Otolith variation in Pacific herring (Clupea pallasii) reflects mitogenomic variation rather than the subspecies classification
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Pacific herring (Clupea pallasii) is divided into three subspecies: two in northeast Europe and one in the north Pacific Ocean. Genetic studies have indicated that the populations in northeast Europe have derived from the northwest Pacific herring recently, or during the last 10–15 kyr, and that they are distinct from the population in the northeast Pacific. In addition, hybridization between the Pacific herring and the Atlantic herring has been documented. Otolith variation has been considered to be largely affected by environmental variation, but here we evaluate whether the genetic differentiation is reflected in otolith shape differences. A clear difference in otolith shape was observed between the genetically differentiated herring species Clupea harengus from the Atlantic and C. pallasii. The otolith shape of C. p. suworowi in the Barents Sea was different from the shape of C. pallasii in northern Norway and C. p. pallasii from the Pacific. Populations of C. p. pallasii, sampled east and west of the Alaska Peninsula, which belong to two genetically different clades of the C. p. pallasii in the Pacific Ocean, show a clear difference in otolith shape. C. p. suworowi and the local C. pallasii peripheral population in Balsfjord in northern Norway are more similar to the northwest Pacific herring (C. p. pallasii) than to the northeast Pacific herring (C. p. pallasii), both genetically and in otolith shape. The Balsfjord population, known to be influenced by introgression of mtDNA from the Atlantic herring, does not show any sign of admixture in otolith shape between the two species. A revised classification, considering the observed genetic and morphological evidence, should rather group the northwest Pacific herring in the Bering Sea together with the European populations of C. pallasii than with the northeast Pacific herring in the Gulf of Alaska.
KeywordsHerring Subspecies Classification Otolith shape
Repeated trans-Arctic interchange of species from the Pacific and the Atlantic Ocean, following climate oscillations during the late Pliocene and Pleistocene, is considered to have played a large role in diversification and speciation among marine species at high latitudes (Vermeij 1991), e.g., in bivalves (Väinölä 2003) and gadoids (Carr et al. 1999). Several molecular studies have shown that some of this diversification is recent or has occurred after the last glacial period as in Theragra finnmarchica in northern Norway (Ursvik et al. 2007) and in Greenland cod, Gadus ogac (Carr et al. 1999). Due to the current changes in temperature, an increased number of trans-Arctic interchange may be expected to occur, resulting in greater connectivity and admixture between populations and species from the Atlantic and the Pacific.
Pacific herring (Clupea pallasii) provides one example of a trans-Arctic species, distributed in the coastal regions of the north Pacific Ocean and in the polar region of northeast Europe, from the Taymyr Peninsula, Russia in the east and west to Balsfjord in northern Norway. The geographic distribution and morphological characteristics within the Pacific herring have led to the description of three subspecies: the nominate subspecies C. pallasii pallasii in the Pacific, the White Sea herring (C. pallasii marisalbi) and the Chesha–Pechora herring (C. pallasii suworowi) of the southeast Barents and Kara Seas in Europe.
The Alaska Peninsula separates the Bering Sea from the northeast Pacific Ocean (Gulf of Alaska) and is considered to have been an obstacle for marine fauna and connectivity of populations in the region. Genetic divergence in mtDNA and microsatellites has been detected between herring occupying each side of the Alaska Peninsula, the northwest Pacific and the northeast Pacific lineages (O’Connell et al. 1998; Liu et al. 2012). Analysis of mtDNA variation by Laakkonen et al. (2013) on European C. pallasii showed that the European samples clustered within the northwest Pacific lineage defining the trans-Arctic group. The divergence between the herring in the northwest Pacific and Barents Sea within the group is recent or has occurred even after the last glacial period 10–15 kyr ago (Laakkonen et al. 2013), and signs of mixing have been reported to have occurred during the comparatively warm years of the 1930–1940s at several Arctic Siberian sites (Svetovidov 1952). The Arctic populations are characterized by low variation within areas, but genetic differentiation has been observed among the populations in the White Sea, in the Pechora Sea east of the White Sea and a strongly bottlenecked peripheral population in Balsfjord in northern Norway (Laakkonen et al. 2013; Semenova et al. 2015). A mixture of local Balsfjord herring and the highly migratory Norwegian spring spawners (Clupea harengus) based on allozymes and mitochondrial markers has also been observed (Jørstad and Pedersen 1986). Hybridization between the two species in northern Norway has been documented, where mitochondrial and nuclear introgression has occurred from Atlantic herring into Pacific herring in northern Norway; 21 % of the C. pallasii individuals in Balsfjord had variants of mtDNA from Atlantic herring (Laakkonen et al. 2015). Atlantic herring has been reported to penetrate the Barents Sea from the west, although they have not been found spawning there (Svetovidov 1952; Jørstad 2004).
