Molluscan Studies

Journal of Molluscan Studies Advance Access published 20 October 2011

Journal of

The Malacological Society of London

Molluscan Studies Journal of Molluscan Studies (2011) 0: 1–8. doi:10.1093/mollus/eyr037

GEOGRAPHIC DISTRIBUTION AND MOLECULAR IDENTIFICATION OF A METAPOPULATION OF BLUE MUSSELS (GENUS MYTILUS) IN NORTHEASTERN NEW ZEALAND 1

Centre for Marine Environmental and Economic Research, School of Biological Sciences, Victoria University of Wellington, PO Box 600, Wellington 6140, New Zealand; and 2 Present address: Department of Biology, Simon Fraser University, 8888 University Drive, Burnaby, BC, Canada V5A 1S6 Correspondence: J.P.A. Gardner; e-mail: [email protected] (Received 12 January 2011; accepted 3 June 2011)

ABSTRACT We report here on the abundance and distribution of a metapopulation of smooth-shelled blue mussels (genus Mytilus) from northeastern New Zealand (NZ), in the latitudinal range 37–358S. Site surveys revealed the presence of blue mussels at 12 of 20 locations on the east coast of the Northland Peninsula, in the Hauraki Gulf and into the Bay of Islands region. Additional material was collected subsequently from three sites, including an estuary location in Auckland, which is the southern-most record of blue mussels in this northern region. This new record of an extensive, but patchily distributed metapopulation in northeastern NZ identifies the existence of a pronounced geographic discontinuity between 378S and 418S in the distribution of Mytilus throughout NZ. Based on its geographic isolation, we suggest that the northeastern metapopulation is genetically isolated from other more southerly distributed blue mussels in NZ. Molecular identification of the mussels was carried out by both nuclear and mitochondrial DNA RFLP assays. While the majority of mussels were identified as native Southern hemisphere M. galloprovincialis, a substantial number of individuals with a nonnative (Northern hemisphere) M. galloprovincialis mitotype were also identified. The identification of this new metapopulation composed of both northern and southern lineage M. galloprovincialis has challenging biosecurity and conservation implications. Because of the pronounced geographic break in the distribution of blue mussels in NZ it may be possible to minimize further spread of the northern lineage mitotype to the rest of the country using an internal borders containment approach. However, based on introductions of (Northern hemisphere) M. galloprovincialis elsewhere in the world, we believe that the most likely outcome in NZ of this bioincursion is extensive interbreeding and introgression between the northern and southern lineages of M. galloprovincialis.

INTRODUCTION Smooth-shelled blue mussels of the genus Mytilus are widely distributed in an anti-tropical fashion throughout many regions of the world (Hilbish, Bayne & Day et al., 2000; Gardner, 2004). These mussels are a key component of the intertidal region and play a major role in transferring energy ( particulates from the water column) to the intertidal benthic community and thereby making secondary production available to predators (Stephenson & Stephenson, 1949; Gardner, 2000, 2002; Rilov & Schiel, 2006; Menge et al., 2007). In many parts of the world, including Europe, North America, South America, Australia, the Far East including Russia, China and Japan, blue mussels are extensively cultivated and form the basis of a large and economically important industry (Bayne, 1976; Smaal, 1991). More recently, the Mediterranean mussel

Mytilus galloprovincialis Lamarck, 1819 has been recognized as an important bioinvasive species based on its prodigious capacity to spread to and establish in areas beyond its native range, and to out-compete native species (Geller, 1999; Lowe et al., 2000; Shinen & Morgan, 2009). Thus, blue mussels are ecologically and economically very important and have been extensively studied wherever they occur. Members of the genus Mytilus are distributed throughout New Zealand (NZ), from the Bay of Islands in the north (358S), as far south as Campbell Island (528S). This includes all offshore (sub-Antarctic) islands, Stewart Island, the South Island and the lower North Island as far north as 418S (Powell, 1955; Morton & Miller, 1968). This distribution spans 178 of latitude (1,800 km) and includes subtropical/warm temperate, cold temperate and subantarctic waters (Gardner, 2004). There is a major discontinuity in

