Otolith microchemistry and diadromy in Patagonian river fishes

Fish collections

Between 2004 and 2011, specimens of Aplochiton zebra, A. taeniatus, A. marinus, Galaxias maculatus, G. platei, Oncorynchus tshawytscha, O. kisutch, O. mykiss, and Salmo trutta were collected using various methods from six locations across a large latitudinal range (39.5–48.0°S) in western Patagonia, Chile (Fig. 1, Table 1). At each location, fish specimens were euthanized by an overdose of anaesthetic solution (tricaine-methanesulfonate MS-222 or clove oil). Due to the difficulties in morphological identification, genetic data were used to identify individuals in the genus Aplochiton to the species level (Alò et al., 2013). The McGill University Animal Care Committee (UACC), Animal Use Protocol No. 5291, approved use and handling of animals.

Otolith preparation

This study used the Strontium (Sr) to Calcium (Ca) molar ratio to infer habitat shifts across salinity gradients of Patagonian fishes. Strontium is particularly useful for reconstructing environmental history of fishes as it replaces Ca in the otolith matrix according to its availability in the fish habitat (Secor & Rooker, 2000; Campana, 2005; Pracheil et al., 2014).

Prior to specimen preservation, sagittal otoliths were extracted and either stored dry in test tubes or in 95% ethanol, as elemental compositions and structures of otoliths are not strongly affected by ethanol for the elements assayed (Proctor & Thresher, 1998). In the laboratory, otoliths were polished, cleaned, and mounted individually on clean glass slides using a thermoplastic cement (Crystalbond™). In order to expose growth rings, 3M™ (fine) and Nanolap^® Technologies (coarse) diamond lapping film wetted with deionized water was used to polish otoliths by hand until a satisfactory sagittal section of annuli was visible (Fowler et al., 1995). For Aplochiton spp., Galaxias spp., S. trutta, O. kisutch and O. mykiss otoliths, a 30-µm and then 3-µm lapping film was used to expose annuli and get a finished polish. O. tshawytscha otoliths required larger lapping film (45 and 60 µm) to reach an appropriate view, but were finished with 3 µm film for increased clarity. For larger otoliths (e.g., O. tshawytscha), it was sometimes necessary to polish on opposite sides to produce a thinner section.

Following polishing, the mounting adhesive was dissolved in a 100% acetone bath and sonicated for 10 min. Larger otoliths were cleaned a second time with acetone as needed. Each otolith was then sonicated twice in Milli-Q water for 5 to 10 min each. Following cleaning, otoliths were rinsed a final time in Milli-Q water, transferred to clean vials and placed in a positive laminar flow hood for 24–48 h to dry (similar to methods of Elsdon & Gillanders (2002)).

Acid-washed porcelain forceps were used to mount clean, dry otoliths on acid-washed microscope slides. Otoliths were grouped according to diameter and mounted 10–28 per slide accordingly. Each otolith was placed within one small drop of fresh Crystalbond melted onto a single side.

Slides were securely kept in acid-washed, sealed petri dishes for transport to Woods Hole Oceanographic Institute (Woods Hole, MA, USA). There, laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) was conducted from October 8th to 11th, 2012 (Aplochiton spp., S. trutta, Galaxias spp., O. kisutch, and O. mykiss) and again from February 4th to 5th, 2013 (O. tshawytscha). Laser ablation was performed with a large format laser ablation cell on a New Wave UP193 (Electro Scientific Industries, Portland, Oregon) short pulse width excimer laser ablation system. This was coupled with a Thermo Finnigan Element2 sector field argon plasma spectrometer (Thermo Electron Corporation, Bremen, Germany) for elemental analysis. The laser was configured for single pass, straight line scanning at a speed of 5 µm per second. The laser beam spot size was 50 µm at 75% intensity and 10 Hz pulse rate. Isotopic activity rates (counts per second) were determined for ⁸⁷Sr and ⁴⁷Ca. Certified standard FEBS-1 (Sturgeon et al., 2005) and a 1% nitric acid (HNO₃) blank were passed through the instrument before and after each block of 4 to 11 otoliths to minimize bias in elemental mass. Each otolith was visualized on screen and the intended ablation transect of each sample was plotted digitally and analysed by ablation with a laser beam (refer to Fig. 2 or supplemental pictures: https://doi.org/10.6084/m9.figshare.6387665.v2 for a visual example).

