Copepods, small aquatic crustaceans, are the most abundant metazoan zooplankton and outnumber every other group of multicellular animals on earth. In spite of ecological and biological importance in aquatic environment, their morphological plasticity, originated from their various lifestyles and their incomparable capacity to adapt to a variety of environments, has made the identification of species challenging, even for expert taxonomists. Molecular approaches to species identification have allowed rapid detection, discrimination, and identification of cryptic or sibling species based on DNA sequence data. We examined sequence variation of a partial mitochondrial cytochrome C oxidase I gene (COI) from 133 copepod individuals collected from the Korean Peninsula, in order to identify and discriminate 94 copepod species covering six copepod orders of Calanoida, Cyclopoida, Harpacticoida, Monstrilloida, Poecilostomatoida and Siphonostomatoida. The results showed that there exists a clear gap with ca. 20 fold difference between the averages of within-specific sequence divergence (2.42%) and that of between-specific sequence divergence (42.79%) in COI, suggesting the plausible utility of this gene in delimitating copepod species. The results showed, with the COI barcoding data among 94 copepod species, that a copepod species could be distinguished from the others very clearly, only with four exceptions as followings: Mesocyclops dissimilis–Mesocyclops pehpeiensis (0.26% K2P distance in percent) and Oithona davisae–Oithona similis (1.1%) in Cyclopoida, Ostrincola japonica–Pseudomyicola spinosus (1.5%) in Poecilostomatoida, and Hatschekia japonica–Caligus quadratus (5.2%) in Siphonostomatoida. Thus, it strongly indicated that COI may be a useful tool in identifying various copepod species and make an initial progress toward the construction of a comprehensive DNA barcode database for copepods inhabiting the Korean Peninsula.
Citation: Baek SY, Jang KH, Choi EH, Ryu SH, Kim SK, Lee JH, et al. (2016) DNA Barcoding of Metazoan Zooplankton Copepods from South Korea. PLoS ONE 11(7): e0157307. https://doi.org/10.1371/journal.pone.0157307
Editor: Jiang-Shiou Hwang, National Taiwan Ocean University, TAIWAN
Received: July 15, 2015; Accepted: May 30, 2016; Published: July 6, 2016
Copyright: © 2016 Baek et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The sequence data examined here are available under the GenBank accession numbers KR048930–KR049062.
Funding: This present work was carried out with the support of “Origin of Biological Diversity of Korea: Molecular Phylogenetic Analyses of Major Korean Taxa” funded by the National Institute of Biological Resources (NIBR No. 2013-02-013), and “Cooperative Research Program for Agriculture Science & Technology Development (National Agricultural Genome Program, Project title: De novo genome annotation for centipede, Scolopendra subspinipes, Project No. PJ010338)” Rural Development Administration, Republic of Korea.
Competing interests: The authors have declared that no competing interests exist.
Copepods are one of the prevalent taxonomic groups among crustaceans, encompassing approximately 14,000 described species worldwide [1, 2, 3], of which about 695 species from 97 families have been known to occur in Korean waters (http://www.kbr.go.kr/home/find/find02001l.do). Their incomparable capability of adaptation to diverse environmental conditions has probably led to their extraordinary morphological and ecological diversity; as a consequence, copepod species are distributed throughout the world and found in nearly every kind of aquatic habitats [2, 4]. In addition, the diversity of copepod species is directly associated with maintaining natural resources as well as nourishing human life, since many of them numerically dominate most planktonic communities [1, 5], play a pivotal function in aquatic food webs , regulate global carbon cycle and climate [7–8] and live as endo- or ectoparasites in many aquatic animals [1, 4, 9]. Despite the ecological and economic significance, little is known about the number of copepod species on earth.
In recent years, because of their ecological importance, a lot of attention has been placed on the estimation of the biodiversity of this subclass Copepoda in marine and freshwater ecosystems [10–13]. The identification and classification of copepods have fundamentally been based on their morphological and anatomical characteristics [1, 2, 4]. However, such conventional ways may have some limitation in precisely estimating the abundance of copepod species in a certain environment, because they are time-consuming and necessitate special training or professional skills. Another difficulty may also be the existence of closely related taxa that are barely distinguishable [12, 14–16]. To make it more difficult, many of copepod species display morphological intraspecific variation corresponding to the habitat types . Consequently, the application of a rapid and promising protocol for the species identification is critically needed for the estimation of copepod diversity.
