Reproductive Biology of Crocodilians

Matthew D Hale, Savannah River Ecology Laboratory, Aiken, SC, United States; and University of Georgia, Athens, GA, United States
Jessica A Cloy-McCoy, College of Charleston, Charleston, SC, United States
Brenna M Doheny and Benjamin B Parrott, Savannah River Ecology Laboratory, Aiken, SC, United States; and University of
Georgia, Athens, GA, United States
© 2018 Elsevier Inc. All rights reserved.

Glossary
Anamniotic Egg-laying vertebrates that do not produce an amniotic egg; fish and amphibians.
Archosaurian Belonging to the amniote lineage Archosauria, which includes dinosaurs, crocodilians, and birds; contrasts with
Lepidosauria, which includes non-crocodilian reptiles (squamates).
Folliculogenesis The process of development, in which a female germ cell matures into a large haploid oocyte, surrounded by
supporting granulosa and thecal cell layers.
Genital ridge Embryonic structure, precursor to the gonad.
Granulosa cell Female somatic support cell that immediately surround oocytes, responsible for estrogen synthesis and
promotion of early ovarian development.
Intromittent organ Phallus or penis, used for sperm delivery in species with internal fertilization.
Lacunae Large spaces or cavities in the medullary region of the ovary of birds, crocodilians, and other reptiles.
Leydig cell Male somatic support cell in the testis, responsible for androgen synthesis.
Multiple paternity Phenomenon of a single clutch of eggs that are fertilized by sperm from more than one father.
Neonate Hatchling crocodilian.
Oocyte Female germ cell, egg.
Pod or crèche Group of hatchlings and/or young crocodilians.
Primordial germ cell (PGC) Germ cells that will migrate into the genital ridge and become either oocytes or spermatogonia.
Sertoli cell Male somatic support cell in the testis, responsible for controlling spermatogonia development and promotion of
early testis development.
Spermatogonial stem cells Germ cells in males that give rise to sperm cells.
Steroidogenic Capable of producing steroid hormone(s).
Testis cord, medullary sex cord Embryonic structure in the testis that will develop into seminiferous tubules.
Theca cell Female somatic support cell that also surround oocytes, derived from granulosa cells, responsible for androgen
synthesis.
Thermosensitive period Window of development during which the embryonic gonad is sensitive to temperature in sex
determination; temperature cues outside this window do not effect sex.
Thermosensitivity Sensitivity to temperature in the context of sex determination during embryogenesis.
TSD Temperature-dependent sex determination, a mode of environmental sex determination where the temperature
experienced during a window of embryogenesis determines sex of offspring.

Natural History of Crocodilian Reproduction

Crocodilians are globally distributed in tropical and subtropical areas, found on every continent excluding Europe and
Antarctica. The International Union for Conservation of Nature recognizes 24 extant species of crocodilians within the order
Crocodylia, with seven of these species listed as endangered or critically endangered. Crocodilians are spread across three
families, including: Alligatoridae, which encompasses alligators and caiman; Crocodilidae, which includes all crocodiles; and
Gavialidae, which includes only a single species, the gharial (Gavialis gangeticus) (although some lines of evidence suggest
that a member of Crocodilidae, Tomistoma schlegelii, or the false gharial, is more closely related to the gharial than to crocodiles).
Members of family Alligatoridae are found predominantly in the Americas, with the single exception being the Chinese alligator
(Alligator sinensis), which is found exclusively in China. In contrast, members of Crocodilidae are found across the Americas, Asia,
and Africa, while the single member of Gavialidae is found only on the Indian subcontinent. Despite their global distribution, all
crocodilians are characterized by a shared reproductive biology and ecology that includes hierarchical social structures, complex
reproductive behaviors, environmental sex determination, and Archosaurian reproductive traits. In this article we review this
shared biology, discussing aspects of crocodilian nesting ecology, sex determination, and the reproductive tract. In the following
section, we begin with a review of selected characteristics of crocodilian reproductive and nesting ecology, describing (1)

646 Encyclopedia of Reproduction, 2nd edition, Volume 6 https://doi.org/10.1016/B978-0-12-809633-8.20612-2

 Reproduction, Exotics/Zoo Animals/Wildlife j Reproductive Biology of Crocodilians 647

crocodilian mating systems and courtship and (2) nesting ecology, focusing on types of nests built by crocodilians and parental
behavior during and after nesting.

