SEASONAL PERIODICITY OF GROWTH AND COMPOSITION
IN VALVES OF DIPLODON CHILENSIS PATAGONICUS
(D’ORBIGNY, 1835)
A.L. SOLDATI 1, D.E. JACOB 1 , B.R. SCHÖNE 1, M.M. BIANCHI 2 AND A. HAJDUK 3
1
Department of Geosciences, Johannes Gutenberg-Universität Mainz, Becherweg 21, D-55099 Mainz, Germany;
INIBIOMA - CONICET, Universidad Nacional del Comahue, Quintral 1250, CP8400 San Carlos de Bariloche, Argentina; and
3
CONICET, Museo de la Patagonia, Centro Cı´vico CP8400, San Carlos de Bariloche, Argentina
2
(Received 6 May 2008; accepted 3 November 2008)
ABSTRACT
Freshwater mussels of the genus Diplodon (Unionida) are common inhabitants of lakes and rivers in
South America, and have slow growth and long life spans. We established the annual periodicity of
incremental shell growth in Diplodon chilensis patagonicus (d’Orbigny, 1835) and calculated growth
rates at different ages, using internal ring counting supported by dyeing methods and d 18O isotope
analyses, in two Patagonian populations (Lago Steffen and its effluent Rı́o Manso Inferior,
Argentina). Longevities of ca. 90 years (Lago Steffen) significantly extend the life spans reported in
the past. Growth rates for old individuals (.30 years) from both lake and river populations average
0.16 mm per year along the axis of minimal growth. We evaluated the seasonal periodicity of minor
and trace elements (Mn, Mg, Sr, Ba, Na, S) in situ by Laser Ablation ICP-MS and Electron Probe
Microanalyser analyses. Line-scans in a valve from Lago Steffen show that Mn, Sr and Ba are preferentially accumulated during the summer, while higher concentrations of Mg are found in the winter
bands. Metal/Ca ratios may serve as long-term archives of environmental variables, e.g. metal concentrations in water, water temperature and primary productivity. Diplodon chilensis patagonicus valves
exhibit excellent characteristics to construct an accurate chronological archive with time windows of
up to around a century, resolving the environmental signal annually and even seasonally.
INTRODUCTION
The soft parts of mussels and ground shells are widely used to
monitor trace metal abundance and bioavailability in ecosystems (e.g. Bertine & Goldberg, 1972; Koide, Lee & Goldberg,
1982; Brooks & Rumsby, 1984; Phillips & Rainbow, 1988;
Odzak et al., 1994). They have also been used successfully to
characterize the environment, for example to detect heavy
metal contamination and radionuclides (e.g. Koide et al., 1982;
Bourgoin, 1990; Hameed et al,. 1993; Lau et al., 1998; Brauer
et al., 2001; Sokolowski, Wolowicz & Hummel, 2007) or to
monitor human impact (e.g. Brown & Luoma, 1995; Puente
et al., 1996; Lau et al., 1998; Szefer et al., 2002; Cardellicchio
et al., 2008). Nevertheless, studies on homogenized material
(tissues or ground shells) do not provide temporal resolution,
but only time-averaged information. In contrast, spatially
resolved data on mussel shells provides information that has
the potential to resolve the time scale annually, seasonally,
fortnightly, daily and even sub-daily (Clark, 1974; Lutz
& Rhoads, 1980).
Bivalve molluscs form their shell by periodic accretion of
calcium carbonate (CaCO3) and organic substances (sugars
and proteins) from the extrapallial fluid at the biomineralization front (Addadi et al., 2006). This fluid is situated between
the outer mantle epithelium and the inner shell surface, and its
composition may be determined by both the composition of
the surrounding water and the metabolic processes in the
mussel (Crenshaw, 1980). Under normal environmental conditions, this process is regulated by rhythmic physiological parameters, known as ‘biological clocks’ (e.g. Palmer, 1970) and
by external cyclic events such as light, tidal or temperature
Correspondence: A.L. Soldati; email: [email protected]
cycles (Thompson, 1975). They can result in distinct periodic
(daily, tidal, seasonal, annual, etc.) ‘growth lines’ in the shell
(Clark, 1974). In addition to these natural cycles and rhythms,
abnormal environmental conditions, such as high summer
temperatures, storms, etc. may also produce ‘disturbance lines’
(Clark, 1974). In many cases growth lines and disturbance
lines may be helpful to create an internal scale to date exactly
the time axis of each part of the shell, i.e to order chronologically the information recorded in the shell (e.g. Rhoads
& Panella, 1970; Hudson et al., 1976).
Variation of environmental parameters such as food supply,
substratum type, salinity, illumination, temperature, concentration of dissolved oxygen or oxygen/carbon dioxide ratio,
among others, may affect growth pattern, shell structure,
mineralogy, isotopic fractionation and chemistry (Rhoads &
Panella, 1970; Bertine & Goldberg, 1972; Meenakshy et al.,
1974; Eisma, Mook & Das, 1976; Carter, 1980; Lutz &
Rhoads, 1980; Brooks & Rumsby, 1984). Thus, shell features,
minor and trace element composition patterns and isotopic
signals may serve as an archive of environmental history (e.g.
