Marine Pollution Bulletin 44 (2002) 204–210 www.elsevier.com/locate/marpolbul

Using genetic techniques to investigate the sources of the invasive alga Caulerpa taxifolia in three new locations in Australia Britta Schaffelke a,*, Nicole Murphy a, Sven Uthicke b a

CSIRO Marine Research, Centre for Research on Marine Introduced Pests (CRIMP), G.P.O. Box 1538, Hobart, TAS 7001, Australia b Australian Institute of Marine Science, PMB 3, Townsville, QLD 4810, Australia

Abstract The invasive green alga Caulerpa taxifolia has gained a high profile due to ‘outbreaks’ in the Mediterranean and California. During the year 2000 three new discrete locations colonised by abundant C. taxifolia were discovered in New South Wales (NSW), Australia. Sequencing of the internal transcribed spacer (ITS) region of the ribosomal DNA was used to explore the source(s) of these new records, which is an important prerequisite for subsequent environmental management responses. Our results indicate that the NSW C. taxifolia originated from several sources and, hence, through different invasion events. For two of the new records (Port Hacking, Careel Bay) it can be excluded that they are derived from the so-called ‘‘aquarium strain’’ of C. taxifolia, closely related to the invasive Mediterranean populations. Port Hacking is likely to have originated from tropical native populations. However, samples from Lake Conjola cannot be sufficiently distinguished with the applied technique from native C. taxifolia in Moreton Bay and the Mediterranean/‘‘aquarium strain’’. Ó 2002 Elsevier Science Ltd. All rights reserved. Keywords: Bioinvasions; Caulerpa taxifolia; Conservation; Environmental impact; Genetics; Resource management

1. Introduction Human-mediated bioinvasions have been recognised as one of the principal threats to the marine environment (e.g., Lubchenco et al., 1991; Vitousek et al., 1997; Huber, 1999). The prevalence of this ‘biological pollution’ is higher in environments, such as ports and marinas (Hewitt, unpub. data), that are subject to potential introduction vectors as well as to physical alteration and chemical pollution. A number of marine introduced species have gained a high profile because of their potential environmental impacts, especially on marine biodiversity by habitat alteration and competition with native species. One of these species is the green macroalga Caulerpa taxifolia (Vahl) C. Agardh. C. taxifolia is assumed to have been introduced to the Mediterranean Sea in the 1980s, and has since spread over about 6000 ha (Meinesz et al., 1995). Prolific growth of Caulerpa along the C^ ote d’Azur, where the introduction was first reported, has been associated with *

Corresponding author. Tel.: +61-3-6232-5407; fax: +61-3-62325485. E-mail address: britta.schaff[email protected] (B. Schaffelke).

urban wastewater pollution (Chisholm et al., 1997). Likely sources of this introduction are considered to be one or several public aquaria that displayed strains of this species, chosen for aquarium use, which subsequently escaped into the Mediterranean in 1984 (Meinesz and Boudouresque, 1996). This strain is now known as the ‘‘aquarium-Mediterranean strain’’ (Jousson et al., 2000). C. taxifolia is a popular ornamental plant in private, public, and commercial aquaria and has been traded over the Internet. It regained international attention in June 2000 after the discovery of its introduction to Californian waters (Kaiser, 2000). C. taxifolia is native to the tropical and subtropical region of Australia with southern distribution limits recorded at Moreton Bay, Southern Queensland (QLD), latitude 28 °S, with records also from the isolated offshore Lord Howe Island, latitude 31 °S (Lucas, 1935; Cribb, 1958; Lewis, 1984; Julie Phillips, pers. comm.). In May 2000, three new discrete locations colonised by abundant C. taxifolia were discovered in embayments in warm-temperate New South Wales (NSW): Port Hacking, Lake Conjola, and Careel Bay (Fig. 1) at latitudes 33–35 °S; up to 800 km further south than the nearest native population.

0025-326X/02/$ - see front matter Ó 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 5 - 3 2 6 X ( 0 1 ) 0 0 2 0 2 - 8

B. Schaffelke et al. / Marine Pollution Bulletin 44 (2002) 204–210

Fig. 1. Sampling locations of Australian isolates. Grey dots denote sampling locations of isolates sequenced by Olsen et al. (1998) and Jousson et al. (2000), respectively.