Mating Dynamics
Hierarchies and male competition
Mating systems of crocodilians are characterized as polygamous male-dominated hierarchies, in which sexually mature
males will compete for and establish territories, permitting access to receptive females. In this system, dominant males will
typically mate with multiple females in a single year. In most crocodilians, environmental cues serve as triggers of breeding
behavior. In subtropical species, increasing spring temperatures can cue reproduction, while equatorial species rely more heavily
on hydrologic cycle periodicity (rainy/dry season transitions). The age and size at sexual maturity for male crocodilians varies by
species, and the time required to reach maturity ranges from 10–18 years, although it can be lesser for species that reach maturity
at smaller sizes. However, intrasexual competition among males promotes systems where only the largest, dominant individuals
will successfully breed each year, leaving smaller males, while technically sexually mature, with few or no breeding opportunities.
For example, male American alligators (Alligator mississippiensis) in some habitats reach maturity at 1.8 m (approximately 6 ft),
but only males >2.7 m (approximately 9 ft) are expected to breed in a given reproductive season (Lance, 1989). In these
competitive interactions, dominant males establish territories and preclude other males from entering and mating with females
in that area. Males of some species will also actively patrol their territory during breeding season to engage competitors.
In contrast to males, female crocodilians typically reach sexual maturity at smaller sizes and most do not engage in the same
intrasexual competition that would preclude younger females from breeding. Although, captive female American alligators have
been reported to aggressively defend their future nest sites against other females, and captive female American crocodiles
(Crocodylus acutus) can form female hierarchies, similar to males. Female American alligators reach sexual maturity at approxi-
mately 1.8 m, however, young females generally produce smaller clutches than older individuals. So, while females might
become sexually mature at 10–16 years of age, their maximum reproductive potential may not be reached until >20 years (Lance,
1989). Additionally, not every mature female (or male) will breed each year. While the likelihood of nesting in a year increases
with body size, estimates of the annual proportion of nesting alligators in Florida range from 16%–58% of adult females in a pop-
ulation (Lance, 1989), and females produce only a single clutch of eggs each year that they breed. However, the reproductive life
span of an individual female can be quite lengthy, with long term data from American alligators suggesting that females can
successfully reproduce for 30–40 years after reaching maturity (Wilkinson et al., 2016).

Courtship behavior
Courtship in crocodilians involves a complex series of behaviors that can include vocal displays and responses and coordinated
physical exchanges. Generally, a male will use a series of vocal displays (bellowing, head slapping, roaring) to attract receptive
females to his territory, to which females may respond with their own vocalizations. Once in close physical proximity, male and
female pairs engage in snout-snout and snout-neck contact and make a series of coughing exhalations. These initial displays are
usually followed by a series of guided, circling swims lead by either sex, riding, additional snout contact, and bubble-blowing. If
courtship is successful and neither party disengages, copulation will usually follow (Garrick and Lang, 1977). The physical act of
copulation can last minutes and entails the insertion of the male penis-like intromittent organ into the cloaca of the female,
permitting gamete transfer and fertilization. Crocodilians also possess two pairs of secretory glands (the gular glands on the
lower jaw and the paracloacal glands in the cloaca) that produce secretions that are thought to be involved in reproduction,
but the exact role of these glands and their secretions is not well understood in the context of courtship behavior, copulation,
or nesting.

