Eos 7d mark 2

Photographs of the H&E stained slides were taken with a Canon EOS 7D Mark II (mfr#9128B002AA) and Canon EF 100 mm f/2.8 L Macro (mfr#3554B002) lens. Slides were placed on a lightbox and photographed at 1:1 magnification with a shutter speed of 1/100 sec, aperture of f8.0, ISO 400 and saved as Canon RAW files. Contrast was adjusted equally for all images with Photoshop Lightroom and then exported as PNG files.

The genitalia preparations were made following standard techniques (Robinson 1976 ). Genitalia were separated from the abdomen, and mostly mounted in ventral aspect, some also in lateral aspect to show structural details not clearly visible in ventral aspect. The abdomen was cut laterally and spread out.
Photographs of adult specimens were taken with a Canon EOS 7D Mark II, MP-E 65 mm EF 100 mm macro lens. Focus stacking was done with Cognisys StackShot and Zerene Stacker, and final image editing with Adobe Photoshop 2021. Images of Meyrick’s adult type specimens in the NHMUK were provided under the museum’s Digital Collection Programme. The genitalia in the research collection of Kari and Timo Nupponen (coll. NUPP) were photographed with a Leica DM1000 microscope and integrated Leica DF295 digital camera. The genitalia in coll. NHMUK were photographed in Sackler Imaging Suite using a Zeiss Axioskop. Most genitalia dissections in both coll. NHMUK and coll. NUPP were photographed in 2–6 images in different focal planes and combined into single images using image-stacking software as implemented in Photoshop 2021. Images were edited in Photoshop 2021 and plates were compiled in CorelDraw 2018. Genitalia figures are not in scale.

Conventional (light) photographs were taken with either a Canon EOS 7D Mark II camera mounted on a BK Lab Plus Lab imaging system (Dun Inc., Charlottesville, VA, USA), a Leica DVM6 microscope (Leica Microsystems GmbH, Wetzlar, Germany), or a Nikon D100 digital camera using a standard copy stand. SEM images were taken with a FEI Quanta 650F variable-pressure field-emission SEM (FEI Company, Hillsboro, OR). The FEI microscope was operated at 15 kV, with a spot size of 2.45 and working distances of about 12 to 15 mm. Notably, the high conductivity of the rocks from McGraths Flat allowed high-resolution SEM imaging without any coating. All microfossils were imaged in situ (i.e., in the matrix) rather than using acid preparation. Micro x-ray fluorescence (μXRF) was undertaken by directly sampling across the surface of a rock containing a fish spine (AM F.146602) using a benchtop M4 Tornado μXRF (Bruker Corp., Billerica, MA). This allowed us to sample both the fossil and matrix for differences in chemistry. The instrument consists of a Rh anode metal-ceramic x-ray tube. The measurement was carried out under vacuum condition (20 mbar). The following parameters were used for elemental mapping analysis: x-ray tube, 45 kV and 600 μA; area mapping point distance, 60 μm; time/pixel, 600 ms; 1 cycle; maximum energy, 40 keV; and maximum pulse throughput, 130 kilo counts per second.

Analysis of salt tolerance in yeast was carried out using a yeast expression vector. PCR primers were designed based on the CDS sequence of the PtrDHN-3 gene as follows: forward primer 5’-ACTCACTATAGGGAATATTA ATGGCTGAGGAAAACAAGAGC-3’ and reverse primer 5’-TAATTACATGATGCGGCCCTCTACTGGGAAGCACTCTCCT-3’. The PtrDHN-3 gene was transferred into the yeast expression vector pYES2 (Carlsbad, CA, USA) using the Infusion enzyme (Zhongmei Taihe, c5891-50, Taihe, China). The constructed expression vector pYES2-PtrDHN-3 was then transformed into INVSC1 yeast cells, with an empty pYES2 plasmid used as a control. The transformed cells were cultivated on an SC-U medium containing 2% glucose in an incubator at 30 °C for three days. Positive clones were then identified by PCR detection. Single-clone cells containing the recombinant DNA (pYES2-PtrDHN-3) and control vector cells were then incubated in an SC-U medium with shaking until an OD of 0.6 was reached. Yeast cells were then diluted 10-, 100- and 1000-fold and spotted on an SC-U medium. For the salt tolerance assay, yeast cells were re-suspended in 3 M NaCl for 24 h then spread on an an SC-U medium and inverted in an incubator at 30 °C for three days. Images were then obtained (Canon EOS 7D MARKII, Japan). As a control, the cultured yeast cells were spotted on an SC-U medium without NaCl treatment.