Spawning patterns in the three-spined stickleback (Gasterosteus aculeatus L.): an evaluation by DNA fingerprinting

We analysed a random sample of 10 three-spined stickleback nests by DNA fingerprinting. DNA from the guardian male and a random subsample of LO fry per nest were probed with pYNZ 132, a human single-locus probe for VNTR-loci (variable number of tandem repeats). On average this probe produced DNA fingerprints of 12 scoreable bands. By comparing the bands present in each individual, we calculated band sharing indices (BSIs) between the guardian male and its fry. The BSls varied between 0.40 and 0.77 with an average (± S.D.) of 0.59 ± 0.09. We therefore conclude that the guardian male was the true father of the fry in all these nests. Once the paternal bands in each fry were determined, we compared the maternal bands among the fry of each nest. Based on the BSIs obtained with these comparisons, we found that one guardian male enticed three females to spawn in its nest, six enticed two females and three enticed one female.     

Microsatellite markers and multiple paternity in a paternal care fish, Hexagrammos otakii

The fertilization success of sneaking behavior in a paternal care fish, Hexagrammos otakii, was estimated using microsatellites as genetic markers. Sneaking behavior was observed using a video camera placed near the nest of a breeding territorial male. Two egg masses fertilized by different patterns of sperm emission by the territorial male and sneaker males were examined for paternity discrimination. When a sneaker emitted a large amount of sperm after sperm emission by the territorial male, the sneaker fertilized more eggs than the territorial male. When the territorial male and a sneaker alternately emitted semen, the territorial male fertilized the most eggs. These results suggest that both the amount of emitted sperm and the timing of sperm emission are important factors in sperm competition. Received: November 18, 1999 / Accepted: January 17, 2000     

Seasonal variation in male alternative reproductive tactics

Genetic parentage analyses reveal considerable diversity in alternative reproductive behaviours (e.g. sneaking) in many taxa. However, little is known about whether these behaviours vary seasonally and between populations. Here, we investigate seasonal variation in male reproductive behaviours in a population of two‐spotted gobies (Gobiusculus flavescens) in Norway. Male two‐spotted gobies guard nests, attract females and care for fertilized eggs. We collected clutches and nest‐guarding males early and late in the breeding season in artificial nests and used microsatellite markers to reconstruct parentage from a subset of offspring from each nest. We hypothesized that mating, reproductive success and sneaking should be more prevalent early in the breeding season when competition for mates among males is predicted to be higher. However, parentage analyses revealed similar values of mating, reproductive success and high frequencies of successful sneaking early (30% of nests) and late (27% of nests) in the season. We also found that multiple females with eggs in the same nest were fertilized by one or more sneaker males, indicating that some males in this population engage in a satellite strategy. We contrast our results to previous work that demonstrates low levels of cuckoldry in a population in Sweden. Our results demonstrate marked stability in both the genetic mating system and male alternative reproductive tactics over the breeding season. However, sneaking rates may vary geographically within a species, likely due to local selection influencing ecological factors encountered at different locations.     

A MICROSATELLITE ASSESSMENT OF SNEAKED FERTILIZATIONS AND EGG THIEVERY IN THE FIFTEENSPINE STICKLEBACK

Attempts by males to steal fertilizations from other males are common in many species. In some sticklebacks, males also are known to steal eggs from the nests of rivals and to carry them back to their own nests. However the genetic consequences of these nest-raiding behaviors seldom have been investigated. Here we assess genetically the prevalence of sneaked fertilizations and egg stealing, and we describe the mating system in a natural population of the fifteenspine stickleback. Six microsatellite markers were developed and employed to assay a total of 1307 embryos from 28 nests. Guardian males and all nest-holding males in the local area also were genotyped for two to six loci Analysis of male genotypes and those of embryos revealed that five of the 28 nests (18%) contained progeny from sneaked fertilizations, and that four of the 24 nests (17%) with resident males contained stolen egg clutches Comparisons of the composite DNA genotypes of nest-holding males against those of inferred sneakers implicated one nest holder as the sneaker of a nest seven meters from his own. Also, the genetic data demonstrated that nests of males frequently contain eggs from multiple females. The multilocus genotypes of inferred mothers indicated that females mate with multiple males, sometimes over distances greater than one kilometer.     

