Adaptive radiation is the diversification of species to different ecological niches and has repeatedly occurred in different salmonid fish of postglacial lakes. In Lake Tinnsjøen, one of the largest and deepest lakes in Norway, the salmonid fish, Arctic charr (Salvelinus alpinus (L.)), has likely radiated within 9700 years after deglaciation into ecologically and genetically segregated Piscivore, Planktivore, Dwarf and Abyssal morphs in the pelagial, littoral, shallow-moderate profundal and deep-profundal habitats. We compared trait variation in the head shape, the eye and olfactory organs, as well as the volumes of five brain regions of these four Arctic charr morphs. We hypothesised that specific habitat characteristics have promoted divergent body, head and brain sizes related to utilized depth differing in environmental constraints (e.g. light, oxygen, pressure, temperature and food quality). The most important ecomorphological variables differentiating morphs were body length, habitat, optic tectum and eye area. The Abyssal morph living in the deepest areas of the lake had the smallest brain region volumes, head and eye size. Comparing the olfactory bulb with the optic tectum in size, it was larger in the Abyssal morph than in the Piscivore morph. The Piscivore and Planktivore morphs that use more illuminated habitats have the largest optic tectum volume, followed by the Dwarf. The observed differences in body size and sensory capacities in terms of vision and olfaction in shallow and deep-water morphs likely relates to foraging and mating habitats in Lake Tinnsjøen. Further seasonal and experimental studies of brain volume in polymorphic species are needed to test the role of plasticity and adaptive evolution behind the observed differences.

We sampled fish using gillnets, traps and baited anchored longlines in August-October 2013 (Østbye et al., 2020). We sampled in four habitats; (i) the pelagial (setting gillnets positioned more than 50 m from shore and 20-30 m depth in midwater using a 12-panel multimesh Nordic series with mesh sizes  in this order of 43, 19.5, 10.0, 55.0, 12.5, 24, 15.5, 35.0, 29.0, 6.3, 5.0 and 10.0 mm and Jensen floating series with mesh size of 13.5, 16.5, 19.5, 22.5, 26.0, 29.0, 35.0, 39.0, 45.0 and 52.0 mm), (ii) the littoral (gillnets within 20 m from the shore using Nordic and Jensen littoral net series), (iii) the shallow-moderate profundal (Jensen littoral net series, traps and hook-line between 20-150 m depth), and (iv) the deep profundal (setting traps > 150 m depth and > 100 m from the shoreline using longlines of 220m long and 3 to 4 mm line with 180 hooks; see more detailed information in Østbye et al., (2020)). In the field, we assigned each individual to one of the four morphs (called field-assigned morphs: FA morphs) based on differences in body and head appearance and coloration. We also measured body length, and determined the sex and maturation stage visually (i.e., mature if the gonads covered more than half of the body cavity length; immature otherwise). We euthanized the fish with an overdose of benzocaine and we preserved the heads in formalin (10% unbuffered).

We had 72 individuals with both genetic and morphological data for each individual. We digitized a set of 30 common anatomical landmarks in tpsDIG2 v.2.22 (Rohlf, 2015) to capture head variation, which we included for landmark-based geometric morphometrics and statistical analyses. In addition, we measured the width (W) and height (H) of the eye in tpsDIG2 to calculate the eye area. We standardized for size with a Generalized Procrustes Analysis (Adams, Rohlf, & Slice, 2004; Zelditch, Swiderski, Sheets, & Fink, 2004) and we calculated the centroid size (i.e. as a measure of size) for each individual to use in further analyses. We determined the age based on otoliths, which are more reliable than scales especially in Arctic charr (Christensen, 1964). Following Pollen et al., (2007), we measured five brain regions: olfactory bulb, telencephalon, optic tectum, cerebellum, and hypothalamus. We measured the width (W) of each brain structure from the dorsal and ventral image of the brain, as well as the length (L) and height (H) from lateral views of the left hemisphere. We used an ellipsoid model to estimate the volume (V = 1/6 π(LWH)) of each brain region (Huber et al., 1997). We dissected the olfactory rosettes and the nasal organ and stored them in 70% ethanol. We measured the width (W) and length (L) using a micrometer under the microscope in order to calculate the surface area of each olfactory rosette (A= 1/4 π(WL)). We also counted the number of olfactory lamellae in each rosette.

We counted (N1) and measured the olfactory organs (1) and measured five brain regions (olfactory bulb (2), telencephalon (3), optic tectum (4), cerebellum (5), and hypothalamus (6). We measured the width (W) of each brain structure from the dorsal and ventral image of the brain, as well as the length (L) and height (H) from lateral views of the left hemisphere. We also measured the the width (D1) and the height (D2) of the eye to calculate the eye area. For the head size, we calculated the centroid size.