Reviews in Fisheries Science, 19(1):52–61, 2011Copyright C©© Taylor and Francis Group, LLCISSN: 1064-1262 printDOI: 10.1080/10641262.2010.533794

Spatial Subdivision and GeneticDiversity in Populations on the Eastand West Coasts of Australia: TheMulti-Faceted Case of Nautiluspompilius (Mollusca, Cephalopoda)

WILLIAM SINCLAIR,1,2 STEPHEN J. NEWMAN,3 GABRIEL M. S. VIANNA,4

STEVEN WILLIAMS,2 and WILLIAM J. ASPDEN2

1Centre for Wildlife Conservation, Faculty of Science and Natural Resources, University of Cumbria, Penrith, United Kingdom2Centre for Environmental Management, CQUniversity, Rockhampton, Queensland, Australia3Western Australian Fisheries and Marine Research Laboratories, Department of Fisheries, Government of Western Australia,North Beach, WA, Australia4Undersea Explorer, Port Douglas, Queensland, Australia

The fragmented distribution of Nautilus pompilius and its biology suggest there will be significant genetic divergence andspatial subdivision between east and west Australian populations. Samples were collected from the northern Great BarrierReef, the Coral Sea, and the Scott Reef off Western Australia. Phylogenetic trees and a minimum spanning tree were developedfrom these data to elucidate evolutionary relationships. These data demonstrate significant evolutionary separation of each ofthe three populations into strongly supported discrete clades matching geographic stratification. Within each of the discretepopulations, genetic variation is evident. Strong inter-population variation is evident, with discrete geographic clades beingrecognized for each extant group. The distinct spatial subdivision between east and west Australian populations of Nautilusare related to geographic and physical isolation over evolutionary time, and this has important fisheries managementimplications. The distinct geographic patterns of genetic structuring demonstrated by these data indicate the existence ofdiscrete eastern and western Australian management units, and as such, these Nautilus populations should be manageddiscretely, as each has a high conservation value containing unique genetic variation.

Keywords Nautilus, coxI, spatial subdivision, Great Barrier Reef, Western Australia, population genetics

INTRODUCTION

The pearly Nautilus (Mollusca, Cephalopoda) is a modernsurvivor of a previously abundant group of shelled cephalopods,and it is a direct descendant of these fossil forms—the nautiloids(Woodruff et al., 1987; Wray et al., 1995). They are the earliestdiverging lineage of this group and are often cited to be “livingfossils” (Boyle and Rodhouse, 2005; Ward, 2008), given thesimilarity of their current form to that of their ancient ancestors;

Address correspondence to Dr. Billy Sinclair, Centre for Wildlife Conserva-tion, Faculty of Science and Natural Resources, University of Cumbria, PenrithCA11 0AH, United Kingdom. E-mail: billy.sinclair@cumbria.ac.uk

they have persisted through a number of mass-extinction events(Strugnell and Lindgren, 2007). However, it has been shown thatcurrent Nautilus species may only have originated between oneand five million years ago (Woodruff et al., 1987). Of the twosubclasses of cephalopods, the Nautiloidea represent a distinctlymonophyletic group, containing the only extant cephalopodswith an external shell (Bonnaud et al., 2004).

While studied nowhere near as extensively as their rela-tives in the Coleoidea—the octopus, squid, and cuttlefish—alimited number of studies of Nautilus biology have been de-scribed (Ward, 1987, Suzuki et al., 2000; Ruth et al., 2002; Basilet al., 2005; Westermann et al., 2005; Moltschaniwskyj et al.,2007).

52

NAUTILUS SUBDIVISION AND GENETIC DIVERSITY 53

Nautilus species are found throughout the southwest PacificOcean at depths ranging from 50–600 m; they are predators andscavengers, feeding on invertebrates and detrital matter settlingon the reef bottom (Carlson et al., 1984; Saunders, 1984; Wardet al., 1984; Hayasaka et al., 1987; Saunders and Landman,1987; Saunders and Ward, 1987; Ward, 1987; Wells et al., 1992).However, as a k-selected species, they exhibit the characteristictraits of low fecundity, late maturity, and relatively long lifespan(Saunders, 1983; Saunders and Landman, 2010).

Saunders recognized five species of Nautilus according to adetermined series of criteria, not including the distinct genusAllonautilus (Saunders, 1987; Ward, 1987; Ward and Saun-ders, 1997). Wray et al. (1995) further examined the geneticand geographic divergence of Nautilus pompilius across itsIndo Pacific distribution, suggesting the presence of three ge-ographically distinct clades as opposed to independent geneticlineages.

The use of molecular techniques to study population struc-ture and to identify discrete populations is now commonplaceacross a wide range of marine species (van Herwerden et al.,2003; Ward and Holmes, 2007). In particular, mitochondrialDNA polymorphisms have become widely used as a popula-tion genetics tool in fisheries biology (Ovenden, 1990; Hebertet al., 2003; Aspden et al., 2006; Pegg et al., 2006). The suit-ability of this technique is related to its maternal mode of inher-itance and relatively rapid rate of evolution (Conseugra et al.,2002).

An increasing number of genetic and evolutionary studieshave been conducted on cephalopod groups, and a number ofthese have utilized mitochondrial DNA gene sequences, such ascoxI (cytochrome oxidase, subunit I), to investigate and resolvegenetic and evolutionary relationships at a range of hierarchicallevels (Anderson, 2000; Lindgren et al., 2005; Strugnell et al.,2005).

Nautilus species are fished for the ornamental shell trade in anumber of locations, and many of these fisheries are being over-exploited, a situation experienced by many k-selected species(Wilkinson, 2008), where high levels of exploitation can resultin diminished population sizes throughout their range (Dunstanet al., 2010). Targeted fishing activities in late-maturing species,such as Nautilus, is recognized as an issue in the conservationand management of such species (Dunstan et al., 2010). Moretargeted management and conservation of these species requiresa detailed understanding of their genetic stock structure andassociated linkages; for Nautilus pompilius, such information isnot available. Other than the studies by Wray et al. (1995) andBonnaud et al. (2004), there are limited reports in the literaturewhere Nautilus DNA sequences have been generated (Merrittet al., 1998; Lindgren et al., 2005).

