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Keywords:

  • DNA barcoding;
  • Gracilaria vermiculophylla;
  • Gracilariaceae;
  • invasive species

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of interest statement
  9. References

As part of an extensive DNA-based floristic survey of marine macroalgae in Canadian waters, an unexpected sequence for a Gracilaria sp. was generated from British Columbia. Before further molecular analyses and corresponding morphological/anatomical observations this mystery sequence was temporarily entered into our database as Gracilaria BCsp. Continued sampling uncovered this species from four additional locations. A timely collaboration with international colleagues introduced sequences from the invasive Gracilaria vermiculophylla into our cytochrome c oxidase I alignments — these a perfect match to BCsp indicating that this species occurs in British Columbia. A discussion of the origin of this taxon in Canadian waters, whether natural or introduced, is provided.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of interest statement
  9. References

As an enterprise, the field of DNA barcoding (Hebert et al. 2003a, b) has embarked on establishing a substantial database of eukaryotic cytochrome c oxidase I (COI) sequences (as well as other markers) with the objective of facilitating the rapid identification of any biological specimen. Ultimately, this endeavour will provide scientists and managers worldwide with a powerful ally in the important task of species identification. An obvious practical outcome will be the ability to identify rapidly and accurately introduced species to an area. A key research initiative of the macroalgal group at the University of New Brunswick is to complete a contemporary floristic account of the marine macroalgae of Canada using the DNA barcode as a preliminary screening tool of species diversity and distribution (Saunders 2008). As part of this mandate, the red algal order Gracilariales was an obvious early target for study because it is currently known to have only three species in Canada. In the Atlantic there is Gracilaria tikvahiae McLachlan (Sears 2002), while in the Pacific Gracilaria pacifica I.A. Abbott and Gracilariopsis andersonii (Grunow) E.Y. Dawson are reported (Abbott & Hollenberg 1976; Gabrielson et al. 2006). Although generally easily distinguished from taxa in other orders (making them easy collection targets for field crews), all of these species are highly plastic in morphology presenting the possibility for cryptic diversity — ideal for assessing the utility of DNA barcoding for species identification and discovery (e.g. Saunders 2005, 2008). During screening of samples from our 2007 collecting season, a ‘new’ species of Gracilaria was uncovered that is not currently recorded in the local flora (Gabrielson et al. 2006). Here we provide molecular and anatomical evidence for putatively introduced populations of Gracilaria vermiculophylla (Ohmi) Papenfuss from five locations in British Columbia.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of interest statement
  9. References

Collection data for the samples included in this study are listed in Table 1. Total DNA was extracted as outlined in Saunders (1993, 2008). The COI-5′ region was polymerase chain reaction (PCR) amplified as outlined in Saunders (2005, 2008) using the forward primers GazF1 (Saunders 2005), GHalF (Saunders 2008), GWSF (5′ TCCCAGTCACGACGTCGT TCAACAAAYCAYAAAGATATYGG 3′) and GWSF5 (5′ ACAAAYCAYAAIGATATYGG 3′) variously combined with the reverse primers GazRI (Saunders 2005), GWSR (5′ GGAAACAGCTATGACCATG GGRTGTCCRAARAAYCARAA3′), GWSR3 (5′ GGAAACAGCTATGACCATG GGRTGTCCAAAIAAYCARAA 3′), GWSR5 (5′ TCAGGRTGNCCIAARAAYCA 3′) and COX1R1 (Saunders 2008). Amplified products were cleaned (Saunders 2005) before sequencing, which used the PE Applied Biosystems BigDye (version 3.0) kit following the manufacturer's protocol (ABI). Forward and reverse sequence reads from the respective PCR primers (sequencing for GWSF used the M13 Forward primer 5′ TCCCAGTCACGACGTCGT 3′, while the M13 Reverse primer 5′ GGAAACAGCTATGACCATG 3′ was used for GWSR and GWSR3) were edited using Sequencher 4.2 (Gene Codes Corporation), and a multiple sequence alignment was constructed in MacClade 4 (version 4.06 for OSX) (Maddison & Maddison 2003). The alignment included 151 taxa (Table 1) with 687 nucleotide positions. Analyses were conducted in paup* 4.0b10 (Swofford 2003) with distances corrected under a general time-reversible model (many models were used, but with no effect on the outcome) and neighbour joining was used to provide a visual display of COI-5′ variation within and between species.

Table 1.  Collection and taxonomic details
Taxon and voucher no.Collection details*GenBank
  • *

    All collections are from Canada unless otherwise indicated. The Portuguese samples were sent by H. Abreau. BC, British Columbia; CA, California; NB, New Brunswick; NS, Nova Scotia; NSW, New South Wales; PEI, Prince Edward Island; RI, Rhode Island; TX, Texas; BCL, B. Clarkston; DMD, D. McDevit; GWS, G. Saunders; HK, H. Kucera; KH, K. Hind; KR, K. Roy; LLG, L. Le Gall; SH, S. Hamsher.

