{"paper_id":"450f2b3b-6292-4b77-a563-5efd756c1dfa","body_text":"Where are extra-pair offspring raised: the mother’s, the father’s or the others’ nest? | 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 Where are extra-pair offspring raised: the mother’s, the father’s or the others’ nest? Chen Wang, Shicheng Chen, Xin Lu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3163972/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 Extra-pair mating prevails among socially monogamous birds with biparental care. Theoretically, females may lay the resultant eggs in their own, their mating partners or other pairs’ nests, accordingly leading to extra-pair paternity (EPP), quasi-parasitism (QP) and extra-pair conspecific brood parasitism (CBP). Empirical evidence showed EPP to be widespread and QP rare, with extra-pair CBP having never been described. In Tibetan ground tits ( Pseudopodoces humilis ), we detected EPP, QP and extra-pair CBP in 54%, 47% and 24% of broods, which involved 20%, 16% and 8% of offspring, respectively. Cuckolding and cuckolded individuals in these events were genetically related and moderate inbreeding occurred between extra-pair partners, whereas social mates formed randomly with respect to relatedness. Promiscuity relative to genetic monogamy significantly increased annual reproductive output of both males and females, and extra-pair offspring did not differ from their within-pair siblings in body mass and heterozygosity. While the much high incidence of promiscuity can be ultimately driven by not only males but also females’ pursuit of increasing reproductive success, it may be proximately promoted by frequent kin ties in association with the species’ habit of burrow nesting and roosting, presumably kin-selected benefits may reduce the cost of arising non-descendant offspring. The fact that less than 30 altricial species have been documented with respect to extra-pair maternity (in contrast to over 360 species for EPP) suggests that more attention should be paid towards QP and extra-pair CBP to get a broader perspective on the evolution of avian mating systems. Extra-pair mating Inbreeding Kin selection Kinship foster care Mating system Relatedness Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Significance Statement Theoretically, females involving in extra-pair mating may lay the resultant eggs in nests of their own or their mating partners, accordingly leading to extra-pair paternity and quasi-parasitism. While a large body of literature about the former has existed, much less attention has been paid to the latter likely because of its low incidence in terms of the proportions of species or nests within a population. The high-level quasi-parasitism found in Tibetan ground tits may be well interpreted with the prevailing nepotism in association with the burrow-nesting and roosting lifestyle if the species. The current results lead to the suggestion that in socially monogamous systems, not only males but also females pursue extra-pair mating to increase reproductive success. This sheds light on the puzzle of why females routinely engage in extra-pair mating. INTRODUCTION Social monogamy with biparental care is the most common mating system in birds (Cockburn 2006 ). Meanwhile, copulations outside the socially monogamous pair bond prevail, as revealed by molecular techniques (Griffith et al. 2002 , 2004 ). For an event of extra-pair mating, females may lay the resultant eggs in either their own or the mating males’ nests, accordingly generating extra-pair paternity (EPP) or maternity, with the latter termed as quasi-parasitism (QP) to distinguish from conspecific brood parasitism (CBP) ― a nest contains eggs not laid by the pair bond. Alternatively, females may lay the eggs fertilized through extra-pair mating in the nests of other breeding pairs and this leads to extra-pair CBP, which is in contrast to within-CBP ― a nest contains eggs laid by other pair bond (Fig. 1 ). Detected in 76% of 364 altricial bird species documented so far with an averaging brood occurrence rate of 37% (Fig. 1 ), EPP is widespread and has been a subject of extensive research over past 30 years (Griffith et al. 2002 ; Brouwer and Griffith 2019 ). These studies have shaped our current view of alternative mating strategy, which argues that socially monogamous males increase their reproductive success by EPP without paying the costs of parental care and accordingly male mate-guarding behaviors against cuckoldry have evolved (Birkhead and Møller 1992 ). Evolution theory suggests that selection also favors females to enhance reproductive output by means of QP and extra-pair CBP. However, empirical studies show that QP occurs rarely, which on average is 8% at the brood level for 22 altricial species examined so far (Fig. 1 ). A review paper based on the findings from 12 species thought that QP is an occasional event and accordingly, few ecological and evolutionary hypotheses have been proposed to explore its adaptive implications (Griffith et al. 2004 ). So far, no attempts have been made to confirm the presence of extra-pair CBP. The scarcity of QP and extra-pair CBP may be attributed to potential limitations to parasitism in terms of behavioral difficulty for a female to lay eggs in other individuals’ nests. For example, a successful parasitism depends on the probability that parasites synchronize their breeding time with potential host females and overcome resistance from the hosts who undertake parental care for non-descendant offspring. Nevertheless, the constraints on parasitism may be mitigated in certain circumstances. Sociality characterized by frequent interactions between breeding individuals should be a factor that can break up the constraints, because it may make females finding synchronously breeding hosts easy. Furthermore, social living is usually kin-based given the prevalence of fine-scale genetic structure of breeding populations (Hatchwell 2010 ). According to kin selection theory (Hamilton 1964 ), indirect fitness accrued from kin interactions such as kinship care for non-descendant offspring can reduce the resistance of hosts, as seen in CBP in waterfowl (Andersson et al. 2018 ). In addition, specific lifestyles in some species such as cavity nesting (Geffen and Yom-Tov 2001 ) may provide convenience for females’ parasitic attempts. It is worth pointing out that empirical examination of QP accounts for only 5% of the existing 619 studies on parentage of altricial birds. The very low taxonomic coverage (28 species) could reduce the opportunity to find more species that exhibit high-level extra-pair maternity. Here we describe the high incidences of EPP, QP and extra-pair CBP in a population of Tibetan ground tits Pseudopodoces humilis (Fig. 2 ). Endemic to alpine meadows of 2800–6000 m elevation in the Tibetan plateau, the birds breed and roost throughout the year in burrows excavated by themselves to buffer against cold, windy and poorly vegetated environments (Ke and Lu 2009 ). Sociality is strong in the study population inhabits northeast Tibet where territoriality is absent, related breeders nest nearby due to dispersal of opposite-sex relatives in collation (Wang and Lu 2014) and cooperative breeding is kin based (Du and Lu 2009 ). In a previous study of the population, we reported a high-level EPP (Wang and Lu 2011 ). The current paper added new data of six years and used more polymorphic microsatellite markers subsequently developed for this species to identify parentages and examine relatedness. Our objects are to 1) provide an overall picture of genetic mating system of this population which covers EPP, QP and extra-pair CBP, 2) reveal drivers behind the prevalence of promiscuity, and 3) highlight the importance to examine QP and extra-pair CBP for more species. MATERIALS AND METHODS Study species Ground tits are a passerine bird inhabiting alpine meadows throughout the Tibetan plateau. They are sexually monomorphic in plumage and slightly dimorphic in body size, with males weighing 40.3 g and females 38.9 g. The birds mainly consume soil invertebrates by digging beneath the soil surface. Breeding occurs between early May and early July. Distance between nearest nest neighbor was 72.1 ± 5.1 m ( n = 231 nests). Each of socially monogamous pairs, including a few (0-13.3%, n = 9 years) with 1–2 helpers, produces a single clutch. Clutch size ranges from 4 to 9 eggs. Females incubate alone for 15–16 days, and nestlings are fed by both parents and helpers if any for 23–25 days until fledging. Territory is lacking during the breeding season, but males keep proximate to their mates who are in the fertile period. Shortly after reproduction, the birds form groups composed of 1 or 2–3 neighboring families. Dispersal and settlement occur in coalition that contains adults and juveniles of both sexes. Consequently, a fine-scale genetic structure is established within the breeding population (Wang and Lu 2014). Field work Data of this study were collected from a ground tit population in the northeastern Tibet (34°14′N, 102°20′E, 3450 m elevation) during the nine breeding seasons (2007–2009, 2011–2012, 2014, 2018–2019, 2022). Details of climates and habitats of the study area have been described elsewhere (Du and Lu 2009 ). Field work of each year followed standard protocols within a 400-ha study plot. We tried to find all ground tit nests and recorded their spatial coordinates with a handheld Garmin GPS unit. We captured adults with a 1 × 1 m mist net around the entrances of nesting burrows, and checked nestlings when they were at least 15 days old by digging a vertical shaft at the end of the nest chamber. These adults and nestlings were marked with a metal and 2–3 colored plastic leg rings to allow individual recognition. For each banded individual, a blood sample of ~ 20 µl was taken via brachial venipuncture. If eggs with unhatched embryos or dead nestlings were occasionally present in the nests, we preserved their muscle samples. We were able to find almost all nests and band almost all individuals nesting within the study plot, relying on the openness of habitats (covered only by short grasses) and the birds’ habit of burrow-nesting and roosting. Family composition and social organization were identified through field observations of banded individuals. During the breeding season, we also evaluated social association between breeders from different breeding pairs by two approaches, that is, daily observation and capture records. The former considered an association event as one witness where at least two members from two or more breeding pairs stayed together without exhibiting overt aggression; in contrast, one solitary event was defined as one witness where only one or both members of a breeding pair were encountered. Investigations were made randomly with respect to breeding pairs at time when all individuals within the population lived in pairs until most breeding pairs entered the incubation period. Capture records were made throughout the breeding season. At dusk, we blocked an active nesting-burrow with a stick, and caught the individuals in the morning and recognized their identity. Because of dispersal of both sexes, individuals in the breeding population turn over annually, with only 7% of banded adults and 4% of banded fledglings being resighted within the study plot in the next year. The pattern differs from the ground tit population in south Tibet where breeders live in permanent territories year around and young males remain on or nearby their natal territories and young females disperse; annual resighting rates of adults and male fledglings are 51% and 20%, respectively (Johannessen et al. 2011 ; Tang et al. 2017 ). Therefore, for most individuals in the population of northeast Tibet, we had only annual data on social behaviors and reproductive parameters. Our dataset included 221 nests where the social parents and their offspring both were sampled, which involved a total of 431 adults