Schöne et al., 2007). Using the incremental growth patterns as
a calendar, the chemical data archived in the shell can be
chronologically aligned, thereby vastly extending the performance of the animal as a long-term water quality and environmental monitor (recent samples), and even as a
palaeoecological and palaeoclimate recorder (fossil samples)
(Rhoads & Lutz, 1980). The potential of bivalve shells for
environmental reconstructions is increasingly recognized, but
most studies have focused on marine species (e.g. Steuber,
1999; Dutton, Lohmann & Zinsmeister, 2002; Schöne et al.,
2002, 2003, 2005b; Schöne, 2003; Steuber et al., 2005; Pearce
& Mann, 2006), while freshwater mussels have been less
frequently investigated (e.g. Nyström et al., 1995; Tevesz,
Journal of Molluscan Studies (2009) 75: 75– 85. Advance Access Publication: 22 December 2008
# The Author 2008. Published by Oxford University Press on behalf of The Malacological Society of London, all rights reserved.
doi:10.1093/mollus/eyn044
A.L. SOLDATI ET AL.
Barrera & Schwelgien, 1996; Wurster & Patterson, 2001;
Schöne et al., 2004; Dunca, Schöne & Mutvei, 2005; Carroll,
Romanek & Paddock, 2006).
In South America, freshwater mussels of the genus Diplodon
(Hyriidae) are the most abundant bivalves in freshwater
bodies on both sides of the South Andean Cordillera, including
the subspecies Diplodon chilensis chilensis (Gray, 1828) and
D. chilensis patagonicus (d’Orbigny, 1835) (Castellanos, 1959a,
1960). The latter is found in Argentina between Mendoza
(328520 S; 688510 W) and Chubut (458510 S; 678280 W), and
includes our study area, the lakes and rivers of the Nahuel
Huapi National Park (Castellanos, 1959b, 1960; Semenas &
Brungi, 2002). Because of its well studied filter-feeding characteristics (Soto & Mena, 1999), soft tissue and digestive glands
of Diplodon have been the target of long-term water quality
monitoring (Soto & Mena, 1999; Lara, Contreras & Encina,
2002) and pollution studies (Ribeiro Guevara et al., 2004,
2005) in the region. To assess the potential of the shells as a
complementary long-term biomonitor for the Patagonian
lakes, however, it is necessary to have a basic understanding of
the relation between the minor and trace elements, their distribution within the shell and the growth pattern (Rosenberg,
1980; Swann, Carriker & Ewart, 1984; Carriker et al., 1996).
A number of studies have evaluated Diplodon shell growth
patterns using mark-and-recapture experiments together with
growth-ring analysis and found longevities of 40 years (Parada
et al., 1989) and average growth rates of 6.9 + 1.7 mm/year
(Valdovinos & Pedreros, 2007) for the Chilean subspecies
Diplodon chilensis chilensis. However, previous studies were
restricted (1) to young specimens, whose valves show well
developed growth rings, but whose high growth rates overestimated the growth in mature specimens, and (2) to the study of
only a few years of growth (generally 1– 5 years) which, in
turn, led to an underestimation of the annual growth variability (Anthony et al., 2001). Furthermore, there have been no
systematic studies determining whether Diplodon shell growth
patterns do indeed record seasonality or if they follow ontogenetic trends in the shell composition of Diplodon species.
The aims of the present study are (1) to evaluate the annual
and/or seasonal periodicity of the shell incremental growth in
Diplodon chilensis patagonicus using stable isotope analysis; (2) to
determine growth rates and life span, especially at late ontogenetic ages; (3) to study the shell composition in situ in
relation to the growth pattern. The results obtained are used
to shed light on the calibration of shell components within a
time scale of high resolution, in order to monitor environmental signals from Diplodon chilensis patagonicus shells over long
time spans.
Sampling
Diplodon chilensis patagonicus (Fig. 1A) were collected alive in
March 2007 from Lago Steffen (11 specimens) and Rı́o Manso
Inferior (3 specimens). The former were found at 3–5 m water
depth, while the latter were found near the river bank in
shallow water. The shells are equivalve, nacreous with a very
thin prismatic layer and have a thick periostracum (Fig. 1).
Immediately after collection the mussels were sacrificed and
the soft parts removed. Water temperature and pH were
recorded in the field. A surface water sample was collected for
stable isotope analysis from Lago Steffen in March 2007 using
an opaque polyethylene bottle and refrigerated at 48C until
measurement.
Sclerochronology
The shells were washed and cleaned with a brush and air
dried. After coating with metal bisphenol-A-epoxy resin
(WIKO, Greussenheim, Germany), the left valves were cut
perpendicular to the growth lines and along the height axis
with an Isomet 1000 low speed saw (Buehler, IL, USA) and
ground on glass plates with 800 and 1200 grit powder and
then polished with 1-mm Al2O2 powder on a Buehler G-cloth.