Gracilaria pacifica
GWS002883Drift; Bamfield, Bradys Beach, BC; 08/06/2005; GWSFJ499534
GWS009064Drift; Bamfield, Bradys Beach, BC; 23/09/2007; GWS & BCLFJ499531
GWS003261Subtidal, 3 m depth; Bamfield, Dixon I., BC; 18/09/2005; GWSFJ499536
GWS010697, GWS010702, GWS010710Low intertidal on cobble, sheltered side; Bamfield, Dixon I., BC; 04/06/2008; GWS & BCLFJ499517–FJ499519
GWS003113Subtidal 6 m, on rock; Bamfield, Scotts Bay, BC; 13/09/2005; J. MortimerFJ499533
GWS008172Subtidal 5 m, on invert; Bamfield, Scotts Bay, BC; 03/06/2007; DMD, BCL, KR & SHFJ499527
GWS010627Subtidal 5 m, on invert; Bamfield, Scotts Bay, BC; 03/06/2008; KH & DMDFJ499516
GWS010561Drift; Bamfield, Trail Head, BC; 02/06/2008; BCL, DMD & KHFJ499515
GWS002826, GWS002837Shallow subtidal, on shell, sheltered side; Bamfield, Wizard I., BC; 07/06/2005; GWSFJ499532, FJ499535
GWS002979Low intertidal, on pebble in sand, sheltered; Cape Beale, Bamfield, BC; 10/06/2005; GWSFJ499524
GWS008592Low intertidal, on rock; Comox Marina Breakwater, BC; 15/06/2007; GWS, BCL, DMD & KRFJ499530
GWS004277Drift; Maple Bay, Vancouver Island, BC; 22/06/2006; GWS, BCL & DMDFJ499525
GWS008399Subtidal, 8 m depth, on rock; Beaver I., Sunshine Coast, BC; 12/06/2007; DMD & SHFJ499528
GWS008406Subtidal, 8 m depth, on rock; Beaver I., Sunshine Coast, BC; 12/06/2007; GWS, BCL & KRFJ499529
GWS009523Subtidal, 8 m depth, on cobble; Nine Mile Point, Sechelt, BC; 16/05/2008; GWS, BCL, DMD & KHFJ499521
GWS009531Subtidal, 2–3 m depth, on cobble; Nine Mile Point, Sechelt, BC; 16/05/2008; GWS, BCL, DMD & KHFJ499510
GWS009577Subtidal, 5 m depth, on rock; Posie Island, Sechelt, BC; 17/05/2008; GWS, BCL, DMD & KHFJ499522
GWS010395, GWS010404Subtidal, 9 m depth, on rock; Savoie Rocks, Hornby Island, BC; 29/05/2008; BCL, DMD & KHFJ499513, FJ499514
GWS010906Low intertidal on pebble; Stephenson Pt., Nanaimo, BC; 07/06/2008; GWS & DMDFJ499520
GWS006588Subtidal, 8 m depth, on rock; Tahsis, Nuchatliz Island, BC; 30/05/2007; BCL, DMD, KR & SHFJ499526
GWS010043Mid intertidal tangled in mud; Tahsis, Brian & Shannon's Beach, BC; 22/05/2008; GWSFJ499523
GWS010203, GWS010204Subtidal, 6 m depth, on cobble in sand; Tahsis, Rosa Harbour, BC; 24/05/2008; GWS & BCLFJ499511, FJ499512
Gracilaria salicornia
GWS002001Subtidal, 2 m depth, on reef flat; Lord Howe I., Neds Beach, NSW, Australia; 29/01/2004; GWSFJ499537
Gracilaria tikvahiae
GWS007982Intertidal on rock; Cap des Caissie, North of Shediac, NB; 17/08/2006; LLG, HK & J. UtgeFJ499548
GWS007993Intertidal on sandstone; St. Thomas, Northumberland Straight, NB; 17/08/2006; LLG, HK & J. UtgeFJ499549
GWS006960On rock; Whycocomagh Picnic Area, bras d’Or Lake, Cape Breton, NS; 09/07/2006; LLG, HK & J. UtgeFJ499542
GWS011644Drift; North Rustico, PEI; 28/07/2008; GWS, DMD, SH & M. BruceFJ499538
GWS011645Subtidal, 8 m depth, on cobble: North Rustico; PEI; 28/07/2008; GWS, DMD, SH & M. BruceFJ499539
GWS009244–GWS009247, GWS009250, GWS009251, GWS009253Oakland Beach, Greenwich Bay, RI, USA; 08/2007; C. ThornberFJ499540–FJ499544, FJ499545, FJ499546, FJ499547
RDW504 FJ499508
Gracilaria vermiculophylla
GWS004314, GWS004315Tangle mats, highly sheltered; Bamfield, Trail Head, BC; 24/06/2006; GWS, BCL & DMDFJ499616, FJ499619
GWS010562Tangle mats, highly sheltered; Bamfield, Trail Head, BC; 02/06/2008; BCL, DMD & KHFJ499556
GWS008547AHigh, on mud, estuarine region, mixed with rhodomelacean; Courtenay Estuary, BC; 15/06/2007; GWSFJ499622
GWS009238, GWS009239High, on mud, estuarine region, mixed with rhodomelacean; Courtenay Estuary, BC; 22/12/2007; BCLFJ499580, FJ499581
GWS010320Upper mid, on mud, estuarine region; Courtenay, BC; 27/05/2008; GWSFJ499554
GWS010321Upper mid, on mud attached to a pebble; Courtenay Estuary, BC; 27/05/2008; GWSFJ499555
GWS009402–GWS009404Mid intertidal on pebble near area of freshwater runoff, rare; Shoreline Park, Port Moody, BC; 12/05/2008; GWSFJ499567–FJ499569
GWS010041, GWS010044, GWS010061,Mid intertidal tangled in mud; Tahsis, Brian & Shannon's Beach, BC; 22/05/2008; GWSFJ499570, FJ499571, FJ499550
GWS010067–GWS010069Low intertidal entangled in barnacles; Tahsis, Brook north of dive jetty, BC; 22/05/2008; GWSFJ499551–FJ499553
