MtDNA barcode identification of fish larvae in the southern Great Barrier Reef , Australia

Australia’s Great Barrier Reef (GBR) is recognised internationally for its importance as a tropical marine fish and coral reef biodiversity reserve, with up to 30 percent of the total reef area now protected as ‘Green Zones’. In 2004, new fisheries rules in the State of Queensland were introduced to prohibit the taking of all regulated reef fish for nine-day closure periods around the new moon in the spring/early summer months (October-December) as a further measure to improve the chances of successful spawning of Lutjanus, Lethrinus , Plectropomus and other reef fish species important to the commercial and recreational fishery. The current study was undertaken at an inshore shallow reef in the southern GBR (approximate latitude of 23 degrees south of the equator), where surface water temperature ranges from 18 degrees in winter to 28 degrees in summer. Larvae were capSCIENTIA MARINA 70S2 October 2006, 7-12, Barcelona (Spain) ISSN: 0214-8358


INTRODUCTION
Australia's Great Barrier Reef (GBR) is recognised internationally for its importance as a tropical marine fish and coral reef biodiversity reserve, with up to 30 percent of the total reef area now protected as 'Green Zones'.In 2004, new fisheries rules in the State of Queensland were introduced to prohibit the taking of all regulated reef fish for nine-day closure periods around the new moon in the spring/early summer months (October-December) as a further measure to improve the chances of successful spawning of Lutjanus, Lethrinus , Plectropomus and other reef fish species important to the commercial and recreational fishery.
RECENT ADVANCES IN THE STUDY OF FISH EGGS AND LARVAE M.P. Olivar and J.J. Govoni (eds.)tured around the new moon periods in spring 2004 by light traps and by plankton nets.Because identification of freshly hatched fish larvae to species level is often difficult due to poorly defined morphological characteristics (Leis and Carson-Ewart, 2004), we were interested in investigating DNA analysis methods that would enable unequivocal identification of larval fish or planktonic eggs.Two distinct approaches were to be explored, both of which involved mitochondrial (mtDNA) sequencing.Our group had been using mtDNA HVR1(Dloop) non-coding sequence to study within-species population diversity for adult reef fish (Aspden et al., 2005) and in this study we sought to compare the suitability of HVR1 sequence data compared with Cytochrome Oxidase subunit 1 (cox1) sequence data for species identification of larvae.The expectation was that lesser within-species variation would be expected for the functional cox1 gene sequences (approximately 650 bp.), whereas more genetic diversity might be expected for the non-coding HVR1 gene.The hypothesis to be tested in the study was whether cox1 sequence data were sufficiently divergent between closely related species to enable unambiguous identification, or whether HVR1 sequence data gave too much variation to be useful as a specific species identifier.We also wished to test a number of the cox1 primer sets for fish reported in the literature to determine which primers gave the best results for reef fish.The study was timely, since Hebert et al. (2003) had initiated an international Barcode of Life project proposing that the cox1 gene sequence could be used to differentiate between most animal species, including fishes.Subsequent to initiating this study, examples of the application of the barcoding concept to adult and larval fish identification have been reported by Ward et al. (2005) and Steinke et al. (2005).

Larvae capture
Larvae were captured using a combination of light traps and plankton net devices.The light traps (900x300x300 mm) were constructed from translucent plastic boxes held together by cable ties.A waterproof pond light (50 W) was fixed into the base of the trap along with a 500 litre per hour electric bilge pump (Rule Corp, USA), thereby facilitat-ing passage of larvae (and eggs) through a grid into the lower section of the trap.The light and pump were powered by a 12 V battery and the trap was suspended vertically 1 metre below the surface beside the research vessel for 2-hour periods.At other times, small zooplankton nets (50 cm diameter), sometimes incorporating a 10 W light, were let out into the 4 km/h tidal current to 0-1 m depth.A dual bongo plankton net of 70 cm diameter (Ocean Instruments, USA) was towed obliquely at a constant speed of 2 knots from a depth of 15 metres to the surface over 10 minutes.Captured larvae and eggs were stored in 80% ethanol / distilled water at 0°C in the field and later sorted with the aid of microscopy.Captures of individual fish larvae types ranged from a single specimen to many hundreds.Typically, groups (n=5 to 20 or more) of the same species were captured within any collection interval within the sampling period.

