A fast HRMA tool to authenticate eight salmonid species in commercial food products.

Atlantic and Pacific salmon are frequently consumed species with very different economic values: farmed Atlantic salmon is cheaper than wild-caught Pacific salmons. Species replacements occur with the high valued Pacific species (Oncorhynchus keta, O. gorbuscha, O. kisutch, O. nerka and O. tshawytscha) substituted by cheaper farmed Atlantic salmon (Salmo salar) and Atlantic salmon by rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta). Here we use High-Resolution Melting Analysis (HRMA) to identify eight salmonid species. We designed primers to generate short amplicons of 72 and 116 bp from the fish barcode genes CO1 and CYTB. The time of analysis was under 70 min, after DNA extraction. Food processing of Atlantic salmon (fresh, "Bellevue", "gravadlax", frozen and smoked) did not impact the HRMA profiles allowing reliable identification. A blind test was conducted by three different institutes, showing correct species identifications irrespective of the laboratory conducting the analysis. Finally, a total of 82 retail samples from three European countries were analyzed and a low substitution rate of 1.2% was found. The developed tool provides a quick way to investigate salmon fraud and contributes to safeguard consumers.

Endangered predators and endangered prey: Seasonal diet of Southern Resident killer whales.

Understanding diet is critical for conservation of endangered predators. Southern Resident killer whales (SRKW) (Orcinus orca) are an endangered population occurring primarily along the outer coast and inland waters of Washington and British Columbia. Insufficient prey has been identified as a factor limiting their recovery, so a clear understanding of their seasonal diet is a high conservation priority. Previous studies have shown that their summer diet in inland waters consists primarily of Chinook salmon (Oncorhynchus tshawytscha), despite that species' rarity compared to some other salmonids. During other times of the year, when occurrence patterns include other portions of their range, their diet remains largely unknown. To address this data gap, we collected feces and prey remains from October to May 2004-2017 in both the Salish Sea and outer coast waters. Using visual and genetic species identification for prey remains and genetic approaches for fecal samples, we characterized the diet of the SRKWs in fall, winter, and spring. Chinook salmon were identified as an important prey item year-round, averaging ~50% of their diet in the fall, increasing to 70-80% in the mid-winter/early spring, and increasing to nearly 100% in the spring. Other salmon species and non-salmonid fishes, also made substantial dietary contributions. The relatively high species diversity in winter suggested a possible lack of Chinook salmon, probably due to seasonally lower densities, based on SRKW's proclivity to selectively consume this species in other seasons. A wide diversity of Chinook salmon stocks were consumed, many of which are also at risk. Although outer coast Chinook samples included 14 stocks, four rivers systems accounted for over 90% of samples, predominantly the Columbia River. Increasing the abundance of Chinook salmon stocks that inhabit the whales' winter range may be an effective conservation strategy for this population.

Fate of MHCII in salmonids following 4WGD.

Major histocompatibility complex (MHC) genes are key players in the adaptive immunity providing a defense against invading pathogens. Although the basic structures are similar when comparing mammalian and teleost MHC class II (MHCII) molecules, there are also clear-cut differences. Based on structural requirements, the teleosts non-classical MHCII molecules do not comply with a function similar to the human HLA-DM and HLA-DO, i.e., assisting in peptide loading and editing of classical MHCII molecules. We have previously studied the evolution of teleost class II genes identifying various lineages and tracing their phylogenetic occurrence back to ancient ray-finned fishes. We found no syntenic MHCII regions shared between cyprinids, salmonids, and neoteleosts, suggesting regional instabilities. Salmonids have experienced a unique whole genome duplication 94 million years ago, providing them with the opportunity to experiment with gene duplicates. Many salmonid genomes have recently become available, and here we set out to investigate how MHCII has evolved in salmonids using Northern pike as a diploid sister phyla, that split from the salmonid lineage prior to the fourth whole genome duplication (4WGD) event. We identified 120 MHCII genes in pike and salmonids, ranging from 11 to 20 genes per species analyzed where DB-group genes had the most expansions. Comparing the MHC of Northern pike with that of Atlantic salmon and other salmonids species provides a tale of gene loss, translocations, and genome rearrangements.

Differentiating salmonid migratory ecotypes through stable isotope analysis of collagen: Archaeological and ecological applications.