SA19591A, SA19591B,SA19591CSobra fango, junto Fucus vesiculosus fijado a piedras; A Coruna: Marisma del Burgo, Culleredo, Ria de A Coruna, Portugal; 09/03/2008; I. BarbaraFJ499572, FJ499573, FJ499574
SA19592A, SA19592B, SA19592CSobra fango, junto a Zostera nolii; Lugo: Marisma de la ria de Foz, Foz, Portugal; 05/04/2008; I. BarbaraFJ499575, FJ499576, FJ499577
GV1, GV2Tangled on mudflat; Ria de Aveiro, Portugal; 28/01/2008; H. AbreuFJ499557, FJ499558
GVPT4Tangled on mudflat; Ria de Aveiro, Portugal; 7/2008; H. AbreuFJ499561
GVPT5–GVPT7Obtained from culture of GVPT1; Ria de Aveiro, Portugal; 03/02/2008; H. AbreuFJ499562–FJ499564
GVPT8–GVPT11Obtained from culture of GVPT4; Ria de Aveiro, Portugal; 03/02/2008; H. AbreuFJ499565, FJ499566, FJ499559, FJ499560
GWS009254–GWS009267, GWS009284, GWS009285Budlong Farm, Greenwich Bay, RI, USA; 10/2007; C. ThornberFJ499601, FJ499603, FJ499605, FJ499606, FJ499594, FJ499608, FJ499609, FJ499611, FJ499612, FJ499613, FJ499615, FJ499617, FJ499620, FJ499578, FJ499584, FJ499588
GWS009240–GWS009243Knowles Way Extension, Pt. Judith Pond, RI, USA; 08/2007; C. ThornberFJ499583, FJ499585, FJ499587, FJ499591
GWS009248, GWS009249, GWS009252Oakland Beach, Greenwich Bay, RI, USA; 08/2007; C. ThornberFJ499590, FJ499597, FJ499599
GWS009268–GWS009283, GWS009286Oakland Beach, Greenwich Bay, RI, USA; 08/2007; C. ThornberFJ499582, FJ499586, FJ499589, FJ499593, FJ499595, FJ499598, FJ499596, FJ499600, FJ499602, FJ499604, FJ499607, FJ499610, FJ499614, FJ499618, FJ499621, FJ499579, FJ499592
Gracilariopsis andersonii
GWS008211, GWS008212, GWS008216, GWS008231, GWS008248Low intertidal in sand; Bamfield, Blowhole, BC; 04/06/2007; DMD, BCL, KR & HKFJ499649, FJ499651, FJ499643, FJ499644, FJ499634
GWS000394Low intertidal, on sand-swept rock; Bamfield, Bradys Beach, BC; 30/04/1998; J.T. HarperFJ499633
GWS000644Low intertidal, on sand-swept rock; Bamfield, Bradys Beach, BC; 04/05/1999; S. DonaldsonFJ499642
GWS002285Low intertidal, on sand-swept rock; Bamfield, Bradys Beach, BC; 07/07/2004; GWSFJ499652
GWS002889, GWS002890Low intertidal, on sand-swept rock; Bamfield, Bradys Beach, BC; 08/06/2005; GWSFJ499655, FJ499653
GWS003218Low intertidal, on sand-swept rock; Bamfield, Bradys Beach, BC; 17/09/2005; GWSFJ499658
GWS004001Low intertidal, on sand-swept rock; Bamfield, Bradys Beach, BC; 15/06/2006; GWS, BCL & DMDFJ499645
GWS009065Drift; Bamfield, Bradys Beach, BC; 23/09/2007; GWS & BCLFJ499637
GWS004121Subtidal, 3 m depth, on cobble; Bamfield, Scotts Bay, BC; 18/06/2006; GWS, BCL & DMDFJ499659
GWS010612–GWS010614Subtidal, 4 m depth, on pebble in sand; Bamfield, Scotts Bay, BC; 03/06/2008; BCL & S. ToewsFJ499629–FJ499631
GWS010623Subtidal, 5 m depth, on rock; Bamfield, Scotts Bay, BC; 03/06/2008; KH & DMDFJ499632
GWS002277Lower intertidal, on Ahnfeltia, sand-swept rock; Bamfield, Tapaltos Beach, BC; 04/07/2004; HK SrFJ499650
GWS010560Drift; Bamfield, Trail Head, BC; 02/06/2008; BCL, DMD & KHFJ499628
GWS002276Mid intertidal mud flats, on pebble, sheltered; Cape Beale, Bamfield, BC; 04/07/2004; GWSFJ499648
GWS002978, GWS002980, GWS002981Mid intertidal mud flats, on pebble in sand, sheltered; Cape Beale, Bamfield, BC; 10/06/2005; GWSFJ499654, FJ499656, FJ499657
GWS004196Subtidal, 6 m depth, on rock; Otter Point, near Sooke, Vancouver Island, BC; 20/06/2006; GWS, BCL & DMDFJ499646
GWS006368Subtidal, 8 m depth, on rock; Otter Point, near Sooke, Vancouver Island, BC; 25/05/2007; DMD, BCL, KR & SHFJ499639
GWS006338Subtidal, 6 m depth, on rock; Pier, Sidney, BC; 24/05/2007; DMD, BCL, KR & SHFJ499638
GWS010380Subtidal, 6 m depth, on rock; Palliser Rock, Comox, BC; 29/05/2008; BCL, DMD & KHFJ499627
GWS008663Mid intertidal on rock; Point Holmes, Comox, BC; 15/06/2007; GWS, BCL, DMD & KRFJ499647
GWS010284, GWS010296Subtidal, 3–4 m depth, on shells & pebbles; Tahsis, Bodega Island, BC; 25/05/2008; GWS & BCLFJ499625, FJ499626
GWS006589, GWS006599Subtidal, 8 m depth, on cobble; Tahsis, Nuchatliz Island, BC; 30/05/2007; BCL, DMD, KR & SHFJ499640, FJ499641
GWS010205Subtidal, 6 m depth, on cobble in sand; Tahsis, Rosa Harbour, BC; 24/05/2008; GWS & BCLFJ499624
GWS009464, GWS009465Subtidal, 6 m depth, on pebbles; Tuwanek, near Sechelt, BC; 15/05/2008; GWS, BCL, DMD & KHFJ499635, FJ499636
Gracilariopsis longissima
Gsp1Mudflat; Ria de Aveiro, Portugal; 06/02/2008; S. BurriFJ499660