and 1170 nestlings. The earlier analyses were based on data from 2006–2009 (Wang and Lu 2011 ). In this study, we excluded those from 2006 when this study was started. In that year, individuals were mostly captured more than one week after fledging through blocking the nesting burrow, a time by which dispersal had occurred as demonstrated in subsequent work (Wang and Lu 2014) so that brood size and parentages were difficult to be identified. Genotyping sex, parentage and relatedness Adults and nestlings were molecularly sexed with two primer pairs, 3007/3112 (Ellegren and Fridolfsson 1997 ) and P2/P8 (Griffiths et al. 1998 ). Both primer pairs produced a consistent result for all individuals. The results were also verified against field observations of individual behaviors and brood patch unique to females, indicating a consistency of 99.6% (1595 of 1601). For a few cases of inconsistency, we adopt the field standard. Individuals were genotyped with a set of 23 microsatellite loci developed for ground tits (20) or their close relatives (3) (Table S1 ). Samples used in a previous study (Wang and Lu 2011 ) were updated with these markers. To assign the genetic parentages of offspring, we ran CERVUS (version 3.0.7) based on the genotype data (Kalinowski et al. 2007 ). This procedure, depending on the difference in likelihood ratio statistics between candidate parents and offspring, may give a quite high accuracy (Queller et al. 1993 ). Specifically, we identified an offspring to derive from extra-pair mating when it had at least two loci that did not match with those of its social parents, by setting the proportion of candidate parents as 98%, total proportion of loci typed as 0.99, mistyping error rates as 0.01 and likelihood calculation error rates as 0.01. All these loci gave an overall exclusionary power of 0.998 for the first parent and 0.999 for the second. A major challenge to produce reliable paternity or maternity when inbreeding occurs is the number of the investigated microsatellite loci. In this study, 23 microsatellite markers (average number of alleles = 22.5; Table S1 ), compared to 16 markers (average number of alleles = 8.5) used by our previous study (Wang and Lu 2011 ), can give highly certain results even the candidate parents were closely related (Thomson et al. 2001 ). Moreover, the high accuracy of parentage analyses may be ensures given that most breeding individuals have been sampled and thus the biological and father or mother of an offspring can be available. Among 1266 sampled young, there were 39 (3.1%) and 44 (3.5%) for which we failed to assign a banded individual as their genetic mother and father, respectively. It was likely that parents of those unassigned young nested nearby the boundaries of the study plot. Genetic relatedness between dyads of individuals was estimated using genotype data under RELATEDNESS v5.0.8 (Wang 2017 ). The estimation takes a value ranging from − 1 to + 1, where 0 denotes random sharing of alleles, while negative and positive values represent less and more allele sharing, respectively. The average of pairwise relatedness coefficients highly corresponds to pedigree-based kinship categories for a group of individuals (first-order kin, r \\(\\approx\\) 0.5; second-order kin, r \\(\\approx\\) 0.25; lower-order kin, r = 0.25 − 0.125; less related, r < 0.125; Queller and Goodnight 1989 ). Paired individuals used to estimate genetic relatedness included dyads of adults with a given relationship such as extra-pair mates and randomly choosing adults from the total samples. In the latter case, the simulation was run 10 000 times. Statistical analyses One-sample t tests were performed to evaluate whether two individuals were genetically related by comparing their relatedness with that between pairs of individuals chosen randomly from the population with the corresponding sexes under the null hypothesis of random mating. We also used independent-samples t tests to compare average offspring production by promiscuous vs. genetically monogamous individuals. Offspring quality was measured with two commonly used two indicators, that is, body mass and individual heterozygosity. The former as a nutrition-related trait that determines post-fledging survival may reflect the consequence of both preference of parental care and competitive ability of nestlings (Roulin and Dreiss 2012 ); the latter may indicate the genetic potential that an individual adapts to environments (Szulkin et al. 2010 ). With a paired-samples t test, we compared the average of each indicator between offspring resulting from extra-pair mating and their within-pair siblings. Statistical analyses were performed with R (R Development Core Team 2021). All tests were two-tailed, setting the significance level at P = 0.05. Values are given as mean ± SE. RESULTS Genetic mating systems For non-cooperative nests, 54%, 47% and 24% were found to contain at least one offspring of EPP, QP and extra-pair CBP, respectively, which corresponded with 20%, 16% and 8% of offspring; within-pair CBP occurred rarely (Table 1 ). For cooperative nests, EPP and QP occurred at rates similar to but extra-pair CBP more than those for non-cooperative nests, being 50%, 44% and 50% in brood and 18%, 23% and 15% in offspring, respectively. Male helpers in 25% nests shared within-group paternity and two female helpers that were present in two different nests shared within-group maternity (Table 1 ). For promiscuous nests, 44% contained one and 56% nests contained more than one, type of extra-pair offspring (Table S2). Table 1 Distribution of extra-pair parentages in the ground tit population. Values provided here are the percentage based on the total samples over 9 breeding seasons for a specific type of extra-pair offspring, with the range of annual variation being in brackets. Brood Offspring Non-cooperatively breeding pair (205 broods, 1171 offspring) EPP 54.1 (11.8–84.6) 20.0 (5.0-31.6) QP 47.3 (34.6–80.0) 15.5 (8.7–25.9) Extra-pair CBP 24.4 (0.0–63.0) 7.9 (0.0-19.6) Within-pair CBP 3.4 (0.0-7.4) 1.0 (0.0-2.1) Cooperatively breeding pair (16 broods, 95 offspring) Extra-group EPP 50.0 (0.0-100.0) 17.9 (0.0-47.8) Within-group EPP 25.0 (0.0–25.0) 5.3 (0.0-7.1) Extra-group QP 43.8 (0.0-100.0) 23.2 (0.0–60.0) Within-group QP 12.5 (0.0–25.0) 3.2 (0.0–13.0) Extra-pair CBP 50.0 (0.0-100.0) 14.7 (0.0-43.5) Within-pair CBP 0 0 At the individual level, 39% and 39% of independently breeding males and females were engaged in extra-pair mating, respectively. Among those multiple-mating males, 41% had one and 59% had more than one, extra-pair partner; the corresponding values for females were 47% and 53% (Table S3). In particular, one male mated with five and each of two females with six individuals outside the pair bond. Dominant breeders in cooperative nests had relatively limited extra-pair partners than breeders in non-cooperative nests. With non-cooperative and cooperative nests combined, 54% of promiscuous males produced one and 46% produced two even three types of extra-pair offspring; the corresponding values for promiscuous females were 57% and 43% (Table S4). Annual reproductive output of promiscuous vs. monogamous breeders Males who participated in extra-pair mating produced significantly more offspring than those who did not do so (independent-samples test, t 218 = 4.68, P < 0.001); the same was true when such a comparison was made between females with and without extra-pair offspring ( t 218 = 4.09, P < 0.001; Fig. 3 ). If removing extra-pair offspring, the number of within-pair offspring of those breeders engaged in promiscuity would be significantly reduced compared to that of breeders with genetic monogamy in both sexes ( t 218 > 2.72, P < 0.007), indicating that multiple-mating significantly increased annual reproductive output. Moreover, promiscuous males did not perform better in annual total offspring productivity than promiscuous females ( t 339 = 0.47, P = 0.64; Fig. 3 ). Relatedness between breeding individuals engaged in extra-pair mating Social pairs associated with EPP and QP formed between genetically unrelated individuals ( r = − 0.011–0.024, n = 90–108), but extra-pair mating took a moderate level of inbreeding (Fig. 4 ). Cuckolding and cuckolded individuals engaged in EPP were kin and those engaged in QP not, but they had relatedness significantly higher than that of randomly chosen dyads from the population ( t 128 = 6.70, P < 0.001). Social partners of cuckolding and cuckolded individuals were unrelated ( r = − 0.023 − 0.009, n = 113–129). Social pairs engaged in an extra-pair CBP event formed between non-kin (–0.018–0.063, n = 57) but extra-pair mating did between kin (Fig. 4 ). Relatedness of parasites vs. hosts for both the same and opposite sexes tended to close, although that of parasite females vs. host males did not reached 0.125 ( t 62 = 6.28, P < 0.001). Other six dyads were unrelated ( r = − 0.002–0.008, n = 57). In 12 within-pair CBP events, social pairs were inbred in parasites and not in hosts ( r = − 0.060). Kinship was present between parasite females and host males but absent between parasite and host males ( r = 0.015). Relatedness between parasite males vs. host females and between parasite and host females tended to be relatives (compared with expected by random, t 11 > 2.81, P < 0.02). Assessment for quality of offspring of different parentage Pairwise comparisons failed to find a statistically significant difference in body mass between extra-pair and their within-pair siblings for EPP, QP and extra-pair CBP (paired-samples t test, P = 0.40–0.80; Fig. 5 a). This was the case for offspring heterozygosity ( P = 0.39–0.89; Fig. 5 b). No statistical differences were evident in these two indicates between parasitized offspring and their siblings in within-pair CBP ( P = 0.40–0.59). These analyses did not include two nests in which parasitized extra- or within-pair CBP offspring were abandoned, likely because they were much younger and smaller than their siblings which had fledged and were cared by parents. Individual ties in relation to extra-pair mating Field observations showed that communal foraging of individuals from more than one breeding pair occurred more often than solitary foraging of individuals from a single breeding pair during the early (20 vs. 14 records; Fig. 6 , Supplementary vides 1) and late (18 vs. 7 records) breeding season. Seven extra-pair copulations were witnessed over a total of 12-h behavioral monitoring for ≥ two neighboring pair bonds that stayed together in their communal foraging grounds. During the pre- and egg-laying period, female breeders inspected the neighbors’ nesting-burrows in daytime suffering no aggression from the owners. Individual captures conducted during the breeding season revealed that female but never male breeders occasionally roosted in the nests of neighboring breeding pairs (13% and 7% of all records during egg-laying or incubation period and nesting period, respectively). Some of these female visitors had offspring (all the cases were proved to be QP) in the nests to which they visited (Table 2 ). They were genetically related with both the female ( r = 0.100-0.157) and male ( r = 0.207–0.230) breeders in both the breeding stages regardless of whether to have or not QP offspring. In a few cases, male breeders were demonstrated to roost in adjacent non-active burrows. Table 2 Frequency distribution of the identity of roosting individuals captured in the nests of focal females during the breeding season, providing evidence for ties between breeders. Individual identity Breeding stage of the focal nest Egg-laying or incubation period ( n = 56 nests) Nestling period ( n = 140 nests) Male breeders 44 134 Female breeders 50 137 Helpers 4 12 Female visitors leaving offspring 6 7 Female visitors without leaving offspring 9 14 Male visitors 0 0 DISCUSSION We revealed that mating system of the ground tits has two distinct features, that is, high-level promiscuity and