The ability to distinguish between different migratory behaviours (e.g., anadromy and potamodromy) in fish can provide important insights into the ecology, evolution, and conservation of many aquatic species. We present a simple stable carbon isotope (δ13C) approach for distinguishing between sockeye (anadromous ocean migrants) and kokanee (potamodromous freshwater residents), two migratory ecotypes of Oncorhynchus nerka (Salmonidae) that is applicable throughout most of their range across coastal regions of the North Pacific Ocean. Analyses of kokanee (n = 239) and sockeye (n = 417) from 87 sites spanning the North Pacific (Russia to California) show that anadromous and potamodromous ecotypes are broadly distinguishable on the basis of the δ13C values of their scale and bone collagen. We present three case studies demonstrating how this approach can address questions in archaeology, archival, and conservation research. Relative to conventional methods for determining migratory status, which typically apply chemical analyses to otoliths or involve genetic analyses of tissues, the δ13C approach outlined here has the benefit of being non-lethal (when applied to scales), cost-effective, widely available commercially, and should be much more broadly accessible for addressing archaeological questions since the recovery of otoliths at archaeological sites is rare.

How "simple" methodological decisions affect interpretation of population structure based on reduced representation library DNA sequencing: A case study using the lake whitefish.

Reduced representation (RRL) sequencing approaches (e.g., RADSeq, genotyping by sequencing) require decisions about how much to invest in genome coverage and sequencing depth, as well as choices of values for adjustable bioinformatics parameters. To empirically explore the importance of these "simple" methodological decisions, we generated two independent sequencing libraries for the same 142 individual lake whitefish (Coregonus clupeaformis) using a nextRAD RRL approach: (1) a larger number of loci at low sequencing depth based on a 9mer (library A); and (2) fewer loci at higher sequencing depth based on a 10mer (library B). The fish were selected from populations with different levels of expected genetic subdivision. Each library was analyzed using the STACKS pipeline followed by three types of population structure assessment (FST, DAPC and ADMIXTURE) with iterative increases in the stringency of sequencing depth and missing data requirements, as well as more specific a priori population maps. Library B was always able to resolve strong population differentiation in all three types of assessment regardless of the selected parameters, largely due to retention of more loci in analyses. In contrast, library A produced more variable results; increasing the minimum sequencing depth threshold (-m) resulted in a reduced number of retained loci, and therefore lost resolution at high -m values for FST and ADMIXTURE, but not DAPC. When detecting fine population differentiation, the population map influenced the number of loci and missing data, which generated artefacts in all downstream analyses tested. Similarly, when examining fine scale population subdivision, library B was robust to changing parameters but library A lost resolution depending on the parameter set. We used library B to examine actual subdivision in our study populations. All three types of analysis found complete subdivision among populations in Lake Huron, ON and Dore Lake, SK, Canada using 10,640 SNP loci. Weak population subdivision was detected in Lake Huron with fish from sites in the north-west, Search Bay, North Point and Hammond Bay, showing slight differentiation. Overall, we show that apparently simple decisions about library construction and bioinformatics parameters can have important impacts on the interpretation of population subdivision. Although potentially more costly on a per-locus basis, early investment in striking a balance between the number of loci and sequencing effort is well worth the reduced genomic coverage for population genetics studies. More conservative stringency settings on STACKS parameters lead to a final dataset that was more consistent and robust when examining both weak and strong population differentiation. Overall, we recommend that researchers approach "simple" methodological decisions with caution, especially when working on non-model species for the first time.

Proteomic Analysis of the Lake Trout (Salvelinus namaycush) Liver Identifies Proteins from Evolutionarily Close and -Distant Fish Relatives.