For anatomical observations, cross-sections were prepared from rehydrated material in a cryostat (CM1850, Leica), stained with 1% aniline blue in 7% acetic acid and subsequently mounted in 40–50% corn syrup. Observations were made on a Leica DFC480 digital camera mounted on a Leica DM5000B light microscope.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of interest statement
  9. References

Molecular results

Genetic barcodes were acquired for 151 representative Gracilariaceae. Sequences ranged from 661–687 bp, depending on the primer combination used, with the exception of isolates SA19591B (633 bp) and GVPT6 (642 bp), which had poor sequence quality near the primers resulting in slightly shorter reads. The 85 Canadian isolates resolved into four rather than the three expected clusters (Fig. 1). These were assignable to Gracilaria pacifica (n = 27), Gracilaria tikvahiae (five isolates from Canada joining seven from Rhode Island and one from Texas (see Table 1); n = 13) and Gracilariopsis andersonii (n = 36). The final cluster contained 17 Canadian collections from five locations that grouped with 16 plants from Portugal and 40 from Rhode Island provided by colleagues for DNA-based identification and putatively considered the invasive Gracilaria vermiculophylla (n = 73). Despite the geographical range of these collections, they had identical COI sequences except for a single substitution in GWS009239, and differed from COI data in GenBank for G. vermiculophylla from Japan by a single substitution over 510 bp available for comparison (e.g. EF434937; Yang et al. 2008). This result is consistent with the collections from international colleagues, as well as rather unexpectedly these specimens from British Columbia, being assignable to G. vermiculophylla (Fig. 1).

image

Figure 1. Unrooted phylogram generated with neighbour-joining analyses from the COI for samples included in this study.

