AGE DETERMINATION AND AGE RELATED FACTORSIN THE TEETH OF WESTERN NORTH ATLANTIC
BOTTLENOSE DOLPHINSALETA A. HOHN
Division of Mammals, Smithsonian Institution, Washington, D.C.

ABSTRACT

Teeth were taken from 120 bottlenose dolphins, Tursiops truncatus, which
had stranded on the mid-Atlantic coast of the United States. The number ofannual growth layer groups (GLGs) for each animal was used to construct a
growth curve. The growth rate of coastal North Atlantic Ocean Tursiops issimilar to other cetaceans in having a high initial rate of growth, with no differences in growth between females and males. In females, the first dentinal
GLG is thickest and is followed by GLGs which become progressively narrower. In males, the second GLG is thicker than the first; GLGs beyond
number two become progressively smaller but at a slower rate than in females. In males and females, the translucent layer makes up proportionallylarger parts of the GLG as the animal ages, but in males the percent translucent layer remains constant at about 50% while in females it continues toincrease up to about 70% of the GLG. These two factors, GLGs width andtranslucent layer width, indicate that the sex and age of the animal influence
the deposition of GLGs. Incremental layers are also present, averaging 12
per GLG, and seem similar to incremental layers described in other marine
mammals. A plot of the relationship of percent growth of the last GLG totime of death suggests that the deposition of GLGs is relatively constant,at least during the first half of the year, and that North Atlantic Ocean
Tursiops give birth in the fall as well as in the spring.
INTRODUCTION

Owen (1945) documented the existence of "concentric layers of dentine" in odontocete teeth and, similarly, the "appearance of opaque striae or conecntric layers"(pg. 512) in polished sections of pinniped teeth. Lankester (1867) also noted alayering pattern in the teeth of a beaked whale, Z,iphius sowerbiensis (=Mesoplodon
bidens), but did not suggest a reason or use for them. In 1950, Scheffer suggested
using these layers as a method of age determination when he found layers in thedentine and cement of a fur seal, Callorhinus ursinus, canines that corresponded to
the known age of seals branded as pups and recovered up to eight years later.
Laws (1952, 1953) suggested using dentinal layers in odontocete teeth as a measureof age when he found layers in sperm whale teeth similar to those in pinniped teeth.
In the following years, dentinal growth layers (growth layer groups) were discovered and used as age indicators in Stenella coeruleoalba (Nishiwaki and Yagi, 1953,
Sci. Rep. Whales Res. Inst.,

No. 32, 1980, 39-66

40

HOHN

1954)i, Berardius bairdii (Omura et al., 1955), and sperm whales (Nishiwaki et al,1958).
In 1959, Sergeant described growth layer groups (GLGs) in the teeth of fourbottlenose dolphins, Tursiops truncatus, from Marine Studios in Florida. Two of theanimals were bor'n and died in captivity; two were wild caught and died in captivity. In each case the number of GLGs in the teeth corresponded with the known
or estimated age of the animals based on their time spent in captivity. The oldest
animal was estimated to be 15.5 years old and had 17 GLGs. He also examineda few teeth from the pilot whale, Globicephala melaena, and one each from the Atlantic whitesided dolphin, Lagenorkynchus acutus, and the common dolphin, Delphinusdelphis. All of the species demonstrated layering in the dentine similar to that of
Tursiops, which was identical to that described by Nishiwaki and Yagi (1953) for
S. coeruleoalba.
Since Sergeant's paper, dentinal GLGs have been used extensively for age determination of odontocetes. Most of the work has centered on direct applicationof the technique, predominantly for species involved in fisheries, for example, sperm
whale (Gambell and Grzeborzewska, 1967; Best, 1970; Gambell, 1977), delphinids(Sergeant, 1962, 1973; Kasuya, 1972, 1974, 1976; Sergeant et al., 1973; Kafaya etal., 1974; Perrin et al., 1976, 1977; Ross, 1977), the white whale (Sergeant, 1962),
Baird's beaked whale (Kasuya, 1977), harbor porpoise (Nielsen, 1973; Gaskin andBlair, 1977) and Dall's porpoise (Kasuya, 1978). However, some aspects of age determination and age related factors visible in the teeth of odontocetes have been
examined but not satisfactorily explained in many species. Some of these include
description and clear identification of GLGs, formation rate of the layers, identification of accessory layers and the more recently discovered incremental layers, andsexual dimorphism.
Since GLGs were first suggested as a means of aging odontocetes much controversy has prevailed over the meaning of these layers, but for the most part theassumption has been that GLGs are deposited annually. However, in a few species evidence based on relative age indices indicates that GLGs are not annual.
In the white whale, Delphinapterus leucas, Sergeant (1962) and Brodie (1969) haveshown circumstantially that more than one GLG is deposited at least during theearly growth of the animal, although Khuzin (1961) felt that only one GLG is formed each year. In the narwhal, Monodon monoceros, a species related to the white
whale, Hay (in press) also feels that more than one GLG is formed. Kleinenberg
and Klevezal (1962) found twice as many dentinal GLGs as maxillary bone layers
in the Black Sea dolphin, Delphinus delphis. In sperm whales, Ohsumi et al. (1963)
determined that one GLG is formed each year based on 11 whales captured sevento 16 years after being tagged, a rate which Best (1970) confirmed when he followed
the growth of the tooth from samples which extended over six months of the year.
Berzin (1961, 1964) and Gambell and Grzegorzwerska (1967), however, concluded
from samples taken throughout the year that two GLGs are deposited annually.
Perrin et al. (1977) suggest three possible hypotheses of rate of deposition of GLGs
in the spinner dolphin, Stenella longirostris, after examining 2500 specimens, none of
Sci. Rep. Whales Res. Inst.,
No. 32, 1980

AGE OF BOTTLENOSE DOLPHIN

41

which is that just one layer is set down per year. They accepted the hypothesisthat 1.5 GLGs are deposited in the first year, while one GLG is deposited in allother years, rather than a rate of 1.5 GLGs per year or 1.5 GLGs per year until
puberty and one GLG per year thereafter.
Direct evidence on the rate of dentinal growth has supported an annual formation rate. Sergeant's (1959) finding that the number of GLGs in Tursiops teeth
correlated with the known age or minimum known age for each animal offered thefirst measure. Similarly, in 1973, Sergeant et al. reported that in three captive
Tursiops from Marin eland of Florida the number of G LGs was approximately equal
to the known age of the animal. Klevezal and Kleinenberg (1967) examined captive and wild specimens from nine orders of mammals, including cetaceans, andconcluded that GLGs are an annual event. Tetracycline injected specimens havealso supported an annual deposition rate of GLGs (Best, 1970; Gurevich and Stewart, in press; Perrin and Myrick, in press).
Divergencies of opinion on the periodicity of GLG formation within a species
probably can be attributed to the presence of accessory layers, thin layers similar
to the hypomineralized layer of the GLG. Klevezal and Kleinenberg (1967) proposed that all odontocetes form one GLG per year but in some species accessory
layers are especially distinct, being almost indistinguishable from an annular layer,
so that it appears as though two GLGs are deposited annually in some species.
More commonly, however, accessory layers are not quite so distinct and may influence the count of GLGs to the extent that repeated counts on the same tooth by
one person or several people tend to give different results (Kasuya et al., 1974; Perrin, 1975; Kasuya, 1977; Kimura, in press; Perrin and Myrick, in press). The
clear distinction between an accessory layer and a GLG boundary layer is still not
defined in some species, for example, the killer whale (Orcinus orca), nor is the causeof accessory layers known.
_
Before the discovery of "annual" growth layers in dentine, research in dental
histology had shown the existence of smaller incremental growth patterns. In 1934,
Schour and Smith showed that in the continuously growing rat incisor 16 micro-·meters of dentine are laid down in 24 hours. In 1935, Schour and Steadman described an incremental stratification, also in the rat incisors, which appear as a succession of dark (well calcified) and light (less calcified) layers, the width of eachpair totaling 16 µm. Further examination (Schour and Hoffman, 1939) showed
the same 16 µm incremental mineralization pattern in 17 other species including
fish, a shark, crocodile, mastodon, and several species of mammals, including man.
These lines were later interpreted as incremental lines of von Ebner. Yilmaz, Newman and Poole (1977) described daily von Ebner lines in pig dentine where eachband consisted of parallel light and dark portions in thin sections of teeth. These
were probably equivalent to 5 µm daily von Ebner lines described by Krauss andJordan (1965) and Newman and Poole (1974). Von Ebner lines have recently
been described from the teeth of Stenella, where about 365 lines were counted within GLGs (Myrick, in press). Although their deposition rate has not been measured directly, the frequency of these von Ebner lines in the dolphin teeth suggests
Sci. Rep. Whales Res. Inst.,
No. 32, 1980

