EVALUATION OF AN EFFECTIVE METHOD TO ESTIMATE AGE OF CAPE FUR SEALS USING GROUND TOOTH SECTIONS

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1 MARINE MAMMAL SCIENCE, 13(4): (October 1997) by the Society for Marine Mammalogy EVALUATION OF AN EFFECTIVE METHOD TO ESTIMATE AGE OF CAPE FUR SEALS USING GROUND TOOTH SECTIONS WESSEL H. OOSTHLJIZEN Sea Fisheries Research Institute, Private Bag X2, Rogge Bay, 8012, Cape Town, South Africa and Department of Zoology and Entomology, University of Pretoria, Pretoria, South Africa ABSTRACT Teeth of known-age Cape fur seals Arctocephalus pusillus pusillus were used to validate age estimated from ground sections. In the canines, dentine growth layer groups (GLGs) reflected age accurately but no reliable readings could be obtained from GLGs in the cementum. Upper canines were the most suitable for age estimation. By contrast, in the postcanines where the cementurn is thicker, only GLGs in the cementum could be used for age determination, but not with the same accuracy as for dentine in the canines. Therefore, it is recommended that GLGs in the dentine be used to determine age in the Cape fur seal. However, pulp cavities in canines closed at about 13 yr and consequently GLGs in the cementum of the postcanines should be used where the pulp cavities of canines are closed. Accurate estimation of age is not possible from the dentine of older animals. Key words: Arctocephalus pusillus pusillus, Cape fur seal, age determination, ground sections, canines, dentine, postcanines, cementum. The validation of age determination methods is essential for accurate age determination and to preclude erroneous estimation of demographic parame- ters (Laws 1962). The ground sectioning of teeth followed by counting of growth layer groups (GLGs) is a commonly used age determination method in several seal species (e.g., Laws 1952, Anas 1970, Payne 1978, Bengtson and Siniff 198 1, Boyd et al. 1990, Mansfield 1991, Boyd and Roberts 1993). GLGs form a repetitive or semirepetitive pattern of parallel incremental growth layers within the formative surface of the hard tissue (Perrin and Myrick 1980). This paper describes age estimation results from ground sections of the canines and postcanines of known-age Cape fur seals, Arctocephalus pusillus pusillus. The technique is validated, the age estimates from upper and lower canines and postcanines are compared and the applicability of such estimates to all age classes is discussed. Other age determination methods such as etch- 683

2 684 MARINE MAMMAL SCIENCE, VOL. 13, NO. 4, 1997 ing, use of thin decalcified and stained sections, and scanning electron microscopy are addressed in detail elsewhere. hfethods Annual tagging of 6-wk-old seal pups commenced in 197 1, and subsequent recovery of tagged animals in later years provided the source of known-age animals (Oosthuizen 1991). There were two main sources of recovery: commercial harvest and drowning in fishing nets. Whole upper (n = 58 females, 90 males) and lower canines (n = 88 females, 90 males), as well as postcanines (n = 19 females, 27 males) of both sexes were extracted from the dead seals and mounted on aluminum blocks with epoxy putty. Only one tooth was used per animal. The teeth were sectioned longitudinally using a modified highresolution industrial circular saw fitted with a 15cm diamond wafering blade (thickness 1.1 mm, grit size ). The root of the tooth was sectioned first and the enamel of the crown last to prevent fracturing of the enamel. A longitudinal section not thinner than 800 p,rn was cut to include the midline. Longitudinal sections as opposed to cross sections were used to increase the accuracy of age estimation, because the cementum and dentine are not deposited evenly along the tooth root (Hewer 1964, Grue and Jensen 1980, Driscoll et ul. 1985). Sections were stored temporarily in 70% alcohol. Owing to their small size, postcanines were ground and not sectioned. Sections were ground to a thickness of ym, the thickness at which the GLGs proved to be the most visible. An Isomer? low-speed saw was used for grinding. The saw blade was replaced with an aluminum disc to which disposable discs of water paper (grit size 320 or 400) were clamped. The catch container was filled with water through which the water paper rotated at controlled speed. Ground sections were stored in absolute alcohol for seven days, then cleared and mounted on glass slides (after Bengtson and Laws 1985). The mounted thin sections were examined under transmitted light through a dissecting microscope at low (7-10X) magnification using polarizing filters. The maximum number of GLGs, in both dentine and cementum, for each tooth was determined five times by a single observer and the mean calcul.ated, as the accuracy of age estimation is improved by multiple readings (Doubleday and Bowen 1980, Bowen et ul. 1983, Mansfield 1991). This mean, rounded to the nearest full year, was used to estimate the age of each seal. Estimates were made without any reference to body parameters, such as body length, which could have influenced the counts. Only one reader was used, in an effort to minimize possible bias between readers, a factor which can lead to statistical errors (see Anas 1970, McLaren and Smith 1985). For convenience, the birth date of all Cape fur seals was taken to be 1 December, the median pupping date (Shaughnessy and Best 1975). For each set of determinations the estimated age was compared with the true age. A weighted least-squares regression procedure (Press et ul. 1992) was used to evaluate the fit of a linear model to the data in each case. On the