Two slabs of 3 and 5 mm thickness were prepared from each
specimen. The thinner sections were used for sclerochronology,
and the 5-mm sections were subjected to isotope and elemental
analyses. Samples for sclerochronology were immersed in
Mutvei’s solution (Schöne et al., 2005a) for 27 min at 388C
and air-dried. This method gently dissolves the calcium carbonate with acetic acid, while preserving the organic matrix
with glutaraldehyde and dyeing the sugars and glycoproteins
of the biominerals with Alcian blue. Organic-rich portions of
the shell are stained deeply blue, facilitating the identification
of growth lines. Digital photomicrographs of the samples were
taken under a reflected-light binocular microscope before and
after etching.
Shell annual-increment lengths along the complete shell
section were measured on these photomicrographs (Fig. 1).
The length of an ‘annual increment’ was defined as the distance between two adjacent growth cessation marks (sharp
blue lines in the etched sample).
The individual age of each mussel was calculated as the sum
of the annual increments until death. The ages of mussels with
an eroded umbo (where the increments are absent or difficult
to see) were reported as minimum ages. Taking the date of
death of the animal (March 2007) as a time anchor point,
each increment length could be chronologically associated to
its deposition year. These well dated and chronologically
ordered incremental length series allowed assignment of a
calendar date to each d 18O sample.
Counting the annual marks also allows assignment of an
‘ontogenetic age’ to each annual increment and reconstruction
of the incremental and cumulative increment length curves vs
the ontogenetic age of the mussel. Shell growth rates are
higher at younger ontogenetic ages so that the cumulative
growth curve is best described as a nonlinear function which
monotonically increases and approaches a final value asymptotically (Figs 4, 6).
To find the best expression for shell growth, each experimental curve was fitted with different exponential and sigmoidal
models. We found that the functions of the Morgan –
Mercer –Flodin (MMF) family were the best descriptors. The
MMF functions (Morgan, Mercer & Flodin, 1975) are sigmoidal functions that are used to describe growth in various fields
(e.g. biology, economy, demography, etc.); applied to this case
they model the cumulative incremental shell length (Y) as a
MATERIAL AND METHODS
Study area
Lago Steffen is a postglacial lake situated at 418300 S 718320 W,
on the west side of the Andean Cordillera. Like many of the
sub-Andean lakes it is a monomictic temperate lake with low
chlorophyll-a concentrations and high water transparency. It is
fed by the Rı́o Manso Superior which drains the Manso
glacier covering the Cerro Tronador and passes through Lago
Mascardi before entering Lago Steffen and exiting the lake as
the Rı́o Manso Inferior, eventually discharging to the Pacific
Ocean. These localities belong to the protected areas within
the Nahuel Huapi National Park, with restricted access, and
therefore human activity is minimal and is concentrated in the
summer months. Several Diplodon chilensis patagonicus populations have been observed by locals in the eastern bay of the
lake as well as downstream, but studies of population densities
or other characteristics have not been published.
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SEASONAL SIGNALS IN PATAGONIAN DIPLODON SHELLS
Figure 1. A. Photograph of a right valve of the shell of Diplodon chilensis patagonicus indicating minimal (I) and maximal (II) growth axes.
B. Photograph of a polished shell section along the minimal growth axis indicating shell structures. C. Enlargement of a part of the shell shown in (B)
with optimized contrast and illumination: black and white parts correspond to annual increments. D. Shell section after treatment with Mutvei’s
solution; growth interruptions (annual marks) are seen as blue lines. The arrow shows one increment length used for sclerochronological analyses.
(500–2000 mm) and thus facilitates sampling with the drill bit.
Sampling was carried out along the middle part of the shell
section only to avoid any possible alteration along the inner
and outer areas. In this way, 225 samples from AS07RM0016
and 108 from AS07LS0030 with weights between 30 and
150 mg were obtained. The samples were analysed following a
protocol for very small sample sizes (Fiebig, Schöne &
Oschmann, 2005) using a Finnigan MAT 253 continuous-flow
mass spectrometer equipped with a GasBench II at the
University of Frankfurt, Germany. d 18O values are reported
relative to the Vienna Pee-Dee Belemnite (VPDB) standard
based on an NBS-19 (d 18O ¼ 22.20) calibrated Carrara
marble value of d 18O ¼ 21.76. The external precision (1 SD)
is better than +0.07 (between 0.03 and 0.07) based on multiple measurements of the Carrara marble standard.
In aragonitic biocarbonates, the water temperatures at
which the CaCO3 was precipitated can be related to the d 18O
value in the shell using the Grossman and Ku equation
(Grossman & Ku, 1986), as modified by Dettman, Reische &
Lohmann (1999):
function of the ontogenetic age of the mussel (X):
Y¼
ða b þ c X d Þ
ðb þ X d Þ
ð1Þ
where a, b, c and d are the fitting constants and X is the variable. The parameters a, b, c and d were adjusted using the commercially available software CurveExpert-1.38 (Microsoft
Corporation, USA).
