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Article << Previous     |     Next >>   Contents Vol 26(6)

DNA barcoding in Nautilus pompilius (Mollusca : Cephalopoda): evolutionary divergence of an ancient species in modern times

Rachel C. Williams A, Stephen J. Newman B and William Sinclair A C D

A Centre for Wildlife Conservation, Faculty of Science and Natural Resources, University of Cumbria, Penrith, CA11 0AH, United Kingdom.
B Western Australian Fisheries and Marine Research Laboratories, Department of Fisheries, Government of Western Australia, PO Box 20, North Beach, WA 6920, Australia.
C Centre for Environmental Management, CQUniversity, Rockhampton, Qld 4702, Australia.
D Corresponding author. Email: billy.sinclair@cumbria.ac.uk



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Invertebrate Systematics 26(6) 548-560 http://dx.doi.org/10.1071/IS12023
Submitted: 11 April 2012  Accepted: 13 September 2012   Published: 19 December 2012


Abstract

DNA barcoding studies to elucidate the evolutionary and dispersal history of the current populations of Nautilus pompilius allow us to develop a greater understanding of their biology, their movement and the systematic relationships between different groups. Phylogenetic analyses were conducted on Australian N. pompilius, and COI sequences were generated for 98 discrete accessions. Sequences from samples collected across the distribution were sourced from GenBank and included in the analyses. Maximum likelihood revealed three distinct clades for N. pompilius: (1) populations sourced from west Australia, Indonesia and the Philippines; (2) populations collected from east Australia and Papua New Guinea; (3) western Pacific accessions from Vanuatu, American Samoa and Fiji, supporting previous findings on the evolutionary divergence of N. pompilius. A minimum spanning tree revealed 49 discrete haplotypes for the 128 accessions, from a total of 16 discrete sampling locations. Population similarity reflects oceanic topographic features, with divergence between populations across the N. pompilius range mirroring geographical separation. This illustrates the success of DNA barcoding as a tool to identify geographic origin, and looks to the future role of such technology in population genetics and evolutionary biology.

Additional keywords: coxI, conservation, population genetics.



Introduction

The collation of evidence from fossils and molecular data demonstrates that in the early Cambrian Period, the major classes of molluscs diverged (Maloof et al. 2010; Smith and Caron 2010; Kröger et al. 2011). Nautiloids mark the first appearance of cephalopods as a separate molluscan entity over 500 million years ago (Jereb and Roper 2005) and remain the only living representatives of the large extinct group of ammonites, belemnites and nautiloids (Holland 1987; Suzuki et al. 2000; Bonnaud et al. 2004). Wray et al. (1995) postulated that the evolution of the ancestral progenitor population of modern Nautilus pompilius (Linneaus, 1751) has divided into three geographically distinct clades: one consisting of western Australian/Indonesian populations, one from the western Pacific and one from eastern Australia/Papua New Guinea. Recent work using a partial sequence of the cytochrome c oxidase subunit I (COI) gene region corroborated these predictions (Sinclair et al. 2011), identifying separation between the western Australian/Indonesian clade and the eastern Australian/Papua New Guinean clade. Conclusions indicate that this separation occurred due to the influence of geography and dispersal capacity (Hewitt 2004; Santos et al. 2006; Silva et al. 2010). The work of Bonacum et al. (2011) on the western Pacific clade also suggested that the divergence demonstrated within this clade appeared to be driven by geographic isolation. Large expanses of open water inhibit their dispersal; their reported inability to travel below ~800 m without imploding due to increasing water pressure (Saunders and Wehman 1977; Kanie et al. 1980; Wani 2004), and their vulnerability to predators when travelling across large expanses of open water, restricts the movement of Nautilus to the reef slopes (O’dor et al. 1993). As an additional limiting factor of their range, temperatures exceeding 25°C can be lethal to Nautilus within several days (Saunders and Spinosa 1979); however, they are able to adapt to adjusting temperatures and can move from 6 to 24°C water in several hours (Carlson et al. 1984). Limited dispersal distances between reefs emphasises the geographical isolation between populations (O’dor et al. 1993). As such, gene flow is restricted, resulting in documented diversification between populations, the pattern of which appears to be occurring along geographic boundaries (Sinclair et al. 2011).

