zappalorti 1991 ; Koirala and Tshering 2021 for exceptions). Riley et al. ( 1985 ) reviewed records of squamate oviposition in ant and termite nests and noted that eggs of amphisbaenians ( Amphisbaena alba , A. kingii , and Leposternon microcephalum ) had been reported exclusively from ant nests. They interpreted this pattern as consistent with a possible obligate association (i.e., exclusive or near-exclusive use) with ant nests for oviposition. However, for amphisbaenians, this inference may not reflect a true functional constraint. As specialized burrowers, amphisbaenians possess morphological and behavioral traits that could allow them to excavate or modify oviposition chambers in the soil, rather than relying solely on pre-existing shelters. This "obligate ant-nest" hypothesis, although cited by later authors (e.g., Azevedo-Ramos and Moutinho 1994 ; Vega 2001 ; Andrade et al. 2006 ), has never been explicitly assessed, largely because natural oviposition sites are rarely documented for this group. Given the many records of amphisbaenians occurring in termite nests (Broadley et al. 1976 ; Vega 2001 ; Pramuk and Alamillo 2003 ; Moreira et al. 2009 ; Duleba and Ferreira 2014 ), and because various other limbless squamates also oviposit in such sites (Riley et al. 1985 ), this idea can reasonably be extended to termite nests as well (FitzSimons 1943 ; Bons and Saint Girons 1963 ). Proposed benefits of ovipositing in ant and termite nests include stable microclimates, protection from predators or microbes, and immediate access to food resources (Hagmann 1907 ; Kopstein 1928 ; Vaz-Ferreira et al. 1970 ; Riley et al. 1985 ; Hood et al. 2020 ). If amphisbaenians truly depend on ant and termite nests as exclusive or near-exclusive oviposition sites, this would imply a high degree of ecological and behavioral specialization, potentially involving coevolutionary mechanisms such as insect tolerance toward eggs or traits that facilitate persistence within active colonies (Baer et al. 2009 ; Sierra-Serrano et al. 2023 ). Testing whether such dependence exists is therefore necessary to distinguish specialization from flexibility in a key reproductive behavior. Here, we evaluate the hypothesis that amphisbaenians obligatorily oviposit in ant and termite nests. First, we describe the rare finding of a natural oviposition site of L. microcephalum —a species previously suggested to oviposit exclusively in ant nests (Riley et al. 1985 )—found in a soil cavity with no evidence of ants or termites. We then compile and critically review published records of amphisbaenian oviposition sites to assess whether ant and termite nests are obligatory for oviposition and whether these sites are used solitarily or communally. If oviposition in ant and termite nests were obligatory, eggs should be restricted to these sites; in contrast, facultative use predicts oviposition across diverse microhabitats. Finally, we discuss the potential costs and benefits that may drive the use of ant and termite nests in amphisbaenians. MATERIAL AND METHODS Original data On 26 February 2011, three eggs were found in a private garden in Praia Grande municipality, São Paulo state, southeastern Brazil (24° 00' 21.6" S, 46° 24' 10.8" W; elevation: 11 m) during routine maintenance. The eggs were collected, placed in a plastic bag with soil from the same site, and transported to the laboratory the following day. Two eggs hatched during transportation. The remaining egg was measured with a digital caliper (to the nearest 0.01 mm), weighed (to the nearest 0.01 g), returned to the bag, and kept at room temperature. Hatchlings were measured (to the nearest 1 mm), weighed (to the nearest 0.01 g), photographed, and deposited in the Reference Collection of the Butantan Institute (IBSP.CRIB) and Museu de Zoologia da Universidade de São Paulo (MZUSP). On 15 December 2007, we manually opened 30 leaf-cutting ant ( Acromyrmex ) nests in a Hevea brasiliensis plantation and one Atta nest in adjacent pasture areas at Fazenda Novo Mundo (22° 19′ 45″ S, 49° 45′ 23″ W), Vera Cruz municipality, São Paulo state, southeastern Brazil. Colonies of Acromyrmex are the ant nests most frequently reported as oviposition sites for squamates (Sacerdote-Velat and Sekits 2023 ). For each nest, we opened chambers as far as feasible and visually searched for eggs or amphisbaenians within the nest structure and adjacent soil. The surveyed area harbors multiple amphisbaenian species (Mott and Vieites 2009 ; Santos 2013 ), and December coincides with the oviposition period reported for amphisbaenians in southeastern Brazil (Santos 2013 ; see also references in Table 1 ). Table 1 Summary of available records on natural oviposition sites of Amphisbaenia. The column 'Information originally provided' reproduces the original account as closely as possible, while the columns 'Nest site' and 'Nest use' represent our standardized interpretation. NA = not available Species Location Information originally provided Nest site Nest use Reference Unidentified amphisbaenid a Rio de Janeiro, Brazil Eggs are laid within fungus chamber of old nests of leaf-cutting (" tanajura ") ants. Ant nest (fungus chamber, Formicidae, Myrmicinae, Attini) NA Tschudi, 1867 ; Brandão and Vanzolini, 1985 Amphisbaena caeca Puerto Rico One egg found beneath a termite nest. Below termite nest Solitary Schmidt, 1920 Amphisbaena caeca Puerto Rico Two eggs and one adult specimen found ~ 7.5 cm under a log beneath an ant's nest on 22 August 1919. One egg (42 × 11 mm) contained a hatchling-sized specimen (86 mm total length) and residual yolk mass (AMNH 13237). Under log Solitary Schmidt, 1920 ; Gans and Alexander, 1962 Amphisbaena darwinii Uruguay Eggs found in layers of humus. Underground (humus layer) NA Vaz-Ferreira et al., 1970 Amphisbaena darwinii Uruguay A clutch of four eggs (mean egg size = 24.7 × 13.0 mm) found within an unoccupied anthill (species unidentified) in a grassland habitat on 30 December 2002. Three eggs hatched on 25–26 January 2003 (hatchling total length = 68, 74, and 85 mm in; ZVC-R 6080). Ant mound Solitary Carreira and Baletta, 2006 Amphisbaena darwinii Argentina A female and a clutch (egg length ~ 20 mm) of two eggs containing near-term embryos were found in a small cavity in the soil dug from a garden in February 1895. Underground cavity Solitary Berg, 1898 Amphisbaena darwinii Argentina A female and a clutch of three eggs (egg length ~ 20 mm) containing near-term embryos were found in a small cavity in the soil dug from a garden in February 1896. Underground cavity Solitary Berg, 1898 Amphisbaena darwinii Argentina Eggs (clutches ranging 6–8 and 17–21) found under logs of an old Eucalyptus crop partially buried in loose soil, which contained two or more species of termites and ants. Eggs and embryonic series housed at UNNEC (see Montero et al., 1999 ). Under log in loose soil (termite and ant presence) Solitary and communal b Montero et al., 1999 ; J. A. Céspedez, pers. comm. Amphisbaena fuliginosa c Brazil A clutch of nine eggs (one egg measuring 28.22 × 14.74 mm) was found on 2 October 2016, partially buried beneath a fallen tree trunk. Eggs hatched on 19–20 October (hatchling size = 122–135 mm total length). An adult female A. fuliginosa was also found in the nest site. Under log Solitary Oliveira and Gomes, 2017 Amphisbaena kingii Argentina One egg (29 × 11 mm) containing a fully developed embryo (75 mm total length) was found in early March 1988 at the base of the fungus chamber of a nest of Acromyrmex silvestrii (MLP S.1097). Ant nest (fungus chamber, Myrmicinae, Attini) Solitary Williams and Wichmann, 1989 Amphisbaena kingii Brazil Several (≥ 2) eggs found in ant nests. Two of these eggs were examined by Gans and Rhodes ( 1964 ); the eggs (30 × 10 and 35 × 10 mm) contained fully developed embryos (113 mm total length; NHMUK 1885.2.3.6-7). Ant nest Probably solitary Boulenger, 1885 ; Gans and Rhodes, 1964 Amphisbaena mertensii Paraguay A gravid female (301 mm SVL; MNHNP 8742) with stomach contents (Coleoptera, Elateridae) was found buried in the softer soil peripheral to a termite ( Cornitermes cumulans ) mound; no eggs recorded. Termite nest (peripheral soil) NA Pramuk and Alamillo, 2003 Leposternon infraorbitale Brazil A clutch of six eggs (egg size = 60 × 25 mm) found on 20 February 1994 in the ground beside a fallen tree in a cacao plantation, 15 cm from the surface and covered in soil and decomposed wood. Hatchlings 150–170 mm total length. Under decomposing wood Solitary Jared et al., 1997 Leposternon microcephalum São Paulo, Brazil A clutch of three eggs (one egg measuring 45.26 × 15.31 mm) found in a cavity 18 cm below the soil surface in a small garden located inside a private residence on 26 February 2011. Two eggs hatched the next day. Hatchlings measured 126, 136, and 142 mm in total length (IBSP.CRIB 295 and 296; MZUSP 103179). Underground Solitary Present study Leposternon microcephalum d Brazil Eggs (unreported number) were taken in March 1893 from a pile of bricks and fragments of roof tiles inhabited by a reasonable size colony of ants ( Camponotus ). Two sets of eggs were found: one with less-developed embryos, another with embryos close to hatching. Two eggs of the older clutch measured 53.5 × 20 and 54.5 × 18 mm. Two other eggs (52 × 12 and 45 × 14 mm) are vouchered as NHMUK 1893.9.30.2–3. Ant nest (Formicinae, Camponotini) Possibly communal Goeldi, 1898 Leposternon microcephalum Brazil A clutch of two eggs was found in an oval (8.0 × 3.5 × 4.5 cm) smooth-walled cavity 23–25 cm below the surface of a truck garden. The soil was sandy-loamy interspersed with hummus. Underground cavity Solitary Engmann, 1927 Leposternon microcephalum e Brazil Two eggs (ZMH 3571) containing partially developed embryos (39 mm total length) collected in an anthill on 17 December 1907. Ant mound Solitary Gans, 1971 Leposternon microcephalum e Brazil Eight eggs (ZMH 3572) containing early embryos collected in an anthill in the spring of 1908. Ant mound Possibly communal (due to the clutch size) Gans, 1971 Rhineura floridana Florida, United States A clutch of two eggs (one egg measuring 38.0 × 8.9 mm) containing fully formed young (93.5 and 112.2 mm in total length) was found after turning up in a spadeful of sandy loam taken from a depth of 20–50 cm. Underground (sandy soil) Solitary Carr, 1949 a Tschudi ( 1867 ) reports A. flavescens and A. fuliginosa inhabiting “tanajura” nests and laying eggs there; however, the original text is ambiguous as to which species (or whether both) oviposit in such sites. Amphisbaena flavescens is now recognized as A. alba , whereas A. fuliginosa does not occur in the region. Moreover, five other amphisbaenian species are currently known from Rio de Janeiro state Guedes et al. ( 2023 ). Given this uncertainty, we list the record as "unidentified amphisbaenid". b Given that A. darwinii typically lays 2–4 eggs (Gallardo 1967 ; Carreira and Baletta 2006 ), these aggregations likely represent oviposition by multiple females (~ 2–11). c Originally published as A. brasiliana but identified as A. fuliginosa by Abecassis et al. ( 2026 ). d Gans ( 1971 ) comments that "it is uncertain that the eggs belong to species microcephalum ". e Identified as L. microcephalum by Gans ( 1971 ). Literature survey To assess the hypothesis of obligatory oviposition in ant or termite nests in amphisbaenians, we searched the literature for records of natural oviposition sites. We searched Google Scholar using combinations of "Amphisbaenia", "amphisbaenian", "worm lizard", genus names, and keywords such as "nest", "oviposition", "egg-laying", and "egg", in Portuguese, Spanish, and English. We extracted descriptions of oviposition microhabitats and classified nesting as solitary or communal when possible. To distinguish between