Euglena: 2013

Evolutionary Divergence of Monodelphis domestica and Myrmecopius fasciatus through Complete Mitochondrial Genome Analysis Kelsey Hermick1, Gabrielle Van Nest1, Michael Terwilliger1, Shannon Wood2, and Victoria Le- gere1.

Department of Biology1 and Ecology2, Susquehanna University, Selinsgrove, PA 17870.

Abstract are characterized by the presence of a marsupium; however, two lack a fully developed marsupium. Myrmecopius fasciatus has a protective flap of skin and Monodelphis domestica does not possess a protective pouch. Through the analysis of the marsu- pium, location of mammae, number of mammae, epipubic bone, and the structure of the pseudo- vaginal canal, we classified Myrmecopius fasciatus and Monodelphis domestica to be no more closely related to the outgroup than any other marsupial. Using MEGA5, Maximum Parsimony and Maximum Likelihood trees were developed to show evolutionary placement of the 27 taxa of interest using complete mitochondrial genome sequences. We interpret our analyses to indicate that the absence of a marsupium is a derived character state for both Myrmecopius fasciatus and Monodelphis domestica. The purpose of this paper is to analyze five morphological characters of the reproductive system in marsupials by phylogenetic analysis, specifically as they relate to Myrmecopius fasciatus and Monodelphis domestica.

Please cite this article as: Hermick, K., G. Van Nest, M. Terwilliger, S. Wood, and T. Legere. 2013. Evolutionary divergence of Monodelphis domestica and Myrmecopius fasciatus through complete mitochondrial genome analysis. Euglena. doi:/euglena. 1(1): 17-25.

Introduction teats differ among several marsupial species (Tyn- Marsupials are an infraclass of Mammalia whose dale-Biscoe 1987). The mammary glands lactate a distinguishing character is the presence of a marsu- nutritious supply of milk to the teats, and the young pium, a protective pouch used to carry underdevel- stay latched onto the teat for several weeks, or in oped offspring (Tyndale-Biscoe 2005). Marsupials some cases months. After the young are attached to give birth to poorly developed fetuses (4-5 weeks the teat, they consume the nutritious milk (Tyndale- old) that then crawl from the birth canal to the mar- Biscoe 2005). Whether the mammae are concealed supium, where they live until they reach maturity by the marsupium or exposed are character states of (Dawson et al.1989). However, while Myrmecopius marsupials. The mammae are often concealed within fasciatus and Monodelphis domestica each lack a the marsupium; however, some mammae are epider- fully developed marsupium, Monodelphis domestica mally exposed. completely lacks a pouch. (Wesierska and Turlejski The epipubic bone is a common morphology 2000). According to Cooper et al. (2003), Myrme- among marsupials, although there are some species copius fasciatus is still considered a marsupial be- that do not exhibit this characteristic, for example, cause the pelt acts as an underdeveloped pouch Notoryctes typhlops (Szalay 1994). The epipubic (Cooper et al. 2003). The purpose of this paper is to bone, or “marsupial bone”, is found in modern mar- analyze five morphological characters of the repro- supials, and other , and functions as a sup- ductive system in marsupials, specifically as they port for the marsupium (Kielan-Jaworowska relate to Myrmecopius fasciatus and Monodelphis 1975). Because this bone is also found in other domestica. mammals, Kielan-Jaworowska (1975) thought that The offspring latch on to a mamma of its mother the original purpose of the epipubic bone was not to until the young are developed enough to function support the marsupium, but to aid in locomotion without full dependence (Tyndale-Biscoe 2005). (Kielan-Jaworowska 1975). Marsupials do not possess the placenta that all other Female marsupials possess two ovaries, two uteri mammals, excluding montrema, possess (Nelson and two vaginal canals (Dawson et al. 1989). In ad- 1978). Therefore, the presence of the marsupium is dition, marsupials possess the character of a third necessary for the young to grow to maturity (Dawson vaginal canal called the pseudovaginal canal that is et al. 1989). The location of mammae and number of specifically for giving birth. Progesterone is a hor-

