Isoform Expression in the Prehensile Tails of Didelphid Marsupials: Functional Differences Between Arboreal and Terrestrial Opossums

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Myosin heavy chain (MHC) isoform expression in the prehensile tails of didelphid marsupials: functional differences between arboreal and terrestrial opossums

Joseph E. Rupert

Submitted in Partial Fulfillment of the Requirements

for the Degree of

Master of Science

in the

Biology

Program

YOUNGSTOWN STATE UNIVERSITY

May, 2013 Myosin heavy chain (MHC) isoform expression in the prehensile tails of didelphid marsupials: functional differences between arboreal and terrestrial opossums

Joseph E. Rupert

I hereby release this thesis to the public. I understand that this thesis will be made available from the OhioLINK ETD Center and the Maag Library Circulation Desk for public access. I also authorize the University or other individuals to make copies of this thesis as needed for scholarly research.

Signature:

Joseph E. Rupert, Student Date

Approvals:

Dr. Michael T. Butcher, Thesis Advisor Date

Dr. Mark D. Womble, Committee Member Date

Dr. Gary R. Walker, Committee Member Date

Dr. Brian De Poy, Interim Dean, School of Graduate Studies Date

Joseph E. Rupert

2013

iii ABSTRACT

Prehensile tails are defined as having the ability to grasp objects and may commonly be used as a fifth appendage during arboreal locomotion. Despite the independent evolution of tail prehensility in numerous mammalian genera, data relating muscle structure, physiology, and function of prehensile tails are largely incomplete. Didelphid marsupials make an excellent model to relate myosin heavy chain (MHC) isoform fiber type with structure/function of caudal muscles, because all opossums have a prehensile tail, but tail function varies widely between arboreal and terrestrial forms. Expanding on our previous study in the Virginia opossum, this investigation tests the hypothesis that arboreal and terrestrial opossums differentially express fast versus slow MHC isoforms, respectively. MHC expression and percent fiber type distribution were determined in the flexor caudae longus (FCL) muscle of

Caluromys derbianus (arboreal) and Monodelphis domestica (terrestrial), using a combination of gel electrophoresis and immuno-histochemistry analyses. C. derbianus expresses three MHC isoforms (1, 2A, 2X) in the FCL that are distributed as 8.2% MHC-1,

2.5% 1/2A, and 89.3% 2A/X hybrid fibers. M. domestica expresses MHC-1, 2A, 2X, and 2B, distributed as 17.2% MHC-1, 0.7% 1/2A, 7.7% 2A, 73.9% 2A/X, and 0.3% 2X/B hybrid fibers. The distribution of similar fiber types differed significantly between species

(P<0.001). Although not statistically significant, C. derbianus was observed to have larger cross-sectional area (CSA) for each corresponding fiber type along with a greater amount of extra-cellular matrix. An overall faster fiber type composition (and larger fibers) in the FCL of an arboreal specialist supports our hypothesis, and correlates with higher muscle force required for tail-hanging and arboreal maneuvering on terminal substrates. Conversely, a broader distribution of highly oxidative fibers is well suited to the tail nest building behaviors of terrestrial opossums.

iv ACKNOWLEDGEMENTS

I sincerely thank my advisor, Dr. Michael Butcher, for all his guidance and mentoring throughout my Thesis research project and Masters Degree. I thank my graduate committee members, Drs. Mark Womble and Gary Walker for critical reviews of my Thesis and their helpful comments. I am grateful to Dr. John VandeBerg (Texas Biomedical Research Institute) for coordinating muscle harvesting in Monodelphis, Dr. Bernal Rodríguez and Eugenia Cordero Schmidt at the Tirimbina Biological Reserve for coordinating trapping (with MINAET) and muscle harvesting in Caluromys, and Suzanne Peurach for assistance with the mammal collections at the National Museum of Natural History (NMNH-Smithsonian Institute). A very special thanks to Andres Moreira for assistance with field collection and muscle biopsy surgeries in Costa Rica. Also, a special thanks to Manuel Rojas (Tirimbina) for help with animal collection, care and handling of Caluromys. Thanks to Laura Kosiorek (YSU) for assistance with data analysis, and Jake Rose and Marc Gorvet for being terrific lab mates. S. Fatteh (Northside Hospital, Youngstown, Ohio) provided access to the cryostat and Dr. Walker (YSU) assisted with protein analyses. The monoclonal antibodies developed by F. Stockdale (S58, F18), S. Schiaffino (SC71, BF-35, BF-F3) and C. Lucas (2F7, 6H1) were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242. Support by University Research Council funding (URC #02-12) and NSF (IOS-1147044). The Department of Biological Sciences at YSU and the entire staff at the Tirimbina Biological Reserve are also gratefully acknowledged.

v DEDICATION

I dedicate my Thesis to my family, especially my father Brian and my mother Colette for their unfaltering support, both emotionally and financially, throughout my academic career and during the completion of this Thesis.

vi TABLE OF CONTENTS

Approval Page ii Copyright Page iii Abstract iv Acknowledgments v Dedication vi Table of Contents vii List of Tables viii List of Figures ix

INTRODUCTION 1

MATERIALS and METHODS 3 Animals 3 Muscle biopsy and Harvesting procedures 3 MHC protein analyses: SDS-PAGE 4 Histochemistry and Immuno-histochemistry 5 Data analysis and Statistics 6

RESULTS 7 Electrophoretic identification of MHC 7 Histological determination of MHC fiber types 7 Fiber type distributions and Fiber size 8

DISCUSSION 10 MHC isoform distribution in opossum tails 10 Fiber CSA and Tail function 12 Prehensile tail structure 14 Conclusions and Future directions 14

REFERENCES 16

APPENDIX 27 Literature review 27

vii LIST OF TABLES

1. Morphometric data for experimental animals. 47

2. Monoclonal antibodies (mAbs) used for IHC analyses and their MHC isoform reaction specificity against the tail muscle FCL from C. derbianus and M. domestica. 48

3. Regional mean distributions (%) of MHC isoform fiber types in the tails of C. derbianus and M. domestica. 49

4. Mean percentage distributions (%) of MHC isoform fiber types in the caudal muscle FCL of C. derbianus, M. domestica, and D. virginiana. 50

5. Regional mean fiber CSA (µm2) of MHC isoform fiber types in the tails of C. derbianus and M. domestica. 51

6. Mean fiber CSA (µm2) of MHC isoform fiber types in the caudal muscle FCL of C. derbianus and M. domestica. 52

viii LIST OF FIGURES

1. Diagram of the tail skeletal anatomy of C. derbianus and M. domestica. 54

2. Silver-stained gels identifying MHC isoforms expressed in the FCL of C. derbianus and M. domestica 56

3. Representative fiber type reactivity for mATPase and IHC throughout the FCL of C. derbianus. 58

4. Representative fiber type reactivity for mATPase and IHC throughout the FCL of M. domestica. 60

5. Mean fiber CSA and minimum diameter of MHC isoform fiber types in the FCL of C. derbianus and M. domestica. 62

ix INTRODUCTION Environmental limitations largely influence the morphology of inhabiting species, resulting in selection for specializations that provide species with advantages for resource acquisition. Habitats rich in natural resources and niche space, such as the neotropical rainforests of Central and South America, provide numerous microhabitats for large scale speciation of mammals (Hortal et al., 2008; Qian, 2010). Didelphid marsupials (opossums) are perhaps the best example of species-specific diversity in mammals native to these neotropical regions. Modern opossums descend from a common South American arboreal ancestor (Szalay, 1994; Cozzuol et al., 2006) and are members of the family Didelphidae, representing 19 genera and more than 100 species (Voss and Jansa, 2009), ranging from the southern-most regions of Argentina to southern Canada (Hershkovitz, 1997). Despite marked diversity in their body size and locomotor behaviors (Cunha and Vieira, 2002), all species of opossums share a crouched limb posture, an opposable hallux, and most notably, a prehensile tail (Nowak, 1999). Function of the prehensile tail is specialized for the requirements of either arboreal or scansorial/terrestrial locomotion. Specifically, the degree of tail prehensility changes with the level of vertical strata occupied within the rainforest (Cunha and Vieira, 2002), ranging from fully-prehensile in arboreal species to semi-prehensile in terrestrial species. A fully-prehensile tail is capable of suspending the body from a substrate (Emmons and Gentry, 1983), whereas a semi-prehensile tail is primarily used for gross manipulation of objects, and lacks the ability to suspend the body (Emmons and Feer, 1990; Bezanson, 1999). Arboreal opossums use the tail for maneuvers such as bridging and jumping (Youlatos, 2008) to traverse gaps between terminal branches in the canopy, and also for added stability during postural behaviors (e.g., foot-hanging and tail-hanging) when feeding (Julien-Laferriere, 1999; Schmitt and Lemelin, 2002). Additionally, they rely on their fully-prehensile tail in the occurrence of falls where it can quickly grasp onto a branch and be used as a substrate to pull the body upward (Lemelin et al., 2003). Arboreal specialists such as Caluromys exclusively dwell, forage, and locomote in the forest canopy (Vieira, 1997; Delciellos and Vieira, 2007, 2009), reaching the forest floor only 1% of their life time budget, typically resulting from falls (Hunsaker, 1977; Charles- Dominique, 1983). In contrast, terrestrial opossums spend most of their life on the forest

1 floor, and primarily use the tail to manipulate materials for nest construction (Layne, 1951; Unger, 1982), as well as for lifting and carrying offspring (Grizimek 1990; Macrini, 2004). Prehensile tails have evolved independently as many as 14 times among 40 extant mammalian genera (Emmons and Gentry, 1983; Emmons and Feer, 1990) and yet, data relating caudal muscle structure, physiology, and function of prehensile tails are largely incomplete (Organ et al., 2009). In our previous report (Hazimihalis et al., 2013), we analyzed myosin heavy chain (MHC) isoform fiber types in the tail of the Virginia opossum (Didelphis virginiana) and found a relatively large distribution of both slow (MHC-1) and fast, oxidative MHC-2A/X hybrid fibers. This finding is consistent with adaptive grasping tasks (e.g., carrying nest building materials) requiring sustained low force contractions. However, no study has analyzed MHC isoforms in the fully- prehensile tail of an arboreal opossum, thus limiting our understanding of how muscle structure and fiber type composition may be specialized for prehension and suspension in the forest canopy. Moreover, determining fiber type in caudal muscle of an arboreal specialist may give insight into the ancestral MHC isoform expression in the prehensile tails of this lineage, as well as relating MHC isoform expression with tail function. This study aims to identify MHC isoform composition in the tail of an arboreal opossum, Caluromys derbianus Waterhouse, 1841, and a closely related terrestrial opossum, Monodelphis domestica Wagner, 1842. C. derbianus is a species of woolly opossum that inhabits both highland and lowland rainforests throughout Central America (Hall and Kelson, 1959), is strictly nocturnal and highly arboreal (Bucher and Fritz, 1977), and uses its fully-prehensile tail extensively for balance and grasping during arboreal maneuvering (Hall and Dalquest, 1963; Bucher and Hoffmann, 1980). Prehension is performed by tail flexion (Lemelin, 1995) and therefore, we focus our analysis on the main flexor muscle to relate MHC isoform composition with tail function in these two species. Based on the observed differences in tail use between arboreal and terrestrial forms, and the findings from our previous study, we expect the caudal muscle fiber type of M. domestica will be similar to D. virginiana, while C. derbianus are hypothesized to express a high percentage of fast MHC isoforms. In particular, the contractile properties of the fast MHC-2X and 2B isoform fiber types are appropriate for

2 high force and the potentially high velocity demands of the prehensile tail for rapid arboreal maneuvers.