diversity of extra-pair offspring identity. So far, the most promiscuous birds are known to be the cooperatively breeding superb fairy-wren Malurus cyaneus in which 95% of broods contained EPP offspring (Mulder et al. 1994 ). The rate of promiscuity in the ground tits is 81%, if all the three types of extra-pair offspring are pooled. More interestingly, the ground tit population has among the highest record of QP in birds (Griffith et al. 2004 ), and extra-pair CBP is a phenomenon never described before. What forces have shaped this genetic mating model? The ultimate driver must be direct fitness benefits from extra-pair mating. Individuals attempt to pass their genes as much as possible. Males of socially monogamous birds do so by mating outside the pair bond and having the resultant offspring raised in nests of the female mating partners (Griffith et al. 2002 ; Brouwer and Griffith 2019 ). However, females engaged in an EPP event cannot increase their offspring production, and good or compatible genes as the potential indirect benefits are less likely (Sardell et al. 2012 ). QP and extra-pair CBP thus should be effective routes for female birds to increase offspring production given that females can produce eggs more than actual clutch size (phenotypic plasticity; Lack 1968 ) and determine where to lay eggs. This may explain the puzzle of why female birds actively seek extra-pair copulations when full fertility can be provided by a single male (Westneat and Stewart 2003 ). Our finding suggests that EPP, QP and extra-pair CBP can be integrated into a framework of extra-pair mating in which individuals of each sex pursues to increase their own reproductive output. Frequent individual ties facilitated by the species’ habit of burrow-nesting and roosting should be the proximate driver of extra-pair mating. Ground tits’ burrows have a chamber that is large enough to allow communal roosting of more than 25 individuals during the non-breeding period (Wang and Lu 2014). In the breeding season, groups break up into socially monogamous pairs and these pairs nest nearby, especially when suitable burrowing habitats are restricted to a small patch (Wang and Lu 2014). Thus, ties between breeders remain, as indicated by the observations that individuals from neighboring pairs fed together and females stayed overnight in neighboring burrows, which should increase opportunities of extra-pair copulations and egg parasitism, because songbirds mostly lay in the early morning (Haftorn 1996 ). Actually, individual ties are the core of two important ecological factors promoting promiscuity ― breeding density and synchrony (Westneat et al. 1990 ; Stutchbury and Morton 1995 ). The frequent individual ties may be thought of as resulting from good ecological conditions the ground tit population experiences (Korb and Heinze 2008 ). Across the species' range, the study area is most abundant in annual rainfall (780 mm in contrast to 300 mm on average), which implies sufficient supply of soil invertebrates, the main food resources of the birds. Indeed, the study population has a larger mean brood size at fledging (6.0) compared to the population in south Tibet with rainfall of 430 mm (Tang et al. 2017 ). The food-rich environments may explain the absence of territoriality in the study population, as expected by a theoretical model showing that well-defined, exclusive territories prevail in populations living in resource-poor environments and high population densities resulting from good habitats lead to the break-down of territoriality (Morrell and Kokko 2005 ). In addition, we also detected high-level promiscuity in four other ground tit populations located in the same climatic zone as the current study population (our unpubl. data). In contrast, the poor environments in the south Tibet could select for limited individual ties because socially monogamous pairs live in permanent territories with cooperative breeding characterized by helpers-at-the-nest. This may explain the rarity of extra-pair mating in the population (Johannessen et al. 2011 ; Tang et al. 2017 ). It has been shown that tropical environments, presumably being favorable, promote EPP at the intraspecific level (Brouwer and Griffith 2019 ). More importantly, individual ties, which are mediated by burrow-nesting and roosting and by favorable ecological conditions in the ground tit population, were kin-based and remain throughout the year. Neighboring families combine shortly after fledging, disperse in kin colony and pairs form small-scale kin structure (Wang and Lu 2014), with breeding females occasionally roosting in kin nests. Thus, inclusive fitness benefits should further facilitate the prevalence of extra-pair mating. It has been well known that the cost of raising non-descendant offspring has promoted the evolution of mate guarding against cuckolding in both males (Birkhead and Møller 1992 ) and females (Andersson et al. 2018 ) and conflict between social partners (Sheldon 2002 ). Kin-selected benefits (Hamilton 1964 ) are expected to lead to a reduction in both mate guarding and sexual conflict, thereby increasing promiscuity. In the ground tit population, two males involved in EPP and two females involved in QP were of genetic similarity. In support of our finding, relatedness between cuckolding and cuckolded males has been detected in a fish (Bose et al. 2019) and two bird species of social monogamy (Huyvaert and Parker 2010 ; Reid et al. 2016 ). In waterfowl, females were found to be less resistant to parasitic attempts by kin (Andersson et al. 2018 ). Inbreeding between extra-pair partners may also contribute to the prevailing promiscuity in the ground tit population. It has been shown that inbreeding can generate kin-selected benefits and maintain co-adapted genes (Avilés and Purcell 2012 ; Szulkin et al. 2013 ; Dorsey and Rosenthal 2022 ). In our system, social pair-bonds form randomly and extra-pair mates were genetically related, as found in some bird species (Arct et al. 2015 ), which suggests the presence of kin recognition based on the strong nepotism. The traditional assumption of inbreeding depression has been considered simplistic, especially when it is between intermediately related individuals (Helgason et al. 2008 ). Indeed, our data demonstrated that inbred and within-pair offspring were of equal quality in terms of body mass and individual heterozygosity. Actually, as a character of social species (Kokko and Ots 2006 ), inbreeding in certain circumstances is considered to constitute a regular part of animal mating systems (Szulkin et al. 2013 ). Taken together, strong kinship in the ground tit population set the stage for high-level promiscuity. The kin-mediated promiscuity, including inbreeding, may in turn increase nepotism within the population through a positive feedback loop. Associations with kin can generate a range of benefits, such as cooperatively breeding (Koenig and Dickinson 2016 ), reduced territory conflict (Bebbington et al. 2017 ) and higher survival and reproductive success (Walmsley et al. 2023 ). Our research adds promiscuity to these benefits. Finally, we argue the potential to detect promiscuity especially QP and extra-pair CBP in more socially monogamous birds. Our reasons are: 1) females increasing reproductive success should be adaptive; 2) in altricial birds studied, the proportion of species with QP (82%) is comparable with that with EPP (83%), and the averaging 8% occurrence rate at the brood level in the former indicates certain adaptive value; 3) eco-social conditions facilitating parasitism such as cavity-nesting (1800–2000 altricial species; van der Hoek et al. 2017) and fine-scale genetic structure (Hatchwell 2010 ; Camerlenghi et al. 2022 ) can be met in many cases, but the low taxonomic coverage of the existing studies on extra-pair maternity of altricial species (< 30, compared to > 360 for EPP) is less likely to include species that meet such conditions; 4) CBP has been increasingly reported in altricial taxa, accounting for 72% of examined species with a mean brood proportion of 17%, in which some of parasitic offspring could derive from extra-pair mating. Therefore, to have a broader perspective on the evolution of mating systems, future work should seek to examine QP and extra-pair CBP for more species. Declarations Acknowledgements We thank B. Du, YH, Li, L.Y. Kang and many volunteers who provided assistance in field data collection and lab analysis. Author contribution X.L. and C.W. designed the study. C.W. and S. C. collected and analyzed the data. X.L. wrote the paper with contributions from C.W. and S.C. Funding This research was supported by the National Natural Science Foundation of China (31830085) and the Second Tibetan Plateau Scientific Expedition and Research program (2019QZKK0501). Data availability The dataset analyzed during the current study is available in the figshare repository, 10.6084/m9.figshare.23666949. Ethics approval Our study was approved by the Law of the People’s Republic of China on the Protection of Wildlife (reference 19881108) and Provisions of the Tibet Autonomous Region for the Protection of Wild Animals and Wild Plants (reference 20091001). The field and lab procedures were approved by the ethics committee of Wuhan University. Competing interests The authors declare no competing interests. Supplementary Information The online version contains supplementary material available at. References Andersson M, Åhlund M, Waldeck P (2018) Brood parasitism, relatedness and sociality: a kinship role in female reproductive tactics. Biol Rev 94:307–327. https://doi.org/10.1111/brv.12455 Arct A, Drobniak SM, Cichoń M (2015) Genetic similarity between mates predicts extrapair paternity — a meta-analysis of bird studies. 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Morrell LJ, Kokko H (2005) Bridging the gap between mechanistic and adaptive explanations of territory formation. Behav Ecol Sociobiol 57: 381–390. https://doi.org/10.1007/s00265-004-0859-5 Mulder RA, Dunn PO, Cockburn A, Cohen KAL, Howell MJ (1994) Helpers liberate female fairy-wrens from constraints on extra-pair mate choice. P Roy Soc Lond B Bio 255:223–229. https://doi.org/10.1098/rspb.1994.0032 Queller DC, Goodnight KF (1989) Estimating relatedness using genetic markers. Evolution 43:258–275. https://doi.org/10.1111/j.1558-5646.1989.tb04226.x Queller DC, Strassmann JE, Hughes CR (1993) Microsatellites and kinship. Trends Ecol Evol 8:285–288. https://doi.org/10.1016/0169-5347(93)90256-O R Core Team (2021) R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. https://www.r-project.org/ Reid JM, Bocedi G, Nietlisbach P, Duthie AB, Wolak ME, Gow EA, Arcese P (2016) Variation in parent-offspring kinship in socially monogamous systems with extra-pair reproduction and inbreeding. Evolution 70:1512–1529. https://doi.org/10.1111/evo.12953 Roulin A, Dreiss AN (2012) Sibling competition and cooperation over parental care. In: Royle NJ, Smiseth PT, Kölliker M (eds) The evolution of parental care. Oxford University Press, Oxford, pp 133–149. Sardell RJ, Arcese P, Keller LF, Reid JM (2012) Are there indirect fitness benefits of female extra-pair reproduction? Lifetime reproductive success of within-pair and extra-pair offspring. Am Nat 179:779–793. https://doi.org/10.1086/665665 Sheldon BC (2002) Relating paternity to paternal care. Philos T Roy Soc B 357:341–350. https://doi.org/10.1098/rstb.2001.0931 Stutchbury BJ, Morton ES (1995) The effect of breeding synchrony on extra-pair mating systems in songbirds. Behaviour 132:675–690. https://www.jstor.org/stable/4535292 Szulkin M, Bierne N, David P (2010) Heterozygosity-fitness correlations: a time for reappraisal. Evolution 64:1202–1217. https://doi.org/10.1111/j.1558-5646.2010.00966.x Szulkin M, Stopher KV, Pemberton JM, Reid JM (2013) Inbreeding avoidance, tolerance, or preference in animals? Trends Ecol Evol 28:205–211. https://doi.org/10.1016/j.tree.2012.10.016 Tang SY, Ke DH, Yu TL, Wang CC, Zhao