Age Determination and
Validation in
Chondrichthyan Fishes
Presented by Kara Baca
and Sharon Homer-Drummond
Methodology
 Age
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determination process:
Collection of hard part samples
Preparation of the hard parts
Examination (age reading)
Assessment of validity and reliability
Interpretation (modeling growth)
Hard parts used
 Vertebral
centra
 Dorsal fin spines
 Neural arches
 Caudal thorns
Vertebral Centra
 Larger,
more
anterior centra
should be used
 Whole or
sectioned
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Vertebrae
should be
sectioned
sagittally
(longitudinally)
Dorsal Fin Spines
 Used
particularly in
dogfish sharks
 Spines from
second dorsal
fin are
preferred
 Used whole or
longitudinally
cut
Neural Arches
 May
be useful for species that have
poorly calcified vertebral centra
 Preliminary studies with sixgill sharks
Caudal Thorns and Other
Structures
Caudal thorns used
with vertebral centra
to determine age in
bathyrajid species
 Evidence for growth
bands found in upper
jaw of the wobbegong
Orectolobus japonicus
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Serra-Pereria et al., 2005
Preparation of Samples (Centra)
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Thaw (if frozen) or wash (if
preserved in alcohol)
Clean excess tissue and
separate into individual
centra
Sectioning typically done
with low-speed diamondbladed saw
After mounting sections to
slides, sand with wet fine-grit
sandpaper to approximately
0.3 to 0.5 mm and air-dry
Binocular dissecting
microscope is generally used
for analysis
Age Determination
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View wide bands
(opaque) separated by
distinct narrow bands
(translucent)
Tend to occur in summer
(wide) and winter
(narrow)
Each pair of
wide/narrow bands
considered to represent
an annual growth cycle
Validity must be tested
Age determination for
spines are nearly
identical
Dorsal fin spines
http://journal.nafo.int/35/21-calis.html
Banding in Neural Arches
 Distinct
bands in
sixgill sharks
 Potential use in
age
determination
Staining & imagery
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Imagery & enhancement
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Staining
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Goldman, 2004
scanning EM (atypical)
steromicroscopy
binocular light microscopy
X-radiograophy (atypical)
X-ray spectrometry
Graphite microtopography
silver nitrate (McFarlane et al.,
2002)
cobalt nitrate (Hoenig and Brown,
1998)
ammonium sulfide (Hoenig and
Brown, 1988)
haemotoxylin
cedar wood oil, alizarin red, crystal
violet, salts (Cu, Fe, & lead)
Histology
Evaluating Precision
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Average percent error (APE)
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May serve as good relative indicator of precision
within and between readers
However, tells which reader was less variable, not
which was better or if either was biased
Goldman’s approach to estimating precision:
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Calculate percent reader agreement (=[No.
agreed/No. read] x 100) within and between readers
for all samples
Calculate percent agreement plus or minus one year
Calculate percent agreement with individuals divided
into appropriate length or disk width groups
Test for bias
Criticism: varies widely among species and ages
within a species
Tests for Bias
 Age
bias plotgraphing one reader
vs. the other and
referencing
equivalence line of
the 2 readers (45°
line through the
origin)
 Chi-square tests of
symmetry, such as
Bowker’s
 Evans-Hoenig test
Back-Calculation
 Method
for describing the growth history
of each individual sampled
 Numerous methods – formulas that follow
hard part or body proportion hypothesis
are recommended
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Proportional relationship between animal
length or disk width and radius of vertebral
centrum or distance from focus to each
annulus within a given centrum
Verification & Validation
Confirmation of age
determination by indeterminate
and determinate methods
Why validation & verification?
Caillet, 1990
Apex Predators website: “Sharks are long-lived animals
that grow very slowly and do not produce many young.
In many parts of the world, sharks are fished
commercially, thus, in order to ensure proper
management of the stocks, age and growth data must be
obtained. With this data, we can determine the longevity
of the species as well as maximum age, age at maturity,
growth rate, and differences in growth between males
and females.”
 The process of evaluating growth zone deposition in
fishes can be divided into verification and validation.