Data analysis

Digital images of each otolith were taken after the laser ablation procedure with a digital Nikon Coolpix P6000 split with a Martin microscope MM Cool mounted on Nikon SMZ800 lenses and illuminated with a NII-LED High Intensity Illuminator (Supplemental pictures: https://doi.org/10.6084/m9.figshare.6387665.v2).

For accuracy, each raw data point was produced from an average of ten consecutive reads. Isotopic intensities were corrected by subtracting the means of the isotopic intensities detected in the blanks. Points below the limit of detection of the same isotope measured in the blanks (mean - 3SD) were eliminated. Isotopic intensities were converted to elemental intensities by taking into account the percent natural occurrence of the isotopes. Strontium was standardized to Ca and converted to molar Sr:Ca ratios using sequential measurements of the standard reference material and the atomic mass of the elements analysed (Wells et al., 2003; Wolff, Johnson & Landress, 2013) (raw data and Python scripts to convert from isotopic intensities to molar ratios provided as Dataset S1). Elemental data and otolith transects were rigorously checked, and outliers caused by recording errors were removed (additive outliers, R Package “tsoutliers” v. 0.6–5, (López-de-Lacalle, 2016)). The ideal double life-history transect obtained ran across each sagittal otolith and through the primordium, thus providing two similar (redundant) patterns related to life history variation, one on either side of the primordium. Interpretations were based on the analyses of both sides of each double transect, if possible. However, in a number of cases, transects were imperfect due to damaged otoliths or inaccurate ablation pathways (Supplemental pictures: https://doi.org/10.6084/m9.figshare.6387665.v2.) In these cases, data were analysed as partial transects that were used to differentiate putatively diadromous or resident signals (Fig. 2).

Classification and Regression Trees (CART, Breiman et al., 1984) were used to detect shifts in elemental ratios across the otolith transect. CART is an alternative to qualitative methods traditionally used to interpret the chronological signal in otolith microchemistry transects (Vignon, 2015). The position along the otolith transect (predictor variable) was recursively partitioned using regression trees in order to differentiate segments of the transect that shared similar mean Sr:Ca values (response variables) (Breiman et al., 1984; Therneau & Atkinson, 1997; De’ath, 2002; Strobl, 2009). CART was implemented in the Tampo library (version 1.0) for R statistical software 3.0.2 (Vignon, 2015).

Summary statistics of molar Sr:Ca ratios were calculated across all individuals. The main goal of this work was to represent movement patterns at a broad scale. Therefore, since shifts in Sr:Ca have been well documented in many species of fish moving across environments with variable Sr (Tzeng & Tsai, 1994; Limburg, 1995; Tzeng, Severin & Wickstrom, 1997; Campana, 1999; Limburg et al., 2001; Chang, Iizuka & Tzeng, 2004; Araya et al., 2014), CART analysis was used in a semi-supervised manner to identify the presence or absence of sudden discontinuities in the Sr:Ca otolith signal (Vignon, 2015). This was done by setting three progressively relaxed conditions to the splitting procedure. A splitting condition is the minimum difference in mean values between consecutive Sr:Ca profile segments beyond which the segments are permanently split in different categories. The three splitting conditions adopted were 0.5, 0.7, and 1.0. The detection of one or more discontinuities or splits in the Sr:Ca signal was interpreted as evidence in favour of diadromy, or otherwise, evidence in favour of residency. When diadromy was inferred, the direction of ontogenetic movements was deduced from differences in segment means; increasing values indicated movements towards the sea, and vice versa.

Further inference about habitat occupancy (freshwaters, estuaries, or the sea) required a visual, heuristic examination of Sr:Ca profiles in relation to (i) the sites of capture, (ii) comparisons with movement patterns inferred from all the species analysed, (iii) assumption of positive correlation between environmental Sr and marine influence (Secor & Rooker, 2000). Finally, all evidence was assembled to make individual inferences about migration patterns suggestive of amphidromy, catadromy, or anadromy. However, determining the precise extent of movements between habitats was beyond the scope of this work. Finally, otolith transect quality affected the confidence of our interpretations; from maximum confidence on inferences from transects that conformed to the hypotheses of the models proposed in Fig. 2, to uncertain interpretations from incomplete or faulty transects.

Native galaxiids

Large elemental shifts in otolith profiles suggested a catadromous life-history for most A. taeniatus analysed (Table 1 and Fig. 5). Even when confined to strictly freshwater habitats, as in Lake Caro, A. taeniatus juveniles showed a mild shift in Sr:Ca suggesting movements between freshwater habitats (Fig. 5, cond. = 0.5), as contrasted with A. zebra or O. mykiss, which showed no such shifts.

