Developmental stage ordering yields greater cranial mineralization sequence resolution than embryo size or days since...

Mineralization sequences of cranial elements (often referred to as ossification sequences) are used for a variety of purposes. Believed to be consistent within species (even though they exhibit some variation) and conserved within lineages, they have been assembled using a variety of developmental timetables-absolute time using developmental days; relative time using either increase in size of embryonic dimensions or developmental staging. The relationship between these developmental timetables is unclear in terms of how mineralization sequences are expressed, although they are generally treated as being able to achieve equivalent levels of resolution. Regardless of the developmental timetable employed, mineralization sequences are replete with ties representing simultaneous mineralization of several elements, even though ties are suspected to be rare. Herein we examine the resolution attainable of cranial mineralization events in the leopard gecko by subjecting the same set of embryos, raised under controlled conditions, to sequence analysis using all three of the above-mentioned timetables, with the working hypotheses being that all three would yield the same level of resolution and that we could improve upon the level of resolution attained for gekkotans so far. We found that developmental stage timetabling yielded far less variability in the determination of mineralization sequence as well as considerably better resolution than those for developmental days or embryonic dimensions. We were able to obtain much greater resolution for the leopard gecko than that so far attained for gekkotans for all three developmental timetables, but especially so when ordering the specimens by developmental stage. Furthermore, we found that subdividing embryonic stages into substages (established using additional morphological features) and assessing intensity of staining of mineralizing elements hold promise for achieving improved levels of resolution. Even so, we were only about 55% successful in resolving the cranial mineralization sequence into a series of unique events, indicating that many such events are so closely spaced in developmental time that apparent simultaneity of element mineralization will be challenging to resolve further.