Multiple paternity
Despite territorial dominance and aggressive intrasexual competition among males, a mounting body of evidence supports the
polygamous nature of crocodilian mating systems. Genetic analyses have uncovered clutches of eggs fertilized by multiple males
(as many as four) in a number of crocodilian species, indicating the possibility that female crocodilians are also promiscuous.
This multiple paternity could be driven by either 1) females mating with more than one male in a single season and using sperm
from multiple mates to fertilize a single clutch of eggs; 2) females mating with a single male each season, but storing
sperm from previous year(s) and using sperm from multiple years to fertilize a single clutch; or 3) both. Multiple paternity is likely
a widespread phenomenon, and has been documented in the American alligator, Chinese alligator, estuarine (or saltwater)
crocodile (Crocodylus porosus), Morelet’s crocodile (Crocodylus moreletii), broad-snouted caiman (Caiman latirostris), Orinoco croco-
dile (Crocodylus intermedius), black caiman (Melanosuchus niger), and spectacled caiman (Caiman crocodilus).

Nesting Ecology
Nesting strategy
Approximately 3 weeks to one month following copulation, females lay a clutch of 20–40 eggs, although clutch size is strongly
influenced by individual body size and age, as well as species. Average clutch size in American alligator in some populations can
range from 40–60 eggs, with the largest clutch ever recorded containing 91 eggs (although this “single” clutch could have been
648 Reproduction, Exotics/Zoo Animals/Wildlife j Reproductive Biology of Crocodilians

produced by two females sharing a single nest). In contrast, the African dwarf crocodile (Osteolamus tetrapsis) regularly produces
clutches of 10 eggs or fewer. Egg size also varies with species; female gharial produce the largest eggs of any crocodilian (160
g), while the Chinese alligator produces much smaller eggs (50 g).
Crocodilian nesting strategy can be classified as either mound-nesters or cavity-nesters, and the choice in strategy seems to be
influenced by habitat type. Species inhabiting forested riparian zones or wetlands regularly build nest mounds consisting of
vegetation and soil, while species inhabiting riverine or marine systems deposit eggs in cavities excavated on sandy beaches. All
species, however, choose nesting sites within close proximity to water. Mound-nesting species include the American and Chinese
alligators, all caiman species, and several crocodiles, including the estuarine, New Guinea (Crocodylus novaguineae), and Orinoco
crocodile, the false gharial and, in certain habitat types, the American and Cuban crocodile (Crocodylus rhombifer) (these species
can build both types of nests). Hole-nesting species include the gharial, the freshwater crocodile (Crocodylus johnstoni) and the
Nile crocodile (Crocodylus niloticus). Generally, members of family Crocodilidae display greater diversity in nesting strategy, as
both mound and cavity nesting is found in crocodiles. In comparison, all members of Alligatoridae are mound nesters and the
single member of Gavialidae, the gharial, is a hole nester.

Temperature and nesting

The temperature eggs experience during incubation is critically important to crocodilians, as all species studied to date exhibit
temperature-dependent sex determination (discussed in greater detail in Section The Crocodilian Sex Determination System).
In this system, sex of the developing embryo is determined by temperature, rather than by genetic factors or sex chromosomes.
Both cool and warm temperatures drive offspring to develop as females, while intermediate temperatures promote the development
of males. The exact range of temperature promoting development of the different sexes varies subtly from species to species, and
exact thermal ranges are not known for all crocodilians. Temperature also influences the time required for eggs to hatch, as warmer
nests will promote faster growth and development of embryos. Eggs typically require anywhere from 60–70 days to hatch once laid,
although this varies among species and may require as long as 90–115 days.

Adult behavior during nesting

During the incubation period, females of many crocodilian species will remain in close proximity to the nest, both guarding it from
predators and waiting to excavate hatchlings. Female American alligators will frequently remain in nearby ponds or holes and
advance upon intruders in defensive displays that include open-mouthed charging and hissing vocalizations. Some female croco-
dilians will also regularly attend the nest, climbing on top and crossing over it several times and will repair the nest if damaged or
disturbed by predators. Nest defense and attendance varies within and among species, and the purpose of the latter (beyond protec-
tion from predators and excavation of young) is not fully understood. Despite nest guarding, predation of nests is still a common
occurrence in most crocodilian species, and both mammals and reptiles represent a persistent threat to nesting success. Flooding
presents an additional natural threat to crocodilian nests, however habitat loss or destruction due to anthropogenic activity likely
presents a much greater existential threat to crocodilian nesting than flooding or predators.
At the conclusion of the incubation period and, in response to collective vocalizations from hatchlings, females of most species
will excavate nests with their mouths and forelimbs to assist hatchlings in exiting the nest. And, in some cases, the maternal female
will grasp unhatched or partially hatched eggs in her jaws and assist in hatching by gently manipulating and cracking eggs with her
teeth. In addition, the female will often carry hatchlings in her mouth to nearby water. Like nest defense and attendance, these
behaviors vary within and among species, but some form of nest attendance or care is likely uniform to all crocodilians. Once
hatching is complete, neonates will frequently remain in groups, termed pods, near the nest site for one year or more, staying in
close proximity to maternal female for protection. Adult care for neonates is most commonly exhibited by the laying female,
however, adult male Gharial will frequently guard large groups (100–1000s), called creches, of mixed-clutch and mixed-year
neonates and juveniles, guarding them for many months after hatching occurs. This behavior is not frequently observed in adult
males of other species, however, and cannibalism of hatchlings and juveniles by adults is common.