Tactic changes in dusky frillgoby Bathygobius fuscus sneaker males: effects of body size and nest availability

Field and laboratory studies were conducted to examine the effects of nest availability and body size on changes in male mating tactics from sneaking to nest‐holding in the dusky frillgoby Bathygobius fuscus. In the field, the body size of nest‐holding males decreased from early to mid‐breeding season, suggesting the possibility of a change in the tactics of sneaker males to nest‐holding. Many sneaker males did not use vacant spawning nests even when size‐matched nests were available, but they continued to reproduce as sneakers. Similarly, in aquarium experiments with available vacant nests, some sneaker males became nest‐holders irrespective of their body size, but some did not. These results showed that nest availability is not a limiting factor for changes in tactics by sneaker males in this species. Because tactic‐unchanged sneaker males were co‐housed with larger nest‐holding males in the tanks, the body size of nearby nest‐holding males may have affected the decision to change tactics for sneaker males. Moreover, smaller individuals among tactic‐changed males tended to spend more time until spawning, probably because they had relatively larger costs and smaller benefits of reproduction as nest‐holding males compared to larger males.     

Mating success of alternative maturation phenotypes in male Atlantic salmon,Salmo salar

Summary Mature male parr successfully fertilized eggs of anadromous female Atlantic salmon, Salmo salar, in competition with anadromous males under simulated natural conditions. Mating situations were created in which mature male parr and anadromous males competing for the opportunity to spawn with an anadromous female differed in allelic forms of the same enzyme. Females deposited eggs into a sequence of 3 or 4 eggs nests. The mean proportion of eggs in a redd fertilized by parr increased with increasing numbers of parr present at a redd, reaching 23% at male parr: anadromous male ratios of 20:1. Single male parr fertilized, on average, 5% of the eggs in a redd. The proportion of eggs in an egg nest fertilized by parr also depended upon the order of egg nest construction, such that parr mating success was highest at the initial nest constructed and lowest at the final nest. Parr have relatively high fertilization success for their size when compared with the smaller maturation phenotype of other salmonids.     

Timing of Spawning and Host-nest Choice for Brood Parasitism by the Japanese Minnow,Pungtungia herzi,on the Japanese Aucha Perch,Siniperca kawamebari

A field study of the breeding ecology of the Japanese aucha perch, Siniperca kawamebari, and brood parasitism by the Japanese minnow, Pungtungia herzi, on nests of the perch was carried out from 1989 to 1991. Observations of perch nests under natural conditions in 1990 showed that brood parasitism by the minnow was concentrated on host nests in which nest owners had just begun their nesting cycle. When spawned in a perch nest with recently spawned perch eggs, parasite eggs always hatched earlier than host eggs. An experiment with imitation perch eggs in 1991 confirmed that changing colour of host eggs was the cue for the parasites to distinguish between different developmental stages of host eggs. Parasite eggs rapidly disappeared without guarding by a host male (Baba et al. 1990). This loss was caused by predation by fishes. Parasite fry left the nest immediately after hatching, so parasite eggs spawned in a host nest in an early stage should be well guarded until they hatch. In the field, minnows deposited their eggs in perch nests which had larger numbers of newly spawned perch eggs. Since the perch males always deserted their nests when their own eggs disappeared, the parasite's choice of host nests with larger numbers of host eggs may ensure survival of the parasite eggs. The timing of egg deposition and choice of host nest by the minnow appear to be adaptive in terms of brood parasitism on nests of the perch.     