Otolith profiles (Figs. 4 and 5) suggested that A. marinus copes with high levels of salinity variation in the Baker River system. Otolith primordia of all specimens of A. marinus showed evidence of higher Sr:Ca ratios at early stages of growth, presumably before the fish entered the estuary (site of capture). Taken together, these data suggest that A. marinus is catadromous.

Results indicated that A. zebra uses a chemically uniform habitat at both collection localities (Fig. 4), although results should be corroborated by future studies because A. zebra individuals assayed were juveniles. Nevertheless, specimens from Tocoihue River appear to have been exposed to marine influence compared to those from Lake Caro (Table S2), suggesting preference for freshwater residency, but capacity for osmoregulation when salinity levels increase.

G. maculatus individuals were sampled from the same site as some specimens of A. zebra (Tocoihue). However, among four specimens assayed, three showed the highest variation in Sr:Ca compared to any other galaxiid species analysed (Fig. 3) with evidence of both catadromous and amphidromous transitions (Table 1). The G. maculatus specimen in Fig. 4 was caught in the lower reach of the Tocoihue River in an area with strong tidal influence. The otolith profile suggests an amphidromous life-cycle with intermediate Sr:Ca levels in the primordium followed by increases in Sr:Ca ratio and subsequent decrease to lower Sr:Ca levels. A fourth specimen of G. maculatus showed no major Sr:Ca fluctuations across the otolith transect (Table S2), suggesting that this individual did not drift out to the ocean during its larval stage.

Only one specimen from the low-elevation coastal Palos Lake was assayed for G. platei, and results indicate freshwater residency as revealed by uniformly low Sr:Ca ratios across the entire otolith transect (Figs. 3 and 4).

Introduced salmonids

This study supports established anadromy in O. tshawytscha in Patagonia, as previously shown by other authors (Ciancio et al., 2005; Correa & Gross, 2008; Arismendi & Soto, 2012; Araya et al., 2014). Data are consistent with changes in Sr:Ca concentration levels that suggest hatching in freshwater, migration to areas with marine influence followed by a return to inland, freshwater areas to spawn (Fig. 4). O. tshawytscha specimens collected from the Simpson River do not show as much variation as other O. tshawytscha from this study most likely because these fish were all juveniles that had not yet migrated.

The two parr O. kisutch analysed revealed one substantial Sr:Ca shift between birth and time of capture. Both otolith profiles showed relatively high Sr:Ca signatures around the core that diminished towards the edges (Fig. 4). These specimens where caught during the summer, about 55 km upstream of the Baker River’s mainstream. The observed pattern is consistent with maternal effects imprinting a marine signature near the core, which is formed during the yolk absorption phase (Kalish, 1990; Volk et al., 2000; Zimmerman & Reeves, 2002). Our results add to the sparse documentation of the establishment of self-sustaining O. kisutch populations in southern Patagonia (Górski et al., 2016). Conversely, O. mykiss exhibited a pattern consistent with freshwater residency and minor Sr:Ca fluctuations over the entire life cycle (Figs. 3 and 5).

Evidence of at least two different life cycle patterns emerged for S. trutta specimens caught at three different locations. The Sr:Ca transect of juveniles from Lake Caro and adults from Lake Palos showed a pattern consistent with continuous residency in freshwater (Figs. 3 and 5) whereas S. trutta from Baker River showed higher values at the primordium (Fig. 4).

Limitations and further studies

Some otolith results may have been influenced by maternal effects or induced by local temporal variation in water chemistry. Further studies may indicate whether the higher Sr:Ca ratios in primordia observed in some species could be attributable to maternal effects or other causes. For example, although the mechanisms are not completely understood, physiological constraints in early ontogeny could increase the rate of Sr absorption into the calcium carbonate matrix of the otolith (De Pontual et al., 2003). Also, as the Baker river system is influenced by a large ice field (Campo de Hielo Norte), high amounts of glacier flour (suspended solids) can contribute to increased salinity levels in water that flows into the estuary (Vargas, Aguayo & Torres, 2011; Marín et al., 2013). These seasonal salinity changes may promote the uptake of Sr into the otolith matrix and confound the assumption of low Sr in freshwater environments (Zimmerman, 2005). Therefore, even though Sr has been traditionally recognized as a very robust marker to discriminate between marine and freshwater environments, several recent studies have indicated that factors such as species-specific variation, environmentally-mediated physiological processes, individual variation and the interaction of different environmental factors can influence Sr uptake into the otolith matrix (Elsdon & Gillanders, 2002; Elsdon & Gillanders, 2004; Gillanders, 2005; Gillanders et al., 2015; Sturrock et al., 2015).

