We identified two distinct types of hatching gland cells in A. callidryas, which differ in size, morphology and distribution on the body and are, for the most part,present through different stages of development, with only a brief period of overlap. We call them ‘early’and ‘late’ HGCs based on their presence at different developmental stages (Fig. 2A, B). In surface view,early HGCs appeared much smaller than epithelial cells and had what appeared to be protrudingmicrovilli (Fig. 2A). They were typically located at the junction of three or four common epithelial cells.In contrast, late HGCs (Fig. 2B) had what appeared to be short microvilli and appeared recessed among and only slightly smaller than common epithelial cells. Neither HGC type was observed in contiguous clusters, although occasionally two HGCs of the same type were in contact. Early HGCs appeared first, and late HGCs remained for a longer duration (Fig. 3), butfor a period at 4 days both HGC types were present,with partial overlap in their spatial distribution on thebody (Fig. 2C–F).We first observed early HGCs at 3.0 days, and within 8 h they were abundant on the snout (Fig.4A–D). Early HGCs also appeared in a patch on top ofthe head (Fig. 4E, F) but were present there for ≤ 16 h. Sibships that were laid on the same night varied in when this top-of-head patch of early HGCs was present (two clutches from 3.33 to 4.0 days; two clutches from 4.0 to 4.67 days). Early HGCs on both the snout and the top of the head disappeared during development regardless of when embryos hatched. Early HGCs weremost abundant in 3-day-old embryos, then declined in abundance beginning at 4 days, as late HGCs began to appear. By 5 days, early HGCs were very sparse, but a few could still be observed on the snout, immediately above the late HGCs (Fig. 5D).