The Crocodilian Sex Determination System

All crocodilian species studied to date display a version of environmental sex determination that is regulated by the temperature that
embryos experience during development. The key aspects of the crocodilian temperature dependent sex determination (TSD)
system are reviewed below and include: (1) developmental timing of thermosensitvity, (2) the crocodilian TSD molecular network
and (3) the influence of hormones on TSD (Fig. 1).

The Developmental Timing of Thermosensitivity

Ferguson and Joanen first described TSD in crocodilians in 1982 and demonstrated the adjustable nature of the alligator sex deter-
mination system (Ferguson and Joanen, 1982). This study along with others established the pattern of TSD in the American alligator
(Alligator mississipiensis) along a gradient of increasing temperatures as female-male-female, with females being produced at extremes
of the viable temperature range (below 31 C and above 34 C) and males produced at intermediate temperatures (32 C)
(Fig. 2(A)). Mixed sex ratios are expected from temperatures between the male and female producing temperatures. Variation in
 Reproduction, Exotics/Zoo Animals/Wildlife j Reproductive Biology of Crocodilians 649

Fig. 1 Comparison of mound and hole nesting in A. mississippiensis and C. acutus. Representative images of an unopened (A) and opened
(C) mound nests of A. mississippiensis in coastal South Carolina (United States). Nests are built from surrounding vegetation and soil, and eggs are
deposited in the center of the mound, covered, and left to develop. For comparison, images of an unopened (covered in a mesh screen; B) and
opened (D) hole nests of C. acutus from the Tempisque Basin (Costa Rica), where the maternal female excavates a cavity in the soil, deposits, and
then covers her eggs. Photos (B, D) Courtesy of Chris Murray (Tennessee Technological University), (C) courtesy of Thomas Rainwater (Clemson
University).

the sex ratios amongst different clutches incubated at identical non-extreme temperatures indicates that relative clutch effects also
contribute to the equation that dictates how temperature impacts crocodilian sex determination (Lang and Andrews, 1994). The
specific period of thermosensitivity in all reptiles displaying TSD is thought to encompass the embryonic stages during which
the developmental trajectory of the bipotential gonad splits resulting in either a testis or an ovary. After the sexual differentiation
of the gonad progresses beyond a specific developmental threshold, the sex of that individual is irreversibly established and the
thermosenstive window is permanently closed. The specific period and associated developmental stages that characterize the
opening of the thermosensitive window are not as clearly defined, likely a result of the practical variation in experimental techniques
and the criteria used to define the window of sensitivity to incubation temperatures.
Knowledge regarding the temporal and developmental features of thermosensitivity originates primarily from detailed studies in
the American alligator and involves thermal shift experimental designs. These design schemes incorporate the incubation of
embryos at male- and female- producing temperatures (MPT and FPT, respectively). Single shift designs involve moving eggs
from one temperature to the other at different developmental stages and are often used to broadly identify the pattern (FMF,
MF, etc.) and the temperature sensitive period (TSP). Double shift designs allow for examination of how sex ratios are affected
by temporary pulses of a MPT against the background of a lower FPT and vice versa during different developmental periods. In
1994, Lang and Andrews applied both single- and double-temperature shift experiments to refine the period of thermosensitivity
in A. mississippiensis and demonstrated that irrespective of incubation temperature, a discrete thermosensitive period that occurs
from embryonic stage 21–24 (approximately 30–45 days of incubation) and encompasses the period of gonadal differentiation
(Lang and Andrews, 1994). The period of development between embryonic stages 21 and 24 has served as the canonical period
650 Reproduction, Exotics/Zoo Animals/Wildlife j Reproductive Biology of Crocodilians