QT, Fan HY, Zhang GY, Wang C, Lu X (2017) Social organisation, demography and genetic mating system of a Tibetan cooperative breeder. Ibis 159:687–692. https://doi.org/10.1111/ibi.12485 Thomson JA, Ayres KL, Pilotti V, Barrett MN, Walker JIH, Debenham PG (2001) Analysis of disputed single-parent/child and sibling relationships using 16 STR loci. Int J Legal Med 115:128-134. https://doi.org/10.1007/s004140100212 Walmsley SF, Boutin S, Dantzer B, Lane JE, Coltman DW, McAdam AG (2023) Benefits of living closer to kin vary by genealogical relationship in a territorial mammal. P Roy Soc Lond B Bio 290: 20221569. https://doi.org/10.1098/rspb.2022.1569 Wang C, Lu X (2011) Female ground tits prefer relatives as extra-pair partners: driven by kin selection? Mol Ecol 20:2851–2863. https://doi.org/10.1111/j.1365-294X.2011.05070.x Wang C, Lu X (2014a) Dispersal in kin coalition throughout the non-breeding season to facilitate fine-scale genetic structure in the breeding season: evidence from a small passerine. Ethology 120:1003–1012. https://doi.org/10.1111/eth.12273 Wang J (2017) Estimating pairwise relatedness in a small sample of individuals. Heredity 119:302–313. https://doi.org/10.1038/hdy.2017.52 Westneat DF, Sherman PW, Morton ML (1990) The ecology and evolution of extra-pair copulations in birds. In: Power DM (ed) Current Ornithology, vol 7. Kluwer Academic/Plenum Publishers, New York, pp 331–369. Westneat DF, Stewart IRK (2003) Extra-pair paternity in birds: Causes, correlates, and conflict. Annu Rev Ecol Evol Syst 34:365–396. https://doi.org/10.1146/annurev.ecolsys.34.011802.132439 Supplementary Files Supp.Tables.docx Socialtiesbetweenneighboringpairsofgroundtits.mp4 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-3163972\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":true,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":221707548,\"identity\":\"55575f80-174f-4e92-8dbb-1de79ec12dc5\",\"order_by\":0,\"name\":\"Chen Wang\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Wuhan University College of Life Sciences\",\"correspondingAuthor\":false,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Chen\",\"middleName\":\"\",\"lastName\":\"Wang\",\"suffix\":\"\"},{\"id\":221707549,\"identity\":\"fdf7e23f-f669-4464-b613-1712cb786038\",\"order_by\":1,\"name\":\"Shicheng Chen\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Wuhan University College of Life Sciences\",\"correspondingAuthor\":false,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Shicheng\",\"middleName\":\"\",\"lastName\":\"Chen\",\"suffix\":\"\"},{\"id\":221707550,\"identity\":\"cdd7cfe1-e6c2-4d0b-9c91-7845d3c05e48\",\"order_by\":2,\"name\":\"Xin Lu\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAApElEQVRIiWNgGAWjYDACdgbGB4wNIFYCsVqYGZgNSNbCJkGaFoPDPGbVvDsOM/Cz5xgw/NxBhBbJZh6z27xnDjNI9rwxYOw9Q4QWfmaglty2wwwGN3IMmBnbiNDCBtRSDNJiT7QWkC3MYFskiNUi2cxWLP23LZ1H4syzgoO9xGgxON688ePMNms5/vbkjQ9+EqMFBnhAxAESNIyCUTAKRsEowAcApj8tygR+o1gAAAAASUVORK5CYII=\",\"orcid\":\"https://orcid.org/0000-0001-7771-0287\",\"institution\":\"Wuhan University College of Life Sciences\",\"correspondingAuthor\":true,\"submittingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Xin\",\"middleName\":\"\",\"lastName\":\"Lu\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2023-07-12 13:38:09\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-3163972/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-3163972/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":40842807,\"identity\":\"204af3fe-84ea-46f8-8051-76f6a9e5f11b\",\"added_by\":\"auto\",\"created_at\":\"2023-07-31 19:22:24\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":57072,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eA schematic illustration of three alternative placements where females lay extra-pair eggs as compared to within-pair CBP, along with their occurrence rates in altricial species. An ellipse represents a social pair -bond and the red line represents extra-pair mating relationship, with the black arrows direct the nest where the extra-pair eggs are deposited. % species = species with extra-pair parentage/all species examined, % broods = mean ± SE of broods with extra-pair parentage (studied populations), % offspring = mean ± SE of offspring with extra-pair parentage (studied populations). In QP, Eastern Kingbirds (\\u003cem\\u003eTyrannus tyrannus\\u003c/em\\u003e) is not included (Griffith et al. 2004), and in CBP, extra- and within-pair mating are not separated.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3163972/v1/0e4bea5d56a3fe174fb36e29.png\"},{\"id\":40842808,\"identity\":\"4c7f97a3-f11a-4774-b6fd-6f32a73551eb\",\"added_by\":\"auto\",\"created_at\":\"2023-07-31 19:22:24\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":480556,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eTibetan ground tits and their habitats. In response to the harsh conditions, the birds nest and roost in burrows constructed by themselves, which consist of an 80-290 cm long tunnel in earth, with a ball-shaped chamber at the tunnel end. Illustration by Y. Chen.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3163972/v1/e90ac26123b53c82adfe75f4.png\"},{\"id\":40842806,\"identity\":\"724ec96a-db26-44fb-ad3b-22aaf1373f80\",\"added_by\":\"auto\",\"created_at\":\"2023-07-31 19:22:24\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":26783,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eThe mean number (± SE) of annual total, within- and extra-pair offspring produced by promiscuous vs. monogamous male and female breeders. A, B and C inside the columns represent total, within- and extra-pair offspring, and sample size is given above SE bars.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3163972/v1/d30e07763d72f5d0116d5692.png\"},{\"id\":40842809,\"identity\":\"31739af9-5365-43ae-8a5f-b95450cf117d\",\"added_by\":\"auto\",\"created_at\":\"2023-07-31 19:22:24\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":75660,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eRelatedness between individuals involved in an event of QP and EPP. The value was compared with the relatedness between dyadic breeders of the same or opposite sex randomly chosen from the study population, with the significant difference shown in bold. Socially paired members are presented with the same colors. Lines represent social relationship between two individuals, with extra-pair mating one highlighted with red.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3163972/v1/61ee07aadedc27ed2a076852.png\"},{\"id\":40843112,\"identity\":\"3a9367f3-26d3-4137-a6cb-83ea85ab71a9\",\"added_by\":\"auto\",\"created_at\":\"2023-07-31 19:30:24\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":40889,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eMean (± SE) of body mass (a) and individual heterozygosity (b) between EPP, QP, extra-pair CBP and within-pair CBP offspring and their within-pair siblings. Sample size is given above SE bars.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3163972/v1/9e3bf6a3bfd713179db681ce.png\"},{\"id\":40842810,\"identity\":\"de2701fe-4761-4b12-b552-b5f4e5dbcd03\",\"added_by\":\"auto\",\"created_at\":\"2023-07-31 19:22:24\",\"extension\":\"png\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":414929,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eSocial ties were frequent between neighboring pairs of ground tits during their pre- and egg-laying period. Photograph by C. Wang.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"6.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3163972/v1/f66fd5f1af8605a40d31521b.png\"},{\"id\":44371882,\"identity\":\"a261b41f-d9ba-4671-bd69-48dbf78b279b\",\"added_by\":\"auto\",\"created_at\":\"2023-10-10 15:56:06\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":1322759,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3163972/v1/542e411f-ea6a-4e77-badc-1adf22482b80.pdf\"},{\"id\":40842805,\"identity\":\"97647e18-1269-4cab-b7de-71de53f9a7b1\",\"added_by\":\"auto\",\"created_at\":\"2023-07-31 19:22:24\",\"extension\":\"docx\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":23051,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"Supp.Tables.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3163972/v1/8a892a7e97d6489b50178c49.docx\"},{\"id\":40842812,\"identity\":\"c6711ee1-5e91-4bc0-8211-53799cff5beb\",\"added_by\":\"auto\",\"created_at\":\"2023-07-31 19:22:25\",\"extension\":\"mp4\",\"order_by\":2,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":6391713,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"Socialtiesbetweenneighboringpairsofgroundtits.mp4\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-3163972/v1/83854ee7cf79fcf81d2e7bcb.mp4\"}],\"financialInterests\":\"\",\"formattedTitle\":\"Where are extra-pair offspring raised: the mother’s, the father’s or the others’ nest?\",\"fulltext\":[{\"header\":\"Significance Statement\",\"content\":\"\\u003cp\\u003eTheoretically, females involving in extra-pair mating may lay the resultant eggs in nests of their own or their mating partners, accordingly leading to extra-pair paternity and quasi-parasitism. While a large body of literature about the former has existed, much less attention has been paid to the latter likely because of its low incidence in terms of the proportions of species or nests within a population. The high-level quasi-parasitism found in Tibetan ground tits may be well interpreted with the prevailing nepotism in association with the burrow-nesting and roosting lifestyle if the species. The current results lead to the suggestion that in socially monogamous systems, not only males but also females pursue extra-pair mating to increase reproductive success. This sheds light on the puzzle of why females routinely engage in extra-pair mating.\\u003c/p\\u003e\"},{\"header\":\"INTRODUCTION\",\"content\":\"\\u003cp\\u003eSocial monogamy with biparental care is the most common mating system in birds (Cockburn \\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e2006\\u003c/span\\u003e). Meanwhile, copulations outside the socially monogamous pair bond prevail, as revealed by molecular techniques (Griffith et al. \\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e2002\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e2004\\u003c/span\\u003e). For an event of extra-pair mating, females may lay the resultant eggs in either their own or the mating males\\u0026rsquo; nests, accordingly generating extra-pair paternity (EPP) or maternity, with the latter termed as quasi-parasitism (QP) to distinguish from conspecific brood parasitism (CBP) ― a nest contains eggs not laid by the pair bond. Alternatively, females may lay the eggs fertilized through extra-pair mating in the nests of other breeding pairs and this leads to extra-pair CBP, which is in contrast to within-CBP ― a nest contains eggs laid by other pair bond (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eDetected in 76% of 364 altricial bird species documented so far with an averaging brood occurrence rate of 37% (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e), EPP is widespread and has been a subject of extensive research over past 30 years (Griffith et al. \\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e2002\\u003c/span\\u003e; Brouwer and Griffith \\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e). These studies have shaped our current view of alternative mating strategy, which argues that socially monogamous males increase their reproductive success by EPP without paying the costs of parental care and accordingly male mate-guarding behaviors against cuckoldry have evolved (Birkhead and M\\u0026oslash;ller \\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e1992\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eEvolution theory suggests that selection also favors females to enhance reproductive output by means of QP and extra-pair CBP. However, empirical studies show that QP occurs rarely, which on average is 8% at the brood level for 22 altricial species examined so far (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e). A review paper based on the findings from 12 species thought that QP is an occasional event and accordingly, few ecological and evolutionary hypotheses have been proposed to explore its adaptive implications (Griffith et al. \\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e2004\\u003c/span\\u003e). So far, no attempts have been made to confirm the presence of extra-pair CBP.