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(Roos, DPZ Gottingen, unpublished).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).