Mental glands and their associated delivery behaviors during courtship are unique to the plethodontid salamanders. Because previous interpretations of the evolution of these features were conducted using older phylogenetic hypotheses, we reanalyzed these traits with newer courtship descriptions and contemporary phylogenetic methods. Using Bayesian ancestral state reconstruction methods that have been developed since the first phylogenetic analyses were conducted in the mid-1990s, we reconstructed mental gland and courtship behavior evolution on a Bayesian phylogeny of the nuclear gene Rag1. The most probable ancestral condition for plethodontids was resolved as presence of a mental gland. Loss of a mental gland occurred in each subfamily and was recovered as the most probable ancestral condition for the Spelerpinae. In contrast, parsimony reconstruction recovered the presence of a mental gland in the ancestor to Spelerpinae with multiple secondary losses. We hypothesize that that absence of a mental gland is possibly ancestral in some clades (i.e., Spelerpinae) and secondary in others (e.g., paedomorphic Eurycea). The most probable ancestral form of the mental gland is likely to be the large pad-type distributed extensively in Plethodontinae and Bolitoglossinae. Desmognathans have the most unique mental glands, occurring in an anterior protrusion or bifurcated form (in Desmognathus wrighti). Fan-shaped mental glands evolved independently in Eurycea and Oedipina. Small pads arose independently in Bolitoglossinae, Plethodontinae, and Spelerpinae. Head-rubbing behavior for mental gland delivery mode was recovered as the most probable and parsimonious ancestral state for the Plethodontidae, with independent losses of this behavior in Plethodontinae and Spelerpinae. Because head-rubbing was observed in outgroups, we hypothesize that head-rubbing behavior predated mental gland evolution. Pulling, snapping, slapping, and biting behaviors evolved independently in the Plethodontinae and Spelerpinae and are not homologous with head-rubbing. All hypotheses of mental gland and courtship evolution invoke homoplasy.

Morphogenesis is an emergent property of biochemical and cellular interactions during development. Genome size and the correlated trait of cell size can influence these interactions through effects on developmental rate and tissue geometry, ultimately driving the evolution of morphology. We tested whether variation in genome and body size is related to morphological variation in the heart and liver using nine species of the salamander genus Plethodon (genome sizes 29–67 gigabases). Our results show that overall organ size is a function of body size, whereas tissue structure changes dramatically with evolutionary increases in genome size. In the heart, increased genome size is correlated with a reduction of myocardia in the ventricle, yielding proportionally less force‐producing mass and greater intertrabecular space. In the liver, increased genome size is correlated with fewer and larger vascular structures, positioning hepatocytes farther from the circulatory vessels that transport key metabolites. Although these structural changes should have obvious impacts on organ function, their effects on organismal performance and fitness may be negligible because low metabolic rates in salamanders relax selective pressure on function of key metabolic organs. Overall, this study suggests large genome and cell size influence the developmental systems involved in heart and liver morphogenesis.

Gladwyn Kingsley Noble was the first investigator to collectively examine courtship glands and correlate their function to the tail-straddling walk in plethodontid salamanders. While mental glands and caudal courtship glands have received the majority of attention since Noble's seminal work, Noble described other glands from Eurycea bislineata that were putatively involved in courtship that have received little or no attention (e.g., glands at the temporal regions of the heads of males). Previous studies demonstrated that heads of males enlarge during the mating season and some studies indicated that the enlargement was because of Noble's previously described temporal glands; however, current consensus of male head enlargement is that skeletal muscle hypertrophy is the cause of the head growth. In this study, we examined male and female E. bislineata throughout the year to test Noble's hypothesis that males possess courtship glands in the integument of the temporal regions of their heads and to assess what underlying tissues are involved with male head enlargement during mating. We found that the temporal regions of male heads change dramatically from non-mating to mating months, exemplified by two-dimensional geometric morphometrics. This variation is a result of M. levator mandibulae externus hypertrophy rather than glandular activity underlying the integument. Although no glandular masses isolated at the temporal regions of the heads result in this dynamic shape change, simple alveolar glands that resemble stereotypical courtship glands are found in the integument of only males from the mating season. These putative courtship glands are scattered amongst mucous and serous glands within the integument around the eyelids to at least the posterior termination of the cranium. The function of these glands is unknown but is probably involved with pheromone production, similar to more thoroughly examined courtship glands.

The skin glands and cloacal morphology of the Korean crevice salamander, Karsenia koreana, were similar to those of other plethodontids. The skin contained mucous, granular, and modified granular glands in varying frequencies and sizes. Males had sexually dimorphic glands in the skin of the chin (mental glands) and the dorsal tail base (caudal courtship glands). On the ventral surface of the tail base, modified granular glands were sexually dimorphic in size, with male glands larger than those in females. The cloacal glands in males, as in other plethodontids, consisted of four eosinophilic gland clusters (dorsal pelvic glands, lateral pelvic glands, caudal pelvic glands, and vent glands) and three basophilic glands (anterior ventral glands, posterior ventral glands, and Kingsbury's glands). In females, the only cloacal gland was the spermatheca, which, as in other plethodontids, was a compound tubulo-alveolar gland in the roof of the cloaca.