REVIEW Treubia 43: 1–16, December 2016 DNA BARCODING: FOUNDATIONS AND APPLICATIONS FOR SOUTHEAST ASIAN FRESHWATER FISHES Hadi Dahruddin1*, Renny Kurnia Hadiaty1, Nicolas Hubert1,2 1Museum Zoologicum Bogoriense, Research Centre for Biology, Indonesian Institute of Sciences (LIPI), Jl. Raya Jakarta - Bogor Km 46, Cibinong 16911, Indonesia 2Institut des Sciences de l’Evolution, CNRS/IRD/UM2-UMR5554, Université de Montpellier, Place Eugène Bataillon, 34095 Montpellier Cedex 05, France *Corresponding author: [email protected] Received: 23 May 2015; Accepted: 2 December 2015 ABSTRACT Identifying and delineating species are the primary tasks of taxonomy. Owing to the decreasing interest of the nations for taxonomy and the inventory of living beings, funds have been drastically decreasing during the last two decades for taxonomic studies. As a consequence, the worldwide pool of taxonomists has dramatically decreased. DNA barcoding, as an automated tool for species delineation and identification, proved to rejuvenate the field of taxonomy and open new perspectives in ecology and conservation. In the present review, we will discuss how DNA barcoding established as a new paradigm in taxonomy and how DNA barcoding has been recently integrated in taxonomic studies. We will further detail the potential applications for species identifications and discuss how DNA barcoding may positively impact the inventory and conservation of living beings, particularly in biodiversity hotspots. We emphasise the benefit of DNA barcoding for the conservation of Southeast Asian freshwater fishes. Key words: biodiversity hotspots, DNA barcoding, integrative taxonomy, species delineation, species identification INTRODUCTION After three decades of inventorying living beings, Earth’s biodiversity, which consist of not less than 10 millions of described species to date, is still poorly known and includes many species that carry potential economic and societal applications that are still to be revealed (Vernooy et al. 2010). Despite the importance of identifying species for either scientific or societal purposes, the interest of the nations in taxonomy and in pursuing the inventory of earth living beings has decreased since its earlier development during 18th century with Carl Linnaeus (Mallet & Willmott 2003). During the second conference of the parties of the Convention on Biological Diversity (CBD) held in Jakarta in 1995, the participant countries have explicitly formulated through the concept of taxonomic impediment, which epitomises the major concern raised by the worldwide community of taxonomists since the 90’s about the increasing disinterest from governments and funding agencies for taxonomy. Unfortunately, several global initiatives such as the Global Taxonomic Initiative (GTI) launched in the context of the CBD early in 2002 failed to embrace a massive adhesion and to help reach the CBD goal to slow-down the pace of species loss by 2010 (www.cbd.int/cop). Several challenges prevented the emergence of a global project, which include the settlement of a universal information system in taxonomy and the digitisation of the collections in national museums, both calling for a more massive investment in taxonomy as a research priority by the nations (De-Carvalho et al. 2005, Agnarsson & Kuntner 2007, Godfray 2007). Another 1 Treubia 43: 1–16, December 2016 challenge is caused by the lack of consensus on the morphological characters to be used by the community of taxonomists, a limit that was to be overcome by the use of DNA sequences due to the universality of the genetic code (Tautz et al. 2002, Blaxter 2003, Tautz et al. 2003, Hebert & Gregory 2005, Godfray 2006 & 2007). Moreover, the ease of access to sequencing facilities was expected by a large community to counterbalance the impact of the taxonomic impediment in conservation and basic biodiversity sciences (Vernooy et al. 2010). Hebert and colleagues (2003) proposed to develop a new paradigm for species identification based on universal molecular markers (i.e. DNA barcoding) with enriched metadata to allow the sustainability and reproducibility of species identification based on DNA sequences. This approach opened new perspectives in taxonomy and conservation by enabling the development of automated molecular identifications that impacted fields as diversified as functional ecology (Smith et al. 2007), taxonomy (Hebert & Gregory 2005, Miller 2007, Smith et al. 2008), biogeography (Fouquet et al. 2007, Kerr et al. 2007, Hubert et al. 2012), conservation (Forest et al. 2007), wildlife forensics (Armstrong & Ball 2005, Wong & Hanner 2008, Holmes et al. 2009, Ardura et al. 2010, Floyd et al. 2010) and biodiversity socio-economics (Stribling 2006, Vernooy et al. 2010). In front of the massive extinction rates at play in nature nowadays, identifying species is an important application of taxonomy and DNA-based taxonomy opened new perspectives (Ubaidillah & Sutrisno 2009, Sutrisno et al. 2013). The objectives of the present review are: (1) to present how DNA barcoding has emerged as a new paradigm for species identification, (2) to discuss how DNA barcoding complement taxonomy, (3) to discuss the potential benefits of using DNA barcoding for the inventory and conservation of Southeast Asia freshwater fishes. Why DNA barcoding emerged as a new perspective in taxonomy? Godfray (2002) stated that using morphological characters for species delineation do not reveal all the diversity of the world biodiversity because it is time-consuming, while funds and specialist taxonomists are few nowadays. Thus, screening phenotypes is often of limited use and molecular methods such as DNA barcoding may open new perspectives (Blaxter 2003). Quoting Mallet and Willmott (2003:59): “Biodiversity is in crisis, and taxonomy is now in vogue again…”. Then, quoting Shimura (2010) in Vernooy et al. (2010:1): “…The science of taxonomy is key to understanding and monitoring biodiversity…”. The last two quotes highlights that conservation biology is a discipline tightly related to the timeframe of taxonomic studies based on morphology (Wilson 2000, Fisher & Smith 2008 in Smith et al. 2008) and both taxonomist and ecologist are responsible of the identification of priority species for conservation plans (Smith et al. 2008). Radulovici et al. (2010) stated that species identification could be conducted quickly, accurately, and at low cost through molecular analysis. 2 Treubia 43: 1–16, December 2016 Dahruddin et al.: DNA barcoding: foundations and applications for Southeast Asian freshwater fishes challenge is caused by the lack of consensus on the morphological characters to be used by the The characterisation and documentation of biodiversity using phenotypes is currently community of taxonomists, a limit that was to be overcome by the use of DNA sequences due to bridled by several limits inherent to morphological characters. First, the morphological variation of the universality of the genetic code (Tautz et al. 2002, Blaxter 2003, Tautz et al. 2003, Hebert & a species often overlaps that of its sister taxa in nature (i.e. morphological characters are similar for Gregory 2005, Godfray 2006 & 2007). Moreover, the ease of access to sequencing facilities was some individuals belonging to different species), which can lead to incorrect identifications or expected by a large community to counterbalance the impact of the taxonomic impediment in species delineations if based on morphological characters only (Pfenninger et al. 2006). Second, the conservation and basic biodiversity sciences (Vernooy et al. 2010). diagnostic morphological characters used for species identification are often defined on adults and Hebert and colleagues (2003) proposed to develop a new paradigm for species identification may be of limited used for the identification of some ontogenetic stages (e.g. larval stages) or based on universal molecular markers (i.e. DNA barcoding) with enriched metadata to allow the particular samples (e.g. fish fillet). By contrast, DNA-based identifications may be applied sustainability and reproducibility of species identification based on DNA sequences. This approach whatever the life stages under scrutiny or available biological materials for identification (Caterino opened new perspectives in taxonomy and conservation by enabling the development of automated & Tishechkin 2006, Pegg et al. 2006). Third, new species have been frequently detected using molecular identifications that impacted fields as diversified as functional ecology (Smith et al. DNA-based methods, sometimes in the absence of diagnostic morphological characters to further 2007), taxonomy (Hebert & Gregory 2005, Miller 2007, Smith et al. 2008), biogeography (Fouquet discriminate them (i.e. cryptic species) (Hebert et al. 2004a, Witt et al. 2006, Smith et al. 2007). et al. 2007, Kerr et al. 2007, Hubert et al. 2012), conservation (Forest et al. 2007), wildlife Tautz et al. (2002) proposed that DNA might offer new perspectives in taxonomy (Hebert et forensics (Armstrong & Ball 2005, Wong & Hanner 2008, Holmes et al. 2009, Ardura et al. 2010, al. 2003, Hebert et al. 2004b, Hebert & Gregory 2005). DNA barcoding is a fast, easy, relatively Floyd et al. 2010) and biodiversity socio-economics (Stribling 2006, Vernooy et al. 2010). inexpensive approach that provides alternative solutions for species difficult to identify because of In front of the massive extinction rates at play in nature nowadays, identifying species is an their morphological similarity (i.e. cryptic species). Cryptic species are actually separated by important application of taxonomy and DNA-based taxonomy opened new perspectives (Ubaidillah reproductive isolation or alternative geographic distribution but they lack morphological differences & Sutrisno 2009, Sutrisno et al. 2013). The objectives of the present review are: (1) to present how or exhibit conflicting individual assignment to the species level if based on morphological DNA barcoding has emerged as a new paradigm for species identification, (2) to discuss how DNA characters. Thus, DNA barcoding may help clarify the biological status of morphological barcoding complement taxonomy, (3) to discuss the potential benefits of using DNA barcoding for variations within and among closely related species, and guide the detection of new diagnostic the inventory and conservation of Southeast Asia freshwater fishes. morphological characters. Why DNA barcoding emerged as a new perspective in taxonomy? DNA barcoding and taxonomy: how they complement each other Godfray (2002) stated that using morphological characters for species delineation do not Species concept and the need for integrative taxonomy reveal all the diversity of the world biodiversity because it is time-consuming, while funds and The main purpose of taxonomy is to delineate species, to explore their boundaries and to specialist taxonomists are few nowadays. Thus, screening phenotypes is often of limited use and develop the knowledge to further assign specimens to nominal species (Mallet & Willmott 2003, molecular methods such as DNA barcoding may open new perspectives (Blaxter 2003). Quoting Seberg et al. 2003, Godfray 2007). Identifying and delineating species is a very important activity Mallet and Willmott (2003:59): “Biodiversity is in crisis, and taxonomy is now in vogue again…”. that has many applications as, for instance, the control of human pathogens, or identifying suitable Then, quoting Shimura (2010) in Vernooy et al. (2010:1): “…The science of taxonomy is key to biological control agent for agricultural pests (Godfray & Knapp 2004, Agnarsson & Kuntner 2007, understanding and monitoring biodiversity…”. The last two quotes highlights that conservation Godfray 2007). Therefore, “Ideally, identification should be easy and efficient because different biology is a discipline tightly related to the timeframe of taxonomic studies based on morphology users, such as pharmacologists, physiologists, conservation biologists and ecologists, need to (Wilson 2000, Fisher & Smith 2008 in Smith et al. 2008) and both taxonomist and ecologist are identify species…. (Dayrat 2005:408)”. responsible of the identification of priority species for conservation plans (Smith et al. 2008). From an historical perspective, several operational species concept have been proposed and Radulovici et al. (2010) stated that species identification could be conducted quickly, accurately, applied: and at low cost through molecular analysis. 2 3 Treubia 43: 1–16, December 2016 1. Morphospecies concept (MSC) According to this concept, the delineation of species is based on the morphological discrimination of specimens that lead to the recognition of morphospecies (Cain 1954 in Dayrat 2005). The variability of morphological characters likely affects this concept, but many species have been described based on this concept (Dayrat 2005). 2. Biological species concept (BSC) Biological species includes interbreeding individuals that produce fertile offsprings. Reproductive isolation can be either based on: (i) isolation, that is the intrinsic reproductive isolation - the absence of interbreeding between heterospecific organisms based on intrinsic properties -, as opposed to extrinsic [geographic] barriers; or (ii) recognition, that is the shared specific mate recognition or the fertilisation system mechanisms by which conspecific organisms, or their gametes, recognise one another for mating and fertilisation (De-Queiroz 2007). 