Summary
Male alternative reproductive tactics have been described in many mating systems. In fishes, these tactics typically involve a territorial male that defends a spawning site or nest and a parasitic male that uses sneaking or female mimicry to steal fertilizations from the territorial male. In this paper, we use molecular genetic markers to examine the success of males that adopt alternative reproductive tactics in two sunfishes, comprising the bluegill (Lepomis macrochirus Rafinesque, 1819) and the pumpkinseed (Lepomis gibbosus (L., 1758)). In sunfishes, the tactics are referred to as parental (territorial male) and cuckolder (parasitic male). We show that cuckoldry rates peak in the second trimester of the breeding season in bluegill, whereas cuckoldry rates are lowest during this period in pumpkinseed. We also show that paternity of parental male bluegill is positively correlated with body condition, but not body length or mass. No relationship between these phenotypic variables and paternity in pumpkinseed was found. We discuss the patterns of cuckoldry in relation to differences between the species in mating opportunities, parental male defence ability, and cuckolder density. Finally, we discuss how the paternity data can be used to differentiate between two mechanisms underlying the expression of alternative reproductive tactics, comprising the condition strategy and alternative strategies.
Methodology
Bluegill (1993) or pumpkinseed (2003) reproductive activity was surveyed daily by swimmers within our study area (~2 km stretch of shoreline) on Lake Opinicon, Ontario, Canada (44°34′N, 76°19′W). For selected nests, a small tissue sample was collected from the tending parental male and a sample of several hundred fry was collected from the nest. Tissue and fry samples were preserved in 95% ethanol for later microsatellite DNA analysis. For bluegill, samples were collected from parental males (n = 60 nests) that spawned during 10 June and 6 July; for pumpkinseed, samples were collected from parental males (n = 15 nests) that spawned during 10 June and 7 July. For all of the pumpkinseed parental males and a subset of the bluegill parental males (n = 16), we also obtained mass (nearest 0.1 g) and total length measurements (nearest millimetre). These measurements were used to calculate Fulton’s condition factor (mass / length3 × 105; Ricker 1975). This factor correlates with the nonpolar lipid density of parental males (Neff and Cargnelli 2004).
The paternity of each nest-tending parental male was determined using three microsatellite loci and the two-sex paternity model (Neff et al. 2000a; Neff 2001). This model determines the proportion of offspring sired by the nest-tending parental male. The remaining offspring are assumed to be sired by the specialized cuckolder males (e.g., Neff and Gross 2001). The microsatellite loci comprised RB7, LMAR10, and RB20 (bluegill) or LMAR14 (pumpkinseed); the primer sequences are published in DeWoody et al. (1998) and Schable et al. (2002). First, DNA was isolated from the samples using a proteinase K (EC 3.4.21.64) digestion (Neff et al. 2000b). DNA concentration was quantified using a spectrophotometer. Second, we used a Whatman Biometra® T1 thermocycler to amplify the microsatellites with the following program: 60s at 92 °C; 7 cycles of 30s at 92 °C, 30s at 54 °C, and 30s at 72 °C; and 28 cycles of 15s at 92 °C, 30s at 54 °C, and 20s at 72 °C. Each 10 µL polymerase chain reaction (PCR) contained ~75 ng of total DNA, 2 mmol/L MgCl2, 1× PCR buffer (Fisher Scientific, Ottawa, Ontario), 0.4 mmol/L of each deoxynucleotide (Fisher Scientific), 0.25 units Taq DNA polymerase (Fisher Scientific), and 0.2 µmol/L of each forward and reverse primer (Invitrogen™ Life Technologies, Eugene, Oregon). PCR product was run following standard protocol for the Ceq. 8000 Genetic Analysis System (Beckman Coulter, Fullerton, California). Genotypes were obtained from a total of 75 parental males and 3724 offspring (mean 50 offspring per nest, range 10–95). The genotypes at these loci did not deviate from Hardy–Weinberg equilibrium. The mean exclusion probabilities for the parentage analysis were 0.90 (range 0.80–1.00) for bluegill and 0.93 (range 0.85–0.98) for pumpkinseed.