Dispersal constraints on marine species can strongly influence the genetic structure of a population (Hewitt 2004; Santos et al. 2006; Wieters et al. 2008; Silva et al. 2010). Marine organisms found in small and isolated populations can experience negative fitness effects as a result of limited connectivity and dispersal (Palumbi 1994; Wieters et al. 2008; Nunes et al. 2009). Isolation can also occur as a result of geography; physical barriers restrict movement and gene flow, causing populations to differentiate by genetic drift (Hindar et al. 1991). Currents and topology can exert a strong influence on the biogeography of the ocean fauna (Neumann 1960; Mitchell 1975; Genin et al. 1986; Maravelias 1999) by acting as barriers to dispersal (Thornhill et al. 2008).

Although physical separation can create a degree of diversity within the species, it also results in small isolated populations, with no means of rapid dispersal; the lifecycle of N. pompilius is not consistent with broadcast spawning dispersal patterns demonstrated by many marine organisms. Nautilus lay their eggs as discreet units attached to benthic substrate, and the resulting zygote is one of the largest known invertebrate eggs, taking ~11 months to hatch (Saunders and Landman 2010). The absence of a planktonic stage and a restrictive temperature regime for hatching success do not support a wide dispersal pattern for the eggs. Newly hatched individuals resemble miniature adults and are able to feed independently of any parental care. Early growth involves the formation of chambers behind the living chamber in which the Nautilus resides (Greenfest-Allen et al. 2010). Young are thought to descend from the shallower depths (~100 m) of spawning (Carlson et al. 1992) to deeper water where their life style mirrors that of adults, reaching sexual maturity after 7–8 years (Saunders and Landman 2010). Due to the entire process of maturation occurring on the same reef, dispersal is limited and genetic differences between individuals on different reefs are gradually accentuated.

The COI gene region is now becoming more widely recognised for this role in genetically distinguishing differences. As a DNA barcoding gene (Hebert et al. 2003a, 2003b), COI is applied in an approach designed to differentiate between morphologically similar species (Barrett and Hebert 2005). The utility of DNA barcoding as an identification tool is growing (COI is the gene fragment commonly used to demonstrate this variation in animals, ITS is the sequence used for identifying fungi and both matK and rbcL sequences are used in plant identification: Hebert et al. 2003a, 2003b). DNA barcoding works by demonstrating that intraspecific genetic variation is exceeded by interspecific genetic variation to such an extent that a separation exists, enabling the assignment of unidentified individuals to their species with a negligible error rate (Doguzhaeva et al. 2007; Wiemers and Fiedler 2007).

Variation between Nautilus and all other cephalopods is both phenotypically and genotypically distinguishable, to the extent that Nautilus DNA samples may not amplify in PCR reactions using primers that work successfully in other cephalopod taxa (Strugnell et al. 2006a). Unlike the more recent evolution of coleoids (Kröger et al. 2011), Nautilus appear to have remained relatively unchanged in the last 200 million years (O’Dor and Webber 1991). This has resulted in a range of phenotypic differences: pin-hole eyes, a leathery hood, a cord-like brain, numerous tentacles without suckers and the absence of the ink sac are all regarded as primitive states and are all exclusively present in Nautilus (Shigeno et al. 2010). These morphological characteristics hold Nautilus as being a unique group within the Cephalopoda. Although identification to distinguish Nautilus from other cephalopods can be ascertained visually, this is not always the case between species and, to a greater extent, within a species.

In combination with assessing levels and extent of genetic variation, the COI barcoding technique could also prove useful in the correction of inaccurate distribution estimates. A single living Nautilus was even reported to have been transported ~2000 km from the Philippine Islands to the coast of Kyushu Island, Japan, by the Kuroshio oceanic current (Tanabe and Hamada 1978; Saunders and Landman 2010). Whilst the capture of a living individual this far from its known distribution is rare, oceanic currents are commonly reported to carry empty Nautilus shells away from their living distribution (Wani and Ikeda 2006; House 2010). Kobayashi (1954) first formalised the concept of post-mortem distribution, emphasising that the occurrence of shells was not representative of their living distribution, several thousand miles away. The knowledge regarding this concept has since developed and continued to inform on both ancient and present day influences of nautiloid dispersal (Manda 2008; Mapes et al. 2010; Schlögl et al. 2011). As a result of post-mortem transportation, the distribution of shells in the Indo-Pacific far exceeds that of living individual’s geographical range; for example, N. pompilius shells have been collected from multiple locations around South Africa (House 2010). The ability to identify the geographic origin of a shell would help to prevent inaccurate distribution maps and also provide information on ocean currents and topography. This would rely on definite evidence of the shell origin; however, in the absence of this shell, drift cannot contribute to data on geographic range. Subsequently, the only conclusive way to identify populations is by live trapping, as demonstrated in this study.

