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Phenotypic and resource use partitioning amongst sympatric, lacustrine brown trout, Salmo trutta Verspoor, Eric; Adams, Colin E.; Greer, R.; Piggott, Camilla; Hooker, Oliver ; Newton, Jason
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Citation for published version (APA): Verspoor, E., Adams, C. E., Greer, R., Piggott, C., Hooker, O., & Newton, J. (2018). Phenotypic and resource use partitioning amongst sympatric, lacustrine brown trout, Salmo trutta. Biological Journal of the Linnean Society, 124(2), 200-212. https://doi.org/10.1093/biolinnean/bly032
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Download date: 30. Sep. 2021 1 Phenotypic and resource use partitioning amongst sympatric,
2 lacustrine brown trout, Salmo trutta (Linnaeus).
3
4 CAMILLA V. H. PIGGOTT1, ERIC VERSPOOR2, RON GREER3, OLIVER HOOKER1, 4 JASON
5 NEWTON5 and COLIN E. ADAMS1
6 1Scottish Centre for Ecology & the Natural Environment, IBAHCM, University of Glasgow,
7 Rowardennan, Glasgow, G63 0AW, Scotland, U.K.
8 2The Rivers and Lochs Institute, University of the Highlands and Islands, Inverness, U.K.
9 3The Old Armoury, Blair Atholl, Scotland, UK
10 4 PR Statistics, 6 Hope Park Crescent, Edinburgh Scotland, UK
11 5 NERC Life Sciences Mass Spectrometry Facility, SUERC, East Kilbride, UK
12
13 Running header: Polymorphism in brown trout
14 Key words: polymorphism, speciation, divergence, evolution, ecotypes
15
16
17 Corresponding Author: Colin Adams. Scottish Centre for Ecology & the Natural Environment,
18 IBAHCM, University of Glasgow, Rowardennan, Glasgow, G63 0AW, Scotland, U.K.
19 Tel: 01360870512 E-mail: [email protected]
20
21
22
23 ABSTRACT
24 Divergence into discrete foraging specialist morphs living in sympatry is relatively well
25 described in lacustrine fishes of the Salmonidae. Although piscivorous forms of Salmo trutta
26 have been widely reported, other trophic foraging specialists are strangely rarely recorded
27 amongst Salmo species. Microsatellite and mitochondrial genetic data segregated Salmo
28 trutta collected from Loch Laidon, Scotland, into four distinct genetic groups. Three groups
29 analysed in this study showed significant differences in body shape, stomach contents,
30 muscle stable isotope signature, gill raker length and spacing, and habitat use. We conclude
31 that the three genetically defined groups comprise: a generalist foraging morph, a pelagic-
32 feeding specialist morph and a profundal macrobenthos feeding morph. The features
33 distinguishing these morphs however, show a degree of overlap. This appears to be only the
34 second record of invertebrate resource use partitioning associated with expression of
35 alternative morphologies in sympatry this species and the first report of a profundal feeding
36 morph. Why such polymorphisms are generally rare in Salmo species still remains unclear,
37 however potential explanations are discussed.
38
39 KEYWORDS: ecotypes – evolutionary – divergence – polymorphism - Salmonidae
40
41 INTRODUCTION
42 The patterns of structuring of phenotypes and genotypes found within a single species in
43 the wild have the potential to provide insights into the very beginnings of the evolutionary
44 processes that may ultimately result in new species (Snorrason & Skulason, 2004). Complex
45 intraspecific structuring is now relatively well documented in many freshwater fishes
46 (Fevolden et al., 2012). It is particularly prevalent amongst fishes from the northern
47 hemisphere that live in freshwater lakes that were glaciated during the Quaternary period
48 (Skulason & Smith, 1996). Where intraspecific structuring occurs between unconnected
49 waters, it is generally thought to be the result of differential selection pressures operating at
50 a local population level (Garant, Forde & Hendry, 2007); the effects of phenotypic plasticity
51 responding to differing local environmental conditions (Adams, Woltering, & Alexander,
52 2003b); the random effects of genetic drift (Frazer & Rusello, 2013); or some combination of
53 these (Adams & Huntingford, 2004; Alexander & Adams, 2004). In a number of places
54 however, the pattern of intraspecific structuring exhibits greater complexity and therefore
55 more complex explanations for the processes resulting in its formation are likely to be
56 required.