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JONATHAN P. A. GARDNER 1 AND KRISTEN M. WESTFALL 1,2

J. P. A. GARDNER AND K. M. WESTFALL

MATERIAL AND METHODS

distribution from 418S to 358S, with only three contemporary valves (two from the Bay of Islands and one from Great Barrier Island) being held in national collections (Gardner, 2004) and with only one published reference to the existence of “. . . interesting and isolated northern records . . .” for this species (Morton & Miller, 1968: 395). For a widespread, ecologically and economically important species this absence of knowledge is surprising and may even represent the last undescribed occurrence at a large spatial scale (100s km of coastline) occurrence of native blue mussels anywhere in the world. The reason for this discontinuity is unclear because much of the region is suited to blue mussels and is occupied by other mussels, including the endemic greenshell mussel Perna canaliculus (Gmelin, 1791) and the little black mussel Limnoperna pulex (Lamarck, 1819), which occur sympatrically with Mytilus species elsewhere in NZ (Morton & Miller, 1968; Gardner, 2004). Several authors have suggested that Mytilus in the Southern Hemisphere may be endemic. Such suggestions have been based on appraisals of fossil valves, molecular phylogenies and/or putatively diagnostic allozyme or molecular markers (McDonald, Seed & Koehn, 1991; Seed, 1992; Sanjuan, Zapata & Alvarez, 1997; Daguin & Borsa, 2000; Hilbish et al., 2000; Gardner, 2004; Ge´rard et al., 2008; Westfall & Gardner, 2010). Mytilus-like valves have been found in the fossil record in NZ dating back to about 1.0 Ma (Gardner, 2004 and references therein) and molecular phylogenetic dating has placed the Southern Hemisphere origin of M. galloprovincialis between 0.84 Ma (Ge´rard et al., 2008) and 1.2 Ma (Hilbish et al., 2000). Gardner (2004) conducted morphometric analyses on three valves from an isolated NZ population in the Bay of Islands. These valves exhibited greatest morphometric affinity to Northern Hemisphere Mytilus edulis Linnaeus, 1758, whereas other NZ blue mussels (contemporary and middens) from 19 offshore and/or remote locations exhibited greatest affinity to M. galloprovincialis. While Gardner (2004) was careful not to ascribe these three valves to M. edulis, he noted that this region of NZ required further examination to elucidate the taxonomic status, possible origin and extent of this disjunct population. One possible explanation for the existence of the northeastern metapopulation in isolation from all other NZ blue mussels is that it has been introduced. Gardner (2004) noted that the area of the Bay of Islands has a long history of shipping; from the earliest days of European arrival in NZ many vessels called in at locations such as Russell (e.g. Hough, 1995). Thus, it is possible that the Bay of Islands population may be of Northern Hemisphere origin. The purpose of the present paper is to describe the spatial distribution and frequency of occurrence at 23 sites of smoothshelled blue mussels in a metapopulation in northeastern NZ. In the context of a Southern Hemisphere investigation of blue mussel taxonomic identity, data for six of the present populations (MB, TB, TP, WA, OA and WI; Fig. 1) have already been published (Westfall & Gardner, 2010). The present paper adds a further nine populations for molecular analysis and a further 17 populations for distribution/abundance data. Expanding the present extremely sparse knowledge about the ecological distribution of blue mussels in northern NZ is important in the context of identifying the geographic distributions of NZ’s coastal taxa. To increase our understanding of Mytilus species in NZ we apply molecular assays to identify individuals to species and also to lineage within the species M. galloprovincialis. This dual nuclear and mitochondrial DNA approach to classification allows us to ascertain the status of the northeastern NZ mussels (native, introduced, hybrids). Finally, our results are discussed briefly in a management context to help conserve NZ’s native marine biota.

Site surveys

Sample collection All samples were collected live, preserved in 70% ethanol and stored at 48C. Additional to the above 14 populations, two other samples were obtained from colleagues: 35 Mytilus from Paihia Beach and 10 Mytilus from Opua Marina. Although these samples were not obtained from a random site survey (as described above), they do provide additional data points of occurrence for blue mussels, as well as an opportunity to determine their taxonomic and genetic diversity. These 16 newly collected or obtained samples were supplemented by samples from six northern populations (MB, TB, WA, TP, OA and WI; Fig. 1) collected by us and surveyed for blue mussels as described above, and by mussels from Maungamaungaroa Estuary, Auckland, which were collected by a colleague (Westfall & Gardner, 2010). In total, we report here information for 23 populations or sites.