Identifying and characterising genetic variation within Nautilus is of increasing importance due to the ongoing harvesting pressure on the species for the ornamental shell trade. Limited legislative protection exists to regulate the collection of Nautilus from the wild and heightened fishing intensity is known to occur in many areas such as the Philippines where no legislation exists to prevent this (Dunstan et al. 2010). In contrast, populations in areas such as the Great Barrier Reef are under protective legislation and populations in the Coral Sea are currently covered by a local agreement with fishermen not to target them (further protective measures are under discussion by Australian regulatory authorities at this time (Marine Bioregional Planning – Coral Sea, see http://www.environment.gov.au/coasts/mbp/coralsea/, accessed 2012, verified 7 November 2012)). The long-term effects of shell collection on the genetic variation of Nautilus are unknown; however, protective legislation is vital to the conservation of genetic diversity and evolutionary processes (Frankham et al. 2004).

With the morphological differences between Nautilus and other cephalopods leaving the COI barcoding system almost redundant at that level, its employment for population separation, origin identification, and geographical ranges could be further developed to benefit our understanding of the evolution and diversity of the species. Here we assess the innovative system of barcoding and its potential to distinguish between populations of the same species, using N. pompilius as a case study.


Methods

Samples were collected from seven reefs (Fig. 1). The Rowley Shoals is a group of atoll-like coral reefs south of the Timor Sea, including Clerke Reef (17°19′S, 119°21′E) and Imperieuse Reef (17°35′S, 118°55′E), on the edge of one of the widest continental shelves in the world. Each atoll covers an area of 80–90 km2 and both Clerke Reef and Imperieuse Reef rise steeply from the surrounding ocean floor. Imperieuse Reef is located 35 km south-west of Clerke Reef and is the most south-westerly of the reefs of the Rowley Shoals. Scott Reef (14°03′S, 121°46′E) is found at the edge of the continental shelf ~265 km off the coast of north-western Australia. Ashmore Reef is located in the Timor Sea (12°11′S, 122°59′E), on the edge of the Australian continental shelf and ~320 km off the Kimberley coast of Australia. East Australian samples were obtained from Osprey Reef (13°53′44″S, 146°33′27″E) and Shark Reef (14°07′59″S, 146°47′52″E) in the Coral Sea, and the Far North Great Barrier Reef (1°39′59″S, 143°58′56″E) in north-western Australia.


 
Fig. 1.  Geographical locations of the sampling sites for Nautilus pompilius. From west Australia: Imperieuse, Clerke, Scott and Ashmore Reefs; east Australia: Shark, Osprey and Great Barrier Reefs; and samples sourced from GenBank: Philippines (Balayan Bay and Pangalao Island), Indonesia (Ambon Strait), Papua New Guinea (Lorengau and Port Moresby), Fiji (Suva), American Samoa (Pago Pago) and Vanuatu. Australian samples were collected from 100–300 m, by attracting individuals into traps using pilchard (Sardinops sagax) or chicken (Gallus gallus domesticus) baits. Captured individuals were kept in a dark, dedicated refrigerated tank (50 L) for a maximum of 15 h at temperatures of 16-19°C. All individuals were released at night at depths of 20–30 m.
 
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Nautilus samples were collected from traps, which have a curved, round or arrowhead design with a diameter of 1500 mm, a height of 600 mm and a mesh size of 50 mm. The traps were set on the reef slope at depths from 100–300 m for 24-h periods as part of a fish-sampling program, and were baited with ~1 kg of pilchards (Sardinops sagax) or chicken (Gallus gallus domesticus). Tentacle samples, 1–2 cm long, were taken from a labial tentacle from each individual collected. Identification photographs and shell measurements were taken before returning each individual back to the reef edge (at ~20 m). Tentacles were initially preserved in 20% DMSO (dimethylsulfoxide), 100 mm EDTA, saturated NaCl solution and stored at 4°C in the field. The tissue was subsequently washed in TE buffer and placed into 80% alcohol preservative for storage, until required for DNA extraction.

