Figure 1. I took this photo of a Tasmanian Devil eating a rabbit at the Perth Zoo in Western Australia. The Tasmanian Devil is a marsupial in the family Dasyuromorphia.

A Marsupial's Story

Evolution and Comparative Anatomy of Marsupial Reproduction

Disclaimer - A complete understanding of ecotourism as a conservation strategy necessitates an understanding of ecology and the environment. Much of the information included here assumes, at minimum, some background in biology. For a more succinct summary of the salient points and an overview of why protecting and researching marsupials is important, scroll down to the Conclusions section. Ecotourism can be used to protect vulnerable species and enable invaluable research to continue. Revenue from ecotourism can help support zoos, wildlife preserves, and breeding programs that in turn contribute to research projects like the ones described below.

Introduction

Figure 2. This is a photo of me with a koala at Caversham Wildlife Park. The koala is a member of the family Diprotodontia. — Figure 2. This is a photo of me with a koala at Caversham Wildlife Park. The koala is a member of the family Diprotodontia.

Australia is the land of the marsupial. Most known for the kangaroo and koala, Australia is home to numerous other species of marsupials including but not limited to possums, wombats, bilbies, and the Tasmanian devil. Figures 1-3 show some of the marsupials I saw along my travels and the morphological diversity of the marsupial infraclass.

The mammalian branch of the tree of life dates back 300-400 million years ago and is currently divided into three main clades: Monotremata, Eutheria, and Metatheria or Marsupialia (Armati et al. 2013, 2). Based on the fossil record of mammalian teeth, the marsupial clade began approximately 95-100 million years ago most likely in Asia (Armati et al. 2013, 3).

Figure 3. This is a photo of me with a kangaroo at Caversham Wildlife Park. The kangaroo is a member of the family Diprotodontia. — Figure 3. This is a photo of me with a kangaroo at Caversham Wildlife Park. The kangaroo is a member of the family Diprotodontia.

Despite their long history, marsupials have only been known to European biologists since the discovery of the America’s and the opossum in the fifteenth century (Armati et al. 2013, 1). In 1758, Linné dubbed the few known opossum species Didelphis, in reference to their two internal uteri, and classified them with the placental mammals in a group including insectivores, armadillos, and pigs and (Armati et al. 2013, 1-2). It was not until Captain Cook’s voyages and settlement of New South Wales, Australia in 1768 that the true diversity and uniqueness of the marsupial family became apparent (Armati et al. 2013, 1). By the end of the eighteenth century, a growing interest in monotremes further problematized the marsupial classification with placental mammals and eventually lead to the modern division of the mammalian branch of life into three clades (Armati et al. 2013, 2). Well before genetic analysis, the three clades were divided primarily based on morphology and reproductive strategy (Armati et al. 2013, 2).

Modern studies of marsupial reproduction can provide useful insights into mammalian evolution both from a genetic and morphological perspective and may aid in the development of new treatment techniques for human pathologies.

Clade/Infraclass Background

The name Marsupial is derived from the Latin for “purse” and references the pouch found on many marsupial species (Armati et al. 2013, 83). While marsupials are most known for their pouches, it is important to note that not all marsupials have a pouch (Armati et al. 2013, 83). Instead of classifying mammals based solely on gross external anatomy, the primary feature used to separate marsupials from monotreme and eutherian mammals is the reproductive strategy unique to each of the three clades. (Armati et al. 2013, 83). All marsupials are characterized by rapid embryonic grow and birth of relatively undeveloped young (Armati et al. 2013, 83). Marsupial offspring primarily develop to independence during the lactation period instead of in utero (eutheria) or in ovo (monotremata) (Armati et al. 2013, 83).

Figure 4-5. Show one possible explanation for the modern distribution of marsupials (shown in orange). These images are from the UCMP and diagram how plate tectonics may have contributed to marsupial dispersion.

Based on fossil evidence, marsupials were once located across the globe, but now are only found in Australia and the Americas (Figure 5, Armati et al. 2013, 3). Despite their modern geographic isolation, the marsupials in South America and Australia are genetically and morphologically similar (UC Museum of Paleontology [UCMP]).