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Distribution, ecology, morphology & anatomy

Gracilaria tikvahiae is reportedly distributed along the Atlantic coast of North America extending into the Caribbean and South America (Guiry & Guiry 2008) and is notoriously plastic in its morphology (Sears 2002). Despite this variability, our collections from all three Maritime Provinces, as well as Rhode Island and Texas, all had similar DNA barcodes indicating that a single species is present. Our collections were generally found in the drift or from the low intertidal to subtidal depths of 1–8 m in sheltered to semi-exposed (deeper collections) warmer-water habitats growing on cobble and rock consistent with published accounts (Bird & McLachlan 1992). Although highly variable in morphology, this appears to be the only species of Gracilariales in eastern Canada and thus anatomical details are not necessary for identification.

Gracilaria pacifica is reported from Baja California to southern British Columbia growing in the mid-intertidal to upper subtidal, commonly associated with sand and rock in sheltered bays and estuaries (Abbott & Hollenberg 1976; Gabrielson et al. 2006). Our collections were strictly from southern British Columbia ranging widely from Tahsis on the west coast of Vancouver Island around the southern tip of the island up to Comox and throughout the Sechelt region on the mainland (Table 1). Trips to northern Vancouver Island in 2003, 2004 and 2006, the Queen Charlotte Islands in 2003, and the Prince Rupert region in 2005 and 2006 failed to yield gracilarialean species. Our collections were found drift, or attached from mid intertidal to subtidal depths of 9 m, with plants typically attached to shells, cobble, or rock, or forming free-floating populations, commonly associated with sandy to silty bottoms, and from sheltered to semi-exposed regions. The morphology of our collections was highly variable with plants ranging from those typically associated with this species (Fig. 2), to plants more reminiscent of Gra. andersonii (Fig. 3) and G. vermiculophylla (Fig. 4), to virtually unbranched subtidal collections that defy morphological identification (Fig. 5). Thalli ranged from eight to upwards of 30 cm in height and varied in colour from yellowish to pinkish, through reddish brown to dark red. Thalli were typically branched to two or three (four) orders with the final order tending towards secund branching (Fig. 2). Branches were typically tapered distally, but only weakly proximally. In cross-section, rehydrated plants ranged from 1 to 2 mm in diameter and were composed of large medullary cells, 100 to 400 µm in diameter, surrounded by an inner cortex of one to three layers of smaller isodiametric cells, and ultimately an outer cortex of one to two layers of oval to elongate cells 7–7.5 by 12.5–17.5 µm (Fig. 6). The transition between medulla and cortex was usually gradual. Tetrasporangia were oval to oblong 25–30 by 35–45 µm.

image

Figure 2–10. Representative Gracilariaceae native to southern British Columbia. Figure 2–6. Gracilaria pacifica. Figure 2–5. Samples GWS010697, GWS010204, GWS010561 and GWS008172, respectively. Scales = a centimetre ruler. Figure 6. Close-up of the cortex. Scale = 50 µm. Figure 7–10. Gracilariopsis andersonii. Figure 7. Sample GWS008248 in typical sandy habitat. Scale = 5 cm. Figures 8 and 9. Samples GWS008211 and GWS010612, respectively. Scales = a centimetre ruler. Figure 10. Close-up of the cortex. Scale = 50 µm.

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Gracilariopsis andersonii is reportedly distributed from Baja California into southern British Columbia, from the mid-intertidal to subtidal depths of 15 m, and generally associated with moderately exposed to exposed sandy habitats, the individuals typically half-buried in sand (Abbott & Hollenberg 1976; Gabrielson et al. 2006). Our collections were commonly on low intertidal rock and partially buried in sand from the moderately exposed sand-swept beaches typically associated with this species, to subtidal collections in slightly more sheltered areas to depths of 8 m. However, we also have intertidal and subtidal collections from more sheltered locations from which G. pacifica was also obtained (Table 1). As described by Gabrielson et al. (2006), it is ‘difficult to distinguish infertile and tetrasporophytic specimens of Gracilaria and Gracilariopsis’, their advice that habitat provides the best indication of which species might be in hand, unfortunately, not passing the DNA barcode test.