Sinclair et al. (2007) were the first study to explore the popu-lation genetics of Nautilus in the Great Barrier Reef (GBR) andreported the discrete populations of Nautilus from the isolatedsea mount reefs of the Coral Sea. Given the fragmented distri-bution of this species, the hypothesis tested here is that therewill be significant genetic divergence between east and west

Australian populations due to geographic and physical isolationover evolutionary time. Data presented in this article will pro-vide important new information, which can help inform futureconservation plans for this species.

MATERIALS AND METHODS

Sampling Sites

Osprey reef (13◦53.44′S, 146◦33.27′E) is a seamount, 195km2 in surface area, in the Coral Sea approximately 200 kmNNE of Cairns (Figure 1). The reef formation at Osprey rangesfrom low-tide breaking reef to coral gardens at 20 m with steepcoral walls dropping to over 1,200 m. Other sampling sitesin this “Coral Sea” group included in this study are SharkReef (14◦05.88′S, 146◦47.02′E), approximately 15 km southof Osprey and separated by depths in excess of 1,000 m, andBougainville Reef (15◦29.00′S, 147◦06.50′E), a further 30 kmsouth of Shark Reef and isolated by similar depths.

A separate “Northern GBR” group of reefs (approximately400 km north of Cairns and 250 km NNW of Osprey Reef)that provided Nautilus samples from the far northern distribu-tion of the species in reefs adjacent to the GBR are South-ern Small Detached Reef (12◦34.71′S, 143◦51.30′E), North-ern Small Detached Reef (1◦39.59′S, 143◦58.56′E), and MantisReef (12◦03.77′S, 143◦55.77′E).

Osprey Reef, unlike the GBR sites, has vertical reef walls thatare protected from the southeasterly trade winds and is, there-fore, accessible throughout the year. GBR sites were accessedfor Nautilus sampling when weather conditions allowed.

Western Australian sampling was conducted at Scott Reefin the eastern Indian Ocean, ∼265 km off the coast of north-western Australia (about 450 km northwest of Broome, West-ern Australia) and ∼360 km south of the nearest islands ofsouthern Indonesia (Figure 1). The Scott Reef complex is foundat the edge of the continental shelf (approximately 14◦03′S,121◦46′E) and is an extensive, 39-km wide macro-tidal reefsystem.

Capture and Release

Eastern Australian Samples

A barrel-shaped trap (90 × 77 cm) constructed from wiremesh (7.5 × 9 cm mesh size) was used. The trap had twoentrance funnels of 23-cm diameter on each end of the barrel.Traps were deployed at 200–300-m depth at dusk and retrieved atdawn; traps were lowered on an 8-mm rope with an attached floatand either set to the seafloor if it is a flat bottom or tethered to thereef if sampling on a vertical wall. Captured Nautilus were keptfor a maximum of 15 hr in a dedicated, dark refrigerated tank(50 L) at temperatures between 16–19◦C before being releasedat night in 20–30-m depths.

reviews in fisheries science vol. 19 1 2011

54 W. SINCLAIR ET AL.

Figure 1 Schematic map illustrating the geographic locations of the sampling sites where Nautilus population samples were obtained (Coral Sea group consistsof Osprey, Shark, and Bougainville Reefs; Northern GBR group consists of Northern Small Detached, Southern Small Detached, and Mantis Reefs; Scott Reef isthe Western Australian reef sampled). Sample animals were collected from between 150–350-m depth in a wire mesh trap with chicken as an attractant. Capturedanimals were stored in fresh sea water at 10◦C for 2 hr before tentacle biopsy samples were taken and stored in 20% DMSO. Animals were released at dusk atdepths of 20–30 m.

Western Australian Samples

Samples at Scott Reef were collected from fish traps. Thefish traps have a curved arrowhead design with a diameter of1,500 mm, a height of 600 mm, and a mesh size of 50 mm. Fishtraps were set on the reef slope at depths from 100–200 m for24-hr periods.

Tissue Sampling and DNA Extraction

The terminal 4–5-mm section from one labial tentacle fromeach animal was collected and preserved initially in 20% DMSO(dimethyl-sulphoxide), 100-mM EDTA, saturated NaCl solu-tion stored at 4◦C in the field. Subsequently, the tissue waswashed in TE and placed into 80% ethanol preservative for stor-age until required for DNA extraction. Total DNA was extractedusing a QIAGEN DNeasy Tissue kit, following the manufac-turer’s instructions (Qiagen Pty. Ltd., Victoria, Australia).

Polymerase Chain Reaction (PCR) Amplification of coxISequences

Polymerase Chain Reaction (PCR) was conducted on ex-tracted DNA samples using previously described primer se-quences (Sinclair et al., 2007). PCR was undertaken in 25-µlreactions, which resulted in the generation of a single ampli-cons fragment, following an amplification protocol compris-ing an initial 2-min incubation at 94◦C, followed by 30 cy-cles of 30 sec at 94◦C, 30 sec at 52◦C, and 2 min at 72◦C,followed finally by 10 min at 72◦C. For each sample, follow-ing PCR and electrophoresis on a 1% agarose gel, an ampli-fied fragment was observed of approximately 600 bp in size.This band was excised and purified (Promega, UK). Threeng of purified DNA was then placed into a sequencing reac-tion according to the manufacturer’s instructions for the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosys-tems, 2001). Both forward and reverse strands were sequenced

reviews in fisheries science vol. 19 1 2011

NAUTILUS SUBDIVISION AND GENETIC DIVERSITY 55

using an ABI3130 genetic analyzer (Applied Biosystems,2001), resulting in 574 bp of sequence information from eachaccession.