Lake trout are used as bioindicators for toxics exposure in the Great Lakes ecosystem. Here the first lake trout (Salvelinus namaycush) liver proteomics study is performed and searched against specific databases: (NCBI and UniProtKB) Salvelinus, Salmonidae, Actinopterygii, and Oncorhynchus mykiss, and the more distant relative, Danio rerio. In the biological replicate 1 (BR1), technical replicate 1 (TR1), (BR1TR1), a large number of lake trout liver proteins are not in the Salvelinus protein database, suggesting that lake trout liver proteins have homology to some proteins from the Salmonidae family and Actinopterygii class, and to Oncorhynchus mykiss and Danio rerio, two more highly studied fish. In the NCBI search, 4194 proteins are identified: 3069 proteins in Actinopterygii, 1617 in Salmonidae, 68 in Salvelinus, 568 in Oncorhynchus mykiss, and 946 in Danio rerio protein databases. Similar results are observed in the UniProtKB searches of BR1RT1, as well as in a technical replicate (BR1TR2), and then in a second biological replicate experiment, with two technical replicates (BR2TR1 and BR2TR2). This study opens the possibility of identifying evolutionary relationships (i.e., adaptive mutations) between various groups (i.e., zebrafish, rainbow trout, Salmonidae, Salvelinus and lake trout) through evolutionary proteomics. Data are available via the PRIDE Q2 (PXD011924).

Population differentiation and genetic diversity of endangered Brachymystax tsinlingensis Li between Yangtze River and Yellow River in China based on mtDNA.

Brachymystax tsinlingensis Li, distributed scatteredly in Qinling Mountains of China, is an ideal material for studies of conservation and phylogeography. In the present study, the genetic variations and phylogeographical patterns of B. tsinlingensis collecting from the Yangtze River (n = 30) and the Yellow River (n = 70) were compared based on 1082-1163 bp control region with partial tRNA and 912 bp cyt b partial sequence of mtDNA. Analysis of variance indicated that a high proportion of the total genetic variance (96.03%) was distributed among populations, supporting strong geographic structuring of mtDNA polymorphism. Ten haplotypes were assigned to two clades that were related to geographic regions. No haplotype shared between geographic populations from the Yangtze River and the Yellow River, and two clades related to geographic regions can be clearly found from phylogenetic analysis by Bayesian approach, one from individuals in the Yangtze River and the other from individuals in the Yellow River, which probably indicated artificial carrying not happened historically. The mitochondrial results revealed an obvious differentiation of genetic structure of B. tsinlingensis between populations of Yellow River and Yangtze River, and the estimated diverge time around 0.20 MY. It was suggested that each of the two evolutionarily distinct groups of B. tsinlingensis should be protected and crossing individuals between two river systems should be avoided for conservation purpose.

Testing the devil's impact on southern Baltic and North Sea basins whitefish (Coregonus spp.) diversity.

BACKGROUND: The diversity and phylogeny of whitefish of the genus Coregonus is complex, and includes many endemic species of high conservation concern. However, because of commercial importance of whitefish fisheries, stockings and translocations have occurred repeatedly, which challenges the identification of local populations as conservation units. This study analyses the phylogenetic relationships of 15 contemporary and two historical populations of lake-resident and anadromous whitefish (Coregonus spp.) from the southern Baltic and North Sea basins. We elucidated the complex history of Lake Schaal (northern Germany) whitefish, for which a local tale suggests that the devil threw whitefish from the Central European Lake Constance into this lake. Studies from the early twentieth century indeed suggested numerous stocking events for Lake Schaal from Lake Constance, from Estonian/Russian Lake Peipsi and from the anadromous whitefish of the Baltic Sea. RESULTS: Analyses of 13 microsatellite markers showed that Lake Constance whitefish are unrelated to any northern Germany whitefish population, including the contemporary whitefish population from Lake Schaal. Comparison with four historical specimens further showed that the native Lake Schaal whitefish (C. holsatus) vanished from the lake, but has survived as a non-native population in the north German Lake Drewitz. The whitefish currently occurring in Lake Schaal and three adjacent lakes are identified as C. maraenoides, introduced from Lake Peipsi. The contemporary anadromous whitefish populations from the Baltic (German and Finnish coast) and the German River Treene (North Sea basin, stocked from Danish River Vida) grouped together, but showed significant genetic differentiation. The 14 historical specimens of C. oxyrinchus from Rivers Rhine and Schelde were assigned to several contemporary whitefish populations, but among them only one specimen was assigned to the contemporary River Treene population. Therefore, we do not support the view that the whitefish from River Vida/Treene are identical with the historical C. oxyrinchus. CONCLUSIONS: Our study demonstrates that lake and anadromous whitefish in the Baltic and North Sea basins reflect a complex phylogeography, which is further blurred by the effects of repeated stocking and translocations. To identify conservation units, the genetic identity of each population has to be scrutinized.