57 Such situations offer opportunities to explore more deeply, early evolutionary processes
58 (Smith & Skulason, 1996; Skulason, Snorrason & Jonsson, 1999). One such situation is where
59 a single waterbody supports two or more clearly defined alternative phenotypic and/or
60 genotypic groups from a single species. Amongst fishes there have been a number of
61 reports of sympatric morphs, ecotypes or forms, particularly, although not solely, from
62 lake-dwelling species (Pakkasmaa & Piironen, 2001; Adams et al., 2016). Where such
63 sympatric morphs exist, evidence of distinct ecological differences between them is also
64 commonly reported (Skulason & Smith, 1995; Skulason et al., 1999; Jonsson & Jonsson,
65 2001). Most frequently, sympatric phenotypic polymorphisms manifest as divergence across
66 the littoral – pelagic habitats (Knudsen et al., 2006) or less frequently across littoral –
67 profundal habitats (Hooker et al., 2016). Such polymorphisms are recognised as an
68 important evolutionary step towards the emergence of new species (Maynard Smith, 1966;
69 Dieckmann & Doebeli, 1999; Snorrason & Skulason, 2004)
70 Amongst the fish genera that are commonly found in recently glaciated fresh waters, there
71 is considerable documented evidence of a common pattern of sympatric polymorphism
72 exhibiting partitioning of feeding resources by foraging specialists (Table I). Such patterns
73 are widely reported amongst fishes of the Salmonidae family (Skulason & Smith, 1995)
74 (Table I). Amongst species of the Salmo genus, piscivorous specialist forms, usually living in
75 sympatry with littoral benthos feeders, are relatively widely reported in Salmo trutta
76 (Hughes et al., 2016). However discrete and sympatric foraging specialisations across the
77 available invertebrate foraging resources now widely reported for other salmonid genera is
78 rarely reported in Salmo (Behnke, 1972).
79 This study describes a rarely reported phenotypic and ecological divergence of three or four
80 serendipitously discovered sympatric populations in the brown trout, Salmo trutta L. 1758.
81 Originally resolved by population genetic analysis (Verspoor et al., submitted), this study
82 tests the following hypotheses, that:
83 1) S. trutta categorised into the three distinct genetic groups are also morphologically
84 segregated.
85 2) Distinct genetic groups exhibit different foraging ecologies.
86 3) Variation in morphology and foraging ecology are linked.
87
88 METHODS
89 STUDY SITE AND SAMPLING METHOD
90 Loch Laidon (56° 39’ 56.65” N, 004° 40’ 4.6667” W) is an oligotrophic lake 283 m above sea
91 level on Rannoch Moor, Perthshire, Scotland. The lake is approximately 8 km long, 4 km2 in
92 area, with a maximum depth of 39 m and a mean depth of 10.7 m (Murray & Pullar, 1910).
93 The catchment is dominated by open heather (Ericaceae) moorland (McLeod, et al., 2005;
94 Shilland, et al. 2011). Campbell (1979) reports S.trutta, Atlantic salmon Salmo salar (L.
95 1758), perch Perca fluviatilis (L. 1758) and Arctic charr Salvelinus alpinus (L. 1758) in Loch
96 Laidon. The contemporary status of this latter species however is uncertain as it has not
97 been collected during recent sampling there (see Verspoor et al. submitted).
98 As a full fish sampling protocol is presented elsewhere (Verspoor et al. submitted) only
99 pertinent details are presented here (Table S1). S. trutta were collected from Loch Laidon
100 during May 2008 using both benthic and pelagic Nordic pattern gillnets, which are non-
101 selective for salmonids between 45 and 495 mm (fork length) (Appelberg et al., 1995).
102 Bathymetric data (Murray & Pullar, 1910) were used to define a random stratified sampling
103 design for four habitats: benthic littoral (< 6m water depth); benthic sub-littoral (6 – 20 m
104 depth); benthic profundal (defined as >20 m depth) and the pelagic habitat, (sampled with a
105 6m deep floating net set from the surface above ca 30 m deep water). All nets were
106 deployed over one night; all S. trutta caught, were killed on site, sampled for genetics
107 (Verspoor et al., submitted) and frozen for further analysis.
108 In the laboratory, specimens were defrosted and digital images taken of the left side of all
109 fish (using a Canon EOS 650D) for geometric morphometric analysis. A background grid
110 provided both a scale for the image and a means to identify image distortion. Images
111 showing significant distortion were not analysed further. Standard length (mm) and weight
112 (g) were measured. A small piece of muscle tissue was taken from the left flank of each fish
113 for analysis of stable isotopes carbon and nitrogen. The first left hand side gill arch was
114 removed and following Bryce (Bryce et al., 2016) the upper arch (h) and lower arch (u)
115 lengths were measured to create a total arch length (a1 = h + u) using electronic callipers
116 (0.05mm accuracy). Gill rakers (g) were counted and the mean spacing between gill rakers
117 (s) calculated (s = a1 ÷ g). The five longest gill rakers were measured using a compound
118 microscope (100-400x magnification) and an ocular micrometer.
119 The stomach contents were removed from each fish, and prey sorted and identified to order
120 or family level. Each prey item recorded was assigned to one of three major habitat
121 categories, profundal benthic, littoral benthic or pelagic, depending on its ecology (Table II).