3. Phylogenetic species concept (PSC) PSC is based on a phylogenetic property of species that is the monophyly (all individuals are derived from a common ancestor that shared derived character states). When considering genetic characteristics, these refer to all alleles of a given gene that are descended from a common ancestral allele, yet not being shared with those of other species (De-Queiroz 2007). Integrative taxonomy or integrated concepts required unifying the properties of each concept for species delineation in order to cope with the diversity of speciation mechanisms and species properties in nature (De-Queiroz 2007). It is now acknowledged that the criteria for species recognition derived from the species concepts are not fundamental properties of the species but clues to be invoked for justifying hypotheses of species delineation (De-Queiroz 2007). Considering the diversity of the mechanisms leading to the emergence of new species, species can exhibit multiple combinations of the criteria defined by the MSC, BSC and PSC. In this context, combining evidences from different sources of biological characters may be expected to provide hypotheses of species delineation that are more robust than those based on a single source of evidence (Pante et al. 2015). This statement calls for an integrated assessment of independent evidence based on genomes, phenotypes and ecology (e.g. Smith et al. 2008). It has been recently proposed that the integration of DNA barcoding as a preliminary step during biodiversity inventories may help speed up the pace of species discovery by avoiding the time consuming sorting of specimens based on their morphological attributes. This approach, recently named as ‘turbo-taxonomy’, proved to open new perspective in taxonomy by streamlining the description of large number of species through the combination of DNA barcodes, concise morphological descriptions and high-resolution digital images (Butcher et al. 2012, Riedel et al. 2013). 4 Treubia 43: 1–16, December 2016 Dahruddin et al.: DNA barcoding: foundations and applications for Southeast Asian freshwater fishes Concepts behind DNA barcoding (repeatability and accessibility of the data) 1. Morphospecies concept (MSC) DNA barcoding is a system designed to provide accurate, fast, and automatable species According to this concept, the delineation of species is based on the morphological identification by using short and standardised gene regions as internal species tag (Hebert & discrimination of specimens that lead to the recognition of morphospecies (Cain 1954 in Dayrat Gregory 2005). DNA barcoding is an accessible method for anyone who wants to use molecular 2005). The variability of morphological characters likely affects this concept, but many species data for species identification either in basic or applied research related to health or medical have been described based on this concept (Dayrat 2005). purposes and even food security (Hebert & Gregory 2005). DNA barcodes data are easily accessible 2. Biological species concept (BSC) through BOLD (Ratnasingham & Hebert 2007), even for researcher focusing on zoogeography or Biological species includes interbreeding individuals that produce fertile offsprings. phylogenetic reconstructions. Reproductive isolation can be either based on: (i) isolation, that is the intrinsic reproductive DNA barcode records are expected to follows the standards in BOLD (Fig. 1), as established isolation - the absence of interbreeding between heterospecific organisms based on intrinsic by Ratnasingham and Hebert (2007) and Hubert et al. (2008), namely: properties -, as opposed to extrinsic [geographic] barriers; or (ii) recognition, that is the shared 1. Name of species specific mate recognition or the fertilisation system mechanisms by which conspecific organisms, 2. Voucher data (catalog number and repository institution) or their gametes, recognise one another for mating and fertilisation (De-Queiroz 2007). 