Parasitic spawning in sand gobies: an experimental assessment of nest-opening size, sneaker male cues, paternity, and filial cannibalism

Sneaking is common in nest-building fish with paternal care,but the role of nest-opening size in protecting against entryby sneaker males has never been tested before. Using the sandgoby (Pomatoschistus minutus), a fish with exclusive paternalcare, experimental manipulations of nest openings provided nosupport for the hypothesis that nest openings serve as physicalor visual defense or that sneaker males prefer to parasitizenests with wide openings. Female mating preference was alsonot influenced by nest-opening size. However, female courtshipbehavior and visibility were important cues for sneaker males.Most sneak entries occurred when the nest holder was occupiedwith courtship, chasing another sneaker male or nest building.In half the cases of observed sneak entry, the sneaker malefertilized eggs, also when sneaking only occurred before spawning.Sneak entry and its duration were good estimates of stolen paternity,but neither sneak entries nor degree of fertilizations werecorrelated with filial cannibalistic behavior. Testes size didnot explain parasitic spawning success in replicates with geneticallydetermined sneaking. However, all sneaker males without breedingcoloration had huge testes and small sperm duct glands, whereasnest-holding males had small testes and large sperm duct glands,and sneaker males with breeding coloration were intermediate.     

Non-destructive sampling of maternal DNA from the external shell of bird eggs

The use of non-destructive sampling methods to collect genetic material from wildlife allows researchers to minimize disturbance. Most avian studies employ capturing and handling of young and parents to draw blood for DNA analysis. In some cases adult female birds are difficult to catch, so maternal genotyping has required collection of contour feathers from nests, or destructive sampling of eggs. Many species do not leave contour feathers in the nest, and destructive sampling has been unreliable due to contamination with embryonic DNA. Alternative field sampling techniques for collection of maternal DNA from birds are therefore desirable. Here we demonstrate that avian maternal DNA can be isolated in a non-invasive and non-destructive way from the external surface of eggs. We used cotton swabs to collect maternal DNA from the external shells of herring gull (Larus argentatus) and Caspian tern (Sterna caspia) eggs. DNA was then amplified by the polymerase chain reaction (PCR) for microsatellite genotyping. We verified that the DNA samples were maternal by comparing microsatellite profiles to those obtained from adults and chicks from the same nests. In 100% of Caspian tern (n=16) and herring gull families (n=12), the egg swabs that amplified matched the maternal microsatellite genotype. In a screening of many nests of both species, we successfully amplified microsatellite markers from 101/115 (88%) egg swabs. Swabs from eggs with blood stains on the shell were more likely to amplify successfully than those from clean eggs. The advantages of this new method include increased parentage assignment/exclusion power, and increased availability of maternal DNA for genotyping of species that do not deposit contour feathers in nests.     

A molecular genetic examination of the mating system of pumpkinseed sunfish reveals high pay-offs for specialized sneakers

Intrasexual variation in reproductive behaviour and morphology are common in nature. Often, such variation appears to result from conditional strategies in which some individuals (e.g. younger males or those in poor condition) adopt a low pay-off phenotype as a 'best of a bad job'. Alternatively, reproductive polymorphisms can be maintained by balancing selection, with male phenotypes having equal fitnesses at equilibrium, but examples from nature are rare. Many species of sunfish (genus Lepomis) are thought to have alternative male reproductive behaviours, but most empirical work has focused on the bluegill sunfish and the mating systems of other sunfish remain poorly understood. We studied a population of pumpkinseed sunfish (Lepomis gibbosus) in upstate New York. Field observations confirm the existence of two male reproductive strategies: 'parentals' were relatively old and large males that maintained nests, and 'sneakers' were relatively young and small males that fertilize eggs by darting into nests of parentals during spawning. The sneaker and parental male strategies appear to be distinct life-history trajectories. Sneaker males represented 39% of the males observed spawning, and sneakers intruded on 43% of all mating attempts. Microsatellite analyses revealed that sneaker males fertilized an average of 15% of the eggs within a nest. This level of paternity by sneaker males appears to be higher than seen in most other fishes, and preliminary analyses suggest that the two male reproductive strategies are maintained as a balanced polymorphism.     

