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Molecular Identification of Atlantic Bluefin Tuna (Thunnus thynnus, Scombridae) Larvae and Development of a DNA Character-Based Identification Key for Mediterranean Scombrids.

Puncher GN, Arrizabalaga H, Alemany F, Cariani A, Oray IK, Karakulak FS, Basilone G, Cuttitta A, Mazzola S, Tinti F - PLoS ONE (2015)

Bottom Line: Misidentification of fish larvae and eggs can lead to inaccurate estimates of stock biomass and productivity which can trigger demands for increased quotas and unsound management conclusions.Several techniques were used to analyze the genetic sequences (sequence alignments using search algorithms, neighbour joining trees, and a genetic character-based identification key) and an extensive comparison of the results is presented.Our results reveal important differences in the accuracy of the taxonomic identifications carried out by different ichthyoplanktologists following morphology-based methods.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Biological, Geological and Environmental Sciences / Laboratory of Genetics and Genomics of Marine Resources and Environment (GenoDREAM), University of Bologna, Ravenna, Italy.

ABSTRACT
The Atlantic bluefin tuna, Thunnus thynnus, is a commercially important species that has been severely over-exploited in the recent past. Although the eastern Atlantic and Mediterranean stock is now showing signs of recovery, its current status remains very uncertain and as a consequence their recovery is dependent upon severe management informed by rigorous scientific research. Monitoring of early life history stages can inform decision makers about the health of the species based upon recruitment and survival rates. Misidentification of fish larvae and eggs can lead to inaccurate estimates of stock biomass and productivity which can trigger demands for increased quotas and unsound management conclusions. Herein we used a molecular approach employing mitochondrial and nuclear genes (CO1 and ITS1, respectively) to identify larvae (n = 188) collected from three spawning areas in the Mediterranean Sea by different institutions working with a regional fisheries management organization. Several techniques were used to analyze the genetic sequences (sequence alignments using search algorithms, neighbour joining trees, and a genetic character-based identification key) and an extensive comparison of the results is presented. During this process various inaccuracies in related publications and online databases were uncovered. Our results reveal important differences in the accuracy of the taxonomic identifications carried out by different ichthyoplanktologists following morphology-based methods. While less than half of larvae provided were bluefin tuna, other dominant taxa were bullet tuna (Auxis rochei), albacore (Thunnus alalunga) and little tunny (Euthynnus alletteratus). We advocate an expansion of expertise for a new generation of morphology-based taxonomists, increased dialogue between morphology-based and molecular taxonomists and increased scrutiny of public sequence databases.

No MeSH data available.


Neighbour-joining phenogram of Mediterranean scombrid reference sequences clustered with number of unknown larvae in parentheses.The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches [67]. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the tree. The evolutionary distances were computed using the Kimura 2-parameter model [65] and are in the units of the number of base differences per site. The analysis involved 280 nucleotide sequences. All ambiguous positions were removed for each sequence pair. There were a total of 612 nucleotide positions in the final dataset.
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pone.0130407.g003: Neighbour-joining phenogram of Mediterranean scombrid reference sequences clustered with number of unknown larvae in parentheses.The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches [67]. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the tree. The evolutionary distances were computed using the Kimura 2-parameter model [65] and are in the units of the number of base differences per site. The analysis involved 280 nucleotide sequences. All ambiguous positions were removed for each sequence pair. There were a total of 612 nucleotide positions in the final dataset.

Mentions: Neighbour joining tree analysis using CO1 sequences produced well-defined clusters of candidate larvae with reference sequences (Figs 2 and 3). All larvae clustered with reference taxa in the same manner as the BLAST and BOLD results. Phenograms containing only the larvae and reference sequences for species found in Mediterranean Sea showed lowest bootstrap probability (BP) for branch nodes separating the true tunas (BFT and albacore) from the other scombrids (BP = 33–35) and a clade containing the true tunas and E. alletteratus from the other species (BP = 30–34). The node containing the A. rochei reference sequences and 74 larvae was the least stable with BPs of 53–59. All other nodes had BPs > 80. Phenograms based on distance matrices using p-distances (Fig 4) had consistently higher BP values than those built with K2P distances (Fig 3). P-distance based phenograms also tended to exclude larval sequences from clusters, as is the case with one larva in the Auxis spp. cluster in Fig 2. When the larval sequences were removed from the alignments, all BP values increased (Fig 4). Predictably, BP values for the A. rochei (ΔBP = 39) and T. thynnus (ΔBP = 17) nodes increased most dramatically, as they were the two clusters to increase in sequence size most dramatically. When T. atlanticus, T. maccoyii, T. obesus and T. albacares reference sequences were included in the alignments, the neighbour-joining tree based on p-distances failed to differentiate clusters for each and combined all with the same cluster alongside the T. thynnus and related larvae sequences (BP = 61; Fig 5). The topology of this same clade in the corresponding K2P-based phenogram changed somewhat but the composition remained the same, albeit the BP value was much lower (BP = 35).


