Research Article |
Corresponding author: Longshan Lin ( linlongshan1974@163.com ) Corresponding author: Yuan Li ( liyuan@tio.org.cn ) Academic editor: Maria Elina Bichuette
© 2020 Liyan Zhang, Jing Zhang, Puqing Song, Shigang Liu, Pan Liu, Cheng Liu, Longshan Lin, Yuan Li.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Zhang L, Zhang J, Song P, Liu S, Liu P, Liu C, Lin L, Li Y (2020) Reidentification of Decapterus macarellus and D. macrosoma (Carangidae) reveals inconsistencies with current morphological taxonomy in China. ZooKeys 995: 81-96. https://doi.org/10.3897/zookeys.995.58092
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Decapterus macarellus and D. macrosoma are economically important pelagic fish species that are widely distributed in tropical and subtropical seas. The two species are often mistakenly identified due to their morphological similarities as described in the Chinese literature on fish identification. In this study, D. macarellus and D. macrosoma samples were collected in the Eastern Indian Ocean and the South China Sea and reidentified using morphological and DNA barcoding techniques. The characteristics that distinguish the two species primarily include the scute coverage of the straight portion of the lateral line (the most indicative characteristic for classification), the shape of the predorsal scaled area and its location relative to the middle axis of the eye, and the shapes of the posterior margin of the maxilla and the posterior margin of the operculum. The results revealed a large number of misidentified sequences among the homologous cytochrome oxidase (COI) sequences of the two species in the NCBI database and that the genus Decapterus may include cryptic species. In terms of genetic structure, the Sundaland has not blocked genetic exchange between D. macarellus populations in the South China Sea and the Eastern Indian Ocean, giving rise to a high level of genetic diversity. In this study, we made corrections to the Chinese classification standards for D. macarellus and D. macrosoma and the erroneous reference sequences in the NCBI database, thereby providing accurate reference points for the future exploration of cryptic species in the genus Decapterus.
DNA barcoding, genetic diversity, mackerel, morphological characteristics, phylogeny, scad, species identification
Fish species of the genus Decapterus in the family Carangidae are pelagic fish widely distributed in tropical and subtropical waters around the world and are generally of high economic value. Fishes of the genus Decapterus present one free finlet behind the second dorsal fin and the anal fin and varying degrees of scute coverage along the straight-line portion of the lateral line but no coverage along the curved portion of the lateral line. These characteristics make the fishes easily distinguishable from other species of the family Carangidae (
Decapterus macrosoma (shortfin scad) and D. macarellus (mackerel scad) are morphologically similar and thus often confused with each other. In Chinese literatures on fish morphological classification, the morphological descriptions of D. macrosoma and D. macarellus are largely incorrect (
The mitochondrial cytochrome oxidase (COI) gene fragment varies little within species but significantly between species; this fragment can be amplified via polymerase chain reaction (PCR) using universal primers and standardized experimental procedures and is thus employed for DNA barcoding, which has been widely accepted and utilized (
In summary, we aimed to reevaluate D. macarellus and D. macrosoma by combining morphological analysis with molecular genetics to discern the major diagnostic morphological characteristics and correct DNA barcoding for identification and to provide a timeline for the differentiation of the two species. The findings of this study can provide a scientific reference for the classification of fishes in China and the identification of Carangidae fishes and a theoretical basis for the protection, utilization, development and management of Decapterus species germplasm resources.
Decapterus macarellus and D. macrosoma samples were collected from the South China Sea (10°N, 110°30'E) and the Eastern Indian Ocean (2°N, 88°E) in July and October 2019, respectively (Fig.
Using the methods of
Genomic DNA was extracted from specimens of both Decapterus species with a Qiagen DNeasy Kit and stored at 4 °C. Using universal primers for the mitochondrial COI gene fragment (F2: 5 '-TCGACTAATCATAAAGATATCGGCAC-3’; R2: 5'-ACTTCAGGGTGACCGAAGAATCAGAA-3') (
To ensure the accuracy of the DNA barcoding for the two Decapterus species, we retrieved all homologous COI gene sequences of the two species from GenBank (Table
Information on haplotype, accession numbers, sequence similarity for the samples and sequences in this study.