Adult fish capture
Representative juvenile and adult fish of known species for this study were caught by line fishing as approved by Queensland Fisheries Regulations.Small pectoral fin clip samples were stored initially in 20% DMSO / saturated brine solution at room temperature, and later washed and transferred into 80% ethanol solution and refrigerated.The field work was sanctioned by Marine Parks Permit G04/12132.1 and CQU Animal Ethics Permit A04/06-160.
The cox1 and HVR1 fragments were amplified as follows.Each PCR reaction mix totalled 25 µl and included 100 µM dNTP, 7.5 pmol of each primer, 1 mM MgCl 2 , 2.5 µl 10X Buffer (Promega), 1.0 unit Taq polymerase (Promega), 17.8 µl sterile MQ water and 1 µl of DNA template (ca. 25 ng).Either a Perkin Elmer GeneAmp PCR System 2400, a BIO-RAD iCycler, or a BIO-RAD Mycylcer thermal cycler was used.Standard PCR conditions varied slightly for the 3 sets of cox1 primers tested in this study.For the tautog and cichlid primers the thermal cycle regime consisted of: 5 min at 95°C for 1 cycle; 1 min at 95°C, 1 min at 47°C, and 1 min at 72°C for 35 cycles; and 5 min at 72°C for 1 cycle.Samples were then held at 4°C until retrieved.The thermal cycle for the Fish-1F and R1 primers consisted of: 2 min at 95°C for 1 cycle; 30 s at 94°C, 30 s at 54°C, and 1 min at 72°C for 35 cycles; and 10 min at 72°C.Samples were then held at 4 o C on the thermocycler until collection.For HVR1 amplification, Touchdown PCR was used.The protocol involved an initial 2 min at 94°C followed by 5 cycles of 30 s at 94°C, 30 s at 45°C and 2 min at 72°C, then 5 cycles with annealing temperature reduced to 43°C, then 25 cycles with annealing temperature reduced to 41°C, followed finally by 10 min at 72°C.
The amplified products and size standards were run on a 1% agarose gel stained with Sybr Green (Astral).The gels were visualised and photographed using a BIO-RAD Gel Documentation Camera.Bands (ca.400 bp for HVR1 and ca.650 bp. for cox1) were excised from the gels and placed into separate Eppendorf tubes.The Promega Wizard SV PCR and Gel Cleanup System was used to extract the DNA from the gel.The amplified products were then sequenced using the BigDye ® Terminator v3.1 Cycle sequencing kit (Applied Biosystems, Inc) following the thermocycler protocol listed by Applied Biosystems and sequenced both in the forward and reverse directions using an ABI Prism 310 genetic analyser or an AB 3130 Genetic Analyser.

Data analysis
For the major Lethrinus and Lutjanus species, 20-30 individual fish samples were sequenced.For larvae that were single or few samples, the sequenc-ing was done in both the forward and reverse directions and the sequences were then checked manually to see that the sequence information was entirely consistent from both directions.Any doubtful base calls were checked manually.If necessary, the sequencing run or the entire DNA extraction/amplification/sequencing reaction was repeated to resolve any doubtful data.

Within species genetic variation of HVR1
Figure 1 shows typical 1-3% within-species variation for mtDNA HVR1 sequences (approximately 420 bp) for reef snapper species Lethrinus miniatus and Lethrinus laticaudis, and for red emperor (Lutjanus sebae) for groups of fish from the same reef location (n=15-25).

Between species genetic variation of HVR1
A matrix of the between species mtDNA HVR1 genetic distances for commercially important reef snapper species L. laticaudis, L. miniatus and L. sebae is shown in Table 1.The data shows between-species HVR1 sequence differences ranging from 17 to 50% among these three species, demonstrating the potential utility of HVR1 sequences for species specific markers.

Primer selectivity for marine fish cox1 sequences
All three primer sets tested for cox1 amplification yielded amplicons having sizes of approximately 650 bp and for which BLAST analysis using GenBank gave partial matches for fish species cytochrome oxidase, or portions of total mitochondrial DNA gene sequences.However, the FishF1 and R1 primer set for cox1 (Ward et al., 2005) consistently gave amplicons which yielded longer length and cleaner sequence data than with either of the other two primer sets tested.The amplicons generated from the same DNA sample for Lutjanus sebae with FishF1/R1 and tautog-BS1 (F/R) showed only 50% sequence similarity when analysed using Chromas v1.45 and BioEdit v7.0.4.1.As discussed by Ward et al. (2005), researchers need to be mindful of pseudo-gene amplification and the tautog primers used might have initiated amplification of shorter nuclear DNA sequences originating from mitochondrial DNA (NUMTs), as discussed by Zhang and Hewitt (1996) and Richly and Lester (2004), or they might have amplified other mitochondrial DNA besides cox1.