Molecular Identification of Atlantic Bluefin Tuna (Thunnus thynnus, Scombridae) Larvae and Development of a DNA Character-Based Identification Key for Mediterranean Scombrids.

Puncher GN, Arrizabalaga H, Alemany F, Cariani A, Oray IK, Karakulak FS, Basilone G, Cuttitta A, Mazzola S, Tinti F - PLoS ONE (2015)

Neighbour-joining phenogram of Mediterranean scombrid reference sequences clustered with number of unknown larvae in parentheses.The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches [67]. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the tree. The evolutionary distances were computed using the Kimura 2-parameter model [65] and are in the units of the number of base differences per site. The analysis involved 280 nucleotide sequences. All ambiguous positions were removed for each sequence pair. There were a total of 612 nucleotide positions in the final dataset.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493144&req=5

pone.0130407.g003: Neighbour-joining phenogram of Mediterranean scombrid reference sequences clustered with number of unknown larvae in parentheses.The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches [67]. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the tree. The evolutionary distances were computed using the Kimura 2-parameter model [65] and are in the units of the number of base differences per site. The analysis involved 280 nucleotide sequences. All ambiguous positions were removed for each sequence pair. There were a total of 612 nucleotide positions in the final dataset.
Mentions: Neighbour joining tree analysis using CO1 sequences produced well-defined clusters of candidate larvae with reference sequences (Figs 2 and 3). All larvae clustered with reference taxa in the same manner as the BLAST and BOLD results. Phenograms containing only the larvae and reference sequences for species found in Mediterranean Sea showed lowest bootstrap probability (BP) for branch nodes separating the true tunas (BFT and albacore) from the other scombrids (BP = 33–35) and a clade containing the true tunas and E. alletteratus from the other species (BP = 30–34). The node containing the A. rochei reference sequences and 74 larvae was the least stable with BPs of 53–59. All other nodes had BPs > 80. Phenograms based on distance matrices using p-distances (Fig 4) had consistently higher BP values than those built with K2P distances (Fig 3). P-distance based phenograms also tended to exclude larval sequences from clusters, as is the case with one larva in the Auxis spp. cluster in Fig 2. When the larval sequences were removed from the alignments, all BP values increased (Fig 4). Predictably, BP values for the A. rochei (ΔBP = 39) and T. thynnus (ΔBP = 17) nodes increased most dramatically, as they were the two clusters to increase in sequence size most dramatically. When T. atlanticus, T. maccoyii, T. obesus and T. albacares reference sequences were included in the alignments, the neighbour-joining tree based on p-distances failed to differentiate clusters for each and combined all with the same cluster alongside the T. thynnus and related larvae sequences (BP = 61; Fig 5). The topology of this same clade in the corresponding K2P-based phenogram changed somewhat but the composition remained the same, albeit the BP value was much lower (BP = 35).

Bottom Line: Misidentification of fish larvae and eggs can lead to inaccurate estimates of stock biomass and productivity which can trigger demands for increased quotas and unsound management conclusions.Several techniques were used to analyze the genetic sequences (sequence alignments using search algorithms, neighbour joining trees, and a genetic character-based identification key) and an extensive comparison of the results is presented.Our results reveal important differences in the accuracy of the taxonomic identifications carried out by different ichthyoplanktologists following morphology-based methods.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Biological, Geological and Environmental Sciences / Laboratory of Genetics and Genomics of Marine Resources and Environment (GenoDREAM), University of Bologna, Ravenna, Italy.

ABSTRACT
The Atlantic bluefin tuna, Thunnus thynnus, is a commercially important species that has been severely over-exploited in the recent past. Although the eastern Atlantic and Mediterranean stock is now showing signs of recovery, its current status remains very uncertain and as a consequence their recovery is dependent upon severe management informed by rigorous scientific research. Monitoring of early life history stages can inform decision makers about the health of the species based upon recruitment and survival rates. Misidentification of fish larvae and eggs can lead to inaccurate estimates of stock biomass and productivity which can trigger demands for increased quotas and unsound management conclusions. Herein we used a molecular approach employing mitochondrial and nuclear genes (CO1 and ITS1, respectively) to identify larvae (n = 188) collected from three spawning areas in the Mediterranean Sea by different institutions working with a regional fisheries management organization. Several techniques were used to analyze the genetic sequences (sequence alignments using search algorithms, neighbour joining trees, and a genetic character-based identification key) and an extensive comparison of the results is presented. During this process various inaccuracies in related publications and online databases were uncovered. Our results reveal important differences in the accuracy of the taxonomic identifications carried out by different ichthyoplanktologists following morphology-based methods. While less than half of larvae provided were bluefin tuna, other dominant taxa were bullet tuna (Auxis rochei), albacore (Thunnus alalunga) and little tunny (Euthynnus alletteratus). We advocate an expansion of expertise for a new generation of morphology-based taxonomists, increased dialogue between morphology-based and molecular taxonomists and increased scrutiny of public sequence databases.

No MeSH data available.