122 We included surface prey in the pelagic category, as those feeding on this prey type would
123 by definition be feeding in the pelagic zone. The frequency of the dominant prey type in the
124 stomachs of fish of each genetic group was estimated as the frequency of occurrence of fish
125 (the relative number of fish) sampled that contained 90% or more of any prey category by
126 number in their stomachs.
127 POPULATION ASSIGNMENT
128 Assignment of individuals to genetic populations was based on individual genotype data for
129 22 microsatelite loci of 172 individual fish which resolved into four genetic populations,
130 three of which are analysed here (more detail in Verspoor et al., submitted). Individuals of
131 the fourth genetic group, characterized by a large size and morphology typical of a
132 piscivorous form (often colloquially referred to as a “ferox”) was not analysed due to the
133 small sample size (N=2). Those analysed were allocated to one of three genetic groups;
134 nominally “Group One”, “Group Two” or “Group Three”.
135
136 STABLE ISOTOPES ANALYSIS
137 White muscle tissue samples from 69 fish, initially frozen at -20oC was later thawed and the
138 epidermal layer was removed. Muscle was then dried at 48oC for 96 h and ground to a fine
139 powder using a pestle and mortar. Samples of 0.7 mg (± 0.1 mg) were loaded into 5 mm tin
140 capsules and analysed for δ15N and δ13C, at the Natural Environment Research Council Life
141 Sciences Mass Spectrometry Facility, East Kilbride, by continuous flow isotope ratio mass
142 spectrometry (CF-IRMS). This system uses an Elementar Pyrocube elemental analyser
143 interfaced with a Delta XP IRMS. The standard deviation of multiple analyses of the internal
144 gelatine standard was 0.1% for both δ15N and δ13C analyses.
145 GEOMETRIC MORPHOMETRIC ANALYSIS
146 Twenty one homologous landmarks were identified on each fish image and digitised using
147 tpsDig2 software (Rohlf, 2013) (Fig. 1). Procrustes superimposition was conducted in
148 MorphoJ and used to remove variation resulting from size, position and orientation of
149 images (Turnbull et al., 2005; Klingenberg, 2011) and to correct for the effect of allometry
150 and lunate-like bending using regression analysis (Valentin et al., 2008; Hooker et al., 2016).
151 Canonical Variate Analysis (CVA) conducted in MorphoJ (Klingenberg, 2011) was used to test
152 for morphometric differences between the three groups defined a priori genetically (Adams,
153 Rohlf & Slice, 2004). Partial Least Squares (PLS) (Two Block PLS) executed in MorphoJ was
154 used to identify patterns of covariation between fish body shape and capture depth, stable
155 isotope signature, genetic group and diet in the absence of a priori genetic grouping
156 (Klingenberg, 2011).
157 STATISTICAL ANALYSIS
158 To remove the effect of allometry on mean gill raker length, mean gap between gill rakers
159 and the total number of gill rakers, these variables were regressed on fish standard length
160 to derive residuals (Reist, 1986; Adams & Huntingford, 2002). The effect of a priori group on
161 gill raker variables, independent of fish size (residuals) was then analysed using generalised
162 linear models (GLM’s), for Gaussian distributed data. The proportion of each prey group in
163 the stomachs of fish from each pre-assigned group, “One”, “Two” or “Three”, was analysed
164 using GLM’s for quasi-binomial-distributed data. Post hoc pairwise comparisons were
165 performed using Tukey’s HSD test. Frequency data were analysed using χ2 and all
166 proportionate data were arcsine transformed before analysis. All analyses were conducted
167 in R (R Development Core Team, 2013).
168
169 RESULTS
170 MORPHOMETRIC AND MERISTIC CHARACTERISTICS
171 Of the 172 fish collected from Loch Laidon, 134 specimens were intact and used in
172 morphometric, meristic and diet analysis. GLM analysis showed that body mass (F2,131 =
173 3.62, P < 0.05) but not body length (F2,131 = 2.77, P > 0.10) was significantly predicted by a
174 priori grouping (Fig. 2A). Group Three fish were significantly lower in mass than Group One
175 fish (p<0.04) and close to statistically significantly lower (P= 0.06) than Group Two fish (Fig.
176 2B). There was no significant effect of group membership on the length weight relationship
177 (F1,131=1.4; P=0.23).
178 Canonical Variance Analysis (CVA) was used to test for significant morphological separation
179 among the three genetic groups. Groups One and Two were significantly different for both
180 Procrustes distance and Mahalanobis distance (0.0238, P = <0.0001 and 2.4208, P = <0.0001,
181 respectively). Groups One and Three were significantly different for both Procrustes
182 distance and Mahalanobis distance (0.0178, P = 0.0001; 2.4491, P = <0.0001, respectively).
