THE COMPARATWE OSTEOLDGY OF
POSTNATAL PEROMYSCUS LEUCOPU'S
AND PERDMYSCUS MAN-lCULATUS BAIRDI

Thesis for the Degree of M. S.
MICHTGAN STATE UNIVERSITY
GORDON L. KIRKLAND, JR.
1968

   

  

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THE COMPARATIVE OSTEOLOGY

0F POSTNAEAL gERggxsgus Lzugogus
AND __BQE_PE YSCUS W W

By

Gordon L9'Kirkland, Jr.

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE
Department of Zoology

1968

ACKNOWLEDGEMENTS

Although it is impossible to acknowledge
individually each person who has aided in the preparation
of this manuscript. certain ones are worthy of recognition
on the basis of their indispensible help. These persons
include: my wife, Carol. who has been a laboratory
assistant. secretary. statistitian, and loyal supporter;
Dr. Edward Price, who while working as a laboratory
assistant in the Department of Zoology, selected over
three hundred specimens of‘zgggngggg graciously
donated by Dr. John.A. King from his research colony;
and Dr. Rollin H. Baker. who took his valuable time to
review this manuscript and to give me encouragement in
its preparation as the major professor on my graduate
committee. I would also like to thank, in addition to
the above, the members of the Department of Zoology at
Michigan State University who provided me with the
laboratory equipment and space needed to perform this
project.

In appreciation to all those formally named in
this manuscript and those denied proper recognition. I
dedicate this research thesis.

ii

TABLE OF CONTENTS

Page
ACKNOWLEGEMENTS. . . . . . . . . . . . . . . . . . . . ii
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . 1
METHODS AND MATERIALS . . . . . . . . . . . . . . . . 4

DESCRIPTION.AND.ANALYSIS o e o a e . o o s . e e o o e 8
Olecranon e e e o e s o e e e e e o o e e o o e 8
Patella o e o o e o o s o o e e e o o e o e o e 11
Distal ossification of the femur. . . . . . . . l4
Ankle 0 e e o o o o o o o o o o e o o o e o e e 19
Wrist......................25
Tibifl'fib‘llae s e e o o o e o o e o e e e e o o 30
SterMI ossification. o e e o e e o o o o e o e 34
vertebral column. . . . . . . . . . . . . . . . 36
Cervical vertebrae. . . . . . . . . . . . . . . 39
Thoracic vertebrae. . . . . . . . . . . . . . . 41
Lumbar vertebrae. . . . . . . . . . . . . . . . 45
Sacral vertebrae. . . . . . . . . . . . . . . . 48
Cauda]. vertebrae. e o o o e o o o e o o o o o e 50

DISCUSSIONXAND CONCLUSION. . o e . o . o o o o o . e e 53

BIBLImRAPHY . . . 0 . . . O . . . . . 0 . C . . . . . 59

iii

INTRODUCTION

Initially this research was concerned with the
perfection of the Alizarin-KOH clearing and staining
technique as a tool for comparing skeletal development in
closely related vertebrates. This technique had been of
interest since I was introduced to it as an undergraduate
at Cornell University in a laboratory methods course
taught by Dr. Lowell D. Uhler. At that time, the process
was represented as a teaching aid for the exhibition of
delicate specimens. This technique has been used for
many years in the study of the genetics of bone deveIOp-
ment in fetal and young rabbits by Crary. Sawin, and
Atkinson (1958), Sawin and Crary (1956), Sawin and
Trask (1965), and Crary and Sawin (1957). Hewever. it
was a paper by Nesslinger (1956) on the bone develop-
ment of young postnatal Virginia opossums which convinced
me of the practicality of using this technique to study
normal bone development. as opposed to the study of

skeletal aberrations.

WWWaMW
manisnlaiua.baizdi were chosen for this study primarily on

the basis of their availability from laboratory stocks.

It might have been wiser to have recognized considerations
other than the ease of procurement, since the differences
between the species were often so slight as to make
differentiation nearly impossible on the basis of skeletal
deveIOpment. Specimens were obtained from the laboratory
of Dr. John A. King and ranged in age from 0-28 days.
Samples of ten per day were taken for each day from 0-7;
every other day 7-21; and every third day from 21-28 days.
This choice of samples was decided upon before any analysis
was done. As will become evident in the analysis of this
material. it would have been advantageous to have made
daily observations throught 14 days. Most structures
appeared during this time, and the second 14 days of life
was devoted mostly to the maturation of the structures which
had previously appeared. Maturation consisted of the final
replacement of cartilage with bone.

Initially, general observations were made of entire
animals at various stages of development. These allowed the
selection of structures which had the most potential as
"key" bone depositional centers. In most cases. observations
were recorded from the first day that at least one individual
displayed the beginnings of bone deposition until the day
that 100% of the age class showed evidences of bone forma-
tion. With this method. I felt that it was possible to

pinpoint the time of appearance. Under this system, a lack

of ossification could be as important as its presence.

The areas chosen for analysis included: the vertebral
column. carpals. patella, olecranon and hind limb (femur,
tibia-fibula, tarsals).

It was haped that, with a calendar of events
derived from this study, it might be possible to determine
the age of young zgggmygggg with the use of x-rays, rather
than having to kill them to use the KOHquizarin method. It
should be possible to identify the various bones well enough
in an x-ray to place the individual in an age category. This
would be helpful if nests of the young were found in the
field, and information on the young or the possible breeding
condition of the mother was desired. Crary and Sawin (1949)
have used x-rays to follow the development of limb bones

in rabbits.

METHODS AND MATERIALS

The clearing and staining method which I used in
preparing my specimens was adapted from.Dr. Lowell T.
Uhler's laboratory manual for Biology 105 at Cornell
University, and that of Nesslinger (1956). Specimens
were stored in 95% alcohol after having the abdominal
cavity opened. Each specimen older than one day was skinned
either before or after being placed in £-2% KOH, this being
necessary in order that small ossifications could be seen.
Otherwise these were obscured by the hair pigments which
are not affected by the KOH clearing process. The KOH
concentration depended upon the age and condition of the
specimen. The solution was changed each day until the long
bones were clearly visible through the flesh. Caution was
observed with 0-2 day individuals since they tended to
disintegrate if left overnight. The cleared specimens
were then placed in a fresh solution of KOH to which
alizarin stain had been added. The concentration was
determined by the color of the solution, lavender being
desired. At this point. Uhler's method was altered by not
mixing the Alizarin Red-S with acetic acid. The Alizarin
stained the bones a purple to red color with the degree of

4

staining controlled by both the concentration and the
length of time exposed.

After the desired color was achieved. the specimens
were then transferred to a solution of 1 part glycerine and
1 part water (distilled). They were left in this until
they sank to the bottom. At this point, they were placed
in jars of 100% glycerine to which a crystal of thymol was
added to prevent mold formation.

The prepared specimens were then observed under a
zoom-scope with magnifications of from 7-30 x. All observa-
tions were qualitative noting the presence or absence of a
structure. although some mention was given to whether a
structure was poorly. moderately, or well developed. With
the constant manipulation which was necessary to make the
observations, I found that the zoom-scape allowed a greater
degree of freedom than was possible with a normal dissecting
scope.

It was possible to locate bones that had not ossified
since the cartilage did not clear as easily as muscle
tissue and appeared from slightly Opaque to transluscent.
This was particularly useful when working with the ankles
and wrists. making it possible to follow ossification in
bones which were fused in later life, as it was possible
to note these bones as separate entities early in develop-

ment.

The specimens proved to be quite resistant to
damage. I found that skinning was much easier after the
clearing and staining process had been completed. By that
time, the skin had become quite soft and easy to remove.
Skinning before preparation while the specimens were still
hardened from the alcohol proved to be disasterous in
several occasions with lost appendages the result.