\\u003c/p\\u003e \\u003cp\\u003eThe scarcity of QP and extra-pair CBP may be attributed to potential limitations to parasitism in terms of behavioral difficulty for a female to lay eggs in other individuals\\u0026rsquo; nests. For example, a successful parasitism depends on the probability that parasites synchronize their breeding time with potential host females and overcome resistance from the hosts who undertake parental care for non-descendant offspring.\\u003c/p\\u003e \\u003cp\\u003eNevertheless, the constraints on parasitism may be mitigated in certain circumstances. Sociality characterized by frequent interactions between breeding individuals should be a factor that can break up the constraints, because it may make females finding synchronously breeding hosts easy. Furthermore, social living is usually kin-based given the prevalence of fine-scale genetic structure of breeding populations (Hatchwell \\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e2010\\u003c/span\\u003e). According to kin selection theory (Hamilton \\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e1964\\u003c/span\\u003e), indirect fitness accrued from kin interactions such as kinship care for non-descendant offspring can reduce the resistance of hosts, as seen in CBP in waterfowl (Andersson et al. \\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e). In addition, specific lifestyles in some species such as cavity nesting (Geffen and Yom-Tov \\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e2001\\u003c/span\\u003e) may provide convenience for females\\u0026rsquo; parasitic attempts. It is worth pointing out that empirical examination of QP accounts for only 5% of the existing 619 studies on parentage of altricial birds. The very low taxonomic coverage (28 species) could reduce the opportunity to find more species that exhibit high-level extra-pair maternity.\\u003c/p\\u003e \\u003cp\\u003eHere we describe the high incidences of EPP, QP and extra-pair CBP in a population of Tibetan ground tits \\u003cem\\u003ePseudopodoces humilis\\u003c/em\\u003e (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e). Endemic to alpine meadows of 2800\\u0026ndash;6000 m elevation in the Tibetan plateau, the birds breed and roost throughout the year in burrows excavated by themselves to buffer against cold, windy and poorly vegetated environments (Ke and Lu \\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e2009\\u003c/span\\u003e). Sociality is strong in the study population inhabits northeast Tibet where territoriality is absent, related breeders nest nearby due to dispersal of opposite-sex relatives in collation (Wang and Lu 2014) and cooperative breeding is kin based (Du and Lu \\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e2009\\u003c/span\\u003e). In a previous study of the population, we reported a high-level EPP (Wang and Lu \\u003cspan citationid=\\\"CR50\\\" class=\\\"CitationRef\\\"\\u003e2011\\u003c/span\\u003e). The current paper added new data of six years and used more polymorphic microsatellite markers subsequently developed for this species to identify parentages and examine relatedness. Our objects are to 1) provide an overall picture of genetic mating system of this population which covers EPP, QP and extra-pair CBP, 2) reveal drivers behind the prevalence of promiscuity, and 3) highlight the importance to examine QP and extra-pair CBP for more species.\\u003c/p\\u003e\"},{\"header\":\"MATERIALS AND METHODS\",\"content\":\"\\u003cp\\u003e \\u003cb\\u003eStudy species\\u003c/b\\u003e \\u003c/p\\u003e \\u003cp\\u003eGround tits are a passerine bird inhabiting alpine meadows throughout the Tibetan plateau. They are sexually monomorphic in plumage and slightly dimorphic in body size, with males weighing 40.3 g and females 38.9 g. The birds mainly consume soil invertebrates by digging beneath the soil surface.\\u003c/p\\u003e \\u003cp\\u003eBreeding occurs between early May and early July. Distance between nearest nest neighbor was 72.1\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;5.1 m (\\u003cem\\u003en\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;231 nests). Each of socially monogamous pairs, including a few (0-13.3%, \\u003cem\\u003en\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;9 years) with 1\\u0026ndash;2 helpers, produces a single clutch. Clutch size ranges from 4 to 9 eggs. Females incubate alone for 15\\u0026ndash;16 days, and nestlings are fed by both parents and helpers if any for 23\\u0026ndash;25 days until fledging. Territory is lacking during the breeding season, but males keep proximate to their mates who are in the fertile period.\\u003c/p\\u003e \\u003cp\\u003eShortly after reproduction, the birds form groups composed of 1 or 2\\u0026ndash;3 neighboring families. Dispersal and settlement occur in coalition that contains adults and juveniles of both sexes. Consequently, a fine-scale genetic structure is established within the breeding population (Wang and Lu 2014).\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eField work\\u003c/b\\u003e \\u003c/p\\u003e \\u003cp\\u003eData of this study were collected from a ground tit population in the northeastern Tibet (34\\u0026deg;14\\u0026prime;N, 102\\u0026deg;20\\u0026prime;E, 3450 m elevation) during the nine breeding seasons (2007\\u0026ndash;2009, 2011\\u0026ndash;2012, 2014, 2018\\u0026ndash;2019, 2022). Details of climates and habitats of the study area have been described elsewhere (Du and Lu \\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e2009\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003e Field work of each year followed standard protocols within a 400-ha study plot. We tried to find all ground tit nests and recorded their spatial coordinates with a handheld Garmin GPS unit. We captured adults with a 1 \\u0026times; 1 m mist net around the entrances of nesting burrows, and checked nestlings when they were at least 15 days old by digging a vertical shaft at the end of the nest chamber. These adults and nestlings were marked with a metal and 2\\u0026ndash;3 colored plastic leg rings to allow individual recognition. For each banded individual, a blood sample of ~\\u0026thinsp;20 \\u0026micro;l was taken via brachial venipuncture. If eggs with unhatched embryos or dead nestlings were occasionally present in the nests, we preserved their muscle samples. We were able to find almost all nests and band almost all individuals nesting within the study plot, relying on the openness of habitats (covered only by short grasses) and the birds\\u0026rsquo; habit of burrow-nesting and roosting.\\u003c/p\\u003e \\u003cp\\u003eFamily composition and social organization were identified through field observations of banded individuals. During the breeding season, we also evaluated social association between breeders from different breeding pairs by two approaches, that is, daily observation and capture records. The former considered an association event as one witness where at least two members from two or more breeding pairs stayed together without exhibiting overt aggression; in contrast, one solitary event was defined as one witness where only one or both members of a breeding pair were encountered. Investigations were made randomly with respect to breeding pairs at time when all individuals within the population lived in pairs until most breeding pairs entered the incubation period. Capture records were made throughout the breeding season. At dusk, we blocked an active nesting-burrow with a stick, and caught the individuals in the morning and recognized their identity.\\u003c/p\\u003e \\u003cp\\u003eBecause of dispersal of both sexes, individuals in the breeding population turn over annually, with only 7% of banded adults and 4% of banded fledglings being resighted within the study plot in the next year. The pattern differs from the ground tit population in south Tibet where breeders live in permanent territories year around and young males remain on or nearby their natal territories and young females disperse; annual resighting rates of adults and male fledglings are 51% and 20%, respectively (Johannessen et al. \\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e2011\\u003c/span\\u003e; Tang et al. \\u003cspan citationid=\\\"CR47\\\" class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e). Therefore, for most individuals in the population of northeast Tibet, we had only annual data on social behaviors and reproductive parameters.\\u003c/p\\u003e \\u003cp\\u003eOur dataset included 221 nests where the social parents and their offspring both were sampled, which involved a total of 431 adults and 1170 nestlings. The earlier analyses were based on data from 2006\\u0026ndash;2009 (Wang and Lu \\u003cspan citationid=\\\"CR50\\\" class=\\\"CitationRef\\\"\\u003e2011\\u003c/span\\u003e). In this study, we excluded those from 2006 when this study was started. In that year, individuals were mostly captured more than one week after fledging through blocking the nesting burrow, a time by which dispersal had occurred as demonstrated in subsequent work (Wang and Lu 2014) so that brood size and parentages were difficult to be identified.\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eGenotyping sex, parentage and relatedness\\u003c/b\\u003e \\u003c/p\\u003e \\u003cp\\u003eAdults and nestlings were molecularly sexed with two primer pairs, 3007/3112 (Ellegren and Fridolfsson \\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e1997\\u003c/span\\u003e) and P2/P8 (Griffiths et al. \\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e1998\\u003c/span\\u003e). Both primer pairs produced a consistent result for all individuals. The results were also verified against field observations of individual behaviors and brood patch unique to females, indicating a consistency of 99.6% (1595 of 1601). For a few cases of inconsistency, we adopt the field standard.\\u003c/p\\u003e \\u003cp\\u003eIndividuals were genotyped with a set of 23 microsatellite loci developed for ground tits (20) or their close relatives (3) (Table \\u003cspan refid=\\\"MOESM1\\\" class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003e). Samples used in a previous study (Wang and Lu \\u003cspan citationid=\\\"CR50\\\" class=\\\"CitationRef\\\"\\u003e2011\\u003c/span\\u003e) were updated with these markers. To assign the genetic parentages of offspring, we ran CERVUS (version 3.0.7) based on the genotype data (Kalinowski et al. \\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e2007\\u003c/span\\u003e). This procedure, depending on the difference in likelihood ratio statistics between candidate parents and offspring, may give a quite high accuracy (Queller et al. \\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e1993\\u003c/span\\u003e). Specifically, we identified an offspring to derive from extra-pair mating when it had at least two loci that did not match with those of its social parents, by setting the proportion of candidate parents as 98%, total proportion of loci typed as 0.99, mistyping error rates as 0.01 and likelihood calculation error rates as 0.01. All these loci gave an overall exclusionary power of 0.998 for the first parent and 0.999 for the second.