The risk of an introduction of non-native C. taxifolia to Australian waters has been recognised by the Australian Quarantine and Inspection Service with the implementation of an import ban of the species in 1996. In order to access Commonwealth and State funds for incursion emergencies (R. Thresher, CRIMP, pers. comm.) it was important to determine whether the new records were an introduction from within Australia or from overseas, in particular the aquarium-Mediterranean strain, introduced by the aquarium trade or by shipping vectors. It was less likely to be a natural range extension, as subsequent surveys have not found any C. taxifolia between the southern limit of the native distribution and the three new records (D. Grey, NSW Fisheries, pers. comm.). The identification of source populations is also essential to determine and manage likely introduction vectors. Holland (2000) emphasised the value of genetic techniques in this assignment of likely geographic sources as well as for other marine bioinvasions research such as taxonomic identifications and spread/dispersal modelling. Jousson et al. (2000) used internal transcribed spacer (ITS region of the ribosomal DNA) sequencing to identify the likely source of the Californian C. taxifolia and showed sequence identity of two Californian isolates and samples from public aquaria and the Mediterranean. The authors called for a rapid eradication of the Californian introduction. ITS sequence identity of a number of Mediterranean isolates and of samples from five public or commercial aquaria was discussed earlier as an indication for the aquarium origin of the Mediterranean C. taxifolia introduction (Jousson et al., 1998). Olsen et al. (1998) used ITS as a marker for interspecific variation in the genus Caulerpa, but subsequently suggested that it may not be suitable for population studies in C. taxifolia because it shows very little variation on a regional scale and is a very conserved (slowevolving) genomic region (Olsen et al., 1999). However, an Australian C. taxifolia isolate was consistently sepa-

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rated from the Mediterranean and overseas aquarium isolates analysed (Olsen et al., 1998), indicating that ITS sequencing may be still useful to resolve intraspecific differences on large biogeographical scales. In addition, ITS sequencing allows comparisons with a large number of published sequences of C. taxifolia, an approach taken by Darius et al. (1999), Jousson et al. (2000) and Meusnier et al. (2001). Re-analyses of available ITS sequences of C. taxifolia and genetic analyses of its associated microbial flora led to the hypothesis that Australian populations are the source of the Mediterranean invasion (Meusnier et al., 2001). This has also been postulated by Wiedenmann et al. (2001). Using analyses of allozyme variation Benzie et al. (2000) found a higher affinity of Mediterranean C. taxifolia to an Australian population from Stradbroke Island (S-QLD) than to isolates from N-QLD. In this report we used sequencing of the ITS region (i) to identify if the three new NSW records of C. taxifolia were similar to the aquarium-Mediterranean strain, (ii) to identify likely source(s) of the NSW introductions, and (iii) to study the relationships between Australian C. taxifolia isolates over a wide geographic range, including published information.

2. Materials and methods Specimens from various locations in Australia (Fig. 1, Table 1) were collected and immediately dried in silica gel. In locations with abundant C. taxifolia (Port Hacking, Lake Conjola, Careel Bay, Moreton Bay) replicate samples were taken, which were separated by at least 10 m up to
15 km. For most collections, voucher specimens are kept in the reference collection of the Centre for Research on Marine Introduced Pests (CRIMP) at the CSIRO Marine Laboratories in Hobart, Australia. Samples were ground using mortar and pestle. Tissue was crushed in 700 ll extraction buffer (50 mM Tris, pH 8.0, 0.7 M NaCl, 10 mM EDTA, 1% hexadecyltrimethylammonium bromide, 0.1% 2-mercaptoethanol) using a pellet pestle mixer (Kontes, Melbourne, Australia), prior to incubation at 65 °C for 1 h with 100 ug ml1 proteinase-k. DNA was extracted using phenol/chloroform, precipitated in ethanol and eluted in 100–200 ll of Milli-Q water (Millipore, Melbourne, Australia). A region of rDNA was amplified by polymerase chain reaction (PCR) in 50 ll volumes using 10–100 ng DNA, 800 lmol dNTPs (Promega, Wisconsin, USA) and 20 pmol of each primer (GeneWorks, Adelaide, Australia). A forward primer located in the 30 region of the small subunit (SSU) (50 -CCTCTGAACCTTCGGGAG-30 ) and reverse primer located in the 50 region of large subunit (LSU) (50 TTCACTCGCCATTACT30 ) (Jousson et al., 1998) were used in PCR. Other PCR reagents were as supplied in a Perkin–Elmer kit

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Table 1 Isolates of C. taxifolia analysed in the present study Locality

