DOI: 10.5061/dryad.j6q573nh3
title: First evidence of hemiclitores in snakes

description: 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.
description: <strong>Specimens and euthanasia</strong>
 We examined female genitalia in ten adult specimens, eight
 frozen, and two fresh-fixed females, across nine species:
 <em>Acanthophis</em> <em>antarcticus</em>,
 <em>Agkistrodon</em> <em>bilineatus</em>,
 <em>Bitis</em> <em>arietans</em>,
 <em>Helicops</em> <em>polylepis</em>,
 <em>Lampropeltis</em> <em>abnorma</em>,
 <em>Morelia</em> <em>spilota</em>,
 <em>Pseudechis</em> <em>colleti</em>,
 <em>Pseudechis</em> <em>weigeli</em>, and
 <em>Pseudonaja ingrami.</em> We also examined the
 micro-anatomy of the male genitalia in an adult and a juvenile specimen
 (<em>Acanthophis</em> <em>antarcticus</em>) (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 <em>A</em>.
 <em>antarcticus</em> 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
 <em>A</em>. <em>antarcticus</em> 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
 <em>A</em>. <em>bilineatus</em>,
 <em>B</em>. <em>arietans</em>,
 <em>M</em>. <em>spilota</em>,
 <em>P</em>. <em>colleti</em>,
 <em>P</em>. <em>weigeli</em>, and
 <em>P</em>. <em>ingrami</em>, were used for
 dissection morphology, and <em>H</em>.
 <em>polylepis</em> and <em>L</em>.
 <em>abnorma</em> were used for diceCT morphology (Table
 S1). <strong>Histology</strong>
 For the female <em>A</em>.
 <em>antarcticus</em>, 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
 <em>A</em>. <em>antarcticus</em> 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). <strong>DiceCT</strong>
 The tail of the female <em>A</em>.
 <em>antarcticus</em> 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. 
description: Histology - Images, no programs needed. CZI data available on
 request - would use ZEN 3.4 (blue eddition) DiceCT -
 Avizo or Dragonfly