The species that were used in this study are
closely related and are difficult to differentiate as
prepared specimens. In the laboratory. these species are
easily distinguished with the bi-colored tail and greyer
pelage ofqggflmgnigglgggg quite evident. There were.
however, certain individuals that were especially difficult
to classify. In the field, these animals are more easily

distinguished on the basis of habitat preference.

Egggmysgg§,123ggpg§ prefers the woodland, whilelfig

magigulgtggqbgirgi is at home in Open fields and is even
found on the Open sandy beaches that border Lake Michigan

(Hamilton, 1939). The differences in habitat preference

in these animals should be noted for as I will point out
later, I feel that this fact may have a bearing on certain
of the differences between these two species. One fact which
I will mention at this point is the type of nest utilized

by these two species. Egggmz§g2§.l§gggpg§ constructs a

nest of leaves and shredded bark usually in a cavity.

§;_manigglg£ug does not build as secure a nest. It is
usually found under a rock or in a depression (Hamilton,
1943). Burt (1957) states that the nest of‘fig‘lggggpug

is more substantial than that of m 2211. LE... M
may, for its nest, seek no shelter other than a partially
buried log or plank on a sandy beach, where shelter is at
a premium (Hamilton, 1943). With shelter at a premium

in areas where.figg‘.bgiggi ranges, it is possible that
predators and other animals would come across nests of this
species more often than those of £3,1295923g. This would
certainly produce differential environmental pressures on

these two species.

DESCRIPTION.AND ANALYSIS

In this section, each of the major areas will be
described and analyzed to determine whether there are any
significant differences in bone development between these
two closely related species. Because of the type of data
collected, it is necessary to use non-parametric statistics
in their analysis. Statistical procedures and tables are
taken from Siegal (1956).

One difficulty in some of the analysis was that,
due to either loss, destruction, or miscounting during
collection, not all of the age classes had equal numbers
of individuals. In order to run a chi-square test, it is
necessary to use equal sample sizes. To allow this, it was
assumed that the specimens were examined in random order.
Thus, "extra" specimens at the ends of the original data

sheets were deleted to provide equal sample sizes.

W

Although it is not a separate bone, the olecranon
is considered in this study because it is a separate center
of ossification. This center appears to be distinct from

the bone deposition in the shaft of the ulna. Bone

8

9

deposition is not present in this area at birth and does not
commence until seen in four day old individuals. Table 1
and Figure 1 follow the course of the ossification in this

structure from 0-7 days.

Table I.-- Olecranon data.

 

 

 

 

 

 

 

 

Days 15.122223: M I
‘0-3 None (of 36) None (of 40)‘
4 None (of 9) 5 (of 11)
5 4 (of 11) 7 (of 10)
6 8 (of 10) 9 (of 10)
7 10 (Of 10) 10 (of 10)
Total 22 (of 76) 31 (of 81)

 

 

 

Differences in the degree of ossification in the
two species occurred on days 4,5, and 6 with 2122:. 22125.11
developed to a greater extent in each case. When these data
are examined using a chi-square test for the total number
of individuals ossified during the three days of differences,
a value of 3.6839 indicates that no significant difference
is present at the .05 level with 2 degrees of difference
(Table 2). However, the value is significant between the

.10 and .20 levels.

10

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Table 2.-- Chi-square analysis of the olecranon data.

 

 

 

 

 

 

 

 

 

 

 

 

 

P, Leugopus jhgh_2§;;gi

Day Obs. Exp. Obs. Exp. 1 _Total
4 o 1.9 5 3.09 T 5
5 5 4.59 7 7.41 12
6 8 6.5 9 10.5 ‘ 17

13 13.0 21 21.0 33
(o- 92..u_-_3..1)3.15-_4._5213.12.;_1.A113

19 3.1 4.59 7.41

2 2
8 - 6 5 (9 - 10.52 = 2
+ % + o x

 

 

3.6839 = x2

 

Eefslla

As in other mammals, the patella is present as a

sesamoid bone in both species.

This structure is late in

ossifying, and no bone is deposited until the seventh day.

Before this time, the patella can be identified as a

shadowy structure representing the unossified tendon lying

immediately distal to the condyles of the femur;

The period of partial representation of this

structure lasted from 7-13 days.

The trend in the appear-

ance of the patella may be seen by noting Table 3 and

12

Figure 2. The development of the patella is quite similar
in the two species. The results of a chi-square test at
the .05 level of significance with three degrees of freedom
indicate no significant difference between the two species”
The chi-square value of .9544 falls between the .70 and .80
levels of significance and indicates a high degree of
similarity between the species in the develogment of the

patella.

Table 3.-- Patella data.

 

 

 

 

 

 

 

 

Days L W P.m. baigdi
0-6 ane ane
7 ' None (oflO) 1 (of 10)
9 3 (of 10) 3 (of 11)
11 7 (of 9) 7 (of 10)
13 7 (of 8) 10 (of 10)
Total - 17 (of 37) 21 (of 41)

 

 

 

.Table 4.-- Chi-square analysis of the patella data.

 

 

 

 

2.1392219. 15mm
Day Obs. Exp. Obs. Exp. Total
‘7 0 .47 1 .53 1

 

 

 

 

 

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Table 4.-- continued.

 

 

 

 

 

 

 

 

 

Day Obs. Exp. Obs. Exp. Total
11 7 6.6 7 7.4 14
13 7 7.1 8 7.9 15
17 17.0 19 19.0 36
1,14
W._11_;_.2313.13_=_2.3312 . - 2 *
.47 .53 2.83 3. 7 _
‘ I
+ .17 - 6.6)2 + (7 - 7.4)2 + {7 - 2.122 + (8 - 7,9)2 =x2 . ,
6.6 7.4 7.1 7.9 I 1
2
.47 .2209 0 9 .0289 .01
.47 T .53 * ‘§T%§" + 3.17 + 6.6 T 7.4 T '771
01
* 7.9 = .9544 = x2

 

Distal Ossifigation.2§ the Femg;

As is characteristic of all mammalian long bones, the
femur of the mouse has a subterminal epiphyseal growth area.
Distal to this area of growth, the condyles of the femur
calcify and are recognizable as being distinct from the
shaft which is also ossifying. Because of its isolated
situation, the distal condyles of the femur present a good
area of study.

The distal end of the femur appears to be formed in
one of three ways. It can appear as a simple arc or line
of bone which has no enlargements. It may also form in a

bilobed or dumbbell form or it may develOp as two separate

15

bony areas which later meet in the midline. There is much
variance in size of this structure between individuals.

No ossification of the distal condyles of the femur
was noted in individuals through three days, with the

exception of one three-day individual in §,m, baigdi in

which the condyle may have begun to ossify, as a faint l
speck of alizarin was present. 3
Four-day individuals were the first to exhibit bone 1
deposition. Three of ten.§&m&'b§i;gi had small dots of F
bones which represented the medial and lateral condyles.
None of the.§& Leucopus showed any signs of bone deposition.
Much the same condition prevailed in the five-day
individuals. §&,Lguggpg§ had no ossifications in nine
specimens. On the other hand, £1.94. m had four of ten
specimens with bone deveIOpments. All of these showed an
advancement over the four-day individuals in that the two
lateral areas of deposition have been joined across the middle
with the medial being the larger.
Bone deposition in.§& Leuggpus occurred first in the
six-day individuals with two specimens exhibiting minute
ossifications while eight lacked ossification. The bone
deposition appeared to be at the same stage as that of the
four day Egg; baigdi, At the same time, £im;,bgi;g1 had all

ten specimens with at least some ossification.

The same discrepency between species was evident in

16

the sevenmday individuals with only three of eight §&_;ggggpg§
showing evidence of bone deposition. All ten specimens of
the other species were well formed.

The ninth day brought the first instance in which a
higher percentage of g; leucgpus were ossified, with all
nine individuals having at least some trace of the distal

5—I
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condyle. In Pama ba d', eleven of twleve were present.