DNA was extracted from the samples using the QIAGEN DNeasy Tissue Kit following the manufacturers’ instructions (Qiagen Pty Ltd, Victoria, Australia). Polymerase chain reaction (PCR) amplifications were conducted using 25-µL reaction volumes. Primer sequences previously demonstrated to be effective in amplifying N. pompilius COI were utilised, as described by Sinclair et al. (2007). The specific PCR protocol used with these samples had an initial 2 min at 94°C followed by 35 cycles of 30 s at 94°C, 30 s at 51°C and 2 min at 72°C, followed finally by 10 min at 72°C. Samples were purified using QIAGEN QIAquick PCR Purification columns following the manufacturers’ instructions. Results were visualised on a 1% agarose gel at each stage in the methodology. Amplified products were sequenced at a commercial sequencing facility at DBS Genomics, Durham University, UK.

Any corrections made to the sequences were carried out manually using Chromas v1.45 Freeware (Technelysium Pty Ltd, Australia). Sequences from samples collected in Papua New Guinea, the Philippines, Indonesia, Vanuatu, Fiji and American Samoa (Fig. 1) were sourced from Bonacum et al. (2011), downloaded via GenBank (GenBank accession numbers: GQ280190–GQ280194, GQ280201–GQ280212, GQ280214–GQ280216, GQ280240–GQ280249) and combined into the data file. The total 128 sequences were aligned using ClustalW in BioEdit v7.0.4.1 Freeware (Ibis Therapeutics, San Diego, CA, USA). Alignment of the sequences required no insertions or deletions (indels). The sections of sequence towards each end of the generated sequences were fully conserved in each individual.

A phylogenetic tree for maximum likelihood was constructed in PAUP* 4.0 Beta 10 Win (Harrison and Langdale 2006), with the TBR (tree-bisection-reconnection) algorithm and rooted on Nautilus macromphalus (GenBank Accession DQ472026). The HKY+G model and gamma-alpha shape parameter of 0.301, as indicated by ModelTest V.3.7, were used (Schneider et al. 2005). Bootstrap and jacknife values were generated (1000 resampling replicates) to provide statistical support.


Results

DNA was extracted from tentacle snips taken from N. pompilius from several sites surrounding Australia. Partial COI sequence information was generated (560 bp) and analysed from a total of 98 samples. In addition, a further 30 N. pompilius COI sequences were mined from GenBank, covering accessions from different geographical locations across the species’ distribution (Bonacum et al. 2011). DNA sequences generated in this study were deposited in GenBank (accession numbers used in this study are shown in Appendix 1). On a GenBank BLAST search, this partial sequence aligned (99.0%) with a N. pompilius COI sequence (GenBank accession: AF120628). Similarly, a specimen identification search on the Barcodes of Life Database produced a 99.4% similarity match with voucher sequences from N. pompilius.

Alignment of the COI partial sequence data obtained from all 128 samples (412 bp) was carried out and a phylogenetic tree representing maximum likelihood was generated (Fig. 2) and rooted on N. macromphalus to assess genetic variation and establish whether geographic origin mirrored genetic separation. Bootstrap and jacknife values were generated and a value greater than 50 indicates that the node is of significance.


 
Fig. 2.  Phylogenetic analyses of N. pompilius COI DNA sequences. Maximum-likelihood analysis generated a consensus tree constructed from the alignment of partial COI sequences of 412 bp from 128 N. pompilius sequences, and rooted against Nautilus macromphalus. Bootstrap values (1000 resampling replicates) higher than 50% are shown above the branches, Jackknife values are shown below. The dark grey bar indicates accessions from the east Australian clade, the light grey bar indicates accessions from the west Australian clade and the black bar indicates accessions from the western Pacific clade.
 
 

Within this tree, the presence of three distinct clades was detected illustrating the biogeographic and evolutionary separation of these accessions. The first clade shows the panmictic nature of the west Australian populations sampled (Ashmore, Imperieuse, Clerke and Scott Reef). These populations show no discrete separation and appear as a single, large panmictic population. Similarly, the Philippine and Indonesian accessions analysed in the dataset fully resolve within this clade. This grouping is strongly supported by both bootstrap (100) and jacknife (98) analysis. There is no further hierarchical separation of accessions within this clade into specific population groups; it maintains the panmictic structure with only limited resolution of discrete accessions.