By anchoring all cumulative growth curves at the point of
death of the animals (March 2007), a cumulative ‘master’
growth curve was calculated. This was done by averaging the
cumulative growth curves of eight specimens older than 60
years of age, collected both from the river and the lake. This
master growth curve enables determination of annual growth
rates for D. chilensis patagonicus that are characteristic for this
locality.
Stable isotope analyses
One specimen of 8.1 cm anterior–posterior length from Lago
Steffen (AS07LS0030) and one 7.1 cm specimen from Rı́o
Manso Inferior (AS07RM0016) were selected for isotopic
analysis. Raman spectroscopy measurements showed that the
valves consist entirely of aragonite, without calcite, so that isotopic differences between different CaCO3 polymorphs (e.g.
Kim et al., 2007) were irrelevant to this study. The nacreous
shell layer was sampled parallel to the growth lines at
intra-annual resolution under a binocular microscope using a
300 mm drill bit, following the axis of minimum shell growth
from the umbo to the ventral margin. We selected the axis of
minimum shell growth for analysis because it is much thicker
Td18 O ½o C ¼ 20:60 4:34 ðd18 Oshell ðd18 Owater 0:27ÞÞ ð2Þ
The d 18Owater of the water sample was measured at the
Advanced Analytical Centre for Environmental Science at the
Savannah River Ecology Laboratory, University of Georgia,
USA and gave a value (+SD) of d 18O ¼ 211.15 + 0.14.
Trace and minor element analyses
Na, Mg, P, Mn, Sr and Ba contents were measured in samples
AS07LS0030 and AS07RM0016 in situ by Laser Ablation
77
A.L. SOLDATI ET AL.
ICP-MS (LA-ICP-MS) using an Agilent 7500ce quadrupole
ICP-MS (Agilent Technologies Inc., Santa Clara, USA)
coupled to a New Wave Research UP123 (New Wave
Research, Fremont, USA) laser ablation system, following
methods described by Jacob (2006). Measurements were
carried out with laser energy densities of 3.22 J/cm2 at 10 Hz
with helium as carrier gas. Line and spot diameters were
15 mm; scanning speed was 10 mm/s. Backgrounds were
measured for 30 s prior to each ablation and 43Ca was used as
the internal standard with calcium concentrations measured
by EPMA (Electron Probe Micro Analyser). NIST SRM 612
glass was used as the external standard and data reduction was
carried out with the commercial software GLITTER 4.0
(Macquarie University, Sydney, Australia). Data for NIST
SRM 612 were taken from the GeoReM database (Jochum &
Nehring, 2006). Detection limits (99% confidence level) for
the measurements were Na, Mg and Mn ¼ 0.1 mg/g, P ¼
0.4 mg/g, Sr ¼ 0.001 mg/g, Ba ¼ 0.006 mg/g and analytical
errors were routinely between 10 and 15% (Jacob, 2006).
BCR-2G (USGS) and NIST SRM 610 were used as secondary
standards. The relative standard deviation (%RSD) obtained
was ,1% for Na, Mg, Mn, Sr and Ba in both reference
materials and 8 and 12%, respectively, for P in BCR-2G and
NIST SRM 610. The deviation from the average published
data (Jochum & Nehring, 2006) is generally between 5 and
10% for all elements except for P in NIST SRM 610 (15%).
Concentrations of Ca and element maps of Mn, Sr, Na, S
and P were measured with a Jeol JXA 8900 RL EPMA by
wavelength dispersive analysis. A beam diameter of 2 mm was
chosen and an area of 600 900 mm was mapped with steps of
3 mm. Standardization was carried out with a range of natural
and synthetic standards and the data were corrected using the
CITZAF procedure (Armstrong, 2005).
Figure 2. d 18O-values in younger parts of sample AS07LS0030. The
light lines in the shell correspond to peaks in d 18O-values (winters).
RESULTS
Seasonal periodicity of shell increments
In polished cross-sections, D. chilensis patagonicus shell increments appear as alternating dark and light pairs (Fig. 1B, C).
All specimens in this study were collected at the end of the
austral summer (March 2007), and have a dark band as the
last band at the ventral margin. The high resolution
d 18O-values show that each of the dark/light pairs in the shell
correlates with a peak in d 18Oshell, which is especially visible in
younger ages of sample AS07LS0030, when growth rates are
high (Fig. 2). Highest d 18Oshell values were observed in the
light bands and lowest values occurred in the dark lines
(Fig. 2). d 18O and water temperature are inversely correlated
and, thus, light bands correspond to colder temperatures
(autumn/winter, i.e. April/August) while dark bands reflect
high ambient temperatures (spring/summer, i.e. September/
March). This result is also supported by sample AS07RM0016
(Fig. 3): the water temperature at which the carbonate of this
valve was precipitated can be reconstructed from the d 18Oshell
values (applying Eq. 2). The resulting temperature (Tshell,
Fig. 3) shows peaks during the summer months and decreases
in the cold season, coinciding with the record of the air temperatures in the region. The seasonal variation in Tshell between
summer and winter in this shell is about 28C (Fig. 3).