\\u003c/p\\u003e \\u003cp\\u003eA major challenge to produce reliable paternity or maternity when inbreeding occurs is the number of the investigated microsatellite loci. In this study, 23 microsatellite markers (average number of alleles\\u0026thinsp;=\\u0026thinsp;22.5; Table \\u003cspan refid=\\\"MOESM1\\\" class=\\\"InternalRef\\\"\\u003eS1\\u003c/span\\u003e), compared to 16 markers (average number of alleles\\u0026thinsp;=\\u0026thinsp;8.5) used by our previous study (Wang and Lu \\u003cspan citationid=\\\"CR50\\\" class=\\\"CitationRef\\\"\\u003e2011\\u003c/span\\u003e), can give highly certain results even the candidate parents were closely related (Thomson et al. \\u003cspan citationid=\\\"CR48\\\" class=\\\"CitationRef\\\"\\u003e2001\\u003c/span\\u003e). Moreover, the high accuracy of parentage analyses may be ensures given that most breeding individuals have been sampled and thus the biological and father or mother of an offspring can be available. Among 1266 sampled young, there were 39 (3.1%) and 44 (3.5%) for which we failed to assign a banded individual as their genetic mother and father, respectively. It was likely that parents of those unassigned young nested nearby the boundaries of the study plot.\\u003c/p\\u003e \\u003cp\\u003eGenetic relatedness between dyads of individuals was estimated using genotype data under RELATEDNESS v5.0.8 (Wang \\u003cspan citationid=\\\"CR52\\\" class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e). The estimation takes a value ranging from \\u0026minus;\\u0026thinsp;1 to +\\u0026thinsp;1, where 0 denotes random sharing of alleles, while negative and positive values represent less and more allele sharing, respectively. The average of pairwise relatedness coefficients highly corresponds to pedigree-based kinship categories for a group of individuals (first-order kin, \\u003cem\\u003er\\u003c/em\\u003e\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\approx\\\\)\\u003c/span\\u003e\\u003c/span\\u003e0.5; second-order kin, \\u003cem\\u003er\\u003c/em\\u003e\\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\approx\\\\)\\u003c/span\\u003e\\u003c/span\\u003e0.25; lower-order kin, \\u003cem\\u003er\\u003c/em\\u003e = 0.25\\u0026thinsp;\\u0026minus;\\u0026thinsp;0.125; less related, \\u003cem\\u003er\\u003c/em\\u003e \\u0026lt; 0.125; Queller and Goodnight \\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e1989\\u003c/span\\u003e). Paired individuals used to estimate genetic relatedness included dyads of adults with a given relationship such as extra-pair mates and randomly choosing adults from the total samples. In the latter case, the simulation was run 10 000 times.\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eStatistical analyses\\u003c/b\\u003e \\u003c/p\\u003e \\u003cp\\u003eOne-sample \\u003cem\\u003et\\u003c/em\\u003e tests were performed to evaluate whether two individuals were genetically related by comparing their relatedness with that between pairs of individuals chosen randomly from the population with the corresponding sexes under the null hypothesis of random mating. We also used independent-samples \\u003cem\\u003et\\u003c/em\\u003e tests to compare average offspring production by promiscuous vs. genetically monogamous individuals.\\u003c/p\\u003e \\u003cp\\u003eOffspring quality was measured with two commonly used two indicators, that is, body mass and individual heterozygosity. The former as a nutrition-related trait that determines post-fledging survival may reflect the consequence of both preference of parental care and competitive ability of nestlings (Roulin and Dreiss \\u003cspan citationid=\\\"CR41\\\" class=\\\"CitationRef\\\"\\u003e2012\\u003c/span\\u003e); the latter may indicate the genetic potential that an individual adapts to environments (Szulkin et al. \\u003cspan citationid=\\\"CR45\\\" class=\\\"CitationRef\\\"\\u003e2010\\u003c/span\\u003e). With a paired-samples \\u003cem\\u003et\\u003c/em\\u003e test, we compared the average of each indicator between offspring resulting from extra-pair mating and their within-pair siblings.\\u003c/p\\u003e \\u003cp\\u003eStatistical analyses were performed with R (R Development Core Team 2021). All tests were two-tailed, setting the significance level at \\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;0.05. Values are given as mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;SE.\\u003c/p\\u003e \"},{\"header\":\"RESULTS\",\"content\":\"\\u003cp\\u003e \\u003cb\\u003eGenetic mating systems\\u003c/b\\u003e \\u003c/p\\u003e \\u003cp\\u003eFor non-cooperative nests, 54%, 47% and 24% were found to contain at least one offspring of EPP, QP and extra-pair CBP, respectively, which corresponded with 20%, 16% and 8% of offspring; within-pair CBP occurred rarely (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e). For cooperative nests, EPP and QP occurred at rates similar to but extra-pair CBP more than those for non-cooperative nests, being 50%, 44% and 50% in brood and 18%, 23% and 15% in offspring, respectively. Male helpers in 25% nests shared within-group paternity and two female helpers that were present in two different nests shared within-group maternity (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eFor promiscuous nests, 44% contained one and 56% nests contained more than one, type of extra-pair offspring (Table S2).\\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\\u003eDistribution of extra-pair parentages in the ground tit population. Values provided here are the percentage based on the total samples over 9 breeding seasons for a specific type of extra-pair offspring, with the range of annual variation being in brackets.\\u003c/p\\u003e \\u003c/div\\u003e \\u003c/caption\\u003e \\u003ccolgroup cols=\\\"3\\\"\\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 \\u003cthead\\u003e \\u003ctr\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u0026nbsp;\\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003eBrood\\u003c/p\\u003e \\u003c/th\\u003e \\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003eOffspring\\u003c/p\\u003e \\u003c/th\\u003e \\u003c/tr\\u003e \\u003c/thead\\u003e \\u003ctbody\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eNon-cooperatively breeding pair\\u003c/p\\u003e \\u003cp\\u003e(205 broods, 1171 offspring)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eEPP\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e54.1 (11.8\\u0026ndash;84.6)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e20.0 (5.0-31.6)\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eQP\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e47.3 (34.6\\u0026ndash;80.0)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e15.5 (8.7\\u0026ndash;25.9)\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eExtra-pair CBP\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e24.4 (0.0\\u0026ndash;63.0)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e7.9 (0.0-19.6)\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eWithin-pair CBP\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e3.4 (0.0-7.4)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e1.0 (0.0-2.1)\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eCooperatively breeding pair\\u003c/p\\u003e \\u003cp\\u003e(16 broods, 95 offspring)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u0026nbsp;\\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eExtra-group EPP\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e50.0 (0.0-100.0)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e17.9 (0.0-47.8)\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eWithin-group EPP\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e25.0 (0.0\\u0026ndash;25.0)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e5.3 (0.0-7.1)\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eExtra-group QP\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e43.8 (0.0-100.0)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e23.2 (0.0\\u0026ndash;60.0)\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eWithin-group QP\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e12.5 (0.0\\u0026ndash;25.0)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e3.2 (0.0\\u0026ndash;13.0)\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eExtra-pair CBP\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e50.0 (0.0-100.0)\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e14.7 (0.0-43.5)\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003ctr\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e \\u003cp\\u003eWithin-pair CBP\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/td\\u003e \\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e \\u003cp\\u003e0\\u003c/p\\u003e \\u003c/td\\u003e \\u003c/tr\\u003e \\u003c/tbody\\u003e \\u003c/colgroup\\u003e \\u003c/table\\u003e\\u003c/div\\u003e \\u003c/p\\u003e \\u003cp\\u003eAt the individual level, 39% and 39% of independently breeding males and females were engaged in extra-pair mating, respectively. Among those multiple-mating males, 41% had one and 59% had more than one, extra-pair partner; the corresponding values for females were 47% and 53% (Table S3). In particular, one male mated with five and each of two females with six individuals outside the pair bond. Dominant breeders in cooperative nests had relatively limited extra-pair partners than breeders in non-cooperative nests. With non-cooperative and cooperative nests combined, 54% of promiscuous males produced one and 46% produced two even three types of extra-pair offspring; the corresponding values for promiscuous females were 57% and 43% (Table S4).\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eAnnual reproductive output of promiscuous vs. monogamous breeders\\u003c/b\\u003e \\u003c/p\\u003e \\u003cp\\u003eMales who participated in extra-pair mating produced significantly more offspring than those who did not do so (independent-samples test, \\u003cem\\u003et\\u003c/em\\u003e\\u003csub\\u003e218\\u003c/sub\\u003e\\u0026thinsp;=\\u0026thinsp;4.68, \\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001); the same was true when such a comparison was made between females with and without extra-pair offspring (\\u003cem\\u003et\\u003c/em\\u003e\\u003csub\\u003e218\\u003c/sub\\u003e\\u0026thinsp;=\\u0026thinsp;4.09, \\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001; Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e). If removing extra-pair offspring, the number of within-pair offspring of those breeders engaged in promiscuity would be significantly reduced compared to that of breeders with genetic monogamy in both sexes (\\u003cem\\u003et\\u003c/em\\u003e\\u003csub\\u003e218\\u003c/sub\\u003e\\u0026thinsp;\\u0026gt;\\u0026thinsp;2.72, \\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.007), indicating that multiple-mating significantly increased annual reproductive output.\\u003c/p\\u003e \\u003cp\\u003eMoreover, promiscuous males did not perform better in annual total offspring productivity than promiscuous females (\\u003cem\\u003et\\u003c/em\\u003e\\u003csub\\u003e339\\u003c/sub\\u003e\\u0026thinsp;=\\u0026thinsp;0.47, \\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;0.64; Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eRelatedness between breeding individuals engaged in extra-pair mating\\u003c/b\\u003e \\u003c/p\\u003e \\u003cp\\u003eSocial pairs associated with EPP and QP formed between genetically unrelated individuals (\\u003cem\\u003er\\u003c/em\\u003e = \\u0026minus;\\u0026thinsp;0.011\\u0026ndash;0.024, \\u003cem\\u003en\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;90\\u0026ndash;108), but extra-pair mating took a moderate level of inbreeding (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e). Cuckolding and cuckolded individuals engaged in EPP were kin and those engaged in QP not, but they had relatedness significantly higher than that of randomly chosen dyads from the population (\\u003cem\\u003et\\u003c/em\\u003e\\u003csub\\u003e128\\u003c/sub\\u003e\\u0026thinsp;=\\u0026thinsp;6.70, \\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001). Social partners of cuckolding and cuckolded individuals were unrelated (\\u003cem\\u003er\\u003c/em\\u003e = \\u0026minus;\\u0026thinsp;0.023\\u0026thinsp;\\u0026minus;\\u0026thinsp;0.009, \\u003cem\\u003en\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;113\\u0026ndash;129).