During courtship, a male P. shermani delivers pheromones to the female's VNO by slapping (2) his mental gland across her nares. (Arnold 1976).
Courtship Behavior & Female Sexual Receptivity

Much of our behavioral work has focused on Plethodon shermani (formerly P. jordani), a plethodontid salamander with complex, stereotyped courtship behavior (Arnold 1976, Houck 1986, Marvin & Hutchinson 1996, Picard 2005, Dyal 2006). 

      Stage 1. Orientation. The male orients to the female and moves towards her. 
      Stage 2. Physical Contact. The male attempts – and is allowed – to make physical contact with the female. Contact typically begins with the male touching his snout to the female’s tail, and then moving up to the female’s body, eventually contacting her head. In some cases, a male will break off contact and face the female (head-to-head).  In this position, the male may move forward rapidly and “slap” his mental gland across her snout (nares included).  This type of pheromone delivery generally is infrequent.
      Stage 3. Tail-straddling Walk. A female will respond to initial male contact by entering into a “tail–straddling walk” (TSW). The female straddles the male’s tail and places her chin on the male’s dorsum; in this position, the pair move forward in tandem (Fig. 3).
      Stage 4. Pheromone Delivery. Delivery (“slapping”, Figs. 4 & 7 video) typically occurs during TSW: the male turns back toward the female and slaps his mental gland (Fig. 5) on her snout . When the male turns back, the female typically lifts her head so that her nares will be contacted by the male’s mental gland. The slapping behavior thus delivers courtship pheromones to the female’s nares. 
      Stage 5. Spermatophore Deposition. While still in TSW, the pair moves forward until the male stops and lowers his cloaca to the substrate to deposit a spermatophore (which consists of a sperm mass that sits atop a gelatinous base). On average, spermatophore deposition takes approximately 7 minutes. 
      Stage 6. Insemination. After spermatophore deposition, the male lifts off of the spermatophore (moving his tail to one side) and proceeds forward (Fig. 6). The female keeps her chin on the male’s tail base and also moves forward until the spermatophore is directly below her cloaca. The female then lowers her cloaca over the spermatophore, lodges the sperm mass in her cloaca, and then lifts off, leaving the base on the substrate (Fig. 8 video).  Following insemination, the female walks away from the male.  Note that insemination is not guaranteed: a female not sufficiently receptive may walk past the spermatophore. In these cases, the spermatophore is a reproductive failure because the female will not retrieve a sperm mass except when she is in TSW.

PHEROMONE EFFECTS ON FEMALE BEHAVIOR During courtship, a male P. shermani salamander delivers pheromones from a submandibular mental gland to the female’s nares. The pheromones are only delivered after courtship has commenced and their purpose is to increase female receptivity and mating success.  These pheromones do not serve as a sexual attractant. We conducted behavioral tests in which the natural delivery of male courtship pheromones was prevented by ablating the courtship (mental) glands of all experimental males in order to control experimentally the amount and type of pheromone stimuli received by each female. We conducted three behavioral tests, each using our standard protocol. In our first test, we used a pheromone solution that was enriched (relative to untreated gland extracts) with the protein component PRF (i.e., PRF accounted for approx. 90% of proteins in this solution). For courting pairs in which the female received this enriched solution, female receptivity was increased (i.e., courtships were completed more quickly) (Houck et al. 1998). In our second behavioral experiment, we similarly staged courtship encounters which demonstrated that a PRF solution that was purified (i.e., no other detectable proteins were present) was effective in increasing female receptivity (Rollmann, Houck & Feldhoff, 1999). In our third experiment, we demonstrated that female response to courtship pheromones from conspecific sympatric males and from heterospecific allopatric males was statistically indistinguishable (Rollmann, Houck & Feldhoff 2003).

EVOLUTIONARY STASIS IN MALE COURTSHIP BEHAVIOR AND MORPHOLOGY The pheromone delivery system of plethodontid salamanders presents a remarkable picture of morphological and behavioral stasis (Houck & Sever 1994, Houck & Arnold 2003). Species in each of the four major lineages share the same, complex system of pheromone delivery by vaccination. During the courtship season, males grow hypertrophied premaxillary teeth and develop a small pad of glandular tissue on their chins (the mental gland). During courtship, the male abrades the female's skin with his premaxillary teeth and rubs secretions from his mental gland into the abraded site. These secretions increase female receptivity and shorten the time to sperm transfer (Houck & Reagan 1990). Because this complex system is found in all four major lineages of plethodontids and in no other salamander family, it was undoubtedly present in the ancestral plethodontid. Thus, because the family itself is approximately 100 million years old (Ruben et al. 1993), the morphological and behavioral elements in the delivery system have been conserved over that entire period. Charlesworth et al. (1982) have persuasively argued that such long term stasis must be a consequence of stabilizing selection. Other mechanisms – such as evolutionary inertia or developmental constraint – may produce short term stasis, but they cannot account for long term stasis.

An equally puzzling example of stasis is found within the genus Plethodon. The forty-one species of this genus found in eastern North America form a monophyletic group with two subdivisions that differ greatly in body size: the small eastern Plethodon and the large eastern Plethodon (Highton & Larson 1979, Larson et al. 1981, Highton 1993, Highton & Peabody 2000). The small eastern Plethodon (12 species) retain the ancestral mode of vaccination delivery of pheromones. The large eastern species (29 species) are a monophyletic group that share an olfactory mode of pheromone delivery. In these species, the males lack protruding premaxillary teeth and possess a large circular mental gland. These species deliver pheromones by slapping their large mental glands over the female's snout during courtship. Immunological and allozyme data indicate that the radiation of large eastern Plethodon – and hence the olfactory mode of delivery – is about 19 million years old (Highton & Larson 1979). Although we do not understand the details of the process that is responsible for this second example of stasis, under the same argument as before, it is probably a consequence of stabilizing selection maintained over a 19 million year period.

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