Several studies have shown that skin pigmentation in vertebrates is a relevant character affecting different biological processes, such as the ornamentation of individuals, female mating preferences and the reproductive success of breeders in terms of offspring quality (Nordeide et al. 2013Nordeide, JT, Kekäläinen, J, Janhunen, M & Kortet, R (2013) Female ornaments revisited - are they correlated with offspring quality? J Anim Ecol, 82(1), 26-38.
[http://dx.doi.org/10.1111/1365-2656.12021] [PMID: 23194443] ). In fishes, for example, its significance has been well documented in relation to the issue of sexual selection, as occurs in wild–type guppy males. In these fish, the male color pattern can determine the female mating preferences, since this character enhances the attractiveness of males during courtship (Kodric-Brown 1985Kodric-Brown, A (1985) Female preference and sexual selection for male coloration in the guppy (Poecilia reticulata). Behav Ecol Sociobiol, 17, 199-205.
[http://dx.doi.org/10.1007/BF00300137] , Houde and Endler 1990Houde, AE & Endler, JA (1990) Correlated evolution of female mating preferences and male color patterns in the guppy Poecilia reticulata. Science, 248(4961), 1405-8.
[http://dx.doi.org/10.1126/science.248.4961.1405] [PMID: 17747527] , Evans et al. 2004Evans, JP, Bisazza, A & Pilastro, A (2004) Female mating preferences for colourful males in a population of guppies subject to high predation. J Fish Biol, 65, 1154-9.
[http://dx.doi.org/10.1111/j.0022-1112.2004.00502.x] , Sathyan et al. 2013). In salmonids, a group that presents a characteristic species–specific pigmentation pattern (Colihueque 2010Colihueque, N (2010) Genetics of salmonid skin pigmentation: clues and prospects for improving the external appearance of farmed salmonids. Rev Fish Biol Fish, 20, 71-86.
[http://dx.doi.org/10.1007/s11160-009-9121-6] ), studies have established that this character has a close relationship with developmental, reproductive and physiological processes. For example, some studies found that skin color intensity (i.e., redness) and body color are associated with offspring quality (Janhunen et al. 2011, Ramstad et al. 2010Ramstad, KM, Woody, CA & Allendorf, FW (2010) Recent local adaptation of sockeye salmon to glacial spawning habitats. Evol Ecol, 24, 391-411.
[http://dx.doi.org/10.1007/s10682-009-9313-5] ), and that the silver skin color is related to the smoltification capacity of different strains (Nichols et al. 2008Nichols, KM, Edo, AF, Wheeler, PA & Thorgaard, GH (2008) The genetic basis of smoltification-related traits in Oncorhynchus mykiss. Genetics, 179(3), 1559-75.
[http://dx.doi.org/10.1534/genetics.107.084251] [PMID: 18562654] ). In addition, it has been reported that, in some species of this group, other skin pigmentation components, particularly the spotting variation, could be related with fish size (Aparicio et al. 2005Aparicio, E, García-Berthou, E, Araguas, RM, Martínez, P & García-Marín, JL (2005) Body pigmentation pattern to assess introgression by hatchery stocks in native Salmo trutta from Mediterranean streams. J Fish Biol, 67, 931-49.
[http://dx.doi.org/10.1111/j.0022-1112.2005.00794.x] ) and with the different environmental features of the rivers inhabited by these populations. In the latter case, reflecting the role of skin pigmentation as a signal status that could determine the spatial arrangement of individuals along the river bed (Penteriani et al. 2015Penteriani, V, Delgado, MM, Clavero, M & Ivanc, M (2015) Spottiness of European graylings (Actinopterygii, Salmonidae) correlates with environmental features and could signal the status of individuals. Italian Journal of Zoology, 82, 202-11.). Further studies have indicated that spotting variation in salmonids can be involved in behavior-related roles, such as stress responsiveness, given that highly spotted individuals are generally more resistant to stress than less spotted fishes (Kittilsen et al. 2009Kittilsen, S, Schjolden, J, Beitnes-Johansen, I, Shaw, JC, Pottinger, TG, Sørensen, C, Braastad, BO, Bakken, M & Overli, O (2009) Melanin-based skin spots reflect stress responsiveness in salmonid fish. Horm Behav, 56(3), 292-8.
[http://dx.doi.org/10.1016/j.yhbeh.2009.06.006] [PMID: 19539629] ). Taken together, these data provide strong evidence that skin pigmentation in salmonids plays an important role in different biological processes and may provide clues to facilitate the analysis of these events.