MATERIALS AND METHODS Animals Six Central American woolly opossums (C. derbianus) were obtained for this study. Animals were live captured at night in the rain forest (Tirimbina Biological Reserve, Costa Rica) using Havahart Easy Set traps (42 x 18 x 18 cm or 60 x 18 x 18 cm) baited with bananas, and fresh muscle tissue was taken from their tails via a biopsy procedure. Five gray short-tailed opossums (M. domestica) were obtained from the Texas

Biomedical Research Institute in San Antonio, TX and euthanized by CO2 asphyxiation followed by cervical vertebral dislocation, and fresh caudal muscle tissue was harvested from their tails immediately post-mortem. All experimental procedures followed protocols approved by the Youngstown State University Animal Care and Use Committee (YSU IACUC: 01-12 and 04-12) and the Costa Rica Environment Ministry (MINAET: DGVS-730-2012). Morphological data for all individuals are in Table 1.

Muscle biopsy and harvesting procedures Muscle biopsies were performed on the tail of four adult C. derbianus using aseptic technique. An intramuscular (IM) dose of 5 mg kg-1 Zolitiel 50 was injected to induce sedation, followed by IM local anesthetic injection of Bupivacaine (diluted to 50% conc.) to the intended muscle biopsy sites on the tail. Opossums were then positioned in lateral recumbency to allow sterile surgical access to the ventrolateral aspect of the right side of the tail. To expose the caudal flexor muscles, small longitudinal incisions were first made through the skin at two locations along the length of the tail: (1) ~16 cm from the base of the tail (beginning of the hairless region) and (2) ~22 cm from the tail base near mid-tail. Our survival surgery protocol only permitted us to incise two sites that excluded the distal region of the tail. The ventral flexor tendon superficial to the caudal flexors (see Hazimihalis et al., 2013: Fig. 1) was then incised and retracted, to gain access to the muscles. A small block of muscle tissue was harvested from m. flexor caudae longus (FCL: Lemelin, 1995; Hazimihalis et al., 2013) at each biopsy site. Fresh muscle blocks were immediately either placed in microcentrifuge tubes or mounted to cork,

3 frozen in liquid nitrogen, and stored at -80°C until analysis. Incisions were closed with absorbable sutures (Vicryl 4-0: Ethicon, Somerville, NJ USA), followed by application of a skin adhesive (Liquid Stitches), and finally treated with an aerosolized antibiotic (Tetracyclin). C. derbianus were allowed to recover 6 h post-procedure before being released back into the rain forest. For adult M. domestica, and two individuals of C. derbianus (one adult female and a juvenile that died prior to muscle biopsy), muscle blocks were harvested from the tail post-mortem. Briefly, the tails were divided into ‘proximal’, ‘transitional’, and ‘distal’ regions (Hansen et al., 1987; Hazimihalis et al., 2013) for muscle dissection (Fig. 1). Small muscle blocks were harvested from the FCL and flash frozen in tubes as described above. Muscle blocks from FCL and whole tail cross-sections were additionally mounted to cork with tissue freezing media (TFM: Triangle Biomedical Sciences, Inc., Durham, NC USA), flash frozen in isopentane cooled in liquid nitrogen, and stored at -80°C until analysis. Muscle blocks mounted to cork were used for histochemistry and immuno- histochemistry (IHC) analyses to provide a percentage distribution of slow and fast MHC fiber types. All samples were shipped cold (on dry ice) to YSU for analysis.

MHC protein analyses: SDS-PAGE Muscle tissue from each tail region was prepared for electrophoresis by directly freezing in liquid nitrogen, grinding them to powder, homogenizing 50 mg of muscle powder in 800 µl (ratio 1:16) of Laemmli buffer with 62.5 mM Tris (pH 6.8), 10% glycerol, 5% β- mercaptoethanol, and 2.3% SDS (Laemmli, 1970), and centrifugation of the muscle homogenates at 13k rpm for 10 min. After centrifugation, the stock supernatant was decanted into sterile microcentrifuge tubes and used to make the protein samples for gel loading. Protein samples were diluted (1:500) to a final protein concentration of ~0.125 µg/µl with gel sample buffer containing 80 mM Tris (pH 6.8), 21.5% glycerol, 50 mM DTT, 2.0% SDS, and 0.1% bromophenol blue (Mizunoya et al., 2008). Samples were then heated (90ºC) for 5 min and either loaded in gels or stored at -20°C. To serve as a MHC band standard, muscle blocks of rat tibialis anterior (TA), extensor digitorum longus (EDL), and soleus (SOL) were harvested, and a combined sample was prepared by the described methods.

4 MHC isoforms were identified on SDS-PAGE gels using established methods (Talmadge and Roy, 1993) performed with slight modifications (Mizunoya et al., 2008) as previously described (Hazimihalis et al., 2013). For densitometry, 1.25 µg of protein together with a known concentration of BSA were run on 4-15% gradient gels (Bio-Rad) and stained with Colloidal Blue (Novex® G-250: Invitrogen). Companion gels were run with higher concentrations of protein (2-5x) for western blots, where unstained gels were transferred to PVDF membranes (Immobilon: EMD Millipore Corp., Billerica, MA USA) in an electrode buffer containing 20% methanol and 0.1% SDS (Bolt and Mahoney, 1997), using a mini-transblot system (Bio-Rad) at a constant 100 mA for 5 h at 4°C (Bolt and Mahoney, 1997; Kohn et al., 2011). The membranes were quickly stained in Ponceau S (Sigma-Aldrich) to check for protein transfer, blocked with a synthetic blocker (CH- blok: Millipore) for 1 h, and then incubated with monoclonal antibodies (mAbs) for 5 h at 4ºC. Antibodies with known immunospecificity against MHC isoforms were purchased (as concentrates) from the Developmental Studies Hybridoma Bank (DSHB, University of Iowa). S58 was specific to the MHC-1 isoform (Miller et al., 1985) and shown to identify MHC-1 fibers in our previous study of Didelphis (Hazimihalis et al., 2013). Three of the mAbs were specific to rat MHC isoforms (Schiaffino et al., 1989): SC71 specific to MHC-2A, BF-35 specific to all MHC isoforms except MHC-2X, and BF-F3 specific to MHC-2B. 2F7 was also specific to the MHC-2A isoform. Lastly, F18, was specific to all fast MHC isoforms. Working dilutions for each antibody ranged between 1:200 and 1:500 (0.002-0.005 µg/ml) depending on the original Ig concentration. Horse- radish peroxidase-adsorbed rabbit (anti-mouse) was used as a secondary antibody (1:10,000; Millipore) and following a 1-2 h incubation, membranes were washed twice in TBS (10 min each), reacted with chemiluminescence HRP substrate (5 min), and imaged with Pharos FX Plus (Quantity One software: Bio-Rad) having a resolution to 50 µm.

Histochemistry and Immuno-histochemistry Transverse serial sections (10 µm) of the muscle blocks were cut on a Leica 1850 cryostat (Leica Microsystems, Buffalo Grove, IL USA) at -20ºC and mounted onto GOLDSEAL slides (Becton Dickinson, Portsmith, NH USA) for histochemistry and charged Superfrost slides (ThermoFisher Scientific Inc., Waltham, MA USA) for IHC. Serial sections were reacted for mATPase activity using modifications (Hermanson et al., 1998)

5 of well established protocols (Brooke and Kaiser, 1970; Guth and Samaha, 1970). See Hazimihalis et al. (2013) for detailed methods. Serial sections of the FCL were reacted against the panel of mAbs specific to slow and fast MHC isoforms (Table 2) used to identify MHC isoforms with western blots. In addition to the mAbs detailed above, serial sections were also reacted against MY32 (Sigma-Aldrich) specific to all fast MHC isoforms in marsupials (Spiegel et al., 2010a), and one antibody (6H1) obtained from DSHB with known specificity to specific fast MHC-2X isoform in marsupials (Lucas et al., 2000). All mAbs were diluted in PBS (pH 7.4). MY32 was diluted 1:400, while antibodies (supernatants) from DSHB were diluted to working Ig concentrations of 2-5 µg/ml. IHC was conducted as previously described (Butcher et al., 2010; Hazimihalis et al., 2013). Briefly, serial sections were blocked, incubated with mAbs for 12-16 h at 4°C and then with a biotinylated rabbit (anti-mouse) secondary antibody for 1 h (washed with PBS), next reacted with streptavidin HRP enzyme conjugate (10 min) followed by a final reaction with DAB chromagen (3-6 min), all using a Histostain Plus kit (Invitrogen). After a rinse in dH2O (10 min), serial sections were counterstained with Mayer modified hematoxylin (Newcomer Supply, Middleton, WI USA) to visualize fiber morphology. Experimental controls were treated with PBS in place of mAbs.

Data analysis and Statistics MHC isoform distributions were quantified from images of IHC-stained serial sections visualized on an Olympus CX31 microscope (Olympus Microscopes, Center Valley, PA USA) and photographed with a SPOT Idea digital camera system (Diagnostic Instruments, Sterling Heights, MI USA). Percentages of each MHC fiber type were calculated based on counts of all fibers from 1-2 sections of muscle from each tail region of each individual. Muscle fibers were classified as hybrids if they showed cross-reaction with two mAbs specific for different MHC isoforms. MHC isoform protein content was quantified by densitometry in Image J (v.1.43: NIH, Washington D.C. USA) using the concentration of BSA to calibrate the protein quantity for each MHC band. In each gel lane (representing a tail region of each individual), band values were summed and then used to calculate a percentage for each MHC isoform. Fiber size was determined by measuring cross-sectional area (CSA: in µm2) and minimum diameter (in µm) of each

6 MHC fiber type from section images imported into Image J. Fiber size was assessed from a subset of fibers from each type classified in each tail region. Additionally, fiber CSA and diameter were used to calculate ratios relating average fiber size (mean CSA across all MHC fiber types) to average tail length in each species. All data are presented as mean (± s.d.). Mean differences in both MHC isoform fiber type distribution and fiber size between C. derbianus and M. domestica were tested by MANOVA followed by Bonferroni post hoc tests, and performed in PASW Statistic 18 (SPSS; IBM). Statistical comparisons were also made with similar data from D. virginiana in our previous study (Hazimihalis et al., 2013). Significance for all tests was accepted at P<0.05.