Salmo macrostigma (Teleostei, Salmonidae): Nothing more than a brown trout (S. trutta) lineage?

We examined specimens of the macrostigma trout Salmo macrostigma, which refers to big black spots on the flanks, to assess whether it is an example of taxonomic inflation within the brown trout Salmo trutta complex. Using new specimens, publicly available data and a mitogenomic protocol to amplify the control and cytochrome b regions of the mitochondrial genome from degraded museum samples, including one syntype specimen, the present study shows that the macrostigma trout is not a valid species. Our results suggest the occurrence of a distinct evolutionary lineage of S. trutta in North Africa and Sicily. The name of the North African lineage is proposed for this lineage, which was found to be sister to the Atlantic lineage of brown trout, S. trutta.

Development of sperm vitrification protocols for two endangered salmonid species: the Adriatic grayling, Thymallus thymallus, and the marble trout, Salmo marmoratus.

Vitrification was applied to the sperm of two endangered fish species of Soca River basin in Slovenia, the Adriatic grayling (Thymallus thymallus) and marble trout (Salmo marmoratus) following testing different cooling devices and vitrifying media. Sperm was collected, diluted in species-specific non-activating media containing cryoprotectants, and vitrified by plunging directly into liquid nitrogen without pre-cooling. Progressive motility, curvilinear velocity, and straightness of fresh and vitrified-warmed sperm were evaluated with computer-assisted sperm analysis (CASA). Fertilization trials were carried out to test the effectiveness of vitrification in the case of grayling. A protocol utilizing a glucose-based extender, 30% cryoprotectants (15% methanol + 15% propylene glycol), 1:1 dilution ratio, and droplets of 2 µl on a Cryotop as cooling device yielded the highest post-thaw motility values for both Adriatic grayling (7.5 ± 6.5%) and marble trout (26.6 ± 15.8%). Viable embryos were produced by fertilizing eggs with vitrified grayling sperm (hatching 13.1 ± 11.7%, control hatching 73.9 ± 10.4%). The vitrification protocol developed in this study can be utilized in the conservation efforts for the two species as an alternative to slow-rate freezing when working in field conditions or when specific equipment necessary for slow-rate freezing is not available.

Interspecific germ cell transplantation: a new light in the conservation of valuable Balkan trout genetic resources?

Interspecific transplantation of germ cells from the brown trout Salmo trutta m. fario and the European grayling Thymallus thymallus into rainbow trout Oncorhynchus mykiss recipients was carried out in order to improve current practices in conservation of genetic resources of endangered salmonid species in the Balkan Peninsula. Current conservation methods mainly include in situ efforts such as the maintenance of purebred individuals in isolated streams and restocking with purebred fingerlings; however, additional ex situ strategies such as surrogate production are needed. Steps required for transplantation such as isolation of high number of viable germ cells and fluorescent labeling of germ cells which are to be transplanted have been optimized. Isolated and labeled brown trout and grayling germ cells were intraperitoneally transplanted into 3 to 5 days post hatch rainbow trout larvae. Survival of the injected larvae was comparable to the controls. Sixty days after transplantation, fluorescently labeled donor cells were detected within the recipient gonads indicating successful incorporation of germ cells (brown trout spermatogonia and oogonia-27%; grayling spermatogonia-28%; grayling oogonia-23%). PCR amplification of donor mtDNA CR fragments within the recipient gonads additionally corroborated the success of incorporation. Overall, the transplantation method demonstrated in this study presents the first step and a possible onset of the application of the germ cell transplantation technology in conservation and revitalization of genetic resources of endangered and endemic species or populations of salmonid fish and thus give rise to new or improved management strategies for such species.

Holobionts and ecological speciation: the intestinal microbiota of lake whitefish species pairs.