DNA Sequence Alignment and Analysis

The sequences representing the coxI region of DNAfrom each individual were aligned manually usingChromas R©v1.45 Freeware (Technelysium Pty. Ltd., Australia)and BioEdit R©v7.0.4.1 Freeware (Ibis Therapeutics, California,USA) using the program default settings. Alignment of the se-quences required no insertion/deletion events (indels). The sec-tions of sequence toward each end of the generated sequenceswere fully conserved in each individual. To compare the data forconsistency, maximum likelihood, neighbor joining, and maxi-mum parsimony, trees were constructed (Harrison and Langdale,2006) in PAUP* 4.0 Beta 10 Win (Swofford, 1998), with the tree-bisection-reconnection (TBR) algorithm. The HKY+G modeland gamma-alpha shape parameter of 0.301, as indicated byModelTest V.3.7, were used (Posada and Crandall, 1998). Sta-tistical support for each analysis was evaluated by the bootstrapprocedure (1,000 resampling replicates). The gamma-alphashape parameter of 0.301 was also applied in calculating pair-wise FSTs (3,024 replicates) in Arlequin V.2.0.1.1 (Schneideret al., 2000). Hierarchical measures of genetic structure were de-termined by AMOVA (as per Excoffier et al., 1992) in ArlequinV.2.0.1.1 (Schneider et al., 2000). The hierarchical structureinvolved grouping the samples into three regions—Scott Reef(WA), the Coral Sea, and the Northern GBR. Within-group andbetween-group genetic distances and standard errors of means(SEMs) were calculated using MEGA V.3.1 using the Tamura-Nei model and 1,000 bootstrap replications (Kumar et al., 2004).A minimum spanning tree (MST) was obtained from the pop-ulation genetic analysis using Arlequin V.2.0.1.1. (Schneideret al., 2000) and was constructed using Sneato (Wooding, 2004;http://www.xmission.com/∼wooding/Sneato/index.html).

RESULTS

DNA Extraction and Partial coxI DNA Sequence Data

DNA was extracted from a number of animals at a range ofdifferent sites in the Coral Sea, the outer edges of the GBR in farnorth Queensland, and from a reef system off Western Australia.Partial coxI sequence information was obtained and analyzedfrom a total of 83 samples: In the Coral Sea group, 23 animalsat Osprey Reef, 9 from Shark Reef, and 2 from BougainvilleReef. From the Northern GBR group, a total of 10 sequenceswere obtained (5 from North Small Detached Reef, 4 fromSouth Small Detached Reef and 1 from Mantis Reef) and 38from Scott Reef Western Australia accessions. Sequences gen-erated in this study were deposited in GenBank under Accessionnumbers EF128174–EF128216 and GQ387444-GQ387481. On

Table 1 Between-site net genetic distances (± SEM) for Nautilus pompilius,calculated with Mega 3.1

Site Scott Reef WA Coral Sea

Coral Sea 0.060 ± 0.010Northern GBR 0.051 ± 0.009 0.020 ± 0.006

a Genbank BLAST search, this partial sequence aligned (93%)with Nautilus pompilius coxI sequence (Genbank accession:AF120628). Similarly, a specimen identification search on theBarcodes of Life Database (www.barcodinglife.com) produceda 99.4% similarity match with voucher sequences from Nautiluspompilius (Ward et al., 2005).

Alignment of the coxI partial sequence data (574 bp) ob-tained from all 83 samples was carried out, with 87 bp (15%)variable sites observed. Of these, 34 points of variation (5.92%)were specific to the accessions from West Australia, and 24(4.18%) were common to both the Coral Sea and the NorthernGBR populations. Eight variable sites (1.39%) were dispersedacross samples from only the Coral Sea, and the remainingthree (0.52%) were specific to individuals from the NorthernGBR group.

Genetic distances within the Scott Reef WA, Coral Sea,and Northern GBR groups were calculated as 0.007 ± 0.002,0.001 ± 0.001, and 0.003 ± 0.002, respectively, with between-group values shown in Table 1. AMOVA results are provided inTable 2, showing indexes of variation among and within pop-ulations, and population pairwise FSTs are shown in Table 3.These analyses indicated significant variation between popu-lations, particularly between the east and west coasts of Aus-tralia. Standard diversity indices for gene diversity was 0.8667± 0.0714 within the Northern GBR group, 0.5526 ± 0.0869 forthe Coral Sea group, and 0.9289 ± 0.0286 for the Scott Reef WAgroup.

Maximum likelihood analysis generated a consensus tree(Figure 2) from an alignment of partial coxI sequences from85 N. pompilius individuals, and rooted against the sequence ofNautilus macromphalus (GenBank Accession DQ472026). Ofthe 574 bp of partial sequence, 487 characters were constant, 33variable characters were parsimony uninformative, and 54 char-acters were parsimony informative, with a consistency indexvalue of 0.9015 and a retention index value of 0.9916.

Within this tree, the presence of three distinct clades wasdetected. These clades, respectively, represented the accessions,

Table 2 AMOVA analysis on Nautilus pompilius from Scott Reef WA, CoralSea, and Northern GBR structured by site

Source of Sum of Variance Percentage ofVariation df Squares Components Variation

Among populations 2 743.711 15.109 Va 93.06Within populations 79 88.947 1.126 Vb 6.94Total 81 832.658 16.235

Fixation index FST: 0.9307; P < 0.000; 1,023 permutations.

reviews in fisheries science vol. 19 1 2011

56 W. SINCLAIR ET AL.

Table 3 Population pairwise FSTs on Nautilus pompilius from Scott ReefWA, Coral Sea, and Northern GBR using the Tamura and Nei distance method(P < 0.000 for each comparison; 3,024 permutations)

Site Scott Reef WA Coral Sea

Coral Sea 0.938Northern GBR 0.895 0.933

which comprised the “West Australia” accessions, the “CoralSea” group of populations, and those in the “Northern GBR”group of populations. These individual clade distinctions werestrongly supported with bootstrap support of 98 and 87 separat-ing the West Australia group from those in the Coral Sea andNorthern GBR groups, respectively. Similarly, the Coral Sea and

Northern GBR group separations were supported by bootstrapvalues of 100 and 88, respectively. Bootstrap values, gener-ated from 1,000 resampling replicates, which are over 50%, areshown above branches. The West Australia clade shows a seriesof within-clade branching steps with levels of support from 61%to 98%, in addition to a number of polytomies (a feature presentin all three clades).