RESULTS Electrophoretic identification of MHC Each region of the FCL in C. derbianus showed expression of three MHC isoform bands: MHC-1, 2A, and 2X (Fig. 2). Fast MHC-2A and MHC-2X bands were predominant and clearly resolved in each individual. Slow MHC-1 was identified in all samples from C. derbianus, although bands for this isoform were comparatively light in their resolution (Fig. 2). The FCL in M. domestica shared expression of the MHC-1, 2A, and 2X isoforms, in addition to the presence of the MHC-2B band. The MHC-2B isoform was clearly expressed in the proximal tail region of male M. domestica, but was absent from the transitional and distal regions, and not expressed in any of the female samples (Fig. 2). Clear separation of 2A and 2X bands was obtained in all gels analyzed, with an overall resolution of >1 cm between the fastest migrating MHC-1 and slowest migrating MHC-2A bands. Histological determination of MHC fiber types Using select mAbs specific for MHC isoforms, three MHC isoform fiber types were identified for C. derbianus, while four were identified for M. domestica. Table 2 summarizes reaction specificity of mAbs with caudal muscle tissue from both species. Fiber types were further distinguished by mATPase reactions in acidic (pH 4.4) and alkaline (pH 10.2) incubations. Figure 3B shows alkaline incubations in C. derbianus with a large numbers of fast fibers stained darkly, slow fibers labile, and slow/fast hybrid

7 fibers stained lightly and circular in shape. Figure 4A shows acid incubations in M. domestica with four different staining intensities (most common) of mATPase activity observed. Staining patterns at pH 4.4 helped verify IHC reactions, where highly acid stable (darkly stained) matched MHC-1 fibers, acid labile matched MHC-2A fibers, moderately acid stable matched MHC-1/2A hybrid fibers, and weakly acid stable (lightly stained) MHC-2A/X hybrid fibers. Pure MHC-1 fibers were identified by strong reactivity against the antibody S58 (Figs. 3C, 4C). S58 consistently reacted with slow MHC-1 isoform fibers in each tail region of both C. derbianus and M. domestica. An abundance of MHC-2A/X hybrid fibers were identified by strong reactions against the antibody SC71 (Figs. 3D, 4D) and BF-35 (Fig. 3E). BF-35 reacted against all muscle fibers in each tail section studied, which indicated a lack of pure MHC-2X fibers, and verified the presence of the 2X isoform in fibers classified as MHC-2A/X hybrids. MHC-1/2A hybrid fibers were identified by matching fibers with moderate acid/alkaline stability and reactions against both S58 and SC71 or S58 and 2F7. Low numbers of slow/fast hybrid fibers were found across regions of the tail in both species, and reaction intensities against S58, SC71, and 2F7 for the same fiber varied slightly with each tail region. Pure MHC-2A fibers were identified in only M. domestica by matching acid labile fibers and reactions against 2F7 and SC71, where 2F7 had a moderate reaction with pure MHC-2A fibers and MHC-2A/X hybrid fibers, and SC71 showed a strong reaction with both of these fiber types (Fig. 4E). Reaction against 6H1 showed no clear ability to identify pure MHC-2X fibers in the FCL of either species. F18 and MY32 both showed moderate reactions against fibers that did not react against S58 (data not shown), however, these mAbs were effective in isolating a few MHC-2X/B hybrid fibers in the proximal tail region of male M. domestica. BF-F3 showed no reaction against muscle fibers from the proximal tail region in M. domestica.

Fiber type distributions and Fiber size MHC isoform fiber type distributions were determined from totals of n=3945 fibers for C. derbianus and n=7968 fibers for M. domestica. In all animals studied, there was a majority of fast, oxidative MHC-2A/X hybrid fibers, though their percent distribution differed significantly (P<0.001) between species, and also varied with tail region in each species (Tables 3, 4). Overall, 89.2% of muscle fibers studied in the FCL of C. derbianus

8 were composed of MHC-2A/X isoforms compared with 75.2% in the tail of M. domestica (Table 4). MHC-1 and MHC-2A fibers were the only pure isoform fiber types identified, and 2A fibers were only found in the FCL of M. domestica, accounting for 9.0 ± 3.8% of its fiber type distribution on average. Slow, oxidative MHC-1 fibers also differed significantly in their percent distribution (P<0.001) between species. Collectively, the FCL was composed of a mean of 8.2 ± 1.3% MHC-1 fibers in C. derbianus compared with a mean of 17.0 ± 3.6% MHC-1 fibers in M. domestica (Table 4). Slow/fast, oxidative MHC-1/2A hybrid fibers were present in low percentages in each species and showed significantly different (P<0.002) distributions in each tail region (Tables 3, 4). The percentage distributions of MHC-1/2A hybrids for the FCL were 2% in C. derbianus and 1% in M. domestica (Table 4). Similarly, low numbers MHC-2X/B fibers were counted in M. domestica and they accounted for only 0.03 ± 0.01% of the total distribution for the entire tail. Means of fiber CSA and diameter for each MHC isoform fiber type identified are shown in Figure 5. Along the tail in the direction cranial to caudal, fiber size (for each fiber type) generally decreased (Table 5), remaining in following order by size: MHC- 2A/X >MHC-1/2A >MHC-1 in C. derbianus; and MHC-2X/B >MHC-2A/X >MHC-2A >MHC-1 >MHC-1/2A in M. domestica. Overall, MHC-2A/X hybrid fibers had a larger mean CSA of 3376.7 ± 1399.6 µm2 in C. derbianus compared with 2899.2 ± 881.5 µm2 in M. domestica (Table 6), although the mean difference was not statistically significant. Mean CSA of MHC-1 fibers was also not significantly different between species (Fig. 5A). The few MHC-2X/B fibers that were measured in the proximal tail region of M. domestica indicated that these fibers were substantially larger in their cross-section size than MHC-2A/X hybrids (4455.1 µm2) (Table 6). Measurements for minimum diameter of muscle fibers were consistent with those of fiber CSA, indicating a good correlation between fiber CSA and diameter. MHC-2A/X hybrid fibers (and MHC-2X/B fibers) had largest minimum diameter followed by MHC-2A, MHC-1, MHC-1/2A hybrids in M. domestica, and MHC-2A/X, MHC-1/2A hybrids, and MHC-1 in C. derbianus. Similar to fiber CSA, minimum fiber diameter means of 53.3 ± 12.9 µm and 47.9 ± 10.3 µm for MHC-2A/X hybrids for C. derbianus and M. domestica, respectively, were more similar,

9 and both were relatively larger than the mean diameters of MHC-1 fibers and MHC-1/2A hybrids in both species (Fig. 5B).

DISCUSSION MHC isoform distribution in opossum tails The findings of this study are similar to those reported for the tail of D. virginiana (Hazimihalis et al., 2013), where both C. derbianus and M. domestica primarily express three MHC isoforms (1, 2A, and 2X), and the FCL muscle is largely composed (>70%) of fast, oxidative MHC-2A/X hybrid fibers. In addition, expression of the fast MHC-2B isoform was found in the proximal tail region of male M. domestica, and is confined to a very small number of MHC-2X/B hybrid fibers. While it was surprising to find MHC-2B in a caudal flexor, and the functional explanation for its presence in the semi-prehensile tail of a terrestrial opossum is unclear, marsupials are known to retain expression of all four conventional MHC isoforms in their skeletal muscles (Lucas et al., 2000). In fact, assorted hindlimb muscles of Australian marsupials are largely composed of pure MHC- 2B fibers (Zhong et al., 2008), and one study found a large distribution of MHC-2X/B hybrids in a caudal extensor muscle of the eastern gray kangaroo (Spiegel et al., 2010b). Kangaroos are considered more derived forms than their South American relatives (Lee and Cockburn, 1985), and while several Australian marsupials possess a prehensile tail (e.g., bettongs and possums), saltatory (i.e., hopping) kangaroos and wallabies do not. The significant presence of the 2B isoform in the tail of kangaroos may provide evidence supporting the hypothesis that a primary composition of fast, glycolytic fibers is ancestral for mammals with long tails (Schilling, 2009). Relatively large distributions of fast, glycolytic fibers also have been found in the caudal muscles of domestic cats (Wada et al., 1994) and rats (Ovalle, 1976). Therefore, we suggest the expression of MHC-2B in caudal muscle of M. domestica may be either an ancestral retention or an ontological retention of a fast, glycolytic fiber type from early in development. As mammalian lineages independently evolved prehensile tails, it is likely that this adaptation coincided with a shift in MHC isoform expression and muscle fiber phenotype. In particular, axial muscles (paravertebral, and possibly caudal muscles) became slower-contracting and more oxidative, as they became stabilizers during

10 locomotion (Schilling, 2009). A change in MHC isoform expression in caudal muscles is further supported by the abundance of fast, oxidative MHC-2A/X hybrid fibers now found in the FCL of three disparate species of didelphids, each with diverse locomotor habits and varying degrees of tail prehensility. The presence of pure MHC-2A fibers in the proximal and transitional tail regions of M. domestica is also consistent with a shift to a more oxidative fiber type composition. Relating this fiber type to use of their semi- prehensile tail for nest building, the behavior of M. domestica differs considerably from that observed in D. virginiana and C. derbianus. Nest construction and remodeling occurs daily in M. domestica (Macrini, 2004), whereas D. virginiana remodel their nest infrequently (Layne, 1951), and C. derbianus may use their tail to carry nesting materials but prefer tree hollows to form their dwelling (Dalloz et al., 2012). MHC-2A fibers have highly oxidative properties for ATP synthesis as well as a higher force and shortening velocity than MHC-1 fibers (Schiaffino and Reggiani, 2011), making them well-suited for prolonged, moderate force contractions involved with lifting, carrying, and manipulating nesting materials. Moreover, the combined percentage of pure MHC-2A and MHC-1 fibers (significantly higher compared to Caluromys) in the FCL correlate well with the extensive nest remodeling of M. domestica. Despite differences in nest building behaviors and presence of MHC-2A fibers, the overall fiber type distribution in the semi-prehensile tail of M. domestica is close to that of the Virginia opossum (Hansen et al., 1987; Hazimihalis et al., 2013), where both species have approximately 20% of slow MHC-1 fibers and a similar (P=0.379) percentage of MHC-2A/X hybrid fibers. In contrast, the fiber type distribution in the fully-prehensile tail of C. derbianus is significantly faster (see Table 3) compared with both M. domestica and D. virginiana, in accordance with our hypothesis. Although their FCL did not have either pure MHC-2X or 2B fibers as predicted, C. derbianus have the lowest percentage of MHC-1 fibers (8.2%) and the highest percentage of MHC-2A/X hybrids (89.2%) among didelphids studied thus far. The differential expression of the MHC-1 isoform may be attributed to less complex nest building in the arboreal C. derbianus, as observed in C. philander (Dalloz et al., 2012), and therefore higher percentages of MHC-1 fibers are more specific to meet the nest building requirements of habitually terrestrial forms. Arboreal opossums extensively use their fully-prehensile tail

11 for balance, and to occasionally perform postural behaviors (e.g., tail-hanging) or locomotor maneuvers such as bridging and cantilevering (Youlatos, 2008; Delciellos and Vieira, 2009b; Dalloz et al., 2012). These behaviors would require the caudal flexors to produce high levels of force (possibly over a short contractile period), as the tail must support the body mass during the maneuver. Relating muscle fiber type with tail- function, a predominantly fast, oxidative FCL with high force and moderate shortening capabilities (i.e., moderate glycolytic properties) is well-suited for the grasping requirements of arboreal locomotion. Similar arboreal habits have been correlated with full prehensility in the tails of non-human primates, which have well-developed caudal flexors capable of producing high force (Lemelin, 1995; Organ et al., 2009). The large distribution of MHC-2A/X hybrid fibers and their relatively high oxidative properties, also seem suitable to meet balance needs and the postural demands of tail suspension.

