Female genitalia are conspicuously overlooked in comparison to their male counterparts, limiting our understanding of sexual reproduction across vertebrate lineages. This study is the first complete description of the clitoris (hemiclitores) in female snakes. We describe morphological variation in size and shape (n = 9 species, 4 families) that is potentially comparable to the male intromittent organs in squamate reptiles (hemipenes). Dissection, DiceCT and histology revealed that, unlike lizard hemiclitores, the snake hemiclitores are non-eversible structures. The two individual hemiclitores are separated medially by connective tissue, forming a triangular structure that extends posteriorly. Histology of the hemiclitores in Australian death adder (Acanthophis antarcticus) showed erectile tissue and strands/bundles of nerves, but no spines (as is found in male hemipenes). These histological features suggest the snake hemiclitores have functional significance in mating and definitively show that the hemiclitores are not underdeveloped hemipenes or scent glands, which have been erroneously indicated in other studies. Our discovery supports that hemiclitores have been retained across squamates and provides preliminary evidence of differences in this structure among snake species, which can be used to further understand systematics, reproductive evolution and ecology across squamate reptiles.

Specimens and euthanasia

We examined female genitalia in ten adult specimens, eight frozen, and two fresh-fixed females, across nine species: Acanthophis antarcticus, Agkistrodon bilineatus, Bitis arietans, Helicops polylepis, Lampropeltis abnorma, Morelia spilota, Pseudechis colleti, Pseudechis weigeli, and Pseudonaja ingrami. We also examined the micro-anatomy of the male genitalia in an adult and a juvenile specimen (Acanthophis antarcticus) (Table S1). The adults were wild caught and were sourced from either Venom Supplies Pty. Ltd., private collections, or the University of Michigan Museum of Zoology (UMMZ). The juvenile A. antarcticus was born at Venom Supplies.

Once euthanised via injection of pentobarbitone, the specimens were immediately frozen at -20°C. Adult female, male, and juvenile male A. antarcticus specimens were used for histology, and an adult female was used for diffusible iodine contrast-enhanced µCT (DiceCT) scanning (Table S1). The adult females of A. bilineatus, B. arietans, M. spilota, P. colleti, P. weigeli, and P. ingrami, were used for dissection morphology, and H. polylepis and L. abnorma were used for diceCT morphology (Table S1).

Histology

For the female A. antarcticus, the tail was dissected dorsally to identify the hemiclitoral structure medial to the two scent glands, posterior to the cloaca. The hemiclitores structure and both scent glands were removed from the tail and fixed in 10% buffered formalin. For both males, the inverted hemipenes structures were removed and preserved in 10% buffered formalin.

The excised genitalia from the A. antarcticus histology specimens were processed and stained for paraffin histology. Each sample was sliced longitudinally with a microtome ten times at 5 µm (first nine slides not stained – 45 µm), once at 10 µm, then once again at 5 µm. The slides were stained in Haematoxylin and Eosin (H&E), Bielschowsky silver, and Masson’s Trichrome, respectively. The slides were scanned using an Axio Scan.Z1 Automated Slide Scanner (Axioscan, Zeiss, Germany) and the ZEN Blue software version 3.4 (Zeiss Zen blue edition, Zeiss, Germany).

DiceCT

The tail of the female A. antarcticus was removed with a transverse amputation just above the posterior lip of the cloaca. The tail of the death adder and the two colubrid full snake diceCT specimens were fixed in 10% buffered formalin, rinsed for 24 h, and transferred into 70% ethanol for at least two weeks. The tail and whole-bodied specimens were transferred into 50% ethanol for 48 h, then into 25% ethanol for 48 h before submersing in 1–1.25% Lugol’s iodine solution (I2 + KI + H2O) for approximately 14 days, as per the following protocol for diceCT (36). Scanning was conducted on the tail prior to and post staining using a SkyScan-1276 Micro-CT (Zeiss, Germany) at the University of Adelaide (Aluminium 1mm filter, 10µm, 90kV, 200µA), and on the whole-bodied specimens on a Nikon Metrology XTH 225ST µCT scanner (Xtect, Tring, UK) at the UMMZ. The 2D tomography slices for each scan were reconstructed in Avizo version 9.2 (Thermo Fisher Scientific, United States) or Volume Graphics Studio Max version 3.2 (Volume Graphics, Heidelberg, Germany) and the hemiclitores were segmented using a thresholding tool. The contrast between soft tissue in the tail was low, but the hemiclitores could clearly be defined by comparing its position with the images of the dissection and histology and by demarcations between the hemiclitoris and the two scent glands. 

Histology - Images, no programs needed. CZI data available on request - would use ZEN 3.4 (blue eddition)

DiceCT - Avizo or Dragonfly