Pangea and plate tectonics help explain this seemingly counterintuitive observation (UCMP). Marsupials were once present all across Pangea (UCMP). When the continents separated, closely related marsupials continued to survive in the areas shown in orange, while the marsupials on the other continents were driven to extinction by eutherian mammals (Figure 4-5, UCMP). As the University of California Museum of Paleontology puts it, “Marsupials didn't need a migration route from one part of the world to another; they rode the continents to their present positions” (UCMP).

Despite their extinction in many parts of the world, marsupials today represent virtually the entire range of placental lifestyles (Armati et al. 2013, 3). However, there are currently no known marsupial bats or cetaceans (Armati et al. 2013).

Phylogeny and Evolution

Marsupials are the sister group to eutherian mammals (Figure 6, Rheede et al. 2005, 593). Together they form the subclass Theria, which is the sister group to the subclass Monotremata (Figure 6, Rheede et al. 2005, 593).

Figure 6. This image was taken from Rheede et al. 2005. It shows the mammalian genetic phylogeny with birds, crocodiles, and turtles as the reference outgroup.

There are seven extant orders of marsupial: Didelphimorphia, Paucituberculata, Microbiotheria, Notoryctemorphia, Dasyuromorphia, Peramelemorphia, and Diprotodontia (Figure 7, Armati et al. 2013, 5).

Didelphimorphia includes the American opossums (Armati et al. 2013, 5). As previously stated, they were the first marsupials discovered by European biologists (Armati et al. 2013, 5). Didelphimorphia range in size from a mouse to large cat and are carnivorous, insectivorous, or omnivorous (Armati et al. 2013, 5). It is hypothesized that Didelphimorphia are most phenotypically like early marsupials of the extant orders (Armati et al. 2013, 5). These species primarily diversified in South America and then eventually invaded North America (Figure 4-5, Armati et al. 2013, 5).

Paucituberculata includes shrew opossums (Armati et al. 2013, 5). These species are found in South America, but their teeth are similar in form to diprotodontia in Australia (Armati et al. 2013, 5-6). As referenced earlier, this is one example of numerous similarities between Australian and South American marsupials that supports dispersion via plate tectonics. Paucituberculata are very diverse and as a result, are not clearly a monophyletic group (Armati et al. 2013, 6).

Microbiotheria includes only one extant species, the Monito del Monte (Armati et al. 2013, 6). The Monito del Monte is also known as the “little bush monkey," colocolo opossum, or Dromiciops gliroides (Armati et al. 2013, 6). Once incorrectly classified as an opossum, the monito del monte is now considered by some to be the missing link between South American and Australasian marsupials (Armati et al. 2013, 6). It is currently found only in South America, but similar fossil forms have been uncovered on Antarctica and Australia (Armati et al. 2013, 6).

Figure 7. This is a table I made showing the classification of marsupials within the tree of life and listing the seven extant orders.

Notoryctemorphia includes insectivorous marsupial moles (Armati et al. 2013, 6-7). This order has an unclear relationship to rest of the marsupial orders, but may be most closely related to the Dasyuromorphia (Armati et al. 2013, 7).

Dasyuromorphia includes Australian carnivorous marsupials (Figure 2, Armati et al. 2013, 6). They are comparable to the Didelphimorphia opossums of America and include the living Tasmanian devil and the now “debatably” extinct Thylacinus, also known as the Tasmanian Wolf or Tasmanian Tiger (Armati et al. 2013, 6). Thylacinus is “debatably” extinct because in Tasmania, it has become a sort of local bigfoot with frequent rumored sighting and conspiracies (ABC Radio 2016).

Peramelemorphia includes bandicoots and bilbies (Armati et al. 2013, 7). These species are very unique and have no similar placental species (Armati et al. 2013, 7). All Peramelemorphia are found in Australia or New Guinea and are either burrowing or terrestrial (Armati et al. 2013, 7). They can be insectivores, omnivores, or herbivores (Armati et al. 2013, 7).

