Euglena: Department of Biology 1 and Ecology 2, Susquehanna University, Selinsgrove, PA

Evolutionary Divergence of Monodelphis domestica and Myrmecopius fasciatus through Complete Mitochondrial Genome Analysis Kelsey Hermick 1, Gabrielle Van Nest 1, Michael Terwilliger 1, Shannon Wood 2, and Victoria Legere 1. Department of Biology 1 and Ecology 2, Susquehanna University, Selinsgrove, PA 17870. Abstract Marsupials are characterized by the presence of a marsupium; however, two marsupial species 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 marsupium, location of mammae, number of mammae, epipubic bone, and the structure of the pseudovaginal 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 Marsupials are an infraclass of Mammalia whose distinguishing character is the presence of a marsupium, a protective pouch used to carry underdeveloped offspring (Tyndale-Biscoe 2005). Marsupials give birth to poorly developed fetuses (4-5 weeks old) that then crawl from the birth canal to the marsupium, where they live until they reach maturity (Dawson et al.1989). However, while Myrmecopius fasciatus and Monodelphis domestica each lack a fully developed marsupium, Monodelphis domestica completely lacks a pouch. (Wesierska and Turlejski 2000). According to Cooper et al. (2003), Myrmecopius fasciatus is still considered a marsupial because the pelt acts as an underdeveloped pouch (Cooper et al. 2003). The purpose of this paper is to analyze five morphological characters of the reproductive system in marsupials, specifically as they relate to Myrmecopius fasciatus and Monodelphis domestica. The offspring latch on to a mamma of its mother until the young are developed enough to function without full dependence (Tyndale-Biscoe 2005). Marsupials do not possess the placenta that all other mammals, excluding montrema, possess (Nelson 1978). Therefore, the presence of the marsupium is necessary for the young to grow to maturity (Dawson et al. 1989). The location of mammae and number of teats differ among several marsupial species (Tyndale-Biscoe 1987). The mammary glands lactate a nutritious supply of milk to the teats, and the young stay latched onto the teat for several weeks, or in some cases months. After the young are attached to the teat, they consume the nutritious milk (Tyndale- Biscoe 2005). Whether the mammae are concealed by the marsupium or exposed are character states of marsupials. The mammae are often concealed within the marsupium; however, some mammae are epidermally exposed. The epipubic bone is a common morphology among marsupials, although there are some species that do not exhibit this characteristic, for example, Notoryctes typhlops (Szalay 1994). The epipubic bone, or marsupial bone, is found in modern marsupials, and other mammals, and functions as a support for the marsupium (Kielan-Jaworowska 1975). Because this bone is also found in other mammals, Kielan-Jaworowska (1975) thought that the original purpose of the epipubic bone was not to support the marsupium, but to aid in locomotion (Kielan-Jaworowska 1975). Female marsupials possess two ovaries, two uteri and two vaginal canals (Dawson et al. 1989). In addition, marsupials possess the character of a third vaginal canal called the pseudovaginal canal that is specifically for giving birth. Progesterone is a hor- 17