3. Collection data (collector, collection date and location with GPS coordinates) 4. Identifier (people who identified of specimen) 3. Phylogenetic species concept (PSC) 5. The order of sequence COI at least 500 bp PSC is based on a phylogenetic property of species that is the monophyly (all individuals are 6. PCR primers used to generate amplicons 7. Traces file (raw electropherograms from sequencing analyses) derived from a common ancestor that shared derived character states). When considering genetic The data elements of a record in BOLD are separated into: (i) a specimen page that includes characteristics, these refer to all alleles of a given gene that are descended from a common ancestral the information about the voucher specimen such as identifier, taxonomy, GPS data and allele, yet not being shared with those of other species (De-Queiroz 2007). photograph, catalog repository and museum catalog number, (ii) a sequence page that includes the Integrative taxonomy or integrated concepts required unifying the properties of each concept DNA barcode sequence, PCR primers and trace files (Hubert et al. 2008). for species delineation in order to cope with the diversity of speciation mechanisms and species Quoting Smith et al. (2008:12364) “…the barcoded specimens are vouchered in permanent properties in nature (De-Queiroz 2007). It is now acknowledged that the criteria for species collections for repeated iterative study and are linked through publicly accessible databases of host recognition derived from the species concepts are not fundamental properties of the species but records and associated metadata…”. Then, quoting Hebert and Gregory (2005:852) “…DNA clues to be invoked for justifying hypotheses of species delineation (De-Queiroz 2007). Considering barcoding allows a day to be envisioned when every curious mind, from professional biologists to the diversity of the mechanisms leading to the emergence of new species, species can exhibit schoolchildren, will have easy access to the names and biological attributes of any species on the multiple combinations of the criteria defined by the MSC, BSC and PSC. In this context, combining planet”. These two quotes highlight the repeatability and accessibility of DNA barcodes data in evidences from different sources of biological characters may be expected to provide hypotheses of BOLD for universal applications involving species identification. species delineation that are more robust than those based on a single source of evidence (Pante et al. 2015). This statement calls for an integrated assessment of independent evidence based on genomes, Benefits of DNA barcoding based on a mitochondrial gene phenotypes and ecology (e.g. Smith et al. 2008). It has been recently proposed that the integration DNA barcoding rely on the COI mitochondrial gene that presents several advantages: (1) the of DNA barcoding as a preliminary step during biodiversity inventories may help speed up the pace mitochondrial genome is present in a large number of copies yielding substantial amounts of of species discovery by avoiding the time consuming sorting of specimens based on their genomic DNA from a variety of extraction methods (Gemeinholzer et al. 2010, Weigt et al. 2012); morphological attributes. This approach, recently named as ‘turbo-taxonomy’, proved to open new (2) the high mutation rate and small effective population size make it often an informative genome perspective in taxonomy by streamlining the description of large number of species through the about evolutionary patterns and processes; (3) multiple primers and new amplification techniques combination of DNA barcodes, concise morphological descriptions and high-resolution digital based on primers cocktail have been developed for the ease of amplification across metazoan images (Butcher et al. 2012, Riedel et al. 2013). lineages (e.g. Ivanova et al. 2007). 4 5 Treubia 43: 1–16, December 2016 h p a gr o ot h p n e m ci e p s a, at d n o cti e oll c y, r o sit o p e r - o bi a n e i pl m a s e u s s n, ti e m ci e p s er h c u o v ut o b a s ail et d g n D includice files). La BOg. tr n e. d ia ( orat cd n reary em of a specimcluding pri ure e in ctod uc Strbar 1. A e N rD gud Fian 6 Treubia 43: 1–16, December 2016 Dahruddin et al.: DNA barcoding: foundations and applications for Southeast Asian freshwater fishes So far, results of DNA barcoding are mostly above 90% of accuracy for species identification that is, DNA barcodes have been estimated to match morphospecies in 90% of the h species analysed, the 10% of failure resulting from the retention of ancestral polymorphism or p a gr o introgressive hybridisation (Hubert et al. 2008, April et al. 2011, Pereira et al. 2013). This result ot h p n highlights that DNA barcoding is a powerful tool for species identification (Pereira et al. 2013). For e m eci some vertebrate taxa, alternative mitochondrial markers have been frequently used in the past, such p s ata, as 12S rRNA and 16S, thus successfully enabling species delineation in several groups such as d n o amphibians (Matsui et al. 2012, Nishikawa et al. 2012). Although, quoting Smith et al. (2008:238) cti e oll “In our preliminary