17 Euglena: 2013 mone secreted from the corpus luteum within the mammals. One major feature is that five tRNA genes uterus that prepares marsupials for parturition by around the origin of the light strand replication are softening the tissues of the pseudovaginal canal rearranged. Another is the anticodon of tRNAasp (Tyndale-Biscoe 2005). The pseudovaginal canal can which is post transcriptionally changed by an RNA either be permanent, meaning that after birth the ca- editing process, altering the coding capac- nal remains open, or transitional, meaning after birth, ity. tRNAasp codes for the anticodon GCC in mar- the tissues within the canal fuse and there is no indi- supials versus that of the GTC anticodon that monot- cation that a canal ever existed (Sweet 1907). remes and placentals code for. The mammalian mi- Marsupials today are found only on three conti- tochondrial genome codes for 22 tRNAs, 2 rRNAs, nents; however, fossil records show that at one point, and 13 proteins. This alteration likely contributed to marsupials were prevalent across the the evolutionary divergence of marsupials from globe. Continental drift during the Oligocene Era (34 eutherians (Janke et al. 1994). to 23 million years before the present) tore the conti- A single morphological character is not suffi- nents apart, and today, 235 species of marsupial can cient in distinguishing between two taxa. Monodel- be found in and 99 species can be found in phis domestica and Myrmecopius fasciatus do not the Americas (Nelson 1978). Throughout the world, possess a physical marsupium; however, other char- marsupials could not compete with other placental acters that these species possess are synapomorphic mammals and began to die out, but in South America with those of species among the marsupial infraclass. and especially Australia, the marsupials had no major Therefore, Monodelphis domestica and Myrmecopius predators and little competition for survival, so they fasciatus both lack a marsupium, but are still classi- were able to flourish and evolve into many forms fied under marsupials because of their reproductive (Nelson 1978). structures and gestation periods (Cooper et al. 2003; There are two major features that distinguish the and Wesierska and Turlejski 2000). mitochondrial genome of marsupials from other

Table 1: Taxa selected were identified by scientific name, order, and common name. Taxa were used for phyloge- netic analysis. The authority for each taxa is also included. There are seven extant orders being investigated from the marsupial infraclass and one order from the placental mammals, Cannis lupus. Complete mitochondrial genome sequences were collected from accession numbers through NCBI. (NCBI 2013) (Redlist 2012) Accession Taxa: Scientific Name Order Common Name Authority Number Lagostrophus fasciatus Banded Hare- Péron & Lesueur, 1807 NC_008447 Macropus robustus Diprotodontia Common Wallaroo Gould, 1841 NC_001794 Lagorchestes hirsutus Diprotodontia Rufous Hare-wallaby Péron & Lesueur, 1807 NC_008136 Petaurus breviceps Diprotodontia Sugar Glider Waterhouse, 1838 NC_008135 Vombatus ursinus Diprotodontia Common Shaw, 1800 NC_003322 Phalanger vestitus Diprotodontia Stein's Cuscus Milne-Edwards, 1877 NC_008137 Potorous tridactylus Diprotodontia Long-Nosed Kerr, 1792 NC_006524 Tarsipes rostratus Diprotodontia Honey Possum Gervais & Verreaux, 1842 NC_006518 Trichosurus vulpecula Diprotodontia Common Brushtail Possum Kerr, 1792 NC_003039 Phascolarctos cinereus Diprotodontia Goldfuss, 1817 NC_008133 Pseudocheirus peregrinus Diprotodontia Common Ring-Tail possum Boddaert, 1785 NC_006519 Monodelphis domestica Didelphimorphia Gray Short-Tailed Wagner, 1842 NC_006299 Metachirus nudicaudatus Didelphimorphia Brown Four-Eyed Opossum E. Geottroy, 1854 NC_006516 delphis Virginiana Didelphimorphia Kerr, 1792 NC_001610 Myrmecobius fasciatus Waterhouse, 1836 NC_011949 cynocephalus Dasyuromorphia Tasmanian tiger Harris, 1808 NC_011944 Dasyurus hallucatus Dasyuromorphia Northern Gould, 1842 NC_007630 Sminthopsis crassicau- Dasyuromorphia Fat-Tailed Gould, 1844 NC_007631 data Sminthopsis douglasi Dasyuromorphia Archer, 1979 NC_006517 harrisii Dasyuromorphia Boitard, 1841 NC_018788 lagotis Greater Bilby Reid, 1837 NC_006520 Isoodon macrourus Peramelemorphia Northern Brown Gould, 1842 NC_002746 gunnii Peramelemorphia Gray, 1838 NC_006521 Caenolestes fuliginosus Dusky Tomes, 1863 NC_005828 Canis lupus dingo Carnivora Dingo Meyer, 1793 NC_008092 Dromiciops gliroides Thomas, 1894 NC_005826 Notoryctes typhlops Notoryctemorphia Southern Marsupial Stirling, 1889 NC_006522