Figure 3. Apparent water temperatures obtained from oxygen isotope
analyses in shell AS07RM0016 (dash-point line, Temperatureshell ).
Records of monthly air temperatures (solid line, Temperatureair) were
obtained from the database of the National Oceanographic and
Atmospheric Administration (airport of S.C. de Bariloche:
41880 53.4100 S, 71890 45.1700 W). Note the difference in scale.
‘annual mark’), which appears only once per year, and can be
used to calibrate the shell time scale annually.
Annual increment lengths (Fig. 1D) measured along the
outer shell surface between two successive annual marks
decreases from the umbo towards the ventral margin (Fig. 4A,
B). The frequency histogram (Fig. 5) shows the typical increment length found over the whole life span of D. chilensis patagonicus: most of the annual increments are smaller than one
millimeter (average increment length (+SD) ¼ 0.35 +
0.43 mm) for lake and river samples. Broadest increments
4.31 mm (lake) and 3.88 mm (river) were typically found in
younger portions of the shells (less than 10 years old, Fig. 4A),
whereas older shell portions showed annual increments as
narrow as 0.02 mm (lake) and 0.05 mm (river).
Individual growth curves calculated from the incremental
lengths (Fig. 4B) can be fitted with sigmoidal functions
Annual growth, growth rates and life span
After immersion in Mutvei’s solution (Fig. 1D), dark (summer)
lines are stained deep blue, while the light (winter) bands are
mottled light blue, differentiating clearly both summer and
winter seasons. This treatment also enhances a sharp organic-rich
line between each winter and summer band (hereafter termed
78
SEASONAL SIGNALS IN PATAGONIAN DIPLODON SHELLS
Figure 6. Growth curve for specimens older than 60 years, from Lago
Steffen (solid grey lines) and from Rı́o Manso Inferior (dashed grey
lines). The thick black line is a sigmoidal curve fitted through all data
points, and represents the theoretical growth curve for individuals born
at around 1930 from this locality.
Figure 4. Incremental length (A) and cumulated incremental length
(B) for shell AS07RM0018 from Rı́o Manso. Data in (B) were fitted
with an MMF sigmoidal function (Morgan et al., 1975). The dashed
line shows the 15th year in the sample; arrow indicates the time of
sampling.
the second portion, showing that the growth rate decreases
after the 15th year of life. River and lake samples older than
60 years show a very similar growth curve and growth rates
(Fig. 6). Growth rates along the axis of minimum growth vary
between 4.31 and 0.02 mm/year; averages are 1.05 mm/year in
younger shell parts (,15 years old) and 0.16 mm/year in older
shell portions (.30 years old). The maximum age observed
among all individuals collected (14 samples) was around 90
years for a specimen of Lake Steffen (9.5 cm anterior– posterior
length). The smaller shells, of about 5 cm anterior –posterior
length, showed between 12 and 15 internal annual marks.
Seasonal variability in shell composition
Element mapping by EPMA in a shell cross section shows the
presence of alternating zones (lines) of high and low trace
element contents (Fig. 7). Sr is present between 1500 and
2500 mg/g, following an alternating pattern of Sr-poor and
Sr-rich zones (Fig. 7B). Mn also shows a heterogenous concentration, alternating between high- (1300 mg/g) and low(700 mg/g) Mn concentration zones (Fig. 7C). Mn-rich and
Sr-rich lines coincide with the dark growth increments
(summers) of the shell cross section (Fig. 7A). Slightly higher S
concentrations than background (.110 mg/g, Fig. 7D) accompany these elements. P and Na are constant at the resolution
of the instrument (ca. 80 and 1660 mg/g, respectively). Mg
and Ba concentrations are below the detection limit of the
microprobe analyses (i.e. 90 and 350 mg/g, respectively).
Line scan analyses allowed precise recording of the elemental
concentrations at mg/g levels with high spatial resolution in the
shell sections. In situ trace element analyses by LA-ICP-MS
within one shell section (AS07LS0030) show strong variability
of Mn, Sr, Ba and Mg concentrations (Fig. 8) that are correlated with the light/dark band pair and therefore represent seasonal signals. Mn peaks appear in summer, coinciding with the
dark bands, while the concentration decreases during winter
(light bands), which is in excellent agreement with the element
maps produced by EPMA (Fig. 7). In AS07LS0030 Sr concentrations are slightly out of phase with respect to Mn (Fig. 8A),
but coincide well with Ba peaks (Fig. 8C), whereas Mg concentrations are in reverse phase to the other three elements
(Fig. 8B), and Na (not shown) shows no cyclicity. The composition of the shells over the complete sampling area (ca. 60 and
25 years within the life span of AS07RM0016 and
Figure 5. Histogram of increment length frequencies for the sampling
sites. A. Rı́o Manso (n ¼ 3 valves; 235 increments). B. Lago Steffen
(n ¼ 10 valves; 482 increments).
(R . 0.9) and can be divided in two regions of approximated
constant slope: one before the 15th year of life and a second
one after the 30th year of life (Figs 4B, 5). The slope of this
curve is steeper in the first portion and generally shallower in
79
A.L. SOLDATI ET AL.