Fiber CSA and Tail function The capability to produce high force is related to the cross-sectional area (CSA) of muscle fibers. In general, we found muscle fiber CSA to increase with progressively faster MHC isoform fiber types. The CSA of MHC-1/2A hybrid fibers is smaller than that of MHC-1 fibers in M. domestica as it was observed in D. virginiana (Hazimihalis et al., 2013); however, this was different from the trend in C. derbianus where MHC-1/2A hybrid fibers have a slightly larger fiber CSA than MHC-1 fibers. While the exact reason for this deviation in terrestrial forms is unknown, an overall slower, more oxidative composition combined with smaller fiber CSA is consistent with adaptive tail-function for nest building. In contrast, C. derbianus have relatively large fiber CSA and a well- developed FCL compared with M. domestica, to which they are more similar in body size than either are to the exceedingly large Didelphis. We also observed that C. derbianus have more extra-cellular matrix than M. domestica (see Fig. 3), and this feature may be related to suspensory behaviors requiring greater tensile strength of the tail. Moreover, arboreal opossums are expected to have larger caudal muscle fibers, as well as longer tail lengths compared with scansorial and terrestrial opossums. On average, the tail length of arboreal forms is approximately 1.4-2 times the body length, whereas in more terrestrial forms, the tail is approximately equivalent to body length, and this relationship appears consistent across numerous didelphid species (Delciellos and Vieira, 2009a). A ratio of

12 average fiber CSA to average tail length, or ‘tail function score’ (TFS), provides another quantitative measure for which to better classify the arboreal, scansorial, or terrestrial habits of didelphids based on their tail properties. If muscle fiber CSA increases relative to increases in tail length, then TFS should be low in arboreal species, intermediate in scansorial species, and high in terrestrial species. Using fiber CSA (average across all MHC isoform fiber types) from this study and similar data from our previous study (Hazimihalis et al., 2013), C. derbianus rank as the most arboreal (TFS = 57), followed by D. virginiana (TFS = 122), and then M. domestica as the most terrestrial (TFS = 346). These rankings agree with previous reports involving locomotor behavior measurements in Caluromys (Lemelin, 1999; Cunha and Vieira, 2002), Didelphis (Jenkins, 1971; Delciellos and Vieira, 2006), and Monodelphis (Lammers and Biknevicius, 2004; Schmitt et al., 2010), and their individual locomotor habit descriptions (Eisenburg and Wilson, 1981; Charles-Dominique, 1983; Nowak, 1999). That said, the lower intermediate range of TFS values for D. virginiana is somewhat surprising given their mildly scansorial habits (Eisenburg and Wilson, 1981). Virginia opossums lose the ability of tail-hanging with maturation, and adults tend to be awkward climbers that only clamber on large diameter substrates (McManus, 1970), where their semi-prehensile tail is of little use. It would be helpful to evaluate muscle fiber type and architecture in didelphids that spend nearly equal time between the forest floor and the rainforest understory (or canopy) to validate the use of our TFS. Future studies are aimed at relating caudal muscle structure and function in more scansorial genera that frequently use the forest understory (e.g., Marmosa, Marmosops, Philander). Likewise, similar data for the tails of non-human primates with a range of tail prehensility would be informative. Although comparative fiber typing data do not currently exist for their caudal muscles, the available evidence indicates caudal muscle fibers are relatively large in primate groups with prehensile tails (e.g., Ateles and Cebus) (Organ et al., 2009). As fiber CSA is observed to increase with faster fiber types, we speculate that species of non-human primates with fully-prehensile tails would also express primarily fast MHC-2A and 2X isoforms and have low TFS values.

Prehensile tail structure

13 Although a detailed understanding of the structure of prehensile tails is lacking, dissection of the tails from species in this study point to two structural differences between fully-prehensile and semi-prehensile tails of didelphids and those of non-human primates. First, a segmented FCL spans the entire length of the tail in both C. derbianus and M. domestica, whereas in arboreal primates with prehensile tails (e.g., Cebus), the belly of FCL is short, spanning only the proximal region of the tail (Lemelin, 1995; Organ et al., 2009). Second, the architecture of caudal muscle tendons is variable, and a relationship can be made with locomotor or adaptive behaviors. In C. derbianus and arboreal primates, the tail has long tendons that, (i) attach to a prominent ventral flexor tendon, (ii) run parallel to the muscle belly, and (iii) span multiple vertebrae (2-3) to insert on the ventrolateral aspect of the cranial end of a caudal vertebrae (Organ et al., 2009; Organ and Lemelin, 2011). In M. domestica, the caudal muscle tendons also attach to a ventral flexor tendon, but they are oriented at acute angles, and span the length of a single vertebrae to insert on the ventral surface (cranial end) of each caudal vertebrae. Interestingly, hemal arches, V-shaped bony projections located ventrally between each articulating pair of caudal vertebrae (see Fig. 1), are observed in arboreal primates with fully-prehensile tails (Organ and Lemelin, 2011) and both species of opossums in this study. The presence of hemal arches along the entire length of the tail allows for increased area of muscle attachment, thus increasing muscular control of the tail during specialized arboreal maneuvers (Organ, 2010), and possibly fine control of the tail for manipulation of nesting materials in terrestrial didelphids. In particular, the segmented FCL and its short, oblique tendons in the semi-prehensile tail of M. domestica may increase the moment arm of the FCL allowing a greater length of the tail to be used for nest building tasks. Again, analyses of the prehensile tails of scansorial species (i.e., those capable of locomoting throughout the floor, understory, and canopy levels of rainforest strata) will allow us to continue to build on this and related hypotheses.

Conclusions and Future directions In conclusion, this study has provided answers to questions posed in our previous study with D. virginiana regarding the large distribution of hybrid fibers in caudal muscle and the fiber type distribution in the tail of a highly arboreal opossum. At present, we have consistently shown the expression of three primary MHC-isoforms (1, 2A, and 2X) in the

14 prehensile tail of three diverse didelphids. A large distribution of MHC-2A/X hybrid fibers has also been observed in all three species studied, regardless of locomotor habit or degree of tail prehensility. From these results (and similar data from the kangaroo), we conclude that an abundance of hybrid fibers may be commonplace in marsupial caudal muscle. Moreover, the novel fiber type data presented for C. derbianus support for our hypothesis that arboreal didelphids have an overall faster caudal muscle composition compared to more terrestrial forms. A significantly higher percentage of slow, oxidative MHC-1 fibers in terrestrial opossums further suggests that the MHC-1 isoform is important for scansorial/terrestrial tail-function, and this difference provides additional evidence of an evolutionary fast-to-slow fiber type shift in didelphids. Quantification of caudal muscle architecture is also necessary for more detailed structure-function comparisons among didelphids, and these data would be important for evaluation of convergent evolution between arboreal didelphids and non-human primates.

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26 APPENDIX Introduction Origin and Speciation Limitations of the environment can pose numerous challenges to animals inhabiting a specific habitat or region and can also require significant morphological specializations. The means by which animals adapt to these selective pressures are commonly observed to be species-specific and may often be mimicked by animals co-inhabiting the same habitat. Understanding the constraints associated with native habitats along with the ancestry of a species is a primary step in determining the reasoning for the observed morphological features. Regions rich in resources such as the rainforests of Central and South America can provide an environment for increased species diversity. Although, while certain environmental factors can be strong forces for determining species richness, the specific history and physiographic characteristics of a region produce notable differences in mammalian assemblages and dictates how they respond to environmental gradients (Hortal, 2008). There are many environmental and physiographic factors that must be considered in any global region. For example, temperature consistency and availability of water are critical factors for species richness. The inclusion of non-climatic influences associated with the evolutionary history and fauna compositions of the region are significant factors as well (Qian, 2010). The neotropic rainforests of Central and South America maintain a diverse assemblage of mammals by receiving an annual average rainfall of 4 m (McDade, 1994) and maintaining a constant temperature range between 20-32ºC (Clark, 2003). The consistent wetness of these mirco-environments provides for diverse flora in which co-inhabiting mammalian species have developed morphological features and strategies in order to maneuver and utilize resources to increase their overall fitness. Didelphid marsupials (opossums) are a prime example of mammalian diversity and specialization in these neotropical regions. Didelphidae is the family of opossums inhabiting the Americas representing 19 genera with more than 100 species, ranging from the southern-most regions of Argentina to southern Canada (one species in North America: Didelphis virginiana) (Nowak, 1991; Hershkovitz, 1997). Didelphids are native to southern South America and represent the largest order of marsupials in the western

27 hemisphere. The appearance of marsupials originated in the Cretaceous-Tertiary Boundary when marsupials and placental mammals (eutherians) diverged from a common mammalian ancestor (Archibald, 1982; Case and Woodburne, 1986; Caroll, 1997). After this evolutionary divergence, these marsupials migrated northward and with aid from the Great American Interchange (65 million years ago), moved into Central and North America (Pascual and Jaurequizar, 1999; Cozzuol et al., 2006). This movement of marsupials throughout the Americas provided the opportunity for increased speciation within the Didelphidae family. However, varying limitations of resources throughout the Americas and the flexibility of both their diet and reproduction strategies determined the success rate of colonization for these invading marsupials. For these reasons, only the species D. virginiana, a derived terrestrial opossum, survived to inhabit North America (Nowak, 1999; Hershkovitz, 1997). Overall, the climatic conditions and abundance of resources found in South America allowed for far greater speciation (over 85 currently recognized species), and most genera became successful by utilizing the natural resources and a multitude of available niche space (Nowak, 1999; Hershkovitz, 1997). Speciation in opossums is evident by the phenotypic characteristics each genera demonstrate. Basic traits, such as fur coloration and thickness, vary in opossums depending on the region they inhabit. Species occupying the northern boundaries of the didelphid range commonly display much larger body sizes, possess a thick underfur, and have a lighter fur coloration with black tips (Grzimek, 1990; Nowak, 1991; Vieira, 1997; Hershkovitz, 1997). These features contrast southern didelphids, which are smaller in body size, have sparse underfur, and usually exhibit medium to dark shades of brown colored furs. Slight differences are also observed in the feet, as northern didelphids commonly show shades of light gray or white (Allan et al., 1901), whereas southern species do not display notable color variation between body fur and their feet. The only feature pertaining to the fur pattern shared by all opossum species, unbiased to region, is the growth of white cheek hairs used for sensory information (McManus, 1970).