42

HOHN

that they represent a daily growth pattern.
In 1970, Scheffer found still a different frequency incremental layer in thin sections of a Dugong tusk. ·He described a pattern consisting of "coarse layers" and "finelayers". Kasuya and Nishiwaki (1978) found the same pattern in captive dugongs
where they defined coarse layers as annual growth layers or GLGs. There were 10
to 15 fine layers within each coarse layer, so the fine layers were suspected to bedeposited on a monthly or lunar cycle. Kasuya (1977) found the "long cycles"(GLGs) in Baird's beaked whale teeth to contain many (11.0 to 13.4) "short cycles".
Myrick (in press) noted incremental layers of the same periodicity in thin sections
from teeth of several species of dolphins. In addition, scanning electron micrographs of a small sample of etched Tursiops teeth also showed subannual incremental
layers, larger than van Ebner lines, which probably represent the same growth pattern seen by Myrick and Kasuya, appearing as a finer mineralization pattern within the mineralization pattern of the GLG (Hohn, in press). Although the deposition rate of these incremental layers has not been measured directly, this circumstantial evidence suggests a "monthly" growth or mineralization cycle which may
be useful for more precise age determination, possibly back-dating the month ofbirth in young animals, or a better understanding of the mineralization patterns in
dentine.
Sexual dimorphisms have also been described in the teeth of some odontocetes. The identification of sexual dimorphism in the growth of teeth has two
advantages. First, skeletal material currently in museum collections that is missing
data on sex may be more useful for systematic or other research if the sex can be
determined from the material on hand, such as the teeth. Secondly, differences in
the growth of teeth or other mineralized tissue probably reflects differences in habits
or intrinsic factors between the sexes. These differences may help explain the causeof formation of growth layers.
Van Utrecht (1969) found a sexual dimorphism in the extent that the neonatalline overlaps the cementum in teeth of the harbor porpoise, Phocoena phocoena, anda small sample of the white-beaked dolphin, Lagenorhynchus albirostris. Although
Nielsen (1972) confirmed this finding in the harbor porpoise, it has not been described in other odontocetes. Another sexual dimorphism in the teeth of Phocoena
is found in the ratio of the width of the opaque layer to the width of the translucent
layer (Gaskin and Blair, 1977). In males, this ratio. remains constant while infemales it decreases steadily with age. Gaskin and Blair (1977) also found differences in the amount of dentine deposited annually, which they attributed to size
differences of the sexes at any given age. Similar or additional sexual di morphisms
in the teeth have not been described in other odontocetes.
The purpose of this study is to describe dentinal GLGs and accessory layers,
including their appearance and position, in western North Atlantic Ocean bottlenose dolphins; to examine age-related changes in GLG deposition and any sexual
dimorphisms in dentinal characteristics; and to construct a growth curve for coastal North Atlantic Ocean bottlenose dolphins.
Sci. Rep. Whales Res. Inst.,
No. 32, 1980

AGE OF BOTILENOSE DOLPHIN

43

MATERIALS AND METHODSThe Sample.

Teeth were collected by the Smithsonian Institution's Marine Mammal Salvage Program (MMSP) from Tursiops stranded along the Atlantic coast of theUnited States between South Carolina and New Jersey. 118 of these animals
have complete data, including sex and total length. Two specimens were missing at least one piece of data and were included for GLG counts but not in otheranalyses.

Preparation of Sections
At least one tooth was taken from each specimen. A medial longitudinal cut
was made on each of the teeth on a Buehler lsomet 11-1180 low speed saw using
a rotating diamond blade with a saw speed of 5 to 6 on a relative scale of 1 to 10(up to 300 rpm), following the method of Hohn (in press). One of the halfsections from each tooth was prepared for scanning electron microscopy. These
were soaked in 5% formic acid for 3 hours, rinsed in water for at least one-halfhour to remove the acid, cleaned in an ultrasonic cleaner with acetone. for 15 to 60
seconds to remove extraneous surface particles, dipped in 70% ethanol to dehydrate
and air dried. These etched sections were examined by scanning electron microscopy (SEM) (Hohn, in press). During an SEM session, a standard set of micrographs was taken which included serial micrographs of the entire tooth at low magnification (15 to 20 X ), requiring up to five micrographs, and higher magnifications(greater than 25 X) for difficult to count, narrow GLGs, GLGs bordering the pulp
cavity, and possible incremental layers. Specimens were prepared for SEM not
more than two or three days before each session to help reduce their absorbing
moisture.
For each tooth, a 150 µm thin section was also prepared. In most cases, thecut surface of the remaining half section was glued to a microscope slide and all
but 150 µm cut off leaving the thin section adhering to the slide. The thin sections
were removed by dissolving the adhesive in acetone, then mounted for examination
on a clean slide.

Data Collection and Anafysis
GLG counts were made for all of the specimens from the SEM micrographs
and from the thin sections using a dissecting microscope with transmitted light (at25 X ). The number of GLGs was plotted against total length for each animal.
The total length of animals in each of the first 3 age classes (GLGs) was comparedfor males and females to examine differences in growth rates. The sample sizes
were too small for statistical analyses for animals with more than four GLGs.
Measurements for GLG and translucent layer widths were taken for 19 females
and 19 males from the thin sections using a dissecting microscope (at 50 X) equipped with an ocular micrometer and a polarizing filter. Measurements of the first
few GLGs were made near the base of the neonatal line on the concave side of theSci. Rep. Whales Res. Inst.,
No. 32, 1980

44

HOHN

tooth. In sections in which it was difficult to measure at this point because of unclear boundaries, the GLG width was determined closer to the crown or tip of the
tooth. The width of the first GLG is approximately the same from the base of theneonatal line to below the crown so a slight change in position did not affect themeasurements. Later layers were measured where they were most clearly defined but in the upper half of the length of the GLG before it becomes compressed in
the root, and below the apical end where the layers are especially wide. Sections
that did not allow determination of boundaries of adjacent G LGs were not included.
Preliminary examination suggested the growth of the tooth by width of GLGs
to be different in males and females. To test this, the width of each GLG was determined for each sex and compared using a Mann-Whitney Rank Sum test. In
addition, the ratio of width of the translucent layer to entire GLG was plotted against GLG number, again considering males and females separately.
The optical density and extent of growth of the last GLG were determined andplotted against time of death for 16 males and 12 females whose teeth had fewer
than four GLGs. Older animals were excluded because the GLGs become too narrow, and sometimes the boundaries too irregular, to get accurate measurements ofa partial layer.
Incremental layers and accessory layers were treated separately. The numberof incremental layers per GLG was determined where all of the increments of a
GLG could be distinguished. The widths of individual increments were measured
by position within different GLGs, i. e., on a ridge or in a groove for GLG 1, 2, or
3, or beyond GLG 3, using the SEM micrographs. The distribution and appearance of irregular accessory layers was noted. For accessory layers that appeared
more regularly, their position within the GLG was measured.
Five variables were tested specifically for possible sexual dimorphism related
to the neonatal line. First, the extent of the neonatal line beyond the bottom of
the enamel was measured in thin sections on the concave and convex side of 42
teeth from mixed males and females. This is similar to the study by van Utrecht(1969). In addition, the width of the prenatal zone at the base of the enamel was
determined, also from thin sections on both sides of the tooth. Finally, the widthof the translucent neonatal line was compared in females and males. Means foreach measurement were compared for males and females using a Student's t-test.
RESULTSAge and GrowthA scatterplot of 64 females, 54 males and two specimens of unknown sex shows
the general growth curve for Tursiops from the western North Atlantic Ocean (Fig.
1). For each of the first few age classes, including neonates, and for a cumulative
average for animals with more than four GLGs, the mean total length is not significantly different for males and females using a Mann-Whitney test (Table 1). The
minimum and mp.ximum total lengths within each age class indicates the large overlap in total length of individuals in different age classes. The decrease in growth,
Sci. Rep. Whales Res. Inst.,
No. 32, 1980