solitary and communal nesting, we used multiple criteria, including developmental stages of embryos within nests, the time interval between hatching events, and whether the number of eggs at a site exceeded the mean clutch size reported for the species (Braz et al. 2008 ). We also compiled clutch size, egg size, offspring size, and incubation duration when available. RESULTS Original data The clutch of three eggs was found in a cavity 18 cm below the soil surface in a small garden (0.9 × 1.6 m) containing ornamental cacti. The garden was located within a private residence and received direct sunlight through a glass brick wall. The garden was searched for additional eggs and for nearby ant or termite nests but none were found. The eggs had whitish, leathery shells (Fig. 1 a). Two eggs hatched within the plastic bag on 27 February 2011, during transport to the laboratory. One hatchling (IBSP.CRIB 295) measured 120 mm snout-vent length (SVL), had a 6 mm tail, and weighed 2.73 g. The other (IBSP.CRIB 296) measured 128 mm SVL, had an 8 mm tail, and weighed 3.52 g. Both hatchlings left substantial unabsorbed residual yolk (1.47 and 1.05 g, respectively) within the eggshells. The remaining egg (45.26 × 15.31 mm, 5.08 g; Fig. 1 a) did not hatch. Dissection revealed a full-term dead embryo (133 mm SVL, 9 mm tail length; MZUSP 103179) with residual yolk. All individuals were identified as L. microcephalum (Fig. 1 b). All 31 excavated ant nests were active, but we found no amphisbaenians and no eggs in any of them. However, we found a blindsnake ( Leptotyphlops sp.) inside an Acromyrmex nest. Literature data Our literature survey identified 18 records of confirmed natural oviposition sites for nine amphisbaenian species from three genera ( Amphisbaena , Leposternon , and Rhineura ) across South, Central, and North America (Table 1 ). One additional report described a gravid A. mertensii inside a termite nest, suggesting—but not confirming—a potential oviposition site (Table 1 ). We treated this record separately from confirmed oviposition sites. Among confirmed records (n = 18), oviposition sites were most often in ant nests (n = 7, 38.9%) and underground cavities (n = 6, 33.3%), followed by sites beneath decaying logs or decomposing wood (n = 4, 22.2%) and termite nests (n = 1, 5.6%). For most species, records are limited to one or two observations. Multiple records were available only for L. microcephalum (n = 5) and A. darwinii (at least 6, as the original sources do not provide an exact count of distinct oviposition records; Table 1 ). In L. microcephalum , oviposition sites were in ant nests (3/5, 60%) and in underground cavities (2/5, 40%). In A. darwinii , half of the records involved underground sites (humus or soil cavities, 3/6), with additional records from beneath a log (at least 2/6) and an ant nest (1/6). Most records indicated solitary nesting (Table 1 ). The only unequivocal cases of communal nesting involved A. darwinii in Argentina, where groups of 6–8 to 17–21 eggs were occasionally found under the same log. Because A. darwinii typically lays 2–4 eggs (Gallardo 1967 ; Carreira and Baletta 2006 ), we interpret this records as oviposition by multiple females (~ 2–11). However, solitary oviposition also occurred in this species (Table 1 ). Two records for L. microcephalum were ambiguous regarding oviposition site use. Goeldi ( 1898 ) described two sets of eggs differing in shapes and developmental stages from a pile of bricks and tiles inhabited by a large Camponotus ant colony, which is consistent with more than one clutch. Similarly, Gans ( 1971 ) reported two clutches (eight and two eggs) of different sizes and developmental stages from the same locality, but we could not determine whether they originated from a single oviposition site. DISCUSSION Facultative use of ant and termite nests for egg-laying Our original observations and literature synthesis do not support the hypothesis that amphisbaenians obligatorily use ant or termite nests for oviposition. Instead, the available evidence indicates that these structures are one option among a broader repertoire of sites. In the two species with multiple records ( L. microcephalum and A. darwinii ), eggs have been found in both ant or termite nests and alternative shelters. This pattern is consistent, though less documented, in other species. This distribution supports facultative rather than exclusive use of ant and termite nests, with females exploiting various shelters that meet incubation requirements. Importantly, the available evidence addresses dependence (i.e., obligatoriness) rather than preference, as the occurrence of eggs outside ant and termite nests indicates these colonies are not required for oviposition. Consistent with this interpretation, we found no eggs or amphisbaenians in 31 excavated ant nests surveyed during the reported oviposition period (Santos 2013 ; see also references in Table 1 ) at a site with documented amphisbaenian occurrences (Mott and Vieites 2009 ; Santos 2013 ). Although these negative results should be interpreted cautiously given detectability constraints, they further support non-exclusivity. While obligate use can be rejected, whether amphisbaenians preferentially select ant and termite nests under certain ecological contexts remains unresolved. The apparent prevalence of these nests in the literature may partly reflect sampling bias, as they are conspicuous, long-lasting, and often searched more intensively than other potential oviposition microhabitats (e.g., soil cavities, burrows, or decaying root systems). Many squamate eggs found in ant and termite nests were discovered incidentally by entomologists during surveys focused on social insects (e.g., Vaz-Ferreira et al. 1970 , 1973 ; Brandão and Vanzolini 1985 ; Bruner et al. 2012 ; Kwapich 2021 ), with dozens to hundreds of nests opened in several cases (e.g., Vaz-Ferreira et al. 1970 , 1973 ; Pramuk and Alamillo 2003 ). This asymmetry in search effort can inflate the perceived frequency of oviposition in ant and termite nests, even when such sites are used facultatively. Similar patterns of non-exclusive use have been reported for many lizards and snakes (reviewed in Riley et al. 1985 ; see also Vitt et al. 1997 ; Khannoon and Evans 2014 ), suggesting that facultative oviposition in ant and termite nests is a recurrent strategy among squamates. Resolving whether these nests are used disproportionately relative to their availability will require standardized surveys across multiple potential nesting microhabitats. Functional hypotheses and trade-offs underlying oviposition in ant and termite nests Although ant and termite nests are not obligatory oviposition sites for amphisbaenians, they are ecologically relevant options whose use has been recurrently explained by several functional hypotheses. These hypotheses propose that ovipositing within these nests may confer advantages such as buffered microclimates, protection against predators or microbes, and immediate access to food for hatchlings (Hagmann 1907 ; Kopstein 1928 ; Vaz-Ferreira et al. 1970 ; Riley et al. 1985 ; Hood et al. 2020 ). However, because these sites are used facultatively, any advantages are expected to be context-dependent and offset by costs, including aggression from resident insects, attraction of predators, constraints on hatchling emergence, and variable antimicrobial environments (Vaz-Ferreira et al. 1970 ; Oliveira and Della Lucia 1993 ). Below, we evaluate these benefits and costs in the context of amphisbaenians, emphasizing how trade-offs among them may shape flexible maternal decisions on oviposition-site choice. Microclimatic stability A frequently proposed explanation for oviposition in ant and termite nests is that they provide buffered and predictable microclimatic conditions that enhance egg survival and offspring performance. These nests can maintain higher and more stable temperature and humidity than adjacent substrates (Vaz-Ferreira et al. 1970 ; Korb and Linsenmair 2000 ; Bollazzi and Roces 2002 ), conditions often assumed to be favorable for squamate eggs (Kopstein 1928 ; Vaz-Ferreira et al. 1970 ; Riley et al. 1985 ; Herrera and Robinson 2000 ; Velásquez–Múnera et al. 2008 ; Baer et al. 2009 ; Rodríguez and Montoya-Lerma 2015 ; Williams et al. 2022 ; Sierra-Serrano et al. 2023 ). This hypothesis is plausible, given the well-established effects of incubation temperature and moisture on offspring traits (While et al. 2018 ; Bell et al. 2025 ). However, Warmer or more stable conditions do not always improve developmental outcomes, and both the direction and magnitude of effects vary among species. In some cases, less variable incubation temperatures produce hatchlings with phenotypes associated with higher survival than more variable regimes (e.g., Patterson and Blouin-Demers 2008 ), whereas in others, differences are minimal or absent (Ji et al. 2003 ; Hao et al. 2006 ; Li et al. 2013 ). Unfortunately, no study has directly tested whether amphisbaenian eggs gain fitness-relevant advantages under the microclimatic conditions typical of ant or termite nests, and therefore, the adaptive value of ovipositing in these environments remains uncertain for amphisbaenians. Controlled comparisons between nest-like conditions and alternative shelters are therefore needed to assess whether microclimatic stability favors repeated, but not exclusive, use of these sites. Protection against predators and microbial attack Another commonly invoked hypothesis is that oviposition within ant and termite nests reduces egg mortality by providing protection against predators and microbial infection. This protection is attributed to both nest architecture and the defensive and hygienic behaviors of resident insects, which may deter egg predators or suppress pathogen growth (Kopstein 1928 ; Vaz-Ferreira et al. 1970 ; Riley et al. 1985 ; Velásquez–Múnera et al. 2008 ). Although such mechanisms are plausible, empirical support for this hypothesis is inconsistent. Ants have been suggested to clean reptile eggs or embedding them in fungus gardens where symbiotic fungi may suppress pathogens (Velásquez–Múnera et al. 2008 ; Baer et al. 2009 ); however, high egg mortality has also been documented within social-insect nests. For example, two-thirds of the tegu lizard ( Tupinambis teguixin ) eggs found in a termite nest were rotten (Herrera and Robinson 2000 ), and mortality has been reported in in termite nests for squamates, including amphisbaenians (Beebe 1944 ; Pramuk and Alamillo 2003 ). Moreover, resident insects can themselves attack and harm hatchlings and adults (Vaz-Ferreira et al. 1970 ; Riley et al. 1986 ; Oliveira and Della Lucia 1993 ). In addition, ant and termite nests often attract vertebrate predators (e.g., tegu lizards: Beebe 1945 ; Avila-Pires 1995 ; snakes: Vaz-Ferreira et al. 1970 , 1973 ; Duleba and Ferreira 2014 ) that prey on eggs or fossorial squamates (Braz et al. 2014 ; Kasperoviczus et al. 2015 ; Rodríguez et al. 2018 ). Therefore, these observations indicate that any protective role of social-insect nests is context-dependent and cannot be assumed a priori. Testing this hypothesis will require direct comparisons of egg survival and infection rates inside and outside active nests, ideally distinguishing effects of insect behavior or nest architecture from those of underground nesting more generally. Immediate access to food resources A further hypothesis is that oviposition within ant or termite nests benefits hatchlings by providing immediate access to abundant food resources (Hagmann 1907 ; Hegh 1922 ; Hood et al. 2020 ). This idea is intuitively appealing for insectivorous reptiles, particularly in subterranean environments where prey availability outside nests may be limited. Its relevance, however, depends