Otolith shape has been used for decades as a population marker for Atlantic herring (Messieh 1972; Messieh et al. 1989; Turan 2000; Burke et al. 2008a, b) and more recently by Libungan et al. (2015a), which employed the same method as in this study. The otolith shape has been found to vary among populations in relation to spawning time of the year where the herring are experiencing different conditions during early developmental stages (Libungan et al. 2015a), despite lack of detectable genetic differentiation (Pampoulie et al. 2015). Otolith shape in Atlantic herring has furthermore been shown to vary among fjord populations along the coast of Norway where neighboring populations are more similar in shape than populations separated by larger distances (Libungan et al. 2015b), suggesting there might also be a genetic basis for the differentiation. The purpose of this study was to investigate whether the variation in otolith shape of herring in Balsfjord in northern Norway and southeast Barents Sea reflects their taxonomic classification into subspecies or the genetic affinities to the Pacific herring and the split between northwest and northeast Pacific known from the studies on mtDNA. If geographic patterns in otolith shape confirm the genetic patterns, a further support of the recent origin of the Arctic population is obtained. Furthermore, by including the Atlantic herring, we are able to analyze the differentiation between the two species and evaluate whether any signs of hybridization occur between them in Balsfjord.
Materials and methods
Samples of Atlantic and Pacific herring (see also Fig. 1)
Longitude (E, W)
Bering Sea, Kuskokwim Bay (Nelson Island)
09, 06, 2006
Gulf of Alaska, Kamishak Bay (Douglas Reef)
01, 05, 2014
08, 08, 2012
23, 01, 2014
10, 03, 2014
11, 03, 2014
23, 04, 2014
14, 02, 2010
19, 02, 2010
24, 02, 2010
24, 02, 2010
10, 06, 1996
11, 06, 1996
11, 06, 1996
19, 02, 2005
19, 02, 2006
22, 02, 2006
Image and data analysis
A digital image of each otolith was captured using either a Leica M60 stereomicroscope with a Leica DFC450 camera and the software Leica Application Suite (LAS Version 4.5) (Leica Micro-systems, Wetzlar, Germany, http://www.leica-microsystems.com) or a Leica MZ95 stereomicroscope (Leica Micro-systems, Wetzlar, Germany) with an Evolution LC-PL A662 camera (MediaCybernetics, Maryland, USA) using the software PixeLINK 3.2 (www.pixelink.com). All statistical analyses were conducted with R (R Core Team 2015) using the R packages ade4 (Dray and Dufour 2007), shapeR (Libungan and Pálsson 2015) and vegan (Oksanen et al. 2013).
Following a method by Libungan et al. (2015a), the variation in otolith shape was examined by plotting the mean shape of each population using the shapeR package (Libungan and Pálsson 2015). The wavelet coefficients, which were 64 in total and represent the otolith shape, were obtained from the digital images using the wavethresh package (Nason 2012) and scaled by adjusting for allometric relationships with fish length as in Lleonart et al. (2000) and Reist (1985), implemented in the shapeR package (Libungan and Pálsson 2015). To inspect the variation in shape within herring groups, the mean and standard deviation of the coefficients was plotted against the angle using plotCI from the gplots package (Warnes et al. 2014). The proportion of variation within groups along the outline was summarized with intraclass correlation (ICC). Temporal stability in otolith shape was analyzed within the Barents Sea sample since there existed samples from 3 years (see Table 1) by applying canonical analysis of principal coordinates (CAP) (Anderson and Willis 2003) and an ANOVA-like permutation test to assess the significance of constraints using 2000 permutations with the vegan package (Oksanen et al. 2013). Otolith shape was then compared among populations with an overall test and also by applying comparisons between all populations to test for regional differences, using the CAP and the ANOVA-like permutation test. The same analyses were used to evaluate differences between age classes and the interaction of age and geographic origin since age is known to have confounding effects on otolith shape (Castonguay et al. 1991).