3 OOSTHUIZEN: ESTIMATING AGE OF CAPE FUR SEALS 685 grounds of the resulting fit, the null hypothesis that the estimated age equalled the true age was rejected at the 95% confidence level when: (1) the intercept and slope were different from zero and one, or (2) the linear model was determined to be inappropriate as explained below. The means of the replicate estimated ages (y;) were fitted to the true ages (Xj) using a maximum likelihood estimator. The likelihood of the set of N estimated ages for any given fit was given by: (Press et al. 1992), where a is the y-intercept of the linear regression, b the slope and cri the standard deviation of the ith mean estimate. For computational convenience, the negative log-likelihood, which is equivalent to twice the x2 statistic, was computed as 2x2 = 5 Flyi - a - bxi12 i=l 2a*. The values of a and b, for which the negative log-likelihood was at a minimum, and their associated variances (SD, and SD,), were computed analytically (Press et al, 1992). For an age determination technique to predict ages accurately, the values of a and b should not be different from 0 and 1 respectively. This was tested using a one-sample t-test (Zar 1984). Models were rejected if 0 < a - t0,050),n SD, or 0 > a + t,,osclj,n SD, or 1 < b - to,oscl,,n SD6 or 1 > b + t0.05(1),n Sk The goodness-of-fit was estimated from the probability (Q) of the x2 value being exceeded by chance. If that probability was very small (Q << 10m3) then a specific fit could be rejected on the grounds that the data were too unlikely to have been observed if that were the true fit. Better fits are those which have the lowest x2 value, with Q typically in the region of 10m3. Values of Q were taken from the table of x2 critical values given in Zar (1984) for 0.5(n - 2) degrees of freedom. Each fit was also tested for the sequential randomness of the residuals. Biases in prediction occur if residuals are serially correlated. The Runs test (Zar 1984) was used to assign a probability to the hypothesis that the residuals were not serially correlated. If this probability did not exceed 0.05, the fit was rejected. RESULTS The GLGs in the dentine of the canines were clear and easy to count (Fig. la), but in the cementum they were either absent or returned unrealistically low estimates of age (Fig. lb). As the lower canines were more curved than the upper, they were cut thicker so as to include the midline. Using polarized light highlighted the GLGs most effectively. The estimated mean ages, plotted against the true age (Fig. 2), provided the parameters a (intercept) and b (slope) with their associated error estimates (Table 1).

4 686 MARINE MAMMAL SCIENCE, VOL. 13, NO. 4, 1997 Figure 1. Longitudinal ground section of a Cape fur seal canine of (a) a 10-yr-old female showing the presence of GLGs in the dentine and (b) a 6-yr-old female showing the absence of GLGs in the cementum. For female lower canines (FLC), the null hypothesis that estimated and true age were equal was accepted (Q > 0.999), although the residuals were serially correlated (Z > 0.001) (Table 1). The residuals showed a slight trend with the estimated age being rather too low than too high (Fig. 3a). There were few errors in age estimation, and the largest difference between the age estimates and the true age was one year (Fig. 3a). For female upper canines (FUC), the null hypothesis was accepted (Q > 0.5) and the residuals were not serially correlated (Z = 0.384) (Table 1). The error in estimating age was small and within one year of the true age, with the exception of two animals (10 and 11 yr old, respectively), the ages of which were underestimated by as much

5 OOSTHUIZEN: ESTIMATING AGE OF CAPE FUR SEALS 687 n a 6 d) Male upper canines TRUE AGE Figwe 2. The means of the replicate estimated ages of the ground sections of Cape fur seal canines fitted to the true ages using a maximum likelihood estimator (solid line) and compared to a line with a slope of one and intercept of zero (broken line). Table 1. Comparison of intercept and slope parameters, standard error estimates, x-square (Q), Runs test (Z) values and probabilities for ground sections, and the sample size (n) of Cape fur seal upper and lower canines of both sexes. Female Female Male Male lower upper lower upper Intercept (a) SE ofa a - 2 (SE) a + 2 (SE) SLOPE (6) SE of b b - 2 (SE) b + 2 (SE) x-square Q Runs test Z n >0.999 >0.5 >0.9 > >O.OOl