Diprotodontia includes kangaroos, wallabies, possums, wombats, and koala (Figure 1 & 3, Armati et al. 2013, 7). It is the most diverse and disparate order and contains most of the commonly known marsupials (Armati et al. 2013, 7). In addition, Diprotodontia also includes gliders and the extinct marsupial lion (Armati et al. 2013, 7).

Figure 8. This image was taken from Nilsson et al. 2010. It shows the phylogenetic relationship between the extant orders and further supports Microbiotheria as the link between South American and Australian marsupials.

Without genetic analysis of basal taxa, it is difficult to parse out analogous and homologous structures of marsupials and determine a complete phylogeny of extant and extinct species (Armati et al. 2013, 8). Additionally, synapomorphic, or derived characteristics, can normally be extremely useful in determining phylogeny from a fossil record; however, since the soft anatomy of the reproductive system is not captured in fossils, it is difficult to determine when exactly marsupials and eutherians branched off (Armati et al. 2013, 9).

Nilsson and colleagues (2010) attempted to address some of these issues by analyzing archaic genomic retroposon insertions in extant marsupial DNA (Nilsson et al. 2010). As shown in Figure 8, they confirmed much of the morphological phylogeny, described earlier, and helped provide further support for Microbiotheria linking the South American and Australia marsupials (Nilsson et al. 2010).

Reproductive Morphology

Relative to the two other mammalian clades, the primary distinguishing feature of a marsupial is the reproductive strategy. As described below, marsupials and eutherian mammals structurally have a lot in common. The fundamental difference is the relative timing and duration of pregnancy, lactation, and development between the two types of therian mammals.

Female

The bulk of research on marsupial reproduction focuses on female anatomy and the development of the embryo.

From a very general perspective, the overall structures of marsupial and eutherian female reproductive tracts have a lot in common (Figure 9, Armati et al. 2013, 90). In both clades, the eggs are released from ovary and pass down oviduct where they may be fertilized by sperm (Armati et al. 2013, 90). If the egg is fertilized, it then travels to the uterus and implants in the uterine wall for development (Armati et al. 2013, 90). During birth, the embryo travels through the cervix into the vagina and then out the urogenital sinus to the exterior (Armati et al. 2013, 90).

However, there are also many important structural differences used to distinguish between marsupial and eutherian mammals. Marsupials have two uteri, two separate cervixes, and three distinct vaginae (Armati et al. 2013, 91). The two lateral and one median vaginae all lead to one common anterior vaginal expansion (Armati et al. 2013, 91). Posterior to the common vaginal expansion is the urogenital sinus used for reproduction, urine, and excrement (Armati et al. 2013, 91).

The divisions of the reproductive tract are partially due to the relative position of the kidneys and bladder. As shown in Figure 9, the bladder is ventral to reproductive tract and kidneys are dorsal (Armati et al. 2013, 91). To connect the urinary tract, the marsupial ureters lie between the two reproductive ducts (Armati et al. 2013, 91). This prevents the ducts and reproductive tract from fusing along the midline during development to form one single vagina and cervix (Armati et al. 2013, 91). This is markedly different from the eutherian reproductive tract in which the ureters do not transverse the reproductive tract and the vagina does not share a urogenital sinus with the bladder (Figure 10, Armati et al. 2013, 91).

Figure 10. This photo was taken from the Northwestern Oncofertility Consortium website. This shows the general structure of a female eutherian reproductive tract. Note the relative simplicity and the reduced number of structures relative to the mars… — Figure 10. This photo was taken from the Northwestern Oncofertility Consortium website. This shows the general structure of a female eutherian reproductive tract. Note the relative simplicity and the reduced number of structures relative to the marsupial.

The birthing process in marsupials also has some common and distinct features with eutherian birth. Similar to eutherian birth, the current hypothesis is that the marsupial fetus stimulates birth by releasing cortisol (Armati et al. 2013, 102). However, unlike in eutherian mammals, the median vagina forms a transient birth canal that opens before each birth and closes up after (Armati et al. 2013, 92).