 

The only individual lacking any development was extremely '
small. [A
All eleven day individuals in both species had the
distal end of the femur ossified.
Table 5 gives a more graphic representation of the
preceeding discussion, which was included only to

illucidate my observations of the trends which were noted.

Table 5.-- Distal ossification of the femur data.

 

 

 

 

 

 

 

 

 

Days fig Ieucgpug figmh,bgi;g1
0-3 None None
4 None (of 10) 3 (of 10)
5 None (of 10) ' 4 (of 10)
6 2 (of 10) 10 (of 10)
7 3 ( of 8) 10 (of 10)
9 9 (of 9 ) 11 (of 12)
11 All All
‘ Total 24 (of 57) 48 (of 62)

 

 

 

17

Differences in the number of ossified femural
condyles occurred in only five of the age classes; 4-9
days. A chimsquare test at the .05 level of significance
with 4 degrees of freedom was applied to the femur data and
produced a value of 11.7609. This was significant at

between the .02 and .01 level (Table 6), indicating a

 

significant difference between the two species as to the

 

number of individuals in each age class with ossified
femural condyles. Figure 3 suggests that 2&m&flbgi;gi had a r
significantly greater number of individuals with ossified
femural condyles than 2‘ Leugoggs on each day. This
figure also suggests that the femural condyles ossify

sooner in Pam, baigdi by a factor-of about one day.

Table 6.-- Chivsquare analysis of the distal condyle of the
femur data.

 

 

 

 

 

 

 

 

P2 Leucogus §;m&,ggiggi

Day Obs. Exp. Obs. Exp. Totali
4 O .90 3 2.10 3
5 0 1.20 4 2180 4
6 2 3.60 10 8.40 12
7 4 4.20 10 9.80 14
I 9 9 5.1 8 11.9 17
15 15.0 35 35.0 50

 

 

 

 

 

 

18

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34m.
43mg

 

 

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.8
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.06
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.8
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uotasotgtsso qatn eIdmss go eSsnueoaeg

 

19

Table 6.-- continued.

 

 

 

2 2 2 2
(9 - -901_. _il_:_2ilQl_ _IQ_:_liZQl_ ._L&_:_2i§Ql.
T 2.10 T 1.20 T 2.80

 

.90
2 2 2
(2 - 3.9022 (10 - §.&2 (4 - 4,2) (10 - 9.82
T 3.60 T 8.4 T 4.2 T 9.8
2 2
.4244)... 8-119 =x2
2 2 \
90 81 1 20 1 44 2.56 2.59 04
596' T TTTE T’TTTEBT' T '2T86' T 3.60_ T 8.4 T“422‘
04 15 21 15 21 _ 2
T ‘978 T TETTTTT T'TTTT9T' — x

 

.90 + 3.857 + 1.20 + .5142 + .7111 + .3047 + .0095 + .004
+ 2.9823 + 1.2781 = 11.7609 = x2

 

Ankle

The deveIOpment of the ankle shows some interesting
differences between the two Species. The ankle consists in
the fully deveIOped condition of eight elements: four
tarsals (#1, 2, 3, 4-5 fused), centrale, tibiale, calca-
neus, and astragalus. The astragalus and the calcaneus are
the largest and the first to develop.

The bones of the ankle in.§§§ggz§gg§ are essentially
the same as those found in the laboratory rat, gagggg
(Rowett, 1957). The five metatarsals are met proximally
by only four distal tarsal elements, the fourth and fifth being
fused. The fused fourth and fifth tarsal lies proximal to the

4th metatarsal and is articulate with the 5th metatarsal on

20

the lateral side by the proximal growth of that meta-
tarsal. The third tarsal is slightly smaller than the
fused 4th and 5th and lies immediately proximal to its
metatarsal. The second is the smallest tarsal element and
occupies the same relative position to its metatarsal as
does the 3rd tarsus. The first tarsal element is elongated
and extends much further distally than any of the others.
The centrale lies just proximal to the second and third
tarsal and articulates to the astragalus proximally and the
first tarsus laterally.

The astragalus and calcaneus are complex in design
and will not be described here. In nearly all cases these
are present at birth. The calcaneus appears to be the
first ankle element to ossify, while the tibiale is the
last element to form, not appearing until about the ninth
day.

At birth, the calcaneus was present in all seven
Egg; baigdi examined. Six of these also had the astragalus
deveIOped (ossified). The situation was nearly the Opposite
in‘£& Legcopus. Of the nine newborn animals examined, only
one had both the astragalus and calcaneus and another had
the calcaneus slightly developed.

In oneoday old individuals. the earlier appearance
of ossification in gym; baigdi continued. All ten.£‘m‘
bgirdi had the calcaneus ossified and only one lacked

21

ossification in the astragalus. although some of the
latter elements appeared as only a cloud of bone. In
2‘ euco u , two of nine lacked ossification in either
bone. Six had both bones ossified and one exhibited only
calcaneus ossification. It was not until the two-day
individuals that ossification was present in all of the
g; Leucopug with only one of seven lacking an astragalus.
The differential size of the astragalus and the calcaneus
was noted as the ossified part of the calcaneus was up to
lk-B times that of the astragalus. Also the first of the
tarsal elements was seen in one of thelggmL baigdi as a
cloud of bone.

The third day was the first time that the calcaneus
and astragalus was found in all ten 2; ngggngg. In
Egg; hairdi, the third day brought on a continued ossifi-
cation of the tarsal elements with four of nine specimens
exhibiting ossification of the first tarsal element. One
of these also had the 4-5 fused element present. One
individual in P. leucopus exhibited an unusual phenomenon
with the 4-5th appearing prior to the first. This situation
was never seen in the other species and was only seen one
other time in fig legcgpus (five days). In all other cases,
the first tarsal preceeded the 4-5th appearance.

Differences between species continued to be evident

in the four day olds. Five of the E; 123392;; showed at

22

least one tarsus; four lacked any tarsal ossification.
On the other hand, Egg; baiggi had only one specimen out
of eleven which lacked some tarsal ossification. All the
rest had at least two, with five having three.
The sequence of ankle bone ossification might be
explored at this point. As stated earlier, the calcaneus 1
appears to be the first bone to ossify with the astragalus T
appearing shortly afterwards. This occurs either before T
birth or at the latest by the third day. By the second or r
third day (fourth in,§"lguggpu§) the tarsal elements begin
to appear. Their sequence of ossification is generally
the first, the 4-5th, and the third, and finally the
second. Some exceptions have been noted previously. The
centrale first appears on the fifth day (Egg; bgiggi).
The tibiale is very late in appearing, the ninth day in
both species, at which time it appears only as a small
speck of bone barely visible at 30 power.
The disparity between the species was evident in
the five-day individuals. All ten.££m& bgigdi had at
least one tarsal element and only two lacked having at
least three. One specimen also possessed a partially
ossified centrale. In §‘_ngggpg§ none of the eleven
individuals had any trace of the centrale and only three
had as many as three tarsals.

The sixth day found eight of eleven P,m, a rd

23

lacking only the tibiale. Two individuals lacked both the
tibiale and the centrale; while one individual had only

the astragalus, calcaneus, and first, third, and 4-5th fused
tarsals present. 2;,lggggpgg had but two of ten individuals
lacking only the tibiale. The rest lacked between three

and four bones.

Data collected on the seven-day individuals exhib-
ited the same trend as witnessed earlier. In.§&m& baigdi
nine of ten specimens lacked only the tibiale, while the
tenth was lacking the tibiale and the centrale. §&,ngggpg§
had six of ten specimens with only the tibiale absent.

The rest lacked the tibiale, the second tarsal and the
centrale.

The ninth day brought the first appearance of the
tibiale with one of nine 2; Leucgpug and one of ten
g‘m& baigdi exhibiting all eight ankle bones. Eight of
the Egg; baigdi lacked only the tibiale and one lacked the
centrale, tibiale, and second tarsal element. Eight
2‘ Lguggpug lacked only the tibiale.