Figure 7. Element distribution maps for Sr (B), Mn (C) and S (D) across several annual marks in a sectioned shell (A, square). The mapping was
carried out with an Electron Probe Microanalyser using a beam size of 2 mm.
Figure 8. A. Linear trends and seasonality of Mn-, Sr-, Mg- and Ba-concentrations measured by LA-ICP-MS in AS07LS0030, using line scans
from the ventral margin (0) to the umbo. Concentrations are normalized to the highest value and separated for better comparison. B. Enlargement
of part of the normalized Mn/Ca- and Mg/Ca-concentrations showing that both elements are in reverse phase in this shell. C. Enlargement of the
normalized Sr/Ca- and Ba/Ca-concentrations showing that both elements are in phase. Mn/Ca is slightly out of phase with Sr/Ca and Ba/Ca (data
not shown). Abbreviation: a.u., arbitrary units.
80
SEASONAL SIGNALS IN PATAGONIAN DIPLODON SHELLS
AS07LS0030, respectively) show a trend of increasing total
trace element uptake with increasing ontogenetic age: higher
concentrations of the metals are accumulated towards the
ventral margin (older ontogenetic age) than near the umbo
and the amplitude of the oscillation increases with increasing
ontogenetic age (Fig. 8A).
temperature (Fig. 3) (although high time-averaging does not
resolve the complete amplitude). Highest temperature values
(lowest d 18Oshell ) are obtained in the dark bands of the shell,
coinciding with the austral summer months, while lower temperatures are found in the light shell bands, coinciding with the
winter. This interpretation is supported by similar findings for
shells from other freshwater molluscs (Dunca & Mutvei, 2001;
Schöne et al., 2004; Dunca et al., 2005).
Due to the sampling method and the variable thickness of
growth increments (i.e. the area between two annual marks),
the resolution of the annual apparent temperature reconstruction varies for each year: samples obtained from larger annual
increments (.1 mm, generally younger than 10 years old)
allow a resolution of ca. 2 months (5– 7 samples per year), and
sometimes even give a 5-week resolution (10 samples per year,
e.g. 1996/97 in AS07RM0016, Fig. 3), while thinner annual
increments (between 1 and 0.1 mm, generally older than 10
years) allow only a 4–6 month resolution (2– 3 points per
sampled year). Thus, the sample set records summer –winter
variability in d 18Owater time-averaged over several weeks or
months.
Comparison of the reconstructed apparent temperatures
from the shells with measured water temperatures (19.88C in
Lago Steffen and 20.28C in Rı́o Manso Inferior, at 1 m depth
in March), shows considerable discrepancy. While the
maximum temperature for Rı́o Manso Inferior reconstructed
from AS07RM0016 is 13.78C, the lacustrine sample gives a
maximum of 15.28C (data not shown). The summer/winter
variability is around 28C for both specimens and is much
smaller than expected in lakes of this region (88C; Barriga,
2006). The possibility of systematic time-averaging in all increments sampled can be excluded, because in younger parts of
the shells some of the bands are clearly separated and the
dimensions of the increments exceed that of the drill bit by a
factor of ten (Fig. 2). Another possibility for this discrepancy
could be an extended period of slow growth during the year
(possibly during the reproductive period) during which little
or no shell material would be secreted by the organism (low
calcification rate). This would prevent recording of the
environmental signal in the shell and effectively dampen the
Tshell temperature curve (Fig. 3). These effects have been
shown for Phacosoma japonicum (Schöne et al., 2003) from Japan
and for Chione fluctifraga from Mexico (Schöne et al., 2006).
However, this hypothesis could not be tested here, because the
samples were obtained in March only, and sampling several
times in a year would be necessary to address this point.
Alternatively, it could be argued that the seasonal d 18O signature in the valves reflects the composition of the water body,
i.e. the influence of precipitation that feeds the rivers and lakes
rather than the water temperatures (Urey et al., 1951). The
effect of changing oxygen isotopic composition of rainwater
during the tropical wet and dry seasons has been conclusively
demonstrated for d 18O profiles in the fossil shells of Diplodon
longulus from the Amazon region of Brazil (Kaandorp et al.,
2005). The limited dataset for seasonal d 18Owater variability in
the region of study, however, precludes thorough evaluation of
this possibility for D. chilensis patagonicus, and will be addressed
in future studies.
Lake vs river environments
The samples obtained from the lake were found living in a
mussel bank, at about 10 m off the lake shore at 5 m depth.
The bottom is a muddy-sandy substrate, with algae and rush
present. The water temperature was 19.88C and the pH 7.0.
The river samples were found living individually (not part of a
bank) at about 1 m from the river bank in quiet waters. The
temperature at that depth was 20.28C and the pH 7.0. The
river samples have a thicker and heavier shell than the lake
samples. Average growth rates in both localities (Fig. 6)
coincide at around 0.16 mm/year, but younger increments in
lake samples are generally wider than in river samples (Fig. 5).