DNA extraction Molecular methods follow those of Westfall & Gardner (2010) and Westfall, Wimberger & Gardner (2010). Approximately 100 mg of mixed posterior adductor muscle and mantle tissue was dissected and total genomic DNA extracted using a modified protocol of the DNeasy Extraction Kit (Qiagen). After tissue digestion with Proteinase K, 50 ml of saturated KCl solution was added to the samples and put on ice for 10 min to solidify the mucopolysaccharides. Centrifugation at 13,000 rpm for 10 min concentrated the solidified mucopolysaccharides to the bottom of the tube and the supernatant was transferred to a clean microcentrifuge tube to complete the DNeasy kit protocol.

Molecular identification of species and lineages Me15/16: The amplified fragments of the Me15/16 PCR assay correspond to species-specific taxonomic status (Inoue et al., 1995). The fragments are 126 bp in M. galloprovincialis, 168 bp 2


A total of 14 sites in northeastern NZ (north of Auckland at 378S) were visited to determine if blue mussels of the genus Mytilus occurred there (Fig. 1, Table 1). Because at the time of surveying there was very limited information about where or even if blue mussels might occur north of Auckland, we deliberately surveyed as many sites of as many different types (soft shore, hard shore, estuarine, volcanic rock, sedimentary rock etc.) as possible to give maximum spatial and habitat coverage. Depending on the state of the tides, two people visited either two or three different sites per day and spent 1 –2 h at each site searching the intertidal zone for blue mussels. Because mussels were either absent or at very low numbers per site the survey approach employed was qualitative. We did not use transects or quadrats (most of which would have returned zero values) but actively searched for blue mussels in locations and habitats that from experience we know are favoured by blue mussels. Under the circumstances, this qualitative approach is the most time- and cost-effective method of describing the occurrence and extent of the metapopulation across multiple sites. Records of blue mussel occurrence were made for all sites. Where possible all structures such as piers, wharves, pilings, buoys and moorings were actively searched. A photo record of each site and its biota was compiled. The survey was specifically targeted at the east coast of the Northland Peninsula and ranged from a southern limit of 36.478S to a northern limit of 35.008S.

MYTILUS METAPOPULATION IN NE NEW ZEALAND

in Mytilus trossulus Gould 1850 and 180 bp in M. edulis (Inoue et al., 1995). PCR conditions were optimized from the published protocol (Inoue et al., 1995) to the following: 948C, 4 min; (948C, 30 s; 568C, 30 s; 728C, 90 s) ! 40; 728C, 4 min; 48C, hold and [MgCl2] ¼ 1.75 mM. Me15/16 RFLP: This assay uses the restriction endonuclease AciI to digest the Me15/16 amplicon for M. galloprovincialis, an assay first introduced by Santaclara et al. (2006). Based on assay and direct sequencing results from this investigation, the M. galloprovincialis amplicon of 126 bp contains a single restriction site for AciI resulting in fragments of 77 and 49 bp. However, in most individuals genotyped as Southern Hemisphere M. galloprovincialis by the 16S RFLP, there is a single-point mutation that causes the cut site for AciI to disappear. Therefore, two fragments are generated for the Northern Hemisphere M. galloprovincialis at 77 and 49 bp, while the Southern M. galloprovincialis Me15/16 amplicon remains uncut at 126 bp. The taxonomic discrimination of the Me15/16 RFLP assay is limited to the Northern and Southern

Hemisphere lineages of M. galloprovincialis. Perfect matches between mitochondrial (16S) and nuclear (Me15/16) taxonomic assignments do not occur in all individuals due to the introgressive hybridizing nature of Mytilus species (see Discussion). Phylogenetic analysis supports Me15/16 RFLP taxonomic assignments from individual mussels where assay results match (with 16S RFLP assay) (results not shown), supporting the use of this assay as a diagnostic tool. Glu-5 0 : The amplified fragments of the Glu-50 assay correspond to taxonomic status, but produce more than one fragment per species (the Glu-50 assay is derived from the same gene as the Me15/16 assay). PCR conditions are provided by Rawson, Joyner & Hilbish (1996). The fragments produced for M. edulis are at 350 and 380 bp. Mytilus trossulus produces a single amplicon at 240 bp. For M. galloprovincialis two amplicons at 300 and 500 bp are produced and additional minor fragments are also sometimes amplified, but these are not considered to be important or informative (e.g. Rawson et al., 1996; Apte et al., 2000). 3