 Verification: “… conforming an age estimate by
comparison with other indeterminate methods”
 Validation: “… proving the accuracy of age estimates by
comparison with a determinate age”
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Some methods of
validation
Chemical tagging of
wild fish
 Mark-recapture of
known-age fish*
 Bomb carbon dating*
 Growth ring
frequency
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distinction between
absolute age (*) &
periodicity
Some methods of
verification
Centrum age analysis
 Relative marginal
increment analysis
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Size mode analysis
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Primarily a verification tool
Progression of discrete length
modes over time
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Size modes taken from a
random sample of all size
classes from a population
Mean or median sizes in age
classes assessed by other means
(e.g.: age classes predicted by
von Bertalanffy growth
function applied to physical
assessment)
Random sample size modes
and mean or median sizes at
age compared
Coincidence supports
assumption of age classes
Monitor discrete length modes
across a given time period at
discrete intervals
Tag-recapture
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Initial in field:
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Capture
Measure
Weigh
Tag
Release
Recapture and assess for growth
in length and/or weight
Compared to von Bertalanffy
growth functions, TET,
oxytetracycline (OTC) or other
methods, based on size changes
between tagging and recapture
file:///D:/age%20validation/Tetracycline%20or%20OTC_files/age1.jpg
Simpfendorder, 2000: dusky
sharks
Chemical marking, tag-recapture & lab
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Validation of absolute age focuses on validating “temporal periodicity
of … growth increment formation”
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Tetracycline (TET): standard for marking free-swimming animals
OTC: marks by binding to Ca2+ & depositing to active calcification sites
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25mg/kg bw IM
+ external tag
highly visible marks in vetebral centra & dorsal fin spines under UV
use body growth & calcification changes to compare ‘time at liberty’ [release]
w/growth band deposition (# rings in vetra centra or spine post-injection = time
hacks)
• lab or captivity
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Tag-recapture + marking
• tag + OTC followed by recapture
• tag + TET followed by recapture
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Other tags
• Fluorescein (not well-evaluated in elasmo.; 5-10mg/kg bw, higher=mortality)
• Calcein (not well-evaluated in elasmo. .; 5-10mg/kg bw, higher=mortality)
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Define age & indiv. growth characteristics
Combining information from mark w/observed growth changes
Tetracycline group
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Broad spectrum Ab’s that block bacterial protein
synthesis by inhibition of aminoacyl-t-RNA binding to
the rRNA A site.
TET = tetracycline
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higher water solubility at pH = 7, than other Ab’s in same
group
OTC = oxytetracycline
Inject TET or OTC at capture while tagging
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25 mg/kg
absorbed & deposited at growth sites
only mark glows under UV
pairs > mark = growth over time
Centrum edge analysis
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Centrum edge analysis:
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Compares opacity (width) &/or translucency (density) of
centrum edge over time
• measure or grade edge
• compare to time of year or season
• detail: analyze Ca2+ & PO4 – at edge by x-ray or electron
microprobe spectrometry
• not used much yet:
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recaptured nurse sharks marked with TET (Carrier and Radtke,
1988)
4 ray species in the Irish Sea marked with TET (Gallagher et al.,
2004)
tiger sharks (Winter and Dudley, 2000)
http://journal.nafo.int/35/10-gallagher.html
Winter and Dudley, 2000
Relative marginal increment analysis
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RMI = MIR (ratio): (VR-Rn)/(Rn-Rn-1)
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Parameters
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MIR – marginal increment ratio
VR = vetebral radius
Rn = distance from center to outer edge of last complete band
Rn-1 = distance from center to outer edge of next-to-last complete band
Direct: plot against month to find band formation trend line
Assess seasonal band &/or ring deposition
• measure margin (growth) area of centrum from last growth ring to
centrum edge
• divide by width of last fully formed annulus (none for age 0)
• plot values against month of capture
• determine periodicity of band formation
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Poss. combine w/stable isotope analysis
• fish work
• no elasmo. work yet
• proved viable in other chondrichthyans
Carlson and Baremore, 2004
Neer et al., 2005: Bull shark growth
Captive rearing
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Laboratory or aquaria
growth
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Particularly useful if poor
ring formation,
calcification, etc.