Surprising similarity of sneaking rates and genetic mating patterns in two populations of sand goby experiencing disparate sexual selection regimes

Molecular markers have proved extremely useful in resolving mating patterns within individual populations of a number of species, but little is known about how genetic mating systems might vary geographically within a species. Here we use microsatellite markers to compare patterns of sneaked fertilization and mating success in two populations of sand goby (Pomatoschistus minutus) that differ dramatically with respect to nest‐site density and the documented nature and intensity of sexual selection. At the Tvärminne site in the Baltic Sea, the microsatellite genotypes of 17 nest‐tending males and mean samples of more than 50 progeny per nest indicated that approximately 35% of the nests contained eggs that had been fertilized by sneaker males. Successful nest holders mated with an average of 3.0 females, and the distribution of mate numbers for these males did not differ significantly from the Poisson expectation. These genetically deduced mating‐system parameters in the Tvärminne population are remarkably similar to those in sand gobies at a distant site adjoining the North Sea. Thus, pronounced differences in the ecological setting and sexual selection regimes in these two populations have not translated into evident differences in cuckoldry rates or other monitored patterns of male mating success. In this case, the ecological setting appears not to be predictive of alternative male mating strategies, a finding of relevance to sexual selection theory.     

Lek-like spawning,parental care and mating periodicity of the triggerfishPseudobalistes flavimarginatus (Balistidae)

Synopsis The triggerfishPseudobalistes flavimarginatus has a lek-like spawning system at Yonge Reef, Great Barrier Reef. Males migrate to a traditional mating ground where they establish territories enclosing nest sites and egg chambers. Females arrive several days later and choose a male for mating. There is biparental care of the fertilized eggs and a strict division of labour. Females appeared to be confined to the nest site by the male. Mating was semi-lunar: eggs were observed in nests several days before the new and full moons on days when high tide occurred near sunset. Differences were observed in the spawning system of another triggerfish (P. fuscus) between the Great Barrier Reef and the Red Sea. Observations of another triggerfish (Balistoides viridescens) are also reported.     

Unravelling the evolution of complex reproductive traits with phenotypic engineering

The social dynamics surrounding courtship, mating and parental care are complex enough when just a single male and female are involved, but for species that employ multiple strategies for achieving fertilization success, the network of interactions among rivals, allies and suitors can be utterly complicated. Such is the case in the ocellated wrasse, Symphodus ocellatus, in which males adopt one of three mating strategies. The large, colourful “nesting males” court females, defend territories and care for fertilized eggs until they hatch. The smaller “satellite males” help the nesting males court females and guard against the third morph, the “sneaker males”, which sneak in when a nesting male is spawning with a female and surreptitiously release sperm. Sneaker males perform no courtship displays nor defend territories, so their reproductive investment is devoted entirely to sperm production. And these alternative male strategies work: 100% of nests contain some eggs fertilized by sneaker and satellite males, despite the fact that parental care is solely the responsibility of nesting males In this issue of Molecular Ecology, work to untangle the proximate mechanisms regulating the reproductive physiology of nesting males and their behaviour towards other males, which impacts the entire social network. Moreover, they describe how variation in neuroendocrine regulation can give rise to variation in reproductive traits, upon which sexual selection can act.     

Polygamous Mating and High Levels of Genetic Variation in Lingcod, Ophiodon elongatus, of the Strait of Georgia, British Columbia

Lingcod, Ophiodon elongatus, is a nest-guarding marine fish of western North America. Breeding occurs in late winter and early spring after males establish territories and guard nest sites therein. Eggs deposited as clutches in the nest site hatch ～7 weeks after fertilization. We evaluated the level of genetic variation in lingcod spawning in the central Strait of Georgia through analysis of microsatellite and mitochondrial D-loop variability in fertilized egg samples collected from guarded clutches. Reconstructed parental genotypes displayed a high level of allelic diversity and observed heterozygosity (83–91%) over five microsatellite loci. Progeny of a single clutch were invariably derived from a single mother and between one and five fathers. Multiple egg samples were collected from inside and outside positions on 13 lingcod egg clutches in February 2002. Fin clip samples provided microsatellite genotypes for six of the nine guardian males. Analysis of between 33 and 306 eggs from each clutch indicated that each of the 13 clutches was produced by a different mother and five of them were sired entirely by the attendant male guardian. Eight clutches were sired by multiple males, with neighboring male guardians frequently involved in clutch fertilization. Known guardian males accounted for at least 78% of observed egg fertilization, although non-territorial males were observed and may have participated in spawning. Egg fertilization by individual males was spatially heterogeneous throughout egg clutches. One male guardian failed to fertilize detectable numbers of eggs in his own or any other clutch within the study area and may have been an adoptive father. The polygynous mating structure of lingcod may help maintain genetic variation in the species.     