on whether hatchlings rely on exogenous food shortly after hatching. Squamates show substantial variation in post-hatching feeding strategies. Some hatchling lizards begin feeding within the first 48 hours and derive little energy from residual yolk (Radder et al. 2007 ; Guo et al. 2023 ), thus rendering immediate food access of limited functional significance. In contrast, other reptiles delay feeding for extended periods and rely largely on residual yolk during early life (Burger 1989 ; Morafka et al. 2000 ; Rowe et al. 2017 ). For amphisbaenians, direct evidence is lacking. Our observations of substantial residual yolk in newly hatched L. microcephalum are consistent with limited early dependence on external food sources. Additionally, postnatal residence within ant nest chambers is suggested to be generally brief in squamates (Vaz-Ferreira et al. 1970 ), limiting opportunities for sustained post-hatching foraging within colonies. These considerations suggest that immediate access to prey is unlikely to be a primary factor influencing oviposition-site choice in amphisbaenians. Targeted data on neonatal feeding behavior and dependence on external food sources are needed to evaluate this hypothesis. Can amphisbaenians build their own nests? It has been assumed that limblessness constrains nest construction in squamates, forcing limbless species to rely primarily on pre-existing structures for oviposition (Packard and Packard 1988 ; Doody et al. 2009 ). However, its relevance to specialized burrowing taxa, such as amphisbaenians, remains uncertain. Our record of L. microcephalum and historical reports for A. darwinii (Berg 1898 ) document oviposition in subterranean cavities without evidence of ants or termites but do not clarify whether these chambers were excavated by the female or were pre-existing. A more informative account describes at least one clutch of L. microcephalum within a smooth, nearly oval underground chamber connected to a distinct access tunnel (Engmann 1927 ). Although active excavation was deemed possible, the author favored the interpretation that the female modified an existing crevice rather than digging the chamber entirely (Engmann 1927 ). Subsequent studies of L. microcephalum burrowing behavior suggest that both scenarios are mechanically feasible. The species uses its reinforced, shovel-like head and muscular body to penetrate compact soils and maintain tunnel integrity (Gans 1969 ; Barros-Filho et al. 2008 ; Hohl et al. 2014 ), which indicates that it possesses the functional capacity to excavate or substantially modify subterranean spaces, including potential oviposition chambers. These observations challenge the assumption that limblessness precludes nest construction and suggest that some amphisbaenians may actively excavate or modify oviposition chambers, expanding the range of nesting strategies available to fossorial squamates. Direct behavioral observations under controlled conditions will be necessary to investigate nest-site preparation behavior, chamber architecture, and soil manipulation during oviposition. Communal oviposition in amphisbaenians Previous reviews found no evidence of communal oviposition in amphisbaenians (Graves and Duvall 1995 ; Doody et al. 2009 ). However, our reexamination of published records indicates that communal oviposition does occur in some South American species, although it appears to be uncommon. Solitary oviposition predominates, yet multiple clutches of A. darwinii have been found together in the same microhabitat (Table 1 ). Communal oviposition in reptiles may arise from social tolerance among females or from aggregation driven by shared responses to limited or favorable nesting sites (Graves and Duvall 1995 ; Doody et al. 2009 ). In A. darwinii , this interpretation is supported by field observations of adults and juveniles sharing refuges, sometimes in close physical contact (Gallardo 1967 ; Borteiro et al. 2013 ). Evidence for communal oviposition in L. microcephalum is more equivocal but compatible with occasional aggregation. Therefore, available evidence suggests that communal oviposition occurs in some amphisbaenian species but is neither frequent nor obligate. The coexistence of solitary and communal nesting within amphisbaenians aligns with flexible, context-dependent oviposition strategies. From a behavioral-ecological perspective, communal nesting in amphisbaenians generates testable hypotheses concerning site limitation, microclimatic advantages, and social tolerance, reinforcing the view that oviposition-site choice in this group reflects adaptive plasticity rather than rigid specialization. Conclusions Our original observations and literature synthesis do not support the hypothesis that amphisbaenians obligatorily oviposit in ant or termite nests. The available evidence indicates that these structures are used facultatively among several oviposition-site options that meet their incubation requirements. Although ant and termite nests may offer advantages such as microclimatic buffering or protection, these benefits are likely context-dependent and can be offset by risks including predation, microbial failure, or insect aggression, favoring flexible rather than exclusive use. Our synthesis also highlights two additional aspects of amphisbaenian reproductive behavior that merit further attention. First, evidence from A. darwinii and more strongly from L. microcephalum suggests that some amphisbaenians may excavate or modify subterranean chambers for oviposition, challenging the assumption that limbless reptiles rely primarily on pre-existing shelters. Second, records of communal nesting in A. darwinii , and possibly in L. microcephalum , expand the known behavioral repertoire of amphisbaenians and further support the view that oviposition strategies in this group are variable and context-dependent. Taken together, these findings indicate that oviposition-site choice in amphisbaenians reflects adaptive plasticity rather than strict specialization or dependence on social insects. Future studies integrating controlled incubation experiments, field surveys across multiple microhabitats, and behavioral observations under natural or semi-natural conditions are necessary to clarify the ecological drivers and adaptive significance of nesting strategies in this group. In particular, combining measures of microhabitat availability with comparable search effort would be especially useful to disentangle preference, availability, and detectability in this system. Declarations Animal ethics Not applicable Competing interests The authors declare no competing interests. FUNDING Lívia Cristina Santos and Selma Maria Almeida-Santos received funding from Fundação de mparo à Pesquisa do Estado de São Paulo; Grant ID 06/58529-3 and 08/58424-2. Author Contribution H.B.B., L.C.S., and S.M.A.S. conceptualized the manuscript.H.B.B. and L.C.S. collected and analyzed the data.H.B.B. wrote the main manuscript text with support from L.C.S. and S.M.A.S.L.C.S. prepared the figures.All authors revised the manuscript. Acknowledgement We thank Cristian Gomes for providing detailed information on the oviposition site described here, Jorge Céspedez, Ricardo Montero, Andressa Oliveira, and Felipe Gomes for providing additional information on nest sites, Luís Roberto Takitane and Tamí Mott for allowing access to their property and for assistance with excavating ant nests. 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Fundación de Historia Natural Félix de Azara, Buenos Aires Williams JD, Wichmann SI (1989) Nueva localidad para Anops kingii Bell (Reptilia: Amphisbaenidae) y Philodryas aestivus (Dumeril, Bibron & Dumeril) (Reptilia: Colubridae) en el este de la provincia de Buenos Aireas. Boletín de la Asociación Herpetológica Argentina 5:12–13 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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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-8864491","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":600670235,"identity":"374ef341-0846-472c-94c1-65d8c5257b8a","order_by":0,"name":"Henrique Bartolomeu Braz","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAv0lEQVRIiWNgGAWjYBACAwYeEGWTAOYlFBCvJS2BgQ2kxYB4LYchWhiI0WLOwHvsw48/5/P45bsTPzwwYJDnFzuAX4tlA1/yzB6e28WSbbybJYAOM5w5O4GAww7wGDPwSNxO3HCMdwNIS4LBbSK0MP4xOAfSsvkH0VqYeRIOgLRsI9KWw3zJzDIHkhNntuVus0gwkCDCL8d7DzO++WOX2M98dvPNHxU28vzSBLQwMKNyJQgoHwWjYBSMglFAFAAAngk/YFFuHKMAAAAASUVORK5CYII=","orcid":"","institution":"Instituto Butantan","correspondingAuthor":true,"prefix":"","firstName":"Henrique","middleName":"Bartolomeu","lastName":"Braz","suffix":""},{"id":600670236,"identity":"c945bb21-483d-420e-adbc-453e668cff29","order_by":1,"name":"Lívia Cristina Santos","email":"","orcid":"","institution":"Instituto Federal de São Paulo","correspondingAuthor":false,"prefix":"","firstName":"Lívia","middleName":"Cristina","lastName":"Santos","suffix":""},{"id":600670237,"identity":"5b42ebca-fc3e-4791-8a09-7eda64b4cba0","order_by":2,"name":"Selma Maria Almeida-Santos","email":"","orcid":"","institution":"Instituto Butantan","correspondingAuthor":false,"prefix":"","firstName":"Selma","middleName":"Maria","lastName":"Almeida-Santos","suffix":""}],"badges":[],"createdAt":"2026-02-12 17:09:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8864491/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8864491/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104063998,"identity":"c354a2f8-8e34-48fd-a993-c4bdb08dc763","added_by":"auto","created_at":"2026-03-06 10:15:13","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":111275,"visible":true,"origin":"","legend":"\u003cp\u003eAn egg (a) and hatchlings (b) from a clutch of the smallhead worm lizard, \u003cem\u003eLeposternon microcephalum\u003c/em\u003e, found in a natural nest in Praia Grande, São Paulo, southeastern Brazil\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8864491/v1/0a868d5ec4502801a9014db7.jpeg"},{"id":104403634,"identity":"eceeacef-0c57-443f-aad6-8d95f2d6035d","added_by":"auto","created_at":"2026-03-11 12:18:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1069081,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8864491/v1/84a52fdb-1827-4770-bd7a-e5a59b940778.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Amphisbaenians facultatively oviposit in ant and termite nests","fulltext":[{"header":"SIGNIFICANCE STATEMENT","content":"\u003cp\u003eHow animals choose where to lay their eggs can strongly influence offspring survival, yet this decision remains poorly understood in secretive, underground species. Worm lizards (amphisbaenians) have long been thought to depend on ant and termite nests for egg-laying, implying rigid ecological specialization. By combining new field observations with a critical reassessment of published records, this study shows that such behavioral decision is not obligatory. Instead, amphisbaenians use social-insect nests as part of a broader and flexible set of nesting options. Our findings highlight oviposition-site choice in these reptiles as a context-dependent maternal decision shaped by trade-offs among environmental conditions, risks, and constraints, rather than fixed dependence on a single nesting strategy. This work reframes amphisbaenian reproduction within a behavioral decision-making framework and underscores the importance of considering plasticity and detectability biases when interpreting nesting ecology in fossorial animals.\u003c/p\u003e"},{"header":"INTRODUCTION","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003e \u003cem\u003e\u0026ldquo;The reason for this association has not yet been determined. Brazilians hold the pious belief that ants take pity on the blind lizard and welcome it into their nests, even bringing it food!