No. of replicates

Collector

Morphology

GenBank accession number

Port Hacking, NSW* Lake Conjola, NSW Careel Bay, NSW Moreton Bay, QLD* Gladstone Harbour, QLD* Arlington Reef, QLD Hastings Reef, QLD Michaelmas Reef, QLD* Sudbury Reef, QLD* Hicks Reef, QLD* Myrmidon Reef, QLD* C. sertularioides* St. Crispin Reef, QLD*

3 5 3 10 1 1 1 1 1 1 4 1

B. Schaffelke M. Miller M. Miller J. Phillips B. Schaffelke S. Uthicke S. Uthicke S. Uthicke S. Uthicke S. Uthicke S. Uthicke S. Uthicke

Delicate Robust, large Robust, large Robust, large Delicate Delicate, small Delicate, small Delicate, small Delicate, small Delicate, small Delicate, small –

AF316358 AF316356 AY 034868 to AY 034872 AF323597 to AF323599 AF316355 AF316353 AF316354 AF323601 AF3236012 AF323596 AY 034873 AF325112

Asterisks indicate that voucher specimens are held in the CRIMP reference collection. The C. sertularioides isolate from St. Crispin Reef has been used as an outgroup. QLD ¼ Queensland, NSW ¼ New South Wales.

(California, USA) and used in the following concentrations; 0.2 Units AmpliTaq Gold, 0.1 mM MgCl2 in buffer (5 mM tris-HCL, 50 mM KCl, pH ¼ 8.3). DNA was denatured at 94 °C for 30 s, primers annealed at 52 °C for 30 s with chains extended at 72 °C for 120 s for 40 cycles, with a final cycle of 72 °C for 5 min (Jousson et al., 1998). The amplified region comprised the 30 SSU, ITS1, 5.8S, ITS2 and 50 LSU and was approximately 750 base pairs in length for all samples. Each PCR product was checked by electrophoresis in a 1% agarose gel. Amplified products were purified using QIA quick spin PCR purification kits (QIAGEN, Chatsworth, USA). DNA concentration was quantified using the GeneQuant pro RNA/DNA fluorometer (Amersham Pharmacia Biotech, Cambridge, UK). PCR products were sequenced using the same primers in BigDye terminator sequencing reactions (ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit, PE Applied Biosystems, California, USA) and run on an automated sequencer (ABI Prism 377 DNA sequencer). Forward and reverse sequences were collected from all samples. Some isolates required cloning before sequencing. C. taxifolia PCR products were inserted into competent bacterial cells using the TOPO TA Cloning kit (Invitrogen, Carlsbad, USA). Colonies were cultured over night in LB medium and plasmid DNA was extracted according to a protocol modified from Birnboim and Doly (1979) using the Plasmid Buffer Set (QIAGEN, Chatsworth, USA). Primers provided in the TOPO TA Cloning kit were used for sequencing. All sequences were manually aligned using Sequence Navigator (Applied Biosystems, Perkin-Elmer, California, USA). For comparison, other published sequences of C. taxifolia were included in the alignment and phylogenetic analyses. These included the following ITS sequences obtained from GenBank (accession numbers in

parentheses): Monaco Aquarium (AJ 007822), Townsville, Australia (AJ 007823) published in Olsen et al. (1998); Martinique Island, Caribbean (AJ 228983), Stuttgart Aquarium, Germany (AJ 228976 and AJ 228977), Le Brusc, France (AJ 228971) published in Jousson et al. (1998); and Carlsbad, USA (AJ 299742), Fraser Island, Australia (AJ 299771 to AJ 299783), Noumea, New Caledonia (AJ 299784), Puerto Rico, USA (AJ 299804), Safaga, Egypt (AJ 299792) published in Jousson et al. (2000). A C. sertularioides (Gmelin) Howe sequence (Table 1) was used as the outgroup. Sequences were analysed with the software packages PHYLIP version 3.57c (Felsenstein, 1995) and DAMBE version 4.0.30 (Xia, 2000). Phylogenetic trees were calculated using maximum parsimony, maximum likelihood (using empirical base frequencies and global rearrangement) and neighbour-joining (two-parameter distance by Kimura, 1980) algorithms. Branches were tested using bootstrap resampling for 1000 replicates. Consensus trees were calculated using both majority rule consensus and strict consensus methods. Trees were drawn using the software package TreeView (Page, 1996).