AGE OF BOTTLENOSE DOLPHIN

45

300
280

••

260

~ 240

2{

~ 220

c:Ji

~

I-

~ 200

z

~ 180
f-

120
100

•••

••

ic:i
l{i)

:;i 160
~ 140

(!)

c:J6

66

~

~

(!)

80 '"-:o!:--:2=--4':---'.5=--8=--170~17
2-1~4~1~6~1~8~2~0~2~2-2~4-2~6--='28

NUMBER OF GLGS
Fig. 1. Scatterplot of the number of growth layer groups (GLGs) to the nearest
half-GLG and total length for 120 western North Atlantic Ocean Tursiops. Circles
are females, triangles are males, diamonds are animals whose sex is unknown.
Solid symbols represent a closed pulp cavity.

TABLE l.

SOME DESCRIPTIVE STATISTICS FOR TOTAL LENGTHSOF TURSIOPS BY NUMBER OF GLGS AND SEX
Total Length (cm)

No. of GLGs

Sex

N

0
(Neonates)

F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F&M

9
9
0
2
6
9
2

0.5

1.5
2
2.5
3
4
More than
4 GLGsSci. Rep. Whales Res. Inst.,
Na. 32, 1980

11
8
2
1
6
8
4
2
24
14
38

Mean

Min.

Max.

116
118

95
110

132
134

153
180
172
184
198
183
196
207
191
210
203
220
220
249
250
250

146
155
143
183

159
200
189
185

168
174
206

205
210
207

205
189
214
216
232
226
226

223
217
227
223
275
284
284

HOHN

46

large ranges in total length, and small sample size for animals with more than four
GLGs allows for the determination of average "adult" total length of approximately 250 cm.

Description of GLGs
For the most part, GLGs in thin sections of teeth of these Tursiops appear as
described in other odontocetes, particularly delphinids and Tursiops (Sergeant, 1959),
as alternating translucent and opaque layers (see Perrin and Myrick, in press).
The prenatal zone is a relatively uniform area easily distinguished by its lack of
substructure, e. g., incremental and accessory layers, seen in post-natal dentine.
700
600
500

0

tl 400

.A

A

:r:

s

0

s 300

(,!j

200
100

.......

o~~~~~~~~~~~~~~~~

0 1 2 3 4 5 6 7 8 910111213141516

GLG NUMBER
Fig. 2. Average width of GLGs for females (circles) and males (triangles), in micrometers. Measurements based on thin sections of teeth from Tursiops.

The neonatal line is a narrow translucent layer usually followed by a narrow, intense opaque layer. The first GLG begins after the neonatal line with a wide,
variably opaque layer riddled with accessory layers, and ends with a narrow translucent layer often less apparent than some of the accessory layers. The second
GLG is similar to the first in having the wide, variably opaque layer followed bya narrow, relatively, but indistinctly, translucent layer. The third and sometimes
fourth GLGs have more distinct opaque and translucent layers and, although accessory layers can still be seen, they are not as overwhelming as in the first two
GLGs. Subsequent GLGs have better demarcated opaque and translucent layers,
less variability in mineral densities within a layer, and fewer accessory layers.
The same basic description is applicable to etched half-sections where opaquelayers are equivalent to grooves and translucent layers are equivalent to ridges.
Sci. Rep. Whales Res. Inst.,
No. 32, 1980

47

AGE OF BOTTLENOSE DOLPHIN

However, the numerous accessory layers in the first few GLGs are usually not asprominent in the etched sections. The exception is when the mineral density differences, normally accentuated by etching, are small across a GLG (usually in thefirst two GLGs), then incremental layers and accessory layers in the opaque zoneof the GLG are almost indistinguishable from the translucent (boundary) layer of
the GLG. This also occurs in thin sections. Furthermore, in etched sections,
there are not clear boundaries between GLGs, i.e., there is not an abrupt end to
the translucent layer or ridge. Rather, the topography of the surface might belikened to rolling hills with a rounded crest and slopes, and valleys inbetween.
This does not allow for clear points or ends of layers from which to make measurements.
In general, the widths of GLGs decrease as the animals get older (Fig. 2).
The first few GLGs are the largest and the last few are the smallest (Table 2).
More specifically, in females, the first GLG is the widest averaging 509 µm, theTABLE 2. SUMMARY OF DESCRIPTIVE STATISTICS FOR TOTAL GLG WIDTHSMEASURED FROM THIN SECTIONS OF TEETH FROM TURSIOPS.
SIGNIFICANT DIFFERENCES DETERMINED USING A MANNWHITNEY RANK SUM TEST. MEASUREMENTS
IN MICROMETERS
GLG No.

2

3

4
5
6

7
8
9

10
11
12
13
14
15

Sex

N

Mean Width

Min.

Max.

F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
M
M
M

19
19
12

509
407
393
480
272
331
285
269
253
241
225
203
194
203
171
181
148
174
114
152
143
181
162
162
133
114
100

418
342
285
380
190
190
228
209
171
152
152
152
152
152
95
133
95
114
76
114
114
133
133
152
95
95
76

570
475
475
608
342
418
380
342
342
380
285
266
266
247
247
228
228
228
190
209
171
209
190
190
171
133
135

Sci. Rep. Whales Res. Inst.,
No. 32, 1980

II

6
10
6
7
6
6
6
6
5
6
5
6
5
6
4
5
2
4
2
4
3
2
4

Significant
Differencesp<.005
p<.05
p<.05

HOHN

48

TABLE 3. SUMMARY OF DESCRIPTIVE STATISTICS FOR TRANSLUCENT LAYERWIDTH DIVIDED BY GLG WIDTH (=PERCENT TRANSLUCENT LAYER) FOR
EACH GLG MEASURED FROM THIN SECTIONS OF TEETH FROMTURSIOPS. SIGNIFICANT DIFFERENCES DETERMINED USINGA MANN-WHITNEY RANK SUM TEST. RESULTS
EXPRESSED AS PERCENTS
GLG No.

2
3
4
5
6
7
8
9
10
11

12
13
14
15

Sex

N

Mean Width-%

F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
F
M
M
M
M

19
19
10
11
6
9
6
6
6
6
6
6
5
6
5
6
5
6
4
6
2
4
2
4
3
2
4

9
10
15
14
39
23
40
33
41
41
48
41
48
45
50
49
57
48
57
45
73
44
71

52
51
47
50

Min.

Max.

