Figure 1. Males in either opposite-sex or same-sex pairs retain their partner preferences for at least four weeks following partner separation.

Additional stats including post-hocs provided in Supplementary file 1. (A) Schematic of a partner preference test (PPT) and timelines for behavioral experiments. Opposite-sex (n=8/cohort) and same-sex (n=8/cohort) pairs were paired for 2 weeks prior to a baseline PPT. Pairs were then either in a remain paired or separated cohort. Remain paired animals stayed with their partners throughout the experiment and underwent PPT 48 hours and 4 weeks after baseline. Separated animals were separated from their partners for either 48 hours (short-term) or 4 weeks (long-term) prior to the endpoint PPT. (B) Partner preference scores (% partner huddle/total huddle) from baseline, short-term, and long-term PPTs of opposite-sex (dark green) and same-sex (light green) paired males. Opposite- and same-sex paired males showed a baseline partner preference that remained evident for both groups after short-term separation and long-term separation (one-way t-tests relative to 50%, *** p<0.001, ** p<0.01). Black dotted line at 50% indicates no preference for partner or novel. (C) Partner (left) and novel (right) huddle duration (seconds) for remain paired animals over time during each PPT (two-way RM-ANOVA). (D) Partner huddle for short-term (left) and long-term (right) separated animals between baseline and the endpoint PPT—48 hours or 4 weeks post-baseline, respectively (two-way RM-ANOVA). (E) Novel huddle for short-term (left) and long-term (right) separated animals between baseline and the endpoint PPT—48 hours or 4 weeks post-baseline respectively (two-way RM-ANOVA).

Figure 1—figure supplement 1. Partner and novel huddle latency is unchanged in both remain paired and separated opposite- and same-sex paired males.

Cumulative event plots for partner (A–C) and novel (D–F) huddle latency in opposite-sex (left, dark green) and same-sex (right, light green) paired males showing the first 10 minutes of the partner preference test. (A) There was no difference in latency to huddle after 6 weeks of paring compared to 2 weeks in opposite sex pairs. This was true for both partner and novel huddle. (B) Same-sex paired males huddle significantly more quickly with their partner at baseline compared to 48 hr post-separation (log-rank test: χ²=4.0, p=0.046) while this change is absent in opposite-sex paired males. (C) Males separated for 4 weeks do not have any changes to their partner huddle latency. (D) As in A, same-sex pairs show no different in huddle latency over time. (E) Males separated for 48 hr have no change to their novel huddle latency. (F) Males separated for 4 weeks have no statistically significant differences between their baseline and endpoint novel huddle latencies.

Figure 1—figure supplement 2. Change scores of partner preference and partner huddle duration for opposite-sex and same-sex paired males.

Change scores were calculated by subtracting the value of either metric (e.g. partner preference or partner huddle duration) at the endpoint PPT minus the baseline PPT. (A) Change scores of percent partner huddle for opposite-sex (left) and same-sex (right) paired males. (B) Change scores of partner huddle duration. Partner huddle duration significantly increased between 4 weeks post baseline only in males that had remained with their opposite-sex partner (t-test against 0%: p=0.000529). Same-sex paired males continued to increase their partner huddle duration after baseline regardless of separation (t-test against 0%: remain paired 48 hr – base p=0.062; remain paired 4 weeks – base p=0.060; separated 4 weeks – base p=0.046).

Figure 1—figure supplement 3. Pearson’s correlation matrices of partner preference test behaviors across time points.

Left: opposite-sex paired males, Right: same-sex paired males. Correlation of PPT metrics for remain paired males (top 2 rows), males separated for 48 hr (3rd row), and males separated for 4 weeks (4th row). Number and color indicate Pearson’s R-value ***p<0.001, **p<0.01, *p<0.05.