The second independent clade (with strong bootstrap support) represents accessions collected from east Australia (Coral Sea reefs and Great Barrier Reef), in addition to accessions sourced from Papua New Guinea which are also resolved in this clade. There is further structuring within this large clade, however, and accessions from the Coral Sea reefs (Osprey and Shark) represent a sister clade to those accessions from the Great Barrier Reef and Papua New Guinea. This division is also strongly supported with bootstrap analysis (99) and supports previous reports of the divergence between these groups (Sinclair et al. 2007, 2011).

Appearing as a third discreet clade are all the western Pacific accessions from Vanuatu, American Samoa and Fiji, which were also strongly supported with bootstrap analysis of 100. Within this clade there was a grouping derived which separated out the accessions from Vanuatu (bootstrap and jacknife values of 100), and those from Fiji and American Samoa (Bonacum et al. 2011), which were clustered together again with bootstrap support (96).

A Minimum Spanning Tree (Fig. 3) constructed using Arlequin (ver. 3.5.1.2) illustrates the frequency of discrete haplotypes found within the samples. From the 128 samples analysed in this study, 49 discrete haplotypes were identified. Each of the three discrete clades are represented by different colours, nodes represent discreet haplotypes, with the k value signifying frequency of individuals represented in a particular node (which therefore have identical haplotypes based on the COI sequencing). In total, 12 haplotypes were found within the east Australian/Papua New Guinean clade, 30 haplotypes within the west Australian/Indonesian/Philippine clade and 7 haplotypes within the western Pacific clade (Table 1). Values between nodes illustrate the number of base pair differences between the individual haplotypes. Within the west Australian/Indonesian/Philippine populations, node values are all relatively small (values of 1.0–2.0 bp predominate, with one outlier at 4.0 bp). Within the east Australian/Papua New Guinean populations node values again average 1.0–2.0 bp, with the largest at 6.0 bp. The western Pacific clade, however, has multiple values of 1.0 bp (from Vanuatu accessions), but also some substantially higher at 17.0 and 18.0 bp (American Samoa and Fiji accessions) – this may be an artefact due to the small sample number at each population.


 
Fig. 3.  Minimum Spanning Tree produced using Arlequin v.3.5.1.2. Nodes represent haplotypes, k values signify the frequency of individuals represented in a particular node, values between the nodes demonstrate base pair differences between haplotypes. Node colour corresponds to the three geographical clades: dark grey nodes are east Australian samples, light grey nodes are west Australian and black nodes represent the western Pacific samples.
 
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Table 1.  Individual Nautilus pompilius accession haplotypes that segregated, based on discrete COI DNA sequence, to form discrete nodes in Arlequin© analysis and MST generation
K values signify the frequency of individuals represented in a particular node

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Between the populations of west Australian/Indonesian/Philippines and those of east Australian/Papua New Guinea, the discriminatory node value is much higher than the general internal node size, at 19.0 bp. Similarly, a node value of 30.0 bp separates the western Pacific clade from the east Australian/Papua New Guinea clade. This illustrates the significant separation of these populations in evolutionary terms, highlighted by the fact that no N. pompilius individual shares an identical haplotype with an individual from a different clade.


Discussion

The Class Cephalopoda (Phylum Mollusca) is highly successful, but cephalopods, as with many taxa, have not escaped population declines as a result of anthropomorphic activity (Moltschaniwskyj et al. 2007; Crook et al. 2009). The overexploitation of marine resources is an ongoing global concern and the finite supplies of marine organisms are now showing dramatic declines (Tittensor et al. 2006). N. pompilius is no exception, being the only extant cephalopod with an external shell has been a key factor in its exploitation and consequent decline. The ornamental shell trade targets Nautilus species and many populations are being overexploited, leading to population fragmentation and isolation, which contributes to diversification (Sinclair et al. 2011).

There is no clear concordance under the current classification of Nautilus, where potentially two to five distinct species are all grouped under a single species – N. pompilius. There is comparatively little knowledge of their population genetics, growth rates, and related population dynamics, which are essential criteria for sustainably managing Nautilus fisheries. Furthermore, the evolutionary division of the different Nautilus species is under question and needs to be redefined (Sinclair et al. 2007).