mone secreted from the corpus luteum within the uterus that prepares marsupials for parturition by softening the tissues of the pseudovaginal canal (Tyndale-Biscoe 2005). The pseudovaginal canal can either be permanent, meaning that after birth the canal remains open, or transitional, meaning after birth, the tissues within the canal fuse and there is no indication that a canal ever existed (Sweet 1907). Marsupials today are found only on three continents; however, fossil records show that at one point, marsupials were prevalent across the globe. Continental drift during the Oligocene Era (34 to 23 million years before the present) tore the continents apart, and today, 235 species of marsupial can be found in Australia and 99 species can be found in the Americas (Nelson 1978). Throughout the world, marsupials could not compete with other placental mammals and began to die out, but in South America and especially Australia, the marsupials had no major predators and little competition for survival, so they were able to flourish and evolve into many forms (Nelson 1978). There are two major features that distinguish the mitochondrial genome of marsupials from other mammals. One major feature is that five trna genes around the origin of the light strand replication are rearranged. Another is the anticodon of trnaasp which is post transcriptionally changed by an RNA editing process, altering the coding capacity. trnaasp codes for the anticodon GCC in marsupials versus that of the GTC anticodon that monotremes and placentals code for. The mammalian mitochondrial genome codes for 22 trnas, 2 rrnas, and 13 proteins. This alteration likely contributed to the evolutionary divergence of marsupials from eutherians (Janke et al. 1994). A single morphological character is not sufficient in distinguishing between two taxa. Monodelphis domestica and Myrmecopius fasciatus do not possess a physical marsupium; however, other characters that these species possess are synapomorphic with those of species among the marsupial infraclass. Therefore, Monodelphis domestica and Myrmecopius fasciatus both lack a marsupium, but are still classified under marsupials because of their reproductive structures and gestation periods (Cooper et al. 2003; and Wesierska and Turlejski 2000). Table 1: Taxa selected were identified by scientific name, order, and common name. Taxa were used for phylogenetic 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) Taxa: Scientific Name Order Common Name Authority Accession Number Lagostrophus fasciatus Diprotodontia Banded Hare- wallaby 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 Wombat Shaw, 1800 NC_003322 Phalanger vestitus Diprotodontia Stein's Cuscus Milne- Edwards, 1877 NC_008137 Potorous tridactylus Diprotodontia Long- Nosed Potoroo 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 Koala Goldfuss, 1817 NC_008133 Pseudocheirus peregrinus Diprotodontia Common Ring- Tail possum Boddaert, 1785 NC_006519 Monodelphis domestica Didelphimorphia Gray Short- Tailed Opossum Wagner, 1842 NC_006299 Metachirus nudicaudatus Didelphimorphia Brown Four- Eyed Opossum E. Geottroy, 1854 NC_006516 delphis Virginiana Didelphimorphia Virginia Opossum Kerr, 1792 NC_001610 Myrmecobius fasciatus Dasyuromorphia Numbat Waterhouse, 1836 NC_011949 Thylacinus cynocephalus Dasyuromorphia Tasmanian tiger Harris, 1808 NC_011944 Dasyurus hallucatus Dasyuromorphia Northern Quoll Gould, 1842 NC_007630 Sminthopsis crassicaudata Dasyuromorphia Fat- Tailed Dunnart Gould, 1844 NC_007631 Sminthopsis douglasi Dasyuromorphia Julia Creek Dunnart Archer, 1979 NC_006517 Sarcophilus harrisii Dasyuromorphia Tasmanian Devil Boitard, 1841 NC_018788 Macrotis lagotis Peramelemorphia Greater Bilby Reid, 1837 NC_006520 Isoodon macrourus Peramelemorphia Northern Brown bandicoot Gould, 1842 NC_002746 Perameles gunnii Peramelemorphia Eastern Barred Bandicoot Gray, 1838 NC_006521 Caenolestes fuliginosus Paucituberculata Dusky Shrew Opossum Tomes, 1863 NC_005828 Canis lupus dingo Carnivora Dingo Meyer, 1793 NC_008092 Dromiciops gliroides Microbiotheria Monito del Monte Thomas, 1894 NC_005826 Notoryctes typhlops Notoryctemorphia Southern Marsupial Mole Stirling, 1889 NC_006522 18