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Materials and Methods Maximum Parsimony (MP), Figure 1-2, trees were The sequences collected from each taxon were run. mitochondrial complete genome (Table 1). The se- To construct Figures 1 and 2, MEGA 5 was used quences were acquired from the NCBI (National with a bootstrap of 1,000 replications (Tamura et. al Center for Biotechnology Information) website. 2011). The figures were constructed and the out Complete mitochondrial genome provided nucleo- group was rooted on both trees. tides, which allowed for the analysis of 15,674 base The evolution of the character states was addressed in pairs. Taxa were selected from the seven extant mar- Figure 3 (the consensus topology tree), which was the supial orders and selected through NCBI (NCBI combination of the ML tree and MP tree (Figure 1-2) 2013). Through the use of complete mitochondrial derived from bootstrap values. Figure 1 supplied genome sequences, we were able to align the DNA supported bootstrap values that aided in the develop- and compare taxa of the Marsupialia infraclass as ment of Figure 3. The major characters were marsu- well as one species from the diverse carnivora or- pium presence, location of mammae, number of der. The ML and MP (Figure 1-2) trees were devel- mammae, epipubic bone, and pseudovaginal canal oped from these sequences. (Table 2). The respective character states of each There were 27 taxa selected and their DNA was taxon are stated in Table 3. The character states that aligned by Clustal W. These elongated branches of had no information recorded or character states that nucleotides were trimmed from the overall sequences were not relevant to that taxon was labeled N/A. to decrease less significant base pair analysis. The These characters were stated to show evolutionary final alignment, after cutting, consisted of 15,674 divergence of Myrmecobius fasciatus and Monodel- base pairs. Then Maximum Likelihood (ML) and phis domestica.

Table 2: Five morphological characters (marsupium presence, location of mammae, number of mammae, epipubic bone, and pseudovaginal canal) and the character states analyzed. Marsupium Present Location of Mammae # of Mammae Epipubic Bone Present Pseudovaginal canal Present Concealed by pouch Less than 4 Present Transitional Not Present Exposed Greater than 5 Not Present Permanent