183 Group Two and Three were not significantly different for Procrustes distance (0.0082, P =
184 0.3716) but were significantly different for Mahalanobis distance (1.8888, P = 0.0001). CV1
185 accounted for 82.4% of the explained variation; the shape change associated with CV1
186 separated Group One from Groups Two and Three. CV2 accounted for 17.6% of the
187 explained variation and separated all three groups, with Group One having a shape
188 intermediate to those of groups Two and Three (Fig. 3). Fish from Group One were
189 characterised by a very deep head and anterior body, a large eye, a longer operculum and a
190 thicker, shorter tail. The majority of fish from Group One also had very pale skin colouration
191 and light red spots over the dorsal surface. Fish from Group Two were characterised by a
192 relatively slender, fusiform body, a narrower head with small eyes, relatively short pectoral
193 fins and a long, well-forked tail. Group Two fish were much darker in colour, especially on
194 the dorsal surface and displayed larger, dark, haloed spots on the dorsal and lateral sides.
195 Fish from Group Three were characterised by a much shorter body, a slightly deeper head
196 and body than Group Two, with slightly larger eyes and long pectoral fins. Group Three fish
197 also exhibited a darker colour on the dorsal but with smaller, less pronounced, dark spot
198 marks. Group Three fish expressed as intermediate in body shape and colouration
199 compared with that of Group One and Two fish.
200 The number of gill rakers was not significantly different between the three groups (F2,131 =
201 1.826, P > 0.10) (Fig. 4A). In contrast both the length of gill rakers (F2,131 = 14.45, p =
202 <0.001) and the mean distance between gill rakers (F2,131 = 10.03, P = <0.001) were both
203 significantly different between genetically defined fish groups. Group Two fish had
204 significantly longer gill rakers than fish from the other two groups which did not differ
205 significantly in length (Fig. 4B). Group Two fish also had a shorter mean distance between
206 rakers than Group One fish (Fig. 4C), Group Three fish were intermediate in raker gap and
207 did not differ significantly from either Group One or Group Two.
208 DIET
209 At least some individuals from each genetic group were found to have prey from each of the
210 three major habitat types in their stomachs (Fig. 5, Fig S1), however the frequency of
211 individuals with stomach contents dominated by each prey type differed significantly
212 between fish groups (χ2 = 35.2; d.f. = 4; p < 0.001). Amongst Group One, 49% of individuals
213 had stomach contents dominated by (defined as more than 90% of prey items) profundal
214 prey (Fig. 6). In Group Two 51% of individuals were dominated by pelagic prey (Fig. 6). In
215 Group Three the pattern was different, with 21% of individuals having stomach contents
216 dominated by profundal prey, 21% by pelagic prey and 14% by littoral prey items (Fig. 6).
217 Analysis of stable isotopes signatures found a significant difference in δ13C between a priori
15 218 groups (F2,131 = 5.547, P = < 0.01) but no significant difference in δ N (F2,131 = 0.2779, P =
219 0.7581). In post hoc pairwise analysis Group One had a significantly lower (more depleted)
220 δ13C (z = 3.273; p = <0.01) than Group Two. Group Three was intermediate in δ13C and not
221 significantly different from either Group One or Group Two (Fig. 7).
222 HABITAT USE
223 The frequency at which fish from different groups were collected from different habitats
224 differed significantly (χ2 = 72.1; d.f.=6; P<0.0001). The frequency of fish collected in benthic
225 set nets compared with pelagic set nets differed significantly (χ2 = 38.4; d.f.=2; P<0.001).
226 Group One and Three fish had a low incidence of capture in pelagic set nets and Group Two
227 fish were approximately equally represented in catches from both pelagic and benthic nets
228 (Fig. S2). For those fish collected in benthic nets, there was a significant difference in the
229 depth of capture between groups (F2,101=22.5; P<0.0001). Group One fish were caught at
230 significantly greater depth (mean+/- S.E. 17.6 +/- 1.6m) compared with the other two
231 groups which did not differ from each other (Group Two & Group Three means 5.4 m (+/-
232 0.49) & 5.5 m (+/- 0.56) respectively; Fig. S3).
233 PLS analysis was conducted without using a priori categorisation of fish into the three
234 genetic groups. PLS 1 explained 98% of variance and was driven by a well-defined axis of
235 δ13C, counter opposing δ15N, however this axis was not correlated with fish shape (P=0.103).
236 In contrast PLS2 explained 1.24% of variation and was highly correlated with fish shape
237 (P<0.001) (Fig. 8). The three genetic groups were clearly defined along this axis with each
238 group expressing as discretely different shapes along a shape continuum. Group One and
239 Group Two present as shape extremes and Group Three as intermediate in shape. Shape
240 was highly correlated with diet, with profundal and pelagic stomach contents at the
241 extremes of the PLS2 continuum (representing Groups One and Two respectively) and
242 generalist stomach contents (Group Three) intermediate to the two extremes (Fig.8). Shape
243 was also highly correlated with depth of capture of individual fish (Fig. 8).