Our results show a significant degree of genetic divergence between the three proposed evolutionary clades, indicative of both movement between reefs, and ancient evolutionary history, as proposed by Wray et al. (1995). Maximum-likelihood analysis (Fig. 2) illustrates the panmictic nature of the west Australian/Indonesian/Philippines clade, showing no clear separation of the different populations. The west Australian populations (Ashmore, Imperieuse, Clerke and Scott Reefs) are all represented as one large intermixed population, indicating the extent of connectivity between the reefs. The Philippine and Indonesian accessions are also interspersed and resolved within the clade. This would indicate that Nautilus from the surrounding seas of the Indonesian archipelago are still, or have been in their recent evolutionary history, sympatrically distributed with the Nautilus surrounding west Australia.

The east Australian/Papua New Guinea clade demonstrates an increase in separation within the clade and appears to divide into two: the first half encompasses samples from Papua New Guinea and the far north Great Barrier Reef, and the second half contains samples from Osprey Reef and Shark Reef. Within the clade containing accessions from Papua New Guinea and the Great Barrier Reef, over 80% of Papua New Guinean samples are clustered together within the clade; the remaining are resolved within the Great Barrier Reef accessions (Fig. 2). This may reflect the recent evolutionary dispersal of accessions from Papua New Guinea moving down towards the east coast of Australia and becoming part of the intrinsic population there. The Osprey/Shark Reef grouping demonstrates a level of similarity indicative of that of a panmictic population (no population separation). When this is considered in terms of the topography of these locations, which, although physically quite close to each other, are separated by water deeper than 1000 m, they would be expected to show patterns indicative of isolated, self-contained populations. Their separation, however, in evolutionary timescales is such that they may not have been isolated from each other for long enough for such trends to have been established within the populations to date. Although it is evident that Papua New Guinea, and Osprey, Shark and far north Great Barrier Reefs form an individual clade, their phylogenetic structure would indicate that there is less connectivity within this geographical clade than that found between the populations from west Australia, suggesting little or no current migration between sites.

The third clade incorporates samples solely from Bonacum et al. (2011); despite emerging as one separate clade, it illustrates a division with 100% statistical support for a divide between the Vanuatu accessions, and those from Fiji and American Samoa. This would indicate that, although their evolutionary distribution has resulted in a higher level of relatedness to each other than to the rest of the dataset, there appears to be no current migration between populations.

All three clades are shown to be more closely related to each other than they are to the outgroup used in this study, N. macromphalus.

The overall phylogeny presented here is a result of ancient distribution and current dispersal patterns, creating a measurable genetic divergence dependent on the degree of separation between populations. Genetic divergence between populations can be avoided with gene flow promulgated by dispersing individuals and multidirectional gene flow (Chesser 1991). The phylogenetic results for west Australia are indicative of individuals moving between populations, whereas the phylogenetic divide within the east Australian clade indicates otherwise. The dispersal ability of the species is determined by the topology between the discussed reefs; the lower depth limit of Nautilus is ~800 m (Saunders and Wehman 1977; Kanie et al. 1980; Wani 2004), with long-term habitat depth suggested to be limited to 300–500 m due to cameral flooding (Saunders and Landman 2010). Water depths surrounding the west Australian reefs show no potential inhibitory effects to movement of N. pompilius, which supports the topology of the maximum-likelihood analysis. During sampling, Clerke Reef had a surrounding depth of 390 m, therefore an individual Nautilus could easily travel the 35-km distance to Imperieuse Reef (O’dor et al. 1993; Wray et al. 1995), which is surrounded by ocean of depth ~320 m. There are also surrounding coral patches, thus there is the likelihood that these areas act as suitable transit ‘corridors,’ connecting the larger reefs (Genin et al. 1986; Rypien et al. 2008).

This topology does not apply to the east Australian clade, where Osprey Reef and the Great Barrier Reef are separated by a distance of ~250 km and by depths of more than 1000 m. Although this distance has been successfully travelled before by Nautilus (Tanabe and Hamada 1978; Saunders and Landman 2010), it is not a regular occurrence; their physiology suggests that the depths were too great to allow benthic travel, which would result in passage through large expanses of open water, thus dramatically reducing their survival rate due to predation (Saunders and Landman 2010). The genetic consequence of their inability to travel large distances over great depths is demonstrated in their phylogenetic separation within the clade. Although one individual is unlikely to travel the maximum dispersal distance of the west Australian reefs (determined by the length of connecting reefs in this area: Wieters et al. 2008), dispersal along partial distances of connecting reefs by individuals is sufficient to prevent the inbreeding problems commonly associated with isolated populations (Madsen et al. 1996; Kuhls et al. 2007; Rypien et al. 2008; Caputo et al. 2009; Griffiths et al. 2009; Santos et al. 2009; Trinkel et al. 2010). This potentially inhibits the development of any significant differences occurring between reefs.