Ordination of the population averages along the first three canonical axes (CAP1, CAP2 and CAP3) was examined graphically with the shape descriptors. Variance within locations was calculated on the shape distances (CAP1 and CAP2) between each individual within each area. To compare the fit of the otolith shape variation to the previous taxonomic classification and to the divergence observed by genetic analyses, the CAP was conducted by partitioning the variation with respect to classification based firstly on the taxonomic split of species: the Norwegian spring spawners (C. harengus) with herring populations within C. pallasii and secondly between the two subspecies C. p. pallasii in the Pacific (Kamishak Bay and Bering Sea herring) with C. p. suworowi in the southeast Barents Sea. Thirdly, the northeast Pacific herring (C. p. pallasii from Kamishak Bay in the Gulf of Alaska) was compared with the trans-Arctic group as described by Laakkonen et al. (2013), comprised of the Bering Sea herring (C. p. pallasii) in the northeast Pacific and Barents Sea herring (C. p. suworowi) in Russia. Lastly, the Balsfjord herring (C. pallasii) in northern Norway, known to have introgressed genetic markers from C. harengus, was compared to its neighboring populations from C. harengus in Norway (NS) and C. p. suworowi from the Barents Sea (BS). Euclidean distances were calculated between the coordinates of the averages of the different population samples for the first four axes, weighted by the contribution of each axis to the overall variation and presented with boxplots.
Main shape features
Multivariate analysis of otolith shape
Otolith shape compared among all herring populations in the present study, between species, subspecies and the genetically distinct groups within C. pallasii
NS versus (BA + BS1 + BS2 + BE + KA)
KA + BE versus BS1 + BS2
Between northeast Pacific and the trans-Arctic lineages
KA versus (BE + BS1 + BS2 + BA)
The results of this study show that otolith shape differs among the Atlantic and Pacific herring species, and variation between the species is larger than within Pacific herring. The C. pallasii herring occupying Balsfjord in northern Norway, C. pallasii suworowi in the Barents Sea and C. p. pallasii from the Bering Sea in the northwest Pacific are more similar to each other than to C. p. pallasii in the Gulf of Alaska in the northeast Pacific. These results are in accordance with previous studies based on genetic variation (Jørstad and Nævdal 1981; Jørstad and Pedersen 1986; Laakkonen et al. 2013, 2015). The Bering Sea herring and the European branch of the Pacific herring are intermediate in otolith shape between the Atlantic herring and the Pacific herring from the Gulf of Alaska.
The difference in the mean otolith shape for the herring populations was different from the patterns observed in previous studies on Atlantic herring (Eggers et al. 2014; Libungan et al. 2015a, b). At the excisura, around the 200° angle, which had the largest variation among Atlantic herring populations (Libungan et al. 2015a), the Norwegian spring spawners at Møre had the inner most shape in this study. A very distinct pattern at the excisura was found, where the Kamishak Bay herring from the Gulf of Alaska had the outermost otolith shape.