Morphometric data
reveals age and growth
Periodicity of growth zone
formation in vetebral
centra
Difficult to determine true
baseline & captivity
effects
www.pac.dfo-mpo.gc.ca
Bomb carbon (radiocarbon) aging
 Tied
to global oceanic rise in 14C following
cold war atomic testing
 Synchronous uptake in marine carbonates
 Dated marker of calcified structures allowing
growth band dating
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Born < 1956: low
Born 1956-1965: higher
Born > 1965: declining
 Regional
identification
 Validation of annulus formation & absolute
age
Campana et al., 2002
Campana et al., 2002
Current status of studies
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Caillet, 1995 was last major review
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115 publications listed in chapter since then (several not included in chapter that were
published late 2004 – mid-2005)
68 newly studied spp. among those
Methods: most studies combine (mult. validation & mult. verification)
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most use structure calcification
• 1°ly vetebral centra (70% of studies reviewed in chap.)
• dorsal spines (7%)
• neural arches (1 study) & jaws (1 study)
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captive growth (9%)
tag-recapture (7%)
embryonic growth (4%)
precision analyses: several growth parameter values
1° reviewed methods = MIR or ratio (50%) + centrum edge
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modal analysis (25 studies, 22 spp.)
tag-recapture (20 studies, 19 spp.; many combined w/OTC marking)
lab growth (21 studies; 18 of which used OTC marking)
bomb carbon study (Natanson et al., 2002, validated pobeagle growth & indicated
shortfin mako growth of 1 ring/yr.)
• combined w/tag-recapture & OTC
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Combining validation & verification seems to be the most robust means
Growth models
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von Bertalanffy (VBGF): fish growth
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size at a given point in time depends on anabolism & catabolism
identification of the growth coefficients are dependent on the given study
easy population comparisons
fitting allows for fine-tuning
problems:
• small sample size: poor parameter estimation
• t0 (theoretical size) should be replaced by L0 (length at birth) as 3rd parameter (more to come)
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Gompertz: larval & early growth
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S-shaped model function (like Ricker logistic function)
also can fit to fine-tune for a given study
growth rate = log of asymptotic disk width or length - actual asymptotic disk width or
length
most commonly used for skates & rays
possibly most appropriate for ovoparous spp.
possibly most appropriate for spp. that grow in V more than weight or L
possibly better estimate captive growth (slowed or atypical growth due to stress)
Best fit functions: choice of model based on length-at-age or weight-at-age
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nonlinear least-squares regression analysis
maximum likelihood function
VBGF
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Traditional: Lt = L∞(1-e-k(t-t0))
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substitute Lo and L for t and t0
Modifications
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Neer et al., 2005 (following Fabens, 1965) to estimate bull
shark growth (modified to fit observed size-at-age data)
• Lt = L∞(1-be-k(t-t0)) = predicted length at time t
•b=
• L∞ = theoretical asymptotic length
• k = growth coefficient
• L0 = length at birth
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compared to fitted models to sex-specific size-at-age data
w/traditional VBGF model
• theoretical longevity estimated at 95% L∞ (5x ln2 k-1)
• growth model parameters derived from least-squares regression
Neer et al., 2005
VBGF growth coefficients (k)
k = average rate to reach maximal length or size from
length at birth
 Studies fitted to VBGF allow estimates of L∞
(asymptotic size), k, & Lo or to
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high k = fast growth rate (0 – 1)
Large variance among spp. & studies
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k in chimaera: 0.05 – 0.47
k in sawfish: 0.07 – 0.08
k in guitarfish, torpedo rays & stingrays: 0.2 – 0.5
k in skates: 0.05 – 0.5
k in whale shark: 0.03 – 0.05
affected by: sample size, aging methods, verification,
validation, & growth and model fitting
Estimating maturity & lifespan
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Tmat estimated by various means & shows great variance among spp. &
studies (sex, spp., method, study)
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chimaeras: 2.9 – 6.0 years
• C. monstrosa: 11.2 – 13.4 years
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sawfish: 10 years
guitarfish, torpedo rays, stingrays: 1 – 9 years
skates: 4 – 13 years
angel shark: 11 years
sixgill shark: 5 – 21 years
carpet sharks: 16 – 25 years
dog sharks: 4 – 45 years
Related to longevity estimates (5x ln2 k-1) ( well duh!)