\u0026rdquo;\u003c/em\u003e (\u003cem\u003eTschudi 1866: 159\u003c/em\u003e)\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e \u003cp\u003eFor oviparous animals in which embryos develop outside the maternal body, oviposition-site choice is a major determinant of reproductive success. In many of these taxa, parental care is limited or absent, so oviposition-site choice becomes a major maternal effect through which mothers influence offspring survival and phenotype (Resetarits \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Refsnider and Janzen \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The physical and biological attributes of the chosen site influence embryonic development and hatching success by modulating temperature, humidity, gas exchange, microbial activity, and predation risk (Refsnider \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Buxton and Sperry \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Sch\u0026uuml;tz and F\u0026uuml;reder \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Fowler et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The relevance of these factors, however, varies widely among taxa and depends on species-specific life-history traits, creating trade-offs among maternal survival, incubation conditions, and offspring performance (Refsnider and Janzen \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Thus, oviposition-site selection is best viewed as a maternal reproductive decision with direct fitness consequences.\u003c/p\u003e \u003cp\u003eIn reptiles, temperature and moisture at the oviposition site can influence embryonic growth, hatching success, and a range of offspring traits, sometimes with long-term effects. Site structure and local biota also affect egg survival by altering predation risk, microbial colonization, and exposure to flooding or desiccation (Refsnider \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Abayarathna and Webb \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Together, these pressures have produced a wide diversity of oviposition strategies that differ in the degree of site preparation, from the use of unmodified, pre-existing shelters (e.g., rock crevices, decaying logs, or colonies of social insects) to actively excavated burrows or constructed nesting structures (Packard and Packard \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e1988\u003c/span\u003e; Refsnider \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Murray et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Variation also occurs in how sites are used. Females may oviposit alone (solitary oviposition) or share sites with other females (communal oviposition). Communal oviposition is often associated with limited availability of suitable sites or with shared benefits such as stable microclimates or reduced predation risk (Graves and Duvall \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Doody et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Despite the relevance of these maternal decisions, current knowledge of reptile nesting ecology and behavior is strongly biased toward large, surface-active taxa (Doody et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Doody and Refsnider \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), leaving fossorial, limbless, and secretive lineages underrepresented in comparative studies.\u003c/p\u003e \u003cp\u003eThis bias is especially pronounced in amphisbaenians (worm lizards), a monophyletic and highly specialized group of ~\u0026thinsp;200 species of fossorial, mostly limbless reptiles (except for the forelimbed \u003cem\u003eBipes\u003c/em\u003e), distributed across tropical and subtropical regions of the Americas, Africa, and parts of the Mediterranean and Middle East (Pough et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Uetz et al. \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Their subterranean lifestyle severely limits direct field observations; consequently, fundamental aspects of their nesting ecology and nesting-related behavior are poorly documented, hindering the identification of general patterns (Andrade et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). This lack of information can also generate detection bias. Conspicuous microhabitats are more likely to be searched and reported, which can inflate the apparent importance of particular oviposition sites. The same limitation can bias inferences about how sites are used. For instance, communal oviposition has not been documented in amphisbaenians; however, given how widespread this behavior is among other squamates (Graves and Duvall \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Doody et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), its apparent absence in amphisbaenians may reflect limited observations rather than a true lack of communal nesting.\u003c/p\u003e \u003cp\u003eFrom a functional perspective, it has been assumed that limblessness constrains nest construction, such that limbless reptiles, including amphisbaenians, rely mainly on pre-existing structures (e.g., decaying logs, rock crevices, insect colonies) for oviposition rather than constructing nests (Packard and Packard \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e1988\u003c/span\u003e; Doody et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; but see Burger and Zappalorti \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1991\u003c/span\u003e; Koirala and Tshering \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2021\u003c/span\u003e for exceptions). Riley et al. (\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e1985\u003c/span\u003e) reviewed records of squamate oviposition in ant and termite nests and noted that eggs of amphisbaenians (\u003cem\u003eAmphisbaena alba\u003c/em\u003e, \u003cem\u003eA. kingii\u003c/em\u003e, and \u003cem\u003eLeposternon microcephalum\u003c/em\u003e) had been reported exclusively from ant nests. They interpreted this pattern as consistent with a possible obligate association (i.e., exclusive or near-exclusive use) with ant nests for oviposition. However, for amphisbaenians, this inference may not reflect a true functional constraint. As specialized burrowers, amphisbaenians possess morphological and behavioral traits that could allow them to excavate or modify oviposition chambers in the soil, rather than relying solely on pre-existing shelters. This \"obligate ant-nest\" hypothesis, although cited by later authors (e.g., Azevedo-Ramos and Moutinho \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Vega \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Andrade et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), has never been explicitly assessed, largely because natural oviposition sites are rarely documented for this group. Given the many records of amphisbaenians occurring in termite nests (Broadley et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1976\u003c/span\u003e; Vega \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Pramuk and Alamillo \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Moreira et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Duleba and Ferreira \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), and because various other limbless squamates also oviposit in such sites (Riley et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e1985\u003c/span\u003e), this idea can reasonably be extended to termite nests as well (FitzSimons \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1943\u003c/span\u003e; Bons and Saint Girons \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1963\u003c/span\u003e). Proposed benefits of ovipositing in ant and termite nests include stable microclimates, protection from predators or microbes, and immediate access to food resources (Hagmann \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e1907\u003c/span\u003e; Kopstein \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e1928\u003c/span\u003e; Vaz-Ferreira et al. \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e1970\u003c/span\u003e; Riley et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e1985\u003c/span\u003e; Hood et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). If amphisbaenians truly depend on ant and termite nests as exclusive or near-exclusive oviposition sites, this would imply a high degree of ecological and behavioral specialization, potentially involving coevolutionary mechanisms such as insect tolerance toward eggs or traits that facilitate persistence within active colonies (Baer et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Sierra-Serrano et al. \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Testing whether such dependence exists is therefore necessary to distinguish specialization from flexibility in a key reproductive behavior.\u003c/p\u003e \u003cp\u003eHere, we evaluate the hypothesis that amphisbaenians obligatorily oviposit in ant and termite nests. First, we describe the rare finding of a natural oviposition site of \u003cem\u003eL. microcephalum\u003c/em\u003e\u0026mdash;a species previously suggested to oviposit exclusively in ant nests (Riley et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e1985\u003c/span\u003e)\u0026mdash;found in a soil cavity with no evidence of ants or termites. We then compile and critically review published records of amphisbaenian oviposition sites to assess whether ant and termite nests are obligatory for oviposition and whether these sites are used solitarily or communally. If oviposition in ant and termite nests were obligatory, eggs should be restricted to these sites; in contrast, facultative use predicts oviposition across diverse microhabitats. Finally, we discuss the potential costs and benefits that may drive the use of ant and termite nests in amphisbaenians.\u003c/p\u003e"},{"header":"MATERIAL AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eOriginal data\u003c/h2\u003e \u003cp\u003eOn 26 February 2011, three eggs were found in a private garden in Praia Grande municipality, S\u0026atilde;o Paulo state, southeastern Brazil (24\u0026deg; 00' 21.6\" S, 46\u0026deg; 24' 10.8\" W; elevation: 11 m) during routine maintenance. The eggs were collected, placed in a plastic bag with soil from the same site, and transported to the laboratory the following day. Two eggs hatched during transportation. The remaining egg was measured with a digital caliper (to the nearest 0.01 mm), weighed (to the nearest 0.01 g), returned to the bag, and kept at room temperature. Hatchlings were measured (to the nearest 1 mm), weighed (to the nearest 0.01 g), photographed, and deposited in the Reference Collection of the Butantan Institute (IBSP.CRIB) and Museu de Zoologia da Universidade de S\u0026atilde;o Paulo (MZUSP).