sampling of Holarctic amphibians — we found that a single mitochondrial gene c y, or DNA barcode correctly identified 94% of species. Amplicons of the 5(cid:397) CO1 region are sit o p straightforward to generate using standard primers designed for insects and vertebrates, we e r - o bi experienced no more difficulty with amphibian samples than with insects (Hebert et al. 2004a, a n e i Smith et al. 2005), fishes (Ward et al. 2005), birds (Hebert et al. 2004b), or bats (Clare et al. pl m a 2007)”. This result highlights that DNA barcoding may be successfully applied for the whole s e u ss vertebrate fauna. n, ti me For a barcoding approach to species identification to succeed, however, within-species DNA ci e p sequences need to be more similar to one another than to sequences in different species. Several s er h c processes, such as pseudogenes ontogenesis, introgressive hybridisation, and retention of ancestral u o v ut polymorphism pose potential difficulties in capturing species boundaries using mtDNA sequences o b a s (Funk & Omland 2003, Pamilo & Nei 1988, Zhang & Hewitt 1996). The detection of mixed ail et d genealogy between closely related species has been previously estimated to occur in nearly 20 g n D includice files). ptheirsc epnetr coefn tthaeg ec acsaens ivna trhye wwiidldel y(F uamnko n&g Opmhylalan,d y2e0t 0s3p)e. cRieesc eansts biganrcmoednint gf asitluudreiess teympipchaallsyi seddo tnhaott La BOg. tr exceed 5 to 10 percent in a large array of organisms (April et al. 2011, Hubert et al. 2012, Kerr et n e. ord iata ( al. 2007). Nevertheless, distinguishing between introgressive hybridisation and the retention of cd n reary ancestral polymorphism call for an integrative assessment of independent sources of evidence em of a specimcluding pri i(nFculnukd i&ng O nmuclalenadr 2D0N03A) . and phenotypes due to the maternal inherence of the mitochondrial genome ure e in uctcod Applications of DNA barcoding in fish biology and conservation in Southeast Asia Strbar 1. A Taxonomy and species delineation: cryptic diversity e N rD gud During the last years, several DNA-based studies highlighted the limits of morphological Fian characters to accurately delineate and uncovered new species as a substantial amount of cryptic diversity has been frequently described and fishes are no exception. Many examples have been described in Indo-Pacific coral reef fishes (Hubert et al. 2012), Indo-Malay Carangidae (Teleosteii: Perciformes) (Jaafar et al. 2012), flathead fishes (Scorpaeniformes: Platycephalidae) (Puckridge 6 7 Treubia 43: 1–16, December 2016 et al. 2013) and gobies from the genus Trimma (Percomorpha, Gobiiformes) (Winterbottom et al. 2014). These studies have enlarged the growing body of evidence suggesting that cryptic diversity may be a much common trend than previously considered as cases of cryptic diversity have been detected in neotropical butterflies (Hebert et al. 2004a), ants of Madagascar (Smith et al. 2005), parasitoid flies and wasps from Central America (Smith et al. 2007, Smith et al. 2008). Worth mentioning, all these studies were based on the use of DNA barcoding as a first step during biodiversity inventories. Identification of early life stages The most prominent benefit of DNA barcoding for species identification lies in the ability to identify early stages that cannot be done by using morphological characters. DNA barcoding proved to be effective for identifying species in juvenile and larvae of Lutjanus cyanopterus in Caribbean beach (Victor et al. 2009), and mantis shrimp larvae from coral reefs in Kimbe Bay, Western Pacific, Papua New Guinea and Red Sea (Barber & Boyce 2006). Along the same line, Hubert and colleagues (2010) collected 46 larvae of Pacific coral reef fishes from the families Holocentridae and Acanturidae, 100% of which were identified to the species level through DNA barcoding (Hubert et al. 2010). Similarly, Ko and colleagues (2013) estimated, based on DNA barcoding, that less than 30% of marine fish larvae were accurately identified to the species level based on morphological characters (Ko et al. 2013). Later, Hubert and colleagues (2015) evidenced that on 1379 coral reef fish larvae sampled in the Pacific, 1264 samples were successfully amplified (92 %) and nearly 90 % can be identified to the species level through DNA barcodes. Market substitution and the ornamental fish trade Expensive fishes for consumption like Tuna are much appreciated worldwide. Substitutions with inexpensive fishes (less flavour, low in nutrients, readily