244
245 DISCUSSION
246 S. trutta from Loch Laidon analysed in this study were assigned to one of three groups on
247 the basis of 22 informative microsatellite loci by cluster analysis although not all with equal
248 certainty (Verspoor et al., submitted). The genetic groups identified overlapped in body size
249 but segregated on the basis of morphology, foraging ecology and in habitat use. The PLS
250 analysis conducted without a priori genetic allocation of group membership showed that
251 body morphology was very strongly correlated with diet, habitat use (depth of capture) and
252 genetic group. There was strong evidence of divergence into three groups on the basis of
253 morphology, diet and to a lesser extent δ13C. Thus hypotheses 1, 2 and 3 are supported.
254 The evidence presented here, combined with that of Verspoor et al. (submitted) points to
255 Group One comprising a genetically distinct profundal benthos foraging specialist. The body
256 form of Group One (deep bodied, larger head and eye and thick short tail); the relatively
257 short and widely spaced gill rakers; the dominant prey taken immediately prior to collection,
258 (Chironomid larvae and Piscidium sp.); the depleted δ13C muscle tissue signature; the
259 habitat of capture for this group are all consistent with descriptions of profundal
260 benthivorous specialists in other salmonid species (Harrod, Mallela & Kahilainen, 2010;
261 Hooker et al., 2016). Hereafter this group is referred to as the “profundal benthivorous”
262 morph.
263 Specialist profundal benthivorous feeding morphs living in sympatry with other foraging
264 specialists have never previously been recorded in the Salmo genus. Where they have been
265 recorded in other genera, there are similarities to features of the profundal benthivorous
266 morph described here. A profundal benthivorous morph of Arctic charr has recently been
267 described from Skogsfjordvatn, Northern Norway, which has a robust, deep and long head,
268 a pale body colouration and larger eyes; it feeds predominantly on Chironomid larvae and
269 Pisidium sp. and is rarely collected at less than 20 m depth (Skoglund et al., 2015). In
270 contrast to the profundal benthivorous morph described here however, the Skogsfjord
271 morph has a maximum body size of about 9 cms compared with 21 cms recorded in S. trutta
272 in this study (Skoglund et al., 2015). A profundal benthovorous Arctic charr from Loch
273 Dughaill, Scotland had similar morphological characters as those described for both
274 Skogsfjordvatn and here for Loch Laidon, it also showed depleted δ13C compared to a
275 plankton feeding morph and did not show evidence of reduced body size (up to 28 cms was
276 recorded) (Skoglund et al., 2015; Hooker et al., 2016).
277 Group Two fish, predominantly fed upon plankton and thus foraged in the pelagic zone and
278 was collected predominantly there in this study (Verspoor et al. submitted). This group
279 differed from the profundal benthivorous morph in expressing a more slender, fusiform
280 body shape, with a narrow head, shorter pectoral fins and a long well-forked caudal fin.
281 They had a darker coloured dorsal surface, dark spots on the flank and gill rakers were
282 longer and more closely spaced than that of the profundal benthivore. The more enriched
283 δ13C muscle tissue signature is consistent with planktonic prey forming a major component
284 of their long-term diet, (Harrod et al., 2010; Hooker et al., 2016). Plankton feeding specialist
285 morphs have been relatively frequently described in sympatric polymorphic systems in
286 salmonids but only once previously in S. trutta, from Loch Melvin, Ireland (Ferguson, 1986).
287 The morphological, colouration and meristic characteristics described here show a strong
288 parallel with those described for planktivorous morphs from other salmonid species
289 (Snorrason et al., 1994; Adams et al., 1998; Fraser, Adams & Huntingford, 1998; Kahilainen
290 et al., 2011; Siwertsson et al., 2013; Hooker et al., 2016) and from plankton feeding S. trutta
291 of Loch Melvin (Cawdery & Ferguson, 1988) . Henceforth this group will be referred to as
292 the planktivorous morph.
293 The genetic group defined a priori as Group Three showed a significantly different but
294 intermediate body shape from the other two groups. Group Three fish had a shorter and
295 deeper body, larger eyes, and longer pectoral fins than the other groups; they were darker
296 in colour on the dorsal surface than the profundal benthivores and showed small, poorly
297 pronounced dark spots. They expressed shorter and more widely spaced gill rakers than the
298 planktivorous morph and had a δ13C signature intermediate to the other two morphs. They
299 were rarely collected from the pelagic zone and were predominantly found close to the
300 bottom near shore in shallow water (Verspoor et al., submitted). The occurrence of
301 individuals with stomach contents comprising littoral, profundal or pelagic origin prey was
302 relatively similar. Taken together, these data suggest that this group comprises a generalist
303 foraging group that does not specialise on a single prey type.