Figure 1. Map of sample locations on the North Island of New Zealand, codes are listed in Table 1. The inset is a detailed map of the Bay of Islands region. Filled circles are sites from which mussels were collected; open circles are sites at which no mussels were found (refer to Table 1 for details).

J. P. A. GARDNER AND K. M. WESTFALL Table 1. Site survey information including presence/absence, number of Mytilus specimens collected, date of collection, geographical coordinates of sites and map codes corresponding to Figure 1. Date

Site

Map code

Latitude, longitude ′

Number of Mytilus collected ′

05.10.05

Tauranga Bay

TA

35800.303 S, 173847.157 E

05.10.05

Matauri Bay

MB

35801.726′ S, 173855.034′ E

05.10.05

Whangaroa Harbour

WH

35802.441′ S, 173843.733′ E

Absent 15

Absent 2

05.10.05

Okura Bay Road, Totara North

OB

35802.449′ S, 173843.769′ E

04.10.05

Te Tii Bay, Te Puna Inlet

TB

35808.768′ S, 174800.258′ E

30

04.10.05

Oihi Bay

OH

35810.172′ S, 174805.710′ E

Absent

Dove’s Bay

DB

35811.789′ S, 174801.860′ E

54

Wharau Road, Kerikeri Inlet

WR

35812.382′ S, 174802.721′ E

112

04.10.05

Scudder’s Beach Road, Kerikeri Inlet

SB

35812.406′ S, 173858.971′ E

Absent

03.10.05

Kerikeri Inlet settlement

KI

35812.434′ S, 174801.743′ E

Absent

06.10.05

Rawhiti

RA

35814.109′ S, 174815.602′ E

12

06.10.05

Russell

RH

35815.571′ S, 174807.195′ E

42

06.10.05

Head of sheltered bay

BI

35815.950′ S, 174813.065′ E

Absent

06.10.05

Taupiri Bay

TP

35816.427′ S, 174817.421′ E

5

06.10.05

Waitangi

WA

35816.718′ S, 174805.383′ E

30

27.01.02

Paihia Beach

PB

35816.820′ S, 174805.464′ E

35

01.12.05

Opua Marina

OM

35818.668′ S, 174807.208′ E

10

07.10.05

Oakura Bay

OA

35822.975′ S, 174820.890′ E

02.10.05

Whangarei Heads

WG

35845.863′ S, 174823.454′ E

07.10.05

Waipu Cove

WC

36801.829′ S, 174830.516′ E

9

13.04.09

Waiheke Island, Hauraki Gulf

WI

36847.272′ S, 175805.264′ E

33

12.04.09

Rangitoto Island, Hauraki Gulf

RI

36847.618′ S, 174853.793′ E

Absent

18.10.06

Maungamaungaroa Estuary, Auckland

ME

36853.284′ S, 174859.219′ E

16S RFLP: Following Westfall et al. (2010) an RFLP assay based on the 16S rRNA gene was used to differentiate between the Southern and Northern Hemisphere monophyletic sister groups of M. galloprovincialis (characterized by haplotype D from Hilbish et al., 2000). There are four taxonomic groups identified by this assay: (1) Southern Hemisphere M. galloprovincialis has fragments at 342, 167 and 28 bp; (2) M. galloprovincialis unique haplotype has fragments at 342 and 195 bp; (3) shared M. edulis/M. galloprovincialis haplotype has fragments at 342, 85, 82 and 28 bp; and (4) M. trossulus has fragments at 370, 85 and 82 bp. For samples displaying the shared M. edulis/M. galloprovincialis haplotype, the Me15/16 marker was applied to determine species status as described by Westfall et al. (2010). The efficacy of the 16S RFLP assay has been demonstrated by matching assay results to phylogenetic placement of 135 sequences (inclusive of all M. edulis complex taxa from locations throughout the world) (Westfall et al., 2010).