The spottiness is a characteristic attribute of the skin pigmentation of rainbow trout, Oncorhynchus mykiss (Walbaum, 1792). In adult individuals of this species, this character is made up of conspicuous small dark spots, mainly distributed on the flank (above and below the lateral line), on the back, including the operculum and the dorsal and caudal fins (Ade 1989Ade, R (1989) Trout and salmon handbookFacts On File, Inc., New York.). The available evidence indicates that the spottiness is not a constant character in rainbow trout, given that it may vary within and between populations, e.g., in number both below the lateral line (Islam et al. 1973Islam, MA, Nose, Y & Yasuda, F (1973) Number and distribution pattern of the black spots on body surface of rainbow trout. Nippon Suisan Gakkaishi, 39, 727-39.
[http://dx.doi.org/10.2331/suisan.39.727] , Tack 1973Tack, E (1973) Züchtungsfragen in der Forellenzucht. Oesterr Fisch, 26, 77-84.) and on the back (Colihueque et al. 2011Colihueque, N, Parraguez, M, Estay, FJ & Diaz, NF (2011) Skin color characterization in rainbow trout by use of computer-based image analysis. N Am J Aquaculture, 73, 249-58.
[http://dx.doi.org/10.1080/15222055.2011.581578] , 2014), or in size on the flanks (Kause et al. 2003Kause, A, Ritola, O, Paananen, T, Eskelinen, U & Mäntysaari, E (2003) Big and beautiful? Quantitative genetic parameters for appearance of large rainbow trout. J Fish Biol, 62, 610-22.
[http://dx.doi.org/10.1046/j.1095-8649.2003.00051.x] ). Thus, it is possible to find trouts within the same stock with a wide variation in the number of dark spots. For example, on the back, which may result in individuals with scarce or no spotting (< 0.5 spots/cm²), in contrast to others that can present a highly spotted skin (> 5.5 spots/cm²) (Colihueque et al. 2011Colihueque, N, Parraguez, M, Estay, FJ & Diaz, NF (2011) Skin color characterization in rainbow trout by use of computer-based image analysis. N Am J Aquaculture, 73, 249-58.
[http://dx.doi.org/10.1080/15222055.2011.581578] ). To date, the origin of this variation in the rainbow trout is unclear, however, it has been reported that the number of dark spots may vary in function of fish size and spawning time for the spottiness below the lateral line (Islam et al. 1973Islam, MA, Nose, Y & Yasuda, F (1973) Number and distribution pattern of the black spots on body surface of rainbow trout. Nippon Suisan Gakkaishi, 39, 727-39.
[http://dx.doi.org/10.2331/suisan.39.727] ), and with age for the spottiness on back (Colihueque et al. 2011Colihueque, N, Parraguez, M, Estay, FJ & Diaz, NF (2011) Skin color characterization in rainbow trout by use of computer-based image analysis. N Am J Aquaculture, 73, 249-58.
[http://dx.doi.org/10.1080/15222055.2011.581578] ). In this species additional evidences also support the possible effects of genetic factors on the variation of the number of dark spots, either below the lateral line (Islam et al. 1973Islam, MA, Nose, Y & Yasuda, F (1973) Number and distribution pattern of the black spots on body surface of rainbow trout. Nippon Suisan Gakkaishi, 39, 727-39.
[http://dx.doi.org/10.2331/suisan.39.727] , Tack 1973Tack, E (1973) Züchtungsfragen in der Forellenzucht. Oesterr Fisch, 26, 77-84.) or on the back (Díaz et al. 2011Diaz, NF, Estay, F, Araneda, C & Colihueque, N (2011) Heritability of Blue Back skin colour and correlations with growth in rainbow trout, Oncorhynchus mykiss. Proceedings of the Australian Society for Fish Biology 2011 ConferenceAustralian Society for Fish Biology, Townsville, Australia.). Most studies known to date on the analysis of the variation of the number of dark spots in rainbow trout have been carried out in cultured populations (Islam et al. 1973Islam, MA, Nose, Y & Yasuda, F (1973) Number and distribution pattern of the black spots on body surface of rainbow trout. Nippon Suisan Gakkaishi, 39, 727-39.
[http://dx.doi.org/10.2331/suisan.39.727] , Tack 1973Tack, E (1973) Züchtungsfragen in der Forellenzucht. Oesterr Fisch, 26, 77-84., Colihueque et al. 2011Colihueque, N, Parraguez, M, Estay, FJ & Diaz, NF (2011) Skin color characterization in rainbow trout by use of computer-based image analysis. N Am J Aquaculture, 73, 249-58.
[http://dx.doi.org/10.1080/15222055.2011.581578] , 2014), being their occurrence in natural or naturalized population of this species largely unknown. In natural populations of salmonid fishes, the evidence indicates a variation of this character is associated with biometric and environmental factors. For example, in brown trout the number of dark spots distributed below the lateral line is positively associated with fish size (Aparicio et al. 2005Aparicio, E, García-Berthou, E, Araguas, RM, Martínez, P & García-Marín, JL (2005) Body pigmentation pattern to assess introgression by hatchery stocks in native Salmo trutta from Mediterranean streams. J Fish Biol, 67, 931-49.
[http://dx.doi.org/10.1111/j.0022-1112.2005.00794.x] ) and in graylings, the spotting level on flank presents a clear interaction with different environmental features of the river that they inhabit (Penteriani et al. 2015Penteriani, V, Delgado, MM, Clavero, M & Ivanc, M (2015) Spottiness of European graylings (Actinopterygii, Salmonidae) correlates with environmental features and could signal the status of individuals. Italian Journal of Zoology, 82, 202-11.).