\u003c/p\u003e \u003cp\u003eOn 15 December 2007, we manually opened 30 leaf-cutting ant (\u003cem\u003eAcromyrmex\u003c/em\u003e) nests in a \u003cem\u003eHevea brasiliensis\u003c/em\u003e plantation and one \u003cem\u003eAtta\u003c/em\u003e nest in adjacent pasture areas at Fazenda Novo Mundo (22\u0026deg; 19\u0026prime; 45\u0026Prime; S, 49\u0026deg; 45\u0026prime; 23\u0026Prime; W), Vera Cruz municipality, S\u0026atilde;o Paulo state, southeastern Brazil. Colonies of \u003cem\u003eAcromyrmex\u003c/em\u003e are the ant nests most frequently reported as oviposition sites for squamates (Sacerdote-Velat and Sekits \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). For each nest, we opened chambers as far as feasible and visually searched for eggs or amphisbaenians within the nest structure and adjacent soil. The surveyed area harbors multiple amphisbaenian species (Mott and Vieites \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Santos \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), and December coincides with the oviposition period reported for amphisbaenians in southeastern Brazil (Santos \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; see also references in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSummary of available records on natural oviposition sites of Amphisbaenia. The column 'Information originally provided' reproduces the original account as closely as possible, while the columns 'Nest site' and 'Nest use' represent our standardized interpretation. NA\u0026thinsp;=\u0026thinsp;not available\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLocation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eInformation originally provided\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNest site\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNest use\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eReference\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eUnidentified amphisbaenid \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRio de Janeiro, Brazil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEggs are laid within fungus chamber of old nests of leaf-cutting (\"\u003cem\u003etanajura\u003c/em\u003e\") ants.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAnt nest (fungus chamber, Formicidae, Myrmicinae, Attini)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eTschudi, \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e1867\u003c/span\u003e; Brand\u0026atilde;o and Vanzolini, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1985\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAmphisbaena caeca\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePuerto Rico\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOne egg found beneath a termite nest.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eBelow termite nest\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSolitary\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSchmidt, \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e1920\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAmphisbaena caeca\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePuerto Rico\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTwo eggs and one adult specimen found\u0026thinsp;~\u0026thinsp;7.5 cm under a log beneath an ant's nest on 22 August 1919. One egg (42 \u0026times; 11 mm) contained a hatchling-sized specimen (86 mm total length) and residual yolk mass (AMNH 13237).\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eUnder log\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSolitary\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSchmidt, \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e1920\u003c/span\u003e; Gans and Alexander, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e1962\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAmphisbaena darwinii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUruguay\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEggs found in layers of humus.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eUnderground (humus layer)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eVaz-Ferreira et al., \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e1970\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAmphisbaena darwinii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUruguay\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA clutch of four eggs (mean egg size\u0026thinsp;=\u0026thinsp;24.7 \u0026times; 13.0 mm) found within an unoccupied anthill (species unidentified) in a grassland habitat on 30 December 2002. Three eggs hatched on 25\u0026ndash;26 January 2003 (hatchling total length\u0026thinsp;=\u0026thinsp;68, 74, and 85 mm in; ZVC-R 6080).\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAnt mound\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSolitary\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCarreira and Baletta, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2006\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAmphisbaena darwinii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eArgentina\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA female and a clutch (egg length\u0026thinsp;~\u0026thinsp;20 mm) of two eggs containing near-term embryos were found in a small cavity in the soil dug from a garden in February 1895.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eUnderground cavity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSolitary\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eBerg, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1898\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAmphisbaena darwinii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eArgentina\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA female and a clutch of three eggs (egg length\u0026thinsp;~\u0026thinsp;20 mm) containing near-term embryos were found in a small cavity in the soil dug from a garden in February 1896.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eUnderground cavity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSolitary\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eBerg, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1898\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAmphisbaena darwinii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eArgentina\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEggs (clutches ranging 6\u0026ndash;8 and 17\u0026ndash;21) found under logs of an old \u003cem\u003eEucalyptus\u003c/em\u003e crop partially buried in loose soil, which contained two or more species of termites and ants. Eggs and embryonic series housed at UNNEC (see Montero et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e1999\u003c/span\u003e).\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eUnder log in loose soil (termite and ant presence)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSolitary and communal \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMontero et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; J. A. C\u0026eacute;spedez, pers. comm.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAmphisbaena fuliginosa\u003c/em\u003e \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBrazil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA clutch of nine eggs (one egg measuring 28.22 \u0026times; 14.74 mm) was found on 2 October 2016, partially buried beneath a fallen tree trunk. Eggs hatched on 19\u0026ndash;20 October (hatchling size\u0026thinsp;=\u0026thinsp;122\u0026ndash;135 mm total length). An adult female \u003cem\u003eA. fuliginosa\u003c/em\u003e was also found in the nest site.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eUnder log\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSolitary\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eOliveira and Gomes, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2017\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAmphisbaena kingii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eArgentina\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eOne egg (29 \u0026times; 11 mm) containing a fully developed embryo (75 mm total length) was found in early March 1988 at the base of the fungus chamber of a nest of \u003cem\u003eAcromyrmex silvestrii\u003c/em\u003e (MLP S.1097).\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAnt nest (fungus chamber, Myrmicinae, Attini)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSolitary\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eWilliams and Wichmann, \u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e1989\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAmphisbaena kingii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBrazil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSeveral (\u0026ge;\u0026thinsp;2) eggs found in ant nests. Two of these eggs were examined by Gans and Rhodes (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1964\u003c/span\u003e); the eggs (30 \u0026times; 10 and 35 \u0026times; 10 mm) contained fully developed embryos (113 mm total length; NHMUK 1885.2.3.6-7).\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAnt nest\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eProbably solitary\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eBoulenger, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1885\u003c/span\u003e; Gans and Rhodes, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1964\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eAmphisbaena mertensii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eParaguay\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA gravid female (301 mm SVL; MNHNP 8742) with stomach contents (Coleoptera, Elateridae) was found buried in the softer soil peripheral to a termite (\u003cem\u003eCornitermes cumulans\u003c/em\u003e) mound; no eggs recorded.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTermite nest (peripheral soil)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePramuk and Alamillo, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2003\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLeposternon infraorbitale\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBrazil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA clutch of six eggs (egg size\u0026thinsp;=\u0026thinsp;60 \u0026times; 25 mm) found on 20 February 1994 in the ground beside a fallen tree in a cacao plantation, 15 cm from the surface and covered in soil and decomposed wood. Hatchlings 150\u0026ndash;170 mm total length.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eUnder decomposing wood\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSolitary\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eJared et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e1997\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLeposternon microcephalum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eS\u0026atilde;o Paulo, Brazil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA clutch of three eggs (one egg measuring 45.26 \u0026times; 15.31 mm) found in a cavity 18 cm below the soil surface in a small garden located inside a private residence on 26 February 2011. Two eggs hatched the next day. Hatchlings measured 126, 136, and 142 mm in total length (IBSP.CRIB 295 and 296; MZUSP 103179).\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eUnderground\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSolitary\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003ePresent study\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLeposternon microcephalum\u003c/em\u003e \u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBrazil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEggs (unreported number) were taken in March 1893 from a pile of bricks and fragments of roof tiles inhabited by a reasonable size colony of ants (\u003cem\u003eCamponotus\u003c/em\u003e). Two sets of eggs were found: one with less-developed embryos, another with embryos close to hatching. Two eggs of the older clutch measured 53.5 \u0026times; 20 and 54.5 \u0026times; 18 mm. Two other eggs (52 \u0026times; 12 and 45 \u0026times; 14 mm) are vouchered as NHMUK 1893.9.30.2\u0026ndash;3.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAnt nest (Formicinae, Camponotini)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePossibly communal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eGoeldi, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1898\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLeposternon microcephalum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBrazil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA clutch of two eggs was found in an oval (8.0 \u0026times; 3.5 \u0026times; 4.5 cm) smooth-walled cavity 23\u0026ndash;25 cm below the surface of a truck garden. The soil was sandy-loamy interspersed with hummus.