Many different genetic markers have been considered to complement those conventional approaches. Mitochondrial cytochrome C oxidase subunit I gene (COI) is the gene offering the most efficient and accurate barcoding method for species-level identification in animal kingdom [18–21], though its efficiency is limited in taxa showing little nucleotide sequence diversity of mitochondrial DNA, such as scleractinian corals and calcarean sponges [22–24]. The partial COI barcoding region, which is ca. 600 bp in length, has been found valuable to reveal cryptic species that may not be possible to resolve the phylogenetic relationships in many copepods [12, 16, 25–27]. Numerous published studies for a variety of copepods have also proved the usefulness of COI in identifying species [28–30]. The COI gene is also effective in investigating phylogenetic relationship among species or higher taxa [27, 30–32]. Whereas COI has been analyzed from many calanoid and cyclopoid copepods, relatively limited genetic information is available for the remaining orders.
In the present study, the COI diversity was investigated from 133 individuals of 94 species of copepods representing six orders, Calanoida, Cyclopoida, Harpacticoida, Monstrilloida, Poecilostomatoida and Siphonostomatoida. Until now, extensive DNA barcoding study has never been done over the six copepod orders. Specifically, COI barcoding has never been attempted in the order Monstrilloida. Primary aims of this study are (i) to test whether COI is a sufficient and promising marker to identify various copepod species and (ii) to create preliminary progress towards the construction of a comprehensive DNA barcode database for identified specimens of copepods inhabiting the Korean Peninsula.
Materials and Methods
Specimens were collected from 2003 to 2014 across freshwater systems, coastal and oceanic areas on and around South Korea (Fig 1). Collection of every sample examined here did not require permission from government authorities, because copepods are an invertebrate animal, for which collecting regulations are not strictly controlled in South Korea. Nevertheless, we received permission from the Ministry of Environment of the Korean government for our sample collection in the present study. Individual specimens were carefully identified based on morphological characters. The entire bodies of all individuals were preserved in 95% ethanol. Species names, GenBank accession numbers and other characteristics of all taxa used in the present study are listed in Table 1.
Ethanol-preserved specimens were rehydrated in distilled water for 5 hours before the procedure of DNA extraction. Genomic DNA was extracted using the QIAamp DNA micro kit (QIAGEN Co. Germany) in accordance with the manufacturer-recommended protocol with an exception that incubation with proteinase K was conducted overnight. For large specimens, the DNA was extracted with the Qiagen DNeasy Blood and Tissue Kit (QIAGEN Co. Germany).
The partial fragment of COI was amplified using the universal COI primer pair, HCO2198 and LCO1490 (Table 2) . For specimens or species that did not amplify with this primer set, different specific forward and/or reverse primers were used (Table 1). The Bio-Rad Dyad Peltier thermal cycler was used to perform amplification using the following parameter: 2 min at 95°C, 34 cycles of 20 sec at 95°C, 40 sec at 42–48°C (Table 1) and 40 sec at 72°C, and 5 min at 72°C. PCR amplification was carried out in a 20μL reaction volume composed of 10–45 ng DNA extract, 0.75 mM of each deoxynucleotide, 0.25 mM of each forward and reverse primer, 3 mM MgCl2, 1 × PCR buffer, and 0.25 units of Taq DNA polymerase (Solgent Co., South Korea). PCR products were tested by electrophoresis on a QIAxcel Advanced (QIAGEN Co., Germany). The PCR products with the expected sized band were purified using QIAquick PCR purification kits (QIAGEN Co. Hilden Germany) along the manufacturer’s protocols. The PCR products were sequenced by the same set of primers used for the PCR amplifications, with ABI PRISM BigDye Terminator system and an ABI3700 automatic sequencer (Genotech Co., South Korea).
Primer sequences are given in 5′ to 3′ direction. Amplification difficulty caused by sequence variation of primer binding sites was resolved with mixed bases; R is a mixture of A and G, Y is a mixture of C and T, W is a mixture of A and T, D is a mixture of G, T and C. References are given for each primer.
Chromatogram evaluation, editing, and assemblage were performed using BioEdit 7.0.9 . The edited sequences were blasted against the GenBank nucleotide database (http://www.ncbi.nlm.nih.gov/). Subsequently, all sequences were aligned using Clustal X ver. 2.0.5 [38–39]. To check for the presence of pseudogenes or nuclear translocated mitochondrial sequences  in the COI dataset, sequences were carefully inspected for whether there were any stop codons or very divergent sequences . The nucleotide sequences were translated to amino acids using EMBOSS Transeq (http://www.ebi.ac.uk/Tools/st/emboss_transeq/) based on the invertebrate mitochondrial genetic code. ClustalX ver. 2.0.5 was used to align each of these translated amino acids sequences with a gap opening of 10 and gap extension penalty of 0.2. The nucleotide sequence was then aligned with the amino acid alignment information using a scripted pipeline (convert-nuaa).