Both the Coral Sea and the Northern GBR clades demon-strate a degree of within-clade branch resolution, but thesedid not show any population-specific groupings of the sam-ples indicative of their discrete reef capture locations. Withinthe Coral Sea clade, there were a number of unresolved steps(containing accessions from Osprey, Shark, and BougainvilleReefs) and some well-supported groupings of individuals fromall sites. A distinct sub-clade contained five accessions from

Northern GroupNorth southern smalldetached, South smalldetached, Mantis reefs

Coral Sea GroupOsprey, Shark andBougainville reefs

Outgroup

West Australia GroupScott Reef

N. macromphalus060702060701060706060715060721060732060703060704060705060735060707060708060725060709060710060711060712060727060713060714060716060717060730060733060734060718060719060720060722060723060724060726060728060729060731060736060737

O-1592O-1594O-1597O-1599O-1600O-1650O-1593O-1604O-1648O-1603O-1606O-1647O-1315O-1490O-1491O-1492O-1493O-1598O-1605O-1649O-1551Shark1S-1737S-1738S-1739S-1740S-1741S-1744O-1495

S-1742S-1745B’ville 1B’ville 2Mantis 4DSSD 2DSSD 3DSSD 10iiSSD 11iiNSD 5iiNSD 9iiNSD 6iiNSD 7ii

060738

O-1595

NSD 8ii

100

6162

65

97

83

9898

61

64

6488

63

87

Northern GroupNorth southern smalldetached, South smalldetached, Mantis reefs

Coral Sea GroupOsprey, Shark andBougainville reefs

Outgroup

West Australia GroupScott Reef

Northern GroupNorth southern smalldetached, South smalldetached, Mantis reefs

Coral Sea GroupOsprey, Shark andBougainville reefs

Outgroup

West Australia GroupScott Reef

N. macromphalus060702060701060706060715060721060732060703060704060705060735060707060708060725060709060710060711060712060727060713060714060716060717060730060733060734060718060719060720060722060723060724060726060728060729060731060736060737

O-1592O-1594O-1597O-1599O-1600O-1650O-1593O-1604O-1648O-1603O-1606O-1647O-1315O-1490O-1491O-1492O-1493O-1598O-1605O-1649O-1551Shark1S-1737S-1738S-1739S-1740S-1741S-1744O-1495

S-1742S-1745B’ville 1B’ville 2Mantis 4DSSD 2DSSD 3DSSD 10iiSSD 11iiNSD 5iiNSD 9iiNSD 6iiNSD 7ii

060738

O-1595

NSD 8ii

100

6162

65

97

83

9898

61

64

6488

63

87

N. macromphalus060702060701060706060715060721060732060703060704060705060735060707060708060725060709060710060711060712060727060713060714060716060717060730060733060734060718060719060720060722060723060724060726060728060729060731060736060737

O-1592O-1594O-1597O-1599O-1600O-1650O-1593O-1604O-1648O-1603O-1606O-1647O-1315O-1490O-1491O-1492O-1493O-1598O-1605O-1649O-1551Shark1S-1737S-1738S-1739S-1740S-1741S-1744O-1495

S-1742S-1745B’ville 1B’ville 2Mantis 4DSSD 2DSSD 3DSSD 10iiSSD 11iiNSD 5iiNSD 9iiNSD 6iiNSD 7ii

060738

O-1595

NSD 8ii

100

6162

65

97

83

9898

61

64

6488

63

87

N. macromphalus060702060701060706060715060721060732060703060704060705060735060707060708060725060709060710060711060712060727060713060714060716060717060730060733060734060718060719060720060722060723060724060726060728060729060731060736060737

O-1592O-1594O-1597O-1599O-1600O-1650O-1593O-1604O-1648O-1603O-1606O-1647O-1315O-1490O-1491O-1492O-1493O-1598O-1605O-1649O-1551Shark1S-1737S-1738S-1739S-1740S-1741S-1744O-1495

S-1742S-1745B’ville 1B’ville 2Mantis 4DSSD 2DSSD 3DSSD 10iiSSD 11iiNSD 5iiNSD 9iiNSD 6iiNSD 7ii

060738

O-1595

NSD 8ii

N. macromphalus060702060701060706060715060721060732060703060704060705060735060707060708060725060709060710060711060712060727060713060714060716060717060730060733060734060718060719060720060722060723060724060726060728060729060731060736060737

O-1592O-1594O-1597O-1599O-1600O-1650O-1593O-1604O-1648O-1603O-1606O-1647O-1315O-1490O-1491O-1492O-1493O-1598O-1605O-1649O-1551Shark1S-1737S-1738S-1739S-1740S-1741S-1744O-1495

S-1742S-1745B’ville 1B’ville 2Mantis 4DSSD 2DSSD 3DSSD 10iiSSD 11iiNSD 5iiNSD 9iiNSD 6iiNSD 7ii

060738

O-1595

NSD 8ii

100

6162

65

97

83

9898

61

64

6488

63

87

100

6162

65

97

83

9898

61

64

6488

63

87

Figure 2 Maximum likelihood analysis generated a consensus tree constructed from the alignment of partial coxI sequences from 85 N. pompilius individualsand rooted against the sequence of Nautilus macromphalus (GenBank Accession DQ472026). Of the 574 bp of partial sequence, 487 characters were constant,33 variable characters were parsimony uninformative, and 54 characters were parsimony informative. Bootstrap values, generated from 1,000 resampling replicates,which are over 50%, are shown above the branches.

reviews in fisheries science vol. 19 1 2011

NAUTILUS SUBDIVISION AND GENETIC DIVERSITY 57

Osprey Reef (O-1593, O-1600, O-1604, O-1648, and O-1650)was identified, which has bootstrap support of 64%. While notvery strongly supported, the group remained consistently linkedin all analyses.

The Northern GBR clade as a whole shows a grouping (64%Bootstrap support) of two accessions from North Small De-tached Reef (NSD 5ii and NSD 9ii), but no further distinctionof the North Small Detached reef accessions was discerned fromthose obtained at Southern Small Detached reef.