Materials and Methods The sequences collected from each taxon were mitochondrial complete genome (Table 1). The sequences were acquired from the NCBI (National Center for Biotechnology Information) website. Complete mitochondrial genome provided nucleotides, which allowed for the analysis of 15,674 base pairs. Taxa were selected from the seven extant marsupial orders and selected through NCBI (NCBI 2013). Through the use of complete mitochondrial genome sequences, we were able to align the DNA and compare taxa of the Marsupialia infraclass as well as one species from the diverse carnivora order. The ML and MP (Figure 1-2) trees were developed from these sequences. There were 27 taxa selected and their DNA was aligned by Clustal W. These elongated branches of nucleotides were trimmed from the overall sequences to decrease less significant base pair analysis. The final alignment, after cutting, consisted of 15,674 base pairs. Then Maximum Likelihood (ML) and Maximum Parsimony (MP), Figure 1-2, trees were run. To construct Figures 1 and 2, MEGA 5 was used with a bootstrap of 1,000 replications (Tamura et. al 2011). The figures were constructed and the out group was rooted on both trees. The evolution of the character states was addressed in Figure 3 (the consensus topology tree), which was the combination of the ML tree and MP tree (Figure 1-2) derived from bootstrap values. Figure 1 supplied supported bootstrap values that aided in the development of Figure 3. The major characters were marsupium presence, location of mammae, number of mammae, epipubic bone, and pseudovaginal canal (Table 2). The respective character states of each taxon are stated in Table 3. The character states that had no information recorded or character states that were not relevant to that taxon was labeled N/A. These characters were stated to show evolutionary divergence of Myrmecobius fasciatus and Monodelphis 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 Figures 1 and 2 place the taxa into similar groups, but the topologies of those groups are very different in each figure. With the exception of the node between the polyphyletic clade Diprotodontia+Microbiotheria and the monophyletic clade Paucituberculata+Peramelemorphia, Figure 1 has bootstrap percentages above 50. The polyphyletic clade labeled Diprotodontia+Microbiotheria has nodes above 60, which moderately supports the grouping of taxon. The monophyletic clade of Paucituberculata + Peramelemorphia orders is strongly supported. In Figure 1 the Didelphimorphia clade is the basal group. The node connecting Diprotodontia + Microbiotheria clade to Paucituberculata+Peramelemorphia clade has a bootstrap of 47, showing weak support. Notoryctemorphia + Dasyuromorphia are a monophyletic clade with moderate support. Clade Diprotodontia, Microbiotheria, Paucituberculata, and Peramelemorphia is a sister clade to Notoryctemorphia and Dasyuromorphia clade. The standard orders of Diprotodontia, Dasyuromorphia, Peramelemorphia, and Didelphimorphia appear to be monophyletic. Figure 2 shows that Peramelemorphia, Microbiotheria, Paucituberculata, and Didelphimorphias have similar characters but are not strongly supported. The paraphyletic clade Diprotodontia is strongly supported, but sister clade Microbiotheria, Dasyuromorphia, Peramelemorphia, and Didelphimorphia Orders are not strongly supported in Figure 2. Notoryctemorphia is also a basal group in Figure 2. Dasyuromorphia and Didelphimorphia both contain species of interest (signified by a star). Figure 1 shows that the clades of Dasyuromorphia and Didelphimorphia are more supported by the bootstrap percentages, when compared to Figure 2. Figure 3 shows that taxa Canis lupus, Myrmecobius fasciatus, and Monodelphis virginiana are characterized under an A1 label. Taxa with a B0 label have concealed mammae and B1 have exposed. The exposure of the mammae may be due to the lack of a marsupium. Taxa with C0 have fewer than four mammae and C1 taxa have more than 5 mammae. The presence of an epipubic bone are D0 and taxa lacking an epipubic bone are D1. Taxa with a transitional pseudovaginal canal are E0 and taxa with a permanent pseudovaginal canal are E1. A primitive character state is represented by 0 states that a majority of marsupials possess. Derived character states are 1 states. The character consensus tree provided viable information to better understand the inheritance or development of a particular character state. The Diprotodontia order exhibits two clades with a derived permanent pseudovaginal canal, while the others maintain the primitive state. 19

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 Mammae Epipubic Bone Pseudovaginal Source Present Mammae 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, (Mun oz- 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 groups, but the topologies of those groups are very different in each figure. With the exception of the node between the polyphyletic clade Diprotodontia+Microbiotheria and the monophyletic clade Paucituberculata+Peramelemorphia, Figure 1 has bootstrap percentages above 50. The polyphyletic clade labeled Diprotodontia+Microbiotheria has nodes above 60, which moderately supports the grouping of taxon. The monophyletic clade of Paucituberculata+Peramelemorphia orders is strongly supported. In Figure 1 the Didelphimorphia clade is the basal group. The node connecting Diprotodontia+Microbiotheria clade to Paucituberculata+Peramelemorphia clade has a bootstrap of 47, showing weak support. Notoryctemorphia+Dasyuromorphia are a monophyletic clade with moderate support. Clade Diprotodontia, Microbiotheria, Paucituberculata, and Peramelemorphia is a sister clade to Notoryctemorphia and Dasyuromorphia clade. The standard orders of Diprotodontia, Dasyuromorphia, Peramelemorphia, and Didelphimorphia appear to be monophyletic. Figure 2 shows that Peramelemorphia, Microbiotheria, Paucituberculata, and Didelphimorphias have similar characters but are not strongly supported. The paraphyletic clade Diprotodontia is strongly supported, but sister clade Microbiotheria, Dasyuromorphia, Peramelemorphia, and Didelphimorphia Orders are not strongly supported in Figure 2. Notoryctemorphia is also a basal group in Figure 2. Dasyuromorphia and Didelphimorphia both contain species of interest (signified by a star). Figure 1 shows that the clades of Dasyuromorphia and Didelphimorphia are more supported by the bootstrap percentages, when compared to Figure 2. Figure 3 shows that taxa Canis lupus, Myrmecobius fasciatus, and Monodelphis virginiana are characterized under an A1 label. Taxa with a B0 label have concealed mammae and B1 have exposed. The exposure of the mammae may be due to the lack of a marsupium. Taxa with C0 have fewer than four 20

