Data from: Hatching plasticity is associated with a more advanced stage at hatching in an Ambystoma with terrestrial eggs

Description

Notes

Methods

All embryos were collected as either an egg mass, constituting a group of presumed half or full siblings surrounded by a jelly layer (ringed salamander), or a clutch of half or full siblings found together in a nest site (marbled salamander). Hereafter, we refer to both of these grouping as clutches. Clutches were kept separate in gas-permeable bags for transport back to Asheville, NC.

We removed jelly from ringed salamander embryos by gently pulling the jelly apart and releasing intact, encapsulated embryos into tap water (aged to allow chlorine to evaporate before use). Marbled salamander embryos are not encased in jelly so were not separated, but were rinsed in the same tap water to remove sediment. 

Each clutch was divided into two treatment groups of 20 embryos. One group was placed in 50 ml specimen jar submerged in ~ 40 ml aged tap water (water treatment) whereas the other group was placed in an identical jar with ~ 1 ml aged tap water to prevent desiccation (air treatment). These two treatments were identical to the "saturated" and "air" treatments in Hale et al. (2016 Evolutionary Ecology). In the water treatment, air was pumped into the jar via an airstone inserted through the jar lid, submerged in the water, and connected through tubing to an aquarium pump. Identical lids with airstones were placed on jars in the air treatment. However, due to difficulty maintaining air pressure from our pumps when connected to a high number of jars, airstones on air treatment jars were not connected to an air pump and lids were left loose on the jars to allow air circulation. All jars were randomly situated in a Conviron CMP6050 growth chamber (Controlled Environments Limited, Manitoba) at 20 °C, 80 % relatively humidity, with a 12 h:12 h light:dark cycle. In the air treatment, embryos were moistened, as needed, to prevent desiccation; embryos rested in a small puddle of water in the bottom of the jar, but were not submerged.

We inspected all embryos under a dissecting microscope daily and recorded the number of live, dead, and hatched embryos. Embryos were scored as dead if the egg membranes were opaque or if the yolk and embryo were collapsed and non-spherical. The latter often occurred in early stage embryos. The minimum and maximum embryonic stage (Harrison, 1969) of live, developing embryos were recorded for each jar and were later converted to median stage to assign the jar a single stage value. The embryonic stage upon hatching was recorded for each individual and the first five hatchlings per jar were euthanized in MS-222 and preserved in Shandon^TM Glyo-Fixx^TM (Richard Allan Scientific, Kalamazoo, MI, USA) before being vacuum dried for 48 h and weighed to the nearest 10 g on a Mettler-Toledo XP2U microbalance (Mettler-Toledo LLC, Columbus, OH, USA).