304 Analysis in this study shows that the foraging specialisms described for the planktivorous
305 and profundal benthivorous groups show some overlap. Stomach contents analysis only
306 gives a snapshot of prey taken very recently and it is certainly possible that the degree of
307 overlap or separation in diet might fluctuate seasonally. The stable isotope analysis however
308 provides an integrated measure of foraging over a much longer period suggesting that there
309 are longer term foraging differences between morphs that remain relatively stable over
310 time. Although highly significantly different, the three groups also overlap in body shape,
311 with the generalist group intermediate in shape. They also overlap in diet; the stomach
312 contents of all three morphs, a snapshot of recent prey choice, showed evidence of foraging
313 on prey from each of the three major habitat types (pelagic, profundal and littoral zones).
314 Similarly, although there were significant differences in habitat use based on benthic and
315 pelagic habitats and depth zones in the benthic habitat, there was also some distributional
316 overlap between morphs (see analysis here but more detail in Verspoor et al., submitted).
317 Additionally, and consistent with earlier reports of this form in Loch Laidon (Campbell,
318 1979), two specialist piscivorous (ferox) trout S. trutta were captured during sampling which
319 belong to one further genetically distinct population (Verspoor et al., submitted). Thus the
320 evidence is that there are four ecologically and morphologically distinct morphs of Salmo
321 trutta in Loch Laidon. Surprisingly there are at least three other examples of sympatric
322 polymorphism the same drainage system as Loch Laidon amongst Arctic charr populations
323 there (Adams et al., 2003; Adams et al., 1998; Fraser et al., 1998; Verspoor, et al., 2010).
324 Examples of lacustrine sympatric phenotypic polymorphisms associated with specialist
325 foraging of littoral macrobenthos and planktonic prey resources have been observed
326 relatively frequently amongst the Salmonidae (Frost, 1965; Svardson, 1979; Snorrason et al.,
327 1994; Adams et al., 1998; Landry & Bernatchez, 2001; Alekseyev et al., 2009; Siwertsson et
328 al., 2010; Kahilainen et al., 2011). Less frequently, but still reported, are exemplars of either
329 a planktonic or a littoral macrobenthos feeding specialist (and sometimes both) being
330 recorded along with a profundal habitat benthivore feeding specialist (Klemetsen et al.,
331 2006; Zimmerman et al., 2006; Siwertsson et al., 2013; Skoglund et al., 2015; Hooker et al.,
332 2016). It is becoming increasingly apparent that this pattern of divergence into two or three
333 specialist pelagic/ littoral/ profundal invertebrate foraging specialists in lakes is variously the
334 result of multiple, independent and parallel evolutionary events, and that they are relatively
335 common (Gíslason et al., 1999; Garduño-Paz et al., 2012; Siwertsson et al., 2013; Gordeeva
336 et al., 2014). However, the above examples derive from only two genera: Salvelinus and
337 Coregonus (see Table I for others). Piscivorous (ferox) and littoral feeding
338 macrobenthivorous forms have relatively frequently been reported in lacustrine S. trutta
339 (Campbell, 1979; Hughes et al., 2016). Despite Salmo being ubiquitous throughout its
340 range, and the best studied salmonid genus, until now, there has only been one clear
341 example of a divergence of foraging specialists based on alterative invertebrate resources
342 in this genus. The S. trutta of Lough Melvin, Ireland partition into three forms differing to
343 some extent in foraging ecology, with a benthic molluscivore (the gillaroo), a plankton
344 feeder (sonaghan) and a piscivore (ferox) (Day, 1887; Ferguson & Mason, 1981). These
345 morphs are sufficiently genetically differentiated to assume that they form separate gene
346 pools (Ferguson & Taggart, 1991).
347 In a few other lakes, genetic differences between sympatric populations have been reported
348 in S. trutta. Two genetically distinct populations in Lake Bunnersjoarna differ in body size
349 but there is no evidence of ecological specialism or more fundamental phenotypic
350 differences between these populations (Allendorf et al., 1976; Ryman, Allendorf & Ståhl,
351 1979; Andersson et al., 2016). Crozier & Ferguson, (1986) identified two genetically distinct
352 groups of S. trutta in Lough Neagh which differ in colouration but any ecological divergence
353 remains untested. Similarly Gratton et al. (2013) report two sympatric Salmo forms
354 (putative species) that differ genetically and in colouration but which showed evidence of
355 hybridisation but ecological divergence has not yet been examined (Gratton et al., 2013). In
356 contrast, expression of alternative life history strategies amongst S. trutta is common. The
357 presence of individuals expressing anadromy and those remaining in fresh water throughout
358 their life cycle is widely reported. Where it occurs, individuals expressing each life history
359 strategy usually comprise a single gene pool (Hindar et al., 1991; Charles et al., 2005).
360 Populations where S. trutta individuals reach a relatively old age, attain large body size, and
361 become piscivorous (ferox trout) have also been widely reported living in sympatry with
362 macrobenthos feeding individuals (Campbell, 1979). It has recently been estimated (Hughes
363 et al., 2016) that this form occurs in at least 192 lakes in Scotland alone. Ferox S. trutta,
364 from lakes in Scotland and Ireland often share a common lineage that is not shared with co-
365 existing macro-benthos feeders. Thus indicating that, at least for some populations, the
366 ferox life history strategy did not arise in sympatry but is more readily expressed in an
367 ancestral lineage that invaded Ireland and Scotland post-glaciation (Duguid et al., 2006).
