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Role of DNA barcoding in marine biodiversity assessment and conservation: An update

More than two third area of our planet is covered by oceans and assessment of marine biodiversity is a challenging task. With the increasing global population, there is a tendency to exploit marine resources for food, energy and other requirements. This puts pressure on the fragile marine environment and necessitates sustainable conservation efforts. Marine species identification using traditional taxonomical methods is often burdened with taxonomic controversies. Here we discuss the comparatively new concept of DNA barcoding and its significance in marine perspective. This molecular technique can be useful in the assessment of cryptic species which is widespread in marine environment and linking the different life cycle stages to the adult which is difficult to accomplish in the marine ecosystem. Other advantages of DNA barcoding include authentication and safety assessment of seafood, wildlife forensics, conservation genetics and detection of invasive alien species (IAS). Global DNA barcoding efforts in the marine habitat include MarBOL, CeDAMar, CMarZ, SHARK-BOL, etc. An overview on DNA barcoding of different marine groups ranging from the microbes to mammals is revealed. In conjugation with newer and faster techniques like high-throughput sequencing, DNA barcoding can serve as an effective modern tool in marine biodiversity assessment and conservation.


An introduction to DNA barcoding
The concept of DNA barcoding has become one of the most important and significant scientific visions in the last decade. As an emerging and effective tool for species identification, the concept of DNA barcoding has gained worldwide popularity. The ground-breaking concept of DNA barcoding was put forward in the year 2003 by Professor Paul Hebert and collaborators serving at University of Guelph, Canada. Mitochondrial cytochrome c oxidase subunit 1 (COI) gene was suggested as unique barcode region for animals (Hebert et al., 2003). This sequence was validated at the 1st International Conference on DNA Barcode of Life. Henceforth, several studies have shown that the sequence diversity in a 650 bp region near the 5 0 region of the COI gene provides strong species level resolution for different animal groups like birds (Yoo et al., 2006;Tavares and Baker, 2008;Schindel et al., 2011), springtails (Hogg and Hebert, 2004), shrimps (Trivedi et al., 2011), fishes (Ward et al., 2005;Yancy et al., 2008;Bhattacharjee et al., 2012;Laskar et al., 2013;Trivedi et al., 2014), tortoise , oysters (Trivedi et al., 2012), mammals (Lim, 2012), spiders (Greenstone et al., 2005), mosquitoes (Cywinska et al., 2006), ticks (Zhang and Zhang, 2014) etc.
The Consortium for the Barcode of Life (CBOL) was established to support worldwide DNA barcoding and subsequently an international online data management system -the Barcode of Life Data Systems (http://www.barcodinglife.org) came into effect. Survey and assessment of genetically diverse organisms of the earth through DNA barcoding is led by CBOL. A milestone in the field of DNA barcoding was achieved by launching of International Barcode of Life Project (iBOL). Canada was the first country to establish national network for DNA barcoding as The Canadian Barcode of Life Network (BOL-NET.ca). Subsequently, several countries and regions have also established barcoding networks as part of the iBOL like Europe (ECBOL; http://www.ecbol.org/), Norway (NorBOL; http://dnabarcoding.no/en/), Mexico (MexBOL; http://www. mexbol.org/) and Japan (JBOLI; http://www.jboli.org/). Besides this, thematic programs like human health (Health-BOL), polar life (PolarBOL) and quarantine and plant pathogens (QBOL, as a part of the ECBOL) are also in place.