The morphology was again highly variable with plants ranging from that typical for this species (Fig. 7) in sand-swept habitats, to plants more reminiscent of G. pacifica and G. vermiculophylla, to sparsely branched plants with hooked appendages (Fig. 8) to virtually unbranched subtidal collections defying assignment to a recognized species (Fig. 9). Thalli ranged from eight to upwards of 50 cm in height, were typically reddish black throughout, or faded yellowish red or green in terminal regions, and were dominantly branched to one order, these branches long and commonly overtopping the principle axis (when visible) and characteristically bearing shorter second (third) order branches. Individual branches were typically tapered distally and proximally. In cross-section, rehydrated plants ranged from 0.8 to 1.6 mm in diameter and were composed of large medullary cells, 100 to 300 (400) µm in diameter, this surrounded rather abruptly (usually) by an inner cortex of two to three layers of smaller isodiametric cells this grading to an outer cortex of two to three layers of oval to elongate cells 5–6.5 by 7.5–16.5 µm (Fig. 10). Whereas the transition between medulla and cortex was usually abrupt, the inner and outer cortex formed more of a continuum. Tetrasporangia were oval to oblong 20–25 by 37–50 µm.

Gracilaria vermiculophylla was rare (Port Moody), occasional (Courtenay and Tahsis) to locally abundant (Bamfield; see Table 1), in the low to upper intertidal zones, commonly associated with freshwater influx, and confined to muddy estuarine habitats and either attached to small pebbles or formed unattached tangled mats. Individual thalli reached from eight to upwards of 20 cm (Figs 11 and 12), but the initial collections of this species were based on much smaller plants, 1–3 cm (Fig. 13), which were overlooked in the field and collected accidentally in a clump of Neorhodomela sp. The plants were typically sparsely and irregularly branched from one to three orders, at times weakly secund, although some thalli typically bore many short spine-like branchlets (Figs 12 and 13). Rehydrated axes from collection GWS009239 were typically terete, 600 µm–1.5 mm in girth mid-thallus, being slightly thicker proximally and tapering distally. Some branch bases were slightly constricted (Fig. 14), and rehydrated thalli were weakly flaccid, purplish-brown to black in colour becoming yellowish in upper regions of larger plants.

image

Figure 11–16. Gracilaria vermiculophylla collections from British Columbia. Figure 11–13. Samples GWS009239, GWS009238 and GWS008547A, respectively. Scales = a centimetre ruler. Figure 14. Rehydrated axis from GWS009239 displaying slight constriction at the point of branching. Scales = a millimetre ruler. Figure 15. Close-up of the sharp transition to the one to two-layered outer cortex. Scale = 50 µm. Figure 16. Hair cell (GWS010321). Scale = 10 µm.

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Typically isodiametric medullary cells reached maximum dimensions centrally, 100–120 (250) µm, reducing gradually in size to a distinct outer cortex of one to two cell layers, the inner isodiametric and composed of cells 7–13 µm in diameter and the outer typically anticlinally elongated 4–7.5 µm wide and 10–15 µm in height (Fig. 15). Isolates commonly had hair cells 5 µm by 50–500 µm, which stained darkly with aniline blue, extending from outer cortical cells (Fig. 16). Tetrasporangia were oblong to oval, 20–30 by 45–50 µm, in rehydrated sections.

Yamamoto (1985; also see Terada & Yamamoto 2002) provides detailed descriptions and keys for the various species of Gracilaria in Japan. Except for being smaller in stature (Japanese thalli 20–60 cm long and 2.5–3 mm in diameter with medullary cells to 500 µm), the British Columbia collections are a reasonable match to G. vermiculophylla.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of interest statement
  9. References

Gracilaria vermiculophylla (Ohmi) Papenfuss was described from Akkeshi Bay in Kushiro Province, Japan (Terada & Yamamoto 2002) and has only recently been recognized as an invasive species, although in hindsight it has been in both North America and Europe for at least a decade. In Europe, Rueness (2005) indicates that the species was first observed on the Brittany coast in 1996 and considered as an unknown Gracilaria putatively introduced with oysters for aquaculture from Japan. Later a similar alga showed up at oyster farms near Roscoff, France, and was tentatively identified as G. vermiculophylla (Mollet et al. 1998). For Pacific North America, an unidentified Gracilaria sp. from Elkorn Slough, California (Goff et al. 1994), is likely the first encounter (Rueness 2005). Bellorin et al. (2002) provided molecular evidence that an unidentified species from Baja California was a match to Goff et al.'s collections, and subsequently confirmed its identity providing the first formal record for G. vermiculophylla from Pacific North America (Bellorin et al. 2004). On the North American Atlantic coast, Gurgel & Fredericq (2004) discuss the possible introduction of a species of Gracilaria from Japan (tentatively identified as G. tenuistipitata C.F. Chang et B.M. Xia) to Virginia, this entity later considered to be G. vermiculophylla by Rueness (2005). A year later, Thomsen et al. (2006) formally reported this species from Virginia indicating that it has been in the area since at least 1998. In the same year, Freshwater et al. (2006) reported this species from North Carolina indicating a spread in distribution.