368 Different fish lineages are known to have different evolutionary rates (Rabosky et al., 2013).
369 It is far from clear however, why for Salmo in general, and Salmo trutta in particular, there
370 are not more reports of foraging specialist diverging across the multiple invertebrate
371 foraging resources living in sympatry as there are for other lake dwelling salmonids. One
372 possibility is that this phenomenon is under-reported from amongst Salmo populations. It is
373 certainly highly likely that there are more polymorphic Salmo populations to be recorded,
374 owever in north-western Europe, Salmo is by far the most studied freshwater fish genus.
375 Under-reporting would thus not easily explain why this phenomenon is more frequently
376 recorded in the less well studied salmonid species. Another possibility is that the high
377 degree of phenotypic plasticity, believed to promote the relatively rapid divergence in some
378 species occupying recently glaciated lakes (Skulason et al., 1999), such those from the
379 Salvelinus (Adams et al., 2003b) and Coregonus genera (Lindsey, 1981), is less pronounced
380 in the genus Salmo. However, the scope for the plastic expression of phenotypic traits has
381 yet to be tested by experimentation in this genus. It is also possible that the genetic
382 architecture of Salmo spp. may be sufficiently different from other salmonid genera to make
383 rapid divergence more difficult. For example, the existence of “genomic islands”, small
384 clusters of linked loci, have been found in locally adapted polymorphisms. It has been
385 suggested that populations with genomic islands may benefit from more rapid evolutionary
386 potential (Khalturin et al., 2009; Schwander et al., 2014) and it is possible that Salmo spp
387 may not possess the appropriate genomic architecture to allow the ecological and
388 morphological divergence seen in other salmonid genera. This may also be linked to a fourth
389 possibility, that S. trutta does not have a fundamental body plan that allows for
390 morphological adaptation to forage efficiently on alternative prey types and are, as a
391 consequence, ecologically constrained. This latter possibility seems highly unlikely as the
392 basic body plan of S. trutta is not markedly different from that of other salmonids.
393 Additionally, at least in some lake habitats, S. trutta is known to outcompete other species,
394 such as Arctic charr (Forseth et al., 2003). The current study illustrates that the species
395 certainly does have the capability to express a range of different morphological adaptations
396 associated with different foraging strategies when the conditions allowing such traits to
397 emerge are manifest. Comparative analysis of the patterns and occurrences of
398 morphological and ecological polymorphisms within and among salmonid genera should
399 thus be a highly informative avenue for study of the evolutionary basis of the
400 polymorphisms described here.
401
402 ACKNOWLEDGEMENTS
403 We thank Jennifer Dodd and for assistance with diet analysis; Travis Van Leeuwen, Andy
404 Ferguson, John Allen and an anonomous referee for useful comments on an early draft of
405 this paper.
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585
586
587
588 FIGURE LEGENDS
589
590 Figure 1. The position of the 21 homologous landmarks used to estimate shape of fish from
591 each of the three genetically defined groups of S. trutta.
592
593 Figure 2. Mean (+ S.E.) for length (A) and weight (B) for each of the three genetically defined
594 groups (1, 2 & 3) of S. trutta. Similar alpha characters indicate no post hoc statistical
595 differences between groups.
596
597 Figure 3. CV1 and CV2 scores for each of the of the three genetically defined groups (1, 2 &
598 3) of S. trutta, from canoncial variate analysis of body shape with 95% confidence ellipses.
599 Wireframes represent the fish shape at the outer most point of each distribution scaled at
600 2:1 to aid visual representation.
601
602 Figure 4. Mean (+ S.E.) for the number of gill rakers on a single gill arch (A), the residual
603 mean length of the gill rakers (corrected for size; B), and the residual distance between gill
604 rakers (C), for the three genetically defined groups (1, 2 & 3) of S. trutta. Similar alpha
605 characters indicate no post hoc statistical differences between groups.
606
607 Figure 5. The proportion of each prey group, pelagic littoral benthic, and profundal benthic,
608 in the stomachs of individuals from the three genetically defined groups (1, 2 & 3) of S.
609 trutta.
610
611 Figure 6. The relative proportions in each of the three a priori defined genetic groups (1, 2 &
612 3) where the abundance of prey items of each of the habitat categories (profundal, pelagic
613 and littoral) exceeded 90% of all prey items in the stomach of an individual. Fish with no
614 detectable prey in their stomachs were excluded (Group One N=55; Group Two N=41;
615 Group Three N=31) (χ2= 35.2; P<0.001).
616
617 Figure 7. Mean (+ S.E.) for stable isotopes a) δ15N and b) δ13C for each of the three
618 genetically defined groups (1, 2 & 3) of S. trutta. Similar alpha characters indicate no post
619 hoc statistical differences between groups.