The appearance of the tibiale was more frequent on
the eleventh day. Six of ten.§gm‘ bairdi had all eight
ankle bones present. The rest lacked only the tibiale.
‘£‘|lggggpg§ had six of nine specimens with all the ankle
bones present, and the others lacked only the tibiale.

Total ossification was achieved in P.m, bai d

24

on the thirteenth day. One of eight P. leucOpus lacked
complete appearance of the ankle bones. Table 7 gives a
graphic representation of the progress of the appearance

of the ankle elements.

Table 7.-- Ankle data.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

W mm

'" Tarsals T I Tarsals
Age # 1 2 3 4-5 Ast Cal Tib Cent # 1 2 3 4-5 Ast Cal Tib Cent
07000012 0 0 70000 6 7 0 0
_ 1 10 o 0 0 0 6 7 0 0 10 o 0 o 0 9 10 0 0
270000 6 7 o 0 70000 7 7 0 0
390000 9 9 0 0 94001 9 9 0 0
494010 9 9 0 0 98557 9 9 0 0
510802 8 10 10 0 0 101048 9 10 10 o 1
6UP 29 9 10 10 0 2 101091310 10 10 0 9
7DB6D10 10 10 0 6 101011010 10 10 0 9
999999 9 9 1 9 99899 9 91 8
1199999 9 9 5 9 99999 9 9 6 9
1388888 8 8 8 8 88888 8 8 8 8

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

The analysis of the ankle was achieved by taking the
mean number of ankle bones present in each species from
birth to the ninth day and testing with a sign test. The

probability of'gg Leucopgg having a higher average number

miiv‘rmuugnun j
I

11‘4

25

of tarsals only once in nine days is .020 and indicates

a significant difference (Table 8). This suggests that
the ankle of £1911. bagdi develops more rapidly. The
figures from Figure 4 indicate that the difference is on
the order of magnitude of one day with the percentages of

§L_lggggpg§ resembling the §,m, baigdi's of the preceeding FT
4
day. -4

Table 8.-- Sign test of the ankle data.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Day £4. 113.999.1125 3.12.1. 125.3111. Sign 13
' o .33 1.81 +¥

71’ 1.45 1.9 +
2 1.81 2.0 +
3 2.1 2.56 +
4 3.0 4.09 +
5 4.09 5.78 +
16 5.2 6.63 +
7 6.2 6.90 +

I 9 7.1 7.0 -

Ln. Rail-£9.1- = 8
2‘ Leucogus = 1
ELLE

The wrist consists of nine elements in both species.

These are arranged in two rows with the distal row composed

26

“334M...
Haj...

.Oaxso can no doauuoamammo 11.8 ouawam

when
0 m o m N H

— _ b

k:

lllll'l'1ll‘lll \

 

 

 

eIdmss Jed sauemeIg JO JeqmnN useu

27

of four carpels with the lateral representing the fused
4th and 5th carpels. The second transverse row is made up
of the five remaining elements: sesamoid, fused radius-
intermedium, centrale, ulnare, and pisiform. At birth

the wrist exists as an unossified structure and bone depo-
sition does not appear to commence until the third day
(two-day olds) when both species exhibit some ossification.
The progress of the bone deposition in the wrist may be

seen in Table 9.

Table 9.-- Wrist data.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

El leasanua ILau.hai£di

Carpals Carpals
Age # 1 2 3 4-5 C R-I U S P # 1 2 3 4-5 C R-I U S'P
‘2 9000110100 9000210100
30000330200 10100761600
49111661530 9545886760
59455855760 9999999990
61099910109090 10010|0101010|1001
71000111010101001 1000'01010101004
98888888885 888|8888|886
11 1013001010 10 10110 10 01010101010111)

 

 

The sequence of appearance of the wrist elements

is slightly less predictable than that of the ankle bones.

28

The centrale, 4th-5th carpal, and ulnare appear first.
Indications from these three-day old individuals suggest
that the 4th and 5th may ossify first on the basis of the
one representation of this element in one individual
(g‘m‘lbgiggi), while the centrale is second on the basis
of two individuals having both this element and the 4th
and 5th. Eight individuals had all three elements out of
the total of eleven three-day olds with some bone present.
The other six bones make their first appearance almost
simultaneously, and thus it is difficult to make a defin-
itive statement as to their sequence of appearance.

The ossification of the wrist apparently occurs at
different rates in the two species, as suggested in
Figure 5. Only in the first day and eleventh day or in
older individuals are the rates of occurrence equal. In
each of the intermediate days, a higher average number
of wrist elements per individual occurs in‘figm&_bgi;gi.

Using the sign test (Siegal, 1956), the results
indicate that the probability of having x = 0 is .008
(Table 10). This value falls below the .05 level of
significance chosen for all analyses in this thesis.
Therefore, there is apparently a significant difference
between the species in the rate of formation of the
wrist elements with 2.12:. 23.1-29.1 having its wrist bones
forming sooner than those of L W.

29

HH

0

_

.umaua on» no soauOOdudmmo ::.m ouowam

when
n e n N H

p b -

 

 

 

qu Jag pat;tsso seuog JO JeqmnN usaw

30

Table 10.-- Sign test of the wrist data.

 

 

 

 

 

 

 

 

 

Day L leucopus* m Mir Sign
2 .33 .50 +
3 .80 2.10 +
4 2.67 5.84 +
5 5.64 7.45 +
6 7.50 8.10 +
7 8.10 8.40 +
9 8.62 8.75 +

 

 

 

 

mmu
Lima-=0
Pr0bo X:- 0:.- .008

 

 

* These figures indicate the mean number of bones per
individual.

Tim-11121113

The tibia and the fibula are closely appressed
bones in many higher vertebrates, so intimately associated
in some species as to become fused along part or all of
their lengths. Partial fusion of these two bones is found
in the genus Egggngggg. In this genus, they are twisted
so as to fold around one another, apparently due to the
twisting of the limbs as they have come to lie under the
body in the mammals. The first signs of fusion of the

tibia and fibula are a series of net-like strands whiCh

31

bridge the gap between the bones starting approximately

1/3 of the distance from the distal end at the area where
the bones first are in close proximity. Growth con-
tinues at the ends and as a result fusion occurs last here.
Since the fusion takes place on he inner faces, it is often
difficult to determine whether fusion has taken place
except with close inspection. The progress of the tibia-

fibula can be seen in Table 11.

Table 11.-- Fusion of tibia-fibula data.

 

 

 

 

 

 

 

 

Days Old £5.12232225 Ihflh.h21£Q1
3 None None
4 None (of 9) 2 (of 10)
5 4 (of 11) 4 (of 10)
6 6 (of 10) 10 (of 10)
7 10 (of 10) 10 (of 10)
Total 20 (of 40) 26 (of 40)

 

 

 

The graph of the fusion of the tibia and fibula
(Figure 6) is basically similar to the graph of the
appearance of the olecranon (Figure 1). In both, the first
appearance of the character in question takes place in
four-day individuals and reaches 100% in the seven-day olds.
On each of the 4th, 5th, and 6th days, the sample of Ehlh.
being; has a higher degree of develOpment than that of

32

.uusnuuwuanau man no cowuuuauammo --.8 museum

 

«.3343. 4M n ------I--II-
«madam.4mum.n

 

 

.oH
you
.om
.oe
.on
.00
.on
.ow
roo

fob:

 

etdmss sea notasotnsso sSsaueoaeg

33

£l_lsasaaua-

A chi-square test run on the data for the fusion of
the tibia-fibula reveals that with four degrees of freedom
the value is 1.707 (Table 12). This figure lies between
the .70 and .80 levels of significance. Therefore, it is
concluded that there is no significant difference in the

rate of appearance of fusion of the tibia and fibula in

the two species.

