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Microhabitat selection is a key component of amphibian breeding biology and can be modulated in response to the features of breeding sites and the presence of predators. Despite invasive alien species being among the major threats to amphibians, there is limited information on the role of invasive species in shaping amphibians’ breeding microhabitat choice. The invasive red swamp crayfish ( Procambarus clarkii)  is a major predator of amphibians’ larvae, including those of the brown frogs  Rana dalmatina  and  Rana latastei . Although qualitative information about the spawning site preferences and breeding microhabitat choice of brown frogs is available in the literature, only a few studies performed quantitative analyses, and the relationship between microhabitat choice and the presence of alien predators has not been investigated yet. The aims of this study were: (1) to characterize the microhabitats selected for clutch deposition by  R. dalmatina  and  R. latastei  and (2) to test if the position and the aggregation of egg clutches differ in sites invaded or not invaded by  P. clarkii . During spring 2017, we surveyed multiple times 15 breeding sites of both brown frogs in Northern Italy; in each site we assessed the features of the microhabitat where each egg clutch was laid, considering its position (distance from the shore, depth of the water column) and the degree of aggregation of clutches. In each site we also assessed the presence/absence of the invasive crayfish and the relative abundance in the breeding period. We detected egg clutches in all sites; the crayfish occurred in eight ponds. Our results showed substantial differences between the spawning microhabitat features of the two brown frogs:  Rana latastei  clutches showed a higher degree of aggregation and were associated with deeper areas of the ponds , while  Rana dalmatina  deposited more spaced out clutches in areas of the ponds that were less deep. For both species, spawning microhabitat features were not significantly different between sites with and without  P. clarkii . Although we did not detect behavioural responses to  P. clarkii  in the choice of spawning microhabitat , additional studies are required to assess whether these frogs modulate other behavioural traits (e.g. during larval development) in response to the invasive predator.

Invasive crayfish does not influence spawning microhabitat selection of brown frogs