1 Absent

4

found at (and therefore assumed to be absent from) 8 of 20 sites (Table 1) despite several apparently suitable habitat qualities (e.g. substrate type, wave exposure).

Molecular identification of species and lineages Me15/16 and Glu-5 0 markers: A total of 49 mussels were genotyped from nine sites in northeastern NZ (Table 2). The Glu-50 marker was used to identify the taxonomic status of three individuals where amplification of Me15/16 was difficult, one from Russell Harbour and two from Rawhiti (both Bay of Islands, northern NZ). These mussels were added to those described by Westfall & Gardner (2010) to give 146 mussels from 15 sites. All individuals were identified as M. galloprovincialis (Table 2). 16S RFLP: The results of this assay revealed the presence of Northern Hemisphere (nonnative) M. galloprovincialis mussels at sites in northeastern NZ. A total of 16 mussels from six sites were successfully genotyped (Table 2). Varying proportions of Northern Hemisphere M. galloprovincialis genotypes (MgN; Table 2) were observed at five of the sites. Combined with the data of Westfall & Gardner (2010) this gave a total of 107 mussels from 10 sites. In the surveyed region, 24 of 73 individuals (¼33%) were of Northern Hemisphere origin. Me15/16 RFLP: A total of 12 individuals from five sites were genotyped. These mussels were added to those described by Westfall & Gardner (2010) to give 103 mussels from nine sites. All individuals exhibited only Southern Hemisphere M. galloprovincialis genotypes at all populations except Waiheke Island, at which Northern hemisphere M. galloprovincialis genotypes were observed (Table 2).

RESULTS Site surveys Combining the new site survey information of the present study with the data of Westfall & Gardner (2010), smooth-shelled blue mussels of the genus Mytilus were collected from 12 of 20 sites. In the Bay of Islands region, blue mussels were found at 10 of 16 sites, north of Auckland at 1 of 2 sites and on islands in the Hauraki Gulf at 1 of 2 sites. The most northerly site at which blue mussels were found was Matauri Bay (35801.7260 S). Mussels were collected from a variety of different environments and habitat types, ranging from soft shore estuarine to exposed rocky reef. At some sites such as Matauri Bay (n ¼ 2 mussels), Taupiri Bay (n ¼ 5) and Oakura Bay (n ¼ 1) the blue mussels occurred at very low numbers. At other sites such as Wharau Road (n ¼ 112), Dove’s Bay (n ¼ 54) and Waiheke Island (n ¼ 33) the mussels were moderately abundant. Mussels were not

DISCUSSION The data presented here build on recent work describing the taxonomy and phylogeography of Southern Hemisphere blue 4


04.10.05 03.10.05

MYTILUS METAPOPULATION IN NE NEW ZEALAND

Region

Bay of Islands

Site (code, Fig. 1)

16S MgN

1

MgS/MgS

2

—

—

Te Tii Bay (TB)

8

MgS/MgS

MgN

22

MgS/MgS

MgS

1

MgS/MgS

MgN

3

MgS/MgS

MgS

2

—

—

Wharau Road (WR)

4

—

MgS

2

—

—

Rawhiti (RA)

4

—

—

Russell Harbour (RH)

3

MgS/MgS

MgS

6

—

—

5

—

—

10

MgS/MgS

MgN

14

MgS/MgS

MgS

6

MgS/MgS

MgS

Paihia Beach (PB)

6

—

—

Opua Marina (OM)

5

—

—

1

MgS/MgS

MgS

Oakura Bay (OA)

1

MgS/MgS

MgN

Waipu Cove (WC)

3

MgS/MgS

MgS

3

—

—

2

MgN/MgN

MgN

Waitangi (WA)

Auckland

Auckland

Me15/16

Matauri Bay (MB)

Taupiri Bay (TP)

Hauraki Gulf

RFLP haplotype

Okura Bay Road (OB)

Dove’s Bay (DB)

North of

n

Waiheke Island (WI)

Maungamaungaroa

1

MgN/MgN

MgS

13

MgS/MgS

MgN

2

MgS/MgS

MgS

9

MgS/MgN

MgN

3

MgS/MgN

MgS

4

—

—

Estuary (ME) n, number of individuals.

mussels at large spatial scales (Westfall & Gardner, 2010) using a newly developed marker system (Westfall et al., 2010) in conjunction with existing diagnostic approaches.