Morphology All didelphid marsupials evolved from a common arboreal ancestor (Szalay, 1994; Lemelin, 1999; Jansa and Voss, 2000; Voss and Jansa, 2009). This shared link between

28 species has led to a number of morphological features that strongly support evidence of arboreal origins. (1) Opossums prominently display a much wider lateral femoral condyle when compared to the medial femoral condyle, and this size difference coincides with the femoral anatomy of marsupials with arboreal ancestry (Muizon and Argot, 2003). The development of a broader lateral femoral condyle increases the mechanical advantage for muscles important in arboreal locomotion and climbing. Arboreal species use the m. iliacus and m. psoas major to flex, externally rotate, and adduct the hindlimb at the hip joint (Flores, 2009; Muizon and Argot, 2003). (2) All opossums possess a powerful opposable hallux on their hindfeet (Muizon and Argot, 2003). This feature is a prominent indicator of arboreal specialization as observed in many arboreal mammals including ancient lineages and derived primates (Lemelin, 1999; Schmitt and Lemelin, 2002; Schmitt et al., 2010). (3) Segmentation of appendages has also created adaptive changes in limb function in opossums and these limb posture modifications help distinguish them from other mammalian clades. The evolution of the mammalian limb is generally characterized by a shift from a two-segment, sprawling limb posture to a three-segment, parasagittal arrangement (Fischer et al., 2002). The parasagittal limb configuration allows for a crouched limb posture typical of smaller mammals (Fischer et al., 2002; Schilling, 2009) and requires that the proximal hindlimb segments be used for propulsion while the distal segments are used for changing center of mass (CoM) during locomotion (Fischer, 1994). The crouched posture also allows opossums to decrease the distance between the CoM and the substrate, thus increasing stability during both arboreal and terrestrial modes of locomotion Schmidt, 1994; Schmitt and Lemelin, 2002; Lammers and Biknevicius, 2004; Demes & Carlson, 2009; Schmitt et al., 2010). Metabolic costs in relation to locomotion in a habitat are important factors that determine morphological features of organisms. This suggests then that body size of opossums may determine the method of locomotion a species will display weighing the metabolic cost of maintaining body size and locomotion method. In actuality this is not the case as terrestrial species vary widely in body size and some genera of arboreal didelphids (e.g., Caluromys) are larger in body size than some terrestrial species (e.g., Monodelphis domestica). Primarily arboreal species, those that dwell, forage, and locomote in the forest canopy (Grizimek, 1990; Hershkovitz, 1997; Vieira, 1997;

29 Delciellos and Vieira, 2006, 2009), do not exceed a body size much larger than Caluromys because the metabolic cost of climbing and maneuvering arboreal substrates increases as body size increases. A higher accuracy method for determining characteristics of locomotion tends to focus on morphological specializations amongst species pertaining to their locomotion and also the unique niche that the species inhabits. All opossum species are primarily classified using the method of locomotion they use and secondarily segregated by the unique niche they inhabit. Opossums can be classified into two groups by their locomotor habit: arboreal and terrestrial. Because of their locomotor versatility many species in South America have become able to co-inhabit various niches, sharing territories and resources and thus insuring their survival. In the neotropic rainforests of South America, species of opossums naturally segregate themselves according to vertical strata within the forest in order to allow for coexistence and resource partitioning between species (Cunha and Vieira, 2002). Arboreal species tend to roam territories vertically, while terrestrial species remain largely on the forest floor and favor stereotypical horizontal paths for locomotion. Related to arboreal versus terrestrial locomotor habits, an important feature within the family Didelphidae is the range of body size observed. Differences in body size are more apparent than differences in body shape, though differences in shape do exist albeit they are subtle (Hildebrand, 1961; Creighton, 1984; Vieira, 1997; Lemelin, 1999; Carvalho 2005; Delciellos and Vieira, 2006). The body size of a species has an effect on its normal home range and thus is an important factor in determining the resources that can be attained. Body size also commonly determines the level of vertical strata that arboreal species in particular, can reach or occupy (Charles-Dominique, 1983; Grand, 1983; Vieira, 1997). Because of this, there are relatively small areas of niche overlap for sharing of specific resources between arboreal and semi-arboreal species at different levels of forest strata (Vieira, 2008; Cunha and Vieira, 2002). In order to reach higher strata within the forest a species requires the ability to efficiently climb and maneuver in an arboreal environment. Arboreal locomotion is very costly with respect to the required metabolic rate needed to produce movement both vertically and horizontally on small diameter substrates. For this reason, the average body size of arboreal species of opossum remains with significant consistency, much smaller than the average body size of their terrestrial relatives.

30 Opossums are considered to possess a general body plan for mammals. As stated previously variations in body shape are subtle and scarce between species of didelphid. One species that displays many of the important characteristics found throughout arboreal didelphids is Caluromys derbianus, commonly called the Woolly Opossum (Lemelin, 1999; Schmitt and Lemelin, 2002; Schmitt et al., 2010). The Caluromys genus is native to the neotropical forests of Central and South America and commonly spends the majority of its time moving and foraging on small, terminal branches high in the canopy, rarely descending to the forest floor (less than 1% of the time) (Hunsaker, 1977; Atramentowicz, 1982; Charles-Dominique, 1983; Rasmussen, 1990). Caluromys derbianus can only be found in Central America, ranging from southern Mexico near Veracruz, and continuing through western Columbia and northern Ecuador (Emmons, 1990; Reid, 1997). As well as being arboreal, C. derbianus is also nocturnal, prefers to remain solitary throughout much of its lifespan (Emmons and Feer, 1990; Reid, 1997), and exhibits a frugivorous diet occasionally feeding on insects, invertebrates, and gums (Charles-Dominique et al., 1983; Atramentowicz, 1982; Emmons, 1997; Reid, 1997). C. derbianus also favors low-lying, viny, and dense mature secondary rainforests, making nests from leaves or in abandoned tree hallows (Emmons and Feer, 1997; Reid, 1997). Terrestrial species differ from that of their arboreal relatives and therefore, terrestrial opossums have made functional adjustments to gait and posture to exploit different niches for survival. For example, they counter instability on small substrates by changes in speed (Schmidt and Andre, 2010) and increased reliance on the forelimbs to support the body weight (Lammers and Biknevicius, 2004). Terrestrial opossums such as M. domestica also employ a lateral sequence walking gait (Lemelin et al., 2003; Lammers and Biknevicius, 2004). This gait is demonstrated when one hindlimb is in contact with a substrate the corresponding forelimb on the same side of the body (ipsilateral) is also touching the substrate; these corresponding limbs are not contacting the substrate when the opposite side fore and hind limbs are touching (Lemelin et al., 2003). While specific gait selection is a main difference that separates terrestrial species from arboreal, there are several physical distinctions as well. Size scaling in terrestrial didelphids varies widely from small body size (e.g., 90g: Monodelphis) to large body size (e.g., >2kg: Didelphis) and this wide range is possible due to frequent locomotion on various broad

31 substrates associated with maneuvering throughout a terrestrial environment (Ladine and Kissell, 1994). The digits of terrestrial opossums also possess a significantly lower prehensile ability (i.e., prehensility) when compared to arboreal species predominantly caused from shorter digits which commonly remain flat to a substrate during locomotion and only possess moderate grasping ability (Lammers and Biknevicius, 2004). The combination of a lateral sequence walking gait, larger body sizes, and less specialized digits cooperatively cause terrestrial opossums to have poor climbing abilities. Arboreal opossums have acquired unique features (both morphological and gait kinematics) that widely separate them from terrestrial forms. To be classified as an arboreal species, the animal must maneuver within and rarely leave the forest canopy (Cunha and Vieira, 2002). Arboreal locomotion requires a high level of resource consumption, and because of this, body size is constrained. In general, arboreal ability decreases as the body size increases. As a group, all arboreal didelphids have relatively low body mass (average range: 90-500g), in turn, a lower body mass helps to offset the high metabolic cost of arboreal locomotion. Specializations for arboreal habit are perhaps evident in the feet of arboreal opossums. Features including broad palm and sole breadth, increased claw length, and increased digit length relative to palm and sole breadth (determined as a ratio) indicate climbing specializations (Vieira, 1997; Lemelin, 1999). During arboreal locomotion, arboreal opossums employ a diagonal sequence walking gait, defined as while one fore limb is extended forward and contacting a substrate the opposite hind limb is also extended forward and touching the substrate; the remaining two limbs a fore and hind limb, on opposite sides of the body, are both extended posteriorly and contacting the substrate (Lemelin et al., 2003; Lammers and Biknevicius, 2004). This specialized gait significantly increases stability during both static and locomotor maneuvering. In an arboreal habitat the majority of time is spent maneuvering and foraging between small diameter substrates and terminal branches located at various heights above the forest floor. As an animal approaches heights in excess of their respective body height, any error in foot placement or judgment in branch stiffness could be catastrophic (i.e., falling) and will most likely result in severe injury or death. For this reason, arboreal species have highly developed prehensile (grasping) feet with relatively longer digits than terrestrial species for improved substrate stability (Lemelin, 1999). In

32 addition, the diagonal sequence gait is also observed to be significantly more exaggerated during arboreal locomotion with increased distance between placements of the limbs (Lemelin et al., 2003). Functionally, this allows the hind foot to remain grasping the substrate for a longer time period throughout the cycling of limbs during locomotion and thereby improving stability. The importance of such arboreal specializations is clearly illustrated by the terrestrial species, M. domestica, which possesses features common to all opossums (e.g., prehensile feet and tail), but lacks the degree of specialization seen in arboreal forms. During climbing behaviors, M. domestica has been shown to experience extreme difficulty remaining on small diameter (<7mm) substrate (Lemelin et al., 2003). Monodelphis experiences these difficulties from the lack of long, significantly prehensile digits and other arboreal specializations such as broad palm and sole breadth as well as the use of a lateral sequence walking gait rather than a diagonal sequence walking gait. Further related to their gaits and stability, arboreal species also have lower peak vertical forces (substrate reaction force) compared to terrestrial species, this is accomplished by branch walking using slower speeds (Schmitt, 1994, 1999, 2003b; Schmitt and Lemelin, 2002, 2005; Lammers and Biknevicius, 2004). When transitioning between branch supports, arboreal opossums employ the use of a crouching maneuver when landing on a subsequent support (Schmitt and Lemelin, 2002), which disperses vertical force over a longer period of time, and results in reduced peak vertical forces. This maneuver is frequently used to lower branch oscillations when landing on or transitioning over branches, which in turn increases stability on small diameter substrates (Demes et al., 2009; Schmitt, 1999; Lammers and Biknevicius, 2004). Lastly, the prehensile tail also plays an important functional role in arboreal locomotion. Arboreal opossums use the tail as an insurance factor against falling and as an instrument to reach hanging food resources such as fruit. These tasks are accomplished through various locomotor and postural behaviors such as feet hanging, bridging and cantilevering between terminal branches, tail hanging, and coiling the tail around substrates when not in motion (standing or branch sitting) (Schmitt and Lemelin, 2002, 2005; Lemelin et al., 2003; Youlatos, 2008). All of these specialized locomotor techniques are performed very rapidly and require short contraction durations and high force and power from the tail muscles. Bridging and cantilevering are two of three specialized maneuvers arboreal

33 opossums employ. Bridging is defined as transitioning between two horizontal substrates where the CoM is shifted above the hindlimbs and the tail is coiled to secure the posterior half of the body to the substrate in a tripod stance. The anterior half of the body is then moved onto the new substrate and the body weight is then carefully shifted to the forelimbs when positioned securely on the new branch. Cantilevering is a behavior used to maneuver between vertical and horizontal substrates, where the hindlimbs and tail are secured to the vertical substrate in a tripod stance with the forelimbs extended to the horizontal substrate. This allows the body weight of the animal to be slowly shifted from vertical substrate to the horizontal substrate. These two maneuvers allow for increased insurance and stability during arboreal locomotion whereas, if the transitioning substrate should fail the tripod stance of the hindlimbs and tail prevent the animal from a catastrophic fall. Lastly, the third specialized maneuver is tail hanging. Tail hanging is demonstrated by rapidly coiling the tail around a substrate and lowering the entire body below the substrate without any additional support from the limbs. Although tail hanging is relatively infrequent (Youlatos, 2008), the behavior is used primarily to acquire and manipulate food sources hanging from branches, and quickly catch a substrate when falling by latching on a branch with a coiling motion of the tail and then using the limbs to pull along the tail to return the body back onto the branch (Youlatos, 2008).