mammae and C1 taxa have more than 5 mammae. The presence of an epipubic bone are D0 and taxa lacking an epipubic bone are D1. Taxa with a transitional pseudovaginal canal are E0 and taxa with a permanent pseudovaginal canal are E1. A primitive character state is represented by 0 states that a majority of marsupials possess. Derived character states are 1 states. The character consensus tree provided viable information to better understand the inheritance or development of a particular character state. The Diprotodontia order exhibits two clades with a derived permanent pseudovaginal canal, while the others maintain the primitive state. Discussion Figures 1 and 2 indicate that Monodelphis domestica is within the order Didelphimorphia and Myrmecobius fasciatus is within the order Dasyuromorphia. Neither tree depicts that Monodelphis domestica nor the Myrmecobius fasciatus segregate from taxa with a marsupium. This means that each lost the marsupium independently. Griffiths et al. (1998) can confirm that Myrmecobius fasciatus is a part of the marsupial infraclass, even though the species lacks the marsupium. The offspring of Myrmecobius fasciatus are born underdeveloped and therefore, it is necessary for the offspring to rely on their mother for nutrients, as in other marsupials (Nelson 1978). Because this pattern is consistent throughout all marsupials, Myrmecobius fasciatus is best classified under the infraclass Marsupialia (Griffiths et al. 1998). Likewise, Wesierska and Turlejski (2000) verify that Monodelphis domestica is characterized as a marsupial because the underdeveloped offspring are reliant on the mother throughout early development (Wesierska and Turlejski 2000). Figure 1 and 2 confirm the interpretations of Griffiths et al (1998) as well as Wesierska and Tureljski (2000) because Myrmecobius fasciatus and Monodelphis domestica are distributed amongst other marsupials that have fully formed marsupium. Figure 3 supports that the marsupium were lost on two separate occasions. 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 branching. 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. 21

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

1994). Figure 3 suggests that the Dasyuromorphia order lost the character of an epipubic bone, however the epipubic bone was lost in Myrmecobius fasciatus but not in the clade. The Order Notoryctemorphia, being its own clade in Figures 1-3, is also characterized by the loss of an epipubic bone. As shown in Figure 2, Notoryctes typhlops is represented as an outgroup, along with Canis lupus. Notoryctes typhlops are categorized as marsupials because it possesses common marsupial morphologies. However, Notoryctes typhlops differs in the structure of the marsupium. This particular species seek out habitats of soft sand, which is why the species has evolved with a backwards-facing pouch that avoids the collection of sand (Glyshaw 2011). Another defining character of marsupials is the pseudovaginal canal, thus the state of this character would further classify particular taxon. The pseudovaginal canal is developed between the two lateral vaginas to provide a passage during the birth of marsupial offspring (Tyndale-Biscoe 1987). Table 3 and Figure 3 show presence of the pseudovaginal canal in the taxa of interest. Lagostrophus fasciatus, Vombatus ursinus, Phascolarctos cinereus, Tarsipes rostratus, Isoodon macrourus, and Perameles gunnii have a permanent pseudovaginal canal. Transitional canals are only present during the birthing process and this character state is seen in most marsupials shown in Table 3, with exception to the six species listed previously. With the consideration of these five morphological characters, we were able to produce Figure 1 and Figure 2 which show that there is no distinguishable difference between Monodelphis domestica and Myrmecobius fasciatus from other marsupials of their orders and that each of the marsupium were lost independently. This is pertinent because even though Monodelphis domestica and Myrmecobius fasciatus lack a marsupium, they are still classified under marsupials because of the unique reproductive system and gestation period that all marsupials share. 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 character state. Stars signify taxa in question. 23

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