Advantages of DNA barcoding in marine perspective
More than 70% of our planet is covered by oceans that have higher biodiversity compared to terrestrial or freshwater ecosystems. The massive marine ecosystem is the habitat for a large number of flora and fauna, both macro and micro. Among the 35 animal phyla, 34 phyla have marine representatives while 14 include exclusively marine animals (Briggs, 1994;Gray, 1997). The occurrence of cryptic species is relatively common in marine ecosystems. Cryptic species are those species that are morphologically similar but genetically distinct. DNA barcoding can be a very effective tool in assessment of these cryptic species. Another problem that persists in the marine and estuarine habitat is the linking of the larval stages with the adult forms. DNA barcoding can accurately link the larval stages of a species in order to unravel the life cycle of different marine species, which is usually difficult and in some cases not possible using the morphological approach. The threat of invasive species to marine biodiversity can be globally assessed through DNA barcoding (Molnar et al., 2008).
The invasive alien species (IAS) poses severe threat and is capable of inflecting huge economic losses. DNA barcoding can be used to quickly and accurately identify the invasive alien species and prompt preventive measures with subsequent regulatory control can be initiated. Barcoding of indicator species can be fruitful in the monitoring and abatement of marine pollution including coastal pollution. One main aim of DNA barcoding initiative is the discovery of new species. DNA barcoding can be used as an important tool for identification, authentication and safety assessment of sea food, particularly for processed, cooked or smoked products. This molecular identification can even allow us to trace the origin of certain products (Galimberti et al., 2013). A study conducted on the Japanese delicacy tuna sushi from different restaurants in USA, revealed the presence of endangered species, fraud and also a health hazard (Lowenstein et al., 2009). An analysis of 254 Canadian seafood samples revealed that 41% of the samples were mislabeled .
DNA barcoding is an important tool in wildlife forensics and conservation. It can be used to identify endangered sea turtles by assessing turtle meat, carcasses or eggs that are illegally traded (Vargas et al., 2009). One important requirement of DNA barcoding is the collection and maintenance of samples as voucher specimens, which allows reliable means of corroborating the identification of the species from which data is accumulated. The voucher specimens provide permanent documentation for investigation of marine biodiversity. DNA barcoding has a great utility in the field of taxonomy (Ali et al., 2014).
DNA barcoding can be very effective for molecular phylogenic studies, geographical distribution and conservation of marine biodiversity. DNA barcoding can be used for pest and disease control as well. With the recent developments in deep sea research and the revelation that several deep sea organisms possess extraordinary pharmaceutical properties, DNA barcoding of deep sea organisms has gained global attention. Census of the Diversity of Abyssal Marine Life (CeDAMar) is devoted to the barcoding of deep sea organisms. The user-friendliness of DNA barcodes is also an added advantage and can be effectively used for marine biodiversity assessment, fisheries management and conservation (Pe´rez1-Huete and Quezada, 2013).

Worldwide DNA barcoding initiative for marine organisms
MarBOL, the Marine Barcode of Life, is an international campaign to barcode marine species. MarBOL (http://www. marinebarcoding.org) is led by an International Steering Committee and an affiliated project of the Census of Marine Life (CoML). CoML is involved in several Ocean Realm Field Projects (Table 1). Already five International Barcode of Life Conferences have been held and the 6th International Barcode of Life Conference is scheduled to be held in Guelph, Ontario, Canada during August 18-22, 2015. Some important marine DNA barcoding conferences are shown in Table 2.

DNA barcoding of marine microbes
Assessment of biodiversity in the microbial world has always been a challenging task. Rapid and accurate identification of the microbes is frequently necessary to prevent the spread of diseases caused by microbes. Protists are eukaryotic microbes which have short generation time and asexual reproductive capability. An ecologically significant group of protists are the dinoflagellates which serve as primary producers, coral symbionts and cause red tides. DNA barcoding of marine environmental samples revealed massive dinoflagellate diversity (Stern et al., 2010).