The rainbow trout was introduced to our country in 1905 (Golusda 1927Golusda, P (1927) Aclimatación y cultivo de especies salmonídeas en Chile. Boletín de la Sociedad de Biología de Concepción-Chile, 1, 80-100.). Currently, several naturalized populations of this species are found from northern to southern Chile inhabiting different river and lakes. In this body of waters, the rainbow trout has importance for recreational fishing purposes. The naturalized populations of rainbow trout inhabiting lakes from southern Chile, called Araucanian lakes, are adapted to particular limnic conditions given that these body waters present special environmental characteristics, such as a) glacial origin with volcanic influence in the soils, b) high deepness, c) thermal stratification, d) high in water transparency, and e) oligotrophic with very low salt/nutrient concentrations (Campos 1984Campos, H (1984) Limnological study of Araucanian lakes (Chile). Verhandlungen Internationale Vereinigung Limnologie, 22, 1319-27., Arismendi et al. 2011Arismendi, I, Sanzana, J & Soto, D (2011) Seasonal age distributions and maturity stage in a naturalized rainbow trout (Oncorhynchus mykiss Walbaum) population in southern Chile reveal an ad-fluvial life history. Annales de Limnologie - International. J Limnol, 47, 133-40.
[http://dx.doi.org/10.1051/limn/2011012] ). In addition, within these lakes, the naturalized populations of rainbow trout coexist with several native fishes and other introduced salmonids, most of them occupying the same habitat (Soto et al. 2006Soto, D, Arismendi, I & Gonzalez, J (2006) Southern Chile, trout and salmon country: Invasion patterns and threats for native species. Rev Chil Hist Nat, 79, 97-117.
[http://dx.doi.org/10.4067/S0716-078X2006000100009] ). Although some studies on the biology of naturalized populations of Araucanian lakes have been reported, such as those referring to the characterization of reproductive parameters and reproductive cycle (Wetzlar 1979Wetzlar, HJ (1979) Beitrage sur biologie und bewirtschaf tung von forellen (Salmo gairdneri und S trutta) in Chile PhD Thesis, Albert-Ludwig Universitat, Contanza, Germany., Soto et al. 2002), there have been no previous studies on their skin pigmentation. These populations provide an ideal opportunity to examine the relationship, for example, between the spottiness and reproductive parameters in nature to identify the factors that may contribute to spotting deviation. The spotting variation could be due to several factors, some of them being the heterozygosis of genes that control the trait, the interaction of individuals with different environmental features, the existence of different ecological pressures, sexual selection, and their correlation with behavioural and physiological traits, or a combination of the above. To assess whether the spotting level on the skin is associated with biometric and reproductive parameters of rainbow trout populations in nature, we investigated the relationship between the number of dark spots below the lateral line with different biometric and reproductive parameters in breeders of this species collected from a naturalized population.

This paper presents the result of the relationship assessed by stepwise multiple regression analyses between the number of dark spots below the lateral line with biometric and reproductive parameters by analysing male and female breeders from a naturalized population of rainbow trout that inhabit a lake from southern Chile. We analyzed breeders from two reproductive seasons to obtain a more complete view of the possible trends that could emerge among these parameters in nature.

The number of dark spots varied widely in both reproductive periods, from 5 to 134 in females and from 0 to 146 in males from the 2007 year, and from 4 to142 in females and from 5 to 230 in males from the 2012 year (Table 3). Examples of male and female breeders of the 2007 year showing the extreme spotting variation are shown in Fig. (2). Moreover, this parameter showed normal distribution in males and females of both reproductive periods (K-S test, P > 0.10) (Fig. 3). Females from the 2007 year, displayed a higher mean number of dark spots than males (39.7 ± 26.1 and 34.6 ± 34, respectively), meanwhile the opposite was observed in females and males in the year 2012 (39.4 ± 29.8 and 43.9 ± 47.9, respectively) (Table 3). However, among these means, there were no significant differences (2-way ANOVA, F_3/112 = 0.24, P > 0.05) either among sex within the same year or among sex between different years. It was noteworthy that the number of dark spots showed more variation in males than females in both years (males CV = 0.98–1.09 vs. females CV = 0.63–0.82). Indeed, in the year 2012 the number of dark spots in females exhibited less range (from 4 to142) than in males (from 5 to 230).

Table 3
Number of dark spots below the lateral line of male and female breeders of naturalized rainbow trout population from southern Chile. Comparison of means within and between sex was not significantly different according to 2-way ANOVA (F_3/112 = 0.24, P > 0.05). N = sample size.