Haplotype | Number | Cited dataset from GenBank | Sequences in this study | |||||
---|---|---|---|---|---|---|---|---|
Accession numbers | Scientific species name | sequence similarity (%) | Corrected species name | ID | Scientific species name | |||
Group 1 | Hap_5 | 63 | HQ560948, HQ564377, HQ564442, JF493340, JF493341, JF493342, JF493343, JF493346, JX261016, JX261033, JX261126, JX261170, JX261203, JX261215, JX261216, JX261243, JX261268, JX261269, JX261389, JX261442, JX261499, JX261514, JX261515, JX261519, JX261629, KF841444, KP856776, KP856777, KP856778, KU943769, KU943771, KU943781, KY371382, KY371387, KY371390, KY371391, KY371392, KY371393, KY371394, KY371396, KY371397, KY371398, KY371399, KY371400, KY371401, MH085881, MH638661, MH638663 | D. macrosoma | 100 | ✓ | B1, B2, B4, B5, B6, B7, B9, B10, B13, B14, B16, B17, B18, B19, B20 | D. macrosoma |
Hap_6 | 6 | JX261160, KY371395, MH638795 | D. macrosoma | 100 | ✓ | B3, B11, B12 | D. macrosoma | |
Hap_7 | 2 | JX260997 | D. macrosoma | 100 | ✓ | B8 | D. macrosoma | |
Hap_8 | 1 | 99.8 | B15 | D. macrosoma | ||||
Hap_9 | 1 | 99.8 | B21 | D. macrosoma | ||||
Hap_20 | 1 | EU514515 | D. macrosoma | 100 | ✓ | |||
Hap_21 | 1 | EU514516 | D. macrosoma | 100 | ✓ | |||
Hap_24 | 1 | HQ564441 | D. macrosoma | 100 | ✓ | |||
Hap_28 | 2 | JF493344, JF493345 | D. macrosoma | 100 | ✓ | |||
Hap_32 | 1 | JX261121 | D. macrosoma | 100 | ✓ | |||
Hap_33 | 4 | JX261134, KC970467, KY371388, KY371389 | D. macrosoma | 100 | ✓ | |||
Hap_34 | 1 | JX261441 | D. macrosoma | 100 | ✓ | |||
Hap_35 | 1 | JX261596 | D. macrosoma | 100 | ✓ | |||
Hap_38 | 2 | KP266782 | D. macrosoma | 100 | ✓ | 7HYS | D. macrosoma | |
Hap_41 | 1 | KU943770 | D. macrosoma | 100 | ✓ | |||
Hap_44 | 2 | KY371383, KY371385 | D. macrosoma | 100 | ✓ | |||
Hap_45 | 2 | KY371384, KY371386 | D. macrosoma | 100 | ✓ | |||
Hap_51 | 1 | KY802095 | D. macrosoma | 100 | ✓ | |||
Hap_54 | 1 | MF541319 | D. macrosoma | 100 | ✓ | |||
Hap_55 | 1 | MF956638 | D. macrosoma | 100 | ✓ | |||
Hap_56 | 1 | MF956639 | D. macrosoma | 100 | ✓ | |||
Hap_59 | 1 | MH638662 | D. macrosoma | 100 | ✓ | |||
Group 2 | Hap_27 | 1 | JF493339 | Decapterusmacarellus | 94.2 | Decapterus sp. 2 | ||
Group 3 | Hap_63 | 1 | MH980014 | Decapterusmacarellus | 96.4 | Decapterus sp. 1 | ||
Group 4 | Hap_1 | 54 | KM986880, KP266765, KU943796, KU943797, KU943798, KY371373, KY371374, KY371376, KY371377, KY371378, KY371380, KY371381, KY570721, KY570723, KY570729, KY570731, KY570733, MF414832, MF414849, MF414876, MH085883, MH085884, MH638676, MH638686, MH638719, MH638731 | D. macarellus | 100 | ✓ | A15, A16, A17, A18, A19, A23, A24, C1, C5, C6, C7, C8, C9, C13, C17, C20, C21, C23, 1CTYS, A4, A10, A11, A12 | D. macarellus |
Group 4 | MH638732, MH638733, MH638755, MH638772, MH638781 | |||||||
Group 4 | Hap_2 | 1 | 99.8 | A20 | D. macarellus | |||
Hap_3 | 8 | KY570726, KY570732, MF414875, MH638794, MN257556 | D. macarellus | 100 | ✓ | A14 A21 C15 | D. macarellus | |
Hap_4 | 1 | 99.8 | A22 | D. macarellus | ||||
Hap_10 | 1 | 99.8 | C2 | D. macarellus | ||||
Hap_11 | 1 | 99.8 | C3 | D. macarellus | ||||
Hap_12 | 1 | 99.8 | C4 | D. macarellus | ||||
Hap_13 | 1 | 99.8 | C10 | D. macarellus | ||||
Hap_14 | 2 | MF541317 | D. macarellus | 100 | ✓ | C11 | D. macarellus | |