DNA barcoding of seagrasses, mangroves and marine phytoplanktons
Seagrasses are important submerged flowering plants that have very noticeable ecological influence on the coastal environment due to their nutrient recycling ability and high primary productivity. Besides this, they contain valuable secondary compounds like phenolic acids which are used in traditional medicines. Rosmarinic acid and zosteric acid obtained from seagrasses are widely used as an antioxidant and effective antifouling agent respectively. Although these marine plants have wide geographical distribution worldwide there is rapid decline in sea grass species and cover globally. It is reported that seagrasses are disappearing at the rate of 110 km 2 per year, since 1980 (Waycott et al., 2009). Hence, there is urgent need for assessment and conservation of seagrasses. Seagrasses perform both, sexual and asexual reproduction, but vegetative reproduction is more common and sexual progenies are short lived. Species identification becomes difficult because the flower as a distinct morphological trait is often unavailable. In such a situation, DNA barcoding can serve as a useful identification tool. Different markers have been used for identification of seagrasses like nuclear ITS for Halophila (Waycott et al., 2002), trnK introns and rbcL for Zostera (Les et al., 2002), ITS1, 5.8S rDNA and ITS2 for Halophila (Uchimura et al., 2008). By using rbcL and matK sequences it was revealed that it is possible to develop DNA barcoding for seagrasses (Lucas et al., 2012).
Mangroves at the intersection of terrestrial, estuarine and near shore marine ecosystem have immense ecological and economic significance. The ecosystem services provided by mangrove forests are worth at least US$1.6 billion per year worldwide (Field et al., 1998;Costanza et al., 1997). This dynamic and unique ecosystem is increasingly threatened and depleted. The conservation of mangroves is of utmost importance in order to maintain the health of this fragile environment. Loss of evolutionary unique species in the mangrove ecosystem has been reported and DNA barcoding provided phylogenetic information for developing unified mangrove management plan worldwide (Daru et al., 2013). The Sunderbans is the single largest block of tidal halophytic mangrove forest listed in the UNESCO world heritage list (http://whc. unesco.org/en/list). It is regarded as the world's largest natural nursery where a large number of marine and estuarine species come to breed and the juveniles stay back to exploit its rich natural resources (Trivedi et al., 2013). In a study conducted in the Sunderbans mangrove ecosystem, molecular methods based on rbcL subunit of RuBisCO enzyme were used for identification of phytoplankton groups lesser than 10 lm size .

DNA barcoding of marine algae
Different species of red marine macro algae are often difficult to identify by using morphological techniques. Two molecular markers namely mitochondrial COI gene and UPA (Universal Plastid Amplicon) domain V of the 23S rRNA gene were used for identification of different species of red alga belonging to the family Kallymeniaceae. Results showed that COI was a more sensitive marker and led to the discovery of a new species Euthora timburtonii (Clarkston and Saunders, 2010). A similar study was conducted involving inter tidal red macro algae in China with three molecular markers -COI, UPA and ITS (nuclear internal transcribed spacer). Although COI was effective to identify species but not all species gave successful amplicons due to lack of universal primers. UPA had effective universal primers but showed problems with closely related species, while ITS was the least effective (Xiaobo et al., 2013).
Gracilariaceae is a red algal family which is commercially important for its use in biotechnology and microbiology research as a phycocolloid agar. Gracilaria species are difficult to identify morphologically and DNA barcoding holds promise in species level identification (Kim et al., 2010). Recently, a novel microalga was isolated and characterized from Indian Ocean which has biofuel potential. In this study 16S rRNA and 23S rRNA were used as barcode (Ahmad et al., 2013). DNA barcoding can be useful as a rapid, sensitive and reliable method for monitoring programs of marine and coastal ecosystems for detecting Harmful Algal Bloom (HAB) species.