Prehensile Tail All opossums possess a relatively long, prehensile tail. To qualify as prehensile, the tail must have the ability to grasp or hold objects, and in some species (i.e., more arboreal opossums), have the capability to suspend the entire body. In general, opossums use their prehensile tail as a fifth appendage for the locomotor behaviors described above (e.g., bridging and cantilevering) (Lemelin et al., 2003; Youlatos, 2008), and for performing adaptive tasks such as object manipulation and transportation of offspring (Macrini, 2004). For example, during nest construction, the prehensile tail is used to manipulate leaves and small branches to develop and form the nest (Gardner, 1973; Macrini, 2004). Although a common feature among all didelphids, the range of functionality of the tail differs markedly depending on the species and habitat. Therefore, opossums can be further distinguished by their degree of tail prehensility as (i) possessing a fully

34 prehensile tail, and (ii) possessing a semi-prehensile tail. Species with fully prehensile tails are capable of manipulating objects with extreme control, and have the ability to maneuver in an arboreal environment on small diameter substrates and terminal branches. Species with semi-prehensile tails have some ability to use their tail to slow the vertical descent between shortly-spaced substrates (i.e., one-half body length), but primarily use their tails for added assistance in carrying materials and offspring (Grzimek, 1990). The majority of opossums possessing a fully prehensile tail are found in Central America and South America. The neotropical rainforests of countries in these regions of the Americas offer dense canopies and many resources at higher elevations (Grzimek 1990; Nowak, 1999; Hershkovitz, 1997). This type of habitat establishes a selective pressure that favors arboreal traits such as a fully prehensile tail. The tail lengths of opossums also vary with species and region, but usually display a directly proportional relationship with body length. For example in the genus Didelphis, the tail length is determined to be equal to the entire length of the head and body giving them a relatively low tail length-to-body length ratio (~1.0) when compared across all species of opossum (Vieira, 1997). In the terrestrial species M. domestica, average tail length is 8 cm, which is approximately one half of the body length (15 cm), giving this species a very low tail length-to-body length ratio (~0.5). This ratio is even lower for Monodelphis females as they display a slightly shorter tail length (7 cm) than males (Macrini, 2004; Fadem and Rayve, 1985). The low tail length-to-body length ratios of terrestrial opossums is in stark contrast to the high ratios observed for arboreal didelphids that have exceedingly long tails relative to their small body sizes. As a comparative example, the arboreal species C. derbianus has an average tail length of 45 cm and a body length of approximately 20 cm, yielding a tail length-to-body length ratio of 2.25 (Delciellos and Vieira, 2006). Tail muscle architecture Detailed descriptions of didelphid caudal muscle architecture are presently unavailable. However, observations from the tail of the Virginia opossum (Kirsch, 1973; Hazimihalis et al., 2013) and personal observations made in relation to this study (unpublished data), provide a foundation for which to compare tail structure across species of Didelphis, Monodelphis and Caluromys. Initially, it is helpful to indicate that the relative shape of proximal, transitional, and distal vertebrae remain similar across the three species

35 observed in this study. However, while shape remains constant the exact number of caudal vertebrae varies widely across species of opossum. Terrestrial species M. domestica and D. virginiana display a range of 19-20 caudal vertebrae (Macrini, 2004) and 20-24 caudal vertebrae, respectively (Argot, 2003), whereas the arboreal C. derbianus displays 27-29 caudal vertebrae (unpublished data). Each species, whether arboreal or terrestrial, possess two main tendons that originate on the superior face of the sacrum and span the entire dorsal side of the tail to insert on the distal-most caudal vertebra. These two tendons are evenly placed along the dorsal side of the tail and are superficial to the main tail extensor m. extensor caudae longus (ECL) attached along the dorsal aspects of the caudal vertebrae. Another shared feature are two additional tendons, one on each side of the tail originating proximolaterally at the base of the tail and traversing the dorsolateral aspects of the tail to insert on the lateral face of the distal caudal vertebrae. These tendons separate of the inferior surface of the ECL and the superior surface of the main tail flexor m. flexor caudae longus (FCL). The four tendons described converge at the distal end of the tail to form one common tendon investing the tail tip. These tendons are referred to as the ‘dorsal extensor tendons’ (Hazimihalis et al., 2013: see Figure 1). Including the ECL and FCL, there are five caudal muscles that each of the three species observed have in common. On the dorsal side of the tail, m. caudofemoralis (CF) and ECL are the two extensor muscles (Kirsch, 1973). CF originates from both the greater trochanter of the femur and the posterior superior iliac spine to insert on the dorsolateral aspects of the proximal caudal vertebrae. Although it is considered a caudal muscle, the main action of CF is hindlimb abduction with a very limited role in tail extension. ECL originates on the superior face of the sacrum and spans the entire length of the dorsal tail traversing the superior surfaces of the transverse processes of each caudal vertebra to ultimately insert on the dorsal surface of the most distal caudal vertebrae. The distal most insertion of the ECL muscle becomes tendinous at the distal tip of the tail. On the ventral side of the tail, m. flexor caudae brevis (FCB) and FCL are two superficial flexor muscles, while m. intertransversari (IT) are the deep flexors that span each caudal vertebra. FCB is located at the proximal end of the tail and is situated deep to the CF and superficial to the FCL. It originates on the posterior portion of the iliac fossa

36 and inserts on the ventrolateral faces of the proximal caudal vertebrae (insertion is where the hairless portion of tail begins with respect to surface anatomy). FCB also inserts on a ‘flexor tendon’ along the ventral side of the tail (Hazimihalis et al., 2013; see Figure 1) giving this relatively short bellied muscle a long moment arm for facilitating the FCL with tail flexion. It was noted that the muscle mass of FCB in a specimen of C. derbianus was smaller than that observed in either M. domestica or D. virginiana. However, the importance of this difference in FCB size and how it relates to tail function between arboreal and terrestrial species is not understood at this time. The FCL originates on the inferior surface of the sacrum and spans the entire length of the ventral tail traversing the inferior surfaces of the transverse processes of each caudal vertebra to ultimately insert on the ventral surface of the distal-most caudal vertebrae. Like the ECL, FCL also becomes tendinous at the distal tip of the tail. Lastly, the intertransversari group are unique muscles that span the entire length of each vertebra (Organ, 2010). The origin of each IT muscle is located at the distal end caudal vertebrae and has an insertion onto the proximal end of the transverse process of the next sequential vertebra. The IT muscles have an important role in tail prehension as function of IT allows for flexion at each vertebral segment articulation helping enable the tail to coil around substrates. All five muscles described have either parallel or unipennate muscle fiber architecture. In each type of muscle architecture displayed, relatively long fascicles the muscle belly from origin to insertion. Much like the difference in FCB size observed between the arboreal and terrestrial species, the arrangement of the ‘ventral flexor tendons’ or aponeuroses also differs among species. In D. virginiana, the ventral aponeurosis is structured as a continuous tendon sheath spanning the entire length of the tail, providing for continuous muscle attachments (Hazimihalis et al., 2013). In C. derbianus, the ventral aponeurosis does not form a sheath, and is instead is arranged as multiple flexor tendons orientated parallel to the muscle fascicles along the entire length of the tail (unpublished data). In M. domestica, the ventral aponeurosis is again different than the two previous descriptions. The ventral aponeurosis in M. domestica is also not a sheath, but rather comprised of multiple flexor tendons similar to the condition in C. derbianus, however, the flexor tendons have an orientation of approximately 20º (unpublished data). The orientation of each of these tendons is more along the midline of the ventral tail where

37 bellies of FCL muscle meet, and each tendon attaches on the proximal end of each caudal vertebrae. The observed variation tail muscle structure may indicate specialization unique to tail function in each species.

Myosin Heavy Chain Isoforms The process of identifying and describing the properties of muscle fibers is known as muscle fiber typing. Historically muscle fibers were broadly classified as either oxidative or glycolytic based on their metabolic properties (Wattenburg and Leong, 1960; Navikoff et al., 1961), and as slow and fast based on ATP incubation reactivity (myosin ATPase assay) in acid and alkaline conditions, respectively ( Guth and Samaha, 1970; Brooke and Kaiser, 1970). The combination of these two analyses is referred to as fiber type histochemistry. Histochemical classification has served as the base standard of fiber typing for several decades, however, the technique is limited by the inability to identify specific myosin heavy chain (MHC) isoforms. As such, histochemical classifications have been restricted to four general muscle fiber ‘types’ in mammals: Type I, Type IIa, Type IIx(d), and Type IIb. Type I fibers are slow-contracting and oxidative in their energy metabolism. Type II fibers are generally fast-contracting and range from oxidative-to-glycolytic energy metabolism, with Type IIa being the slowest and most oxidative and Type IIb being the fastest and most glycolytic (Sciote and Rowlerson, 1998; Kohn et al., 2011; Zhong et al., 2001). Identification of specific MHC isoforms is important because these protein sub-units determine the contractile properties of muscle fibers and by extension, determine the contractile performance of entire muscles (Sciote and Rowlerson, 1998; Kohn et al., 2011; Zhong, 2001; Sokoloff, 2010). Three analyses are frequently used to properly identify individual MHC isoforms, including Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE), Immuno-Blotting (Western Blot), and Immuno- histochemistry (IHC). Analysis with SDS-PAGE uses electrophoresis to separate MHC within a polyacrylamide gel based on the molecular weight of each isoform, with the lighter isoforms migrating through the gel farther than the heavier isoforms (Talmadge and Roy, 1993; Mizunoya et al., 2008). The Western Blot enables the MHC isoforms separated using SDS-PAGE to be transferred onto a PVDF membrane that is

38 subsequently incubated with monoclonal antibodies (mAbs) specific to individual MHC isoforms, allowing precise identification and conformation of each individual isoform found using SDS-PAGE (Kohn et al., 2007, 2011). IHC analysis identifies the MHC isoform composition expressed in single muscle fibers by reacting serial sections of fresh muscle tissue against mAbs specific to a MHC isoform allowing individual fibers to be classified based on their immunospecificity (Sciote and Rowlerson, 1998; Lucas et al., 2000; Spiegel et al., 2010a). Specifically, the MHC isoforms expressed within a muscle determine the rate at which ATP is hydrolyzed by the myosin ATPase (i.e., myosin heads) and thus the maximum shortening velocity (Vmax) and power output of a muscle fiber (Rowlerson, 1994; Schiaffino and Reggiani, 1996). There are four types of MHC isoforms that are commonly expressed in mammalian skeletal muscles (listed in order of the lowest-to- highest Vmax): MHC-1, MHC-2A, MHC-2X and MHC-2B (Toniolo 2005; Kohn et al., 2011; Zhang et al, 2010; Zhong, 2001). The four histochemical fiber types (by their metabolic properties and myosin ATPase rate) are intended to correspond to MHC muscle fibers expressing the specific isoforms of the same name. MHC-1 isoform fibers isoforms are slow, oxidative and produce low force that can be sustained without fatigue. MHC-2A isoform fibers are fast, highly oxidative and produce higher force and power than MHC-1 fibers. MHC-2X isoform fibers are fast, moderately oxidative/glycolytic and produce higher force and power than MHC-2A fibers. Lastly, MHC-2B isoform fibers are fast, highly glycolytic and produce the highest amount of force and power of any fiber type, but fatigue easily (Schiaffino and Reggiani, 2011; Sciote and Rowlerson, 1998; Kohn et al., 2011; Zhong et al., 2008). Therefore, the more oxidative fibers (MHC-1 and MHC-2A) can produce force over a long duration whereas more glycolytic fibers (MHC- 2X and MHC-2B) can produce high force for a short duration. Marsupials have been shown to express each of the four MHC isoforms in their limb muscles (Lucas et al., 2000; Zhong et al., 2001). This finding is significant as marsupials do not appear to show the same body size dependence on MHC isoform expression as observed in eutherian (i.e., placental) mammals. Only relatively small eutherian mammals, including most members of the Family Rodentia and Leporidae express fast, glycolytic MHC-2B fibers in their limb muscles (Bottinelli and Reggiani, 1991; Galler et