6 688 MARINE MAMMAL SCIENCE, VOL. 13, NO. 4, a) Female lower canines b) Female upper canines $... I....I..., -1 t I j 4.,7.,2..3.., '...' N=58 Y?I LT 5 _ c) Male lower canines I I I I I I, ,7.qg q * ~8.2~ j...,...,.. -I N=QO N=QO I I I I I I 1 I I I I t I TRUE AGE Figwe 3. Residuals of actual age minus estimated age from ground sections of Cape fur seal canines for each year class. as three years (Fig. 3b). Such underestimation can be attributed to poorly prepared sections (not ground to the correct thickness). The errors in age estimates also exhibited a slight tendency to be rather too low than too high (Fig. 3b). In male lower canines (MLC), the null hypothesis was accepted on all grounds (Q > 0.9) and residuals were not serially correlated (2 = 0.113) (Table 1). The residuals showed no obvious trend and indicated that the error in age determination was always within one year of the true age (Fig. 3~). In male upper canines (MUC) the null hypothesis was accepted (Q > 0.975) and the residuals were not serially correlated (Z = 0.288) (Table 1). The error in estimating the age was always within one year of the true age (Fig. 3d). In the postcanines, the dentine GLGs could not be used to determine age because the pulp cavity closes at an early age and prevents the further formation of GLGs (Fig. 4a). However, the GLGs in the cementum were clearly visible and were used to estimate age (Fig. 4b). The null hypothesis for age estimated from cementum GLGs versus true age was rejected for male postcanines (MPC) (Q < O.OOl), but not for female postcanines (FPC) (Q > 0.1) (Fig. 5 and Table 2). The residuals were not

7 OOSTHUIZEN: ESTIMATING AGE OF CAPE FUR SEALS 689 Figure 4. Longitudinal ground section of a Cape fur seal postcanine of (a) an 11-yrold female showing the absence of GLGs in the dentine and (b) a 10-yr-old male showing the presence of GLGs in the cementum. serially correlated for either males (Z = 0.643) or females (Z = 0.861) (Table 2). For both sexes the residuals showed a trend of the estimated age being too low (Fig. 6). The largest difference between the estimated age and the true age was 2 yr for females and 3 yr for males (Fig. 6). DISCUSSION GLGs in the dentine of Cape fur seal canines are clearly observable and can be used to estimate age accurately, as is the case for other species such as

8 690 MARINE MAMMAL SCIENCE, VOL. 13, NO. 4, TRUE AGE Figure 5. The means of the replicate estimated ages of the ground sections of Cape fur seal postcanines fitted to the true ages using a maximum likelihood estimator (solid line) and compared to a line with a slope of one and intercept of zero (broken line). southern elephant seals Mirounga leonina (Laws 1952, 1953), northern fur seals Callorhinus ursinus (Kenyon et al. 1954, Fiscus et al. 1964), leopard seals Hydrurga leptonyx (Laws 1957), crabeater seals Lobodon carcinophagus (Laws :l958), ringed seais Phoca hispida (McLaren 1958), harp seals Phoca groenlandica (Bowen et al. 1983), subantarctic fur seals Arctocephalas tropicalis (Bester 1990) and antarctic fur seals Arctocephalus gazella (Boyd and Roberts 1993). In coatrast, the cementum of the canines was too thin for reliable age determination, as has also been reported for the northern fur seal (Kenyon and Fiscus 1963). The method of grinding down canine sections allows the midline to be followed along the curvature of the tooth (Watts and Gaskin 1989). As with antarctic fur seals (McCann 1993), the upper canines are preferred to the lower, which are larger but more curved. Table 2. Comparison of intercept and slope parameters, standard error est.imates, x-square (Q), Runs test (2) values and probabilities, and sample size (n) for ground sections of Cape fur seal postcanines of both sexes. Female Male Intercept (a) SE ofa a - 2 (SE) a + 2 (SE) SLOPE (b) SE ofb b - 2 (SE) b + 2 (SE) x-square Q >O.l <O.OOl Runs test Z n 19 27