available, low price) are tempting and Tuna fishes are no exception. After testing the identity of samples of “White Tuna Sushi” through DNA barcoding in North American market, Wong & Hanner (2008) detected that the sold fillet were derived from the fish Oreochromis mossambicus instead of Thunnus alalunga or “White tuna or Albacore tuna”. For safety and economic reasons, the certification of appropriate labeling for fisheries products is required. Recent studies highlighted that DNA barcoding may be efficiently used for the regulation of the fisheries market and detection of market substitution (Ardura et al. 2010, Haye et al. 2012, Maralit et al. 2013, Cutarelli 2014). Alternatively, ornamental fishes are much appreciated by the public as pet animals due to less space required space than the other domesticated animals; an aquarium of only 30 cm size can be readily used to enjoy ornamental fishes. Many tropical ornamental fishes display beautiful colours and are popular (Veiga et al. 2014), thus provide important sources of incomes. The international ornamental fish market has drastically increased during the recent years. This pressure 8 Treubia 43: 1–16, December 2016 Dahruddin et al.: DNA barcoding: foundations and applications for Southeast Asian freshwater fishes et al. 2013) and gobies from the genus Trimma (Percomorpha, Gobiiformes) (Winterbottom et al. urges for the regulation of this market to protect native species and to promote more sustainable 2014). These studies have enlarged the growing body of evidence suggesting that cryptic diversity practices. Many ornamental fishes fall in the category of endemic and threatened in the IUCN Red may be a much common trend than previously considered as cases of cryptic diversity have been List. Conservation of ornamental fishes is a concern in order to avoid their extinction in natural detected in neotropical butterflies (Hebert et al. 2004a), ants of Madagascar (Smith et al. 2005), habitat (Raghavan et al. 2013). One important element of the regulation is the effective parasitoid flies and wasps from Central America (Smith et al. 2007, Smith et al. 2008). Worth identification of the species. For this purpose, DNA barcoding has proven to meet the requirement mentioning, all these studies were based on the use of DNA barcoding as a first step during as shown in the following studies that successfully identify species each in a promising success biodiversity inventories. percentage: 98% of the 391 species of Indo-Pacific coral reef fishes analyzed by Steinke et al. (2009); 90–99% of the 172 cyprinid fish species examined by Collins et al. (2012); and 60% (6 Identification of early life stages species) of the 10 species of Hyphessobrycon, which were altogether represented by 158 specimens The most prominent benefit of DNA barcoding for species identification lies in the ability to observed by Paz et al. (2014), were easily distinguishable by DNA barcoding: H. bentosi, H. identify early stages that cannot be done by using morphological characters. DNA barcoding proved copelandi, H. eques, H. epicharis, H. pulchripinnis, and H. sweglesi. to be effective for identifying species in juvenile and larvae of Lutjanus cyanopterus in Caribbean DNA barcoding enables to identify species for the purpose of certifying the labeling of beach (Victor et al. 2009), and mantis shrimp larvae from coral reefs in Kimbe Bay, Western consumed fisheries products for consumption as well as verifying the species identification in the Pacific, Papua New Guinea and Red Sea (Barber & Boyce 2006). Along the same line, Hubert and export-import business of the international ornamental fish trade. Exporting countries can increase colleagues (2010) collected 46 larvae of Pacific coral reef fishes from the families Holocentridae their incomes by improving the competitiveness of their fisheries products with the certification and and Acanturidae, 100% of which were identified to the species level through DNA barcoding the accurate labeling. Importing countries can significantly reduce the loss due to misidentification (Hubert et al. 2010). Similarly, Ko and colleagues (2013) estimated, based on DNA barcoding, that simply by using DNA barcoding (Steinke et al. 2009, Collins et al. 2012, Raghavan et al. 2013). less than 30% of marine fish larvae were accurately identified to the species level based on Parties associated with the ornamental fish trade regulations are collectors, wholesalers and morphological characters (Ko et al. 2013). Later, Hubert and colleagues (2015) evidenced that on retailers, as well as regulatory control agencies, all of which will undoubtedly benefit from the 1379 coral reef fish larvae sampled in the Pacific, 1264 samples were successfully amplified (92 %) identification services available from a comprehensive DNA barcoding framework (Steinke et al. and nearly 90 % can be identified to the species level through DNA barcodes. 