39 al., 1997; Oleg, 2004). However, MHC-2B are expressed in extraocular (and possibly laryngeal) muscles of numerous mammals regardless of body size (Toniolo et al., 2005; Rhee and Hoh, 2008; McLoon, 2011). With the exception of these specialized fast muscles, the general rule of muscle shortening velocity scaling is that as mammals increase in body size their muscles become slower contracting and less powerful (Rome et al., 1990; Seow and Ford, 1991; Pelligrino et al., 2003; Marx et al., 2006; Toniolo et al., 2007). Marsupials being a basal mammalian lineage (metatherians), retain expression of all four MHC isoforms at least in the skeletal muscles of the Australian forms (e.g., kangaroos, wallabies, possums, etc.) which display a variety locomotor habits (Zhong et al., 2001). For example, kangaroos and wallabies are saltators and hop as their mode for running, while possums and a variety of marsupials climb and maneuver in trees during arboreal locomotion. Didelphid marsupials walk and trot terrestrially (e.g., Didelphis and Monodelphis), and walk and clamber while moving arboreally (Schmitt and Lemelin, 2002; Youlatos, 2008). At this time, it is not known if the South American forms also express fast MHC-2B fibers in their skeletal muscles. The method of locomotion an animal uses directly influences the composition of slow and fast skeletal muscle fiber types, and thus their muscle contractile properties. Muscles are specialized to meet the performance needs of the species within its native habitat. Specifically, the expression of MHC isoforms in muscle fibers reflects the functional specialization of muscles. Specializations of muscles include properties such as muscle architecture (pennation), physiological cross-sectional area (PCSA), maximum shortening velocity (Vmax), and maximum power output (W). These properties define the amount of force and work a muscle can produce and sustain. In addition, performance requirements for force, work and power will vary with the constraints of the niche that a species occupies. Therefore, specializations of muscles directly reflect the relationship between evolution and functional behavior, understood as a structure-function relationship, and also implies the metabolic cost associated with a functional behavior (Zhong et al., 2001). The ability to minimize the metabolic cost of muscle force is an important factor for survival, and is directly related to the evolution of a species. Few studies have investigated fiber types (Peters et al., 1984; Hansen et al., 1987) and MHC isoform composition (Sciote and Rowlerson, 1998; Hazimihalis et al., 2013) in

40 didelphid marsupial skeletal muscles. Fiber type analyses of limb muscles in D. virginiana have been limited to the use of mATPase histochemistry to broadly classify fiber types as either slow or fast. In addition to limb muscles, Hansen et al. (1987) further reported the tail of D. virginiana to be composed of 25% slow (Type I) and 75% fast (Type II) fibers. In our previous study of MHC we used a combination of mATPase histochemistry, SDS-PAGE and IHC techniques to determine isoform composition in the caudal muscles of the tail of D. virginiana and found 21% MHC-1 fibers, 4% MHC-1/2A hybrid fibers and 75% MHC-2A/X hybrid fibers (Hazimihalis et al., 2013). These data are in accord with the results of Hansen et al. (1987), however, provide important detail about the sub-divisions of fast MHC fiber types needed to more accurately interpret tail function. Beyond the Virginia opossum, fiber typing in didelphids has only otherwise been attempted in the species M. domestica, where MHC isoform fiber types in limb muscles were determined by IHC (Sciote and Rowlerson, 1998). The hindlimb muscles in M. domestica were indicated to possess four MHC isoforms including 1, 2A, 2X, and an isoform which reacted similar to MHC-2B, but could not be confirmed as a pure fiber type. Interestingly, like the tail of D. virginiana, the MHC composition in the hindlimb of M. domestica shows a majority of fast 2A/2X isoforms (Sciote and Rowlerson, 1998). Caudal muscle fiber typing Relatively little is understood about structure and function of caudal muscles in mammalian tails. Limited studies of caudal muscle fiber typing have largely focused on rodents and felines, and have primarily relied on mATPase histochemical classification of muscle fibers. Furthermore, with the exception of one study (Hazimihalis et al., 2013), no other investigation has identified MHC isoform fiber types in the specialized prehensile tails of some marsupials and non-human primates. Caudal muscle fiber typing in rodents indicates that the rat possesses a large distribution fast (Type II) fibers (Ovalle, 1976), and similar results have recently been shown for the mouse (Zhang et al., 2010). Muscle properties data for rats and mice are often viewed as ancestral or basal model for comparison with other species of placental (eutherian) mammals. This is because the evolution of small eutherian mammals has been marked by changes in muscle function with modified locomotor postures, and consequently, changes in fiber type composition, especially in the paravertebral and caudal muscles (Schilling, 2009). Combining muscle

41 data from mice, rats and other small eutherians (e.g., shrews), it has been hypothesized that the ancestral condition for muscle fibers in the mammalian tail is to be fast- contracting (Schilling, 2005, 2009). Consistent with this hypothesis, the domestic cat has been shown to have a large distribution of fast (Type II) fibers throughout the six caudal muscles comprising the tail (Wada et al., 1994). For example, the muscles involved in precise movements using tail flexion and abduction are composed of >80% fast fiber types. With regard to feline locomotion, the tail is held in an upright or erect posture by the ECL, and this muscle was reported to have a relatively large percentage (38.6%) of slow (Type I) fibers (Wada et al., 1994). Muscles involved with maintaining tail and or body posture would be expected to be composed of predominately slow MHC-1 fibers. In a similar way, deep paravertebral muscles that function as axial stabilizers, in addition to some proximal caudal muscles in a number of small eutherians, have a highly oxidative fiber type composition indicating a role in prolonged contractions (Schilling, 2009). Evolutionary changes in muscle function and composition of fiber types presently observed in the tail of eutherians has direct implications for interpretation of caudal muscle fiber types in didelphid marsupials. Assuming a fast (more glycolytic) fiber type composition is the ancestral trait, and allowing for changes in tail function throughout evolution, terrestrial species of opossums are derived compared with their arboreal ancestors (Voss and Jansa, 2009) and therefore, shifts in MHC isoform expression are predicted to occur in caudal muscles. This phenomenon may be most evident in the highly derived species D. virginiana, a terrestrial scampering animal that uses its tail in an adaptive manner to carry nesting materials instead of an appendage for climbing (McManus, 1970). For this reason, it is possible that in the transitional region of tail, the main prehensile caudal muscle FCL was found to have a relatively large distribution of slow, oxidative MHC-1 fibers, instead of high percentages of fast fiber required for arboreal maneuvers (Hazimihalis et al., 2013). It is also possible for species to experience a loss of function in relation to a specialized muscle as a result of environmental changes throughout evolution. In this scenario, a species may be locked to an environment which no longer requires the use of a specialized structure and thus muscle may shift back to the ancestral condition of fast fiber types. As an example, kangaroos are extremely derived and isolated species of marsupials that can only be found in Australia, and yet all have a

42 common ancestor native to the rainforests of South America (Vandeberg and Williams, 2010). Extreme differences in environmental conditions between the rainforests of South America and the Australian outback signify a strong selection pressure for loss of prehensile function in the tail of kangaroos. Muscle fiber type composition in the tail of the eastern grey kangaroo (Macropus giganteus) substantiates a change in tail function. A prominent tail extensor muscle was shown to composed of >90% fast fiber types (Spiegel et al., 2002). Of these fast fibers, MHC-2X and MHC-2B were the predominant isoforms expressed, and they were contained in fast hybrid fibers (Spiegel et al., 2010b). The large distribution of hybrid fiber types reported for kangaroo tails suggests a reversal to the ancestral condition (i.e., fast, more glycolytic fibers) in these Australian marsupials. Ontogeny of caudal muscle fiber types Examination of fiber types throughout ontogeny is another aspect to consider when evaluating changes in muscle fiber type resulting from alterations in function, and the adaptation of specialized structures. Briefly, muscle tissue is generated during embryonic development when myoblasts emerge from mesenchyme-borne precursor cells (Wirth- Dzieciolowska, 2011), and these myoblasts are then incited to exit the cell cycle and begin differentiation by specific extracellular signals (Rehfeldt, 2000). Myotubes are the precursors to muscle fibers (i.e., muscle cells) and must be formed prior to fiber formation. Myoblasts that have started differentiation, synthesize muscle specific proteins known as myogenic regulatory factors (MRFs) to start the formation of polynucleated myotubes; during myogenesis formation of muscle fibers from myotubes is controlled by genes activated by the presence of specific MRFs (Wirth-Dzieciolowska, 2011). Throughout the early stages of myogenesis and through the final stages of differentiation, myogenic genes are continuously influencing muscle fiber development. Notably, MyoD is the main family of genes influencing skeletal muscle development (Wirth- Dzieciolowska, 2011) where the MYF-5 gene controls early myoblast determination, and both the MYOG and MYF-6 genes are active during the final differentiation of myoblasts (Sakuma et al., 1999). The ongoing control of MYF-6 indicates a constant influence through the late developmental stages of muscle tissue. Muscle fibers have also been shown to undergo changes in fiber type when functional requirements begin to be placed on developing muscle tissue (Close, 1972). Ongoing development and alterations in

43 developing muscle tissue all occur within an Extracellular Matrix (ECM). This ECM is composed of materials required for myogenesis including macromolecules, glycoproteins, and proteoglycans (Frantz et al., 2010). The ECM also functions in cell differentiation by relaying biochemical and biomechanical signals to developing muscle tissue through the use of substances called integrins (Van der Flier and Sonnenberg, 2001). Through this process of differentiation, it becomes important to identify MHC isoform expression across adolescence to maturity to understand how MHC isoform fiber types are influenced by extracellular biochemical and biomechanical stimuli. Ontogenetic studies of caudal muscle fiber types are very limited. Changes in histochemical fiber types with development have been reported in the tail of rats. At birth, histochemical analysis on cross-sections of the entire tail showed a nearly homogeneous distribution of fast, glycolytic fibers. At 21 days post-natal, the fiber type distribution changed to 72% of fast, glycolytic fibers while the remaining 28% of muscles fibers had intermediate staining patterns that could not be conclusively determined as solely fast or slow fibers (Ovalle, 1976). The changes in muscle fiber type were attributed to the development of nerve endings contacting the developing muscle fibers, which provided the necessary sensory information for intended tail function. Nerve-to-muscle interaction (i.e., strengthening synaptic connections) correlates developmentally with changes in muscle fiber type (Ovalle, 1976; Zhang et al., 2010). From these data, it is expected that a similar interaction between nervous tissue and caudal muscle tissue in a common terrestrial opossum (e.g., D. virginiana and M. domestica) will influence MHC isoform expression to optimize tail muscle contractile properties for object manipulation and offspring transportation functions. This change is likely to occur early in adolescent development. In particular, M. domestica has been observed to lack nest building capabilities prior to 3 months of age and after this critical period of development, nest building behavior is demonstrated (John Vandeberg, pers comm). This early absence of tail function may be attributed to the immature development of caudal muscle fibers or incomplete expression of functional MHC isoforms.