Table 12.-- Chi-square analysis of the tibia-fibula data.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

.21.lsussaua 21ml.aairdi
Day Obs. Exp. Obs. Exp. Total
4 0 .769 2 1.23 2
5 4 3.077 4 4.923 8
6 6 6.154 10 9.846 16
10 10.0 16 16.0 26
0- 692._1L=_1_.2;12_ +_i€1_3_.._;2)_ M113
. 69 1. 3 07 4.923
- 9 2 (19 - 9.84622 -
T T'éTTETE'T’ T 9.846 ‘ *2
.5229 5 929 II851929 .923719
T5785§ T 1.23 T “T57877‘T 4. 923 T 6.159

+ 1222119 = x2
9.746

 

.769 + .482 + .2768 + .173 + .00385 + .0024 = 1.707 = x2

 

34

mm

The ossification of the sternum is a highly
eccentric phenomenon, involving a great deal of individual
variation. There are usually six sternal elements
(sternebrae) which develOp embryologically from paired
centers, as documented by Little (1937), and are either
partially or entirely fused at birth. Seven true ribs are
attached to the sternum along its length. In certain
individuals, however, a seventh element appears between
the 6th and 7th ribs and separates the 5th and 6th
sternebrae. This "extra" element was noted as being
present in 28.7% of the.2dg"b§i;gi_and 14.7% of the
Llam-

Even though the sternebrae fuse early, they Often
keep the appearance of having been two distinct rods for
some time after birth, up to the third day in some
individuals. The first element, the manubrium, is often
bifurcated at the anterior end. The xiphisternum, on the
other hand, is rarely noted as having developed from two
rods and was not divided in any of the 300 specimens used.

The fifth sternal element showed the greatest
variation in size and shape. It was usually smaller, often
distorted, and frequently sections were missing. There were
also instances of co-ossification between various sternal

elements .

35

No statistical analysis was done to compare the
number of individuals with or without complete fusion
since only three days were involved and on one of these
the figures from the two species were identical with only
one individual difference on the other two days. It was
evident that there was no difference.

The difference between the two species in the
development of the 7th sternal element was analyzed to
determine if there was a significant difference in the
occurrence of the "extra" element. The Wilcoxin Matched
Pairs Rank-Sign Test (Siegal, 1956) was chosen to analyze
these data with a .05 level of significance. The data
indicated that there was a significant difference between
the two species (Table 13). The value of 14 with an
N = 12 fell exactly at the .05 level.

Table 13.-- Wilcoxin matched pairs rank-sign test of the

 

 

 

 

 

 

 

 

W-
Rank
with
Day # W Masai Difference Ranks less
freq.
sign
0 9 0 O 0 0
1 10 1 0 -1 2 -2
2 9 1 2 +1 2
3 10 2 2 0 0
4 9 0 6 +6 12

 

 

 

 

 

 

 

36

Table 13.-- continued.

 

 

 

 

 

 

 

 

 

 

 

 

 

Rank—-
with
Day # 2‘ Leugoggs ‘figmg‘ggiggi Difference Ranks #32:.
sign
5 10 2 4 +2 6
/ 6 10 2 2 0 0
7 10 4 2 -2 6 -6
r 9 9 3 1 -2 6 -6
11 9 l 4 +3 9
13 8 1 3 +2 6
15 10 2 4 +2 6
18 7 0 4 +4 11
21 9 1 4 +3 9.5
24 7 0 1 +1 2
136 20 39 14

 

 

 

 

 

 

 

mm

The vertebral column presents an interesting
aspect of this study because although it is basically one unit,
it is divided into five morphologically and functionally
distinct units. For this reason, it is possible to
glean information from this structure by analyzing its five
regions: cervical, thoracic, lumbar, sacral, and caudal,
in addition to studying it as a whole.

The aspect of the vertebral column chosen for study

was the fusion of the dorsal portion of the two lateral

37

arches. This was a good area of study for several reasons.
First, this fusion could be studied in all sections of the
column. Second, it provided an easily recognizable
quantitative measure of growth: being either fused or not.
Third, it was felt that this dorsal fusion was a good

index of bone growth in the vertebral column. In all cases,
information was gathered only on the status of the fusion
of the dorsal aspect of the vertebral column.

The vertebral column in the two species is essentially
identical and is similar to that of the laboratory mouse,
Mg§_mg§gglu§ (Wirtschafter, 1960). There are of course
seven cervicals, including an atlas and an.axis. The
remaining vertebrae are divided into thirteen thoracics,
six lumbar, three sacral, and approximately 25 caudals.
Since there are differences between the sections con-
cerning the times of fusion, each will be discussed sepa-
rately.

The vertebral column, taken as a whole, appears to be
equal in its rate of development in the two species. A
chi-square test at the 5% level Of significance with 7
degrees of freedom produces a value of 13.9831 (Table 14).
To be significant, the value would have to be at least
14.07. The chi-square value for the vertebral column lies
between the .05 and the .10 level. This agrees with the

individual components, in which only the thoracic vertebrae

38

showed a significant difference between the two species,
with m. baigdi showing significantly more rapid fusion.
The other sections revealed no differences between the

species.

Table 14.-- Chi-square analysis of the vertebralczolumn.

 

 

 

 

 

 

 

 

 

 

 

 

£3. euc 8 M4. 12212.91

Day Obs. Exp. Obs. Exp. Total
3 5 4.32 4 4.68 9 #4
4 17 14.39 13 15.61 30

5 46 38.37 34 41.63 80
I6 71 65.71 66 71.29 137
47 92 109.84 137 119.16 229
‘9 188 178.44 184 193.56 372
11 202 216.11 250 235.19 452
13 252 245.11 259 265.89 511
Total 873 872.99 947 946.91 1820

 

 

 

 

 

 

2 2
1.9822 1.9812 (2,6122 (2,6122 (7,632 7
4.32 T 4.68 T 14.39 T 15.61 T 38.37 T 'T41T63'

2 2 2 2 2
9 9 (17.842 112.941 9
TTM+65J T421247 .29 T 109.84 T 119.16 T T73)” 8.44

+ (9.5922 + (14,8122 I (14,8122 + (9.8912 + (6,8922 3 x2
193.56 216.81 235.19 245.11 265.89

 

39

Table 14.-- continued.

 

 

,4624 8 6,8121 58,2169 58 169
4. 32 T 4.39 T 15.61 T 38. 37 T 41.63
27.9841 318.2656 318.2656 91.3939

T '85‘71TT‘ T 71.29 109. 84 119.16 T 178. 44

91 3936 216 33 1 16 6 4 4

193. 56 + '2T6T8TT‘ T '235f15‘l: T T2457TTT

42.4721 _ 2
T 265.89 - x

 

.1071 + .0988 + .4733 + .4363 + 1.5172 + 1.3984 + .4258
+ .3925 + 2.8975 + 2.6709 + .5121 + 4721 + 1.0116 + .9325
+ .1936 + .1785 = 13.7181 = x2

 

Esrzisal.!sriearas

The cervicals were the last vertebrae to initiate
fusion. The first fusions occurred on the seventh day in
.figgg.b§i;_i. On that day, two of ten individuals had at
least one vertebrae fused, the 7th in one and the 1st plus
3-7th in the other. Unfortunately, as has been mentioned
previously, no eight day individuals were collected. By
the ninth day, the cervicals were well fused in both species.
Seven of eleven specimens in 23.3.1.1. m and four of ten in
£‘_lggggpg§ exhibited fusion. Of these eleven individuals,
all had the first fused: no other single vertebrae was as
well represented in the fused state.

On the eleventh day, all ten of the P,m, being;

 

40

showed at least some cervical fusion with two being
completely fused. Six of eight individuals in'gL euc
exhibited cervical fusions with three being completely fused.
Thirteen-day individuals tended to show complete fusion
with one of eight lacking such in.£“L§uggpg§, a small
individual with #2-5 not fused. Nine (of ten).£ug"h§1;g1
were completely fused with only one individual lacking
fusion in the axis. Fifteen day specimens were all completely
fused.