Marsupial offspring are also a dramatically different in size relative to offspring of eutherian mammals (Armati et al. 2013, 101). Kangaroos have some of the largest marsupial neonates which only weigh up to 800mg (Armati et al. 2013, 101). Tarsipes newborns are the smallest and weigh under 5mg or about the size of a grain of rice (Armati et al. 2013, 101). After birth, the tiny newborn marsupial climbs unaided from the urogenital sinus to the mother’s teat (Armati et al. 2013, 101). While their exact method of locating the teat is currently unknown, the newborns are speculated to rely on smell and gravity for guidance (Armati et al. 2013, 101).

Once attached to the mother’s teat, the newborn suckles for the remainder of its development (Figure 11, Armati et al. 2013, 102). In general, the lactation stage of marsupial development lasts two to three times as long as in utero development (Armati et al. 2013, 102).

Most commonly, the mother’s teats are located within the pouch and remainder of embryo development will occur inside (Armati et al. 2013, 92). However, despite being named for their pouches, not all marsupial species even have a pouch (Armati et al. 2013, 92). For example, several species of South America opossum lack a pouch entirely and carry their young attached to their nipple until they are large enough to stay in a nest. (Armati et al. 2013, 92).

Even among marsupials with pouches, there is some variably in pouch form (Armati et al. 2013, 92). Most marsupials, including the kangaroo family, have a pouch with an anterior opening (Armati et al. 2013, 92). In contrast, wombat pouches are morphologically similar to kangaroo pouches but open posteriorly (Armati et al. 2013, 92). It is hypothesized that this difference is evolved to keep dirt out of the pouches of burrowing marsupials (Armati et al. 2013, 92).

Figure 11. This photo was taken from Lemay and Nicholas 2013. It shows a newborn tammar wallaby suckling on its mother’s teat. Note the corner of the photographer’s thumb as a size reference.

As the newborn develops, the concentration of the milk from the teat changes to meet the changing developmental needs of the newborn (Armati et al. 2013, 102). Lactation occurs in three main phases (Armati et al. 2013, 102). In phase 1, the marsupial mammary glands release a clear fluid rich in proteins and antibodies that is suspected to protect the newborn from infection (Armati et al. 2013, 102). This form of milk is similar to colostrum released by eutherian mammals in the late stages of pregnancy (Armati et al. 2013, 102). In phase 2 of marsupial lactation, there is a progressive increase in the concentration of milk solids from 10% to up to 40% (Armati et al. 2013, 102). These solids are primarily lipids and proteins (Armati et al. 2013, 102). Phase 2 does not have an equivalent in eutherian lactation (Armati et al. 2013, 102). In phase 3, there is an increase in lipid concentration, a decrease in carbohydrates, and the release of late-lactation specific proteins occurs (Armati et al. 2013, 102). The majority of eutherian lactation is equivalent to phase 3 in marsupials (Armati et al. 2013, 102).

The endocrine regulation of reproduction is also similar in marsupial and eutherian mammals (Armati et al. 2013, 93). Much like eutherian mammals, marsupials rely on cycles of Gonadotropin-releasing hormone (GnRH), follicle stimulating hormone (FSH), luteinizing hormone (LH), estrogen, and progesterone to stimulate ovulation and promote maturation (Armati et al. 2013, 93). Additionally, in all therian mammals, lactation is stimulated by prolactin (Armati et al. 2013, 93).

However, in most species, marsupials have a special hormonal adaptation to regulate the timing of consecutive pregnancies (Armati et al. 2013, 97). This adaptation, called diapause, occurs when the suckling of a newborn stimulates the release of prolactin (Armati et al. 2013, 97). The prolactin prevents the full development of the corpus luteum, which as a result the decreases progesterone secretion and uterine stimulation (Armati et al. 2013, 97). The interaction of these hormones causes the embryo to temporarily halt in the blastocyst phase without growth or mitotic activity (Armati et al. 2013, 97). Diapause lasts until weaning when the prolactin levels begin to decrease to normal levels (Armati et al. 2013, 97).