\\u003c/p\\u003e \\u003cp\\u003eSocial pairs engaged in an extra-pair CBP event formed between non-kin (\\u0026ndash;0.018\\u0026ndash;0.063, \\u003cem\\u003en\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;57) but extra-pair mating did between kin (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e). Relatedness of parasites vs. hosts for both the same and opposite sexes tended to close, although that of parasite females vs. host males did not reached 0.125 (\\u003cem\\u003et\\u003c/em\\u003e\\u003csub\\u003e62\\u003c/sub\\u003e\\u0026thinsp;=\\u0026thinsp;6.28, \\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001). Other six dyads were unrelated (\\u003cem\\u003er\\u003c/em\\u003e = \\u0026minus;\\u0026thinsp;0.002\\u0026ndash;0.008, \\u003cem\\u003en\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;57).\\u003c/p\\u003e \\u003cp\\u003eIn 12 within-pair CBP events, social pairs were inbred in parasites and not in hosts (\\u003cem\\u003er\\u003c/em\\u003e = \\u0026minus;\\u0026thinsp;0.060). Kinship was present between parasite females and host males but absent between parasite and host males (\\u003cem\\u003er\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;0.015). Relatedness between parasite males vs. host females and between parasite and host females tended to be relatives (compared with expected by random, \\u003cem\\u003et\\u003c/em\\u003e\\u003csub\\u003e11\\u003c/sub\\u003e\\u0026thinsp;\\u0026gt;\\u0026thinsp;2.81, \\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.02).\\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eAssessment for quality of offspring of different parentage\\u003c/b\\u003e \\u003c/p\\u003e \\u003cp\\u003ePairwise comparisons failed to find a statistically significant difference in body mass between extra-pair and their within-pair siblings for EPP, QP and extra-pair CBP (paired-samples \\u003cem\\u003et\\u003c/em\\u003e test, \\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;0.40\\u0026ndash;0.80; Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003ea). This was the case for offspring heterozygosity (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;0.39\\u0026ndash;0.89; Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eb). No statistical differences were evident in these two indicates between parasitized offspring and their siblings in within-pair CBP (\\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;0.40\\u0026ndash;0.59). These analyses did not include two nests in which parasitized extra- or within-pair CBP offspring were abandoned, likely because they were much younger and smaller than their siblings which had fledged and were cared by parents.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003e \\u003cb\\u003eIndividual ties in relation to extra-pair mating\\u003c/b\\u003e \\u003c/p\\u003e \\u003cp\\u003eField observations showed that communal foraging of individuals from more than one breeding pair occurred more often than solitary foraging of individuals from a single breeding pair during the early (20 vs. 14 records; Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003e, Supplementary vides 1) and late (18 vs. 7 records) breeding season. Seven extra-pair copulations were witnessed over a total of 12-h behavioral monitoring for \\u0026ge;\\u0026thinsp;two neighboring pair bonds that stayed together in their communal foraging grounds.\\u003c/p\\u003e\\u003cp\\u003eDuring the pre- and egg-laying period, female breeders inspected the neighbors\\u0026rsquo; nesting-burrows in daytime suffering no aggression from the owners. Individual captures conducted during the breeding season revealed that female but never male breeders occasionally roosted in the nests of neighboring breeding pairs (13% and 7% of all records during egg-laying or incubation period and nesting period, respectively). Some of these female visitors had offspring (all the cases were proved to be QP) in the nests to which they visited (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e). They were genetically related with both the female (\\u003cem\\u003er\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;0.100-0.157) and male (\\u003cem\\u003er\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;0.207\\u0026ndash;0.230) breeders in both the breeding stages regardless of whether to have or not QP offspring. In a few cases, male breeders were demonstrated to roost in adjacent non-active burrows.\\u003c/p\\u003e \\n\\u003cdiv class=\\\"gridtable\\\"\\u003e\\n \\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\"\\u003e\\n \\u003ctable id=\\\"Tab3\\\" border=\\\"1\\\"\\u003e\\n \\u003ccaption language=\\\"En\\\"\\u003e\\n \\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 2\\u003c/div\\u003e\\n \\u003cdiv class=\\\"CaptionContent\\\"\\u003e\\n \\u003cp\\u003eFrequency distribution of the identity of roosting individuals captured in the nests of focal females during the breeding season, providing evidence for ties between breeders.\\u003c/p\\u003e\\n \\u003c/div\\u003e\\n \\u003c/caption\\u003e\\n \\u003cthead\\u003e\\n \\u003ctr\\u003e\\n \\u003cth align=\\\"left\\\" rowspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003eIndividual identity\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\" colspan=\\\"2\\\"\\u003e\\n \\u003cp\\u003eBreeding stage of the focal nest\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eEgg-laying or incubation period\\u003c/p\\u003e\\n \\u003cp\\u003e(\\u003cem\\u003en\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;56 nests)\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003cth align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eNestling period\\u003c/p\\u003e\\n \\u003cp\\u003e(\\u003cem\\u003en\\u003c/em\\u003e\\u0026thinsp;=\\u0026thinsp;140 nests)\\u003c/p\\u003e\\n \\u003c/th\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/thead\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eMale breeders\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e44\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e134\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eFemale breeders\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e50\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e137\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eHelpers\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e4\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e12\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eFemale visitors leaving offspring\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e6\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e7\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eFemale visitors without leaving offspring\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e9\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e14\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd align=\\\"left\\\"\\u003e\\n \\u003cp\\u003eMale visitors\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e0\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd align=\\\"char\\\"\\u003e\\n \\u003cp\\u003e0\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n \\u003c/table\\u003e\\n \\u003c/div\\u003e\\n\\u003c/div\\u003e\"},{\"header\":\"DISCUSSION\",\"content\":\"\\u003cp\\u003eWe revealed that mating system of the ground tits has two distinct features, that is, high-level promiscuity and diversity of extra-pair offspring identity. So far, the most promiscuous birds are known to be the cooperatively breeding superb fairy-wren \\u003cem\\u003eMalurus cyaneus\\u003c/em\\u003e in which 95% of broods contained EPP offspring (Mulder et al. \\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e1994\\u003c/span\\u003e). The rate of promiscuity in the ground tits is 81%, if all the three types of extra-pair offspring are pooled. More interestingly, the ground tit population has among the highest record of QP in birds (Griffith et al. \\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e2004\\u003c/span\\u003e), and extra-pair CBP is a phenomenon never described before.\\u003c/p\\u003e \\u003cp\\u003eWhat forces have shaped this genetic mating model? The ultimate driver must be direct fitness benefits from extra-pair mating. Individuals attempt to pass their genes as much as possible. Males of socially monogamous birds do so by mating outside the pair bond and having the resultant offspring raised in nests of the female mating partners (Griffith et al. \\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e2002\\u003c/span\\u003e; Brouwer and Griffith \\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e). However, females engaged in an EPP event cannot increase their offspring production, and good or compatible genes as the potential indirect benefits are less likely (Sardell et al. \\u003cspan citationid=\\\"CR42\\\" class=\\\"CitationRef\\\"\\u003e2012\\u003c/span\\u003e). QP and extra-pair CBP thus should be effective routes for female birds to increase offspring production given that females can produce eggs more than actual clutch size (phenotypic plasticity; Lack \\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e1968\\u003c/span\\u003e) and determine where to lay eggs. This may explain the puzzle of why female birds actively seek extra-pair copulations when full fertility can be provided by a single male (Westneat and Stewart \\u003cspan citationid=\\\"CR54\\\" class=\\\"CitationRef\\\"\\u003e2003\\u003c/span\\u003e). Our finding suggests that EPP, QP and extra-pair CBP can be integrated into a framework of extra-pair mating in which individuals of each sex pursues to increase their own reproductive output.\\u003c/p\\u003e \\u003cp\\u003eFrequent individual ties facilitated by the species\\u0026rsquo; habit of burrow-nesting and roosting should be the proximate driver of extra-pair mating. Ground tits\\u0026rsquo; burrows have a chamber that is large enough to allow communal roosting of more than 25 individuals during the non-breeding period (Wang and Lu 2014). In the breeding season, groups break up into socially monogamous pairs and these pairs nest nearby, especially when suitable burrowing habitats are restricted to a small patch (Wang and Lu 2014). Thus, ties between breeders remain, as indicated by the observations that individuals from neighboring pairs fed together and females stayed overnight in neighboring burrows, which should increase opportunities of extra-pair copulations and egg parasitism, because songbirds mostly lay in the early morning (Haftorn \\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e1996\\u003c/span\\u003e). Actually, individual ties are the core of two important ecological factors promoting promiscuity ― breeding density and synchrony (Westneat et al. \\u003cspan citationid=\\\"CR53\\\" class=\\\"CitationRef\\\"\\u003e1990\\u003c/span\\u003e; Stutchbury and Morton \\u003cspan citationid=\\\"CR44\\\" class=\\\"CitationRef\\\"\\u003e1995\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eThe frequent individual ties may be thought of as resulting from good ecological conditions the ground tit population experiences (Korb and Heinze \\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e2008\\u003c/span\\u003e). Across the species' range, the study area is most abundant in annual rainfall (780 mm in contrast to 300 mm on average), which implies sufficient supply of soil invertebrates, the main food resources of the birds. Indeed, the study population has a larger mean brood size at fledging (6.0) compared to the population in south Tibet with rainfall of 430 mm (Tang et al. \\u003cspan citationid=\\\"CR47\\\" class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e). The food-rich environments may explain the absence of territoriality in the study population, as expected by a theoretical model showing that well-defined, exclusive territories prevail