4
4
4
18
5
17
13
22
10
33
14
38
36
33
23
40
33
50
27
67
27
70
38
50
43
50

17
14
33
32
67
50
60
50
56
75
36
56
58
50
60
57
71
67
67
56
78
57
71
63
52
51
51

Significant
Differences

p<.10

p<.10
p<.05
p<.05

second is smaller at 393 µm, with subsequent GLGs decreasing in width. An exception is a slight increase in size at GLGs 11 and 12 which might be an artifactof small sample sizes (N=2). In males, the trend is similar except that the second
GLG, which is 480 µm, is larger than the first, which is 407 µm. The third GLG
is smaller than the first and subsequent GLGs follow the same pattern as in theteeth of females. Again, there is a slight increase in size at GLG 11. Measurements of the widths of GLGs I, 2 and 3 are statistically significantly different between males and females (Table 2).
The width of the translucent layer follows the opposite pattern. GLGs set
down in older animals have wider translucent layers or the width of the translucent layer to the entire GLG (percent translucent layer) increases (Table 3). In
females there is a leveling off of percent translucent layer at GLGs 6 through 8,
then an increase for subsequent GLGs. In males, the percent translucent layer
Sci. Rep. Whales Res. Inst.,
No. 32, 1980

49

AGE OF BOTTLENOSE DOLPHIN

increases initially, then remains fairly constant after GLG 4. This gives GLGs in
the teeth of male Tursiops a more even appearance in size and spacing, which isespecially apparent in SEM micrographs (Plate I, Fig. 1).
The translucent layer widths between females and males are statistically significantly different at GLGs 1, 3, 6, and 12. The percent translucent layer is significantly different between females and males at GLGs 3, 10, 11, and 12 (Table 3).
There are no measurements for females beyond GLG 12.
80
(')

(')

70
60
f-

(')

50

(')

zw

(')

...

~ ...

(')

......

......

...

...

Cl(')@ ...

~ 40
w

...

a..

30
20
10
0

~

o 1 2 3 4 5 6 7 8 91011121314151617
GLG NUMBER

Fig. 3. Average percent translucent layer for each GLG.
triangles are males.

Circles are females,

Last GLG
A plot of percent growth of the last layer against time of death for 28 female
and male Tursiops shows most of the points to set roughly on a line representing
the expected growth of the current GLG if the deposition of dentine is constant andthe new GLG begins in late January or early February (Fig. 4, Table 4 ). For thecluster of 14 points which occurs during February and March, with four additional
points in May, the extent of growth of the GLGs increases proportionally with timealong this line. Some of the points which do not fit this line fall on or close to another line representing the expected tooth growth of animals whose new GLG
begins in September. However, there are only six specimens representing thisgroup.
In most cases the last layer is opaque (Table 5 ). The translucent laye~ borders the pulp cavity in four of the specimens, and one of these is a translucent accessory layer and not a GLG boundary layer. Two of the GLG translucent layers
Sci. Rep. Whales Res. Inst.,
No. 32, 1980

HOHN

50

100
/

90
80

/

b.

/
/

:::c

/

/

/

/

6

/

70

/

6

f-

s0 60

A

f-

/

a:
~ 50

/

/

(!)

/

(!)

/

/
/

/

zUJ 40

/

(.)

a: 30
UJ

(!)

a..

20
/

10
0

/

/

/

/

/

/
/

1

2

3

4

5

6

7

8

9

/

10 11 12MONTH

Fig. 4. Scatterplot of the percent growth of the last GLG in teeth of young Tursiops
on approximate date of death. Percent growth calculated for each animal from
actual width of the last layer divided by the average total width of the GLG. So]jd
line presents expected growth of the GLG throughout the year if the GLG begins
in lateJan.-early Feb. and grows at a constant rate. Hatched line represents thesame for GLGs which begin in September. Circles are females, triangles are
males. Hollow symbols indicate an opaque last layer, solid symbols represent atranslucent last layer.

occur in late January to early February and one occurs in September. The accessory layer that falls in mid-March is an animal whose dentinal growth pattern falls
on the line for GLGs which begin in the fall.

Non-GLG Layered Structures
In addition to GLGs three layered structures can be differentiated in the teethof Tursiops. Incremental layers are relatively evenly spaced layers which are foundthroughout the GLGs and are most easily seen in SEM micrographs of etched sections. Accessory layers are layers of opposite mineral density that would be expected within a GLG with an even mineral density. The third structure is similar to an accessory layer because it appears in a layer of opposite mineral densityof the dentine adjacent to it.
Incremental layers could be counted and measured from some of the SEM
micrographs (Plate I, Fig. 2). The average number of incremental layers per GLG
is 11.8 for three GLGs from 39 specimens (Table 5 ). Of the three GLGs in which
incremental layers could fairly consistently be counted, GLG 2 was the easiest for
counting. Incremental layers are rarely visible in the compressed GLGs set down
Sci. Rep. Whales Res. Inst.,

No. 32, 1980

AGE OF BOTTLENOSE DOLPHIN

51

TABLE 4. PERCENT GROWTH OF THE LAST FORMED GLG. GROWTH OF THE
LAST GLG AND TIME OF YEAR OF DEATH FOR SOME TURSIOPS MALES
AND FEMALES. PERCENT LAST LAYER CALCULATED FROMEXPECTED AVERAGE SIZE OF THE FULLY FORMED GLG.
OPTICAL DENSITY OF THE DENTINE BORDERING THE
PULP CAVITY IS BASED ON THIN SECTIONSUSING TRANSMITTED LIGHT
Catalogue
No.
Females504565504590504403CWP094
SEAN3331
504539504550504592504399504583395179504528504549Males
504578
JGM401504536504553504567504218395790504535504561504591504290504122504551504765CWP084504291504313% Growth
Last GLG

Approx. Dateof Death

OpticalDensity

2
2
2
2
2
2
2

31
26
26
37
50
56
0
14
28
21
63
0
21

late Feb
mid-March
15 May22 Maymid-Auglate Dec
early Feb
30 March
15 Mayearly Mar
4 Nov
early Dec
mid-Feb

opaqueopaqueopaqueopaqueopaqueopaquetranslucent
opaqueopaqueopaqueopaquetranslucent
opaque005480late Feb
14 Dec21 Janmid-Feblate Feb
I March
22 Mayearly Janearly Marearly Mar
mid-June
21 Sept
11 Feb
14 Marlate Mar
27 June3 Septtranslucent*opaqueopaqueopaqueopaqueopaqueopaqueopaqueopaqueopaqueopaqueopaqueopaqueopaqueopaqueopaquetranslucent

No. ofComplete GLGs

9
21
17
25
92
2
2
2
2
3
3
3
3
3

77
25
35
60
13
13
44
71
0

* translucent accessory layer.

in the teeth of "old" animals, e.g., beyond GLG 10, in etched sections. Even in
the first few GLGs incremental layers were not always clear enough to count. On
the etched surface of the tooth, they were most clear on the "slopes" rather thanon the top of a ridge, where one prominent very wide increment often turned out
to be two, or in the grooves, where it is was "dark". The incremental layers havethe same three-dimensional pattern as GLGs and are only visible when there is acontrast between the ridges and grooves of adjacent increments.
Sci. Rep. Whales Res. Inst.,
No. 32, 1980

52

HOHN
TABLE 5. NUMBER ·OF INCREMENTAL LAYERS PER GLG
IN TURSIOPS DENTINE
Catalogue No.
29147250412229143129140329140212277
291462CWP088

GLG l
12-13
9
12

191456504534504864504726291498291466395430395179504561
JGM39929142614

Means
Std. dev.

