Genetic evidence from offspring allows us to describe new aspects of reproductive behavior in A. sordidus
at Johnston Atoll. Previous studies have observed that the same males return to defend their nest sites [25
]. This study used genetic methods to confirm that males utilize the same nests over multiple nesting cycles but also documented that some males practice alternative reproductive tactics such as reproductive parasitism through sneaked fertilizations, which have not been observed or reported previously. Like previous studies, this study documented multiple clutches being deposited in a nest. However, the number of females spawning in a clutch and the population was not known. Here we provide genetic evidence as to the identity and number of females spawning within a clutch as well as multiple clutches within a nest, also over multiple reproductive cycles. Additionally, the use of female genotypes provided new evidence on female spawning frequency and behavior including serial polyandry.
3.1. Mating System
A promiscuous mating system in A. sordidus
, a damselfish with male parental care, was defined using genetic data. Promiscuous mating systems in fishes are common [29
], but the occurrence and genetic documentation of female multiple mating in species with male parental care varies [6
]. This study found evidence of multiple matings for both sexes over several reproductive cycles. Males were polygynous within and between cycles, as compared to multiple mating females that were primarily monandrous within, but polyandrous between cycles, with 13 of 14 multiple mating females switching mates. While the data suggest that majority of females (60 of 74) only spawned once during the 3.5 month period, it cannot be ruled out that some of these females likely spawned elsewhere or remained unidentified in mixed or unsampled clutches. However, individuals that spawned only one time may also have been smaller females without sufficient body condition for spawning multiple times.
Uncertainty due to low numbers of offspring sampled per clutch and potential errors in maternal assignments resulted in low power for the detection of both multiple maternal contributors and parasitic males. While these results are consistent with spawning observations, they provide an initial, but likely lower end estimate for the occurrence of these behaviors.
3.2. Male Reproductive Behavior
Alternative reproductive tactics (ARTs) [30
] occur when males attempt to steal fertilizations from other males monopolizing reproductive opportunities. In nest tending species, reproductive parasites exploit the investment of territory holders or “bourgeois” males who expend energy in obtaining and defending resources and offspring [31
]. Parasitic spawning was not previously documented for this species. Such behavior benefits the “parasitic male” who gain fertilizations while not expending energy in caring for offspring or defending a territory. Our results showed that up to 14.7% of clutches contained offspring inconsistent with paternity by the territorial male indicating that the nest tending male participated in alloparental care. Two guardian males were identified as parasites spawning in other nests, suggesting that guardian males sneak fertilizations while their neighbors spawned. In both cases, the parasitic guardian male had not yet obtained a clutch but was present at his nest site. As the males were not guarding embryos, this allowed them to leave their nest and sneak fertilizations with a spawning neighbor. In the case of male F2, this neighbor was less than 2.5 m away within the same metal structure. While the sampling method covered large portions of a clutch and larval collection was essentially random within a large pool of larvae, the low numbers genotyped per clutch may underestimate the level of parasitism occurring. On the other hand, some mismatches may also have resulted from genotyping error, overestimating the occurrence of parasitic spawning. Future work, to determine precise levels of reproductive parasitism and the reproductive success of both parasitic and guardian males, should employ additional markers and analyses of larger numbers of offspring. Larger sample sizes might also resolve whether the single locus mismatches in some offspring were typing errors, or true mismatches due to parasitism. However, as a first estimate of the percentage of clutches parasitized, 14.7% is comparable to lower end estimates from other nest tending species [19
]. If the additional nine clutches with single locus mismatches are included as incidences of parasitism, the estimate of clutches parasitized increases to 21.7%. While it is significant that this study documented A. sordidus
males practicing ARTs, the total effect on the reproductive success of the nest tender as well as the parasitic male was not quantifiable.
The genetic data indicate that parasitic A. sordidus
males were successful at fertilizing eggs within territorial male’s nests and that some of these males were identified as adjacent or nearby territory holders, but there were additional unidentified males (i.e., non-territory holders) who may have been unsuccessful in the competition for nest sites. These parasite-intruders might gain access to the nest through streaking (rapidly releasing gametes while rushing the spawning pair). In the damselfish Chromis chromis
this occurs as many as 2.8 times per ten minutes during spawning [17
], and cuckoldry was observed in every nest sampled [33
]. Other tactics the parasite might employ to gain access to fertilizations include stealth in the form of drab coloration that is similar to female coloration [31
]. Such deceit through female mimicry was also observed in male C. chromis
]. The advantage of using these tactics is that the parasitic male gains fertilizations without the cost of defending a territory or providing parental care.
Consistent with spawning observations, our genetic evidence indicated that the majority of A. sordidus
nests contained clutches spawned by a single female, but there is also genetic evidence that some clutches have multiple maternal contributors. Individual females spawned 76.7% of the individual clutches within a male’s nest, with up to four additional clutches being spawned in the nest during the same spawning cycle. As spawning events lasted several hours and only one clutch was laid in a nest per day, sequential spawning with only one female may serve as an adaptation against female mimic sneaker males. For example, Stegastes nigricans
will only spawn sequentially with females, chasing off a second female trying to enter the nest site [34
]. However, evidence of multiple females contributing to a clutch, might suggest either a distraction or an opportunity for males to mimic females as a means to gain access to the nest.