9 OOSTHUIZEN: ESTIMATING AGE OF CAPE FUR SEALS a) Female postcanlnes 4 3 b) Male postcanines 3: 20 (j,.j..s..* *.. f -1 1 I u N= TRUE AGE N=27 I I I I I Figure 6. Residuals of actual age minus estimated age from ground sections of Cape fur seal postcanines for each year class. In the postcanines of Cape fur seals, GLGs were visible only in the thick cementum layer, which can therefore be used in age determination, as for crabeater seals (Bengtson and Siniff 1981, Bengtson and Laws 1985), Antarctic fur seals (Boyd et al. 1990), Weddell seals Leptonychotes weddelli, leopard seals (McCann 1993), and northern elephant seals Mirounga angustirostris (Klevezal and Stewart 1994). However, in the Cape fur seal, estimated ages were not as accurate as those from the dentine of canines. Interpretation of the GLGs was uncomplicated, but readable sections were not obtained from all teeth. The main cause of misinterpretation was poorly prepared sections, in which either the teeth were not cut along the midline or not ground to the correct thickness. Where the midline was missed, the accessory layers appeared wider, resulting in misinterpretation of layers (Hohn et al, 1989). This is especially so in older animals where the annual growth is reduced and annuli are narrow (Fiscus et al. 1964). In sections ground to an incorrect thickness, the GLGs did not show clearly, as in two females of 10 and 11 yr old, where ages were underestimated by as much as three years. Therefore, to reduce reading error, all poorly prepared sections should be rejected in any subsequent determination of age. Conclusions It is concluded that GLGs in the dentine of upper canines are preferred for age determination in the Cape fur seal. This is mainly because their greater size results in wider spacing of GLGs, making them easier to interpret than other teeth (Laws 1953, Roberts 1978). However, the pulp cavity in Cape fur seals is totally occluded at around 13 yr of age, thereby preventing any further age estimation from dentinal GLGs. Consequently, cemental GLGs in the postcanines, which are not spatially limited as in dentine (Morris 1972, 1978; Harris 1978), should be used to predict age in older animals with closed pulp cavities. Such prediction is less accurate than that made using the upper canines.

10 692 MARINEMAMMALSCIENCE,VOL. 13,NO. 4, 1997 ACKNOWLEDGMENTS My special thanks go to Drs Marthan Bester, Jeremy David, and Denzil Miller for their critical reading of the manuscript and to Mr Colin Attwood who derived the statistical tests. This study would not have been possible without the known-age animals collected, and I thank everybody involved in this collection. Finally, I thank Nola Summers for her technical assistance. LITERATURE CITED ANAS, R. E Accuracy in assigning ages to fur seals. Journal of Wildlife Management BENGTSON, J. L., AND D. B. SINIFF Reproductive aspects of female crabeater seals (Lobodon carcinophagus) along the Antarctic peninsula. Canadian Journal of Zoology 59: BENGTSON, J. L., AND R. M. LAWS Trends in crabeater seal age at maturity: An insight into Antarctic marine interactions. Pages in W. R. Siegfried, P. R. Condy and R. M. Laws, eds. Antarctic nutrient cycles and food webs. Springer-Verlag, Berlin. BESTER, M. N Reproduction in the male sub-antarctic fur seal Arctocephalus tropicalis. Journal of Zoology (London) 222: BOWEN, W. D., D. E. SERGEANT AND T. ORITSLAND Validation of age estimation in the harp seal, Phoca groenlandica, using dentinal annuli. Canadian Journal of Fisheries and Aquatic Sciences 40: BOYD, I. L., AND J. P. ROBERTS Tooth growth in male Antarctic fu.r seals (Arctocephalus gazella) from South Georgia: An indicator of long-term growth history. Journal of Zoology (London) 229: BOYD, I. L., N.J. LUNN, P. ROTHERY AND J. I?. CROXALL Age distribution of breeding female Antarctic fur seals in relation to changes in population growth rate. Canadian Journal of Zoology DOUBLEDAY, W. G., AND W. D. BOWEN Inconsistencies in reading the age of harp seals (Pagophilus groenlandicus) teeth, their consequences, and a means of reducing resulting biases. North Atlantic Fisheries Organization Scientific Council Report 80/X1/160, Serial Number N247. [Available from NAFC, DFO, P 0. Box 5667, St. John s, Nfld., Canada AlC 5X1.) DRISCOLL, K. M., G. S. JONES AND F. NICHY An efficient method by which to determine age of carnivores, using dentine rings. Journal of Zoology (London) 205: FISCUS, C. H., G. A. BAINES AND F. WILKE Pelagic fur seal investigations, Alaska waters, U.S. Fish and Wildlife Service Special Scientific Report, Fisheries pp. GRUE, H., AND B. JENSEN Review of the formation of incremental lines in tooth cementum of terrestrial mammals. Danish Review of Game Biology 11: HARRIS, S Age determination in the red fox (Vulpes vulpes): An evaluation of technique efficiency as applied to a sample of suburban foxes. Journal of Zoology (London) 184: HEWER, H. R The determination of age, sexual maturity, longevity and a lifetable in the grey seal, (Halichoerus grypus). Proceedings of the Zoological Society of London 142: HOHN, A. A., M. D. SCOTT, S. W. RANDALL, J. C. SWEENEY AND A. B. IRVINE Growth layers in teeth from known-age, free-ranging bottlenose dolphins. Marine Mammal Science 5: KENYON, K. W., AND C. H. FISCUS Age determination in the Hawaiian monk seal. Journal of Mammalogy 44:

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