Genetic distances within species, genera, families and orders were calculated in MEGA 6 using Kimura two-parameter (K2P) models  for the alignments. Unrooted neighbor-Joining (NJ) trees were established using MEGA under the K2P evolutionary model with 1,000 bootstrapping replicates. The cluster analysis was shown in a radial tree topology, with node confidence values supported only by greater than 50% values.
The partial COI sequences from 133 individuals of 94 copepod species were determined and aligned. Although the size of the COI fragments amplified in the present study varied from 650 to 1,024 bp, the nucleotides at both ends were trimmed to only use high-quality, well matched data. A final sequences alignment of 575 bp was used in the analyses. Among the sequences, no sign of indels was revealed. Neither frame-shift mutations nor premature stop codons were detected during translation of the sequences into amino acids, supporting evidence that all of the sequences used were functional. Among the 575 bp of COI, 425 (74%) were polymorphic, of which 395 (69%) were parsimoniously informative. The average GC contents of all the sequences analyzed were 37.7%.
Mean divergences at various taxonomic levels are given in Table 3. As expected, the genetic divergence increases with higher taxonomic rank: 0.62% to 2.42% within species, 2.42% to 36.95% within genus, 13.00% to 56.94% within family, and 32.61% to 56.94% within order. Across copepod samples (N = 133), mean K2P divergence was 2.42% within species, 15.85% within genus, 24.22% within family, and 42.69% within order (Table 3). K2P distances within genus were highly variable, ranging from 2.42 (Siphonostomatoida) to 36.95 (Monstrilloida), though this type of comparison may not be reliable due to highly different sample sizes among copepod orders examined in this study (Table 3). Although these distance variability ranges were partially overlapped among specific, generic, familial and ordinal levels (Fig 2), it is likely that they were significantly different at a level sufficient to distinguish one copepod species from others.
The horizontal axis represents intervals of genetic distance in percentage and the vertical axis is the number of individuals associated with each distance interval. The flat box indicates zero value.
The COI genetic distances within and between species of the six copepod orders were summarized in Table 4 and Fig 3 (Refer to S1–S12 Tables and S1–S6 Figs). Within-species K2P distances ranged from 0.00% to 17.14% (Table 4), whereas between-species K2P distance from 0.17% to 96.53% (Table 4). There exists a clear gap with ca. 20 fold difference between the averages of within-species sequence divergence (2.42%) and between-species sequence divergence (42.79%) in COI, as shown in Table 4 and Fig 3, suggesting that the results of the present DNA barcoding of copepods could be effective in delimitating species. When we compared the COI barcoding data among 94 copepod species examined here, in most of them, a species could be distinguished from the others very clearly, only with the exceptions of four cases: Mesocyclops dissimilis–Mesocyclops pehpeiensis (0.26% K2P distance in percent) and Oithona davisae–Oithona similis (1.1%) in Cyclopoida, Ostrincola japonica–Pseudomyicola spinosus (1.5%) in Poecilostomatoida, and Hatschekia japonica–Caligus quadratus (5.2%) in Siphonostomatoida. A color heatmap representing the distribution of pairwise sequence divergence among 133 copepod individuals examined in this study showed comparatively and clearly greater values in Monstrilloida indicated by a darker color (Fig 4).
‘W’ indicates genetic diversity within species and ‘B’ indicates that between species. The plot summarizes median (central bar), position of the upper and lower quartiles (central box), value of minimum (lower bar), and value of maximum (upper bar).
The phylogenetic analysis of COI barcode sequences by a neighbor-joining method yielded an unrooted tree displayed in radial shape (Fig 5), which confidently showed a monophyletic clustering of individuals within a species in most of the copepod species examined here, albeit with the four exceptions indicated with asterisks (*) on the tree. In the four exceptional cases of M. dissimilis–M. pehpeiensis and O. davisae–O. similis in Cyclopoida, O. japonica–P. spinosus in Poecilostomatoida, and H. japonica–C. quadratus in Siphonostomatoida, the two closely related species were not clearly distinguished, respectively. Such exceptions are coincident with their lower between-species K2P distances inferred from the COI barcoding data. Also, each of the copepod orders with multiple genera and families formed a monophyletic clade. However, we could not find significant bootstrap support values in most basal nodes of the tree, suggesting a lack of phylogenetic signals of partial COI at higher taxonomic levels (Fig 5).