The sequence of fusion is difficult to present in
detail: however, it appears that the axis fuses first
with the others fusing later. The order of the others
cannot be precisely determined, but the 7th appears to be
the last to fuse in general.

As the cervicals were the last vertebrae to initiate
fusion, the number of possible comparisons was reduced
to four days: 7, 9, 11, and 13. It should be noted
that the only major vertebral difference between the two
species occurred on the seventh day when,§‘g&‘bgiggi had
a total of eight fused cervicals, while §;,1§ggggg§ had
none.

A chi-square test at the .05 level of significance
and three degrees of freedom produced a value of 7.261
(Table 15). This did not indicate any significant difference

between the two species since the value lay between the

41

.05 and .10 level. Therefore, the cervicals were con-

cluded to have fused at the same rates in both species.

Table 15.-- Chi-square analysis of the cervical vertebrae.

 

 

 

 

 

 

 

 

 

£5.1222222e Thau.asindi
Day Obs . Exp . Obs . Exp . Total
7 0 3.68 8 4.32 8
9 19 19.31 23 22.69 42
’11 31 30.35 35 35.65 66
13 53 49.66 55 58.34 108
103 103.0 121 121.0 224

 

 

 

 

 

2
MMM.W,M.M
.68 4. 32 19. 31 22.69 30.35 35.65

#fifi

 

153823 Wt—ffir%%+ *3“?-
+2318‘E-T‘H-3-815ii“

 

3.68 + 3.135 + .004 + .004 + .013 + .011 + .224 + .191 7.261

ll
>1

Tharssis.!sr£shrae

Dorsal fusion in the thoracic vertebrae made its first

appearance in both species on the sixth day. One individual

42

in nine in 2‘,ngggpg§ had the 7-8-9th fused. One of
eleven in.£‘m“b§i;gi had the 6th-11th fused. The number
of individuals with fused thoracics was increased on the
seventh day when‘gg leucopug had two of ten individuals
with thoracic fusions, while‘gdmgqbgigdi had seven of ten
individuals with at least one vertebrae fused.

By the ninth day, almost all individuals had at least
some dorsal fusion in the thoracic areas with nine of ten
individuals in L 1.229222% and nine of eleven in m
lbgizgi. Three specimens in thelfig manigulaggs group had
all the thoracic vertebrae fused, while none of the
,Bg‘ngggpgg specimens had proceeded that far. Individuals
from the eleventh day all had some fusion in the thoracic
area. Four of ten individuals in'zgmg‘bgiggi had complete
dorsal fusion: twolfiL‘leuggpgg had reached full fusion.

It is difficult to analyze the direction of fusion
in the thoracic area but it appears that the middle
vertebrae (#3-11) are the first to become fused. In general,
the 13th is the last to fuse. Evidence for this is that
none of the six-day individuals had #1-2 or #12-13 fused.

In fact, the first day that the 13th becomes fused is
probably the eighth or ninth day since the first record of
the 13th being fused occurred on the ninth day. That is only
one out of a total.of 21 specimens for both species.

2‘,ngggfi§§ did not have the 13th fused until the 11th

43

day when three individuals showed that condition.

The thoracic vertebrae were the only ones which
exhibited a significant difference between the two species.
The data suggested that this might be the case since
m m had more fused thoracics in each age class
from 6 - 13 days. A chi-square test on the total number
of vertebrae fused each day proved to be significant at
the .05 level of significance with four degrees of freedom
(Table 16). The chi-square value of 16.117 fall between
the .01 and .001 levels of significance.

Table 16.--Chi-square analysis of the thoracic vertebrae.

 

 

 

 

 

 

 

 

 

am A 2.211.112.1291.

Day Obs. Exp. Obs. Exp. Total
6 3 6.32 11 7.68 14
7 15 27.52 46 33.48 61
9 71 64.96 73 79.04 144

11 88 87.07 105 105.93 193

13 100 91.13 102 110.87 202

277 277.0 337 337.0 614

 

 

 

 

 

1.1.3213 1.13.32). 11125213 1.1.2.222L11L25123
27. 52 33. 48 64.96

2
+%+%+jfi%+%+%=

 

44

Table 16.-- continued.

 

11,0224 11,0224 I 15637504 + 156,7504 + 36,4816
27.52

6.32 + 7.68 33.48 64.96
36.3819 8649 18649 2816269 2816769 2
+ 79.04 *"'87.67' + 105.93 91.13 * 110.87 = x

 

1.744 + 1.435 + 5.695 + 4.632 + .561 + .451 + .009 + .008
+ .863 + .709 = 16.117 = x2

 

To determine whether the difference lay in the number
of individuals with fused vertebrae or the number of
vertebrae per individual, a chi-square test was run on the
number of individuals with fused thoracics on each day.
Using the .05 level of significance and four degrees of
freedom, the chi-square value was 2.585 (Table 17). which
fell between the .50 and .70 level of significance.

Table 17.--Chi-square analysis of the thoracic vertebrae.

 

 

 

 

 

 

 

 

 

 

e on 2.23M

Day Obs. Exp. Obs. Exp. Total
,6 1 .94 1 1.06 2
_ 7 2 4.21 7 4.79 9
9 9 7.95 8 9.05 17
11 9 8.42 9 9.58 18
13 8 7.48 8 8.52 16
29 29.0 _ 33 33.0 ‘62

 

 

 

 

 

45

Table 17.-- continued.

 

 

2 2 2
(.0922 06 2 (21212 21 (11052
.94 + .06 * 4.21 * Léf79l" * 7.95
2 2 2 2 2
(11052 (.582 (.582 1.221. _ 2
* + * 4.7.221.“ * 8.52 - x

 

+ 9.05 8.42 9.52
19036 1 10036 418841 418841 111025 111025
.94 1.06 * 4.21 + 4.79 + 7.95 * 9.05 .
.2363 3364 12704 12204 _ 2
+ 8.42 *"‘9752‘ + 7.48 + 8.52 - x

I

.003 + .003 + 1.16 + 1.1019 + .138 + .121 +.039 +.035
+ .036 + .031 = 2.585 . x2

 

The results indicate that the differences between
species is due to the number of fused vertebrae per
individual and not due ot g‘m‘12gigdi having significantly

more individuals with fused vertebrae.

Lumbar vertebrae

First signs of lumbar fusion occurred on the fifth
day in both species. figh12§13gi had four of ten individuals'
and £3,1ggggpgg had two of eleven. In both species only
#5-6 were fused. Six-day individuals exhibited much the same
pattern, with eight of nine in £&_1ggggpg§ and eight of 3
eleven in12gmL1béiggi showing fusion. Hewever, of these
sixteen total individuals, only one of each species had

the 4th lumbar vertebrae fused. None had #1-3 fused.

46

Little change was evident between the sixth and

seventh day individuals. The percentages were 70% (7/10)

for £‘_Lguggpgg and 80% (8/10) for £3E;.221£_1- Of these,
five of the L W and only one of the h Leugoggg

had other than the 5th and 6th fused. The third was present

in one of the L Leucgpgs and three of the E; W.
One nine-day individual in.£‘_m§gigglggg§ had all

the lumbar fused. At that age, all ten of the £‘_lggggpgg
and nine of eleven.§‘,manigglg§g§ had some fusion.

Even in some of the thirteen-day individuals, there were
many individuals which lacked complete fusion of the dorsal
aspect of the lumbar vertebrae.

There is a definite trend in both species to have
the 5th and 6th lumbars fused long before the rest. It
is possible that this may be a functional adaptation due
to the proximity of the sacral vertebrae.

The number of lumbar vertebrae was almost identical
in each species over the days sampled (4 - 13). A chi-
square test was run on the data at the .05 level of
significance with six degrees of freedom, to test for the
possibility of significant differences. The value of the
chi-square of 3.205 was significant between the .70 and
.80 levels (Table 18). From this result. it was concluded
that there was no significant difference between the two

species in the number of fused lumbar vertebrae.

