Collection of own additional data from two free-ranging populations in Poland and Germany
We added data we collected ourselves from two populations: Radolfzell, in the vicinity of Lake Constance (Germany; 47.764345, 8.997449; data published in Lázaro et al. (2018a); and Gugny, in the Biebrza National Park (Poland, 53.347487, 22.589436; new data). All handling and sampling methods in Poland were approved by the Ministry of Environment (DLP-III-4102-42/2607/14/MD, DLP-III.4102.136.2016.AK).
We captured shrews with wooden live traps (PPUH A. Marcinkiewicz, Rajgród, Poland) baited with mealworms and checked at 2-h intervals. In Radolfzell we trapped monthly from December 2013 to July 2016. In Gugny we trapped at the estimated peak periods of the morphological change cycle, in February, June and July 2014, May 2015 and May 2016. Immediately after capture, shrews were weighed (± 0.01 g) and carried to the laboratory where they were euthanized with anaesthesia overdose (Isoflurane) and perfused transcardially with phosphate-buffered saline (PBS) followed by 4% formaldehyde in PBS. Then we extracted the skull and used a digital caliper (± 0.01 mm) to obtain braincase height, skull length, and braincase width as described above for museum specimens. After this, we extracted the brain and weighed it (±0.001 g). We size corrected brain mass by the maxillary tooth row length, which does not change seasonally and we obtained from post-mortem X-ray images of the skulls. This size-correction factor had been used for brain and brain regions volume in previous work (see Lázaro et al. (2017) for details).
We classified individuals as summer juvenile, winter subadult or adult based on the degree of gonadal development, capture date and degree of tooth wear (Pankakoski, 1989; Churchfield, 1990). For adults, sex can be directly determined visually. For immature individuals (juveniles and subadults) for which we could not directly check the gonads during dissection, we determined sex with a PCR-based gonosomal sexing method (Roos, DPZ Gottingen, unpublished). For this, we extracted DNA from tail tip samples with a standard DNeasy kit (Qiagen, GmbH, Hilden).