Does paternal care influence mate preference? Male and female mating behavior in Threespine Stickleback ecotypes that differ markedly in parental care

**Background:** Sexual selection often targets body size, with larger individuals gaining advantages in contests and mate choice. Parental care may influence the strength and direction of these preferences, as caring males might be more selective about mates. On Canada's East Coast, two sympatric ecotypes of Threespine Stickleback (Gasterosteus aculeatus) differ markedly in parental care: common males provide paternal care for 7+ days, while white males disperse eggs after fertilization and provide no care. The ecotypes are genetically distinct but occasionally hybridize. This study tested whether males investing more in parental care (common ecotype) show stronger mate selectivity, and examined female preferences across ecotypes and body sizes.

**Methods:** Fish were collected in June 2020 from three Nova Scotia sites: Rainbow Haven Estuary, Antigonish Landing, and Crossing Road. A total of 120 fish were used in 35 trials (18 with common males, 17 with white males). Each trial had a focal male with nesting material and two females (one common, one white) in separate compartments. Trials had two 30-minute stages: a partitioned stage where males could see but not reach females, and an interaction stage where each female was released sequentially for 15 minutes. Male preference was measured as proportion of time near each female (partitioned stage) and courtship frequency (zig-zagging, biting, leading, nest poking, plus dorsal pricking for common males and side fanning for white males). Female response was a binary variable (head-up, follow, or nest inspect). Body size (standard length) was recorded post-trial. Statistical analyses used GLMMs with AICc model selection.

**Key Results:** Male preference was associated with female size. For proportion of time near females, all top five models included female size and female ecotype, with positive coefficients for size (pseudo-R² = 0.20–0.32). For courtship frequency, the top model (AICc = 874.2, ωi = 0.619, pseudo-R² = 0.12) included interactions of male ecotype × female ecotype and male ecotype × female size. White males courted common females more intensely with increasing female size, while common males courted larger females but without a continuous linear relationship. For female response, multiple models fit; common females were less likely to respond overall but were more likely to respond to smaller white males (pseudo-R² = 0.11 for common female × white male subset). White females showed no clear preference. Larger females of both ecotypes were more likely to perform head-up displays (pseudo-R² = 0.13). Male courtship frequency was weakly associated with male size—smaller males courted more (pseudo-R² = 0.06). Size matching analysis showed males courted females larger than themselves more, and females were more likely to respond to relatively larger males (pseudo-R² = 0.21). Ten spawning events occurred (28.6% of trials): 5 pure crosses (1 common × common, 4 white × white) and 5 hybrid crosses (4 common male × white female, 1 white male × common female). Pure crosses averaged 3.93 mm size difference; hybrid crosses averaged 6.61 mm.

**Clinical Implications:** This study provides evidence that male mate choice in Threespine Stickleback is primarily driven by female body size rather than by the presence or absence of paternal care. Both caring (common) and non-caring (white) males preferred larger females, contradicting the hypothesis that parental investment increases male selectivity. The finding that hybrid matings occurred as frequently as pure crosses (5 of 10 spawnings each) challenges previous reports of complete assortative mating in this system, suggesting that reproductive isolation between ecotypes may be weaker than previously thought. These results contribute to understanding how mating systems, body size, and parental care interact to shape sexual selection and speciation.