 

 

47

Table 18.-- Chi-square analysis of the lumbar vertebrae.

 

 

 

 

 

 

.., .v
,_v-—

 

 

 

 

 

 

 

L euco us 2.1.23 122.1521
Day Obs. Exp. Obs. Exp. Total
4 1 .92 1 1.08 2
5 4 5.07 7 5.93 11
6 14 11.52 11 13.48 25
7 15 17.50 23 20.50 38
9 28 26.71 30 31.29 58
11 33 36.38 46 42.62 79
13 45 41.91 46 49.09 91
140 140.0 164 163.99 304

 

 

 

_.f5\ '4--.’-.".'

 

 

1._12:3§:. 2 + 23,0912 (310922 2
36.38 + 42.62 41.91 + 49. 09 = x

 

‘ 4996!; 1.1459 1,1449 6,1504 6,1504
.92 * 1. 08 + 5. 07 * 5. 93 + 11.52 + 13.48

++fi%%-+-%6333-++391L+41%%l 11%.... + W

+ 91548; 9.5481 = x2
41.91 49. 09

in;

.006 + .005 + .225 + .193 + .533 + .456 + .356 + .304
+ .062 + .053 + .314 + .268 + .227 + .194 = 3.205 = x2

 

48

Sacral vertebrae

The sacral vertebrae consist of three elements
which fuse to form a solid base of attachment for the
ilium. The sacrals, along with the caudals. begin to
exhibit dorsal fusions on the third day. In each species,
one individual (of nine in‘gL leucopus and eleven in

:7

f I
L3

it .‘

£‘_magigglg;u§) had the 2nd and 3rd fused. Four-day
individuals in‘g“ngggpg§ exhibited fusions in the 2nd or
3rd or both. §h,mggigglg£g§ had two of eleven with the

L3 ‘— .._
O

Wale‘zf‘ mmflv . Tm: ‘3].

2nd and 3rd fused.
The first sacral did not beome fused until the
fifth day' when one individual from each species had all
the sacrals fused. In addition. seven of eleven'gLIngggpgg
had at least one fusion.while four of ten B‘nggigglggng
had a similar condition. By the sixth day, only two of
twenty individuals lacked any fusion in the sacrum.
On the seventh day and ninth day, all £1,1gnsgggg
had fusion of at least one sacral vertebrae,‘while.§dnL
being; lacked one individual on each day. Hewever. on the
eleventh day,1§L1ngggpn§ lacked ossification in two out of
nine individuals, while LE2 25.13311 had all with fusions.
The 2nd and the 3rd sacral vertebrae appear to
ossify first followed by the first. The 2nd and 3rd first
appear on the third day. The first does not appear until the

fifth day in both species. The first never appears alone.

49

As with the other divisions of the vertebral column,
the data from the sacral vertebrae were analyzed with a
chi-square test with the hope of establishing the presence
or absence of significant differences between the species.
The data did not suggest that there was any significant
difference since each age class had not more than a total
difference of six fused vertebrae. The results of the
test at the .05 level of significance with six degrees of
freedom was a chi-square value of 3.294 (Table19). This

figure was significant at_between the .70 and .80 levels.

Table 19.--Chi-square analysis of the sacral vertebrae.

 

 

 

 

 

 

 

 

 

 

 

 

him 2.1mm
Day Obs. Exp. Obs. Exp. Total
3 2 2.09 2 1.91 4
4 8 6.28 4 5.72 12
5 15 13.61 11 12.39 26
6 22 20.41 17 18.59 39
7 25 26.69 26 24.31 51
9 30 28.79 25 26.21 55
11 21 25.12 27 22.88 48
123 122.89 112 112.01 235

 

 

 

 

 

 

8.

50

Table 19.-- continued.

2 2 2 2 2
9 09 (1172) (11392
Lil-52.0 4‘ 417+ + 6.28 "’ #5. 2 "' 13.61
2 2 2 2 .2
1 9 9 59 (1,692 (1,69)
* '127391' +"S%0?Z1' +“18?59l' * 26.69 + 24.31

2 2 2
(112122 (11212 (41122 (41122 2
+ 28.79 + 26.21 + 25.12 22.88 = x
1 081 219584 2 95 93 1 9
298%" + ‘¥T9T + 6.28 *“5T72§&' + '13761"’+"127391

5 8 41

+ ‘207411 +"18¥§%l‘ 1,.333331_ + 24. 1 *“28%g9’
114641 16 97 1619244 _ 2

+ 26.21 +"'2371%é' 22.88 7 x

 

 

 

 

.003 + .004 + .471 + .517 + .141 + .155 + .123 + .135
+ .107 + .117 + .050 + .055 +.675 + .741 = 3.294 = x2

A

Apparently there is no difference between the

species in the number of sacral vertebrae fused on each day.

932331.232323233
The caudal vertebrae present an unusual situation
in that not all of them posses neural arches. Only the
first five or six have neural arches while the remainder
of the 25 or 26 vertebrae consist only of the centrum.
In examining the tail, it was found that caudals

showed first fusion in the three-day individuals, with one

of nine L Wand two of eleven m M fused.

51

Each species had two four-day individuals with fused caudals
out of the same number of specimens as at three days.
Eight of eleven'gg Leucopgg showed caudal fusion in
five-day individuals. These ranged in number from one to
four in number in both species. By the sixth day, all
eleven m band; were fused and eight of nine E; W h
were fused. All individuals were fused on the seventh day. 71-1
The analysis of the caudal vertebrae was done on
only the first four arched vertebrae. A chi-square test was r
run with a .05 level of significance and six degrees of i
freedom and indicated no significant developmental differ-
ences between the two species. The chi-square value was

3.011 which fell between the .80 and .90 levels (Table 20).

Table 20.-- Chi-square analysis of the caudal vertebrae.

 

 

 

 

 

 

 

 

 

 

 

 

22121122228. 12.11.331.231.

Day Obs. Exp. Obs. Exp. Total
3 3 2.63 2 2.37 5
4 8 8.43 8 7.57 16
5 27 23.71 18 21.29 45
6 32 31.09 27 27.91 59
7 37 37.41 34 33.59 71
9 40 38.47 33 34.53 73

11 29 34.25 36 30.75 65

176 175.99 158 158.00 334

 

 

 

 

 

52

Table 20.-- continued.
2 3.3213 13.1.13 12133131422913
2.63 + 2. 37 + 28.43 + 7. 57 23. 71
42 .412
37.1"33.59

* 7%‘79l'+ 30. 909 +
%§?%%—+-%z-?%%3 “3:333- + %=

 

4Q

.1369 + :1369 + I1849 + I1839 10.8241 10 ,§24 1
2.63 2.37 8. 43 7. 57 23.71 21. 29

+ 30. 09 +’ ‘27§91 +‘ ‘l7filT 5933. + 23333;
$§3§+13639§L= 2

.052 + .057 + .021 + .024 + .456 + .508 + .027 + .027
+ .029 + .004 + .005 + .060 + .068 + .804 + .896 = 3.011

+ 34.53

' In .1.
. . . .|
r-.—

 

DISCUSSION AND CONCLUSION

As might be expected from a comparative study of
two closely related forms, the results of this study
indicate that there are few significant differences in
bone development between £12.11. m and L W. The
fact that some were found substantiates the belief that the
Alizarin-ROB clearing and staining technique is an
adequate tool in this type of research for comparisons at
the sub-generic level. In addition, the results of this
research suggest that the differences between the two
species in the rates of bone develOpment might at least
partially mirror the dissimilar environments of the two
species and the differential selective pressures inherent
therein.

