Comparative spermatozoa ultrastructure of neotropical grass frogs (genus Leptodactylus) with comments on anuran reproductive modes and phylogeny

The sperm cell ultrastructure provides important insights into fertilization mechanism and phylogenetic relationships. However, it has rarely been included in phylogenetic analysis of anurans. This work aims to characterize the ultrastructure of the mature spermatozoa in the genus Leptodactylus, to relate the observed variations with the different reproductive strategies and define characters to optimize in a previously published cladogram of this genus. We studied 24 species distributed into four groups: L. fuscus, L. latrans, L. melanonotus and L. pentadactylus where morphological characters were coded and optimized using the parsimony method. The analyzed spermatozoa have three conspicuous regions: head, middle zone and tail. Regarding the head, a short and thin acrosomal vesicle is observed in species of the groups L. fuscus and L. pentadactylus, but it is long and thick in most of the studied species of the groups L. latrans and L. melanonotus. Furthermore, nuclear lacunae are observed in most species. In relation to the middle zone, an organelle-free cytoplasmic region was observed in species of the groups L. fuscus, L. latrans and L. melanonotus, but it was absent in species of the L. pentadactylus group. Most of the studied species showed a mitochondrial collar and cytoplasmic canal. Finally, in the tail all of the species have an axoneme, an undulating membrane, and an axial fiber, but only some have a juxtaxonemal fiber. Results show the thick acrosomal vesicle as a synapomorphy of the clade L. latrans + L. melanonotus. The presence of nuclear lacunae and the absence of a sub-acrosomal cone optimizes as a putative synapomorphy of Leptodactylidae. In conclusion, we observed a relationship between reproductive strategies and spermatozoa morphology, which also supports the phylogenetic relationships in this genus.