Fig. (2) Images showing the wide spotting variation below the lateral line in breeders of a naturalized rainbow trout population from year 2007. A male with low spotted skin (< 20 dark spots), B male with high spotted skin (>120 dark spots), C female with low spotted skin (< 20 dark spots), D female with high spotted skin (>120 dark spots).

Fig. (3) Distribution of the number of dark spots below the lateral line in breeders of a naturalized rainbow trout population from southern Chile. (A) Males and females breeders of the spawning season from year the 2007, (B) males and females breeders of the spawning season from the year 2012.

Stepwise multiple regression for biometric parameters revealed a significant relationship, ranging from medium to large effects, in broodstocks during year 2007. Thus, results indicated a statistically significant relationship, and medium to large effects, between NS-BLL and BW for males (P < 0.01, d = 1.71) and NS-BLL and TL for females (P < 0.05, d = 0.73) (Table 4). BW in males presented a medium negative correlation with NS-BLL (β = -0.6496), while TL in females showed a weak positive association with NS-BLL (β = 0.3434). R² indicated that the model explained 42.1% and 11.7% of the variability in the NS-BLL of males and females, respectively (Table 5). Bootstrap correction of P-values of Spearman correlation showed that only the relationship between NS-BLL and BW for males (r_s = -0.5166, P = 0.0337, P_BOOTS = 0.0371) was significant at P < 0.05. Thus, BW in males had the greatest influence on the response variable NS-BLL. For males 2012 no significant correlations were registered between NS-BLL and any biometric parameters (P > 0.05). In the case of BW a negative correlation was found (β = -0.2794), but without significance (exact P-value = 0.1860). Stepwise multiple regression for reproductive parameters revealed a significant relationship for females during the years 2007 and 2012 (Table 4). This analysis indicated the selection of only one of the six independent variables tested in the model, which had a significant association, and large effects, with NS-BLL that corresponded to ED for females in year 2007 (P < 0.01, d =1.0) and SPT for females in year 2012 (P < 0.01, d = 0.96). In both cases, the correlations were positives and medium between the response variable and the independent variable (β = 0.4487 and β = 0.4309, respectively). R² indicated that 20.1% and 18.5% of variations in the response variable NS-BLL occurred because of changes in the independent variables ED and SPT, respectively (Table 5). Bootstrap correction of P-values of Spearman correlation showed that the relationships between NS-BLL and ED for females year 2007 (r_s = 0.5328, P = 0.0011, P_BOOTS = 0.0021), and NS-BLL and SPT for females year 2012 (r_s = 0.4166, P = 0.0067, P_BOOTS = 0.0113) were significants at P < 0.05. This result indicates that ED and SPT are important factors in the response variable NS-BLL . The equations of the fitted models were

Females year 2007:

NS-BLL = -0.9010 + 0.4461 x ED

Females year 2012:

NS-BLL = -3.8494 + 0.0186 x SPT

Table 4
Final stepwise multiple regression models. Models show the effect of the biometric and reproductive variables on the number of dark spots below the lateral line in male and female broodstocks of a naturalized population of rainbow trout from southern Chile. SE = standard error of the estimate, β = regression coefficient, P = p-value with exact p-value indicated in parenthesis.

Table 5
Model summary of the stepwise multiple regression. R = coefficient of multiple correlation, R² = coefficient of multiple determination, SE = standard error of the estimate, P = p-value with exact p-value indicated in parenthesis.