Results Dasyuromorphia, Peramelemorphia, and Didelphi- Figures 1 and 2 place the taxa into similar morphia Orders are not strongly supported in Figure groups, but the topologies of those groups are very 2. Notoryctemorphia is also a basal group in Figure different in each figure. With the exception of the 2. Dasyuromorphia and Didelphimorphia both con- node between the polyphyletic clade Diprotodon- tain species of interest (signified by a star). Figure 1 tia+Microbiotheria and the monophyletic clade Pau- shows that the clades of Dasyuromorphia and Didel- cituberculata+Peramelemorphia, Figure 1 has boot- phimorphia are more supported by the bootstrap per- strap percentages above 50. The polyphyletic clade centages, when compared to Figure 2. labeled Diprotodontia+Microbiotheria has nodes Figure 3 shows that taxa Canis lupus, Myrmeco- above 60, which moderately supports the grouping of bius fasciatus, and Monodelphis virginiana are char- taxon. The monophyletic clade of Paucituberculata + acterized under an A1 label. Taxa with a B0 label Peramelemorphia orders is strongly supported. In have concealed mammae and B1 have exposed. The Figure 1 the Didelphimorphia clade is the basal exposure of the mammae may be due to the lack of a group. The node connecting Diprotodontia + Micro- marsupium. Taxa with C0 have fewer than four biotheria clade to Paucituberculata+Peramelemorphia mammae and C1 taxa have more than 5 mammae. clade has a bootstrap of 47, showing weak support. The presence of an epipubic bone are D0 and taxa Notoryctemorphia + Dasyuromorphia are a mono- lacking an epipubic bone are D1. Taxa with a transi- phyletic clade with moderate support. Clade Dipro- tional pseudovaginal canal are E0 and taxa with a todontia, Microbiotheria, Paucituberculata, and permanent pseudovaginal canal are E1. A primitive Peramelemorphia is a sister clade to Notoryctemor- character state is represented by 0 states that a major- phia and Dasyuromorphia clade. The standard orders ity of marsupials possess. Derived character states of Diprotodontia, Dasyuromorphia, Peramelemor- are 1 states. The character consensus tree provided phia, and Didelphimorphia appear to be mono- viable information to better understand the inheri- phyletic. Figure 2 shows that Peramelemorphia, Mi- tance or development of a particular character state. crobiotheria, Paucituberculata, and Didelphimorphias The Diprotodontia order exhibits two clades with a have similar characters but are not strongly sup- derived permanent pseudovaginal canal, while the ported. The paraphyletic clade Diprotodontia is others maintain the primitive state. strongly supported, but sister clade Microbiotheria,

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Table 3: Character taxon matrix of Table 1 taxa and Table 2 the characters. The characters are present marsupium, mammae location, number of mammae, present epipubic bone and pseudovaginal canal. Taxa Marsupium Location of # of Mam- Epipubic Bone Pseudovaginal Source Present Mammae mae Present canal Lagostrophus fasciatus Present Concealed 4 Present Transitional 3, 5, 18, 30 Macropus robustus Present Concealed 4 Present Transitional 30 Lagorchestes hirsutus Present Concealed 4 Present Permanent 1, 3, 30 Petaurus breviceps Present Concealed 4 Present Transitional 26, 30 Vombatus ursinus Present Concealed 2 Present Permanent 30, 32 Phalanger vestitus Present Concealed 4 Present Transitional 5, 25, 30 Potorous tridactylus Present Concealed 4 Present Transitional 27, 30 Tarsipes rostratus Present Concealed 4 Present Permanent 23, 30 Trichosurus vulpecula Present Concealed 2 Present Transitional 7, 21,30 Phascolarctos cinereus Present Concealed 2 Present Transitional 11, 13, 22, 30 Pseudocheirus peregrinus Present Concealed 4 Present Transitional 18, 28, 29, 30 Monodelphis domestica Not Present Exposed 22 Present Transitional 19, 30 Metachirus nudicaudatus Present Exposed 9 Present Transitional 2, 20, 30 Didelphis virginiana Present Concealed 11 to 17 Present Transitional 14, 30 Myrmecobius fasciatus Not Present Concealed 4 Present N/A 4, 30 Thylacinus cynocephalus Present Concealed 4 Not Present Transitional 12, 30 Dasyurus hallucatus Present Concealed 6 Present Transitional 5, 30 Sminthopsis crassicaudata Present Concealed 8 to 10 Present Transitional 10, 30 Sminthopsis douglasi Present Concealed 4 Present Transitional 30, 31 Sarcophilus harrisii Present Concealed 4 Present Transitional 17, 30 Macrotis lagotis Present Concealed 8 Present Transitional 15, 30, 33 Isoodon macrourus Present Concealed 8 Present Permanent 7, 30 Perameles gunnii Present Concealed 8 Present Permanent 15, 30 Caenolestes fuliginosus Present Concealed 4 Present Transitional 16, 30 Dromiciops gliroides Present Concealed 4 Present Transitional 17, 30 Notoryctes typhlops Present Concealed 2 Not Present N/A 8, 9, 28, 30 Canis lupus dingo Not Present N/A N/A Not Present N/A 24, 30 Source Guide: (Adkins 2007)=1, (Bies 2002)=2, (Chedid)=3, (Cooper et al. 2003)=4, (Dawson et al. 1989)=5, (Fishman 2000)=6, (Gemmell et al. 1988)=7, (Glyshaw 2011)=8, (Graham 2000)=9, (Griffiths 1989)=10, (Hanger and Heath 1991)=11, (Jones and Stoddard 1998)=12, (Karlen and Krubritzer 2007)=13, (Kerr 1792)=14, (Lan- caster 2001)=15, (Meyer and Zardoya 2003)=16, (Munoz-Pedreros et al. 2005)=17, (Nowak 1991)=18, (Nowak 1999)=19, (Redford and Eisenburg 1992)=20, (Reilly et al. 2010)=21, (Rodger et al. 2009)=22, (Saunders and Hinds 1997)=23, (Scalter 1984)=24, (Sharman et al. 1970)=25, (Smith 1973)=26, (Sweet 1907)=27, (Szalay 1994)=28, (Thomson and Owen 1964)=29, (Tyndale-Biscoe 1987)=30, (Wells 1989)=31, (Wesierska and Turlejski 2000)=32, (Wooley et al. 2002)=33, (Wund and Myers 2000)=34

