Martin D. Garlovsky1,14, Emma Whittington2,14, Tomas Albrecht3,4, Henry Arenas-Castro5, Dean M. Castillo6, Graeme L. Keais7, Erica L. Larson8, Leonie C. Moyle9, Melissa Plakke10, Radka Reifová4, Rhonda R. Snook11, Murielle Ålund12 and Alexandra A.-T. Weber13,14 1Applied Zoology, Faculty of Biology, Technische Universität Dresden, Dresden 01062, Germany 2Natural History Museum, University of Oslo, 0318 Oslo, Norway 3Institute of Vertebrate Biology, Czech Academy of Sciences, Brno 60365, Czech Republic 4Department of Zoology, Faculty of Science, Charles University, Prague 128 00, Czech Republic 5School of Biological Sciences, University of Queensland, St Lucia 4072, Queensland, Australia 6Department of Biological Sciences, Miami University, Hamilton, Ohio 45011, USA 7Department of Botany and Biodiversity Research Centre, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada 8Department of Biological Sciences, University of Denver, Denver, Colorado 80208, USA 9Department of Biology, Indiana University Bloomington, Indiana 47405, USA 10Division of Science, Mathematics, and Technology, Governors State University, University Park, Illinois 60484, USA 11Department of Zoology, Stockholm University, Stockholm 109 61, Sweden 12Department of Ecology and Genetics, Animal Ecology, Uppsala University, Uppsala 75236, Sweden 13Department of Aquatic Ecology, Swiss Federal Institute of Aquatic Science and Technology (Eawag), Dübendorf 8600, Zürich, Switzerland Correspondence: martin.garlovskytu-dresden.de; emawhittgmail.com; alexandra.webereawag.ch

↵14 Co-lead authors contributed equally; all other authors listed alphabetically.

How barriers to gene flow arise and are maintained are key questions in evolutionary biology. Speciation research has mainly focused on barriers that occur either before mating or after zygote formation. In comparison, postmating prezygotic (PMPZ) isolation—a barrier that acts after gamete release but before zygote formation—is less frequently investigated but may hold a unique role in generating biodiversity. Here we discuss the distinctive features of PMPZ isolation, including the primary drivers and molecular mechanisms underpinning PMPZ isolation. We then present the first comprehensive survey of PMPZ isolation research, revealing that it is a widespread form of prezygotic isolation across eukaryotes. The survey also exposes obstacles in studying PMPZ isolation, in part attributable to the challenges involved in directly measuring PMPZ isolation and uncovering its causal mechanisms. Finally, we identify outstanding knowledge gaps and provide recommendations for improving future research on PMPZ isolation. This will allow us to better understand the nature of this often-neglected reproductive barrier and its contribution to speciation.