This study suggests that considerable variation in migratory life history may exist in Chilean fishes, but its inferential scope is restricted by the limited number of samples, which were collected for other research purposes (see Correa & Hendry, 2012; Correa, Bravo & Hendry, 2012; Alò et al., 2013), and by lack of water chemistry samples. The interpretations given for diadromous and resident patterns are limited to the observed Sr:Ca shifts in the otolith profiles and should be considered carefully, especially when referring to the extent of movement between different habitats. Species-specific reference values for Sr:Ca ratios and a more comprehensive sampling will be needed to quantify the extent of fish movements within Chilean continental waters in more detail. Ideally, a variety of different field techniques (natural markers such as stable isotopes, otoliths, statoliths for lampreys, or scales; as well as molecular markers, tagging, trapping and tracking) and laboratory methods (e.g., movement physiology, swimming performance, metabolism) (see Dingle, 2014) should be used to characterize daily and seasonal migration patterns. Incorporation of this knowledge could improve design and operation of fish passage structures to benefit native fishes (Laborde et al., 2016). Currently, information on fish life history for native fishes in Chile and elsewhere in the temperate Southern Hemisphere is lacking, and fish passage design is generally based solely on professional judgment (Wilkes et al., 2018).

Conservation issues

This study could help refine the conservation priorities for freshwater fishes in southern Chile. Given high endemism and the likelihood of dependence on diadromous behaviour, potential threats to fishes from fragmentation of river-to-estuary networks are correspondingly high. Hydroelectric power development causes loss of hydrological connectivity and alteration of the river flow regime, disproportionately affecting fishes with migratory life histories (Fullerton et al., 2010). Comparative otolith microchemistry results underscore the variation in life history strategies that should be accounted for when planning to manipulate water-flow for hydroelectric developments. Diadromous species depend on the habitat diversity and complexity created by unobstructed watersheds and are locally extirpated when barriers preclude movement to essential habitat. Additionally, anthropogenic barriers and alterations to water flow (e.g., hydropeaking) may also negatively affect landlocked populations because such structures disrupt successful reproduction, recruitment and habitat quality (Alò & Turner, 2005; Garcia et al., 2011). Current hydropower capacity in Chile (∼6.000 megawatts of energy connected to the central grid) is expected to increase (∼11.000 megawatts by the year 2020) building approximately 900 additional hydroelectric power plants due to legislative privileges given to hydroelectric investors such as (i) water allocation rights favouring in-stream productive uses; (ii) ease for hydropower investors to acquire riverside land; (iii) specific tax easements and deferments for hydroelectric investors; and iv) taxes imposed for “ecological” in-stream uses (Prieto & Bauer, 2012; Santana et al., 2014; Toledo, 2014). Preliminary studies have identified most of the hydroelectric potential in the south-eastern sector of the country, in the sub-basins with high elevations and discharge (Santana et al., 2014). Development is slated in basins that harbour the majority of native fish species diversity.

Ongoing spread of exotic species threatens native species through negative interactions including predation, competition, behavioural inhibition and homogenization (Correa & Gross, 2008; Penaluna, Arismendi & Soto, 2009; Correa & Hendry, 2012; Correa, Bravo & Hendry, 2012; Habit et al., 2012; Arismendi et al., 2014; Vargas, Arismendi & Gomez-Uchida, 2015). In particular, establishment of migration runs of O. tshawytscha and O. kisutch could trigger additional threats such as the shift of significant amounts of marine-derived nutrients to previously oligotrophic environments (Helfield & Naiman, 2001; Arismendi & Soto, 2012) and increased competition for limited resources with the native diadromous species. Non-native salmon and trout are also likely to be negatively affected by future hydroelectric dams. Additional hydropower development will almost certainly impact a flourishing tourism industry supported by salmonid recreational fisheries (Arismendi & Nahuelhual, 2007; Vigliano, Alonso & Aquaculture, 2007).

The evolutionary processes that allowed dispersal and colonization of Patagonian fishes are influenced by the region’s unique geography, climate, and geological processes. To ensure conservation of native freshwater, diadromous, and commercially relevant sport fisheries, managers will have to carefully designate and protect critical habitats, and in many cases mitigate obstruction of river flows imposed by dams with appropriate fish passage structures (Wilkes, Mckenzie & Webb, 2018). Long-term monitoring should also be a priority to understand the broad impacts of hydropower development on aquatic biodiversity.