Figures 1 and 2 place the taxa into similar Dasyuromorphia, Peramelemorphia, and Didelphi- groups, but the topologies of those groups are very morphia appear to be monophyletic. Figure 2 shows different in each figure. With the exception of the that Peramelemorphia, Microbiotheria, Paucitubercu- node between the polyphyletic clade Diprotodon- lata, and Didelphimorphias have similar characters tia+Microbiotheria and the monophyletic clade Pau- but are not strongly supported. The paraphyletic cituberculata+Peramelemorphia, Figure 1 has boot- clade Diprotodontia is strongly supported, but sister strap percentages above 50. The polyphyletic clade clade Microbiotheria, Dasyuromorphia, Perame- labeled Diprotodontia+Microbiotheria has nodes lemorphia, and Didelphimorphia Orders are not above 60, which moderately supports the grouping of strongly supported in Figure 2. Notoryctemorphia is taxon. The monophyletic clade of Paucitubercu- also a basal group in Figure 2. Dasyuromorphia lata+Peramelemorphia orders is strongly sup- and Didelphimorphia both contain species of interest ported. In Figure 1 the Didelphimorphia clade is the (signified by a star). Figure 1 shows that the clades basal group. The node connecting Diprotodon- of Dasyuromorphia and Didelphimorphia are more tia+Microbiotheria clade to Paucitubercu- supported by the bootstrap percentages, when com- lata+Peramelemorphia clade has a bootstrap of 47, pared to Figure 2. showing weak support. Notoryctemor- Figure 3 shows that taxa Canis lupus, Myrmeco- phia+Dasyuromorphia are a monophyletic clade with bius fasciatus, and Monodelphis virginiana are char- moderate support. Clade Diprotodontia, Microbiothe- acterized under an A1 label. Taxa with a B0 label ria, Paucituberculata, and Peramelemorphia is a sister have concealed mammae and B1 have exposed. The clade to Notoryctemorphia and Dasyuromorphia exposure of the mammae may be due to the lack of a clade. The standard orders of Diprotodontia, marsupium. Taxa with C0 have fewer than four