Here we provide molecular evidence for five populations of G. vermiculophylla in British Columbia. This contrasts current perspectives on Gracilariaceae in Canada, for which only two nonparasitic species, Gracilaria pacifica I.A. Abbott and Gracilariopsis andersonii (Grunow) E.Y. Dawson), are reported in British Columbia (Gabrielson et al. 2006) and a single species, Gracilaria tikvahiae McLachlan, from Atlantic waters (Sears 2002). Whereas populations of G. vermiculophylla in Virginia (Thomsen et al. 2006) and Europe (Rueness 2005) are widely considered as introduced, those from Mexico and California, when confirmed, were not explicitly regarded as exotic, presumably considered more as an extension in our knowledge of this species’ natural distribution (Bellorin et al. 2004). It thus remains to be established whether or not G. vermiculophylla is native or introduced to British Columbia.

Although we know little about the status and distribution of this species in British Columbia, many factors lend support to the notion that G. vermiculophylla is introduced to the Pacific Coast of North America as first posited by Rueness (2005). First, multiple collecting trips by my research group resulting in some 4000 collections over the past three years (in addition to many trips before the current DNA barcoding floristic work) have uncovered this species from only five locations (although further surveys are warranted). Indeed, the extensive algal surveys throughout British Columbia by Scagel and Widdowson and their many colleagues from c. 1957 to 1990 (see Gabrielson et al. 2006) failed to detect this species. This is possibly attributable in part to the highly plastic nature of the gracilarialean taxa in British Columbia (documented above), morphologically distinct only when growing in their ‘typical’ habitat and differing in only subtle aspects of vegetative anatomy. Second, this species is negatively buoyant (Thomsen et al. 2006), which would greatly impede natural dispersal across the Pacific. Third, Wallentinus et al. (2004) indicate that the alga is hardy, surviving at least 3 weeks in salinities as low as 2 psu, and establishing that plants kept for 5 months in plastic bags in darkness at 8 °C could resume growth. Finally, this species is known to break easily and regenerate from the small fragments, is resistant to desiccation and grazing, and is not influenced by either high or low levels of light and nutrients (Thomsen & McGlathery 2007).

The relationship of putatively introduced populations in California and Mexico relative to the British Columbia populations is unknown. Given the highly disjunct distribution of the sites, it is likely that separate events were responsible for these introductions. However, introduction at one site with secondary transfers along the coast by local shipping or movement of species for aquaculture remain possibilities. Only detailed studies into the true range and distribution of this species along the Pacific coast of North America will resolve this issue, and indeed whether or not this species is native or in fact introduced to these waters.

Rueness (2005) indicates that although the vector of this species into Europe is uncertain, the association of populations in Brittany with oyster farms suggests a connection with aquaculture, with secondary invasions likely a result of local shipping. Thomsen et al. (2006, and references therein) consider that it probably also arrived in Virginia attached to oysters because it is an avid recruiter onto bivalve shells and has an ability to recover from fragmentation. There have been few reports of invasive seaweeds along the western coast of North America (e.g. see Miller (2004) and Williams (2007)) these introductions attributed variously to shipping and oyster aquaculture. Perhaps the most infamous algal introduction into British Columbia is Sargassum muticum (Yendo) Fensholt, which was reportedly brought with oysters from Japan — one of the earliest records being in 1941 from Comox adjacent our Courtenay site (Scagel 1956, 1957). This vector is also highly likely for G. vermiculophylla in British Columbia, although international shipping cannot be ruled out. Interestingly, Druehl (1973) predicted that, following the shipment by air of oyster product for aquaculture from British Columbia to France in the spring of 1972, Sargassum would establish in Europe. Druehl's words proved prophetic with the discovery of Sargassum along the coast of Britain only 1 month after his article was published (Farnham et al. 1973). The possibility that G. vermiculopylla made the same stepping-stone passage through British Columbia to Europe is worthy of investigation.

In a recent report, Miller (2004) indicates that only 12 species of seaweeds have likely been introduced to California, with three of these also in British Columbia and likely introduced as a consequence of oyster aquaculture. This report potentially adds one more to that number. However, Druehl (personal communication) recently indicated that Mazzaella japonica (Mikami) Hommersand may also be present in the waters on the east coast of Vancouver Island. We had in our database an unknown Mazzaella sp. from an area adjacent to Courtenay. In light of Druehl's comments, I took the time to identify this taxon and confirmed that it is Mazzaella japonica— perhaps another exotic species to add to the list of plants introduced via oyster aquaculture (bringing the total from three to five). Apparently, the true level of seaweed introductions as a result of this careless practice has yet to be fully appreciated.