Distributional occurrence of blue mussels in northeastern New Zealand We report the occurrence at low abundance of smooth-shelled blue mussels at multiple sites in northeastern NZ. The collection of blue mussels from an estuary in Auckland and our findings of Mytilus at 12 of 20 sites indicate that these mussels are not uncommon at a large spatial scale (in the region 378S to 358S) on the east coast of the North Island of NZ. We have added extensive new site information to the limited information from northeastern NZ reported by Westfall & Gardner (2010). We were unable, because of time constraints, to survey sites on the west coast of the North Island or to extend our survey north of 358S. Thus, our surveys most probably do not cover the full spatial extent of the distribution of these blue mussels. We anticipate further fieldwork to address this point. Nonetheless, our new data provide evidence of an undescribed metapopulation of blue mussels in northeastern NZ. Blue mussels were collected from a variety of different environments and habitat types, ranging from soft shore 5


estuarine to exposed rocky reef. At some sites such as Matauri Bay (n ¼ 2 mussels), Taupiri Bay (n ¼ 5) and Oakura Bay (n ¼ 1) the blue mussels were rare (total search period of 4 h at each site), indicating very small site-specific population sizes. Given their rarity, these mussels may well be best considered to be opportunistic settlers. However, despite their rarity, these individuals were .30 mm shell length, indicating that, at least under certain conditions, these sites can support blue mussels beyond a single recruitment event. At other sites such as Wharau Road (n ¼ 112), Dove’s Bay (n ¼ 54) and Waiheke Island (n ¼ 33) the mussels occurred at greater abundance (although not at levels of abundance recorded elsewhere in southern NZ or other parts of the world, e.g. Stephenson & Stephenson, 1949; Gardner, 2000) were often large in size and well established. The size range of mussels encountered at these sites and the number of individuals suggest that more than one successful recruitment event had occurred during the last 36 months and that the site-specific environments are conducive to the maintenance of mussel populations. Blue mussels were also collected from sites such as Te Tii Point, which is characterized by very high levels of water column turbidity and extensive patches of fine sand/mud overlaying a rocky reef. Mussels were collected from the outer reef edge in less silty conditions, but also were found in a semi-infaunal state attached to small rocks/large pebbles or to oyster (Crassostrea gigas) shells. At many sites, mussels were only collected from small rock pools rather than from rock exposed to air at low tide, suggesting that rock pools may act as ecological refugia for mussels at these locations. Inevitably for a rare species, detailed sampling of microhabitat type was not possible given the very low numbers of individuals encountered at most sites and the paucity of meaningful environmental data that such numbers would generate. Blue mussels were absent from sites such as Whangarei Heads (soft shore, estuarine habitat, with many C. gigas and numerous gastropods such as Nerita melanotragus, Turbo smaragdus and Diloma aethiops), Kerikeri Inlet settlement (volcanic rock outcroppings covered with C. gigas and numerous gastropods), and Scudder’s Beach Road (estuarine environment, very soft fine sand/mud, a C. gigas) (Fig. 1). Sites such as Oihi Bay (large sedimentary rock outcropping, poorly developed intertidal community, small patch of Limnoperna pulex, at the top of the shore) and Tauranga Bay (sedimentary rocky outcroppings at north and south end of beach, with moderately welldeveloped intertidal community) had no detected blue mussels on apparently appropriate hard substrates for attachment. These northern NZ populations on the east coast of the Northland Peninsula and extending as far south as the Hauraki Gulf and Auckland are geographically isolated from the majority of blue mussel populations in NZ, which occur with increasing frequency further south (Morton & Miller, 1968; Gardner, 2004). Our data identify the existence of a pronounced distributional break (geographic discontinuity) between intertidal regions of 378S and 418S in the distribution of blue mussels in NZ. As such, we suggest that the northeastern populations constitute a metapopulation in the sense that there is likely to be genetic connectivity among them, but there may well be reduced or even completely absent genetic connectivity (i.e. in the sense of gene flow in the form of larval exchange) between them and populations further south. A microsatellite analysis of population genetic structure among NZ blue mussel populations is currently underway to test this hypothesis. Previous records of contemporary Mytilus species in northern NZ are restricted to three valves from two sites (Gardner, 2004). Our identification of blue mussels at many more sites in northeastern NZ than previously recognized is an unusual report of a new distributional occurrence for a species which is

Table 2. Haplotype information for Mytilus collected from northern New Zealand (sites are ordered north to south).