Skin pigmentation in fishes has been related to biological and environmental parameters to try to understand if the variation of this character, either within or among populations, has a biological significance. To date these studies have been able to clarify the participation of this phenotype in several processes, for example, in predation avoidance (Endler 1980Endler, JA (1980) Natural selection on color patterns in Poecilia reticulata. Evolution, 34(1), 76-91.
[http://dx.doi.org/10.1111/j.1558-5646.1980.tb04790.x] [PMID: 28563214] ), in female mating preferences (Kodric-Brown 1985Kodric-Brown, A (1985) Female preference and sexual selection for male coloration in the guppy (Poecilia reticulata). Behav Ecol Sociobiol, 17, 199-205.
[http://dx.doi.org/10.1007/BF00300137] , Houde and Endler 1990Houde, AE & Endler, JA (1990) Correlated evolution of female mating preferences and male color patterns in the guppy Poecilia reticulata. Science, 248(4961), 1405-8.
[http://dx.doi.org/10.1126/science.248.4961.1405] [PMID: 17747527] , Evans et al. 2004Evans, JP, Bisazza, A & Pilastro, A (2004) Female mating preferences for colourful males in a population of guppies subject to high predation. J Fish Biol, 65, 1154-9.
[http://dx.doi.org/10.1111/j.0022-1112.2004.00502.x] ), in phenotypic plasticity that may facilitate invasions in nature (Westley et al. 2013Westley, PA, Stanley, R & Fleming, IA (2013) Experimental tests for heritable morphological color plasticity in non-native brown trout (Salmo trutta) populations. PLoS One, 8(11), e80401.
[http://dx.doi.org/10.1371/journal.pone.0080401] [PMID: 24260385] ), in cryptic camouflage (Donnelly and Dill 1984Donnelly, WA & Dill, LM (1984) Evidence for crypsis in coho salmon, Oncorhynchus kisutch (Walbaum), parr: Substrate colour preference and achromatic reflectance. J Fish Biol, 25, 183-95.
[http://dx.doi.org/10.1111/j.1095-8649.1984.tb04865.x] , Donnelly and Whoriskey 1993Donnelly, W & Whoriskey, F (1993) Transplantation of Atlantic salmon (Salmo salar) and crypsis breakdow. Can Spec Publ Fish Aquat Sci, 118, 25-34.), in status signals related to the spatial arrangement of individuals along the river bed (Penteriani et al. 2015Penteriani, V, Delgado, MM, Clavero, M & Ivanc, M (2015) Spottiness of European graylings (Actinopterygii, Salmonidae) correlates with environmental features and could signal the status of individuals. Italian Journal of Zoology, 82, 202-11.), and, also, in stress response of individuals (Kittilsen et al. 2009Kittilsen, S, Schjolden, J, Beitnes-Johansen, I, Shaw, JC, Pottinger, TG, Sørensen, C, Braastad, BO, Bakken, M & Overli, O (2009) Melanin-based skin spots reflect stress responsiveness in salmonid fish. Horm Behav, 56(3), 292-8.
[http://dx.doi.org/10.1016/j.yhbeh.2009.06.006] [PMID: 19539629] ). In spite of this progress, few studies have been conducted on the role of skin pigmentation in relation to the reproductive success of broodstocks, although the scarce data available on this issue suggest its possible role in spawning displays and reproductive success (Wedekind et al. 2008Wedekind, C, Jacob, A, Evanno, G, Nusslé, S & Müller, R (2008) Viability of brown trout embryos positively linked to melanin-based but negatively to carotenoid-based colours of their fathers. Proc Biol Sci, 275(1644), 1737-44.
[http://dx.doi.org/10.1098/rspb.2008.0072] [PMID: 18445560] , Janhunen et al. 2011). This study contributes to understanding the influence of skin pigmentation, particularly skin spottiness, on reproductive and biometric patterns observed in two naturalized populations of rainbow trout. Through this analysis, we provide new data on the possible trends that could emerge among these parameters in nature, in which particular intrapopulation interactions may have taken place in response to different ecological factors.

In this study, we support that below the lateral line (area B) the number of dark spots is also a variable character in naturalized populations of this species from southern Chile. Thus, for example in the 2007 stock, this parameter varied from 4 to 142 and from 5 to 230 dark spots in females and males, respectively. As far as we know, to date no data on naturalized population from Chile and other countries has been published on this character to compare our results. However, taken into account the data available for the cultured population of this species, our results have a good concordance. Indeed, Islam et al. (1973)Islam, MA, Nose, Y & Yasuda, F (1973) Number and distribution pattern of the black spots on body surface of rainbow trout. Nippon Suisan Gakkaishi, 39, 727-39.
[http://dx.doi.org/10.2331/suisan.39.727] reported hatchery populations from Japan with a number of dark spots in the area B ranging either from 6 to 338, or from 12 to 316. In addition, our data also agree with reports from the other cultured population, as is the case of a German population where this character varied from 0 to 250 (Tack 1973Tack, E (1973) Züchtungsfragen in der Forellenzucht. Oesterr Fisch, 26, 77-84.). In summary, our data along with previous studies indicate that the number of dark spots below the lateral line of the rainbow trout is mostly a polymorphic character, a phenomena that are displayed by cultured and naturalized populations of this species.