20 Euglena: 2013 mammae and C1 taxa have more than 5 mam- Griffiths et al. (1998) can confirm that Myrmecobius mae. The presence of an epipubic bone are D0 and fasciatus is a part of the marsupial infraclass, even taxa lacking an epipubic bone are D1. Taxa with a though the species lacks the marsupium. The off- transitional pseudovaginal canal are E0 and taxa with spring of Myrmecobius fasciatus are born underde- a permanent pseudovaginal canal are E1. A primitive veloped and therefore, it is necessary for the off- character state is represented by 0 states that a major- spring to rely on their mother for nutrients, as in other ity of marsupials possess. Derived character states marsupials (Nelson 1978). Because this pattern is are 1 states. The character consensus tree provided consistent throughout all marsupials, Myrmecobius viable information to better understand the inheri- fasciatus is best classified under the infraclass Mar- tance or development of a particular character supialia (Griffiths et al. 1998). Likewise, Wesierska state. The Diprotodontia order exhibits two clades and Turlejski (2000) verify that Monodelphis domes- with a derived permanent pseudovaginal canal, while tica is characterized as a marsupial because the un- the others maintain the primitive state. derdeveloped offspring are reliant on the mother throughout early development (Wesierska and Turle- Discussion jski 2000). Figure 1 and 2 confirm the interpretations Figures 1 and 2 indicate that Monodelphis do- of Griffiths et al (1998) as well as Wesierska and mestica is within the order Didelphimorphia and Tureljski (2000) because Myrmecobius fasciatus and Myrmecobius fasciatus is within the order Dasyuro- Monodelphis domestica are distributed amongst other morphia. Neither tree depicts that Monodelphis do- marsupials that have fully formed marsupium. Fig- mestica nor the Myrmecobius fasciatus segregate ure 3 supports that the marsupium were lost on two from taxa with a marsupium. This means that each separate occasions. lost the marsupium independently.

Figure 1: A cladogram generated from the Maximum Likelihood method based on the Tamura-Nei model. The bootstrap consensus tree, inferred from 1,000 replicates, is taken to represent the evolutionary history of the taxa analyzed (Felsenstein 1985). Branches with less than 50% bootstrap replicates represent poorly supported branch- ing. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test are shown next to the branches (Hall 2011). Evolutionary analyses were conducted in MEGA5 (Tamura et al. 2011). The taxon are grouped by order classification. Stars signify taxa of interest.

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Figure 2: A cladogram generated from the Maximum Parsimony method. The bootstrap method estimated the reliability of the tree. The number of bootstrap replications was 1,000. Branches corresponding to divisions repro- duced in less than 50% bootstrap replicates are unreliable. The percentage of replicate trees that the associated taxa are clustered together in is based off the bootstrap test and is shown next to the branches (Hall 2011). There were a total of 15, 674 base pairs used for analysis. Evolutionary analyses were conducted in MEGA5 (Tamura et al. 2011). Stars signify taxa of interest.

Further analysis was done on the reproductive Microbiotheria, Paucituberculata, and Diprotodontia system to investigate other contributing characters to orders, seen in Figure 3. The number of mammae the marsupials (Table 2). The location of mammae would contribute to the classification of Myrmeco- almost directly correlates to marsupium pres- bius fasciatus and Monodelphis domestica because ence. The location of the mammae can either be con- most orders of marsupials appear to have similar cealed by the protective pouch or exposed (Nowak number of mammae (Tyndale-Biscoe 1987). 1999). With respect to only the taxa of interest, ex- Of the 26 species examined, two species either posure meant that the mammae had no protective had no presence of a marsupium or an altered version pouch. The young would simply hang from the teat of a productive pouch. Myrmecobius fasciatus has a until it was functionally stable (Tyndale-Biscoe protective flap of skin for the young while Monodel- 1987). Within each species of marsupials, the mam- phis domestica has no protective pouch or marsupium mae that the female possess vary in num- (Cooper et al. 2003; and Nowak 1999). This is why bers. Generally, they do not directly correlate to the some species are limited to the exposure of their number of offspring. For example, Monodelphis mammae. domestica possess 22 mammae (Table 3). Table 3 The epipubic bone generally is found in most depicts the common character state of 4 mammae or marsupials, but Thylacinus cynocephalus and No- less. This character state was consistent among the toryctes typhlops lack the epipubic bone (Szalay