BACKGROUND: It is well established that symbionts have considerable impact on their host, yet the investigation of the possible role of the holobiont in the host's speciation process is still in its infancy. In this study, we compared the intestinal microbiota among five sympatric pairs of dwarf (limnetic) and normal (benthic) lake whitefish Coregonus clupeaformis representing a continuum in the early stage of ecological speciation. We sequenced the 16s rRNA gene V3-V4 regions of the intestinal microbiota present in a total of 108 wild sympatric dwarf and normal whitefish as well as the water bacterial community from five lakes to (i) test for differences between the whitefish intestinal microbiota and the water bacterial community and (ii) test for parallelism in the intestinal microbiota of dwarf and normal whitefish. RESULTS: The water bacterial community was distinct from the intestinal microbiota, indicating that intestinal microbiota did not reflect the environment, but rather the intrinsic properties of the host microbiota. Our results revealed a strong influence of the host (dwarf or normal) on the intestinal microbiota with pronounced conservation of the core intestinal microbiota (mean ~ 44% of shared genera). However, no clear evidence for parallelism was observed, whereby non-parallel differences between dwarf and normal whitefish were observed in three of the lakes while similar taxonomic composition was observed for the two other species pairs. CONCLUSIONS: This absence of parallelism across dwarf vs. normal whitefish microbiota highlighted the complexity of the holobiont and suggests that the direction of selection could be different between the host and its microbiota.

Inferring phylogenetic structure, hybridization and divergence times within Salmoninae (Teleostei: Salmonidae) using RAD-sequencing.

Phylogenetic studies focusing on Salmonidae have revealed significant obstacles in trying to clarify some interspecific relationships within the Salmoninae subfamily, due to a limited number of markers typed, conflicting phylogenetic signals and ancient hybridization events. To infer reliable phylogenetic relationships, evaluate several putative scenarios of ancient hybridization, and estimate divergence times within Salmoninae, we applied restriction-site associated DNA sequencing (RAD-seq) to 43 samples, including 26 genetic lineages across 21 species, largely representing the subfamily, with an emphasis on the genus Salvelinus. We identified 28,402 loci and 28,363 putatively unlinked SNPs, which were used in downstream analyses. Using an iterative k-means partitioned dataset and a Maximum Likelihood approach; we generated a well-supported phylogeny, providing clear answers to several previous phylogenetic uncertainties. We detected several significant introgression signals, presumably ancient, in the genus Salvelinus. The most recent common ancestor of Salmonidae dates back to approximately 58.9MY ago (50.8-64 MY) and the crown age of Salmoninae was estimated to be 37.7 MY (35.2-40.8 MY) using a Bayesian molecular dating analysis with a relaxed molecular clock. The divergence among genera of the subfamily occurred between the late Eocene and middle of the Miocene (≈38-11 MY) such as the divergence between the genus Oncorhynchus and Salvelinus, which we estimated to 21.2 MY ago (95% HPD: 19.8-23.0 MY), while species diversification took place mainly during the Neogene (≈22-1.5 MY), with more than half of these events occurring in the last 10 MY.

Complete mitochondrial genome of Coregonus muksum.

The complete mitochondrial genome of Coregonus muksum was determined in this study. The mitogenome is 16 736 bp in length and contains one D-loop region, 2 ribosomal RNA genes, 22 transfer RNA genes, and 13 protein-coding genes. The overall base composition of the heavy strand is 26.82% for A, 29.50% for C, 18.04% for G, and 25.68% for T. The percentage of G + C content is 47.54%. This is the first time of the mitochondrial genome sequencing for Coregonus muksum.

Global Salmonidae introductions reveal stronger ecological effects of changing intraspecific compared to interspecific diversity.

The introduction of organisms within the native range of wild conspecifics is a widespread phenomenon and locally modifies patterns in intraspecific diversity. However, our knowledge of the resulting ecological effects, as opposed to those caused by invasion-induced changes in interspecific diversity, is still limited. Here, we investigated the ecological effects of native and non-native invaders across levels of biological organisations and recipient organisms using the global and long history introductions of salmonids. Our meta-analysis demonstrated that the global effects of native species introductions exceeded those induced by non-native invaders. The impacts of native invaders were primarily manifested at the individual level on wild conspecifics, but remained largely unexplored on other native organisms and at the community and ecosystem levels. Overlooked and poorly appreciated, quantifying the impacts of native invaders has important implications because human-assisted introductions of domesticated organisms are ubiquitous and likely to proliferate in the future.

Toll-like receptors in maraena whitefish: Evolutionary relationship among salmonid fishes and patterns of response to Aeromonas salmonicida.