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eUnderground cavity\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSolitary\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eEngmann, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1927\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLeposternon microcephalum\u003c/em\u003e \u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBrazil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTwo eggs (ZMH 3571) containing partially developed embryos (39 mm total length) collected in an anthill on 17 December 1907.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAnt mound\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSolitary\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eGans, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1971\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLeposternon microcephalum\u003c/em\u003e \u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBrazil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eEight eggs (ZMH 3572) containing early embryos collected in an anthill in the spring of 1908.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAnt mound\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePossibly communal (due to the clutch size)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eGans, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1971\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eRhineura floridana\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFlorida, United States\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eA clutch of two eggs (one egg measuring 38.0 \u0026times; 8.9 mm) containing fully formed young (93.5 and 112.2 mm in total length) was found after turning up in a spadeful of sandy loam taken from a depth of 20\u0026ndash;50 cm.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eUnderground (sandy soil)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSolitary\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCarr, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1949\u003c/span\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003csup\u003ea\u003c/sup\u003e Tschudi (\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e1867\u003c/span\u003e) reports \u003cem\u003eA. flavescens\u003c/em\u003e and \u003cem\u003eA. fuliginosa\u003c/em\u003e inhabiting \u0026ldquo;tanajura\u0026rdquo; nests and laying eggs there; however, the original text is ambiguous as to which species (or whether both) oviposit in such sites. \u003cem\u003eAmphisbaena flavescens\u003c/em\u003e is now recognized as \u003cem\u003eA. alba\u003c/em\u003e, whereas \u003cem\u003eA. fuliginosa\u003c/em\u003e does not occur in the region. Moreover, five other amphisbaenian species are currently known from Rio de Janeiro state Guedes et al. (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Given this uncertainty, we list the record as \"unidentified amphisbaenid\".\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003csup\u003eb\u003c/sup\u003e Given that \u003cem\u003eA. darwinii\u003c/em\u003e typically lays 2\u0026ndash;4 eggs (Gallardo \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1967\u003c/span\u003e; Carreira and Baletta \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), these aggregations likely represent oviposition by multiple females (~\u0026thinsp;2\u0026ndash;11).\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003csup\u003ec\u003c/sup\u003e Originally published as \u003cem\u003eA. brasiliana\u003c/em\u003e but identified as \u003cem\u003eA. fuliginosa\u003c/em\u003e by Abecassis et al. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2026\u003c/span\u003e).\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003csup\u003ed\u003c/sup\u003e Gans (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1971\u003c/span\u003e) comments that \"it is uncertain that the eggs belong to species \u003cem\u003emicrocephalum\u003c/em\u003e\".\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"6\"\u003e\u003csup\u003ee\u003c/sup\u003e Identified as \u003cem\u003eL. microcephalum\u003c/em\u003e by Gans (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1971\u003c/span\u003e).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eLiterature survey\u003c/h3\u003e\n\u003cp\u003eTo assess the hypothesis of obligatory oviposition in ant or termite nests in amphisbaenians, we searched the literature for records of natural oviposition sites. We searched Google Scholar using combinations of \"Amphisbaenia\", \"amphisbaenian\", \"worm lizard\", genus names, and keywords such as \"nest\", \"oviposition\", \"egg-laying\", and \"egg\", in Portuguese, Spanish, and English. We extracted descriptions of oviposition microhabitats and classified nesting as solitary or communal when possible. To distinguish between solitary and communal nesting, we used multiple criteria, including developmental stages of embryos within nests, the time interval between hatching events, and whether the number of eggs at a site exceeded the mean clutch size reported for the species (Braz et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). We also compiled clutch size, egg size, offspring size, and incubation duration when available.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eOriginal data\u003c/h2\u003e \u003cp\u003eThe clutch of three eggs was found in a cavity 18 cm below the soil surface in a small garden (0.9 \u0026times; 1.6 m) containing ornamental cacti. The garden was located within a private residence and received direct sunlight through a glass brick wall. The garden was searched for additional eggs and for nearby ant or termite nests but none were found. The eggs had whitish, leathery shells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). Two eggs hatched within the plastic bag on 27 February 2011, during transport to the laboratory. One hatchling (IBSP.CRIB 295) measured 120 mm snout-vent length (SVL), had a 6 mm tail, and weighed 2.73 g. The other (IBSP.CRIB 296) measured 128 mm SVL, had an 8 mm tail, and weighed 3.52 g. Both hatchlings left substantial unabsorbed residual yolk (1.47 and 1.05 g, respectively) within the eggshells. The remaining egg (45.26 \u0026times; 15.31 mm, 5.08 g; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea) did not hatch. Dissection revealed a full-term dead embryo (133 mm SVL, 9 mm tail length; MZUSP 103179) with residual yolk. All individuals were identified as \u003cem\u003eL. microcephalum\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). All 31 excavated ant nests were active, but we found no amphisbaenians and no eggs in any of them. However, we found a blindsnake (\u003cem\u003eLeptotyphlops\u003c/em\u003e sp.) inside an \u003cem\u003eAcromyrmex\u003c/em\u003e nest.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eLiterature data\u003c/h3\u003e\n\u003cp\u003eOur literature survey identified 18 records of confirmed natural oviposition sites for nine amphisbaenian species from three genera (\u003cem\u003eAmphisbaena\u003c/em\u003e, \u003cem\u003eLeposternon\u003c/em\u003e, and \u003cem\u003eRhineura\u003c/em\u003e) across South, Central, and North America (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). One additional report described a gravid \u003cem\u003eA. mertensii\u003c/em\u003e inside a termite nest, suggesting\u0026mdash;but not confirming\u0026mdash;a potential oviposition site (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). We treated this record separately from confirmed oviposition sites.\u003c/p\u003e \u003cp\u003eAmong confirmed records (n\u0026thinsp;=\u0026thinsp;18), oviposition sites were most often in ant nests (n\u0026thinsp;=\u0026thinsp;7, 38.9%) and underground cavities (n\u0026thinsp;=\u0026thinsp;6, 33.3%), followed by sites beneath decaying logs or decomposing wood (n\u0026thinsp;=\u0026thinsp;4, 22.2%) and termite nests (n\u0026thinsp;=\u0026thinsp;1, 5.6%). For most species, records are limited to one or two observations. Multiple records were available only for \u003cem\u003eL. microcephalum\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;5) and \u003cem\u003eA. darwinii\u003c/em\u003e (at least 6, as the original sources do not provide an exact count of distinct oviposition records; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In \u003cem\u003eL. microcephalum\u003c/em\u003e, oviposition sites were in ant nests (3/5, 60%) and in underground cavities (2/5, 40%). In \u003cem\u003eA. darwinii\u003c/em\u003e, half of the records involved underground sites (humus or soil cavities, 3/6), with additional records from beneath a log (at least 2/6) and an ant nest (1/6).\u003c/p\u003e \u003cp\u003eMost records indicated solitary nesting (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The only unequivocal cases of communal nesting involved \u003cem\u003eA. darwinii\u003c/em\u003e in Argentina, where groups of 6\u0026ndash;8 to 17\u0026ndash;21 eggs were occasionally found under the same log. Because \u003cem\u003eA. darwinii\u003c/em\u003e typically lays 2\u0026ndash;4 eggs (Gallardo \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1967\u003c/span\u003e; Carreira and Baletta \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), we interpret this records as oviposition by multiple females (~\u0026thinsp;2\u0026ndash;11). However, solitary oviposition also occurred in this species (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Two records for \u003cem\u003eL. microcephalum\u003c/em\u003e were ambiguous regarding oviposition site use. Goeldi (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1898\u003c/span\u003e) described two sets of eggs differing in shapes and developmental stages from a pile of bricks and tiles inhabited by a large \u003cem\u003eCamponotus\u003c/em\u003e ant colony, which is consistent with more than one clutch. Similarly, Gans (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1971\u003c/span\u003e) reported two clutches (eight and two eggs) of different sizes and developmental stages from the same locality, but we could not determine whether they originated from a single oviposition site.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eFacultative use of ant and termite nests for egg-laying\u003c/h2\u003e \u003cp\u003eOur original observations and literature synthesis do not support the hypothesis that amphisbaenians obligatorily use ant or termite nests for oviposition. Instead, the available evidence indicates that these structures are one option among a broader repertoire of sites. In the two species with multiple records (\u003cem\u003eL. microcephalum\u003c/em\u003e and \u003cem\u003eA. darwinii\u003c/em\u003e), eggs have been found in both ant or termite nests and alternative shelters. This pattern is consistent, though less documented, in other species. This distribution supports facultative rather than exclusive use of ant and termite nests, with females exploiting various shelters that meet incubation requirements.\u003c/p\u003e \u003cp\u003eImportantly, the available evidence addresses dependence (i.e., obligatoriness) rather than preference, as the occurrence of eggs outside ant and termite nests indicates these colonies are not required for oviposition. Consistent with this interpretation, we found no eggs or amphisbaenians in 31 excavated ant nests surveyed during the reported oviposition period (Santos \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; see also references in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) at a site with documented amphisbaenian occurrences (Mott and Vieites \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Santos \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Although these negative results should be interpreted cautiously given detectability constraints, they further support non-exclusivity.