2009). Market substitution and the ornamental fish trade Expensive fishes for consumption like Tuna are much appreciated worldwide. Substitutions Perspectives with inexpensive fishes (less flavour, low in nutrients, readily available, low price) are tempting and Biodiversity hotspots and the taxonomic impediment: the example of fishes Tuna fishes are no exception. After testing the identity of samples of “White Tuna Sushi” through Biodiversity is not evenly distributed in the world, some parts of which have a higher DNA barcoding in North American market, Wong & Hanner (2008) detected that the sold fillet number of endemic species that are impaired primarily by human activities. Such areas are were derived from the fish Oreochromis mossambicus instead of Thunnus alalunga or “White tuna classified as ”biodiversity hotspots” (Myers et al. 2000) and constitute absolute priorities for or Albacore tuna”. For safety and economic reasons, the certification of appropriate labeling for conservation purposes (Sechrest et al. 2002). To date, 25 hotspots are recognised worldwide based fisheries products is required. Recent studies highlighted that DNA barcoding may be efficiently on the number of endemic species in plants and four groups of vertebrates including mammals, used for the regulation of the fisheries market and detection of market substitution (Ardura et al. birds, reptiles and ampibia (Fig. 2). Among those 25 hotspots, two are present in Indonesia, namely 2010, Haye et al. 2012, Maralit et al. 2013, Cutarelli 2014). Sundaland and Wallacea. In Sundaland, a total of 701 species of endemic vertebrates live in Alternatively, ornamental fishes are much appreciated by the public as pet animals due to protected areas covering 90000 km2, while it adds up to 529 species in 20415 km2 correspondingly less space required space than the other domesticated animals; an aquarium of only 30 cm size can in Wallacea. For the four groups of vertebrate, Sundaland has a high percentage of endemic be readily used to enjoy ornamental fishes. Many tropical ornamental fishes display beautiful vertebrates species namely 2.6%, which is higher than Brazil, one of the highest biodiversity colours and are popular (Veiga et al. 2014), thus provide important sources of incomes. The countries in the world with 2.1%. Globally, Sundaland is the third hotspot in terms of endemism, international ornamental fish market has drastically increased during the recent years. This pressure while the most diverse Amazonian hotspots is the fourth (Myers et al. 2000). The exponential 8 9 Treubia 43: 1–16, December 2016 growth of human impacts on ecosystems has called considerable attention on the role of biodiversity in maintaining ecosystem services on which a growing human population depends. Ecosystems provide a wide variety of services including food resources (e.g. fisheries) and incomes (e.g. ecotourism) for millions of people, flood control (e.g. forest cover) and waste detoxification (e.g. nitrogen cycle). Recent meta-analyses of threats, however, evidenced that Indonesian hotspots are the world most endangered to date (Orme et al. 2005, Lamoureux et al. 2006, Hoffman et al. 2010). Fishes account for more than 50% of entire vertebrate diversity with 32.900 fish species described worldwide (Nelson 2006, Froese & Pauly 2014). Kottelat & Whitten (1996) proposed biodiversity hotspots based of freshwater fish species endemism in Sundaland and Wallacea (Fig. 3). Kottelat & Whitten (1996) stated that Indonesia has the highest number of freshwater fish species among the Asian countries and second worldwide after Brazil with 1216 species (Froese & Pauly 2014). Endemic freshwater fish species in both Sundaland and Wallacea altogether (i.e. Sumatra, Borneo, Java and Sulawesi) add up to 243 species (Kottelat et al. 1993). Given the high endemic diversity and multiple threats on freshwater fishes, the accumulation of new studies is likely to increase the number of endemic species and provide clues for the definition of new biodiversity hotspots for fishes (e.g. Kadarusman et al. 2012). Figure 2. The 25 world hotspots (Myers 2000). 10