The mean concentration of trace elements measured by
LA-ICP-MS in both, the river sample (AS07RM0016) and
the lake sample (AS07LS0030, Fig. 9), are identical within
analytical error except for Mn, which is higher in the lacustrine sample (418 + 39 mg/g) than in that from the river
(117 + 56 mg/g).
DISCUSSION
Seasonal periodicity of growth increments
A number of studies have described the growth periodicity in
valves of Diplodon chilensis chilensis. Parada et al. (1989), for
example, identified dark/light band pairs as 1 year and
reported a decreased rate of shell growth during gametogenesis
in the summer months, resulting in the formation of prominent
external concentric growth rings as well as internal dark
bands. These findings are supported for the other subspecies
D. chilensis patagonicus in our study. First, after treatment with
Mutvei-solution, D. chilensis patagonicus shells show strongly
dyed zones at the end of each white line (winter) and prior to
the next black line (summer); this is a result of an accumulation of more organic than mineral material, is in accordance
with the hypothesis of a slowing of growth during reproduction. Secondly, oxygen isotopic measurements of the shell
increments yield apparent water temperatures that qualitatively reproduce the seasonal changes observed in air
Annual growth, growth rates and life span
The analysis of internal growth rings by sclerochronology, as
opposed to external growth rings, offers a number of critical
advantages for long-lived bivalve species: (1) all growth rings,
independent of their state of development can be reliably visualized by the etch-dye treatment with Mutvei’s solution; and
(2) determination of the growth rate can be carried out over
the complete life span of the specimen and is not restricted to
Figure 9. Average concentrations of trace elements measured with
LA-ICP-MS. Error bars are the standard deviation for n ¼ 11
(AS07LS0030) and n ¼ 12 (AS07RM0016).
81
A.L. SOLDATI ET AL.
young individuals that show higher-than-average growth rates.
The treatment with Mutvei solution enhanced a single sharp
blue line at the beginning of each dark band of the sectioned
valve, the annual mark. Alcian blue, one of the principal components of Mutvei solution, binds preferentially to glycoproteins and sugars (Schöne et al., 2005a), and the intense
colour of the annual mark shows that this region is enriched in
organic material. Thus, the annual marks represent phases of
extremely slow growth (or even interruption of the mineral
deposition) during the end of the winter and the beginning of
the summer. Studies of Diplodon population dynamics (Parada,
Peredo & Gallardo, 1990; Semenas & Brungi, 2002) and
density (Lara & Parada, 1991), reproductive cycle (Peredo &
Parada, 1986; Parada, Peredo & Gallardo, 1987; Parada et al.,
1990; Semenas & Brungi, 2002) and embryonic stage
(Gluzman de Pascar, 1973; Semenas & Brungi, 2002) have
shown that reproduction is seasonal in this genus, coinciding
with the generation of an annual growth mark.
Growth curves (Fig. 4) can be reconstructed from the etched
and dyed sections. The time axis of each D. chilensis patagonicus
shell can then be calibrated by counting the blue annual
marks from the umbo (time ¼ 0) to the ventral margin. The
last band at the ventral margin corresponds to the time
immediately preceding the death of the animal, while the
period between two adjacent marks records the time between
two successive summers.
Diplodon chilensis patagonicus specimens from Lago Steffen
show a life span of around 90 years while those from Rı́o
Manso are around 70 years, which extends previous life span
estimations for this species (Parada et al., 1989). An even
longer life span is possible, analogous to observations for other
lacustrine unionid mussels, which have been reported to reach
ages in excess of a century (Anthony et al., 2001). Although
our statistics are not enough to characterize both populations,
we observed the general trend that lake populations have
higher longevities than the river specimens, in agreement with
Parada et al. (1990). Although D. chilensis patagonicus growth
rates between lake and river differ for young specimens, those
of older samples are similar (Fig. 6). Life span and growth
rates are highly correlated with geographic and limnological
conditions (Valdovinos & Pedreros, 2007), and differences in
eutrophication, nutrients and water temperature may be the
principal factors accounting for the differences observed
between our samples and those of the Chilean populations
(Parada et al., 1989). Recently, Valdovinos & Pedreros (2007)
have studied internal growth rings in D. chilensis chilensis
employing an aragonite staining method, and validated the
correspondence between internal and external banding in
young specimens. The mean annual growth rate for 5-year-old
D. chilensis chilensis specimens averaged at 6.9 + 1.7 mm/year
(along the maximal growth axis). However, Fig. 5 shows that
the growth rate decreases rapidly after maturity, thus growth
rates determined for young individuals severely overestimate
the average lifetime growth rate. Growth rates (along the axis
of minimum growth) calculated from our data vary between
4.31 and 0.02 mm/year. In comparison, Parada et al. (1989)
calculated growth curves for D. chilensis chilensis in two populations using an exponential growth model, resulting in growth
rates for 5-year-old specimens of 4.18 and 4.96 mm/year and
for 30-year-old shells of 0.002 and 0.007 mm/year along the
axis of maximum growth. The largest rates calculated from our
data are similar to the growth rates for younger specimens of
D. chilensis chilensis (Parada et al., 1989; Valdovinos & Pedreros,
2007), allowing for discrepancies due to the use of the maximal
growth axis in these studies instead of the minimal growth axis
used here. Older samples (.30 years old) present incremental
growth rates in the order of 0.16 mm/year and in some years
are extremely slow growing (0.02 mm/year). These rates are
one order of magnitude larger than the values reported for
older specimens of D. chilensis chilensis (Parada et al., 1989).