GLG 2
12
13

GLG 3
12

>3 GLGs
10-11

11

13
10

8-9
11-12
9

12
13
12

14
12-13

11
11

11
11
9
12
11

11

12
11-12
10
10
11-12

10-12
10

12
11
11

12.2
1.6

15

11

Summary
11.8
1.2

11.3
l. l

10.8
1.2

The widths of the incremental layers varied slightly (Table 6). Their mean
width in GLG 1 was 26 µm and 23 µm, for ridges and grooves, respectively, 28 µm
and 26 µm in GLG 2, 27 µm and 24 µm in GLG 3, and 23 µm and 20 µm for measurements made from GLGs later than three. It appears, in general, that increments in the grooves are smaller than those on the ridges in the same GLG and thatthe increments become smaller with successive GLGs.
Conversely, accessory layers tend to be variable in position and intensity, but
are usually thin translucent layers or lines within the opaque layer of the GLG.
In addition, in each of the first two GLGs there is an especially wide, prominent
layer about halfway through the GLG (Plate II, Fig. 1). In the first GLG, thisaccessory layer occurs an average of 50% through the GLG in females and 64% of
the width of the GLG in males. It occurs approximately in the middle of thesecond GLG in females and males. In some teeth these accessory layers are more
conspicuous than the GLG boundary layer. This is particularly true since in thefirst two GLGs the translucent boundary layer is very narrow. In GLGs beyond
number three, accessory layers become less frequent and the center, wide accessory
layer is not as pronounced. Accessory layers are not found in the compressed GLGs
set down in old animals.
A third non-GLG layered structure might also be called an accessory layer.
These layers occur adjacent to any accessory or GLG boundary layer as very inSci. Rep. Whales Res. Inst.,
No. 32, 1980

AGE OF BOTTLENOSE DOLPHIN

53

TABLE 6. WIDTHS OF INCREMENTAL LAYERS IN TURSIOPS DENTINEMEASURED FROM SEM MICROGRAPHS OF ETCHED SECTIONS.
MEASUREMENTS ARE SEPARATED BY THEIR LOCATIONWITHIN A GLG AND BY GLG NUMBER.
MEASUREMENTS IN MICROMETERS
Cata!.
No.

29147250412229143129140329140212277
291462

GLG 1

GLG 2

GLG 3

Ridge

GrooveRidge

GrooveRidge

Groove

Ridge

38.8

35.4

34.6

25.4

26.6

20.3

29.8

20.0
25.0

26.0

28.9
24.0

36.3

23.9

34.5

31.8
27.5

25.8
32.0
29.3

32.5

25.0
20.0
30.0

25.0
32.5

Groove

17.5
22.8

22.5

31.5

30.8
16.8
20.3

CWP088291456504534504864504726291498291475291466395179504561
JGM399504532291426395430>3 GLGs

22.1
22.5

18.9
15.0

19.8
20.3
18.4
31.0

14.3

20.2
35.7
15.2
22.8

14.4
47.7
29.9

31.3

16.3

28.6
18.0

40.0
15.1

11.9

20.0
28.6

17 .1
16.7

28.6
29.2
29.8

20.0

26.6

28.0

27.8
22.5

23.9
7 .0

23.0
4.7

20.0
SummaryMeans
26.2
Std. Dev.
7.5
Overall mean
Std. Dev.

23.4
8.4

27.6
8.7

26.2
5.7

26.6
9.2

20.2

26.1
7.0

tense narrow bands of the opposite mineral density as the structure they abutt.
Most often, this layer is opaque and follows a translucent layer. It is very noticeable following the neonatal line, the wide central accessory layer, and some of theirregular accessory layers throughout the GLGs. When this structure occurs bythin translucent boundary layers, it is sometimes more prominent than the translucent layer so more easily used as the boundary layer (Plate II, Fig. 2).

Sexual Dimorphism
Measurements of four of the five variables specifically tested for possible sexual dimorphism similar to that found by van Utrecht (1969) proved not to be significantly different (Table 7). The difference in the fifth variable, neonatal line
width, was highly significant (p<.005) using a t-test with males having a wider
neonatal line than females.
Sci. Rep. Whales Res. Inst .•No. 32, 1980

54

HOHN
TABLE 7.

SUMMARY OF MEASUREMENTS FOR POSSIBLE SEXUALDIMORPHISMS IN THE TEETH OF TURSIOPS.
MEASUREMENTS IN MICROMETERS

Variable
Extent neonatal line extends
beyond enamel, convex side*
Extent neonatal line extends
beyond enamel, concave side*
Width of prenatal zone, convex
side*
Width of prenatal zone, concave
side*
Neonatal line width convex
side**

Sex

N

Mean

S.E.

F
M
F
M
F
M
F
M
F
M

13
18

418.0
471.8
429.2

64.2
52.5
54.4

405.3

53.3

58.5

4.4

54.4

4.3

21

54.6
48.1
27.0

5.2
4.6
0.8

22

37.3

1.3

17
18
13
18
16

18

* Not significantly different using Student's t-test.
** Significant at p< .005.

DISCUSSION

Age and Growth
The growth rate of Tursiops is similar to that of many other odontocetes.
During its first year, an average western North Atlantic Tursiops increases its lengthby 53%, approximately the same increase found for the same age animals of Stenella attenuata (Perrin et al., 1976), S. coeruleoalba (Kasuya, 1972, 1976), Globicephalamelaena (Sergeant, 1962), Tursiops from Florida (Sergeant et al., 1973), and Tursiops
from South Africa (Ross, 1977). The rate of growth decreased during the second
year of life, averaging only a 10% increase in total length in these Tursiops and an
average of 10 to 15% in the other species mentioned above. After three GLGs thegrowth rate slows cbnsiderably.
Cetaceans have been described as having high rates of growth while they areyoung (Bryden, 1972). However, there is a great deal of variability in total lengths
of individuals in any age class. In S. longirostris, animals with one GLG range fromabout 100 cm to 135 cm in total length, those with two GLGs range from 110 to145 cm, with equivalently large variation in older animals (Perrin et al., 1977). In
pilot whales, G. melaena, neonates range from 165 to 195 cm in total length (Sergeant, 1962). A sample of harbor porpoises from the Bay of Fundy ranged from75 to 105 cm at birth, 105 to 130 cm at one year, 118 to 135 cm at two years, 125
to 145 cm at three years, again continuing this variation in length within each age
category for older animals (Gaskin and Blair, 1977). The large variability andoverlap of different aged animals illustrates that, although it is useful to construct
growth curves and use mean total lengths for examining growth rates, it is not reliable to use total length to determine the age of an individual.
In the western North Atlantic Tursiops an especially large range in total length
exists in each age class. This may be due to strandings of individuals from two
different population of Tursiops, coastal and offshore, where the offshore animals
Sci. Rep. Whales Res. Inst.,
No. 32, 1980

AGE OF BOTTLENOSE DOLPHIN

55

seem to be larger than those found along the coast CJ. G. Mead, unpub. data).
At this time, however, it is not possible to differentiate to which population a young
stranded animal belonged, so all specimens less than 280 cm have been included.
Differences in adult total length or rate of growth between males and females
have been found in some cetaceans. When it occurs, generally males are larger
than females as adults in odontocetes (e.g. pilot whales and sperm whales), whilein mysticetes males are smaller than females as adults (e.g. humpbacks and fin
whales) (Bryden, 1972). But delphinids generally have not been found to be dimorphic in adult total length (True, 1889; Sergeant et al., 1973). The results fromthis study seem to support this.
Two studies have been published on the growth rate of wild Tursiops. Sergeant et al. (1973) looked at Tursiops from Florida waters. Although they reported
birth to occur at 100 cm, they present no data for neonates. The growth curve,
based on 22 males and 24 females, is similar to that of western North Atlantic
Tursiops but with younger animals (less than five GLGs) apparently smaller in
Florida. The apparent difference between the two groups may be due to differences in our definition of GLGs. The asymtotic length of males from Florida wasfound to be 270 cm and that of females to be 250 cm. The coastal Tursiops in thisstudy appear to have the same asymptotic length for males and females at 250 to
260 cm. Ross (1977) gives some biology of T. aduncas from South African waters.
The age of birth ranged from about 84 cm to 112 cm. Females reached sexual
maturity at a smaller length than males. The largest female was 252 cm and thelargest male 254 cm which makes them smaller than the western North Atlantic
Tursiops. Although the Tursiops from Florida waters might be the same type as
those from further north along the coast, those from Africa are probably not.