DNA barcoding of marine zooplanktons
Zooplanktons have great ecological significance and represent 15 animal groups (phyla). Therefore, DNA barcoding of zooplanktons is an important aspect of modern ecological studies. Census for Marine Zooplanktons (CMarZ) is devoted to the study of global zooplankton assemblages. The DNA Barcoding Centers of CMarZ are located in UConn (USA), Bremer- Gulf of Maine Area Program GOMA 10 International Census of Marine Microbes ICOMM 11 Natural Geography in Shore Areas NaGISA 12 Pacific Ocean Shelf Tracking POST 13 Tagging of Pacific Pelagics TOPP haven (Germany), ORI (Japan), Qingdao (China) and Goa (India). Fig. 1 shows the five CMarZ barcoding centers of the world. Barcode analysis using COI gene involving 52 specimens of 14 species of chaetognaths could successfully discriminate different species of chaetognaths across the phylum. The average K2P distance within species was 0.0145. Among the marine zooplanktons the copepods are one of the most systematically complex and ecologically significant groups with more than 2500 species. Several studies have been conducted on this diverse group. The occurrence of cryptic species is widespread among the copepods which necessitates more DNA barcoding studies. Some important publications on DNA barcoding of marine copepods are shown in Table 4.
Since it is difficult to identify the different chaetognath species based on morphological characters, especially with those preserved in alcohol, DNA barcoding can be very effective to resolve this problem . A study was conducted with Neocalanus copepods involving four marker genes namely COI, 12S, nuclear ITS, and 28S. The results showed that although all the four markers could identify distinctly all the species but distinction of the form variants was only confirmed by the COI sequences (Machida and Tsuda, 2010). DNA sequence variation of a 575 base-pair region of 28S rDNA, from North and South Atlantic regions could accurately and reliably identify the three species of Oithona, an ecologically important copepod species (Cepeda et al., 2012).

DNA barcoding of marine invertebrates
The pteropods which belong to the phylum Mollusca and class Gastropoda are of unique research interest due to their vulnerability to ocean acidification. Barcoding of Diacavolinia pteropods indicated that the Atlantic specimens comprise a single monophyletic species and show probable species-level divergence between Atlantic and Pacific populations (Maas et al., 2013). DNA barcoding comprising 227 species of Canadian marine mollusks indicated possible cases of overlooked species (Layton et al., 2014). DNA barcoding projects should be developed for megadiverse groups such as mollusks to facilitate species discovery and conservation (Puillandre et al., 2009). A study involving 315 specimens from around 60 venerid species showed that DNA barcoding can be very effective in species delimitation (Chen et al., 2011). Marine oysters are bivalves that have great economic significance. Identification of oysters largely based on phenotypic characters like shell morphology is problematic due to the taxonomic controversies. Shell morphology, used as a primary distinguishing feature is greatly affected by habitat (Tack et al., 1992). In such cases, molecular identification proves to be useful (Table 4).
Echinoderms are exclusively marine animals. DNA barcoding of 191 echinoderm species belonging to five classes was undertaken. Based on shallow intraspecific versus deep congeneric divergences 97.9% specimens were assigned to known species (Ward et al., 2008a). Sponges have canal system inside the body and possess pharmaceutical properties. Sponge Barcoding Project, http://www.spongebarcoding.org is a global initiative. A DNA barcoding workflow capable of analyzing large sponge collections has been developed through this project (Vargas et al., 2012). Nematodes are known for their role as indicator of anthropogenic stress in the marine ecosystems. In the nematodes, 18S gene was able to amplify across several taxa and showed identification success rate of 97% (Bhadury et al. (2006)). Universal primers for diverse group of marine metazoan invertebrates are available (Folmer et al., 1994;Lobo et al., 2013) (Table 4).

DNA barcoding of lower chordates
Ascidians are filter-feeding marine urochordates which are regarded as model organisms used to study complex biological processes. They are used to study the transcriptional control of embryonic development, mechanism of metal accumulation, evolution of the immune system, conservation of gene regulatory networks in chordates, development of heart, etc. (Holland and Gibson-Brown, 2003;Trivedi et al., 2003;Satoh et al., 2003;Stolfi and Christiaen, 2012;Tolkin and Christiaen, 2012;Razy-Krajka et al., 2014). The genome of  Role of DNA barcoding in marine biodiversity assessment and conservation an ascidian species Ciona intestinalis is the smallest of any experimentally manipulable chordate, as a consequence it is used in genome analysis studies. COI gene analysis of Ciona specimens from New Zealand revealed for the first time, the existence of solitary ascidian Ciona savignyi in the Southern Hemisphere (Smith et al., 2012). A new ascidian species belonging to the genus Diplosoma has been revealed through DNA barcoding in the Ryukyu Archipelago of Japan (Hirose and Hirose, 2009).