Seasonal variability in shell composition
Chemical parameters, such as stable isotope ratios (Figs 2, 3)
and trace element concentrations (Figs 7, 8) show a pronounced seasonality in D. chilensis patagonicus valves. As discussed in the previous sections, d 18Oshell vary between summer
and winter with lowest values recorded in summer and consistently higher d 18O values in winter, i.e. recording the temperature cyclicity (Figs 2, 3). In situ trace element analyses show a
similar behaviour in molar metal/Ca ratios for Mn, Sr, Ba and
Mg (Fig. 8), which correlates with the seasonal growth bands
in the shells (Fig. 7). In AS07LS0030 the Mg/Ca ratios show
minima during summers, while Mn/Ca, Sr/Ca and Ba/Ca
ratios are at their maxima and, overall, the mean and amplitude of the oscillation increase with increasing ontogenetic age
(Fig. 8). These findings agree well with those for other bivalve
species, whose valves have shown to be excellent biomonitors
for certain environmental variables (e.g. Toland et al., 2000;
Van der Putten et al., 2000). Microlaminations in the valves of
freshwater hyriid Hyridella depressa, for example, were demonstrated to be a long-term monitor for Mn concentration in the
water (Jeffree et al., 1995). Langlet et al. (2006) showed that
seasonal trends of Mn/Ca ratios in valves of the freshwater
bivalve Pleiodon spekii matched the seasonal increase in
chlorophyll-a concentrations in surface waters that records
seasonal upwelling in Lake Tanganyika and are associated
with the monsoon climate system. Chlorophyll-a contents in
the water bodies in our sampling locality, the Nahuel Huapi
National Park, are also reported to oscillate seasonally
with higher concentrations in summer than in winter
(C. Queimaliños, Personal Communication), which may
explain the higher Mn/Ca ratios in D. chilensis patagonicus
valves during the warmer months.
Generally, we observed average Sr contents of around
650 mg/g, with Sr/Ca ratio maxima in the warmer months,
minima during the cold season, and seasonal variations of ca.
150 mg/g amplitude (Fig. 9). Variations in Sr contents are
commonly found associated with growth increments in mollusc
shells. Although there have been many attempts to use the
Sr/Ca ratio as biomonitor for water temperature and salinity,
its extended use is controversial because the Sr/Ca ratio seems
to be affected by vital (Gillikin et al., 2006b) and kinetic effects
(Lorrain et al., 2005) in many species. Indeed Sr/Ca ratios
have been found to correlate with the carbonate precipitation
rate in marine Pecten maximus, Saxidomus giganteus, Conus ermineus
and Mercenaria mercenaria shells (Thorn, Cerrato & Rivers,
1995; Gillikin et al., 2006b; Sosdian et al., 2006), although these
ratios vary in response to seasonal changes in Arctica islandica
(Toland et al., 2000).
Ba/Ca ratios follow the same cyclicity as the Sr/Ca signals
(Fig. 8C). Ba shows a mean concentration of 30 mg/g, increasing in summer and decreasing in winter, with very variable
annual amplitudes (10 –40 mg/g). Ba/Ca in bivalve shells have
been related to particulate and dissolved Ba in water (Gillikin
et al., 2006a). Its seasonal variability was associated with the
primary productivity, coincident for example with the annual
algal biomass maximum and the spring-phytoplankton bloom
in the sea for Mytilus edulis (Van der Putten et al., 2000;
Gillikin et al., 2006a), Mercenaria mercenaria (Stecher et al., 1996)
and Arctica islandica (Toland et al., 2000) among others. In the
case of D. chilensis patagonicus, algae or phytoplankton bloom
are a plausible cause for the periodicity of both Sr and Ba
signals, and this is supported by the synchronicity of both
elements (Fig. 8C). Nevertheless, more work is required in D.
chilensis patagonicus to asses whether this could be an effect of
82
SEASONAL SIGNALS IN PATAGONIAN DIPLODON SHELLS
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Mg concentrations in D. chilensis patagonicus shells vary seasonally around a mean of 17 mg/g by as much as 5 mg/g
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ACKNOWLEDGEMENTS
The authors are grateful to Prof. Liliana Semenas
(Universidad Nacional del Comahue) for providing bibliography
on Diplodon. Benita Witt is acknowledged for the sample
preparation and the Nahuel Huapi National Park for allowing
sampling. Two anonymous reviewers helped to improve the
manuscript and are gratefully acknowledged. This study was
supported by the Geocycles Cluster, Johannes GutenbergUniversität Mainz and is publication number 468.
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