Fig. 2 The thermosensitive period of sex determination in crocodilians. (A) Several crocodilian species display the female-male-female (FMF)
pattern of sex determination. Extreme temperatures (below 32 C and above 34 C) produce primarily females and intermediate temperatures (between
32 and 34 C) produce primarily males. (B) The thermosensitive window of sex determination in crocodilians initiates as early as stage 15 of
embryonic development and persists until the period of gonadal differentiation (stage 24).

of thermosensitivity in the American alligator and numerous studies have incorporated this specific period into designs aimed at
characterizing the origins and mechanisms of temperature dependent sex determination. Subsequent thermal shift experiments,
however, have challenged the discrete nature of this window and show that the thermosensitive period for the American alligator
initiates by embryonic stage 15 of development, six stages prior than previously reported (Mccoy et al., 2015) (Fig. 2(B)). This
finding further highlights the context dependent nature of the temperature dependent sex determination system in crocodilians.

The Crocodilian TSD Molecular Network

The morphological features of the adult testis and ovary display a remarkable degree of conservation across amniotes. This observed
morphological conservation has prompted targeted investigations aimed at elucidating the molecular cascade that translates
temperature into sex. Many of the key regulatory molecules involved in mammalian sex determination also appear to regulate
sex determination in crocodilians. Sex determining region Y (SRY)-box 9 (SOX9) mRNA is expressed in alligator embryos and
its abundance increases in gonads incubated at the MPT. SOX9 is a conserved transcription factor that promotes Sertoli cell differ-
entiation and testis development. In mammals, SOX9 is directly downstream of the male determining factor, SRY. Reptiles and
other non-mammals, however, lack SRY and the precise mechanisms by which SOX9 is regulated in these species remain elusive.
Temperature-induced alternative splicing is involved in Indian mugger (Crocodilus palustris) gonadal sex determination and results in
a female version of the Sox9 transcript that is not translated into a functional protein, thereby limiting its ability to induce testis
development (Agrawal et al., 2009). Further investigations are needed to determine how prevalent this mechanism is across other
crocodilian species. Anti-M}ullerian hormone (AMH), another key regulator of testis differentiation, is also expressed in alligator
during the thermosensitive period. The regulatory mechanisms that govern the expression of AMH during testis development
remain unclear for American alligator in ovo, although SOX9 can induce AMH transcription in vitro.
Conserved genes involved in ovarian differentiation have also been identified in crocodilians including aromatase (CYP19A1),
forkhead box L2 (FOXL2), and R-spondin 1 (RSPO1). The sequencing and assembly of the American alligator genome has helped to
overcome many of the technical challenges associated with genetic studies of non-traditional model organisms and will provide
additional insights into the puzzle of temperature dependent sex determination.

The Influence of Hormones on Crocodilian TSD

It is evident that hormones also play central roles in promoting ovarian differentiation in TSD. In cases where estradiol is
applied exogenously to eggs incubated at MPT, the process of testis differentiation is completely overridden and females are
 Reproduction, Exotics/Zoo Animals/Wildlife j Reproductive Biology of Crocodilians 651

produced. In the American alligator, estrogen activation of estrogen receptor-alpha (ESR1) appears to be sufficient, but not necessary
for instigating the most upstream steps of ovarian development based in part on the expression profile of aromatase, the catalytic
enzyme responsible for estrogen production, which is not detected until after the first morphological signs of gonadal differentia-
tion occur (Parrott et al., 2014). Many persistent contaminants found in the environment are categorized as endocrine disrupting
chemicals (EDCs) due to their ability to impact the endogenous endocrine system. Exposure to EDCs during hormone-sensitive
developmental windows can promote permanent alterations in reproductive system function. The nature of the crocodilian sex
determination system makes it inherently sensitive to environmental factors (temperature, humidity, contaminants, etc.). This
feature, along with other key aspects of crocodilian life history, warrant these species as exceptional wildlife sentinels for environ-
mental health.