Diapause is thought to function to either align reproductive cycles with seasonal changes or halt pregnancy in response to poor environmental conditions (Armati et al. 2013, 97). However, there is some debate on how the molecular uterine signaling mechanism operates in response to hormonal signals (Armati et al. 2013, 99). One theory argues that diapause requires a uterine signal to stop development and without the signal unhalted development will occur as default (Armati et al. 2013, 99). The other theory argues for the opposite mechanism (Armati et al. 2013, 99). Supporters of this theory believe diapause is the default developmental pathway and unhalted development requires a uterine signal to prevent diapause (Armati et al. 2013, 99). To further complicate the matter, some evidence indicates the signaling pathway may vary across marsupial orders (Armati et al. 2013, 97). With little definitive knowledge of marsupial diapause, additional research is necessary (Armati et al. 2013, 97).

Male

While much of the research and interest in marsupial reproduction focuses on female anatomy, a complete reproductive morphology review must also include male reproductive anatomy.

Figure 12. This image was taken from Paris et al. 2005. The parts on the images are labeled as follows: Ag, accessory glands; Bl, bladder; Cg, Cowper’s gland; Cp, crus penis; E, epididymis; Pe, penis; Pr, prostate; R, rectum; Te, testis; Ub, urethra… — Figure 12. This image was taken from Paris et al. 2005. The parts on the images are labeled as follows: Ag, accessory glands; Bl, bladder; Cg, Cowper’s gland; Cp, crus penis; E, epididymis; Pe, penis; Pr, prostate; R, rectum; Te, testis; Ub, urethral bulb.

The marsupial male reproductive system is similar to eutherian male reproduction in most respects (Armati et al. 2013, 83). Sperm develops in the testes and is stored in the epididymis (Armati et al. 2013, 83). Next, it is passes down the vas deferens to the urethra and into the female vagina via the penis (Armati et al. 2013, 83). Additionally, in most eutherian and marsupial species, males are larger than females (Armati et al. 2013, 83).

Despite the many similarities, there are a few key points of distinction between the two classes of therian mammals. In marsupials, the scrotum is in front of the penis rather than behind it (Armati et al. 2013, 84). A few marsupial species, primarily burrowing ones, lack a pendulous scrotum all together and instead have internal testes (Armati et al. 2013, 85). Additionally, penile anatomy is varied across marsupials (Armati et al. 2013, 86). In most species, the penis is divided into two halves which may aid in insemination of the two lateral vaginae of the females (Armati et al. 2013, 86).

Marsupial sperm also have unique adaptations. Unlike Eutheria sperm which use fructose as the primary energy source, many marsupials rely on N-acetylglucosamine (Armati et al. 2013, 85).

Figure 13. This image was take from Pizzari and Foster 2008. These photos show the unusual flattened acrosome in marsupial sperm and the associated pairing (B) and unpairing (C) behavior.

The shape of the sperm acrosome is also different in marsupials relative to both eutherian and monotreme mammals (Armati et al. 2013, 87). The acrosome is a membrane bound vesicle on the head of the sperm that aids in penetration of the egg (Armati et al. 2013, 87). In marsupials, the acrosome is flattened and lies on one side, as opposed to wrapping around the whole head of the sperm (Figure 13, Armati et al. 2013, 87-88). During development, the angle between the head and midpiece of the sperm increase and becomes closer to 180 degrees as the sperm ages (Armati et al. 2013, 88).

In some South American marsupials, during sperm maturation, two sperm attach at the acrosome to form a pair with two separate tails (Figure 13, Armati et al. 2013, 88). At fertilization, the sperm separate so only one fertilizes the egg (Armati et al. 2013, 88). The purpose of this is unknown but it may aid in directional movement of the sperm pair (Armati et al. 2013, 88).

One additional fun fact: the male honey-possum has the longest sperm of any mammal and its testes make up 4% of its total body weight (Armati et al. 2013, 85)!

Evolution of Diapause

As previously described in the “Reproductive Morphology” section above, embryonic diapause is the arrest of embryo development in the blastula stage which temporarily delays implantation in the uterus (Ptak et al. 2012, 1).