The similarities between the two species were more
numerous than the differences. The rates of ossification
of the olecranon. patella, vertebral column (entire),
cervical vertebrae, sacral vertebrae, lumbar vertebrae,
caudal vertebrae, and the fusion of the tibia-fibula
exhibited no significant differences between the two
species. The degree of similarity between the species in
the various characters studied were not consistent and as

53

54

can be seen in the results of the statistical analyses,
the probabilities of the characters not being significantly
different ranged from 5-10% to 80-90%.

Those areas which exhibited significant differences
in bone development were the ankle, wrist, femural
condyles, and the thoracic vertebrae. The probabilities
that these structures were in actuality not significantly
different ranged between .02 and .001 indicating that
these structures were highly different. There is a
possibility that the two species differed in other areas
of bone development. but of the areas examined, only
these four showed differences between the species.

It was inferred earlier that these disparities in
ossification between Lg; 25.11.221- and L W may be the
result of differences in their habitats, as reflected in
differential selective pressure. This cannot be proven
with the results of this research; it can only be presented
as a logical possibility. Further evidence for this con-
tention could be obtained from an analysis of a series of
individuals of the woodland deer mouse, L W
5%. This species is very closely related to £3.93.
251391; however, it lives in habitats very'similar to
those of 22112399233. Were this sub-species to mimic the
bone develOpment of 2L11§gggpg§,more closely than that of
La m, credence could be attributed to the suggestion

55

that the more rapid development of the grassland forms
was due to the selective pressure favoring early mobility.

At this point, it might be noted that three of the
four structures with significant differences are directly
associated with locomotion (wrist, ankle. and femural
condyles). This compares with three structures directly
associated with locomotion which do no exhibit differences
(patella, olecranon, and tibia-fibula). In those areas
not associated with locomotion only one area, the thoracic
vertebrae, shows significant differences, compared with
five which do not. This indicates that skeletal elements
associated with locomotion show more variability than
those which are not, and thus may be influenced by
environmental pressures.

In considering certain structures, a number of
elements were lumped to produce an entity. such as the wrist.
However. if all the elements are considered separately.
there are a total of twenty-six. An interesting trend
may be seen in the days which each of these elements
first appears. Of the twenty-six characters studied,
LE... m had eleven which ossified before the corresponding
structures in1£212ggggpgg. Fifteen elements appeared
on the same day. while, in no case did a bone from‘fi‘

lggggpgg ever appear prior to a corresponding one in‘LJgL
23;;g1. In a sign test run on these data, the probability

56

that there is no significant difference between species
when 2pm, baigdi preceeds 22H2gg§gpg§ eleven times to none
is .000 (Siegal, 1956), as seen in Table 21. This

definitely suggests that ossification is tending to take

place in.£‘m‘ baggdi faster than in.£‘ 12392235.

Table 21.-- Days when ossification first takes place in
each species.

 

 

helm 231232929122

 

Ole cranon * 4 5

 

Distal ossification of
femur‘*

 

Ratella.*

 

Tibia-Fibula *

 

Astragalus

 

Calcaneus

 

Tarsal 1 *

 

Tarsal 2 *

 

Tarsal 3

 

Tarsal 4-5

 

Centrale'*

 

Tibiale *

 

Cervical vertebrae

 

Thoracic vertebrae

 

#MMVUIWkUINOOJ-‘Vk
nl-‘U'IMQO‘pr‘J-‘OOUIOG

Lumbar vertebrae

 

 

 

57

Table 21.-- continued.

 

 

 

 

 

 

 

 

 

 

 

 

 

§3g31bairdi lg; leuggpug
Sacral vertebrae 3 3
Caudal vertebrae 3 3
Ulnare 2 2
Pisiform * 6 7
Sesamoid 4 4
Centrale 2 2
Fused R-I * 3 5
Carpal 1 * 4 5
Carpal 2 4 4
Carpal 3 4 4
Carpal 4-5 2 2

 

 

 

* Indicates structure first appearing on different days.

 

The manifestation of the seventh sternal element
in both species represents an anomalous condition.
Skeletal variations are very common in mammals, parti-
cularly in island forms. There is no significant difference
between the species, even though.£&m‘ baigdi possessed this
structure almost twice as often as §‘_lggggpg§ (28.7%
vs. 14.7%).

The results of this research are of importance in

laying the groundwork for the study of some of the other

species in the genus Pergmyggug, particularly 2&m‘,g£gg;l;§.

58

Members of this genus have been studied intensively for
many years with the hape of determining ecological and
phylogenetic relationships. The KOH-Alizarin clearing
and staining technique may add a new avenue of research
in this area of Mammalogy.

The determination of an aging schedule is somewhat
tenuous. The great amount of variability within the
species precludes a high degree of precision. In all
cases an underestimation of age would result, as there are
a few individuals which exhibit bone development at least
one day prior to the majority. Since an aging table would
have to be based on the minimum ages of individuals
exhibiting ossified structures, an underestimation of
age would be likely.

Thus, this thesis presents the results of what
might be termed a pilot study for a more detailed research
project in the comparative osteology of the genus
Egggmxgggg. It establishes the methods and procedures
which may be used in analyzing other species, as well as
pointing to certain pitfalls which are inherent in this

type of study.

B IBL IOGRA PHY

Burt, William H., 1957, Magm32§12j13hg Greag Lgkgg
Regggn, University of Michigan Press, Ann Arbor,

Crary, D.D. and P.B. Sawin, 1949, "Morphogenic Studies in
the Rabbit VI. Genetic Factors Influencing the
Ossification Patterns of the Limbs", Genetics 34:
508-23.

. 1957, "Morphogenic Studies on the Rabbit XVIII.
Growth of Ossification Centers of the vertebral

Centra During the 2181: Day". as mm 82312139
127. 131-50.

, and Nancy Atkinson, 1958, "Morphogenic Studies of
the Rabbit XXI. The Nature of Disproportionate
Dwarfism Induced by the‘Qg Gene Revealed by Early

Fetal Ossification Pattern", Ameg$ggg13233351121
Angggmy 103(1): 69-98.

Hamilton, William J., Jr., 1939, Amgg;g§ghg§gmglg, McGraw-
Hill Book Co., New Yerk, 434 pp.

_- 1943.1112W23383mm “331313.
Cornell University Press, Ithaca, New York, 432 pp.

Hyman. Libbie H.. 1922. 291323233323 233323122113 M.
University of Chicago Press, Chicago. 544 pp.

Little. Malcomb Eu 1937. 32mm 12:. 3129. W.
Farrar and Rinehart Co., New York, 488 pp.

Nesslinger, Carlita L., 1956, "Ossification Centers and
Skeletal DeveloPment in Postnatal Virginia Opossum",

1£gggg§l1gfi Mammalggy 37: 382-94.

Rowett. H-Gan 1957. 2133933328 311.1333: in. 18.2 831;.
Rinehart and Co., New York, 64pp.

59

Sawin,

Sawin,

60

P.B. and Crary, D.D., 1956, "Mbrphogenic Studies
of the Rabbit XVI. Quantitative Racial Differences
in Ossification Pattern of the vertebrae of Embryos
as an.Approach to Basic Principles of Mammalian

0 wth". 3211:2191 MW
12‘? 62.-.?” 2‘

P.B. and Muriel Trask, 1965, "Morphogenic Studies
of the Rabbit XXXV. Pleiotropic Effects of the
Dachs Gene and the Radient Growth Pattern”, ggggngl

9;,flggphglggy 1171 87-114.

Siegal, Sidney, 1956, N

3.123333311333113
fighgxig;gl1§g;§ngg§, McGraw-Hill Book Co., New
York, 312 pp.

Wirtschafter, Zolton Tillson, 1960, Ihg_§gng§;§ g;

11911333113133.2118 3
Springfield, Illinois, 6 pp.

11113
A;1§§,.Thomas Co.,

 

llHIHIIIIIIHIIWIIUWIIHNIIlilllllllllHlHllll‘lli‘Il’l