The economic consequences are very real — the Gracilariaceae are the World's main source of agar (Rice & Bird 1990; Oliveira et al. 2000). Morphologically convergent species may have different agar yields and qualities necessitating accurate identification for utilization and marketing of product (Bellorin et al. 2004). Invasive species, overlooked or not, can have real consequences for local ecosystems and the economies dependent on them. For example, Freshwater et al. (2006) indicate that populations of Gracilaria vermiculophylla in North Carolina have become a problem for both commercial fisheries and for industries drawing water from the Cape Fear River. Beyond economics, the ecological impacts of invasive species can also be significant. Invasions of alien species are considered a major threat to biodiversity because these species can often out-compete local species, which for macroalgae can alter nursery habitat for fishes and invertebrates, reduce the penetration of light, modify biogeochemical cycles, and ultimately suffocate or even drift away with some species of shellfish (this having obvious economic impacts as well) (Thomsen et al. 2007, and references therein). Thomsen et al. (2007) discuss that the impact on ecosystems associated with invasive species is assumed to increase with the abundance of the alien. Gracilaria vermiculophylla is dominant in some Virginia communities representing more than 80% of the submerged macrophyte biomass (Thomsen et al. 2006). Considering the impact on human related ecosystem services in neighbouring North Carolina (Freshwater et al. 2006), it is likely that damage to the ecosystem itself is significant and warrants considerable study. Our sites varied widely in estimated abundance of this species, but at Bamfield it formed extensive mats appearing to dominate the algal biomass. In this location, there is clearly potential for ecological impacts and further studies are warranted.

As discussed above, species identification for the Gracilariaceae, especially of the terete forms, is difficult owing to the plasticity and overall morphological convergence of these taxa (Bellorin et al. 2004; Rueness 2005). Cryptic species and taxonomic chaos abound with identifications and distinctions difficult with almost all forms of taxonomic tools except for DNA analyses and, where applicable, crossability tests (Bellorin et al. 2004). In this light, it is not surprising that species, whether introduced or natural to an area, are commonly overlooked in floristic and ecological surveys. Thomsen et al. (2006) suggest that ‘To avoid future taxonomic confusion, we encourage researchers to create silica-gel, air dried, and/or herbarium presses as voucher specimens so that the correct identification can be confirmed using morphological and molecular analysis. Such precautions need to be undertaken to avoid similar cryptic invasions ...’. A further stipulation would be the development of a single marker as advocated by DNA barcoding (Hebert et al. 2003a, b; Saunders 2005, 2008). Multiple molecular markers are indeed essential to confirm the results of a single marker such as the 5′COI, and to complete phylogenetic assessments, but only a single marker is necessary, indeed preferred, for rapidly screening biological specimens for species assignments. Gracilaria vermiculophylla is a case in point. In using a laboratory's gene of choice, or in trying to have comparative data for what was available in the literature Goff et al. (1994; collections from California) sequenced the nuclear internal transcribed ribosomal (ITS) and the plastid Rubisco spacer regions, Bellorin et al. (2002, 2004; collections from Mexico, they also sequenced bona fide G. vermiculophylla isolates, recognizing a match to that species, but not the global invasiveness of this species) the ITS, and Gurgel and Fredericq (2004; for collections from the eastern USA) the rbcL (not comparable with any of the previous studies). Rueness (2005) generated sufficient rbcL and Rubisco spacer data to link all of the previous records together providing the first comprehensive distributional picture for this apparently highly invasive species, and also added the cox2–3 spacer to the list of available data, which was subsequently used by Thomsen et al. (2006) to compare their data to that of Rueness (2005). Chasing after molecular markers on a project-by-project basis is not a problem for the contemporary systematist, but does not lend itself to rapid species identifications by nonspecialists (ecologists, managers, etc.) interested only in the identity of a specimen. A species-rich DNA barcode database and a single COI sequence back in the mid-1990s would have facilitated identification a decade earlier and flagged this species as an invasive. This knowledge may have facilitated an early implementation of protocols to slow the spread of this species and may have prevented some of the secondary introductions throughout European waters (see Thomsen et al. 2007, p. 70).

Finally, I would add to this the importance of research funding in support of floristic monitoring by systematists trained in the use of both traditional taxonomic and contemporary molecular tools. All of the molecular databases in the world will fail to uncover invasive species, extensions in range boundaries, and endangered species if trained systematists are not funded to be out in nature monitoring species occurrence in our ecosystems.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of interest statement
  9. References

All of the collectors listed in Table 1 are acknowledged for their critical involvement in this project, as are Alexandra Johnson and Bethany Herrmann for generating the sequence data used in this report. The Bamfield Marine Sciences Centre is thanked for hosting much of the field component of this research. This research was supported through funding to the Canadian Barcode of Life Network from Genome Canada through the Ontario Genomics Institute, NSERC and other sponsors listed at http://www.BOLNET.ca. Additional support was provided by the Canada Research Chair Program, the Canada Foundation for Innovation and the New Brunswick Innovation Foundation.

Conflict of interest statement

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of interest statement
  9. References
  • The authors have no conflict of interest to declare and note that the funders of this research had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Conflict of interest statement
  9. References
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