DNA barcoding of marine fishes
Marine fish is an important source of protein, vitamin D, vitamin B 12 , iodine, selenium and omega-3 fatty acids. Marine fisheries sector has a very significant contribution in food security and economic welfare. Proper identification of fish species is important for management of fisheries and authentication of food products. DNA barcoding allows fast and efficient means of fish identification. Two main global barcoding initiatives for fish are FISH-BOL (http://www.fishbol.org) and SHARK-BOL (http://www.sharkbol.org). DNA barcoding is useful not only for the identification of whole fish but also for the identification of larvae, eggs, fillets, fins or other fragments of the body which are difficult to identify based on morphology. This molecular technique was used to identify shark fins that were confiscated from illegal fishers in Australia (Holmes et al., 2009). Demand for ornamental fish is rapidly increasing globally. COI gene analysis of 391 ornamental fish species from 8 coral reef locations revealed that most (98%) of these species belonged to distinct barcode clusters (Steinke et al., 2009a,b). Some important publications on DNA barcoding of marine fishes are depicted in Table 3.

DNA barcoding of marine reptiles
As compared to fishes there is less information on the DNA barcoding of reptiles. First large scale DNA barcoding of reptiles (including Squamata and Testudines) was conducted with 468 specimens from biodiversity hotspot of Madagascar. In this study 41-48 new (undescribed) species were identified thereby indicating the utility of DNA barcoding in biodiversity assessment. This study also revealed that the average interspecific genetic distance within families was 13.4% in Boidae and 29.8% in Gekkonidae (Nagy et al., 2012). A study conducted on the Brazilian sea turtles revealed that species-specific COI barcode tags can be used for identifying each of the marine turtle species that were investigated (Vargas et al., 2009). In a separate study DNA barcoding was done for globally threatened marine turtles. This study showed that DNA barcoding is not only a powerful tool for species identification but also can play a vital role in wildlife forensics and conservation genetics (Naro-Maciel et al., 2009).

DNA barcoding of sea birds
The All Birds Barcoding Initiative (ABBI) was launched in September 2005 with the aim of barcoding approximately 10,000 known species of world birds. DNA barcoding studies revealed that there are hundreds of undescribed avian species. Seabirds inhabit the marine environment either directly or indirectly and spend most of their life in the sea but cannot be considered as sea creatures because they do not have the ability to use the dissolved oxygen in sea water. Sea birds include Gulls, Terns, Brown Booby, Carp Plover, Swans, Spoonbill, Ardeidae, etc. The seabirds depend on the marine environment for food or use the sandy beaches, tidal flats or mangrove environment for food and nesting. DNA barcoding analysis was conducted with 387 individuals of 147 species of birds (including seabirds) from the Netherlands, which is ornithologically one of the best covered countries (Aliabadian et al., 2013). Due to depletion of mangrove forests, ocean acidification, rising salinity and sea level elevation, more efforts are needed for DNA barcoding of sea birds and their conservation. Caribbean and western central Atlantic fishes Weigt et al., 2012 4 Antarctic fishes Dettai et al. (2011) 5 Arctic marine fishes Mecklenburg et al. (2011) 6 Marine and brackish water fishes from Argentina Mabragan~a et al. (2011) 7 Marine fishes from Japan (Zhang and Hanner, 2011) 8 Marine fishes of China (Zhang, 2011) 9 European marine fishes (Kochzius et al., 2010) 10 Marine fishes of India (Lakra et al., 2010) 11 Campaign to barcode all fishes (Ward et al., 2009) 12 Coral reef fishes (Steinke et al., 2009a) 13 Indo-Pacific, Australian and South African Marine Fishes (Zemlak et al.,. 2009) 14 North American marine fishes (Steinke et al., 2009b) 15 Salmon and trout species from North America (Rasmussen et al., 2009) 16 North-east Atlantic deep-water sharks (Moura et al., 2008) 17 Sharks and rays of Australia (Ward et al., 2008b) 18 Fish larvae in Great barrier Reef, Australia (Pegg et al., 2006) 19 Marine fishes of Australia (Ward et al., 2005) Bucklin et al.(2010b) Zooplankton diversity analysis through single-gene sequencing of a community sample Machida et al. (2009) Comparison of molecular species identification for North Sea calanoid copepods (Crustacea) using proteome fingerprints and DNA sequences Laakmann et al. (2013) Comparison of morphological and molecular traits for species identification and taxonomic grouping of oncaeid copepods Bo¨ttger-Schnack and Machida (2011) Morphological and molecular phylogenetic analysis of evolutionary lineages within Clausocalanus (Copepoda: Calanoida) Bucklin and Frost (2009) Dissimilarity of species and forms of planktonic Neocalanus copepods using mitochondrial COI, 12S, nuclear ITS, and 28S gene sequences