Figure 14. This image was taken from Ptak et al. 2012. It shows their experimental design for inducing diapause. Group 1 is the experimental group and Groups 2-4 are controls

Among vertebrates, diapause is most common in birds, fish, and marsupials (Ptak et al. 2012, 1). While it is represented in eutherian mammals, it is not known to be widespread (Ptak et al. 2012, 1). Marsupials and some members of the families Rodentia and Insectivora do conduct facultative diapause, which occurs due to unfavorable environmental conditions or maternally driven stimuli (Ptak et al. 2012, 1). Less frequently, some mammals perform obligate diapause, which by definition occurs in all pregnancies in the species and normally functions to align reproduction with season fluctuations (Ptak et al. 2012, 1). However, even in mammals with obligate diapause, the duration of the diapause is flexible and based on the environment (Ptak et al. 2012, 1). This variability problematizes the distinction between the facultative and obligate forms (Ptak et al. 2012, 1).

According to Ptak and colleagues (2012), if our prior classifications of facultative and obligate diapause do not fall neatly into two categories, then perhaps we may also be incorrectly distinguishing between diapausing or nondiapausing species (Ptak et al. 2012, 2). Maybe diapause has not been observed across eutherian mammals only because the right combination of environmental factors has yet to trigger it (Ptak et al. 2012, 2). Moreover, if diapause is common throughout mammalian taxa, then this suggests diapause is an evolutionary conserved trait and not a derived trait as was commonly accepted (Ptak et al. 2012, 2).

To test this theory, Ptak and colleagues (2012) attempted to induce diapause in blastocysts of a nondiapausing species (Ptak et al. 2012, 2). They removed blastocysts from sheep and inserted them into the uteri of pseudo-pregnant mice with induced diapause conditions (Ptak et al. 2012, 2). Adding the blastocysts to a diapause environment successfully induced diapause in the developing embryo of a nondiapausing species (Ptak et al. 2012, 2). To further demonstrate that the diapause did not permanently halt growth, Ptak and colleagues then transferred the blastocysts to surrogate sheep and found the development resumed normally and the pregnancies went to term (Ptak et al. 2012, 3). As an added benefit, diapause embryos were found to have lower cell death than the controls (Ptak et al. 2012, 3).

Across species, Ptak and colleagues noted three similar traits in diapause (Ptak et al. 2012, 4). First, they noted no physiological differences between diapause embryos (Ptak et al. 2012, 4). Second, all diapause embryos were halted at the same stage during the blastocyst (Ptak et al. 2012, 4). Third, as shown by this experiment, there is no species specificity in the uterine conditions producing diapause (Ptak et al. 2012, 4). Ptak and colleagues use these observations to hypothesize that mammalian diapause is an evolutionary conserved trait across therian mammals (Ptak et al. 2012, 5).

Conclusions

The study above illuminates one of many important reasons to study marsupial reproductive morphology. As the sister group to marsupials, eutherian mammals, including humans, will inevitably share some conserved reproductive traits with our pouched cousins. By studying reproductive morphology in the context of evolution, not only will we be able to better understand marsupial branch of the tree of life, but we will also uncover new insights into the derived and conserved features of eutherian reproduction, including human reproduction.

From a practical and applied scientific perspective, studying marsupial reproduction may also provide insights into new treatment strategies for human reproductive or developmental disorders. For example, the diapause study by Ptak and colleagues (2012) may prove useful in isolating stem cells, facilitating DNA repair in vivo, and as a strategy for human family planning.

The Milk Genomics Consortium, further argues that studying kangaroo lactation may be useful for developing treatments for human preemies (Lemay and Nicholas 2013). In particular, the antibacterials cathelicidin and WFDC-2 are only present during marsupial early lactation and weaning which suggests they function to protect the newborn from external pathogens (Lemay and Nicholas 2013). Unfortunately, one cannot simply feed a human baby with kangaroo milk, but finding comparable proteins in human milk may aid in the development of lifesaving pharmaceuticals for underdeveloped infants (Lemay and Nicholas 2013).

While some big strides have been taken towards understanding marsupial reproduction, a lot is still unknown. As previously stated, the soft anatomy of the marsupial reproductive system cannot be captured in the stone of the fossil record (Armati et al. 2013, 3). Marsupials in the fossil record are normally identified by the number and form of their teeth (Armati et al. 2013, 3). However, based on the current phenotypic diversity of the marsupial form, I hypothesize that this method is perhaps insufficient to establish a complete and accurate marsupial fossil record or to determine the timing of reproductive divergence between marsupial and eutherian mammals.