The variation in the number of dark spots in the naturalized population of rainbow trout studied here is an interesting result that will require more in depth investigation to clarify their origin. However, this variation appears to be not sex-linked given that males and females analyzed in this work from both reproductive periods did not present significant differences in this parameter. Other factors, mostly studied in cultured populations of this species, although not analyzed in this work, may involve the effect of genetic, environmental and even, behavioural factors. For instance, the strain-specific variation in the number of dark spots below the lateral line (Islam et al. 1973Islam, MA, Nose, Y & Yasuda, F (1973) Number and distribution pattern of the black spots on body surface of rainbow trout. Nippon Suisan Gakkaishi, 39, 727-39.
[http://dx.doi.org/10.2331/suisan.39.727] ), and the transmission of this character to the offspring in experimental crosses of progenitors with contrasting number of dark spots (Tack 1973Tack, E (1973) Züchtungsfragen in der Forellenzucht. Oesterr Fisch, 26, 77-84.), represent findings that support the effect of genetic factors in rainbow trout. In line with these evidences, more recent data obtained in this species also indicate the effect of a polygenetic determination for the dark spotting on the skin, whose heritability (h²) could reach a value ranging from h² = 0.16 (Díaz et al. 2011Diaz, NF, Estay, F, Araneda, C & Colihueque, N (2011) Heritability of Blue Back skin colour and correlations with growth in rainbow trout, Oncorhynchus mykiss. Proceedings of the Australian Society for Fish Biology 2011 ConferenceAustralian Society for Fish Biology, Townsville, Australia.) to h² = 0.45 (Kause et al. 2003Kause, A, Ritola, O, Paananen, T, Eskelinen, U & Mäntysaari, E (2003) Big and beautiful? Quantitative genetic parameters for appearance of large rainbow trout. J Fish Biol, 62, 610-22.
[http://dx.doi.org/10.1046/j.1095-8649.2003.00051.x] ). In addition, the action of environmental factors on the spotting level in rainbow trout would also occur based on the evidence documented in other salmonids. Particularly, in the brown trout, where it has been demonstrated that the variance of the number of dark spots below the lateral line could be subjected to a significant environmental effect (Blanc et al. 1982Blanc, J, Poisson, H & Vibert, R (1982) Variabilité génétique de la ponctuation noire sur la truitelle Fario (Salmo trutta L.). Ann Genet Sel Anim, 14(2), 225-36.
[http://dx.doi.org/10.1186/1297-9686-14-2-225] [PMID: 22896233] ), and in graylings, whose spotting level in the flank is positively associated with particular environmental features of the river, such as water velocity, water depth and water transparency (Penteriani et al. 2015Penteriani, V, Delgado, MM, Clavero, M & Ivanc, M (2015) Spottiness of European graylings (Actinopterygii, Salmonidae) correlates with environmental features and could signal the status of individuals. Italian Journal of Zoology, 82, 202-11.). Other studies even support the effect of behavioural factors, given that in rainbow trout and Atlantic salmon the variation in spots number is related to the stress response of individuals (Kittilsen et al. 2009Kittilsen, S, Schjolden, J, Beitnes-Johansen, I, Shaw, JC, Pottinger, TG, Sørensen, C, Braastad, BO, Bakken, M & Overli, O (2009) Melanin-based skin spots reflect stress responsiveness in salmonid fish. Horm Behav, 56(3), 292-8.
[http://dx.doi.org/10.1016/j.yhbeh.2009.06.006] [PMID: 19539629] ), being this capacity, higher in spotted fish than in non-spotted fish.

In females of the year 2007, we found a weak positive association between the number of dark spots below the lateral line and total length. This pattern is discordant with previous studies since, in rainbow trout, a clear relationship among these variables has been described for specimens above a certain size (> 30 cm) (Islam et al. 1973Islam, MA, Nose, Y & Yasuda, F (1973) Number and distribution pattern of the black spots on body surface of rainbow trout. Nippon Suisan Gakkaishi, 39, 727-39.