620
621
622
623
624 Figure 8. Two Block Partial Least Squares analysis of shape data. PLS1 (98% of variance
625 explained) is dominated by an axis comprising variation in δ15N counter opposing δ13C. This
626 axis does not correlate with body shape. PLS2 (1.2% variance explained) provides an axis
627 which separates body shape along a continuum which is also correlated with the major diet
628 groups, depth of capture and the three genetic groups of trout. The arrow heads indicate
629 the extreme end of axes that in some cases overlap.
630
631
632
633
634
635 TABLES
636 Table 1. A non-exhaustive list of exemplars of populations of lacustrine fish species
637 exhibiting sympatric polymorphisms and resource partitioning. * For additional references
638 on Artic charr polymorphisms see Hooker et al. 2016.
Common Species References name Arctic charr Salvelinus alpinus Snorrason et al., 1994; Adams et al., 1998; Jonsson & Jonsson, 2001; Alekseyev et al., 2009; Garduño-Paz et al., 2012; Hooker et al., 2016* Brook charr Salvelinus Bourke, 1997; Bertrand, Marcogliese & Magnan, 2008 fontinalis Lake charr Salvelinus Zimmerman et al., 2006; Eshenroder, 2008; Chavarie, namaycush Howland & Tonn, 2013; Chavarie et al., 2014 Dolly Varden Salvelinus malma Senchukova et al., 2013 charr Whitefish Coregonus Svardson, 1979; Amundsen, 1988; Kahilainen, lavaretus Lehtonen & Könönen, 2003; Amundsen, Knudsen & Klemetsen, 2004; Østbye et al., 2006 Lake whitefish Coregonus Bernatchez et al., 1996; Landry, Vincent & clupeaformis Bernatchez, 2007; Rogers & Bernatchez, 2007 Rainbow smelt Osmerus mordax Taylor & Bentzen, 1993 3-spined Gasterosteus McPhail, 1984, 1993; Kristjánsson, Skúlason & stickleback aculatus Noakes, 2002; Olafsdottir, Snorrason & Ritchie, 2007 Eurasian perch Perca fluviatilis Svanbäck & Eklöv, 2002; Svanback & Eklov, 2003 Roach Rutilus rutilus Faulks et al., 2015 Bluegill Lepomis Ehlinger & Wilson, 1988; Ehlinger, 1990 sunfish macrochirus Pumpkinseed Lepomis gibbosus Robinson et al., 1993; Robinson & Wilson, 1996 639
640
641
642 Table 2. Prey item groups in fish stomachs and their allocation to profundal benthic, littoral
643 benthic, or pelagic habitats depending on their ecology.
644 645 Profundal benthic Littoral benthic Pelagic Chironomidae Trichoptera (larvae) Ephemeroptera 646 (adult) Piscidium spp. Ephemeroptera Diptera (adult) 647 (nymph) Neuroptera (larva) Apidae (adult) 648 Gyrinidae (larva) Coleoptera (adult) Annelida Plecoptera (adult) 649 Dytiscidae (larva) Leptodora spp. Plecoptera (larva) Daphnia spp. 650 Nematoda Other winged insect Formicidae 651
652 653
654
655 Supplementary material
656 Supplementary figure legends
657 Figure S1. The proportion (%) of each a priori defined group of S. trutta containing specific
658 prey items in its stomach.
659 Figure S2. The relative proportion (%) of each of the three genetically defined groups of S.
660 trutta collected by gill net in pelagic and benthic habitats. Frequency analysis: (χ2 = 38.4;
661 d.f.=2; P<0.001).
662 Figure S3. Mean (+ S.E.) for depth of capture for the three genetically defined groups (1, 2 &
663 3) of S. trutta. Group One fish were caught at significantly greater depth compared with the
664 other two groups, which did not differ from each other.
665
Table S1. Fish collection locations and depth zones in Loch Laidon. Nordic pattern nets
(littoral, sub-littoral and profundal) were set on the lake bottom. Nordic pattern pelagic nets were set in the pelagic zone extending from the surface down to 6 m over deep water.
Latitude Longitude Depth (m) Habitat type 56° 38.865'N 4° 39.166'W 0-3 Littoral 56° 38.867'N 4° 39.147'W 3-6 Littoral
56° 38.911'N 4° 39.124'W 6-12 Sub-littoral 56° 38.867'N 4° 38.789'W 12-20 Sub-littoral 56° 38.912'N 4° 38.782'W 20-35 Profundal 56° 39.208'N 4° 38.290'W 35-50 Profundal
56° 40.699'N 4° 35.765'W 6-12 Sub-littoral
56° 40.718'N 4° 35.704'W 3-6 Littoral 56° 40.929'N 4° 35.397'W 0-3 Littoral 56° 39.278'N 4° 38.197'W Surface Pelagic