J. P. A. GARDNER AND K. M. WESTFALL widely studied and about which much is known in terms of its ecology and distribution. While we can speculate why the occurrence and distribution of blue mussels in northeastern NZ was unrecorded ( poor general knowledge about the coastal marine biota of northeastern NZ; blue mussels present in areas where presence was not previously detected), we cannot know for sure. Regardless, what is interesting in an ecological sense is why there are not more blue mussels in northeastern NZ given that the intertidal habitat at many sites is apparently suitable for them, that blue mussels have been described from comparable latitudes elsewhere in the world (reviewed by Koehn, 1991; Gosling, 1992; Seed, 1992) and that blue mussels generally, and M. galloprovincialis in particular, are such successful invasive species with the ability to establish new populations and to out-compete and/or displace native biota (Geller, 1999; Apte et al., 2000; Lowe et al., 2000; Elliott et al., 2008). The occurrence of moderate-sized (.30 mm length) and reasonably well-established mussels (likely to be 12 –24 months of age) at many sites, even if only as isolated individuals, indicates that blue mussel larvae arrive at these shores, can settle and recruit successfully, and can survive for ecologically meaningful periods of time. This raises the possibility that we may be witnessing the establishment of a new Mytilus metapopulation in a region where none previously existed. Alternatively, ecological conditions in this region may be borderline for blue mussels and our report of the metapopulation may reflect the historical situation in which small, isolated populations have existed, but gone unrecognized, for a long period of time. Only further monitoring of these populations can answer this.

documented among hybrids within the M. edulis species complex in North America (Rawson & Hilbish, 1995; Rawson, Agrawal & Hilbish, 1999) and Europe (Bierne et al., 2003; Kijewski et al., 2006). Although varying degrees of introgression characterize specific hybrid zones and may also occur asymmetrically (i.e. from one species to another but not vice versa) (Rawson & Hilbish, 1998), the exchange of any genetic background may enable exploitation of marginal habitats not familiar to the parental species and, hence, possible niche expansion for taxonomically intermediate individuals. Due to their close taxonomic proximity and the introgressive hybridization precedence set in the M. edulis species complex, there is a strong possibility that introgressive hybridization is occurring among hemispheric lineages of M. galloprovincialis present in northeastern NZ. The consequences of such introgressive hybridization to habitat exploitation and niche expansion need to be explored.