[http://dx.doi.org/10.2331/suisan.39.727] ). Our finding also disagrees with reports from other salmonid fishes, such as the brown trout, since total length in this species is significantly associated with the number of dark spots located below the lateral line (Aparicio et al. 2005Aparicio, E, García-Berthou, E, Araguas, RM, Martínez, P & García-Marín, JL (2005) Body pigmentation pattern to assess introgression by hatchery stocks in native Salmo trutta from Mediterranean streams. J Fish Biol, 67, 931-49.
[http://dx.doi.org/10.1111/j.0022-1112.2005.00794.x] ), with those distributed in the ventral zone (Blanc et al. 1982Blanc, J, Poisson, H & Vibert, R (1982) Variabilité génétique de la ponctuation noire sur la truitelle Fario (Salmo trutta L.). Ann Genet Sel Anim, 14(2), 225-36.
[http://dx.doi.org/10.1186/1297-9686-14-2-225] [PMID: 22896233] ), and with the total number of red spots on the body (Kocabas et al. 2011Kocabas, M, Kayim, M, Can, E, Ates, M, Kutluyer, F & Aksu, Ö (2011) Spotting pattern features in the brown trout (Salmo trutta macrostigma, T., 1954) population. Sci Res Essays, 6, 5021-4.). In contrast, the males analyzed in this study, especially those of the year 2007, exhibited a strong inverse relationship between both parameters, i.e., larger specimens tend to have a lower number of dark spots below the lateral line than small ones. It is possible that this association may be related to the different development stages or ages of males analyzed if we consider that the spotting level decrease across the development. However, the available evidence in the rainbow trout indicates that on the contrary, this interpretation would not be consistent given that the spotting level of this species increases steadily across the development, such as have been observed at juvenile stage (Colihueque 2011). Therefore, it is likely that other factors could be involved in the origin of this pattern, being environmental factors an interesting hypothesis to be considered in the light of the evidence obtained in other salmonids. For example, in natural populations of graylings, Penteriani et al. (2015)Penteriani, V, Delgado, MM, Clavero, M & Ivanc, M (2015) Spottiness of European graylings (Actinopterygii, Salmonidae) correlates with environmental features and could signal the status of individuals. Italian Journal of Zoology, 82, 202-11. have supported the interaction of the spotting level on flank with different environmental features of the river where they inhabit, such as water velocity, water depth and water transparency. Indeed, individuals with high spottiness level exhibit a positive and significant association to fast waters or with greater depths. Interestingly, these interactions also reveal that the spotting level may constitute a status signal related to the spatial arrangement of individuals along the river bed. Although further analyses on this issue are required to clarify the possible effects of environmental factors on the spotting level in naturalized populations of rainbow trout, in our case it should be noted that specimen analyzed were collected at spawning season from the small inlet streams of Calafquén Lake during their migration period. As it has been observed in this species (Knapp and Vredenburg 1996Knapp, RA & Vredenburg, VT (1996) Spawning by California Golden trout: characteristics of spawning fish, seasonal and daily timing, redd characteristics, and microhabitat preferences. Trans Am Fish Soc, 125, 519-31.
[http://dx.doi.org/10.1577/1548-8659(1996)125<0519:SBCGTC>2.3.CO;2] ), the spawning behaviour is a complex process that involves the interaction of spawning females with particular characteristics of the river to construct their redds, such as water velocity, water depth, and substrate size. Moreover, in this process the interaction of females with males is also important for fertilization success, especially, if we keep in mind that in natural conditions ripe males are 3-9 times more abundant than ripe females (Knapp and Vredenburg 1996Knapp, RA & Vredenburg, VT (1996) Spawning by California Golden trout: characteristics of spawning fish, seasonal and daily timing, redd characteristics, and microhabitat preferences. Trans Am Fish Soc, 125, 519-31.
[http://dx.doi.org/10.1577/1548-8659(1996)125<0519:SBCGTC>2.3.CO;2] ). Thus, it is possible that the spotting variation of males that is negatively associated with size may constitute some class of signal to address the interaction by spawning females.