The analysis was done with Kimura-2-Parameter (K2P) distance matrix and 1,000 bootstrapping replicates. Branches supported with less than 50% bootstrap values were collapsed. The rate variation among sites was modeled with a gamma distribution. The asterisks indicate four species pairs, within each of which the two closely related species are not distinguished from each other based on the COI DNA barcoding marker.
This study examined sequence variation of partial COI sequences and its utility as a DNA barcoding marker to identify and discriminate copepod species from six different copepod orders including Calanoida, Cyclopoida, Harpacticoida, Monstrilloida, Poecilostomatoida and Siphonostomatoida collected from the Korean Peninsula. Our results provide novel data with a wide sample range over the six copepod orders to confirm the validity of COI barcoding for copepod species identification. The ratio 21.9 of between-species to within-species sequence variation is more than twice of the threshold (= 10.0) proposed by Hebert et al. (2004) as a potential species’ boundary .
However, in the four unexpected cases of M. dissimilis–M. pehpeiensis and O. davisae–O. similis in Cyclopoida, O. japonica–P. spinosus in Poecilostomatoida, and H. japonica–C. quadratus in Siphonostomatoida, the COI marker did not provide clear-cut resolution of species identification. As an extreme example, three sequences determined from the two individuals of Oithona similis and one individual of Oithona davisae turned out to be almost identical (1.1% K2P distance in percent), while the two species are easily classified by distinctive morphological characters. Likewise, the other three cases had extremely lower between-species K2P distances (0.3–5.2%). Through further studies, it is necessary to be examined whether COI marker is appropriate for distinguishing such closely related species or not. On the other hand, COI sequences of Paracalanus parvus showed a relatively large difference among the three individuals within the species (S1 Table), although they formed a monophyletic groups (Fig 5). P. parvus has been known as a cosmopolitan copepod species and often confused with other morphologically similar species. Accordingly, multiple cryptic species could be involved with respect to the species, as mentioned in , and thus it is possible that this species may be a member of a species complex. If more detailed DNA barcoding work is done with multiple individuals from a variety of collection sites, the implication of high sequence similarity of COI shown in those copepod species could be clearly interpreted.
The present analyses revealed that the higher taxonomic rank of copepods, the more divergent the COI sequence variation is. Such tendency implied that the COI maker could be a powerful tool for confirmation of species identification as well as examination of copepod classification system based on morphological taxonomy of copepods (Tables 3 and 4, Figs 2–4).
Interestingly, between-species diversity (mean 64.67) of the order Monstrilloida and within-species diversity (mean 4.26) of the order Cyclopoida showed the highest values of genetic distances compared to those of the other orders (Table 4, Figs 3 and 4). Within-species diversity shown in cyclopoids may be due to much larger sample size and diversity examined here. High degree of between-species diversity shown in monstrilloid copepods may be closely related to their parasitic lifestyle. The order Monstrilloida is a unique and puzzling group, known as endo-parasites of polychaetes and mollusks during larval stages, though they become free-living and non-feeding plankton in their adult stage [45–46]. Parasitic montrilloid species often causes considerable difficulty in taxonomic classification due to their ambiguity of morphological characters: their mouthparts are highly reduced or nearly absent in their adult stage. One of the most important difficulties is to match monstrilloid males to their females. The only reliable method to link the sexes of a species is the confirmation of particular apomorphies shared by both sexes, by finding both sexes in the same host or as a pre-copulatory male-female pair in the plankton, or by using molecular identification . Thus, the resultant divergence of monstrilloid COI sequences presented here could be helpful for understanding accelerated evolutionary rate of these parasitic copepod species, and also for designing suitable PCR primers to successfully amplify the COI barcode for molecular identification of monstrilloid copepod species.
It should be noted that one of the most fundamental problems encountered with DNA barcoding of copepods is the lack of a stable universal COI primer set and insufficient reference sequences. During the study, frequent PCR failures have repetitively occurred with some universal primers for most of copepods examined here. It may not be surprising if we take into account the fact that taxonomically broad copepods may have an enormous degree of COI sequence divergence.
Although DNA nucleotide sequences or deduced protein amino acid sequences from complete mitochondrial genomes have been frequently used to elucidate enigmatic arthropod phylogeny in higher taxonomical levels above order [47–53], it is generally known that the COI barcode marker, which is ca. 500–600 bp in length, does not contain enough phylogenetic signal for higher taxonomical levels. Rather, it can be more informative for questions related to population differentiation or cryptic speciation [18–19, 54–60]. Despite the weak resolution of the COI marker in familial- and ordinal-level phylogenetic relationships , the COI-based NJ tree (Fig 5) can be quite meaningful in terms of evidently showing the monophylies of most of the copepod species examined here as well as conveniently providing us with an overview of the COI barcoding results of 133 individuals from 94 copepod species including the six different orders at a glance.