Additionally, marsupials are currently being used to find genetically conserved genes and make inferences about mammalian ancestors (Armati et al. 2013, 24). In order to accurately use this data, it is imperative that scientists continue to very carefully to assess the morphology of both therian clades in the context of genetics and distinguish between analogous and homologous structures. For example, the study by Ptak and colleagues (2012) is weakened by the lack of current genetic data on the conservation of diapause across mammals.

Comparative anatomical studies of extant species will continue to be a necessary part of understanding mammalian evolution and the marsupial role in biogeographical history. Despite some challenges in studying marsupial reproduction, modern studies of our phylogenetic sister group can provide useful insights into mammalian evolution and may aid in the development of new treatment techniques for human pathologies.

As an increasing number of species are negatively impacted by human activities, it is imperative that we find new ways to preserve biodiversity and prevent extinction. Ecotourism can be an effective way to protect native flora and fauna and enable important biological research to continue for generations.

Resources

ABC Radio Adelaide By Brett Williamson. "Is There a Tasmanian Tiger Living in the Adelaide Hills?" ABC News. N.p., 07 Sept. 2016. Web. 09 Mar. 2017.

Armati, Patricia J., Chris Dickman, and Ian Hume, eds. Marsupials. Cambridge: Cambridge UP, 2013. Print.

"Geography." University of California Museum of Paleontology. University of California Berkeley, n.d. Web. 09 Mar. 2017.

"LABELED-FEMALE-REPRODUCTIVE-SYSTEM-DIAGRAM.JPG." The Oncofertility Consortium. Northwestern, n.d. Web. 09 Mar. 2017.

Lemay, Danielle, and Kevin Nicholas. "Kangaroo Tips for Human Preemies." International Milk Genomics Consortium. International Milk Genomics Consortium, n.d. Web. 09 Mar. 2017.

"Marsupial Mammals." University of California Museum of Paleontology. University of California Berkeley, n.d. Web. 05 Mar. 2017.

"Marsupials." Vertebrate Diversity. University College London, n.d. Web. 05 Mar. 2017.

Nilsson, Maria A., Gennady Churakov, Mirjam Sommer, Ngoc Van Tran, Anja Zemann, Jurgen Brosius, and Jurgen Schmitz. "Tracking Marsupial Evolution Using Archaic Genomic Retroposon Insertions." PLoS Biology 8.7 (2010): n. pag. Web.

Paris, D. B B P, D. A. Taggart, G. Shaw, P. D. Temple-Smith, and M. B. Renfree. "Changes in Semen Quality and Morphology of the Reproductive Tract of the Male Tammar Wallaby Parallel Seasonal Breeding Activity in the Female." Reproduction 130.3 (2005): 367-78. Web.

Pizzari, Tommaso, and Kevin R. Foster. "Sperm Sociality: Cooperation, Altruism, and Spite." PLoS Biology 6.5 (2008): n. pag. Web.

Ptak, Grazyna E., Emanuela Tacconi, Marta Czernik, Paola Toschi, Jacek A. Modlinski, and Pasqualino Loi. "Embryonic Diapause Is Conserved across Mammals." PLoS ONE 7.3 (2012): n. pag. Web.

Rheede, T. Van, Trijntje Bastiaans, David N. Boone, S. Blair Hedges, Wilfried W. de Jong, and Ole Madsen. "The Platypus Is in Its Place: Nuclear Genes and Indels Confirm the Sister Group Relation of Monotremes and Therians." Molecular Biology and Evolution 23.3 (2005): 587-97. Web.

Szalay, Frederick. Evolutionary History of the Marsupials and an Analysis of Osteological Characters. Place of Publication Not Identified: Cambridge Univ, 2006. Print.

Tyndale-Biscoe, C. H. Life of Marsupials. N.p.: CSIRO, 2005. Print.

Vogelnest, Larry. "Marsupialia (Marsupials)." Veterian Key. N.p., 27 Aug. 2016. Web. 09 Mar. 2017.