The identification of invasive Northern Hemisphere mitotypes in the Bay of Islands region presents two main conservation problems. The first is to try to minimize the spread of the Northern mitotype throughout the region. Realistically, this is unlikely to be achieved, both because of the biology of mussels (external fertilization; millions of larvae produced by a population; long-lived pelagic larval dispersal phase), and because of the extensive movement of personal watercraft and other possible vectors within the region. The second is to prevent gene flow between the northern region and other regions of NZ, which may be more achievable if an active biosecurity approach based on the use of internal borders (Forrest, Gardner & Taylor, 2009) is applied. However, any approach to preventing movement of the northern mitotype further south is likely to be patchily successful at best, and is already compromised, at least in part, by the occurrence of the northern mitotype at low frequency (much lower than reported here in the north of NZ) at a few southern NZ locations (Westfall & Gardner, 2010). Extensive documentation of natural occurrences of sympatry and of the effects of accidental introductions highlights two main points. First, the competitive and dominant nature of Northern Hemisphere M. galloprovincialis in an ecological sense, as it often out-competes and displaces other intertidal taxa, including native blue mussels (e.g. Wilkins, Fujino & Gosling, 1983; Grant & Cherry, 1985; Lee & Morton, 1985; Geller, Carlton & Power, 1994; Geller, 1999; Schneider & Helmuth, 2007; Elliott et al., 2008; Hanekom, 2008; Shinen & Morgan, 2009). Second, the extensive interbreeding that occurs among conspecifics, which may be accompanied by varying degrees of introgression (e.g. Skibinski, 1983; McDonald & Koehn, 1988; McDonald et al., 1991; Vaïno¨la¨ & Hvilsom, 1991; Hilbish, Bayne & Day, 1994; Gardner, 1996; Suchanek et al., 1997; Rawson et al., 1999; Bierne et al., 2002; Brannock, Wethey & Hilbish, 2009; Doherty, Brophy & Gosling, 2009). At the moment it is too soon to know what the likely ecological or molecular outcomes may be as a consequence of the identification of the Northern Hemisphere mitotype in NZ. However, its co-occurrence with the Southern Hemisphere mitotype in NZ will provide the first opportunity to test for fitness differences (biochemical, physiological) between the two lineages to start to predict likely outcomes of their interactions (e.g. Gardner & Skibinski 1988; Gardner & Skibinski 1990a, b, 1991; Buckley, Owen & Hofmann, 2001; Bierne et al., 2006; Toro, Thompson & Innes, 2006; Schneider & Helmuth, 2007; Dutton & Hofmann, 2008; Elliott et al., 2008; Shields, Barnes & Heath, 2008; Dias et al., 2009; Shinen & Morgan, 2009). While it may not be possible to contain this

Molecular identification of species and lineages Identifications of species and lineages depend to a large extent on the power of the molecular markers to accurately and consistently differentiate among the respective groups. Inevitably, such diagnostic power increases with the advent of new technology and new markers. Based on the latest (but perhaps not the ultimate) molecular markers employed here, the nuclear Me15/16 assay identified all mussels from all populations except Waiheke Island as Southern Hemisphere M. galloprovincialis, whereas the mitochondrial marker (16S RFLP) identified a mix of Northern and Southern Hemisphere M. galloprovincialis (ratio of 20:56). Proportions of Northern to Southern Hemisphere lineages based on the 16S RFLP assay varied substantially among sites, ranging from 0 to 60% (Table 2). These data confirm the existence of Southern Hemisphere endemic blue mussels in northern NZ and confirm the occurrence of Northern Hemisphere blue mussels occurring sympatrically in NZ with their Southern Hemisphere counterparts. For all populations in the Bay of Islands area, we have observed a pronounced discordance between the Me15/16 nuclear DNA RFLP assay (all mussels have Southern Hemisphere M. galloprovincialis genotypes) and the 16S mtDNA RFLP assay [Northern Hemisphere (introduced) or Southern Hemisphere (native) M. galloprovincialis mitotypes]. This discordance between nuclear and mitochondrial DNA markers suggests one of two possibilities. First, the 16S RFLP assay itself is not perfectly diagnostic. Although the 16S RFLP is a newly developed assay and mitochondrial genes can be sensitive to homoplasy, a large spatial scale phylogenetic test across globally distributed locations and concurrence with previously published molecular work (Hilbish et al., 2000; Westfall & Gardner, 2010) suggests that the assay is indeed diagnostic. The second possibility is introgression among genomes during the hybridization process. When a hybrid is backcrossed with a pure parental species, introgression of the parental genome may take place within the progeny. This phenomenon is well 6


Biosecurity and management implications

MYTILUS METAPOPULATION IN NE NEW ZEALAND incursion of nonnative blue mussels, it may be possible to model the likely outcome and use this event to make predictions about achievable distributional patterns. Such knowledge may be useful in the wider context of the Northern Hemisphere mitotype of M. galloprovincialis having recently been reported in other Southern Hemisphere countries such as Australia and Chile, but not in Argentina or the Falkland Islands (Daguin & Borsa 2000; Ge´rard et al., 2008; Westfall & Gardner, 2010).

ACKNOWLEDGEMENTS We thank Lisa Bognuda for help in the field and Ann Wood for assistance and advice in the lab. This research was funded in part by Biosecurity NZ and the Centre for Marine Environmental and Economic Research of Victoria University of Wellington. We thank Charles Bedford for assistance with collecting mussels (Maungamaungaroa Estuary, Auckland).

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