Body Size Correlates with Fertilization Success but not Gonad Size in Grass Goby Territorial Males

In fish species with alternative male mating tactics, sperm competition typically occurs when small males that are unsuccessful in direct contests steal fertilization opportunities from large dominant males. In the grass goby Zosterisessor ophiocephalus, large territorial males defend and court females from nest sites, while small sneaker males obtain matings by sneaking into nests. Parentage assignment of 688 eggs from 8 different nests sampled in the 2003–2004 breeding season revealed a high level of sperm competition. Fertilization success of territorial males was very high but in all nests sneakers also contributed to the progeny. In territorial males, fertilization success correlated positively with male body size. Gonadal investment was explored in a sample of 126 grass gobies collected during the period 1995–1996 in the same area (61 territorial males and 65 sneakers). Correlation between body weight and testis weight was positive and significant for sneaker males, while correlation was virtually equal to zero in territorial males. That body size in territorial males is correlated with fertilization success but not gonad size suggests that males allocate much more energy into growth and relatively little into sperm production once the needed size to become territorial is attained. The increased paternity of larger territorial males might be due to a more effective defense of the nest in comparison with smaller territorial males.     

Maternal investment in a bee species with facultative nest guarding and males heavier than females

1. Maternal investment can be influenced by several factors, especially maternal quality and possibilities for future reproduction. Mass provisioning Hymenoptera are an excellent group for measuring maternal investment because mothers distribute food sources to each brood cell for each offspring separately. Generally in aculeate Hymenoptera, larger females produce larger offspring and invest more in female offspring than in male offspring. 2. This study investigated patterns of maternal investment in Ceratina chalcites, which has an uncommon type of sexual size dimorphism in Hymenoptera: on average, males are heavier than females. It was found that larger females produce a significantly higher proportion of male offspring, as males are the costlier sex in this species. 3. Facultative nest guarding by females was observed. Females can guard offspring until adulthood, as is typical for bees of genus Ceratina (34.43% of nests); however, in the majority of cases (65.56% of nests), females plug and abandon the nest. Significant differences were found in the amount of investment between guarded and unguarded nests. Guarded nests had a greater number of provisioned brood cells and a higher proportion of male offspring. It is suggested that mothers have two facultative strategies – either she makes a large investment in the offspring of one nest or she abandons the first nest and carries out a second nesting elsewhere.     

The genetic mating system of spotted sunfish (Lepomis punctatus): mate numbers and the influence of male reproductive parasites

In nest-building fish species, mature males often exhibit one of two alternative reproductive behaviours. Bourgeois males build nests, court females, and guard their eggs. Parasitic cuckolders attempt to steal fertilizations from bourgeois males and do not invest in parental care. Previous evidence from the bluegill sunfish (Lepomis macrochirus) suggests that adult males are morphologically specialized for these two tactics. Here, we used microsatellite markers to determine genetic parentage in a natural population of the spotted sunfish (L. punctatus) that also displayed both bourgeois and parasitic male morphs. As gauged by relative investments in gonadal vs. somatic tissues, between 5 and 15% of the mature adult males were parasites. Multi-locus genotypes were generated for more than 1400 embryos in 30 nests, their nest-guardian males, and for other adults in the population. Progeny in approximately 57% of the nests were sired exclusively by the guardian male, but the remaining nests contained embryos resulting from cuckoldry as well. Overall, the frequency of offspring resulting from stolen fertilizations was only 1.3%, indicating that the great majority of paternity is by bourgeois nesting males. With regard to maternity, 87% of the nests had at least three dams, and computer simulations estimate that about 7.2 dams spawned per nest.     