The results presented here support the work of Wray et al. (1995) in demonstrating the presence of three distinct clades within Nautilus evolution. We expand on this hypothesis to reveal further separation in their more recent evolutionary history. Despite this separation, populations within each clade continue to show greater similarity to each other than they do to populations of another clade, demonstrating that the process of diversification has been occurring for longer between the clades than it has within them.

Recent work (Bonacum et al. 2011) suggests that the living Nautilus lineage originated around New Guinea, potentially only two million years ago, and one lineage of Nautilus voyaged from New Guinea to the more easterly archipelagos of New Caledonia, Fiji and Samoa, whereas another travelled to Australia, the Philippines and the South China Sea. Samples from potential progenitor populations in Papua New Guinea and the Philippines have allowed clearer elucidation of evolutionary divergence further back along evolutionary time. The varied lineage of each N. pompilius clade, and the current separation into smaller discrete populations, has resulted in genetic sequences unique to specific evolutionary clades. Results show that highly conserved regions have remained unchanged, still enabling species identification; however, variable regions are no longer generic across the species.

As an identification tool, DNA barcoding using the COI sequence has proved successful in distinguishing between clades but migration between populations has prevented population-level (individual reefs) identification of accessions. COI could therefore prove effective in reducing the scope for the plausible geographic origins of a sample found through post-mortem transportation. Although the tissue of the living animal is often no longer located within the retrieved shell, a sample could be taken from the remaining siphuncle tissue within the shell (Strugnell et al. 2006b). DNA barcoding would, at this present time, be unable to distinguish between an unprotected specimen taken from Papua New Guinea, and a protected specimen taken from the Great Barrier Reef, due to both populations belonging to the same geographic clade.

In earlier work, Dunstan et al. (2010) indicated that market forces are driving the ongoing development of the Nautilus fishing industry and called for an assessment of Nautilus species by the IUCN, potentially to have it categorised as globally endangered. Here we present data generated by DNA barcoding that supports the inherent vulnerability of N. pompilius populations to fishing pressure due to the discrete evolutionary history of each of the three identified clades and the current populations they represent. If these current populations in these areas were targeted by commercial fishermen, the risk of losing a unique genetic resource (potentially representing unique species and subspecies) is extreme.


Future work

Future work could expand on the use of barcoding to determine the origins of empty shells through the use of other barcoding sequences to assess whether a higher level of variation is displayed between populations. This could incorporate the development of population-specific identifiers for discrete sections of the COI sequence, potentially through the use of single nucleotide polymorphisms as population-specific markers. This will require investment in the initial start-up process, it will provide a useful mechanism by which to address questions such as identity establishment for samples of unknown provenance to determine whether they have been illegally fished – thereby helping to corroborate and enact any protective legislation on Nautilus.

Further research is needed into the effects of fishing efforts on the overall structure of the current Nautilus populations and how this impacts the genetic and evolutionary diversity across the distribution. Such work will determine whether and where a sustainable fishery for Nautilus could be established while maintaining the range of genetic variation within the Nautilus group as a whole.

The continuing development of new, more powerful molecular technologies has opened up the genome of the world’s flora and fauna to unprecedented scrutiny. By harnessing this technology, as has been done with DNA barcoding projects, we can utilise it to help understand the diversity of life around us. The range of life found in the world’s oceans is dropping through a range of factors, not least anthropogenic, and as a priority, we must strive to understand what we can within as short a time as possible. Unique species such as Nautilus represent a flagship, iconic lifeform about which we know so little, yet DNA barcoding has helped to show the evolutionary pathways of dispersal in this ancient species, has shown the degree of genetic variation contained within the species, and may even be a deciding factor in reclassifying N. pompilius into discrete species or subspecies.



Acknowledgements

This work was funded in part by The Centre for Wildlife Conservation, Faculty of Science and Natural Resources, University of Cumbria, United Kingdom. The Department of Fisheries, Government of Western Australia, provided logistical support. The authors thank Tony, Liz, Euan and Douglas for their help in recording all the tissue samples used in the study. The authors sincerely thank their colleagues Dr Christine McPhie and Professor Graham Pegg for their constructive comments and assistance in the preparation of this manuscript.


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Appendix 1.  GenBank accession numbers used in this study

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