Subsequent generation of consensus trees from a neighbor-joining analysis and from a maximum parsimony analysisin PAUP∗ 4.0 constructed from the coxI sequence alignment(above) demonstrated the same general topology. The West Aus-tralia, Coral Sea, and Northern GBR individual clades, distinc-tions were supported with bootstrap support of 100% in boththese analyses, generated from 1,000 resampling replicates.

The MST alignment identified the presence of 31 haplo-types spread across the 82 Nautilus accessions sampled. Fromthe Coral Sea accessions, 5 haplotypes were identified, fromthe Northern GBR a further 5 haplotypes were shown, and 21discrete haplotypes in the samples from Scott Reef in westernAustralia. A total of eleven synapomorphic substitutions wereidentified, which unite the Coral Sea populations as a distinctclade from those found in the Northern GBR clade. A further 27synapomorphic substitutions identify the West Australian cladeas distinct from those on the Northern GBR. This alignmentsupported the phylogenetic separation of the data set by max-imum likelihood, maximum parsimony, and neighbor-joininganalysis.

DISCUSSION

Nautilus pompilius (Mollusca, Cephalopoda) are reef preda-tors and scavengers that inhabit depths up to 600 m on coralreef walls of the tropical Indo Pacific. Demand for their ornateshell has led to heavy targeting by fisheries across their dis-tribution, and little is known about the biology of this iconicanimal. There is comparatively little knowledge of their popu-lation genetics, growth rates, and related population dynamics,which are essential criteria for sustainably managing Nautilusfisheries. Furthermore, the evolutionary division of the differ-ent Nautilus species is under question and needs to be redefined(Sinclair et al., 2007). DNA sequence analysis was used to assesslevels of genetic diversity and evolutionary divergence betweendiscrete Nautilus populations found on the Northern GBR, theisolated seamounts of the Coral Sea, and from reefs off WesternAustralia. Earlier work reported significant differences betweenanimals found in the GBR compared to those in the Coral Sea,resolving into two discrete clades (Sinclair et al., 2007).

In this study, partial DNA sequencing of the coxI gene re-gion yielded data that was aligned for samples from the GBR andCoral Sea populations in addition to samples from Western Aus-tralia. Phylogenetic trees from maximum likelihood, neighbor-

joining and maximum parsimony analysis all demonstrated thesame gross topology in the consensus trees they produced. Eachshowed the presence of three distinct clades representative ofeach of the three geographic populations under study, each sup-ported by high bootstrap values. Consensus trees also demon-strated strong retention and consistency indices. AMOVA resultsalso strongly supported three different populations, with partic-ularly strong support for the separation of east and west Aus-tralian populations. MST analyses suggests that both east andwest coast populations are genetically distinct and that the eastcoast stocks (Northern GBR and Coral Sea) have been diverginggenetically and show significant genetic separation. This east–west divergence across northern Australia is now evident ina number of taxa (e.g., starfish [Linckia laevigata], Williamsand Benzie, 1998; coral trout [Plectropomus leopardus],van Herwerden et al., 2009; olive sea snake [Aipysurus lae-vis], Lukoscheck et al., 2007). While there is no contemporarysolid barrier to geneflow between eastern and western Australia,historically, there was a physical barrier (the Torres Strait landbridge). The Torres Strait land bridge has been documented as abarrier associated with genetic differentiation for those tropicalmarine species that display an east–west divergence.

There is no clear concordance under the current classificationof Nautilus, where potentially two to five distinct species are allgrouped under a single species—Nautilus pompilius. Based onthe data shown here, there is the distinct possibility for reclas-sification of this species into discrete sub-species. Ward (2008)has indicated that the population from the Coral Sea is currentlyunder consideration as a “dwarf” sub-species relative to the re-maining N. pompilius groups; the data presented here wouldsuggest that the West Australia group may also be considered adiscrete sub-species. Alternatively, are they not just sub-stocks?The data herein suggest that there are at least three discrete ge-netic stocks in Australia for management purposes (GBR, CoralSea, and WA). The level of genetic diversity between stocks sug-gests that individuals from the eastern and western Australianstocks of Nautilus do not intersect or overlap, thereby limitinggene flow. Given the large number of synapomorphies separat-ing eastern and western Australian stocks, there is no evidenceof contemporary gene flow between these populations, nor isthere any evidence to support gene flow between the discretepopulations of the Northern GBR and Coral Sea on the eastcoast.

The genetic diversity within populations may suggest that(i) sequence divergence accumulates slowly in Nautilus (notquantified here) and (ii) dispersal between populations ishighly restricted. With the geographic separation of the popula-tions studied here and the geophysical conditions surroundingeach population location, it is highly unlikely that mixing ofpopulations would occur. This phylogenetic analyses suggestthat Nautilus represents a polyphyletic group of currently diverg-ing lineages. The samples examined here appear to representthree divergent populations. The bio-geographic groupings ofthe samples in the phylogenetic and MSTs (Figures 2 and 3) in-dicate that evolutionary diversification is underway, with each of

reviews in fisheries science vol. 19 1 2011

58 W. SINCLAIR ET AL.

Figure 3 Minimum spanning tree (MST) with number of substitutions between haplotypes indicated on connectors. Connectors with no number, indicate asubstitution value of 1.0. Locations sampled are represented by different colored fills: Black symbols represent the Coral Sea group (Osprey Shark and Bougainvillereefs); Light Grey symbols represent the Northern GBR group (North small Detached, South Small Detached, and Mantis reefs); Dark Grey symbols represent theWest Australia group (Scott Reef).

the three distinct geographic locations represented by a distinctphylogenetic clade. Each of these clades could share a commonevolutionary ancestry (in spite of their current 3,000-km sepa-ration). This supports the hypothesis of Wray et al. (1995) andillustrates the increasing separation at the genetic level of thesepopulations.