Toll-like receptors (TLRs) interact directly with particular pathogenic structures and are thus highly important to innate immunity. The present manuscript characterises a suite of 14 TLRs in maraena whitefish (Coregonus maraena), a salmonid species with increasing importance for aquaculture. Whitefish TLRs were structurally and evolutionary analysed. The results revealed a close relationship with TLRs from salmonid fish species rainbow trout and Atlantic salmon. Profiling the baseline expression of TLR genes in whitefish indicated that mainly members of the TLR11 family were highly expressed across all investigated tissues. A stimulation model with inactivated Aeromonas salmonicida was used to induce inflammation in the peritoneal cavity of whitefish. This bacterial challenge induced the expression of pro-inflammatory cytokine genes and evoked a strong influx of granulated cells of myeloid origin into the peritoneal cavity. As a likely consequence, the abundance of TLR-encoding transcripts increased moderately in peritoneal cells, with the highest levels of transcripts encoding non-mammalian TLR22a and a soluble TLR5 variant. In the course of inflammation, the proportion of granulated cells increased in peripheral blood accompanied by elevated TLR copy numbers in spleen and simultaneously reduced TLR copy numbers in head kidney at day 3 post-stimulation. Altogether, the present study provides in-vivo evidence for relatively modest TLR response patterns, but marked trafficking of myeloid cells as an immunophysiological consequence of A. salmonicida inflammation in whitefish. The present results contribute to improved understanding of the host-pathogen interaction in salmonid fish.

Real-time PCR identification of lake whitefish Coregonus clupeaformis in the Laurentian Great Lakes.

The purpose of this study was to develop a real-time PCR assay to specifically identify lake whitefish Coregonus clupeaformis in larval fish assemblages based on a 122 bp amplicon from the mitochondrial genome. The efficiency of the reaction, as calculated from the standard curve, was 90.77% with the standard curve having an r(2) value of 0.998. Specificity of the assay provided single melt peak in a melt-curve analysis and amplification of only the target species. The assay successfully identified target DNA in as low as 0.1% proportion of a DNA mixture. This assay was designed on the portable Smart Cycler II platform and can be used in both field and laboratory settings to successfully identify C. clupeaformis.

Complete mitochondrial genome of Coregonus autumnalis.

The complete mitochondrial genome of Coregonus autumnalis is determined in this study. The mitogenome is 16,736 bp in length and contains 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes and a D-loop region. The overall base composition of the H-strand is 26.73% A, 29.44% C, 18.14% for G and 25.69% for T. The G+C content is 47.58%. In this article, the mitochondrial genome sequencing for Coregonus autumnalis is carried out for the first time.

Complete mitochondrial genome of Coregonus cluncaformis.

The complete mitochondrial genome of Coregonus cluncaformis was determined in this study. The mitogenome is 16 736 bp in length and contains 1D-loop region, 2 ribosomal RNA genes, 22 transfer RNA genes, and 13 protein-coding genes. The overall base composition of the heavy strand is 26.79% for A, 29.49% for C, 18.07% for G and 25.66% for T, The percentage of G + C content is 47.56%. This is the first time of the mitochondrial genome sequencing for C. cluncaformis in this study.

Comparative analysis of complete mitochondrial genomes suggests that relaxed purifying selection is driving high nonsynonymous evolutionary rate of the NADH2 gene in whitefish (Coregonus ssp.).

Several studies have recently reported evidence for positive selection acting on the mitochondrial genome (mitogenome), emphasizing its potential role in adaptive divergence and speciation. In this study we searched 107 full mitogenomes of recently diverged species and lineages of whitefish (Coregonus ssp.) for signals of positive selection. These salmonids show several distinct morphological and ecological differences that may be associated with energetics and therefore potentially positive selection at the mitogenome level. We found that purifying selection and genetic drift were the predominant evolutionary forces acting on the analyzed mitogenomes. However, the NADH dehydrogenase 2 gene (ND2) showed a highly elevated dN/dS ratio compared to the other mitochondrial genes, which was significantly higher in whitefish compared to other salmonids. We therefore further examined nonsynonymous evolution in ND2 by (i) mapping amino acid changes to a protein model structure which showed that they were located away from key functional residues of the protein, (ii) locating them in the sequences of other species of fish (Salmonidae, Anguillidae, Scombridae and Percidae) only to find pronounced overlap of nonsynonymous regions. We thus conclude that relaxed purifying selection is driving the evolution of ND2 by affecting mostly regions that have lower functional relevance.