GLG Widths
As a dolphin tooth grows and deposits more dentine, the pulp cavity becomesincreasingly smaller until it finally occludes and no more dentine deposition canoccur. Correspondingly, as the pulp cavity becomes smaller, the GLGs become
narrower. This decrease in GLG width could be solely the result of the new forming GLG being compressed into a smaller pulp area. If this is the case then each
GLG would be a little smaller than its preceding GLG and a plot of the relationship between GLG width and number would be approximately a straight line witha negative slope. If this relationship follows a different pattern then it would suggest that there are other factors influencing the deposition or mineralization ofdentine.
There are some general patterns that can be found in a plot of average GLG
width against GLG number that suggest that physiological factors affected by theage or sex of the animals are important in GLG growth. These patterns can bevisualized in Fig. 2. First, there are significant differences between males andfemales in the widths of the first two or three GLGs. It is particularly interestingthat the second GLG is, on the average, wider than the first GLG in teeth frommales, and that the third GLG decreases so that it is narrower than the first. SecSci. Rep. Whales Res. Inst.,
No. 32, 1980

56

HOHN

ondly, the changes in width of the first three GLGs is large, but subsequent GLGs
show a marked decrease in this rate, i.e., GLGs following number three are notmuch narrower than their adjacent preceding GLG. This appears to be an affectof age of the animals since it occurs in females and males. Again it is interestingthat this abrupt decrease in GLG deposition rate occurs at the same time that thegrowth curve of Tursiops (Fig. 1) shows a noticeable decrease in rate of growth.
Thirdly, the average width of each GLG is larger in females than in males from
GLGs four to six and smaller in GLGs which follow. The effect is an almost constant decrease in width of GLGs four through ten in females. In males, the rate ofchange in width of GLGs appears smaller than in females for GLGs six through ten.
In an etched section of tooth, these GLGs appear evenly spaced and sized acrossthe surface and almost can be considered diagnostic to determine teeth from males.
Similarly, the characteristic constant decrease in size of GLGs in teeth from females
is apparent in etched sections. Perrin et al. (1977) plotted the percent decrease inwidth from one GLG to its adjacent GLG in the spinner dolphin beginning at GLG
3 and continuing to GLG 16. They found that each GLG average more than 95%
of the width of the preceding GLG. They did not consider females and males separately, which in Tursiops teeth gives different results, as discussed above. A progressive decrease in width of GLGs has also been described from the harbor porpoise (Nielsen, 1972; Gaskin and Blair, 1977), striped dolphin (Kasuya, 1972), andthe pilot whale (Sergeant, 1962). Christensen (1973) found an initial decrease inbottlenose whales followed by constant thickness after the fourth GLG. Except for
Gaskin and Blair (1977), measurements of widths were not given, nor were specificdifferences by sex or age noted. The mean widths of GLGs given by Gaskin andBlair (1977) show that GLGs are wider in teeth from female harbor porpoises thanthose from males.
The translucent layer can also be used as an indicator of physiologicalchanges affecting GLGs. The absolute value of the width of the translucent layer
is probably not an important parameter by itself, except to help determine the timespan over which it is deposited, because it is dependent on the GLG number andwidth. The percent translucent layer is a more useful quantity. As with GLG
width, the trend in percent translucent layer is different in females and males although significant differences occur only in GLGs 3 and 10 through 12. In females, the percent translucent layer increases almost continuously with age.
Beyond GLG 10 more than half of the GLG consists of the translucent layer, reaching about 70%. This trend appears as an initial rapid increase in percent translucent layer which levels almost as a plateau (between GLGs 3 through 8) then increases rapidly again after GLG 8. This is noticeably different than males where
an initial rapid increase in percent translucent layer leads to a nearly constant level,with some fluctuation. The translucent layer comprises only about 50% of the
GLG after GLG 8. The change in percent translucent layer around GLG 8
through 10 in females and males may be related to physiological changes associated with the onset of sexual maturity, since this seems to be approximately the age
at which the animals become sexually mature (Harrison, 1969; Ridgway, 1972).
Sci. Rep. Whales Res. Inst.,
No. 32, 1980

AGE OF BOTTLENOSE DOLPHIN

57

The changes at GLGs 3 to 4 may be influenced by the decreasing growth rate atthat age. The large percent translucent layer in females beyond age ten can be
speculated about with even less certainty. The most obvious explanation since
there are such large differences between males and females would be that the wide
translucent layer, which is hypomineralized (Hohn, in press), is the result of a calcium drain caused by reproduction. At this time, there is no direct evidence forthis. Gaskin and Blair (1977) noticed a progressive increase in proportional thickness of the translucent layer with age in harbor porpoises but only in females, sothey attributed it to calcium mobilization. They constructed a regression line
through a scatterplot of the proportion of opaque layer to translucent layer but did
not mention specific differences at any age. However, their results, along withmine, indicate that percent translucent layer is worth examining in other species
for possible dimorphism and explanation for the formation of the layers.
Last GLG
Most of the strandings of Tursiops occurs during February and March. Since
calving also occurs during this season, it should be about this time when the neonatal line appears in the teeth. If the deposition of dentine is fairly constant, animals
born in the late winter to early spring would have completed each GLG during thatseason in successive years and then started another GLG. Each month more of
the new GLG would have accumulated following a hypothetical line representing
constant growth by percent GLG formed throughout the year, one twelfth or 8%monthly, as in Fig. 4. Most of the 28 animals examined for percent growth of thelast GLG follow this hypothetical line, at least during the spring of the year whenmost of the animals strand. However, this line begins in January or early February, earlier than when neonates are generally found. By late February or March,
10 to 20% of the new GLG has already formed, the amount that would be expected
at a constant deposition rate of dentine with the new GLG beginning in January.
This would suggest that the end of the previous GLG, which is the translucent layer,
occurs in December or January. Scatter around this line is probably due to individual variation in time of birth and actual widths of GLGs, and possibly to abnormal physiological states of the animals before they strand.
Tursiops may have two seasons of parity: one in the spring and another in thefall (Townsend, 1914; Harrison, 1969; Ridgway, 1972; Ross, 1977). In particular,
Townsend (1914) believed that North Atlantic Ocean Tursiops give birth during
both seasons. Ross (1977) has found stranded neonates in the fall and spring whichsuggests more than one or an extended breeding season. But neonates have notbeen found through the MMSP during the fall, even though many are picked up
during the spring. However, ifthe growth of the tooth can be used as an indicatorof season of birth, i.e., animals born in the spring have formed one GLG the following spring as previously discussed, then Tursiops which have formed 50% of a
GLG in the spring must have started that GLG during the fall. Similarly, ananimal whose last GLG ends in the fall, probably also began deposition of GLGs
during the fall. A hypothetical line drawn to represent the expected growth of teSci. Rep. Whales Res. Inst.,
No. 32, 1980

