Complexity in prezygotic mating behavior can contribute to the emergence of sexual incompatibility and reproductive isolation. In this study, we performed behavioral tests with two tidepool copepod species of the genus Tigriopus to explore the possibility of precopulatory behavioral isolation. We found that interspecific mating attempts failed prior to genital contact and that this failure occurred at different behavioral steps between reciprocal pairings. Our results suggest that prezygotic barriers may exist at multiple points of the behavioral process on both male and female sides, possibly due to interspecific differences in mate-recognition cues used at those “checkpoints”. While many copepod species are known to show unique precopulatory mate-guarding behavior, the potential contribution of prezygotic behavioral factors to their isolation is not widely recognized. The pattern of sequential mate-guarding behaviors may have allowed diversification of precopulatory communication and contributed to the evolutionary diversity of the Tigriopus copepods.

The .xlsx file registered here contains original datasets for Figures 4, 5, 6, and 7 and Table 1 in our manuscript.

The data were obtained by video and microscopic observations of interspecific and intraspecific mating behavior of tidepool copepods, Tigriopus californicus and Tigriopus japonicus. For detailed methods, see the published manuscript as well as Tsuboko-Ishii S, Burton RS. Individual Culturing of Tigriopus Copepods and Quantitative Analysis of Their Mate-guarding Behavior. J Vis Exp. 2018 Sep 26;(139):58378 (doi: 10.3791/58378).

• Figure 4. Difference in frequency of approaches by males between conspecific and interspecific pairs. Each datapoint was obtained from one tested pair. One approach is defined as a swift (<0.5 s) chase or pounce by a male.
• Figure 5. Difference in mean velocity of captured females between conspecific and interspecific pairs. Each datapoint was obtained from one observed attempt. 
• Figure 6. Difference in paring duration between conspecific and interspecific pairs. Each datapoint was obtained from one tested pair.
• Figure 7. Duration of each step of prezygotic behavioral process in conspecific and interspecific pairs. Datapoints represent average duration of each behavioral step per tested pair. Pair formation: time spent from body contact to either initiation of crawl down or termination of pairing, whichever the earlier.

This .xlsx file was created with Microsoft Excel for Mac (version 16.75).

• Figure 4. Difference in frequency of approaches by males between conspecific and interspecific pairs. Each datapoint was obtained from one tested pair. One approach is defined as a swift (<0.5 s) chase or pounce by a male.
  • Fig 4A. Frequency of approaches by T. californicus males toward conspecific (T. californicus) and heterospecific (T. japonicus) females. The unit is times/min (number of approaches per free time).
  • Fig 4B. Frequency of approaches by T. japonicus males toward conspecific (T. japonicus) and heterospecific (T. californicus) females. The unit is times/min (number of approaches per free time).
• Figure 5. Difference in mean velocity of captured females between conspecific and interspecific pairs. Each datapoint was obtained from one observed attempt. 
  • Fig 5A. Mean velocity of T. californicus females against conspecific (T. californicus) and heterospecific (T. japonicus) males. The unit is mm/s.
  • Fig 5B. Mean velocity of T. japonicus females against conspecific (T. japonicus) and heterospecific (T. californicus) males. The unit is mm/s.
• Figure 6. Difference in paring duration between conspecific and interspecific pairs. Each datapoint was obtained from one tested pair.
  • Fig 6A. Average pairing duration of T. californicus females with conspecific (T. californicus) and heterospecific (T. japonicus) males. The unit is seconds.
  • Fig 6B. Avarage pairing duration of T. japonicus females with conspecific (T. japonicus) and heterospecific (T. californicus) males. The unit is seconds.
• Figure 7. Duration of each step of prezygotic behavioral process in conspecific and interspecific pairs. Datapoints represent average duration of each behavioral step per tested pair. Pair formation: time spent from body contact to either initiation of crawl down or termination of pairing, whichever the earlier.
  
  • Fig 7A. Average duration of the pair formation process: time spent from body contact to either initiation of crawl down or termination of pairing, whichever the earlier. The unit is seconds.
  • Fig 7B. Average duration of the crawl down by males. The unit is seconds.
  • Fig 7C. Average duration of the dorsal bend by males. The unit is seconds.
  • Fig 7D. Average duration of the genital press by males. The unit is seconds.
  • Fig 7E. Average duration of the whole pairing. The unit is seconds.