Hap_15 | 2 | 99.8 | C12 C18 | D. macarellus | ||||
Hap_16 | 3 | KY371375 | D. macarellus | 100 | ✓ | C14 C22 | D. macarellus | |
Hap_17 | 1 | 99.8 | C16 | D. macarellus | ||||
Hap_18 | 1 | 99.8 | C19 | D. macarellus | ||||
Hap_19 | 3 | KY570727, MH638687 | D. macarellus | 100 | ✓ | C24 | D. macarellus | |
Hap_23 | 1 | HQ564302 | D. macarellus | 100 | ✓ | |||
Hap_25 | 1 | JF493337 | D. macarellus | 100 | ✓ | |||
Hap_26 | 1 | JF493338 | D. macarellus | 100 | ✓ | |||
Hap_36 | 1 | KF009585 | D. macarellus | 100 | ✓ | |||
Hap_42 | 3 | KY371372, MH638698 | D. macarellus | 100 | ✓ | A9 | D. macarellus | |
Hap_43 | 2 | KY371379, MH085882 | D. macarellus | 100 | ✓ | |||
Hap_46 | 1 | KY570722 | D. macarellus | 100 | ✓ | |||
Hap_47 | 1 | KY570724 | D. macarellus | 100 | ✓ | |||
Hap_48 | 1 | KY570725 | D. macarellus | 100 | ✓ | |||
Hap_49 | 1 | KY570728 | D. macarellus | 100 | ✓ | |||
Hap_50 | 2 | KY570730, MH638739 | D. macarellus | 100 | ✓ | |||
Hap_52 | 2 | MF414851, MH638756 | D. macarellus | 100 | ✓ | |||
Hap_53 | 1 | MF414877 | D. macarellus | 100 | ✓ | |||
Hap_57 | 1 | MH119969 | D. macarellus | 100 | ✓ | |||
Hap_58 | 1 | MH119978 | D. macarellus | 100 | ✓ | |||
Hap_60 | 1 | MH638714 | D. macarellus | 100 | ✓ | |||
Hap_61 | 1 | MH638749 | D. macarellus | 100 | ✓ | |||
Hap_62 | 1 | MH638771 | D. macarellus | 100 | ✓ | |||
Hap_64 | 1 | 99.8 | 17CTYS | D. macarellus | ||||
Hap_65 | 1 | 99.8 | A1 | D. macarellus | ||||
Hap_66 | 1 | 99.8 | A2 | D. macarellus | ||||
Hap_67 | 1 | 99.6 | A3 | D. macarellus | ||||
Hap_68 | 1 | 99.8 | A5 | D. macarellus | ||||
Hap_69 | 1 | 99.8 | A6 | D. macarellus | ||||
Hap_70 | 1 | 99.8 | A7 | D. macarellus | ||||
Hap_71 | 1 | 99.8 | A8 | D. macarellus | ||||
Hap_72 | 1 | 99.8 | A13 | D. macarellus | ||||
Group 5 | Hap_40 | 2 | KT326329, MF541318 | D. macrosoma | 100 | D. russelli | ||
Group 6 | Hap_31 | 1 | JQ681500 | D. macarellus | 100 | D. maruadsi | ||
Hap_37 | 6 | KT718513, KT718514, KT718515, KT718516, KT718519 | D. macarellus | 100 | D. maruadsi | KP266752 | D. maruadsi | |
Group 7 | Hap_39 | 1 | 100 | KP267655 | T. japonicus | |||
Group 8 | Hap_22 | 1 | EU514517 | D. macarellus | 100 | S. crumenophthalmus | ||
Hap_29 | 2 | JQ431681, KJ202148 | D. macarellus | 100 | S. crumenophthalmus | |||
Hap_30 | 1 | JQ431682 | D. macarellus | 100 | S. crumenophthalmus |
Due to a lack of fossil records for fishes from the genus Decapterus, it is impossible to precisely determine the timing of their differentiation. In this study, the divergence time of investigated fishes was estimated based on a nucleotide site divergence rate of 1.2% per million years (
To determine whether the Decapterus species from the two sides of the Sundaland have differentiated, we assessed the genetic diversity and genetic structure of D. macrosoma and D. macarellus based on the acquired COI sequences. Specifically, diversity parameters and unrooted minimum spanning tree (MST) data were analyzed using ARLEQUIN software (
Based on the correct classification of D. macarellus and D. macrosoma, countable and measurable characteristics were determined for 50 individuals from each population (Table
Comparison of countable and measurable characteristics of D. macarellus and D. macrosoma.