The relationship between skin color and reproductive parameters in rainbow trout has received little attention. In vertebrates, including fishes, this approximation has been analyzed in the context of the effect of female ornamentation on offspring quality, and therefore, as a factor affecting fitness (reviewed by Nordeide et al. 2013Nordeide, JT, Kekäläinen, J, Janhunen, M & Kortet, R (2013) Female ornaments revisited - are they correlated with offspring quality? J Anim Ecol, 82(1), 26-38.
[http://dx.doi.org/10.1111/1365-2656.12021] [PMID: 23194443] ). These studies indicate that different vertebrate ornamentation attributes (e.g., plumage coloration, size of black feather spots, skin color and body color) are related positively or negatively at different levels; for example, with the survival and body mass of the offspring and with the size, mass and color of eggs. In particular, the effects of spotting level on fitness were well documented in Rana pipiens several years ago (Merrel 1972). It has also been observed that a particular female ornamentation may affect the female’s own reproductive parameters, such as spawning time and fecundity (Roulin et al. 2001Roulin, A, Dijkstra, C, Riols, C & Ducrest, A (2001) Female- and male-specific signals of quality in the barn owl. J Evol Biol, 14, 255-66.
[http://dx.doi.org/10.1046/j.1420-9101.2001.00274.x] , Janhunen et al. 2011). In our case, results indicating that the existence of some reproductive parameters in female rainbow trout broodstocks, particularly egg diameter and spawning time, were significantly associated with the number of dark spots below the lateral line, suggest that this skin color attribute may be linked with female fitness. Thus, the spotting level could be interpreted as an indicator that reflects the reproductive quality of females, especially if we take into account that egg diameter (Brooks et al. 1997Brooks, S, Tyler, CR & Sumpter, JP (1997) Egg quality in fish: what makes a good egg? Rev Fish Biol Fish, 7, 387-416.
[http://dx.doi.org/10.1023/A:1018400130692] ) and spawning time (Siitonnen and Gall 1989, Quinn et al. 2002Quinn, TP, Peterson, JA, Gallucci, VF, Hershberger, WK & Brannon, EL (2002) Artificial selection and environmental change: Countervailing factors affecting the timing of spawning by Coho and Chinook salmon. Trans Am Fish Soc, 131, 591-8.
[http://dx.doi.org/10.1577/1548-8659(2002)131<0591:ASAECC>2.0.CO;2] ) are important traits for salmonid reproduction. In addition, since our study analyzed a naturalized population of rainbow trout, it is possible that ecological factors may be involved in the origin of this relationship. This possibility is not unlikely, in view of ecological studies on different fishes which indicate that greater reproduction success in females under natural conditions appears to depend on egg size, since larger eggs provide survival and growth advantages to the offspring during their first few days of life (Brooks et al. 1997Brooks, S, Tyler, CR & Sumpter, JP (1997) Egg quality in fish: what makes a good egg? Rev Fish Biol Fish, 7, 387-416.
[http://dx.doi.org/10.1023/A:1018400130692] ). In addition, our findings on the association between spottiness and spawning time, agree with other previous studies that reveal the existence of this relationship in cultured populations of rainbow trout (Islam et al. 1973Islam, MA, Nose, Y & Yasuda, F (1973) Number and distribution pattern of the black spots on body surface of rainbow trout. Nippon Suisan Gakkaishi, 39, 727-39.
[http://dx.doi.org/10.2331/suisan.39.727] ). They would also concur with data available on other vertebrates, such as the barn owl, where this association occurs in relation to the size of dark spots (Roulin et al. 2001Roulin, A, Dijkstra, C, Riols, C & Ducrest, A (2001) Female- and male-specific signals of quality in the barn owl. J Evol Biol, 14, 255-66.
[http://dx.doi.org/10.1046/j.1420-9101.2001.00274.x] ). However, in contrast to Islam et al. (1973)Islam, MA, Nose, Y & Yasuda, F (1973) Number and distribution pattern of the black spots on body surface of rainbow trout. Nippon Suisan Gakkaishi, 39, 727-39.
[http://dx.doi.org/10.2331/suisan.39.727] , we observed a positive relationship between the numbers of dark spots below the lateral line and spawning time, i.e., females with higher numbers of dark spots tend to spawn later in the reproductive season than females with lower numbers of spots. Although, based on the present data, it is difficult to clarify the origin of this discordance between both data sets, it is possible that strain differences, management conditions, selection or genetic factors may explain, in part, these results. This is not unlikely, given that Islam et al. (1973)Islam, MA, Nose, Y & Yasuda, F (1973) Number and distribution pattern of the black spots on body surface of rainbow trout. Nippon Suisan Gakkaishi, 39, 727-39.
[http://dx.doi.org/10.2331/suisan.39.727] studied cultured strains in Japan, which that are usually subject to intense management in fish farms and, in contrast to a free-living population, are reared under controlled environmental conditions. These conditions are very different to those experienced by the naturalized populations of rainbow trout studied in this work, given that they inhabit a lake in southern Chile (Calafquén Lake), with particular limnic conditions related to special water characteristics (Campos 1984Campos, H (1984) Limnological study of Araucanian lakes (Chile). Verhandlungen Internationale Vereinigung Limnologie, 22, 1319-27., Arismendi et al. 2011Arismendi, I, Sanzana, J & Soto, D (2011) Seasonal age distributions and maturity stage in a naturalized rainbow trout (Oncorhynchus mykiss Walbaum) population in southern Chile reveal an ad-fluvial life history. Annales de Limnologie - International. J Limnol, 47, 133-40.
[http://dx.doi.org/10.1051/limn/2011012] ). In the case of selection and genetic factors, their effect on spawning time and spottiness would be expected to produce considerable variation among strains. This is possible given that, in rainbow trout, spawning time (Siitonnen and Gall 1989, Su et al. 1999Su, G-S, Liljedahl, L-E & Gall, GA (1999) Estimates of phenotypic and genetic parameters for within-season date and age at spawning of female rainbow trout. Aquaculture, 171, 209-20.
[http://dx.doi.org/10.1016/S0044-8486(98)00494-3] ) and spotting level (Kause et al. 2003Kause, A, Ritola, O, Paananen, T, Eskelinen, U & Mäntysaari, E (2003) Big and beautiful? Quantitative genetic parameters for appearance of large rainbow trout. J Fish Biol, 62, 610-22.
[http://dx.doi.org/10.1046/j.1095-8649.2003.00051.x] ) characters have a significant genetic determination, and therefore, may respond to selection pressure, which would alter their distribution in populations. In fact, Kause et al. (2003)Kause, A, Ritola, O, Paananen, T, Eskelinen, U & Mäntysaari, E (2003) Big and beautiful? Quantitative genetic parameters for appearance of large rainbow trout. J Fish Biol, 62, 610-22.
[http://dx.doi.org/10.1046/j.1095-8649.2003.00051.x] have demonstrated that through artificial selection of the spottiness level of a cultured rainbow trout strain, it is possible to respond rapidly to selection pressure after a few generations.

Further studies on spotting variation in other naturalized populations of rainbow trout from Chile will be important to support the relationship of this parameter with biometric and reproductive parameters reported in this work. The analysis of the spotting variation in these populations, for example, according to the habitat characteristics, it will also be useful to add more data to clarify this issue.

No significant differences were observed in the mean number of dark spots below the lateral line between males and females in both reproductive seasons. This result suggests that spottiness below the lateral line is not sex-specific. Stepwise multiple regression analyses revealed that the body weight of males from year 2007 has a significant negative influence on the number of dark spots below the lateral line, while the reproductive variables egg diameter and spawning time of females from years 2007 and 2012, respectively, also have a relevant positive influence on this variable response. Overall, these results suggest that some biometric and reproductive variables may influence the skin spot variation of rainbow trout broodstocks living in natural environments.