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1994). Figure 3 suggests that the Dasyuromorphia Figure 3 show presence of the pseudovaginal canal in order lost the character of an epipubic bone, however the taxa of interest. Lagostrophus fasciatus, Vom- the epipubic bone was lost in Myrmecobius fasciatus batus ursinus, Phascolarctos cinereus, Tarsipes ros- but not in the clade. The Order Notoryctemorphia, tratus, Isoodon macrourus, and Perameles gunnii being its own clade in Figures 1-3, is also character- have a permanent pseudovaginal canal. Transitional ized by the loss of an epipubic bone. As shown in canals are only present during the birthing process Figure 2, Notoryctes typhlops is represented as an and this character state is seen in most marsupials outgroup, along with Canis lupus. Notoryctes ty- shown in Table 3, with exception to the six species phlops are categorized as marsupials because it pos- listed previously. sesses common marsupial morphologies. However, With the consideration of these five morphologi- Notoryctes typhlops differs in the structure of the cal characters, we were able to produce Figure 1 and marsupium. This particular species seek out habitats Figure 2 which show that there is no distinguishable of soft sand, which is why the species has evolved difference between Monodelphis domestica and with a backwards-facing pouch that avoids the collec- Myrmecobius fasciatus from other marsupials of their tion of sand (Glyshaw 2011). orders and that each of the marsupium were lost in- Another defining character of marsupials is the dependently. This is pertinent because even though pseudovaginal canal, thus the state of this character Monodelphis domestica and Myrmecobius fasciatus would further classify particular taxon. The pseudo- lack a marsupium, they are still classified under mar- vaginal canal is developed between the two lateral supials because of the unique reproductive system vaginas to provide a passage during the birth of mar- and gestation period that all marsupials share. supial offspring (Tyndale-Biscoe 1987). Table 3 and

Figure 3: The character consensus tree was produced using the Maximum Likelihood tree and the character taxon matrix. Taxa possessing primitive character states were assigned 0. Taxa with derived character states were labeled 1. They key provides colored coated lettering, which correspond to the five morphological characters of interest. Taxa branches residing above letter-number label possess the labeled state and taxon below exhibit opposing charac- ter state. Stars signify taxa in question.

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Literature Cited Hall, B. G. 2011. Phylogenetic Trees Made Easy. Adkins, J. 2007. Lagorchestes hirsutus. Di- Sinauer. Maryland. versity Web. Museum of Zoology. University of Hanger, J. J. and T. J. Heath. 1991. Topography of Michigan. the major superficial lymph nodes and their ef- tos cinereus). Journal of Anatomy. 177: 67-73. Bies, L. 2002. Metachirus nudicaudatus. Animal Janke, A., G. Feldmaier-Fuchs, W. Kelley Thomas, Diversity Web Museum of Zoology. University A. von Haeseler, and S. Paabo. 1994. The mar- of Michigan. supial mitochondrial genome and the evolution Jaworowska, Z. K. 1975. Possible occurrence of mar- Cardillo, M., O. R. P. Bininda-Emonds, E. Boakes, supial bones in eutharian mammals. and A. Purvis. 2004. A species-level phyloge- Nature. 225: 698-699. netic supertree of marsupials. The Zoological Jones, M. E. and D. M. Stoddard. 1998. Reconstruc- Society of London. 264: 11-31. tion of the predatory behavior of the extinct mar- Chedid, K. 2006. Lagostrophus fasciatus. Animal supial (Thylacinus cynocephalus). Diversity Web. Museum of Zoology. University Journal of Zoology. 246: 239-246. of Michigan. Karlen, S. J. and L. Krubritzer. 2007. The functional cortex: evidence for parallel evolution across Cooper, C.E., G. E. Walsberg, and P. C. Withers. mammals. Progress in Neurobiology. 82(3): 122- 2003. Biophysical properties of the pelt of a di- 141. urnal marsupial, the numbat (Myrmecobius fas- Kerr, R. 1792. The animal kingdom, or zoological ciatus), and its role in thermoregulation. The system, of the celebrated Sir Charles Linnaeus. Journal of Experimental Biology. 206: 2771- Class I. Mammalia. London. pp 193. 2777. Lancaster, E. 2001. Perameles gunnii. Animal Diver- Dawson. T. J., E. Finch, L. Freedman, I. D. Hume, sity Web. Museum of Zoology. University of M. B. Renfree, and P. D. Temple-Smith. 1989. Michigan. Morphology and physiology of the .

