Af s micro nikkor lens

A recent study focusing on Crocidura species from Sulawesi showed that skull size, rostral length and skull width are useful criteria for species discrimination^{48 (link)}. All three are captured in the complete outline of the skull in dorsal view, using skull centroid size as a proxy for overall size. Due to the small size, the fragile nature, and the high likeliness of Crocidura specimens, using the outlines allowed the inclusion of more individuals while capturing the variability in overall skull shape. In addition, the use of an automated procedure avoided the biases caused by manual landmark placement^{49 (link)}. 433 genotyped individuals of the C. poensis species complex were photographed using a NIKON D5600 and a 60 mm AF-S Micro NIKKOR lens. The individuals and their information are displayed in SM2. Specimens were assigned an age group using a combination between suture fuse of the basioccipital and basisphenoid bones and tooth wear (Supplementary Fig. 2), to verify the possible interaction between species and age effects on shape. The outline curves and a single homologous landmark were used in the morphometrics protocol detailed in SM1, outputting symmetrical Procrustes coordinates, centroid size (which was log transformed), and PC axes retaining 90% of total shape variability.

Following injection procedures, each filter was allowed to air dry for increased microsphere opacity. Photographs of each filter were captured using a DSLR camera (Nikon D500 with 85 mm AFS Micro NIKKOR lens, Nikon Corporation, Tokyo, Japan). Filters were backlit with the use of LED lights and an optical diffuser. A custom MATLAB application, developed through extensive adaptations of an open-source application (“FindCirclesGUI” by Brett Shoelson [22 ]), was used to automate the counting of microspheres in each image (Fig. 9). While most microspheres deposited on a single layer, microspheres depositing in multiple layers were distinguished by their darker color in relation to adjacent microspheres. In these cases, quantities of visible microspheres were doubled to account for stacking.Fig. 9

Quantification of microspheres with MATLAB-based image analysis: a GUI, b enlarged view of top center region of original image, and c detection of microspheres in region shown in b. Circled region denotes microspheres arranged in multiple layers