DNA barcoding of marine mammals
Mammalia Barcode of Life (http://www.mammaliabol.org) is devoted to barcoding of mammals including the marine mammals. A study conducted along the French Atlantic coast demonstrated that DNA barcoding in conjunction with a stranding network can be used in monitoring marine mammal diversity (Alfonsi et al., 2013).

Criticisms of DNA barcoding
Initially the concept of DNA barcoding invoked criticisms from traditional taxonomists (Will and Rubinoff, 2004;DeSalle et al., 2005;Will et al., 2005;Rubinoff et al., 2006). DNA barcoding has some limitations, like low resolutions in the cases of recently diverged species, species complexes and hybrids. The presence of pseudogenes and mitochondrial introgression is highlighted by some researchers (Song et al., 2008;Kemppainen et al., 2009). Reproductive isolation, an important aspect for the biological species concept is difficult to investigate in the marine ecosystems. A study involving cosmopolitan marine bryozoan revealed that divergent barcode clusters corresponded to reproductively isolated groups, thereby establishing a link between biological species concept and DNA barcoding (Go´mez et al., 2007). The integration of morphological, ecological and physiological data with DNA barcode data will improve species discovery and identification process (Waugh, 2007;Padial et al., 2010). Some shortcomings of integrating DNA barcode data were revealed (Goldstein and DeSalle, 2011). Collins and Cruickshank (2013) assessed seven deficiencies and outlined potential improvements on each of them. Seven shortcomings in the experimental design addressed by these workers are as follows: 1. Failure to test clear hypotheses.
2. Inadequate a prior identification of specimens.
3. The use of the term 'species identification'. 4. Inappropriate use of neighbor-joining trees. 5. Inappropriate use of bootstrap resampling. 6. Inappropriate use of fixed distance thresholds. 7. Incorrectly interpreting the barcoding gap.
During DNA barcoding, care has to be taken to address these issues. Several studies have shown that finding a universal barcode for all life forms is more difficult than initially supposed.

Future of marine DNA barcoding
Genomic studies in conjugation with DNA barcoding can be very effective in assessment of global biodiversity. Recently, Centre for Biodiversity Genomics (CBG) was established at University of Guelph, Canada, as a global hub for DNA barcoding. With the advancement of high-throughput sequencing, it can be useful in DNA barcoding of bulk environmental samples. Environmental barcoding was very effective in analyzing zooplankton samples collected from Equatorial Pacific Ocean (Machida et al., 2009). Screening of cDNA libraries can reduce artifacts caused by pseudogenes or mispriming. Real Time Quantitative PCR (qPCR) can be very useful in assessing the species diversity and abundance particularly for unsorted and bulk samples. A taxonomic approach of integrating DNA sequences with morphological characters will achieve higher efficiency in species identification. With the development of newer and faster techniques, DNA barcoding holds great promise in the assessment, analysis and conservation of marine biodiversity.

Conclusions
Most DNA barcodings are focused on animals and more effort is needed on the barcoding of plants and protists. One main reason may be the lack of universal barcode gene in plants that makes the situation comparatively tricky. Despite of some limitations, DNA barcoding approach can be used for survey of marine biodiversity and prioritizing conservation strategies. In conclusion it can be said that DNA barcoding can play a very significant role in assessment and conservation of biodiversity in the massive and diverse marine ecosystem.