58

HOHN

eth which begin GLGs in the fall should parallel the equivalent line for animals
whose GLGs begin in the spring, but off-set by approximately six months. Thishypothetical line, represented in Fig. 2, has several points which fall on or close toit. This would suggest that these animals were also born in the fall rather than in
the spring as we would have anticipated based on stranding records. This may
provide evidence to support Townsend's (1914) idea of two seasons of calving for
Tursiops in the North Atlantic Ocean.
Although growth layers in teeth and bone have been used for about 30 years
for age determination, there have been only speculative suggestions as to the causeof the rhythmic deposition of the layers. The environmental or endocrinologicalmechanisms influencing or regulating the mineralization pattern in hard tissue is
unknown. In several species of odontocetes, the optical or mineral density of the
GLG layer bordering the pulp cavity when an animal dies has been used as an indicator of the physiological or nutritional state of the animal. Sergeant (1959)
found the "clear zone" (translucent layer) to begin in February and occur throughApril in captive Tursiops from Florida. Sergeant et al. (1973) said they could not
determine the density of the last layer in mostly wild Tursiops from Florida basedon their sample size (N=62). Sergeant (1962) found the opaque zone to occur adjacent to the pulp cavity in pilot whales, Globicephala melaena, from Canadian waters
caught in the summer. In a mass stranding of pilot whales, G. macrorhynchus, in South
Carolina during mid-October, the opaque layer bordered the pulp cavity (unpub.
data). Nishiwaki and Yagi (1954) found that the well stained (=opaque) layers
occur in December and May through June in S. coeruleoalba. Similarly, the opaquelayer borders the pulp cavity in June through February in harbor porpoises fromthe Bay of Fundy (Gaskin and Blair, 1977), in the summer in Baird's beaked whales
from the North Pacific (Kasuya, 1977), in May and June in bottlenose whales fromthe North Atlantic (Christensen, 1973), and in June through November in dugongs
from the equatorial and North Pacific (Kasuya and Nishiwaki, 1978). In summary, the opaque layer appears to border the pulp cavity at least during the summer months, and usually longer, in most species. Conversely, the translucent layer
is more often at that point during the winter or early spring. In Tursiops, the translucent layers, as accessory layers or GLG boundary layers, are deposited in the fall
(September, N=l), and in late January, early February, or March (N=3). However, all the animals examined were young which is when the translucent layer isvery narrow. As males and females age, a higher proportion of each layer becomestranslucent, so the time of deposition of each layer of a GLG would depend on theage of the animal, at least in Tursiops. This, again, assumes that the dentine continues to be deposited at a constant rate and only the mineralization pattern changes. The GLG number at which previous researchers determined the optical or
mineral density of the layer bordering the pulp cavity, or changes in the relative
amount of translucent dentine, has not been mentioned. The collection of enoughdata of this kind may help us to understand the cause of layering.

Sci. Rep. Whales Res. Inst.,
No. 32, 1980

AGE OF BOTTLENOSE DOLPHIN

59

Non-GLG Layered Structures
Incremental layers. It is difficult to count incremental layers from SEM micrographs because the small mineral changes throughout an increment and between
increments does not always produce enough contrast in etched sections to make individual increments clear (Plate III). In addition, increments are difficult to seeon the "back side" of a ridge or in a groove where it is "dark" because fewer electrons reach the collecting plate. The effect is probably that counts are slight underestimates of actual numbers of incremental layers.
Partially for the same reasons, it may not be accurate to measure widths of incremental layers from etched sections. The etching process produces the ridge andgroove pattern in GLGs and in incremental layers, so there is no longer a flat surface. This is compounded by the tilting of the sections in the SEM chamber. Although the quantitative effect of these factors is unknown, the average number ofincrements per GLG (12) multiplied by the average width of the increments (26µm) is not equivalent to the average width of the first three GLGs measured fromthin sections (approximately 420 pm). Measurements are probably an underestimate of actual sizes of increments. SEM of etched sections, however, seems toprovide the best way to visualize incremental layers.
The appearance of incremental layers in Tursiops dentine supports the idea of
Schour and others that dentine grows or is mineralized in spurts or increments.
The frequency of approximately 12 increments in one annular layer or GLG suggests that this sub-annual growth is similar to that described in dugongs (Kasuya
and Nishiwaki, 1978), Baird's beaked whale (Kasuya, 1977), and dolphins of thegenus Stenella (Myrick, in press). However, the width of the increments has notbeen determined for those species. The average size (26 µm) of incremental layers
in Tursiops dentine seems much too large to represent daily von Ebner's lines of 5
pm width described by Krauss and Jordan (1965) and Newman and Poole (1974)
in human teeth, or von Ebner's lines in pig dentine (Yilmaz, Newman, and Poole,1977). If incremental lines in Tursiops are approximately monthly, daily increments would have to average 0.87 µm, measured from SEM micrographs. Myrick(in press) has found daily increments in dolphin dentine, but has not determined
the width of either these von Ebner's lines or the "monthly" growth increments.
Schour and Hoffman (1939) measured the "calcification" rhythm in 17 species ofvertebrates and found it to recur at intervals of 16 µm (no time interval was mentioned). They were counting accentuations (light or more calcified areas) whichalternated with dark areas. It is possible that "monthly" increments in Tursiops,
which are the result of mineral differences, are similar to the pattern found in othervertebrates by Schour and Hoffman (1939).
One of two basic assumptions seems to be made when considering the deposition or mineralization of dentine. The first presumes the increments are timeregulated by some extrinsic or intrinsic factor so that the common denominator isthe number of increments set down during a given period of time. The second
assumption is that these processes continue at a constant rate regardless of the age
or the species of the animals. The latter would seems to be supported by SchourSci. Rep. Whales Res. Inst.,
No. 32, 1980

60

HOHN

and Hoffman's (1939) observations of the 16 µm calcification rhythm and researchers looking at constant sizes ofvon Ebner's lines (Krauss and Jordon, 1965; Newman
and Poole, 1974). However, the authors do not specify the age of the dentine
where the measurements were made, e.g., immediately subjacent to the enamel orseveral years growth away from the enamel. Nor do they mention making a seriesof measurements throughout each tooth, except over short periods of time such asdays or weeks. Although my measurements of incremental layers are subject toerror, as discussed previously, the trends suggest that the increments decrease insize in GLGs set down later in an animal's life. Increments in GLGs beyond
number three are smaller than those in the first three GLGs. This may be an artifact of etched sections. However, in order for the number of increments to remain
the same in all of the GLGs, the increments must become narrower as the GLGs
become narrower; again with the assumption that the number of incremental layers
is relatively constant, which seems to be more supported by the data from Tursiops
teeth.
The assumption of constant size of increments also seems to be disputed by
the differences in width of incremental layers on the ridges and grooves in etched
sections. Increments in the grooves measure, on average, smaller than incrementson the ridges and slopes of .the GLGs. This size difference may be a result of thecollapse of the tissue when the mineral is removed or it may be a real difference inwidths of increments in the hypomineralized and hypermineralized parts of the
GLG. This should be checked in untreated dentine, possibly in thin sections using
a petrographic microscope (Myrick, in press).
It is tenable that the mineralization or deposition of dentine could be regulated by monthly or lunar factors, that this produces the relatively constant numberof increments per GLG in Tursiops teeth and possibly all species, that the size ofthe increments changes progressively with the age of the animal and possibly between the hypo- and hypermineralized layers of the GLGs, and that the incremental layers can be used to gain more information about the biology of species and thegrowth of mineralized tissue. However, this must be measured by direct marking
of individuals, e.g., tetracycline, or by the examination of many more species before any conclusions are drawn.
Accessory layers. There appears to be two kinds of accessory layers as they havebeen described formally or referred to in the literature. The first are the narrow
accessory layers which appear irregularly in GLGs. These are approximately thesame size as incremental layers and can be distinguished from incremental layers
only because their mineral density, seen as optical density, differs appreciably fromthat of the GLG layer in which they occur. The second kind are the very prominent, wider than incremental layer accessory layers which occur regularly, approximately in the center of the GLG. It is undoubtedly these prominent accessory layers
which are responsible for the debate about number of GLGs set down annually inodontocete teeth. An unexperienced person would almost assuredly count theseregular accessory layers as GLGs. For example, Leatherwood et al. (1978) counted
five GLGs in thin sections of teeth from a young Tursiops (USNM 504122). In
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the same animal I counted a little more than two GLGs (Plate 5). The SEM micrograph shows only two GLGs (although the first prominent ridge is an accessory
layer - the first translucent layer is hardly visible). Similarly all of their countsof GLGs from seven Tursiops used in both studies are in disagreement with mine,with their counts from the teeth of young animals doubling mine. Kleinenberg
and Klevezal (from Klevezal and Kleinenberg, 1967) described an annual deposition of two GLGs in the teeth of the Black Sea dolphin, Delphinus delphis, which
they later (1967) attributed to the presence of this accessory layer. In general,they agreed that GLGs are annual but this prominent center accessory layer often
gives the impression that two GLGs are deposited.
Myrick (in press) refers to incremental layers in Stenella teeth as accessory layers. It seems that incremental layers are visible due to their mineralization cycle
within GLGs. If this cycle is subject to extrinsic or intrinsic influences then incremental layers will be more obvious within a GLG when the expected mineral density is disrupted. This disruption would make the incremental layer appear as oneof the narrow, irregular accessory layers. In the first two GLGs where the translucent layer is very narrow, these accessory layers would and do make it difficult
to determine where the GLG ends. However, incremental layers by themselves
do not usually interfere in the process of counting GLGs in Tursiops teeth, especiallyusing SEM.
Regardless of the kind of accessory layer, accessory layers make counting GLGsvery confusing in the first few GLGs. A person experienced in "reading" odontocete teeth can probably estimate very close to the actual number of GLGs in a tooth
section, but identifying the exact end of a GLG becomes very difficult in someteeth. There is also a great deal of variability between animals. Accurate age determination of some species of odontocetes, e.g. Tursiops, depends on experience,from looking at many teeth from all ages of the animals to accurately identify accessory layers before trying to count GLGs.
Small scale mineral changes. The narrow opaque accessory-type layers which
follow translucent layers appear to be the result of abrupt small-scale changes in thedentine. Irving and Weinmann ( 1948) described a similar occurrence in rat incisors.
They injected rats with strontium to observe the effects on calcification in the teeth
and found that the strontium caused a hypocalcified layer (16 µm wide) to occur.
Immediately following this layer, a very evident narrow "calciotraumatic" line wasfound which sharply demarcated the hypocalcified dentine from the normal, calcified dentine. They attributed this line to be caused by a shock to calcium metabolism in the rat by the strontium. The same effect was found in rats after administration of calciferol or parathyroid hormone (Schour and Ham, 1934; Schouret al., 1934), orimmediately following parathyroidectomy (Schour et al., 1937), where
the parathyroid helps regulate mineralization processes. In addition, Kronfeld andSchour (1939) found the calciotraumatic line to occur following the neonatal line.
It may be this same calciotraumatic line that is present in thin sections of Tursiops
teeth following the neonatal line and other translucent zones.
There has been some question about the mineral density of the narrow layers
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HOHN

