Data for: Evolutionary transition from surface to subterranean living in Australian water beetles (Coleoptera, Dytiscidae) through adaptive and relaxed selection

Summary of contents:

• Zip Folder "R Scripts" containing the R code for the analyses
• Three scripts called: antenna_legs.R; dorsal.R; triplets.R
• Zip Folder "raw data" containing the measurement data (linear and landmarks)
• Zip folder "ZYX Dytiscids Photographs - beetles dorsal views" containing the photographs from which measurements were taken - Folder is 5.64GB (9.3GB uncompresed)
• Zip folder "ZYX Dytiscids Photographs - beetles linear measuring photos" containing the photographs from which measurements were taken - Folder is 21.12GB (38.9GB uncompresed)
  

Samples overview and ecology

A total of 362 specimens were measured from the South Australian Museum (SAMA) Entomology collection, plus 13 published drawings (Watts & Humphreys, 2003, 2004, 2006, 2009). This comprised 121 species (108 from SAMA and 13 from camera-lucida drawings) in Dytiscidae (Coleoptera), including 31 ‘surface’ aquatic diving species, 88 ‘subterranean’ aquifer-inhabiting species, and four ‘interstitial’ species (Figure 1). These classifications refer to their ecology: surface refers to diving dytiscid species that inhabit aboveground stream- or pond-related habitats; subterranean refers to species that live in underground aquifers (calcrete/ fractured rock); interstitial species reside in ephemeral water bodies such as seasonal drying streams. A species list, including authorities for each species, is included in Supplementary File 1. The selected dytiscid species are distributed across genera Limbodessus Guignot, 1939, Paroster Sharp, 1882, Allodessus Guignot, 1953, Gibbidessus Watts 1978, Neobidessodes Hendrich & Balke, 2009 and Uvarus Guignot, 1939, in Dytiscidae, with over 90% of species distributed in genera Limbodessus and Paroster. Among the 13 species sampled from drawings, five are in Limbodessus, five in Paroster, two in Neobidessodes, one in Exocelina Broun, 1886. Species sampled from drawings were used for the aquifer triplet analysis only, detailed below.

Digital imaging

Photographs of the dorsal view of the whole animal and close-up images of the antennae and the legs were taken with one Auto Montage system, which used algorithms to merge multiple photos in different focuses into one high-quality image. The imaging system was a Leica M205C microscope on a vertical track operated by Leica Application Suite v3.8.0 attached to a Leica DFC500 camera. From the series of images with incremental focus, a stacked montage image was automatically produced in the Leica Application Suite v3.8.0 (Figure 2).

To maintain consistency and reduce the impact of specimen presentation orientations, all specimens were imaged with the scutellum or the anterior end of the middle suture of the elytra being the uppermost point. No colour calibration was used. No optical control was used except an automatic exposure time regulator at its default value. The specimens were presented as flat to the plane of the camera as possible to avoid horizontal tilts.

To capture as many details of the specimen as possible, we used optimising steps for the multifocus within Leica Application Suite v3.8.0. A step was defined as each focus plane. Each step varied between 0.01mm to 0.05mm. The outcome was high-resolution (4080*3072 pixels) TIFF image files with scale bars.

Morphometrics

Two morphometric methods were used to capture the morphological variation among specimens. We quantified the morphology in the body outline of the specimens by using geometric morphometrics (landmark-based measurements). As the landmarking required a dorsal view of the beetles, we used 255 specimens of 99 species that were dorsally orientated and presented. Morphology of the three pairs of limbs and antennae were quantified using linear measurements for 107 species, each represented by one specimen.

The body shape dataset used both fixed homologous landmarks identifiable on all specimens and semi-landmarks that constitute a homologous curve (Zelditch et al., 2004). Since male dytiscids were slightly larger than females, male specimens were chosen where possible to remove bias caused by potential allometry. Multiple specimens were selected to represent their species. However, some species were represented by a single specimen. For example, Paroster extraordinarius, the first and only known stygobiotic dytiscid species found in South Australia (Leys et al., 2010), was represented by one holotype specimen. In summary, a set of 71 landmarks were chosen to capture the outline shape of the dytiscids, illustrated in Figure 2 with definition described in Table 1. We used the software tpsDIG2 v.2.32 (Rohlf, 2021) to manually place the fixed landmarks and semi-landmarks on the photographs (Figure 2). The landmarks, represented by two-dimensional coordinates, were exported in the .TPS file format.

The body length, and dimensions of the limbs and antennae comprised a total of 17 measurements (in mm) recorded from the photographs with scale bars (Figure 2, Table 2). When photographing, the length of the body, antennae and tarsi were recorded on-site using the default measuring tool implemented within Leica Application Suite v3.8.0. The length and width of legs were later measured using the measuring tool in the GIMP v2.10.32 software (The GIMP Development Team, 2022). Both dorsally and ventrally presented specimens were used to capture the size of the features. As the antennae were usually curled out of the plane of the camera, up to three images were taken at different angles. The length of the antennae was recorded by the sum of the length of the eleven segments – the scape, the pedicel and nine flagellomeres.

As the coxae of dytiscids are fused medially to the sternum and the trochanters are not always presented in the same orientation with the rest of the legs, both coxae and trochanters were not recorded, leaving only femur, tibia, and tarsus (Figure 2, Table 2).