Sitting ducklings: Timing of hatch,nest departure,and predation risk for dabbling duck broods

For ground‐nesting waterfowl, the timing of egg hatch and duckling departure from the nest may be influenced by the risk of predation at the nest and en route to wetlands and constrained by the time required for ducklings to imprint on the hen and be physically able to leave the nest. We determined the timing of hatch, nest departure, and predation on dabbling duck broods using small video cameras placed at the nests of mallard (Anas platyrhynchos; n = 26), gadwall (Mareca strepera; n = 24), and cinnamon teal (Anas cyanoptera; n = 5). Mallard eggs began to hatch throughout the day and night, whereas gadwall eggs generally started to hatch during daylight hours (mean 7.5 hr after dawn). Among all species, duckling departure from the nest occurred during daylight (98%), and 53% of hens typically left the nest with their broods 1–4 hr after dawn. For mallard and gadwall, we identified three strategies for the timing of nest departure: (a) 9% of broods left the nest the same day that eggs began to hatch (6–12 hr later), (b) 81% of broods left the nest the day after eggs began to hatch, and (c) 10% of broods waited 2 days to depart the nest after eggs began to hatch, leaving the nest just after the second dawn (27–42 hr later). Overall, eggs were depredated at 10% of nests with cameras in the 2 days prior to hatch and ducklings were depredated at 15% of nests with cameras before leaving the nest. Our results suggest that broods prefer to depart the nest early in the morning, which may best balance developmental constraints with predation risk both at the nest and en route to wetlands.     

Reproductive ecology of the jacky dragon (Amphibolurus muricatus): an agamid lizard with temperature‐dependent sex determination

Abstract The jacky dragon, Amphibolurus muricatus (White, ex Shaw 1790) is a medium sized agamid lizard from the southeast of Australia. Laboratory incubation trials show that this species possesses temperature‐dependent sex determination. Both high and low incubation temperatures produced all female offspring, while varying proportions of males hatched at intermediate temperatures. Females may lay several clutches containing from three to nine eggs during the spring and summer. We report the first field nest temperature recordings for a squamate reptile with temperature‐dependent sex determination. Hatchling sex is determined by nest temperatures that are due to the combination of daily and seasonal weather conditions, together with maternal nest site selection. Over the prolonged egg‐laying season, mean nest temperatures steadily increase. This suggests that hatchling sex is best predicted by the date of egg laying, and that sex ratios from field nests will vary over the course of the breeding season. Lizards hatching from eggs laid in the spring (October) experience a longer growing season and should reach a larger body size by the beginning of their first reproductive season, compared to lizards from eggs laid in late summer (February). Adult male A. muricatus attain a greater maximum body size and have relatively larger heads than females, possibly as a consequence of sexual selection due to male‐male competition for territories and mates. If reproductive success in males increases with larger body size, then early hatching males may obtain a greater fitness benefit as adults, compared to males that hatch in late summer. We hypothesize that early season nests should produce male‐biased sex ratios, and that this provides an adaptive explanation for temperature‐dependent sex determination in A. muricatus.     

Filial cannibalism in the barred-chin blenny Rhabdoblennius ellipes: males do not preferentially eat young eggs

Female barred-chin blenny Rhabdoblennius ellipes prefer to mate with males tending young eggs, possibly to reduce the risk of male selective filial cannibalism against young eggs in the nests. To test this hypothesis, we conducted aquarium experiments with nests containing eggs at different developmental stages. All egg-tending males consumed both young and old eggs. However, the observed and expected number of consumed young eggs did not differ, indicating that R. ellipes males do not selectively cannibalize young eggs in nests with eggs of mixed developmental stages. Relatively small differences in egg age within a nest may explain this nonselectivity in this species.