in odontocete teeth, for example, the neonatal line. The consensus is that they aretranslucent and hypomineralized. But Nielsen (1972), Klevezal and Kleinenberg(1967), and Kasuya (1976) have suggested that the narrow layers are translucent but
hypermineralized for some species. The wide layers are then relatively hypomineralized, so the relationship between mineral and optical densities is different from thatof other odontocetes. Because of this, Nielsen (1972) has suggested that opticaldensity cannot be used as an indicator of mineral density. However, it may bethat where the translucent layer is very narrow, the opaque "calciotraumatic" line
is more apparent and is considered the layer. In some tooth sections of Tursiops it
is easier to use this opaque line than the preceding translucent layer to follow a
GLG down the extent of the tooth. However, the neonatal is still considered to betranslucent and hypomineralized and subsequent boundary layers are translucent
and hypomineralized. The calciotraumatic line needs more examination for its
occurrence and effect in odontocete teeth.

Sexual Dimorphisms
The sexually dimorphic trait found by van Utrecht (1969), i.e. the greater
extent of the neonatal line in males, in teeth from 20 harbor porpoises, Phocoena
phocoena, is not significantly different in the teeth of male and female Tursiops. Van
Utrecht also mentioned that the same trait was applicable to Lagenorhynchus albirostris, a delphinid, but this was based on only three females and three males. Teethof species from the family Phocoenidae are considerably different from those of thefamily Delphinidae. Instead of the nested "cone" appearance of GLGs in Tursiops teeth, the GLGs in Phocoena are bulbous in the crown or spatulate end, thencontinue in the narrower root parallel to the long axis, as in the handle of a spatula.
The difference in growth between phocoenid teeth and delphinid teeth presumably
allows for any dimorphic growth of the neonatal line relative to the top of the cement in Phocoena but not in Tursiops.
The second trait examined for possible differences between sexes of Tursiops
seems related to the first. If the neonatal line was to extend further down the rootof the tooth in male Tursiops as it does in male Phocoena, it might be because there
was a greater amount of prenatal dentine in males. Having more prenatal dentine
could have suggested that the neonatal line was deposited at a later time in males
or that the tooth began development earlier. But neither trait was significant soshould not be used to identify an animal of unknown sex in Tursiops.
The greater extent of the neonatal line could also be related to the widthof the neonatal line. If the measurements were made from the outer edge of theneonatal line, a wider neonatal line would extend its length if the deposition of theneonatal line was initiated at the same time in males and females. In the case of
Tursiops, the width of the neonatal line is significantly different in males and females. However, the greater neonatal line width in males is coupled with insignificant differences in either the extent of the neonatal line or width of the prenatalzone. This is probably because the neonatal line is very narrow, especially relative to its length, and the 10 µm mean difference in this width between males andSci. Rep. Whales Res. Inst.,
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AGE OF BOTTLENOSE DOLPHIN

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females does not significantly alter the overall measurement of the extent of the neonatal line. In addition, it is very difficult to accurately measure the neonatal line
width because it is so small and, although not difficult to identify, its boundaries
are not always clear. Even though the width of the neonatal line proved to be asignificantly different sexually dimorphic trait, I would not recommend that it beused as the sole identifier of the sex of an unknown specimen of Tursiops.
ACKNOWLEDGMENTS
A great deal of help came from the staff at the National Museum of Natural
History (NMNH). Mary Jacque Mann of the SEM lab was invaluable for her
skill with the SEM. Victor Krantz, NMNH photo lab, took the photomicrographs.
Charley Potter helped gather specimens and read parts of the draft. Eugenie
Clark, University of Maryland, gave me continuous support and offered suggestions on the manuscript. James G. Mead, curator of marine mammals, NMNH,
gave me the use of research materials and equipment, data he had been collecting
for many years, financial support as the research required, and discussed many aspects of this work with me as it progressed. To him I am especially grateful.
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EXPLANATION OF PLATESPLATE I
Fig. 1. SEM micrograph of etched longitudinal section of tooth from an old maleTursiops showing relative evenness in GLG width, translucent layer width, andspacing of GLGs. Magnification: X 17.
Fig. 2. Etched longitudinal section of tooth from a 2.5 year old male Tursiops.
The small layers running parallel to the GLGs are incremental layers. Althoughmore than two GLGs are present, the first is deceptively masked so that the actual
boundary layer, marked by the arrow, is not the first prominent ridge. The beginning of the second prominent ridge serves as the boundary for GLG 2. nlneonatal line, al-accessory layer, PC-pulp cavity
Original magnification: X 18.
PLATE II
Fig. I. Longitudinal thin section (transmitted light) of a young Tursiops tooth.
The dark evident line following the neonatal line (nl) may be a " calciotraumaticline". One GLG is complete, the second is beginning. The center accessory
layer (al) is very evident and could easily be mistaken for an additional GLG.
Magnification: X 18.
Fig. 2. Longitudinal thin section of a tooth from a male which could have from 1.5
to 6 GLGs. This animal is probably just over 3 three years old. The GLGs aremarked by arrows. Notice the accessory layers which occur centrally in the first
two GLGs.
nl-neonatal line
Magnification: X 12.
PLATE III
Fig. 1. Etched section of same 2.5 year old male in Plate II, Fig. 2 at a higher
magnification. Notice especially that it is difficult to separate individual
incremental layers (il) because two adjacent layers with small mineral density
differences appear as one.
nl-neonatal line, al-accessory layer
Magnification: X 50.

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