It is known that mitochondrial genes evolve unusually rapidly in some copepods compared to those of other arthropods , with some closely related copepod species exhibiting unexpected gene order rearrangements [34, 62–64]. The previously known COI sequences are limited to a very small portion of copepods, which actually impedes the design of universal primers. Hopefully, as COI data of copepods grow, development of universal primers specific to copepods might be possible. Such group specific oligonucleotide sequences might be desirable to minimize contamination due to non-copepod PCR amplification, known as “the peril of universal primers” .
In summary, the present study including 133 individuals of 94 copepod species is the first attempt to establish a DNA barcoding system for a half dozen orders, which is the broadest survey yet reported in the literatures. It was found that a high degree of COI sequence divergence among most species was clearly sufficient for species identification of copepods in most cases. Thus, it is concluded that COI can serve as a standard, powerful molecular marker for DNA barcoding of copepod species, even though universal PCR primers specific to COI for copepods should be developed through further studies.
S1 Fig. Distribution of pairwise genetic distances (= Kimura-2-parameter, K2P) estimated from COI nucleotide sequences of 16 calanoid species (N = 39).
S2 Fig. Distribution of pairwise genetic distances (= Kimura-2-parameter, K2P) estimated from COI nucleotide sequences of 17 cyclopoid species (N = 25).
S3 Fig. Distribution of pairwise genetic distances (= Kimura-2-parameter, K2P) estimated from COI nucleotide sequences of 9 monstrilloid species (N = 15).
S4 Fig. Distribution of pairwise genetic distances (= Kimura-2-parameter, K2P) estimated from COI nucleotide sequences of 12 harpacticoid species (N = 14).
S5 Fig. Distribution of pairwise genetic distances (= Kimura-2-parameter, K2P) estimated from COI nucleotide sequences of 29 poecilostomatoid species (N = 33).
S6 Fig. Distribution of pairwise genetic distances (= Kimura-2-parameter, K2P) estimated from COI nucleotide sequences of 11 siphonostomatoid species (N = 16).
S1 Table. Mean genetic distances within each species estimated from COI nucleotide sequences of 16 calanoid species (N = 39) based on Kimura-2-parameter distances.
S2 Table. Kimura-2-parameter pairwise distances between species estimated from COI nucleotide sequences of 16 calanoid species.
S3 Table. Mean genetic distances within each species estimated from COI nucleotide sequences of 17 cyclopoid species (N = 25) based on Kimura-2-parameter distances.
S4 Table. Kimura-2-parameter pairwise distances between species estimated from COI nucleotide sequences of 17 cyclopoid species.
S5 Table. Mean genetic distances within each species estimated from COI nucleotide sequences of 9 monstrilloid species (N = 15) based on Kimura-2-parameter distances.
S6 Table. Kimura-2-parameter pairwise distances between species estimated from COI nucleotide sequences of 9 monstrilloid species.
S7 Table. Mean genetic distances within each species estimated from COI nucleotide sequences of 12 harpacticoid species (N = 14) based on Kimura-2-parameter distances.
S8 Table. Kimura-2-parameter pairwise distances between species e estimated from COI nucleotide sequences of 12 harpacticoid species.
S9 Table. Mean genetic distances within each species estimated from COI nucleotide sequences of 29 poecilostomatoid species (N = 33) based on Kimura-2-parameter distances.
S10 Table. Kimura-2-parameter pairwise distances between species estimated from COI nucleotide sequences of 29 poecilostomatoid species.
S11 Table. Mean genetic distances within each species estimated from COI nucleotide sequences of 11 siphonostomatoid species (N = 16) based on Kimura-2-parameter distances.
The authors would like to thank to Prof. Ho-Young Suk, Yeungnam Univ. Kyungsan, Kyungpook Province for his kind advice and help with data analyses on the manuscript.
Conceived and designed the experiments: SYB EHC SKK KHJ JMJ MHK CYC IHK UWH. Performed the experiments: SYB EHC JML JHL CYC IHK. Analyzed the data: SYB EHC YJL YSL JSH UWH. Contributed reagents/materials/analysis tools: SHR KHJ SKK UWH. Wrote the paper: SYB EHC BAVM UWH.
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