The Crocodilian Reproductive System

Gonadogenesis
As in other vertebrates, the gonads of crocodilians originate as a genital ridge, develop into bipotential gonads, and after sex deter-
mination differentiate into testes in males and ovaries in females. Primordial germ cell (PGC) specification and migration has not
yet been observed in crocodilians due to experimental difficulties, but based on their shared Archosaurian lineage they are expected
to follow the patterns seen in birds; namely, PGC specification follows the predetermined mode and the cells migrate through the
bloodstream to arrive in the genital ridge at Ferguson stage 2 of development (Kohno et al., 2014).
In Alligator mississippiensis, gonads develop in complex with the adrenal gland and the embryonic kidney (gonadal-adrenal-
mesonephros complex, or GAM). The germinal epithelium and underlying medullary sex cords are observable by stage 12,
and by stage 20 the bipotential gonads exhibit a distinct cortex containing PGCs. The earliest signs of gonadal sexual differen-
tiation occur during the thermosensitive window of TSD, with observable presumptive Sertoli cells at stage 21 in the gonads
of embryos incubated at the MPT, and germ cell proliferation in the gonadal cortex at stage 22 in embryos incubated at the
FPT (Smith and Joss, 1993).
Sexually dimorphic gonadal differentiation continues throughout the rest of embryonic development. In the testes, the germinal
epithelium diminishes and germ cells disperse into the medulla, where seminiferous cords become more organized, with well-
defined Sertoli cells (Smith and Joss, 1993). In contrast, the ovarian cortex becomes more defined, characterized by proliferating
germ cells, while lacunae form in the fragmenting medulla (Smith and Joss, 1993). Oocytes develop in the cortex, enclosed by gran-
ulosa cells and steroidogenic theca cells. The caudal portion of the ovary, known as the medullary rest, does not follow this pattern
but instead exhibits the dense arrangement of medullary sex cords seen in the testis (Forbes, 1940).

Adult Gonadal Structure and Function

Full maturation of the gonads of crocodilians, as with birds, continues post-hatching. Unlike birds, however, crocodilian ovaries
maintain a reserve of cortical oogonial nests rather than converting all oogonia into primary oocytes during gonadal maturation,
and thus can produce new follicles throughout their lifetime (Kohno et al., 2014). The lacunae that develop in the medullary stroma
are characterized by a framework of collagen and smooth muscle, and may function to facilitate follicular expansion. The signaling
processes guiding folliculogenesis in crocodilians have not yet been fully elucidated, but developmental exposure to environmental
estrogens disrupts the process, resulting in multi-oocytic follicles (Guillette et al., 1994).
The adult crocodilian testis exhibits the tubular morphology characteristic of amniotes, with highly convoluted seminiferous
tubules lined with an epithelium of Sertoli cells. The testosterone-generating Leydig cells of the testis localize around cord structures
and mature sperm are typically replenished throughout reproductive life due to the persistence of mitotic spermatogonial stem cells.
In A. mississippiensis, spermatogenesis begins in early spring, and germ cells mature in a single cohort to produce one continuous
spermiation event during the breeding season from May-June, which differs from the consistent spatial developmental pattern
of spermatogenesis observed in other amniotes. This temporal pattern is reminiscent of spermatogenesis in anamniotic amphibians,
but was also documented in two other seasonally breeding reptile species, the slider turtle (Trachymes scripta) and the European wall
lizard (Podarcis muralis), suggesting that the testes of crocodilians and these other reptiles are transitional between the anamniotic
and amniotic testis (Gribbins et al., 2006).