\u003c/p\u003e \u003cp\u003eWhile obligate use can be rejected, whether amphisbaenians preferentially select ant and termite nests under certain ecological contexts remains unresolved. The apparent prevalence of these nests in the literature may partly reflect sampling bias, as they are conspicuous, long-lasting, and often searched more intensively than other potential oviposition microhabitats (e.g., soil cavities, burrows, or decaying root systems). Many squamate eggs found in ant and termite nests were discovered incidentally by entomologists during surveys focused on social insects (e.g., Vaz-Ferreira et al. \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e1970\u003c/span\u003e, \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e1973\u003c/span\u003e; Brand\u0026atilde;o and Vanzolini \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1985\u003c/span\u003e; Bruner et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Kwapich \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), with dozens to hundreds of nests opened in several cases (e.g., Vaz-Ferreira et al. \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e1970\u003c/span\u003e, \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e1973\u003c/span\u003e; Pramuk and Alamillo \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). This asymmetry in search effort can inflate the perceived frequency of oviposition in ant and termite nests, even when such sites are used facultatively. Similar patterns of non-exclusive use have been reported for many lizards and snakes (reviewed in Riley et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e1985\u003c/span\u003e; see also Vitt et al. \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Khannoon and Evans \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), suggesting that facultative oviposition in ant and termite nests is a recurrent strategy among squamates. Resolving whether these nests are used disproportionately relative to their availability will require standardized surveys across multiple potential nesting microhabitats.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eFunctional hypotheses and trade-offs underlying oviposition in ant and termite nests\u003c/h3\u003e\n\u003cp\u003eAlthough ant and termite nests are not obligatory oviposition sites for amphisbaenians, they are ecologically relevant options whose use has been recurrently explained by several functional hypotheses. These hypotheses propose that ovipositing within these nests may confer advantages such as buffered microclimates, protection against predators or microbes, and immediate access to food for hatchlings (Hagmann \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e1907\u003c/span\u003e; Kopstein \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e1928\u003c/span\u003e; Vaz-Ferreira et al. \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e1970\u003c/span\u003e; Riley et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e1985\u003c/span\u003e; Hood et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, because these sites are used facultatively, any advantages are expected to be context-dependent and offset by costs, including aggression from resident insects, attraction of predators, constraints on hatchling emergence, and variable antimicrobial environments (Vaz-Ferreira et al. \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e1970\u003c/span\u003e; Oliveira and Della Lucia \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). Below, we evaluate these benefits and costs in the context of amphisbaenians, emphasizing how trade-offs among them may shape flexible maternal decisions on oviposition-site choice.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMicroclimatic stability\u003c/h2\u003e \u003cp\u003eA frequently proposed explanation for oviposition in ant and termite nests is that they provide buffered and predictable microclimatic conditions that enhance egg survival and offspring performance. These nests can maintain higher and more stable temperature and humidity than adjacent substrates (Vaz-Ferreira et al. \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e1970\u003c/span\u003e; Korb and Linsenmair \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Bollazzi and Roces \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), conditions often assumed to be favorable for squamate eggs (Kopstein \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e1928\u003c/span\u003e; Vaz-Ferreira et al. \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e1970\u003c/span\u003e; Riley et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e1985\u003c/span\u003e; Herrera and Robinson \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Vel\u0026aacute;squez\u0026ndash;M\u0026uacute;nera et al. \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Baer et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Rodr\u0026iacute;guez and Montoya-Lerma \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Williams et al. \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Sierra-Serrano et al. \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). This hypothesis is plausible, given the well-established effects of incubation temperature and moisture on offspring traits (While et al. \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Bell et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). However, Warmer or more stable conditions do not always improve developmental outcomes, and both the direction and magnitude of effects vary among species. In some cases, less variable incubation temperatures produce hatchlings with phenotypes associated with higher survival than more variable regimes (e.g., Patterson and Blouin-Demers \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), whereas in others, differences are minimal or absent (Ji et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Hao et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Unfortunately, no study has directly tested whether amphisbaenian eggs gain fitness-relevant advantages under the microclimatic conditions typical of ant or termite nests, and therefore, the adaptive value of ovipositing in these environments remains uncertain for amphisbaenians. Controlled comparisons between nest-like conditions and alternative shelters are therefore needed to assess whether microclimatic stability favors repeated, but not exclusive, use of these sites.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eProtection against predators and microbial attack\u003c/h2\u003e \u003cp\u003eAnother commonly invoked hypothesis is that oviposition within ant and termite nests reduces egg mortality by providing protection against predators and microbial infection. This protection is attributed to both nest architecture and the defensive and hygienic behaviors of resident insects, which may deter egg predators or suppress pathogen growth (Kopstein \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e1928\u003c/span\u003e; Vaz-Ferreira et al. \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e1970\u003c/span\u003e; Riley et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e1985\u003c/span\u003e; Vel\u0026aacute;squez\u0026ndash;M\u0026uacute;nera et al. \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Although such mechanisms are plausible, empirical support for this hypothesis is inconsistent. Ants have been suggested to clean reptile eggs or embedding them in fungus gardens where symbiotic fungi may suppress pathogens (Vel\u0026aacute;squez\u0026ndash;M\u0026uacute;nera et al. \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Baer et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2009\u003c/span\u003e); however, high egg mortality has also been documented within social-insect nests. For example, two-thirds of the tegu lizard (\u003cem\u003eTupinambis teguixin\u003c/em\u003e) eggs found in a termite nest were rotten (Herrera and Robinson \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2000\u003c/span\u003e), and mortality has been reported in in termite nests for squamates, including amphisbaenians (Beebe \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1944\u003c/span\u003e; Pramuk and Alamillo \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Moreover, resident insects can themselves attack and harm hatchlings and adults (Vaz-Ferreira et al. \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e1970\u003c/span\u003e; Riley et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e1986\u003c/span\u003e; Oliveira and Della Lucia \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). In addition, ant and termite nests often attract vertebrate predators (e.g., tegu lizards: Beebe \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1945\u003c/span\u003e; Avila-Pires \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; snakes: Vaz-Ferreira et al. \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e1970\u003c/span\u003e, \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e1973\u003c/span\u003e; Duleba and Ferreira \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) that prey on eggs or fossorial squamates (Braz et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Kasperoviczus et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Rodr\u0026iacute;guez et al. \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Therefore, these observations indicate that any protective role of social-insect nests is context-dependent and cannot be assumed a priori. Testing this hypothesis will require direct comparisons of egg survival and infection rates inside and outside active nests, ideally distinguishing effects of insect behavior or nest architecture from those of underground nesting more generally.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eImmediate access to food resources\u003c/h2\u003e \u003cp\u003eA further hypothesis is that oviposition within ant or termite nests benefits hatchlings by providing immediate access to abundant food resources (Hagmann \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e1907\u003c/span\u003e; Hegh \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e1922\u003c/span\u003e; Hood et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). This idea is intuitively appealing for insectivorous reptiles, particularly in subterranean environments where prey availability outside nests may be limited. Its relevance, however, depends on whether hatchlings rely on exogenous food shortly after hatching. Squamates show substantial variation in post-hatching feeding strategies. Some hatchling lizards begin feeding within the first 48 hours and derive little energy from residual yolk (Radder et al. \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Guo et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), thus rendering immediate food access of limited functional significance. In contrast, other reptiles delay feeding for extended periods and rely largely on residual yolk during early life (Burger \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1989\u003c/span\u003e; Morafka et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Rowe et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). For amphisbaenians, direct evidence is lacking. Our observations of substantial residual yolk in newly hatched \u003cem\u003eL. microcephalum\u003c/em\u003e are consistent with limited early dependence on external food sources. Additionally, postnatal residence within ant nest chambers is suggested to be generally brief in squamates (Vaz-Ferreira et al. \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e1970\u003c/span\u003e), limiting opportunities for sustained post-hatching foraging within colonies. These considerations suggest that immediate access to prey is unlikely to be a primary factor influencing oviposition-site choice in amphisbaenians. Targeted data on neonatal feeding behavior and dependence on external food sources are needed to evaluate this hypothesis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eCan amphisbaenians build their own nests?