3. Results The alignment comprised 720 nucleotides. To allow comparative analyses with published sequences, the alignment length was reduced to 644 bases. The sequence divergence between C. taxifolia and the outgroup C. sertularioides was 11.4% (0.5 standard deviation, S.D.). The alignment of C. taxifolia isolates alone included 107 variable and 66 parsimony-informative sites (40 transversions and 26 transitions) and substitution saturation was not indicated (p < 0:001). The average G þ C content was 44.3% (0.4).

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PCR produced single, well-defined bands and for most isolates direct sequencing resulted in sequences of good quality. One or two ambiguous alleles were found in some sequences (Lake Conjola, Port Hacking, Gladstone, Arlington Reef and two Moreton Bay isolates). This, however, did not affect the outcome of the phylogenetic analyses. Direct sequencing of Careel Bay isolates produced multiple overlying sequences. These were separated by cloning before re-sequencing, which resulted in five different genotypes. The three isolates cloned contained two, three, and four different genotypes per individual, respectively. Sequences of replicate samples from Port Hacking and Lake Conjola were identical within each of these locations. Replicate isolates were also analysed from Moreton Bay and Myrmidon Reef. Moreton Bay isolates (n ¼ 10) had four, and Myrmidon Reef samples (n ¼ 4) had three different genotypes, respectively. Coral reef isolates from the Northern Great Barrier Reef (GBR) had a large insert of 28 bases in the ITS1. An identical insert is found in published sequences of C. taxifolia from other tropical reef regions (Jousson et al., 1998, 2000), three of which we included in our alignment. The phylogenetic analyses showed that the C. taxifolia isolates from each of the three new NSW locations were consistently clustered in distinct clades, supported by bootstrap values above 50% (Fig. 2). The clade comprising isolates from Lake Conjola (NSW), Moreton Bay (S-QLD), and published sequences from Fraser Island, the Mediterranean, California and overseas aquaria, is separated from all other sequences. Port Hacking (NSW) isolates cluster with samples from inshore areas of tropical Queensland (Gladstone Harbour, Townsville), one clone of the Careel Bay (NSW) isolates, and published sequences from Fraser Island, Indonesia, and New Caledonia. Four clones of the Careel Bay isolates form a clade separated from other sequences. The isolates from six Northern GBR reefs and published sequences from Puerto Rico, Martinique and the Red Sea also form a well-supported clade. Sequence divergence within the clades ranged from low levels in the ‘‘Mediterranean Clade’’ (0.14%) and ‘‘Reef Clade’’ (0.56%) to higher levels in the ‘‘Tropical Inshore Clade’’ (1.44%) and the ‘‘Careel Bay Clade’’ (1.18%). Sequences previously published in Jousson et al. (2000) from a presumably native population on Fraser Island (located between Moreton Bay and Gladstone) showed intermediate levels of sequence divergence (1.03%). With few exceptions, these samples do not cluster consistently with any of the four well-supported clades. Sequence divergence between the four clades ranged from 1.67% to 2.50%. Neighbour joining (Fig. 2), maximum parsimony, and maximum likelihood analyses gave similar tree topologies.

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Fig. 2. Consensus tree for C. taxifolia and the outgroup C. sertularioides after Neighbour-Joining analyses of ITS r-DNA sequences. Numbers indicate the percentage bootstrap values (1000 replicates). Scale bar and forklengths represent Kimura two-parameter genetic distance. Isolates in bold print have been sequenced in the present study, numbers in parentheses indicate replicate samples. (1) sequences from Olsen et al. (1998), (2) sequences from Jousson et al. (1998), (3) sequences from Jousson et al. (2000).

4. Discussion The ITS sequences of the three new NSW records of C. taxifolia consistently clustered into three different clades. This indicates that they were derived from different source regions. Sequence identity within Port Hacking and Lake Conjola indicates founder effects, consistent with potential introductions. However, several Moreton Bay samples and isolates from four reefs