Adult Gonadal Structure and Function

Full maturation of the gonads of crocodilians, as with birds, continues post-hatching. Unlike birds, however, crocodilian ovaries
maintain a reserve of cortical oogonial nests rather than converting all oogonia into primary oocytes during gonadal maturation,
and thus can produce new follicles throughout their lifetime (Kohno et al., 2014). The lacunae that develop in the medullary stroma
are characterized by a framework of collagen and smooth muscle, and may function to facilitate follicular expansion. The signaling
processes guiding folliculogenesis in crocodilians have not yet been fully elucidated, but developmental exposure to environmental
estrogens disrupts the process, resulting in multi-oocytic follicles (Guillette et al., 1994).
The adult crocodilian testis exhibits the tubular morphology characteristic of amniotes, with highly convoluted seminiferous
tubules lined with an epithelium of Sertoli cells. The testosterone-generating Leydig cells of the testis localize around cord structures
652 Reproduction, Exotics/Zoo Animals/Wildlife j Reproductive Biology of Crocodilians

and mature sperm are typically replenished throughout reproductive life due to the persistence of spermatogonial stem cells. In
A. mississippiensis, spermatogenesis begins in early spring, and germ cells mature in a single cohort to produce one continuous sper-
miation event during the breeding season from May-June, which differs from the consistent spatial developmental pattern of sper-
matogenesis observed in other amniotes, including birds. This temporal pattern is reminiscent of spermatogenesis in anamniotic
amphibians, but was also documented in two other seasonally breeding reptile species, the slider turtle (Trachymes scripta) and
the European wall lizard (Podarcis muralis), suggesting that the testes of crocodilians and these other reptiles are transitional between
the anamniotic and amniotic testis (Gribbins et al., 2006).

Reproductive Tract Structure and Function

The crocodilian female reproductive tract, or oviduct, is highly functionally and structurally regionalized. Whereas the oviducts of
other reptiles produce both the fibrous proteins that form the eggshell membrane and the outer calcareous layer of the eggshell in
one uterine region sequentially, crocodilians and other Archosaurs have two spatially distinct regions, complete with distinct glan-
dular structures (Palmer and Guillette, 1992). Thus crocodilian eggs can be packaged in an “assembly line” fashion, an evolutionary
adaptation that has been seen as an innovation allowing for flight in birds, although birds process eggs through their oviducts one at
a time, whereas crocodilians process an entire clutch at once.
When reproductively active, the oviduct is highly responsive to estrogen stimulation, undergoing dramatic increases in size and
extensive glandular development for producing the necessary secretory products for proper egg packaging (Palmer and Guillette,
1992). As in other vertebrates, the oviducts originate from the embryonic Müllerian ducts. After gonadal sex determination, Mülle-
rian ducts regress in males due to the influence of AMH, but they are fully retained in females, and unlike in birds, both mature into
functional adult oviducts.
In contrast to female reproductive structures and function, the male reproductive tract has not been well studied in crocodilians.
However, male extra-gonadal reproductive structures are generally similar to mammals and birds, with the ductus deferens linking
the testes to the phallus for gamete transfer (although the crocodilian phallus is distinct from both mammalian and avian
structures). The crocodilian ductus deferens serves to collect, store, and deliver sperm from the epididymis and seminiferous tubules
within the testes during breeding season. Crocodilians appear to lack a dedicated accessory secretory gland for production of
seminal fluid. However, evidence is beginning to emerge that multiple regions of the male reproductive tract may contribute to
seminal secretions. In spectacled caiman, the morphological and histochemical properties of the epididymis and ductus deferens
suggest its involvement in the production and modification of seminal fluid, and structural characteristics at the gross morpholog-
ical, cellular, and ultrastructural levels are highly similar to birds, more so than to other reptiles, supporting the Archosaurian lineage
of crocodilians. (Guerrero et al., 2004).
These findings in caiman have been corroborated by recent work in the American alligator that have reported the presence of
exocrine glands in the male phallus that likely contribute secretions during reproduction to form seminal fluid. The crocodilian
phallus is itself a highly specialized and complex structure, characterized by three distinct morphological regions: the tip, cuff,
and base, and an open groove, termed the sulcus or penile groove, for sperm delivery. During copulation, it is likely that increased
blood flow to vascular regions surrounding the sulcus close it into a complete tube, while coordinated smooth muscle
contractions promote sperm movement into the female reproductive tract (Moore et al., 2012). Unlike mammalian or avian
penile structures, most of the crocodilian phallus is rigid, containing dense collagenous regions, and only a small region is erec-
tile. The phallus is also retained within the cloaca until copulation occurs, when it is everted for intromission into the female
(Moore and Kelly, 2015).