Recent work by Bonacum et al. (2007) suggested that liv-ing Nautilus lineage originated around New Guinea, potentiallyonly two million years ago; it also suggested that one lineageof Nautilus voyaged from New Guinea to the more easterlyarchipelagos of New Caledonia, Fiji, and Samoa, while anothermade its way to Australia, Palau, the Philippines, and the SouthChina Sea. The further divergence of this Australia clade is ev-ident from the results presented here. The discrete associationof individuals from the Northern GBR group may represent anancestral group to those in the Coral Sea, as suggested previ-ously (Sinclair et al., 2007). The ancestral relationships betweenthe Northern GBR group and those from Western Australia mayalso indicate evolutionary divergence, but further investigationswould be required to ascertain which would be a possible pro-genitor population.

The number of genetically distinct individuals surviving in anendangered species is critical in terms of the amount of diversity

and evolutionary potential (Rossetto et al., 1999). Because oftheir depth limitations, and also because they rarely swim outof sight of the bottom and not in open water over depths thatwould cause them to implode, they do not easily travel to distantplaces (O’Dor et al., 1993). Hence, their behavior and biologycontribute to their separation and divergence. Perhaps fallingsea levels during previous glaciation events allowed Nautilusindividuals to travel further afield, and/or rare “sweepstake”events allowed the spread of these animals to new habitats.

The populations examined here represent a localized demo-graphic of the Australia/Papua New Guinea evolutionary lineidentified by Wray et al. (1995) as opposed to the larger geo-graphic scale they outlined. Within this scale, the genetic di-vergence can be resolved between geographically disjunct lin-eages, and as such, this study represents a microcosm of theevolutionary patterns and processes that the greater evolution-ary lineage has been under over time and goes some way toresolving the segregation of populations at this scale. Whileeach of these populations is discrete and isolated (surroundedby deep, open ocean), the possibility of movement betweenanimals of the different populations in this Australian/PapuaNew Guinean lineage still cannot be discounted, as it has beenproposed that all populations in this lineage are likely to share a

reviews in fisheries science vol. 19 1 2011

NAUTILUS SUBDIVISION AND GENETIC DIVERSITY 59

common ancestor from their founder population (Hughes et al.,2002; McRoberts et al., 2002). It has also been suggested thatthere would be movement of animals between geographicallyadjacent populations (Swan and Saunders, 1987). Similarly, thepotential for long-distance transport and cool-water survival ofNautilus has been postulated (O’Dor et al., 1993), and it may bethat such processes and opportunities become more prevalentwith the current changing patterns of climate and water dynam-ics. Furthermore, data from this study suggest that at the scale ofatoll reefs or island groups, there is population subdivision—thiscould be tested at the Rowley Shoals reef system by compar-ing reefs less than 50 km apart with those at Scott Reef that is500 km away.

The production and analysis of molecular datasets, such aspresented here, is of particular value when dealing with a uniquespecies inhabiting an often inaccessible habitat, which is in-creasingly being put under pressure both from a changing en-vironment and from the increasingly targeted nature of fishingpressure that is being applied to these isolated and discrete pop-ulations. Nautilus fishing to supply the ornamental shell tradeis on the increase (Dunstan et al., 2010), and already reducedcatch rates across large areas indicate potential population de-clines at the local level are being experienced by fishermen.This has the knock-on-effect of increased fishing effort beingapplied to already reduced populations, leading to the targetingand exploitation of new unexploited fishing areas to maintainthe increasing demand.

Dunstan et al (2010) indicated that market forces are driv-ing the ongoing development of the Nautilus fishing industryin the Philippines, and they have called for the assessment ofNautilus species by the IUCN, potentially to have it includedwith a Red List categorization as globally endangered. The datapresented in this article supports the inherent vulnerability ofNautilus populations to fishing pressure and illustrates that inwaters around Australia, the degree of genetic variation is sopronounced that if populations in these areas were to be tar-geted by commercial fishermen, there is a high risk of losingsome of the unique genetic resources in these disparate popula-tions (possibly even unique species and sub-species within theNautilus group).

Earlier work (Sinclair et al., 2007) indicated that there wasgenetic variation between Nautilus populations in the Coral Seaand those on the Northern GBR; this study indicates that thelevel of genetic variation reported by Sinclair et al. (2007) isdwarfed by the degree of genetic variation apparent betweenpopulations on the east and west coasts of Australia, a patternthat needs to be explored more completely both at the inter- andintra-population level. Research is required into the effects offishing efforts on the overall structure of Nautilus populations indiscrete geographic locations and into the degree of genetic di-vergence among the ancient lineages of these animals. Only bygaining such information is there hope to establish a regulatedand sustainable fishery for Nautilus and to maintain the range ofgenetic variation within the Nautilus group as a whole. Deter-mining the effects of fisheries, changing environmental impacts,

and changing climate will have on the ongoing survival (and ba-sic biology) of one of the oceans most enigmatic and iconicinhabitants is required to ensure the sustainable development ofany fisheries for these species.

ACKNOWLEDGMENTS

The authors are indebted to the biologists and crew of Un-dersea Explorer—in particular, Louise, Chris, Brendon, Karl,and Andy—for their help in obtaining and recording all the tis-sue samples used in this study and to both Euan and Douglasin recording samples. The authors sincerely thank their col-leagues Dr. Christine McPhie and Professor Graham Pegg fortheir constructive comments and assistance in the preparationof this manuscript. Craig Skepper and Ben Rome are gratefullyacknowledged for their assistance in the collection of WesternAustralia samples used in this study. This work was funded inpart by CQUniversity via a Faculty of Sciences, Engineering andHealth Faculty Research Grant (awarded to BS). Animal cap-ture and release procedures were carried out under the auspicesof CQUniversity ethics permit number A08/04-231.

REFERENCES

Anderson, F. E. Phylogeny and historical biogeography of the lolig-inid squids (Mollusca: Cephalopoda) based on mitochondrial DNAsequence data. Mol. Phylogenet. Evolut., 15: 191–214 (2000).

Applied Biosystems. Primer Express Software v2.0 applications-basedprimer design software user’s manual. Foster City, CA: AppliedBiosystems (2001).

Aspden, W., G. Pegg, L. Briskey, and W. Sinclair. Species–specific PCRprimers for the mitochondrial genome control region hypervariableregion 1 of the reef fish Lutjanus sebae. Mol. Ecol. Notes, 6: 499–501(2006).