Parameters | D. macrosoma | D. macarellus | |
---|---|---|---|
South China Sea (N = 50) | South China Sea (N = 50) | Eastern Indian Ocean (N = 50) | |
dorsal fin | VII~VIII, I-31~35+1 | VIII, I-30~35+1 | VII~VIII, I-30~36+1 |
pectoral fin | 20~23 | 20~23 | 20~24 |
pelvic fin | I-5~6 | I-5~6 | I-5~6 |
anal fin | II, I-26~30+1 | II, I-26~30+1 | II, I-27~30+1 |
caudal fin | 15~18 | 16~18 | 16~17 |
scute | 24~38 | 25~36 | 24~38 |
vertebrae | 23~26 | 23~25 | 24~26 |
body weight (g) | 9.8~24.4 | 7.1~23.9 | 17.2~27.7 |
body length (mm) | 92.1~119.3 | 20.6~114.3 | 108.2~127.3 |
fork length (mm) | 104.3~128.4 | 29.3~125.1 | 114.5~134.6 |
Combining the findings of previous studies (
The 652 bp COI gene fragments from both D. macarellus and D. macrosoma were amplified using the F2 and R2 primers, and D. macarellus exhibits a higher level of genetic diversity than that of D. macrosoma. The haplotype diversity (h) and the nucleotide diversity (π) were 0.862 ± 0.067 and 0.0037 ± 0.0023, respectively, for D. macarellus from the Eastern Indian Ocean; 0.797 ± 0.086 and 0.0030 ± 0.0019, respectively, for D. macarellus from the South China Sea; and 0.486 ± 0.124 and 0.0008 ± 0.0007, respectively, for D. macrosoma from the South China Sea. The MST constructed based on the COI sequences of the two fish species (Fig.
After annotating and aligning all the sequences retrieved from GenBank and gained in this study, a 534 bp target fragment was obtained that hosted 142 mutation sites, including 24 single-nucleotide polymorphisms, 118 parsimony-informative sites, and no insertions/deletions. The A+T content was 51.7%, slightly higher than the G+C content, revealing an AT preference. The NJ tree was constructed using all studied sequences with D. maruadsi and T. japonicus as outgroups (Fig.
Based on a 1.2% nucleotide divergence rate per million years, we estimated the divergence time of the species (Table
Biodiversity is an important material basis and condition for human survival and sustainable development and usually encompasses species diversity, genetic diversity, ecosystem diversity, and landscape diversity. To study biodiversity, we must first accurately identify the existing species; only with this approach do follow-up studies make sense. For example, both D. macrosoma and D. macarellus are economically important species in China, but due to historical reasons, the domestic literature on the identification of these two species has been confused, with the species descriptions from China contradictory to those from international literature. In this study, using samples collected in the Eastern Indian Ocean and the South China Sea, we re-examined the two Decapterus species from the perspectives of morphology and molecular genetics and provided their major morphological diagnostic characteristics and correct DNA barcoding.
The comparison of countable and measurable characteristics between the two species showed that most of the characteristics are identical or significantly overlapping, making it impossible to distinguish the two species, whereas some directly observable morphological characteristics allow differentiation of the two species (
Comparison of major morphological diagnostic characteristics of D. macarellus and D. macrosoma.
D. macarellus | D. macrosoma | |
---|---|---|
straight-line portion of the lateral line covered with scutes | posterior end, approximately 1/2 | majority in the rear, approximately 3/4 |
external morphological characteristics of scutes | the highest scute is approximately half the eye diameter | no particular external characteristics |
whether the predorsal scaled area reaches the middle of the eye | reaching or extending past | not reaching |
shape of the predorsal scales | “∩” | “m” |
shape of the posterior end of the maxilla | convex and round | truncated |
shape of the posterior margin of the operculum | oblique | straight |
The DNA barcoding technique has been repeatedly applied for species identification and has successfully revealed the “cryptic biodiversity” in many taxa (
Genetic distance of COI gene among (below the diagonal) and within (on the diagonal) groups, and the divergence time between groups (above the diagonal).