The samples from the Barents Sea (C. p. suworowi) were collected at different times of the year, the 1996 sample in June and the 2005–2006 samples were both from February. Shape differences were detected in a comparison between the 1996 sample and the 2005–2006 samples pooled. Herring in the southeast Barents Sea are reported to spawn on average in July (Semenova et al. 2015). Herring occupying nearby oceans, from the White Sea (C. p. marisalbi), southwest of the sampling area in the Barents Sea spawns in spring/early summer in March–June (Semenova et al. 2013, 2015), while herring occupying the Kara Sea (C. p. suworowi), east of the Barents Sea, spawns in late summer in August (Semenova et al. 2015). Even though the samples from the Barents Sea were sampled in different seasons (February and June), the majority of the herring from each sample were maturing (stage 4), which indicates a mixture of herring populations occupying this region, with one population spawning in spring and the other during late summer. Since the herring were close to spawning, the population sampled in February might have been White Sea herring migrating to their respective spawning grounds during the time of sampling. Since genetic variation exists between spawning groups of White Sea and Barents Sea herring at four allozyme loci (Semenova et al. 2009), further investigations are needed to see whether the same pattern of divergence is observed with otolith shape. Comparisons of the species C. harengus (Norwegian spring spawners from W-Norway) and C. pallasii from Balsfjord, Barents Sea, Bering Sea and Kamishak Bay in the Gulf of Alaska yielded the highest F value (53.83, Table 2), while a comparison of Kamishak Bay herring in the Gulf of Alaska with the trans-Arctic group of herring from the Barents Sea, Balsfjord and Bering Sea (Laakkonen et al. 2013) had a considerably lower F value (27.17) and thus more divergence in otolith shape, as might be expected, at the species level than intraspecies level. Differentiation in otolith shape between the C. pallasii subspecies was less than among populations within C. pallasii based on the genetic lineages of the northeast and the trans-Arctic group (Laakkonen et al. 2013).
Studies on genetic variation have shown that the more southerly distributed herring groups, such as the large Norwegian spring spawners and herring in the northeast Pacific, harbor more genetic variation than the northern populations in accordance with their population sizes and even bottlenecks in populations following the colonization of the Barents Sea and northern Norway (Laakkonen et al. 2013). In otolith variation, we observe a similar pattern, where the smallest variation was in the Bering Sea and Barents Sea herring. Higher variance could be expected in the Balsfjord population as a result of hybridization (Laakkonen et al. 2015), but this was not the case in the present study.
Similar amphiboreal distribution as in the Pacific herring is known for other species which may reflect emigration of Pacific herring into the Atlantic after the last glacial period of the Ice Age. The two gadoid genera, Eleginus and Theragra, E. navaga and T. finnmarchica species are known in the northeast Atlantic and E. gracilis and T. chalcogramma in the north Pacific (Christiansen et al. 2005), where the Theragra species have been classified as a single species based on mtDNA variation (Ursvik et al. 2007). Pacific cod (Gadus macrocephalus) and Greenland cod (G. ogac) are also closely related and may be classified as a single species (Carr et al. 1999), and the circumpolar capelin (Mallotus villosus) (Vilhjálmsson 1994) shows signs of trans-Arctic migration (Præbel et al. 2013). For Atlantic and Pacific herring, the diversification between the species is clear both genetically and in the morphology of the otoliths despite introgression. Also, populations of Pacific herring which are separated both by large geographic distances and barriers along the coast of northern Norway and the Alaska Peninsula are clearly distinguishable genetically and in otolith shape.
Further studies are needed to resolve the patterns within C. pallasii from northern Europe. The Barents Sea sample, which was intermediate in shape between the two herring samples from the Pacific, did not contain spawning fish and could therefore be a mixture of populations. Thus, analysis of samples from spawning populations of C. p. suworowi and also from C. p. marisalbi in the White Sea is warranted. Additional samples from the coast between Balsfjord and the Barents Sea might also be needed to evaluate whether the Balsfjord population has been shaped by the known genetic introgression and/or its small effective population size (Laakkonen et al. 2015).
It is apparent, as pointed out by Laakkonen et al. (2013), that the pattern does not comply with the current subspecies division within C. pallasii. A revised classification, considering the observed genetic and morphological evidence, should rather distinguish the northwest Pacific population occupying the Bering Sea together with the European populations of C. pallasii than with the northeast Pacific herring, occupying the Gulf of Alaska.
Torstein Pedersen at the University of Tromsø is thanked for providing the samples from Balsfjord in Norway. Ole Ingar Paulsen at the Institute of Marine Research in Norway is thanked for allozyme analysis, splitting out Norwegian spring spawning herring (C. harengus) from Balsfjord herring (C. pallasii) in Balsfjord. This work was funded by the Assistant teacher’s grant of the University of Iceland.
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