Basil, J. A., I. Bahctinova, K. Kuroiwa, N. Lee, D. Mims, M. Preis,and C. Soucier. The function of the rhinophore and the tentacles ofNautilus pompilius L. (Cephalopoda, Nautiloidea) in orientation toodour. Marine Freshwater Behav. Physiol., 38: 209–221 (2005).

Bonacum, J., N. H. Landman, R. H. Mapes, M. M. White, A. J. White,and J. Irlam. The evolutionary radiation of modern Nautilus. SeventhInternational Symposium Cephalopods – Present and Past, Sapporo,Japan (2007 September).

Bonnaud, L., C. Ozouf-Costaz, and R. Boucher-Rodoni. A molecularand karyological approach to the taxonomy of Nautilus. ComptesRendus Biol., 327: 133–138 (2004).

Boyle P., and P. Rodhouse. Cephalopods: Ecology and fisheries. Ox-ford, UK: Blackwell Science (2005).

Carlson, B. A., J. N. McNibben, and M. V. DeGruy. Telemetric inves-tigation of vertical migration of Nautilus belauensis in Palau. Pacif.Sci., 38: 183–188 (1984).

Conseugra, S., C. Garcia de Leaniz, A. Serido, M. Gonzalez-Morales,L. G. Straus, D. Knox, and E. Verspoor. Mitochondrial DNAvariation in Pleistocene and modern Atlantic salmon from the Iberianglacial refugium. Mol. Ecol., 11: 2037–2048 (2002).

reviews in fisheries science vol. 19 1 2011

60 W. SINCLAIR ET AL.

Dunstan, A, O. Alanis, and J. Marshall. Nautilus pompilius fishingand population decline in the Philippines: A comparison with anunexploited Australian Nautilus population. Fisheries Res., DOI:10.1016/j.fishres.201006015 (2010).

Excoffier, L., P. E. Smouse, and J. M. Quattro. Analysis of molecularvariance inferred from metric distances among DNA haplotypes:Application to human mitochondrial DNA restriction data. Genetics,131: 479–491 (1992).

Harrison, C. J., and J. A. Langdale. A step by step guide to phylogenyreconstruction. Plant J., 45: 561–572 (2006).

Hayasaka, S., K. Oki, K. Tanabe, T. Saisho, and A. Shinomiya. On thehabitat of Nautilus pompilius in Taon Strait (Philippines) and FijiIslands, pp. 179–200. In: Nautilus: The biology and paleobiologyof a living fossil (Saunders, W.B., and N.H. Landman, Eds). NewYork: Plenum Press (1987).

Hebert, P. D. N., A. Cywinska, S. L. Ball, and J. R. de Waard. Biologicalidentifications through DNA barcodes. Proc. R. Soc. Lond. B, 270:313–322 (2003).

Hughes, T. P., D. R. Bellwood, and S. R. Connolly. Biodiversityhotspots, centres of endemicity, and the conservation of coral reefs.Ecol. Lett., 5: 775–784 (2002).

Kumar, S., K. Tamura, and M. Nei. MEGA3: Integrated software formolecular evolutionary genetics analysis and sequence alignment.Briefings Bioinformat., 5: 150–163 (2004).

Lindgren, A. R., O. N. Katugin, E. Amezquita, and M. K. Nishiguchi.Evolutionary relationships among squids of the family Gonatidae(Mollusca: Cephalopoda) inferred from three mitochondrial loci.Mol. Phylogenet. Evolut., 36: 101–111 (2005).

Lukoschek, V., M. Waycott, and H. Marsh. Phylogeography of the olivesea snake, Aipysurus laevis (Hydrophiinae) indicates Pleistocenerange expansion around northern Australia but low contemporarygene flow. Mol. Ecol., 16: 3406–3422 (2007).

McRoberts, C. M., C. J. McClean, J. E. N. Veron, J. P. Hawkins, G.R. Allen, D. E. McAllister, C. G. Mittermeir, F. W. Schueler, M.Spalding, F. Wells, C. Vynne, and T. B. Werner. Marine biodiversityhotspots and conservation priorities for tropical reefs. Science, 295:1280–1284 (2002).

Merritt, T. J. S., L. Shi, M. C. Chase, M. A. Rex, R. J. Etter, and J.M. Quattro. Universal cytochrome b primers facilitate intraspecificstudies in molluscan taxa. Marine Mol. Biol. Biotechnol., 7: 7–11(1998).

Moltschaniwskyj, N. A., K. Hall, M. Lipinski, J. E. Marian, V. Miske,M. Nishiguchi, M. Sakai, D. Shulman, B. Sinclair, D. Sinn, M.Staudinger, R. Villanueva, and K. Warnke. Ethical and welfare con-siderations when using Cephalopods as experimental animals. Rev.Fish Biol. Fisheries, 17: 455–476 (2007).

O’Dor, R. K., J. Forsythe, D. M. Webber, J. Wells, and M. J. Wells.Activity levels of Nautilus in the wild. Nature, 362: 626–627 (1993).

Ovenden, J. Mitochondrial DNA and marine stock assessment—a re-view. Aust. J. Mar. Freshwater Res., 41: 835–853 (1990).

Pegg, G. G., B. Sinclair, L. Briskey, and W. Aspden. MtDNA barcodeidentification of fish larvae in the southern Great Barrier Reef—Australia. Scientia Marina, 70S2: 7–12 (2006).

Posada, D., and K. A. Crandall. Modeltest: Testing the model of DNAsubstitution. Bioinformatics, 14: 817–818 (1998).

Rossetto, M., P. K. Weaver, and K. W. Dixon. Use of RAPD anal-ysis in devising conservation strategies for the rare and endan-gered Grevillea scapigera (Proteaceae). Mol. Ecol., 4: 321–329(1995).

Ruth, P., H. Schmidtberg, B. Westermann, and R. Schipp. The sensoryepithelium of the tentacles and the rhinophore of Nautilus pompiliusL. (Cephalopoda, Nautiloidea). J. Morphol., 251: 239–255 (2002).