in populations living in resource-poor environments and high population densities resulting from good habitats lead to the break-down of territoriality (Morrell and Kokko \\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e2005\\u003c/span\\u003e). In addition, we also detected high-level promiscuity in four other ground tit populations located in the same climatic zone as the current study population (our unpubl. data). In contrast, the poor environments in the south Tibet could select for limited individual ties because socially monogamous pairs live in permanent territories with cooperative breeding characterized by helpers-at-the-nest. This may explain the rarity of extra-pair mating in the population (Johannessen et al. \\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e2011\\u003c/span\\u003e; Tang et al. \\u003cspan citationid=\\\"CR47\\\" class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e). It has been shown that tropical environments, presumably being favorable, promote EPP at the intraspecific level (Brouwer and Griffith \\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eMore importantly, individual ties, which are mediated by burrow-nesting and roosting and by favorable ecological conditions in the ground tit population, were kin-based and remain throughout the year. Neighboring families combine shortly after fledging, disperse in kin colony and pairs form small-scale kin structure (Wang and Lu 2014), with breeding females occasionally roosting in kin nests. Thus, inclusive fitness benefits should further facilitate the prevalence of extra-pair mating. It has been well known that the cost of raising non-descendant offspring has promoted the evolution of mate guarding against cuckolding in both males (Birkhead and M\\u0026oslash;ller \\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e1992\\u003c/span\\u003e) and females (Andersson et al. \\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e) and conflict between social partners (Sheldon \\u003cspan citationid=\\\"CR43\\\" class=\\\"CitationRef\\\"\\u003e2002\\u003c/span\\u003e). Kin-selected benefits (Hamilton \\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e1964\\u003c/span\\u003e) are expected to lead to a reduction in both mate guarding and sexual conflict, thereby increasing promiscuity. In the ground tit population, two males involved in EPP and two females involved in QP were of genetic similarity. In support of our finding, relatedness between cuckolding and cuckolded males has been detected in a fish (Bose et al. 2019) and two bird species of social monogamy (Huyvaert and Parker \\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e2010\\u003c/span\\u003e; Reid et al. \\u003cspan citationid=\\\"CR40\\\" class=\\\"CitationRef\\\"\\u003e2016\\u003c/span\\u003e). In waterfowl, females were found to be less resistant to parasitic attempts by kin (Andersson et al. \\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eInbreeding between extra-pair partners may also contribute to the prevailing promiscuity in the ground tit population. It has been shown that inbreeding can generate kin-selected benefits and maintain co-adapted genes (Avil\\u0026eacute;s and Purcell \\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e2012\\u003c/span\\u003e; Szulkin et al. \\u003cspan citationid=\\\"CR46\\\" class=\\\"CitationRef\\\"\\u003e2013\\u003c/span\\u003e; Dorsey and Rosenthal \\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e). In our system, social pair-bonds form randomly and extra-pair mates were genetically related, as found in some bird species (Arct et al. \\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e2015\\u003c/span\\u003e), which suggests the presence of kin recognition based on the strong nepotism. The traditional assumption of inbreeding depression has been considered simplistic, especially when it is between intermediately related individuals (Helgason et al. \\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e2008\\u003c/span\\u003e). Indeed, our data demonstrated that inbred and within-pair offspring were of equal quality in terms of body mass and individual heterozygosity. Actually, as a character of social species (Kokko and Ots \\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e2006\\u003c/span\\u003e), inbreeding in certain circumstances is considered to constitute a regular part of animal mating systems (Szulkin et al. \\u003cspan citationid=\\\"CR46\\\" class=\\\"CitationRef\\\"\\u003e2013\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eTaken together, strong kinship in the ground tit population set the stage for high-level promiscuity. The kin-mediated promiscuity, including inbreeding, may in turn increase nepotism within the population through a positive feedback loop. Associations with kin can generate a range of benefits, such as cooperatively breeding (Koenig and Dickinson \\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e2016\\u003c/span\\u003e), reduced territory conflict (Bebbington et al. \\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e) and higher survival and reproductive success (Walmsley et al. \\u003cspan citationid=\\\"CR49\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e). Our research adds promiscuity to these benefits.\\u003c/p\\u003e \\u003cp\\u003eFinally, we argue the potential to detect promiscuity especially QP and extra-pair CBP in more socially monogamous birds. Our reasons are: 1) females increasing reproductive success should be adaptive; 2) in altricial birds studied, the proportion of species with QP (82%) is comparable with that with EPP (83%), and the averaging 8% occurrence rate at the brood level in the former indicates certain adaptive value; 3) eco-social conditions facilitating parasitism such as cavity-nesting (1800\\u0026ndash;2000 altricial species; van der Hoek et al. 2017) and fine-scale genetic structure (Hatchwell \\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e2010\\u003c/span\\u003e; Camerlenghi et al. \\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e) can be met in many cases, but the low taxonomic coverage of the existing studies on extra-pair maternity of altricial species (\\u0026lt;\\u0026thinsp;30, compared to \\u0026gt;\\u0026thinsp;360 for EPP) is less likely to include species that meet such conditions; 4) CBP has been increasingly reported in altricial taxa, accounting for 72% of examined species with a mean brood proportion of 17%, in which some of parasitic offspring could derive from extra-pair mating. Therefore, to have a broader perspective on the evolution of mating systems, future work should seek to examine QP and extra-pair CBP for more species.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgements\\u0026nbsp;\\u003c/strong\\u003eWe thank B. Du, YH, Li, L.Y. Kang and many volunteers who provided assistance in field data collection and lab analysis.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthor contribution\\u0026nbsp;\\u003c/strong\\u003eX.L. and C.W. designed the study. C.W. and S. C. collected and analyzed the data. X.L. wrote the paper with contributions from C.W. and S.C.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFunding\\u003c/strong\\u003e This research was supported by the National Natural Science Foundation of China (31830085) and the Second Tibetan Plateau Scientific Expedition and Research program (2019QZKK0501).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eData availability\\u003c/strong\\u003e The dataset analyzed during the current study is available in the figshare repository, 10.6084/m9.figshare.23666949.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eEthics approval\\u0026nbsp;\\u003c/strong\\u003eOur study was approved by the Law of the People\\u0026rsquo;s Republic of China on the Protection of Wildlife (reference 19881108) and Provisions of the Tibet Autonomous Region for the Protection of Wild Animals and Wild Plants (reference 20091001). The field and lab procedures were approved by the ethics committee of Wuhan University.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCompeting interests\\u003c/strong\\u003e The authors declare no competing interests.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eSupplementary Information\\u003c/strong\\u003e The online version contains supplementary material available at.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n \\u003cli\\u003eAndersson M, \\u0026Aring;hlund M, Waldeck P (2018) Brood parasitism, relatedness and sociality: a kinship role in female reproductive tactics. Biol Rev 94:307\\u0026ndash;327. https://doi.org/10.1111/brv.12455\\u003c/li\\u003e\\n \\u003cli\\u003eArct A, Drobniak SM, Cichoń M (2015) Genetic similarity between mates predicts extrapair paternity \\u0026mdash; a meta-analysis of bird studies. 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Annu Rev Ecol Evol Syst 34:365\\u0026ndash;396. https://doi.org/10.1146/annurev.ecolsys.34.011802.132439\\u003c/li\\u003e\\n\\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\":\"info@researchsquare.com\",\"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\":\"Extra-pair mating, Inbreeding, Kin selection, Kinship foster care, Mating system, Relatedness\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-3163972/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-3163972/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eExtra-pair mating prevails among socially monogamous birds with biparental care. Theoretically, females may lay the resultant eggs in their own, their mating partners or other pairs\\u0026rsquo; nests, accordingly leading to extra-pair paternity (EPP), quasi-parasitism (QP) and extra-pair conspecific brood parasitism (CBP). Empirical evidence showed EPP to be widespread and QP rare, with extra-pair CBP having never been described. In Tibetan ground tits (\\u003cem\\u003ePseudopodoces humilis\\u003c/em\\u003e), we detected EPP, QP and extra-pair CBP in 54%, 47% and 24% of broods, which involved 20%, 16% and 8% of offspring, respectively. Cuckolding and cuckolded individuals in these events were genetically related and moderate inbreeding occurred between extra-pair partners, whereas social mates formed randomly with respect to relatedness. Promiscuity relative to genetic monogamy significantly increased annual reproductive output of both males and females, and extra-pair offspring did not differ from their within-pair siblings in body mass and heterozygosity. While the much high incidence of promiscuity can be ultimately driven by not only males but also females\\u0026rsquo; pursuit of increasing reproductive success, it may be proximately promoted by frequent kin ties in association with the species\\u0026rsquo; habit of burrow nesting and roosting, presumably kin-selected benefits may reduce the cost of arising non-descendant offspring. The fact that less than 30 altricial species have been documented with respect to extra-pair maternity (in contrast to over 360 species for EPP) suggests that more attention should be paid towards QP and extra-pair CBP to get a broader perspective on the evolution of avian mating systems.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Where are extra-pair offspring raised: the mother’s, the father’s or the others’ nest?\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2023-07-31 19:22:19\",\"doi\":\"10.21203/rs.3.rs-3163972/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"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\":\"e669bada-3823-43e0-8f93-670a89b680d4\",\"owner\":[],\"postedDate\":\"July 31st, 2023\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2023-10-10T15:47:59+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2023-07-31 19:22:19\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-3163972\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-3163972\",\"identity\":\"rs-3163972\",\"version\":[\"v1\"]},\"buildId\":\"cBFmMYwuxLRRLfASyISRj\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}