Inferring Sex from Osteohistology in Eocene Penguins (Aves, Sphenisciformes) from Antarctica

Sexual dimorphism is a major source of intraspecific variation that might complicate the taxonomic interpretation of fossil penguins, potentially leading to an overestimation of species diversity when sex-related morphological variation is misinterpreted as taxonomic differences. Bone histology and microanatomy provide an independent line of evidence for sexual differentiation, as they preserve records of physiological processes associated with skeletal development and reproduction. In this study, we analysed the osteohistology and microanatomy of 37 thin sections from appendicular elements of Eocene Antarctic penguins from Marambio (Seymour) Island, including Delphinornis, Marambiornis, Anthropornis, Palaeeudyptes, Archaeospheniscus, Notodyptes, and undetermined specimens. The histological observations focused on bone matrix organisation across tissue regions, vascularisation, secondary remodelling, medullary microstructure, and the condition of the inner circumferential layer (ICL), particularly in the hindlimbs, to assess its possible significance for sex determination. No evidence of medullary bone was identified in any specimen, probably reflecting the transient nature and low preservation potential of this reproductive tissue. However, variation in ICL development and remodelling revealed three microstructural states associated with the reproductive cycle in extant penguins. Remodelled ICLs, interpreted as evidence of females that had completed at least one reproductive cycle, were identified in Delphinornis arctowskii, Anthropornis nordenskjoeldi and Palaeeudyptes gunnari. The presence of an unremodelled ICL, as observed in Anthropornis grandis, does not allow reliable sex determination because it may correspond either to males or to females outside the oviposition period. Likewise, the lack of ICL in Delphinornis larseni and Palaeeudyptes klekowskii could indicate that they are females. Comparisons among skeletal elements further indicate that hindlimbs preserve more informative histological signals than forelimbs, whose microanatomy is more strongly modified by biomechanical adaptations related to wing-propelled diving. These findings show that osteohistological and microanatomical evidence can substantially improve the recognition of sex-related variation in fossil penguins, providing a framework for interpreting morphological diversity. Incorporating these criteria into systematic studies may reduce taxonomic bias and refine the delineation of fossil species within Sphenisciformes.