Saunders, W. B. Natural rates of growth and longevity of Nautilusbelawensis. Paleobiology, 9: 280–288 (1983).

Saunders, W. B. The role and status of Nautilus in its natural habitat:Evidence from deep-water remote camera photosequences. Paleo-biology, 12: 469–486 (1984).

Saunders, W. B. The species of Nautilus, pp. 35–52. In: Nautilus: Thebiology and paleobiology of a living fossil (Saunders, W. B., and N.H. Landman, Eds.). New York: Plenum Press (1987).

Saunders, W. B., and N. H. Landman. Eds. Nautilus: The biology andpaleobiology of a living fossil. New York: Plenum Press (1987).

Saunders, W. B., and N. H. Landman. Eds. Nautilus: The biology andpaleobiology of a living fossil, reprint with additions (Topics inGeobiology). New York: Springer (2010).

Saunders, W. B., and P. D. Ward. Sympatric occurrence of living Nau-tilus (N. pompilius and N. stenomphalus) on the Great Barrier Reef,Australia. Nautilus, 101: 188–193 (1987).

Schneider, S., D. Roessli, and L. Excoffier. Arlequin: A software forpopulation genetics data analysis. Ver. 2.000. Geneva, Switzerland:Genetics and Biometry Lab, Department of Anthropology, Univer-sity of Geneva (2000).

Sinclair, W., L. Briskey, W. Aspden, and G. G. Pegg. Genetic diversityof isolated populations of Nautilus pompilius in the Coral Sea. Rev.Fish Biol. Fisheries, 17: 223–235 (2007).

Strugnell, J., M. Norman, J. Jackson, A. J. Drummond, and A.Cooper. Molecular phylogeny of coleoid cephalopods (Mollusca:Cephalopoda) using a multi-gene approach; the effect of data par-titioning on resolving phylogenies in a Bayesian framework. Mol.Phylogenet. Evolut., 37: 426–441 (2005).

Strugnell, J. M., and A. R. Lindgren. A barcode of life database for theCephalopoda? Considerations and concerns. Rev. Fish Biol. Fish-eries, 17: 337–344 (2007).

Suzuki, T., H. Fukuta, H. Nagato, and M. Umekawa. Arginine kinasefrom Nautilus pompilius, a living fossil. J. Biol. Chem., 275: 23884–23890 (2000).

Swan, A. R. H., and W.B. Saunders. Morphological variation in Nau-tilus from Papua-New Guinea, pp. 85–103. In: Nautilus: The biol-ogy and paleobiology of a living fossil (Saunders, W. B., and N. H.Landman, Eds.). New York: Plenum Press (1987).

Swofford, D. PAUP*V4.0b10win phylogenetic analysis using parsi-mony. Sunderland, MA: Smithsonian Institution, Sinauer Associates(1998).

van Herwerden, L., J. A. H. Benzie, and C. A. Davies. Microsatellitevariation and population genetic structure of red throat emperor(Lethrinus miniatus) in the Great Barrier Reef, Australia. J. FishBiol., 62: 987–999 (2003).

van Herwerden, L., J. H. Choat, S. J. Newman, M. Lerray, and G.Hillesroy. Complex patterns of population structure and recruitmentof Plectropomus leopardus (Pisces: Serranidae) in the Indo west Pa-cific: Implications for fisheries management. Mar. Biol., 156: 1595–1607 (2009).

Ward, P. Chamber of secrets. New Scientist, 2650: 40–43 (2008).Ward, P. D. The natural history of Nautilus. London: Allen and Unwin

Press (1987).Ward, P. D., B. Carlson, M. Weekly, and B. Brumbaugh. Remote

telemetry of daily vertical and horizontal movement of Nautilusin Palau. Nature, 309: 248–252 (1984).

reviews in fisheries science vol. 19 1 2011

NAUTILUS SUBDIVISION AND GENETIC DIVERSITY 61

Ward, R. D., and B. H. Holmes. An analysis of nucleotide andamino acid variability in the barcode region of cytochromecoxidase I (cox1) in fishes. Mol. Ecol. Notes, 7: 899–907(2007).

Ward R., D., and W. B. Saunders. Allonautilus: A newgenus of living Nautiloid cewphalopod and its bearingo nphlogeny of the Nautilida. J. Paleontol., 71: 1054–1064(1997).

Ward, R. D., T. S. Zemlak, B. H. Innes, P. R. Last, and P. D. Hebert.DNA barcoding of Australia’s fish species. Phil. Trans. R. Soc. B,360: 1847–1857 (2005).

Wells, M. J., J. Wells, and R. K. O’Dor. Life at low oxygen ten-sions: The behaviour and physiology of Nautilus pompilius andthe biology of extinct forms. J. Mar. Biol. Assoc. Uk. 72: 313:328(1992).

Wooding, S. Natural selection: Sign, sign, everywhere a sign. Curr.Biol., 14: R700–R701 (2004).

Westermann, B., H. Schmidtberg, and K. Beuerlein. Functionalmorphology of the mantle of Nautilus pompilius (Mollusca,Cephalopoda). J. Morphol., 264: 277–285 (2005).

Wilkinson, C. (Ed.). Status of coral reefs of the world: 2008.Townsville, Australia: Global Coral Reef Monitoring Network andReef and Rainforest Research Centre (2008).

Williams, S. T., and J. A. H. Benzie. Evidence of a biogeographic breakbetween populations of a high dispersal starfish: Congruent regionswithin the Indo-West Pacific defined by color morphs, mtDNA, andallozyme data. Evolution, 52: 87–99 (1998).

Woodruff, D. S., M. P. Carpenter, W. B. Saunders, and P. D. Ward.Genetic variation and phylogeny in Nautilus, pp. 65–83. In: Nautilus:The biology and paleobiology of a living fossil (Saunders, W. B.,and N. H. Landman, Eds.). New York: Plenum Press (1987).

Wray, C. G., N. H. Landman, W. B. Saunders, and J. Bonacum. Geneticdivergence and geographic diversification in Nautilus. Paleobiology,21: 220–228 (1995).

reviews in fisheries science vol. 19 1 2011