\u003c/h2\u003e \u003cp\u003eIt has been assumed that limblessness constrains nest construction in squamates, forcing limbless species to rely primarily on pre-existing structures for oviposition (Packard and Packard \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e1988\u003c/span\u003e; Doody et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). However, its relevance to specialized burrowing taxa, such as amphisbaenians, remains uncertain. Our record of \u003cem\u003eL. microcephalum\u003c/em\u003e and historical reports for \u003cem\u003eA. darwinii\u003c/em\u003e (Berg \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1898\u003c/span\u003e) document oviposition in subterranean cavities without evidence of ants or termites but do not clarify whether these chambers were excavated by the female or were pre-existing. A more informative account describes at least one clutch of \u003cem\u003eL. microcephalum\u003c/em\u003e within a smooth, nearly oval underground chamber connected to a distinct access tunnel (Engmann \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1927\u003c/span\u003e). Although active excavation was deemed possible, the author favored the interpretation that the female modified an existing crevice rather than digging the chamber entirely (Engmann \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1927\u003c/span\u003e). Subsequent studies of \u003cem\u003eL. microcephalum\u003c/em\u003e burrowing behavior suggest that both scenarios are mechanically feasible. The species uses its reinforced, shovel-like head and muscular body to penetrate compact soils and maintain tunnel integrity (Gans \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1969\u003c/span\u003e; Barros-Filho et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Hohl et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), which indicates that it possesses the functional capacity to excavate or substantially modify subterranean spaces, including potential oviposition chambers. These observations challenge the assumption that limblessness precludes nest construction and suggest that some amphisbaenians may actively excavate or modify oviposition chambers, expanding the range of nesting strategies available to fossorial squamates. Direct behavioral observations under controlled conditions will be necessary to investigate nest-site preparation behavior, chamber architecture, and soil manipulation during oviposition.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eCommunal oviposition in amphisbaenians\u003c/h2\u003e \u003cp\u003ePrevious reviews found no evidence of communal oviposition in amphisbaenians (Graves and Duvall \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Doody et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). However, our reexamination of published records indicates that communal oviposition does occur in some South American species, although it appears to be uncommon. Solitary oviposition predominates, yet multiple clutches of \u003cem\u003eA. darwinii\u003c/em\u003e have been found together in the same microhabitat (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Communal oviposition in reptiles may arise from social tolerance among females or from aggregation driven by shared responses to limited or favorable nesting sites (Graves and Duvall \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Doody et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). In \u003cem\u003eA. darwinii\u003c/em\u003e, this interpretation is supported by field observations of adults and juveniles sharing refuges, sometimes in close physical contact (Gallardo \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1967\u003c/span\u003e; Borteiro et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Evidence for communal oviposition \u003cem\u003ein L. microcephalum\u003c/em\u003e is more equivocal but compatible with occasional aggregation. Therefore, available evidence suggests that communal oviposition occurs in some amphisbaenian species but is neither frequent nor obligate. The coexistence of solitary and communal nesting within amphisbaenians aligns with flexible, context-dependent oviposition strategies. From a behavioral-ecological perspective, communal nesting in amphisbaenians generates testable hypotheses concerning site limitation, microclimatic advantages, and social tolerance, reinforcing the view that oviposition-site choice in this group reflects adaptive plasticity rather than rigid specialization.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur original observations and literature synthesis do not support the hypothesis that amphisbaenians obligatorily oviposit in ant or termite nests. The available evidence indicates that these structures are used facultatively among several oviposition-site options that meet their incubation requirements. Although ant and termite nests may offer advantages such as microclimatic buffering or protection, these benefits are likely context-dependent and can be offset by risks including predation, microbial failure, or insect aggression, favoring flexible rather than exclusive use.\u003c/p\u003e \u003cp\u003eOur synthesis also highlights two additional aspects of amphisbaenian reproductive behavior that merit further attention. First, evidence from \u003cem\u003eA. darwinii\u003c/em\u003e and more strongly from \u003cem\u003eL. microcephalum\u003c/em\u003e suggests that some amphisbaenians may excavate or modify subterranean chambers for oviposition, challenging the assumption that limbless reptiles rely primarily on pre-existing shelters. Second, records of communal nesting in \u003cem\u003eA. darwinii\u003c/em\u003e, and possibly in \u003cem\u003eL. microcephalum\u003c/em\u003e, expand the known behavioral repertoire of amphisbaenians and further support the view that oviposition strategies in this group are variable and context-dependent.\u003c/p\u003e \u003cp\u003eTaken together, these findings indicate that oviposition-site choice in amphisbaenians reflects adaptive plasticity rather than strict specialization or dependence on social insects. Future studies integrating controlled incubation experiments, field surveys across multiple microhabitats, and behavioral observations under natural or semi-natural conditions are necessary to clarify the ecological drivers and adaptive significance of nesting strategies in this group. In particular, combining measures of microhabitat availability with comparable search effort would be especially useful to disentangle preference, availability, and detectability in this system.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAnimal ethics\u003c/h2\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003ch2\u003eFUNDING\u003c/h2\u003e\n\u003cp\u003eL\u0026iacute;via Cristina Santos and Selma Maria Almeida-Santos received funding from Funda\u0026ccedil;\u0026atilde;o de mparo \u0026agrave; Pesquisa do Estado de S\u0026atilde;o Paulo; Grant ID 06/58529-3 and 08/58424-2.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eH.B.B., L.C.S., and S.M.A.S. conceptualized the manuscript.H.B.B. and L.C.S. collected and analyzed the data.H.B.B. wrote the main manuscript text with support from L.C.S. and S.M.A.S.L.C.S. prepared the figures.All authors revised the manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eWe thank Cristian Gomes for providing detailed information on the oviposition site described here, Jorge C\u0026eacute;spedez, Ricardo Montero, Andressa Oliveira, and Felipe Gomes for providing additional information on nest sites, Lu\u0026iacute;s Roberto Takitane and Tam\u0026iacute; Mott for allowing access to their property and for assistance with excavating ant nests. We also thank S\u0026iacute;ria Ribeiro, Roberta Graboski, and Tam\u0026iacute; Mott for confirming the taxonomic identity of some species, Dione Seripierri and the Carl Gans Collections and Charitable Fund for providing access to some references, and Valdir J. Germano for assistance in the laboratory. LCS received a master\u0026rsquo;s fellowship (06/58529-3) and a doctoral fellowship (08/58424-2) from the Funda\u0026ccedil;\u0026atilde;o de Amparo \u0026agrave; Pesquisa do Estado de S\u0026atilde;o Paulo (FAPESP).\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eAll data supporting the findings of this study are available within the paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbayarathna T, Webb JK (2022) Consequences of oviposition site choice for geckos in changing environments. 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Bolet\u0026iacute;n de la Asociaci\u0026oacute;n Herpetol\u0026oacute;gica Argentina 5:12\u0026ndash;13\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"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":"oviposition-site choice, maternal decision-making, behavioral plasticity, fossorial reptiles","lastPublishedDoi":"10.21203/rs.3.rs-8864491/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8864491/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOviposition-site selection is a key maternal decision in oviparous animals, involving trade-offs among incubation conditions, offspring performance, and maternal constraints. In amphisbaenians (worm lizards), a clade of highly specialized fossorial reptiles, oviposition has long been assumed to occur obligatorily in ant and termite nests, implying strong ecological specialization. Here, we re-evaluate this assumption using new field observations and a critical synthesis of published records. We describe a natural oviposition site of the smallhead worm lizard (\u003cem\u003eLeposternon microcephalum\u003c/em\u003e) located in a soil cavity with no evidence of ants or termites and review 18 records from nine amphisbaenian species. Across species, eggs occur not only in ant and termite nests but also in subterranean cavities and decaying logs, indicating facultative rather than exclusive use of social-insect nests. Field inspections of 31 ant nests during the oviposition season yielded no eggs or amphisbaenians, further supporting non-obligate use. Additional evidence suggests some species excavate or modify underground chambers and that at least one species oviposits communally. These findings challenge the view of strict dependence on ant and termite nests and instead support oviposition-site choice in amphisbaenians as a flexible, context-dependent maternal behavior shaped by ecological trade-offs rather than rigid specialization. Standardized surveys across microhabitats and experimental tests of incubation environments are needed to clarify how availability, costs, and benefits interact to shape nesting decisions in fossorial reptiles.\u003c/p\u003e","manuscriptTitle":"Amphisbaenians facultatively oviposit in ant and termite nests","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-06 10:15:03","doi":"10.21203/rs.3.rs-8864491/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":"71b3ad75-4a7d-4b1e-b0a5-404277e5f1b5","owner":[],"postedDate":"March 6th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-06T10:15:03+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-06 10:15:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8864491","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8864491","identity":"rs-8864491","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}