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chimaeras: 5 - 10 years
• C. monstrosa: 29 years
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sawfish: 30 - 44 years
guitarfish, torpedo rays, stingrays: 1 – 9 years
skates: 9 - 50 years
angel shark: 35 years
carpet sharks: 9-35 years
dog sharks: 12 - 70 years
Back to why
 Better
knowledge = better estimation of
growth potential
 Better estimation of growth potential =
better management strategies
 Real consequences mean that precise,
careful, redundant work is needed
Key references and citations
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Goldman, K.J. 2004. Chapter 6: Age and growth of elasmobranch fishes. unidentified book.
Serra-Pereira, B., Figueiredo, I., Bordalo-Machado, P., Farias, I., Moura, T. and Gordo, L.S. 2005. Age and growth of Raja
clavata Linnaeus, 1758 – evaluation of ageing precision using different types of caudal denticles. Elasmobranch Fish. Sci.
ICES CM. N-17:1-10.
Gallagher, M.J., Nolan, C.P. and Jeal, F. 2004. Age, growth and maturity of the commercially important ray species from the
Irish Sea. e-journal NW Atl. Fish. Sci. V35(art.10):http://journal.nafo.int/35/10-gallagher.html.
Carlson, J.K. and Baremore, I.E. 2005. Growth dynamics of the spinner shark (Carcharhinus brevipinna) off the United States
southeast and Gulf of Mexico coasts: a comparison of methods. Fish. Bull. 103:280-291.
Natason, L.J., Casey, J.G. and Kohler, N.E. 1999. Growth of the tiger shark, Galeocerdo cuvier, in the western North Atlantic
based on tag returns and length frequencies; and a note on the effects of tagging. Fish. Bull. 97:944-953.
McFarlane, G.A., King, J.R. and Sauders, M.W. 2002. Preliminary study on the use of neural arches in the age determination
of bluntnose sixgill sharks (Hexanchus griseus) Fish. Bull. 100:861-864.
Gburski, C.M. 2005. Ageing procedures for the big skate (Raja binoculata), longnose skate (Raja rhina), Alaska skate
(Bathyraja parmifera), Aleutian skate (Bathyraja aleutica) and Berring skate (Bathyraja interrupta) at the Alaska Fisheries
Science Center. In submission.
Calis, E., Jackson, E.H., Nolan, C.P. and Jeal, F. 2005. Preliminary age and growth estimates of the rabbitfish, Chimaera
monstrosa, with implications for future resource management. e-journal NW Atl. Fish. Sci.
V35(art.21):http://journal.nafo.int/35/21-calis.html
Simpfendorfer, C.A. 2000. Growth rates of juvenile dusky sharks, Carcharhinus obscurus (Lesueur, 1818), from southwestern
Australia estimated from tag-recapture data. Fish. Bull. 98:811-822.
Wintner, S.P. and Dudley, S.F.J. 2000. Age and growth estimates for the tiger shark, Galeocerdo cuvier, from the east coast of
South Africa. Mar. Freshwater Res. 51:43-53.
Neer, J.A., Thompson, B.A. and Carlson, J.K. 2005. Age and growth of the Carcharhinus leucas in the northern Gulf of
Mexico: incorporating variability in size at birth. J. Fish. Biol. 67:370-383.,
Campana, S.E., Natanson, L.J. and Myklevoll, S. 2002. Bomb dating and age determination of large pelagic sharks. Can. J.
Fish. Aquat. Sci. 59:450-455.
Conrath, C.L., Gelsleichter, J. and Musick, J.A. 2002. Age and growth of the smooth dogfish (Mustelus canis) in the
northwest Atlantic Ocean. Fish. Bull. 100:674-682.
www.pac.dfo.-mpo.gc.ca. Age determination of elasmobranchs.
www.psrc.mlml.calstate.edu. Age growth and demographic studies.
www.flmnh.ufl.edu. Pacific shark research center. Moss Landing Marine Laboratories.
www.na.nefsc.noaa.gov. Apex predators: age and growth in sharks.