3 Materials and Methods
3.1 Snail shells pretreatment and sampling strategy
The entire shell was firstly cleaned with distilled water, and the soil
particles attached to the shell surface were brushed using a toothbrush,
and then the shell was placed in a drying oven and heated at 60 °C for
12 hours. The relatively large shells were chosen for sampling along the
growth band. Firstly, weremoved the residual clay cements on the surface
of shells using a dental drill, then cleaned the shells using a
ultrasonic utility for multiple times, and finally dried the shells in
an oven. The three dimension of each shell (i.e.,shell height, width and
height of shell mouth) was measured using a ruler. For intra-shell
sampling, we use a micro drill to take powders from the shell lip till
apex at 1-2 mm interval along the growth direction of the snail (Figure
2). The drill bit was soaked in diluted hydrochloric acid solution after
each sample to remove residual carbonate powder on it.
For the carbon and oxygen isotope analyses of the whole shell, about 10
shells were combined according to the availability of snail shells in
each horizon. This can ensure the measured data to represent a general
and average environment condition under which land snails lived. After
the shell was cleaned and dried for the first time, it was broken into
fragments. The clay cement attached to each shell fragment was
physically removed, and then the fragments were further cleaned using an
ultrasonic utility. After very clean shell fragments were obtained, we
dried them in an oven at 60 °C. Finally, we ground them into powders and
homogenized using a mortar and pestle.
3.2 Stable isotope analyses
The carbon and oxygen isotopic analyses of the snail shell powder were
performed on the GasBench II multifunctional gas preparation system
coupled with the Delta V Plus isotope ratio mass spectrometer (Thermo
Fisher). A 100µg carbonate powder reacted with 100%
H3PO4 at 72 °C for 1 hour. The generated
CO2 passed through two NAFIONTM water traps to remove
trace water and passed through a PoraPlot Q chromatography column at 45
°C to separate with other impurities. After that, the
CO2 was introduced into the isotope ratio mass
spectrometer to measure the carbon and oxygen isotope ratios. Both
carbon and oxygen isotope data are reported relative to the VPDB. The
standards used for data correction and calibration were GBW4416
(δ13CVPDB=1.61‰,
δ18OVPDB=-11.59‰) and NBS19
(δ13CVPDB=1.95‰,
δ18OVPDB =-2.20‰). The analytical
precision of carbon and oxygen isotopes is 0.06‰ and 0.10‰,
respectively. Detailed analytical method can be found in Wang et al.
(2019).
4 Results
4.1 Carbon and oxygen isotopes of whole shell for two species land
snails
The variation range of δ18OVPDB for
cold-aridiphilous C. pulveratrix was -2.16‰ to -8.13‰, and the
average value was -5.03‰. The maximum value of
δ18OVPDB was at the depth of 1.1 m in
the profile, which corresponds to MIS2, while the minimum value of
δ18OVPDB was at the depth of 11.7m,
which belongs to MIS7. The δ18OVPDBvalue for sub-humidiphilous M. yantaiensis ranged from -7.34‰ to
-9.71‰, with an average of -8.43‰. The maximum
δ18OVPDB value was at 4.6 m (MIS4) and
the minimum at 11.6 m (MIS7).
The δ13CVPDB for C. pulveratrixranged from -3.17‰ to -6.62‰ with an average of -4.81‰. The maximum
δ13C was at the depth of 10.1 m in the profile, which
belongs to MIS6 whereas the minimum δ13C was at 6.6m,
which corresponds to the MIS5. The range of
δ13CVPDB for M. yantaiensis was
between -3.05‰ and -5.03‰, and the average value was -3.95‰. The maximum
δ13CVPDB for M. yantaiensisshowed at 3.4 m (MIS3) whereas the minimum
δ13CVPDB occurred at 12 m (MIS7).
4.2 Carbon and oxygen isotope changes along the growth band of
individual shell
In the MIS3 and MIS5, intra-shell
δ18OVPDB variation for C.
pulveratrix was from -12.3‰ to 0.2‰, and the variation of
δ13CVPDB was between -6.9‰ and -4.9‰.
In contrast, of the intra-shell variation of
δ18OVPDB for M. yantaiensis was
relatively small, i.e., from -10.1‰ to -5.9‰. The intra-shell
δ13CVPDB ranged from -7.7‰ to -4.8‰
(Table 1).
During the MIS4 and MIS6 stages, the intra-shell
δ18OVPDB and
δ13CVPDB for C. pulveratrixvaried from -12.0‰ to -3.5‰ and from -7.8‰ to -2.6‰, respectively. The
corresponding intra-shell variations for M. yanntaiensis were
much larger, i.e., from -13.3‰ to -1.7‰ for
δ18OVPDB and from -11.7 to -0.6‰ for
δ13CVPDB (Table 1).
The cold-aridiphilous C. pulveratrix had a shell height of 1.1 to
1.5 cm, a shell lip height of 0.7 to 0.85 cm, and a shell lip width of
0.6 to 0.8 cm. In contrast, the sub-humidiphilous M. yantaiensishad shell height ranging from 0.55 to 0.95 cm, shell lip height ranging
from 0.3 to 0.4 cm, and shell lip width ranging from 0.35 to 0.5 cm
(Table 2). Obviously, the shell of C. pulveratrix was
significantly larger than that of M. yantaiensis . As a result,
the intra-shell sampling number for C. pulveratrix was larger
than that for M. yantaiensis .
Table 2 Statistics for Intra-shell δ18O and
δ13C variations of two species at various MIS stages.