44 Objectives and Hypotheses MHC isoforms are the primary determinants for muscle contractile properties and in turn, proper classification of muscles is based on the type of MHC isoform expressed in the muscle fibers. Studies of fiber types and function are well documented for the limb muscles of mammals, however, few investigators have worked to describe the contractile properties of distal extremity muscles such as those in the mammalian tail. Furthermore, the rare studies that have examined caudal muscles were mostly limited to histochemical analyses which lacked the ability to identify specific MHC isoforms (Ovalle, 1976; Hansen et al., 1987; Wada et al., 1994). Didelphid marsupials represent an excellent model for expanding our understanding of the relationship between MHC isoforms and muscle function, as all opossums display a prehensile tail, as well as a range of locomotor behaviors from arboreal to terrestrial across species. Arboreal species of opossum spend the majority of their time foraging and moving throughout the forest canopy high above the forest floor (Vieira and Cunha, 2008). In these opossums, the tail is frequently used as an additional appendage for performing a number of quick climbing maneuvers such as cantilevering and bridging to transition between branches (Schmitt and Lemelin, 2002). Arboreal opossums can also use the prehensile tail to fully suspend their body below a branch to gather and manipulate food (Schmitt and Lemelin, 2002; Youlatos, 2008). In general, these types of arboreal maneuvers require rapid production of muscle force. The caudal muscles in arboreal species used for quick maneuvering (i.e., cantilevering and bridging) on tree branches and fall prevention are hypothesized to express MHC-2X and MHC-2B isoforms as these faster fiber types can generate the high force and power required to perform these specialized tail functions. In contrast, the prehensile tail of terrestrial species is most commonly used for carrying and manipulating nesting materials such as sticks and leaves, and also for maneuvering offspring (Layne, 1951; Unger, 1982). These adaptive tail functions involve sustained slow muscle contractions of low force. Caudal muscles used in nest building and offspring manipulation tasks are hypothesized to express MHC-1 and MHC-2A isoforms as these slower fiber types would be most efficient in performing these tail functions. The distribution of MHC isoforms expressed in the caudal muscles should be

45 adapted to meet the contractile requirements for the specific tail functions observed between arboreal and terrestrial species of opossums. The results observed will be essential to our understanding muscle specialization and species adaptation for survival. This study seeks to answer three research questions: (i) what is the MHC isoform composition in the caudal musculature of terrestrial (Monodelphis domestica) and arboreal (Caluromys derbianus) opossums, and how does it relate to tail function, (ii) has MHC isoform composition in the tail musculature of didelphids changed with adaptive use and evolution, and (iii) does MHC isoform composition of caudal muscles change with normal ontogeny? Muscle fiber typing results from the tail muscles of the arboreal C. derbianus will represent the ancestral condition for opossums, as all species are believed to have evolved from a common arboreal ancestor (Szalay, 1994; Voss and Jansa, 2009). Importantly, this study extends our previous study (Hazimahalis et al., 2013) where it was found that the prehensile tail of the terrestrial D. virginiana is composed of MHC-1 and MHC-2A/2X isoforms. The lack of fast MHC-2B fibers emphasizes the slower, more oxidative contractile properties of the caudal muscles in this terrestrial species. These findings support the hypothesis that terrestrial species require slow oxidative muscles to perform object manipulation tasks. Furthermore, discovering a difference in MHC isoforms from fast, glycolytic in an arboreal species to slow, oxidative in terrestrial species would support the hypothesis that fast caudal muscles are the ancestral condition from which MHC isoform composition changed as adaptive functions of prehensile tails have evolved.

46 Table 1. Morphometric data for experimental animals.

Body Hallux Head-Body Tail Opossum Sex mass length length length Relative Tail (g) (cm) (cm) (cm) Length C. derbianus Cd76 F 370 1.0 24 43.5 1.81 Cd77 F 395 1.2 28 47.0 1.68 *Cd79a F 220 1.0 21 40.0 1.90 Cd79b M 310 1.0 21 42.5 2.02 Cd79c M 410 2.0 27 45.0 1.66 Cd710 M 515 1.5 27 46.0 1.70 370 (99.3) 1.3 (0.4) 25 (3.1) 44 (2.5) 1.8 (0.1)

M. domestica Md1 M 126 1.5 7.0 9.5 1.36 Md2 F 76.6 1.4 6.9 7.5 1.09 Md3 F 83.3 1.9 6.8 7.5 1.10 Md4 F 86.5 1.4 6.5 7.2 1.11 Md5 M 132 1.6 7.6 8.3 1.09 101 (25.9) 1.5 (0.2) 7.0 (0.4) 8.0 (0.9) 1.1 (0.1) Values in bold are mean and (s.d.) in parentheses *juvenile; all other individuals were adults

47 Table 2. Monoclonal antibodies (mAbs) used for western blot and IHC analyses and their MHC isoform reaction specificity against the caudal muscle from C. derbianus and M. domestica.

Antibody Slow, MHC-1 Fast (MHC) MHC-2A MHC-2X MHC-2B S58 ++ MY32 + F18 + 2F7 + SC71 ++ + 6H1 +/- BF-35 ++ ++ +/- BF-F3 - (+) positive reaction against muscle MHC antigen (++) strong positive reaction against muscle MHC antigen (+/-) weak reaction against muscle MHC antigen (-) no reaction against muscle MHC antigen

N/A 82.0 (4.3) 2A/X domestica.

. M 1.5 N/A (1.6) 1/2A Distal and and

us 1 n N/A 15.8 (4.9) a

C. derbi 89.8 75.5 (0.8) (1.7) 2A/X of

tails -- 2A 7.0 (2.0)

1.9 0.6 (0.6) (0.5) Transitional 1/2A

1 8.1 for each species 16.8 (0.6) (0.7)

-- 0.03 2X/B (0.01)

across all animals

s ) 88.7 68.1 (3.1 (5.4) 2A/X

(%) of MHC isoformthe in fiber types

)

s.d. tail region -- 2A all 12.2 (2.6) r Proximal

istributions istributions d 3.1 2.2 (1.7) (2.3) 1/2A

1 8.2 18.4 (1.8) (4.0)

3945) 7968) Species n= n= total number of fibers counted fo ( ( C. derbianus C. M. domestica M.

Table 3. Regional mean mean 3. Regional Table n= in parentheses is the standard deviation (

49 Table 4. Mean percentage distributions (%) of MHC isoform fiber types in the caudal muscle FCL of C. derbianus, M. domestica, and D. virginiana.

MHC Isoform Fiber Type Species 1* 1/2A* 2A 2A/X 2X/B n C. derbianus 8.2 (1.3) 2.6 (1.4) -- 89.2 (2.3)† -- 3945

M. domestica 17.0 (3.6) 1.3 (1.5) 9.0 (3.8) 75.2 (7.0) 0.03 (0.01) 7968 D. virginiana 23.1 (1.0) 4.4 (0.6) -- 72.5 (0.8) -- 6640

F-value 86.2 44.3 160.7 P <0.001 <0.002 <0.001 n= total fibers counted for percent fiber type calculations in parentheses is the standard deviation (s.d.) * denotes significant difference among all species †denotes a significant difference D. virginiana data from Hazimihalis et al. (2013).

N/A 2A/X 2365.6 (749.4)

N/A 1/2A 1923.5 Distal

(1083.4)

1 N/A 1610.5 (784.4)

M. domestica.

and and 2A/X 2555.3 3122.7 (742.9) (735.0)

-- 2A 2896.3 (752.9)

C. derbianus

A Transitional 1/2 1307.0 2293.7 (305.6) (752.9)

1 1299.1 1852.7 (900.2) (1020.3)

for each species -- 2X/B 4455.1 (596.3)

2A/X 4380.1 3123.7 (924.6) (1359.2) across all animals

) of MHC isoform fiber types in the tails of) of MHCtails isoformthe in fiber types -- 2 2A m

2587.2 (711.6) ) µ Proximal tail region s.d.

all

for 1/2A 2741.2 1625.6 (819.3) (514.6)

measured

1 2098.4 2251.9 (1020.3) (1152.2)

)

ianus Regional mean fiber CSA ( mean Regional 1227 3134) Species n= (n= ( C. derb C. M. domestica M. total number of fibers

Table 5. Table n= in parentheses is the standard deviation (

51 Table 6. Mean fiber CSA (µm2) of MHC isoform fiber types in the caudal muscle FCL of C. derbianus and M. domestica. MHC Isoform Fiber Type Species 1 1/2A 2A 2A/X 2X/B

1888.8 2306.1 3376.7 C. derbianus -- -- (957.2) (965.6) (1399.6)

1969.1 1787.8 2769.4 2899.2 4455.1 M. domestica (1026.1) (763.2) (750.8) (881.5) (596.3) in parentheses is the standard deviation (s.d.)

52 Figure 1. Diagram of the tail skeletal anatomy of C. derbianus (left) and M. domestica (right). Shown is a right lateral view of each tail indicating ‘proximal’, ‘transitional’, and ‘distal’ tail regions from which muscle blocks were harvested from the muscle flexor caudae longus (FCL). The FCL (center inset) is segmented and attaches along the ventrolateral aspect of each caudal vertebrae. Hemal arches (for attachment of ventral flexor muscle tendons) are also present on each caudal vertebrae along the entire length of the tail in both species. Scale bar = 1 cm.

53 54 Figure 2. Silver-stained gels identifying MHC isoforms expressed in the FCL of C. derbianus (upper row) and M. domestica (lower row) (A) MHC isoform identity and band migration patterns were confirmed by comparison with a combination muscle homogenate from rat (TA, EDL, SOL), and biceps femoris (BF) from M. domestica. Female samples (F) and male samples (M). Gel lanes are bracketed by MHC isoform expression in the proximal, transitional, and distal tail regions. MHC-2B was found only in proximal region of male M. domestica.

56 Figure 3. Representative fiber type reactivity for mATPase and IHC throughout the FCL of C. derbianus. Serial cross-sections reacted for mATPase after (A) acid incubation (pH 4.4) and (B) alkaline incubation (pH 10.2), and against a panel of mAbs specific to MHC isoforms: (C) S58 (anti MHC-1), (D) SC71 (anti MHC-2A, 2X), (E) BF-35 (anti MHC-1, 2A, 2B) and (F) 2F7 (anti MHC-2A). Slow MHC-1 fibers reacted strongly against S58. Fast, oxidative MHC-2A/X hybrid fibers reacted strongly against SC71, moderately against 2F7, and all fibers in each section analyzed reacted against BF-35 indicating a lack of pure MHC-2X fibers. Dark staining of large numbers of MHC-2A/X hybrid fibers after alkaline incubation confirmed reaction against mAbs. Fibers labeled with an asterisk (*) are the same fiber of reference in each serial section. Scale bar = 100 µm.

58 Figure 4. Representative fiber type reactivity for mATPase and IHC throughout the FCL of M. domestica. Serial cross-sections reacted for mATPase after (A) acid incubation (pH 4.4) and (B) alkaline incubation (pH 10.2), and against mAbs (C) S58, (D) SC71, and (E) BF-35 (anti MHC-1, 2A, 2B). MHC fiber type identity by reaction against mAbs S58, SC71, and BF-35 are the same as those in Fig. 3. Four fiber types were confirmed after acid incubation, and MHC-2A fibers matched the acid labile fibers at pH 4.4. Fibers labeled with an asterisk (*) are the same fiber of reference in each set of serial sections. Scale bar = 100 µm.

60 Figure 5. Mean fiber CSA (A) and minimum diameter (B) of MHC isoform fiber types in the FCL of C. derbianus and M. domestica. CSA and diameter were measured from totals of n=1227 fibers (C. derbianus) and n=3134 fibers (M. domestica), and averaged across the proximal, transitional and distal tail regions. Error bars are standard deviations (s.d.).