Factors influencing the occurrence of male ARTs include female preference for nests that already contain eggs [35
] spawning synchrony and nest density [37
]. Nest “piracy” provides a method for males to take advantage of female’s preference for nests containing embryos without expending energy on territory maintenance or courtship [41
]. As there were no clutches where none of the offspring matched the guardian male, there was no evidence for nest takeovers.
are temporarily territorial pomacentrids with males present at their nest sites only during reproductive activities [25
]. Spawning was not highly synchronized and nest densities were also low as compared to other temporarily territorial species [40
]. Therefore intrusion rates may be reduced, as neighboring males may not be present during a given spawning bout, or are too far away, reducing the opportunities for reproductive parasitism [17
]. However, males will take advantage of spawning neighbors, as there were two instances of territorial males being identified as parasitic males in their neighbors’ nests. As many of the parasitic spawners were not identified, it is likely that the majority of stolen fertilizations were from smaller, non-territorial males who are unable to compete for nest sites [31
Distribution of nesting resources and population size also influence mating systems. Competition for limited nest sites found primarily on man-made debris appears to result in increased mating opportunities for the males able to defend these sites. Reproductive output also varied between the guardian males suggesting factors beyond availability of nesting resources also influence reproductive success.
The group of males studied here was found in the largest aggregation or colony of A. sordidus
within the atoll [27
]. While surveys identified 40 nesting sites at this location, they were not used consistently every year or even every spawning cycle indicating the sites may vary in quality. Additional surveys around the island and other reef areas within the atoll, only found solitary individuals [27
]. Study site surveys conducted during daily dives, observed very few (1–3 individuals) A. sordidus
in the colony between reproductive cycles. Therefore it appears that the site functions primarily as a reproductive arena or aggregation site as opposed to permanent territories for the fish. The overall number of males competing for these sites is not known but the genetic analysis suggests a minimum of 36 reproductive parasites or sneaker males were detected in offspring samples. There were at minimum, 74 individual females spawning in the nest sites defended by these 10 guardian males. This female biased operational sex ratio (OSR) reflects resource competition for nesting sites among males, but may also suggest that mating competition could occur between females attempting to access limited oviposition sites [43
]. The physical space needed for one clutch can be quite large, with the smallest clutch documented measuring 22 × 30 cm, so that multiple clutches require much more defendable space [27
]. At Johnston, the availability of oviposition sites may influence female A. sordidus
movements. As so few non-territory holding individuals were observed in the study site on a daily basis, it is possible that females migrated from other locations to find suitable mates and/or oviposition sites.
3.3. Female Reproductive Behavior
Spawning females left their entire clutch with one male during a single spawning bout, but some females were identified spawning in additional cycles. Two of the females spawned three times during the study. There were too few multiple spawning females to accurately determine the spawn interval, but the minimum time detected between two clutches spawned by one female was 14 days. Within-day polyandrous matings were not detected in A. sordidus
. Within-day polyandrous matings do occur in other pomacentrids, accounting for <6%, 0% to 1.2%, 2.9% to 4.1% and <15% of all matings in Stegastes nigricans
, S. dorsopunicans, Dascyllus aruanus,
and Amblyglyphidodon leucogaster,
]. However, like A. sordidus
, female Chromis notata
generally mate with a single male per spawning day [46
We documented sequential polyandry for A. sordidus
females with 13 of the 14 multiple spawning females changing mates at least one time. Only one female spawned two times with the same male. Other pomacentrids including Stegastes nigricans, S. dorsopunicans, Dascyllus aruanus,
and Amblyglyphidodon leucogaster
also change their mates frequently over the season [12
]. In contrast, female Stegastes partitus
show high mate fidelity only changing mates due to nest loss or failure [47
]. It is likely than many of the other females spawned multiple times in nests that were not sampled and that some may have contributed to the nests with multiple female contributors and were not detected due to small number of offspring sampled. To further understand female spawning behavior and frequency, it will require larger offspring samples collected from all nests. Determining if females migrate between spawning “aggregations” would also require additional sampling at these location.
Factors influencing female choice include male size [49
], territory quality and defense [37
], courtship rate/intensity [16
], and the presence and survival of eggs [48
]. Future analysis will examine the influence of nest density and spawning synchrony in the preference of female A. sordidus
for some males (or the territories they were able to secure) over others, as evident through highly variable male reproductive success. Specifically, males receiving the highest number of clutches also appear to have more of the multiple spawning females returning to spawn within their nests. So, if some males were preferred over others due to better territories or better offspring care, why would multiple mating females choose to mate with other males? Genetic bet-hedging is a female strategy whereby having offspring sired by more than one male allows females to reduce errors made assessing mates under variable environmental conditions (genetic diversity hypothesis), or due to a females ability to discriminate between male genotypes (good genes hypothesis) [53
]. Since female reproductive behaviors vary, ultimately influencing mating systems and male reproductive success, it is therefore important to consider both male and female reproductive ecology and their interactions in order to gain an understanding of the evolution of mating systems [7