Species discrimination using cox1 sequence comparisons
Nearest neighbour analyses of the cox1 sequences generated in this study were conducted with the bar-code of life fish ID database for Australian fishes (available at www.barcodinglife.org),yielding either identification for fish species cox1 sequences already in the database, or logical closest related species in all cases.This analysis and the recent report by Ward et al. (2005) strongly support the contention that cox1 sequences are highly useful for identification of marine fish species.

DISCUSSION
The question of fish and fish egg identification using DNA analysis methods is being pursued by many laboratories around the world.The use of modern molecular genetics techniques combined with taxonomic expertise provides a very powerful approach to solving existing taxonomic dilemmas, and allows new insights into the relatedness and evolution of fish species.
In a recent study (Aspden et al., 2005), our group utilised HVR1 sequences to study the population differences for red throat emperor Lethrinus miniatus across its distribution along the East and West Australian coasts.In the present study we compared the within species HVR1 sequence variation for two other commercial reef fish species (Lethrinus laticaudis and Lutjanus sebae) compared with L. miniatus (Fig. 1).With only 1-3% variation within species, but up to 50% difference between species (Table 1), it therefore seemed feasible to explore using HVR1 sequences to develop a putative taxonomic identification tree for reef fish species.An example partial tree construct using neighbour joining analysis for fish HVR1 sequences is shown in Figure 2. Fish from the same genus (see the Lethrinid and Lutjanid examples shown) all align closely, suggesting that though HVR1 is a non-coding genetic sequence, this gene marker is remarkably useful for distinguishing between closely related species.Some of the larvae accessions from the 2004 collection for this study are also shown in Figure 2   heads Atherinomorus (L14) are also readily identified by HVR1 sequence comparisons.While encouraged by these preliminary data proving the usefulness of HVR1 sequence comparisons for species identification, the international Barcode of Life project initiated by Hebert et al. (2003) suggested that another mitochondrial gene sequence, namely cytochrome oxidase subunit 1 (cox1), be used as the gene marker of choice for species discrimination.As it is a structural gene encoding a functional respiratory chain enzyme, it might be expected that cox1 sequence data would show fewer between-species differences, and this might compromise discrimination between closely related species.For this study, the efficacy of three different published primer pairs for amplification of cox1 in fish tissue samples was compared, namely FishF1, FishR1 (Ward et al., 2005); cichlidBS1(F), cichlidBS2(R) (Sparks, 2003); and tautog-BS1(F), tautog-BS2(R) (Orbacz and Gaffney, 2000).While all of the primer sets studied yielded PCR amplicons of approximately 650 bp from fish tissue DNA extracts with varying success, the FishF1, FishR1 recently reported by Ward et al. (2005) for DNA barcoding of Australian fishes gave the most consistent results across a range of species tested and are therefore recommended to other workers (the authors thank Dr Ward for a pre-publication copy of his paper which assisted this study).Future workers in this field are advised to consider some of the cox1 DNA sequence data available in the public domain with caution, since some of the reported sequences may in fact be of genomic origin (Ward et al., 2005;Zhang and Hewitt 1996;Richly and Lester 2004), or of other mitochondrial origin.For all fish species tested in this study (including all commercially important Plectropomus, Epinephelus, Lethrinis, and Lutjanus examples), the cox1 sequences generated using the FishF1, FishR1 primer set gave reproducible sequences for each species that allowed ready species discrimination.

CONCLUSIONS
We conclude that the DNA Barcode approach for fish identification appears valid and that while HVR1 or cox1 mtDNA sequence data both appear useful for this purpose, cox1 should be used in future studies as the marker of choice since a large international database for fish identification is presently being constructed.The cox1 amplification and sequencing method allows identification of planktonic larval fish and fish eggs through comparison to DNA from authenticated adult fish specimens, and therefore provides a major new advance for fisheries biologists, taxonomists and fisheries regulators.
and are indicated with an L prefix.As examples, L04 and L11 were closely aligned to Engraulis japonicus and are proposed to be the Australian anchovy Engraulis australis.The analysis suggests that L09 is a Lethrinus species, most likely a larval form of the spangled emperor Lethrinus nebulosus, while L03, L08, L13 and L24 are Siganus species.Tropical pelagic species such as hardy SCI.MAR., 70S2, October 2006, 7-12.ISSN: 0214-8358 10 • G.G. PEGG et al.

TABLE 1 .
-Between-species HVR1 mean genetic distances for example reef snapper species calculated using MEGA v3.1 with Tamura-Nei model.