References

Agrawal, R., Wessely, O., Anand, A., Singh, L., & Aggarwal, R. K. (2009). Male-specific expression of Sox9 during gonad development of crocodile and mouse is mediated by
alternative splicing of its proline-glutamine-alanine rich domain. The FEBS Journal, 276, 4184–4196.
Ferguson, M. W. J., & Joanen, T. (1982). Temperature of egg incubation determines sex in Alligator mississippiensis. Nature, 296, 850–852.
Forbes, T. R. (1940). Studies on the reproductive system of the alligator. VI. Further observations on heterosexual structures in the female alligator. Anatomical Record, 77,
343–365.
Garrick, L. D., & Lang, J. W. (1977). Social signals and behaviors in adult alligators and crocodiles. American Zoologist, 17, 225–239.
Gribbins, K. M., et al. (2006). Cytological evaluation of the germ cell development strategy within the testis of the American alligator, Alligator mississippiensis. Acta Zoologica,
87(1), 59–69.
Guerrero, S. M., et al. (2004). Morphology of the male reproductive duct system of Caiman crocodilus (Crocodylia, Alligatoridae). Annals of Anatomy, 186(3), 235–245.
Guillette, L. J., Gross, T. S., JR, Masson, G. R., et al. (1994). Developmental abnormalities of the gonad and abnormal sex hormone concentrations in juvenile alligators from
contaminated and control lakes in Florida. Environmental Health Perspectives, 102, 680–688.
Kohno, S., Parrott, B. B., Yatsu, R., et al. (2014). Gonadal differentiation in reptiles exhibiting environmental sex determination. Sexual Development, 8, 208–226.
Lance, V. A. (1989). Reproductive cycle of the American alligator. American Zoologist, 29(3), 999–1018.
Lang, J. W., & Andrews, H. V. (1994). Temperature-dependent sex determination in crocodilians. Journal of Experimental Zoology, 270, 28–44.
Mccoy, J. A., Parrott, B. B., Rainwater, T. R., Wilkinson, P. M., & Guillette, L. J., JR (2015). Incubation history prior to the canonical thermosensitive period determines sex in the
American alligator. Reproduction, 150, 279–287.
Moore, B. C., & Kelly, D. A. (2015). Histological Investigation of the adult alligator phallic sulcus. South American Journal of Herpetology, 10(1), 32–40. Available at: http://www.
bioone.org/doi/10.2994/SAJH-D-14-00037.1.
 Reproduction, Exotics/Zoo Animals/Wildlife j Reproductive Biology of Crocodilians 653

Moore, B. C., Mathavan, K., & Guillette, L. J. (2012). Morphology and histochemistry of juvenile male American alligator (Alligator mississippiensis) phallus. Anatomical Record,
295(2), 328–337.
Palmer, B. D., & Guillette, L. J., JR (1992). Alligators provide evidence for the evolution of an archosaurian mode of oviparity. Biology of Reproduction, 46, 39–47.
Parrott, B. B., Kohno, S., Cloy-Mccoy, J. A., & Guillette, L. J., JR (2014). Differential incubation temperatures result in dimorphic DNA methylation patterning of the SOX9 and
aromatase promoters in gonads of alligator (Alligator mississippiensis) embryos. Biology of Reproduction, 90, 2.
Smith, C. A., & Joss, J. M. P. (1993). Gonadal sex differentiation in Alligator mississippiensis, a species with temperature-dependent sex determination. Cell Tissue Research, 273,
149–162.
Wilkinson, P. M., et al. (2016). Determinate growth and reproductive lifespan in the American alligator (Alligator mississippiensis): Evidence from long-term recaptures. Copeia,
104(4), 843–852. Available at: http://www.bioone.org/doi/10.1643/CH-16-430.