Group 1 | Group 2 | Group 3 | Group 4 | Group 5 | Group 6 | Group 7 | Group 8 | |
---|---|---|---|---|---|---|---|---|
Decapterus macarellus | 0.005 | 5.92 | 6.33 | 5.25 | 7.17 | 7.67 | 10.25 | 14.75 |
Decapterus sp. 2 | 0.071 | 0 | 5.67 | 5.17 | 8.17 | 7.42 | 9.92 | 15.92 |
Decapterus sp. 1 | 0.076 | 0.068 | 0 | 3.00 | 7.92 | 7.75 | 10.08 | 16.50 |
Decapterus macrosoma | 0.063 | 0.062 | 0.036 | 0.007 | 7.50 | 7.58 | 11.58 | 16.00 |
Decapterus russelli | 0.086 | 0.098 | 0.095 | 0.09 | 0 | 2.58 | 7.75 | 14.50 |
Decapterus maruadsi | 0.092 | 0.089 | 0.093 | 0.091 | 0.031 | 0.002 | 8.17 | 14.67 |
Trachurus japonicus | 0.123 | 0.119 | 0.121 | 0.139 | 0.093 | 0.098 | 0 | 12.33 |
Selar crumenophthalmus | 0.177 | 0.191 | 0.198 | 0.192 | 0.174 | 0.176 | 0.148 | 0.009 |
We estimated the timing of divergence within the genus Decapterus to be in the early Miocene Epoch to the late Pliocene Epoch based on the COI nucleotide site divergence rate, which provides a rough timeline for the evolution of species in the family Carangidae. The species in Carangidae originated through differentiation via geographical isolation and adaptive evolution during the diffusion process (
Decapterus macarellus shows significantly higher genetic diversity than D. macrosoma and additional mutation characteristics, suggesting that it has higher adaptability, most likely related to its wider distribution. At the level of the COI gene, the genetic differentiation appeared in P. chinensis (
Currently, the shortage of experienced taxonomists capable of completing and updating the descriptions and cataloging work of biodiversity is a major challenge for the scientific community. Species classified by external morphological characteristics are referred to as morphospecies (
Initially, species classification primarily depended on the experience of the taxonomist and the accuracy of the literature. However, taxonomists do not necessarily have a background in genetics, whereas geneticists lack expertise in species identification and are unaware of the classification characteristics of the species, resulting in a rift between the two methods. Only by combining the two methods and using DNA barcoding technology as a new identification method enabling the disciplines to complement each other is it possible to classify species rapidly and accurately based on correctly identified morphological characteristics. For example, by combining morphological characteristics and DNA barcoding technology,
In summary, when identifying fish species, marine biologists need to understand the research status of different taxonomic categories of the fish at home and abroad to ensure the validity of morphological classification. The findings of this study have implications for the classification and evolution of fish species in the genus Decapterus and for the conservation of species diversity.
Decapterus macarellus and D. macrosoma in the Eastern Indian Ocean and the South China Sea waters were collected and reidentified using morphological and DNA barcoding techniques. The results showed that the morphological diagnostic characteristics of the two species primarily include the scute coverage of the straight portion of the lateral line (the most indicative characteristic for classification), the shape of the predorsal scaled area and its relative location to the middle axis of the eye, and the shapes of the posterior margin of the maxilla and the posterior margin of the operculum. Molecular analysis revealed that both the two species have high genetic diversity, and no genetic differentiation in D. macarellus from the South China Sea and the Eastern Indian Ocean was detected. By comparing the COI sequences obtained in this study and those homologous sequences downloaded from GenBank, we speculated that the genus Decapterus may include cryptic species and corrected a number of erroneous referenced sequences in the NCBI database.
The present study could not have been performed without assistance from Mrs Xing Miao, Zhijin Yang, Liangming Wang, Ran Zhang during the sample collection. We also thank the anonymous reviewers for their helpful comments. This work was supported by the National Programme on Global Change and Air-Sea Interaction (GASI-02-SCS-YD sum/spr/aut, GASI-01-EIND-YD01aut/02aut).