Otolith microchemistry techniques and analyses
To collect fish for otolith analyses, ten juveniles that had recently
returned from their pelagic developing habitat to a fluvial environment
were collected from each coastal stream and lake tributary via backpack
electric fishing (Kainga EFM300; NIWA Instrument Systems), euthanized
using an overdose of anesthetic, and preserved in 90% ethanol. Sagittal
otoliths were then retrieved in the laboratory. Otolith surfaces were
cleaned by sonication in ultra-pure water for 30 s, allowed to air dry,
then mounted on a standard glass microscope slide covered with double
sided sticky tape (Warburton, Reid, Stirling, & Closs, 2017). Larval
otolith trace element signatures were obtained by depth profiling laser
ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) as
described in Warburton et al. (2017). Depth profiling was
completed at the University of Otago Centre for Trace Element Analysis
on an Agilent 7500cs ICP-MS coupled to a Resonetics (now ASI) 155
RESOlution M-50 laser ablation system powered by a Coherent 193 nm ArF
excimer laser. During depth profiling, the laser was repeatedly fired at
the same spot on the surface of the otolith, ‘drilling’ through the
otolith and obtaining a complete life-history profile from otolith
surface (point of capture) through to the core (hatch). Possible
down-hole effects associated with depth profiling were minimized
following the recommendations of Warburton et al. (2017). A 75 µm spot
size was used at a firing rate of 10 Hz tuned to capture an 11-element
suite (Ca, Ba, Sr, Rb, Mn, Li, Cu, Ni, B, Mg, Al) with a sample fluence
of 2.5 ± 0.1 Jcm-2. Condensation and contamination
were minimized by completing ablations in a chamber containing an
atmosphere of pure helium gas. Possible machine drift was accounted for
by taking a 20-s gas blank at the start of each ablation, and standards
(NIST 610, NIST 612, MACS-3) were run bracketing every 10 samples.
Standard values for NIST were obtained from Jochum et al. (2011) and the
standard value for MACS-3 was obtained from Chen et al. (2011). The
chance of hitting otolith cores was maximized by mounting slides in a
sampling cell and using a 400x video microscope to view otoliths.
Data were processed using the Trace_Elements and Trace_Elements_IS
data reduction schemes in IOLITE version 2.5 (Paton, Hellstrom, Paul,
Woodhead, & Hergt, 2011), with 43Ca set as the
internal standard, resulting in elemental data expressed as a molar
ratio of mols element/mols Ca. NIST 610 was set as the calibration
standard, while NIST 612 (Jochum et al., 2011) and MACS-3 (Chen et al.,
2011) were used as reference materials. To interpret otolith signatures,
the otolith core was identified by a spike in Mn (Ruttenberg et al.,
2005), and the 7–10 µm of drilling depth before this spike was selected
as the larval development period, as indicated by traces (see
Supplementary material, Appendix 1, Fig. A1). Elemental concentrations
within this period were averaged to use as a standardized, comparable
larval development value. Otolith trace element signatures lacking Mn
spikes were excluded from further analyses due to the likelihood of
having missed the natal core region, and this resulted in final sample
sizes of 47 (coastal), 44 (Wanaka), and 47 (Wakatipu). Marine versus
lake development was determined by interpreting patterns in Ba and Sr.
High Sr and low Ba are characteristic of marine development, and the
opposite is generally true in freshwater (Campana, 1999; Warburton et
al., 2017).
Statistical methods similar to Hogan et al. (2014) were used to
determine population structuring. To determine whether a linear
discriminant analysis (LDA) could be used for evidence of population
structure in otolith signatures, boxplots for each element were used to
determine which elements were most likely to show variation. A
six-element suite (Sr, Ba, Rb, Mg, Cu, Ni) was selected based on these
box plots and likelihood of element stability in the otolith (see
Campana, 1999). An LDA was then run on these six elements in the R
statistical environment using the MASS package (R Core Team, 2015;
Ripley et al., 2011) to examine clustering of otolith micro-chemical
signatures that would indicate discrete larval development pools. To
determine the geographic scale on which population structuring occurred,
an LDA was first run at the broad-scale system level (i.e. attempting to
reclassify individuals only to the three broad-scale habitats of
‘Coast’, ‘Wanaka’, and ‘Wakatipu’). To determine to what extent, if any,
populations are structured within-system, additional LDAs were then run
at a finer scale, i.e. within each lake or coastal region by site. See
Fig. 1 for sampling sites and a priori regional classification.
Re-classification rate was determined using training and test data where
half of the samples were used to determine clustering and the other half
then used for re-classification.