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Redford and Eisenburg. 1992. Metachirus nudicau- ters. Cambridge University Press. New York. 57- datus. Animal Diversity Web. Museum of Zool- 59. ogy. University of Michigan. < Tamura K,, D. Peterson, N. Peterson, G. Stecher, M. http://animaldiversity.ummz.umich.edu/accounts Nei,, and S. Kumar. 2011. MEGA5: molecular /Metachirus_nudicaudatus/> evolutionary genetics analysis using maximum Reilly, S. M. and T. D. White. 2003. Hypaxial motor likelihood, evolutionary distance, and maximum patterns and the function of epipubic bones in parsimony methods. Molecular Biology and primitive mammals. Science. 299: 400-402. Evolution. 28: 2731-2739. Reilly, S. M., E. J. McElroy, and T.D. White, A. R. Thomson, J. A., and W. H. Owen. 1964. A field Biknevicius, and M. B. Bennet. 2010. Abdomi- study of the Australian Ringtail Possum Pseudo- nal muscle and epipubic bone function during cheirus peregrinus (marsupialia: phalangeridae). locomotion in Australian Possums: insights to Ecological Monographs. 34: 27-52. basal mammalian conditions and eutherian-like Tyndale-Biscoe, H. 2005. Life of Marsupials. Ligare. tendencies in Trichosurus. Journal of Morphol- Sydney. ogy. 271: 438-450. Tyndale-Biscoe, H. and M. Renfree. 1987. Reproduc- Rodger, J. C., D. P. Paris, N. A. Czarny, M. S. Harris, tive physiology of marsupials. Cambridge Uni- F. C. Molinia, D. A. Taggart, C. D. Allen, and S. versity. New York. 7-445. D. Johnson. 2009. Artificial insemination in Wells, R. T. 1989. Vombatidae. Fauna of Australia. marsupials. Theriogenology. 71(1): 176-189. 32: 1-20. Saunders, N. R. and L. A. Hinds. 1997. Marsupial Wesierska, M. and K. Turlejski. 2000. Spontaneous Biology. University of New South Wales. pp behavior of the gray short-tailed opossum 169. (Monodelphis domestica) in the elevated plus- Scalter, W. L. 1984. The geography of mammals. No. maze: comparison with Long-Evans rats. Journal II the Australian Region. The Geographical of Experimental Biology. 60: 479-487. Journal. 4(1): 35-52. Wooley, P. A., M. F. Patterson, G. M. Stephenson, Sharman, G. B., E. S. Robinson, S. Walton, and P. J. and D. G. Stephenson. 2002. The ilio- Berger. 1970. Sex chromosomes and reproduc- marsupialis muscle in the dasyurid marsupial tive anatomy of some intersexual marsupials. Sminthopsis douglasi: form, function and fiber- The Journal of Society for Reproduction and type profiles in females with and without suck- Fertility. 21: 57-68. ling young. The Journal of Experimental Biol- Smith. M. J. 1973. Petaurus breviceps. Mammalian ogy. 205: 3775-3781. Species. 30: 1-5. Wund, M. and P. Myers. 2000. Metatheria. Animal Sweet. G. 1907. The skin, hair, and reproductive or- Diversity Web. Museum of Zoology. Univer- gans of Notoryctes. 325-344. sity of Michigan. Szalay, F. S. 1994. Evolutionary history of the mar-

Submitted 22 February 2013 Accepted 13 March 2013

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