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were also identical, and prevalent asexual reproduction may be an additional factor for this low genetic diversity. In contrast, the remarkable sequence similarity between isolates from coral reef regions over a large geographical area (Australia, the Caribbean, the Red Sea) cannot be explained by asexual reproduction and will require further analyses. In Mediterranean C. taxifolia only male gametes have been observed and this strain is apparently only vegetatively spreading (Zuljevic and Antolic, 2000). Sexual reproduction in Caulerpa species appears to be a stochastic event (Clifton and Clifton, 1999) and fertility in Mediterranean C. taxifolia has only been observed in temperatures above 25 °C (Zuljevic and Antolic, 2000). It is possible that low water temperatures in Moreton Bay and Lake Conjola may inhibit sexual reproduction in situ. In contrast, samples from Fraser Island, located only about 250 km north of Moreton Bay, showed a high genetic diversity. This could be due to algae in this location frequently experiencing temperatures above the critical threshold for sexual reproduction, or to overlapping distributional ranges and mixing of Moreton Bay and N-QLD inshore populations. The finding that several Fraser Island isolates clustered consistently near samples from these two regions supports the latter hypothesis. The high sequence similarity between C. taxifolia from Port Hacking (NSW) and Gladstone (0.18% sequence difference) or Townsville (1.09%), respectively, indicated that natural populations from inshore areas of tropical QLD are the most likely sources for this new NSW record. Four of the five clones of the Careel Bay samples formed a separate clade with no apparent relationship to other locations investigated. However, one clone was associated with the clade containing tropical QLD, Port Hacking, Fraser Island, Noumea and Jakarta samples. Although the exact source of the Careel Bay C. taxifolia cannot be pinpointed, it is safe to conclude that they are distinctly different to the Moreton Bay/Mediterranean/ California/aquarium clade. This is important information for resource management and conservation because a so-called aquarium strain of C. taxifolia, referring to the Mediterranean invasion, is on the interim species list to trigger a marine pest incursion emergency response in Australia (R. Thresher, pers. comm.). This strain has also been publicised as the ‘‘killer algae’’ (Meinesz, 1999). The large clade including isolates from Lake Conjola (NSW), the two Moreton Bay sites, and the published samples from the Mediterranean, overseas aquaria, and California can either not be further resolved with ITS sequencing or there are no actual differences. This is comparable to results of Darius et al. (1999), Jousson et al. (2000), and Wiedenmann et al. (2001). Consequently, the source of the new Lake Conjola record cannot be identified with certainty. The lack of genetic

variability between samples from a large biogeographic range may indicate limitations of the technique rather than the real relationships. However, a translocation from Moreton Bay to NSW is more likely because of the geographical proximity and a high volume of recreational boat traffic, a potential distribution vector (Sant et al., 1996). C. taxifolia and other species of the genus Caulerpa are known to show highly variable morphologies depending on environmental conditions such as light, nutrients, and temperature (Chisholm et al., 1995; Calvert, 1976; Ohba and Enomoto, 1987). However, Benzie et al. (2000) and the current study indicate some genetic control over the phenotypes, as morphology correlates with genotype. C. taxifolia in the coastal areas of North to Central QLD and from Port Hacking has a delicate morphology with narrow stolons, fronds, and pinnules (Benzie et al., 2000; Table 1, this study). In contrast, C. taxifolia in the Mediterranean is generally quite large with broader stolons, fronds, and pinnules, although variable depending on depth (larger in low light) and season (Meinesz et al., 1995). Samples from Moreton Bay, Lake Conjola and Careel Bay in the present study display a similar robust morphology. The coral reef isolates display a fine morphology with very small fronds. Rather than controlling individual morphology, local water quality conditions may affect growth in length of the C. taxifolia thallus, and, hence, the expansion of a colony. C. taxifolia can utilise nutrients and carbonsources from the sediment by uptake through the rhizoids and associated bacteria (Chisholm et al., 1996), even in eutrophied, anoxic sediments (Chisholm and Jaubert, 1997). This enables the alga to grow in areas where photosynthesis is light-limited. Sediment nutrient enrichment has been shown to increase growth of Mediterranean C. taxifolia (Ceccherelli and Cinelli, 1997). Preen (1993) and Pillen et al. (1998) suggest a recent increase in abundance of C. taxifolia in Moreton Bay. Older records, however, indicate that this species has been common in the eastern part of the bay since at least the 1970s (J. Phillips, pers. comm.). Parts of Moreton Bay have had serious water quality problems, which have led to the decline of seagrass beds, frequent algal blooms (Anon., 1998) and potentially to a prolific growth of C. taxifolia. A similar situation applies to Lake Conjola, an intermittently closing coastal lake. Eutrophied conditions were observed in the late 1990s, when anecdotal reports already attested the presence of C. taxifolia, and have since been abated by flushing the lake (D. Grey, pers. comm.). Compared to the incursions of C. taxifolia in the Mediterranean and California, the situation in Australia is more complex because several native populations of C. taxifolia exist along the tropical and subtropical coasts. However, we were able to identify that three

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recently introduced populations resulted from as many different sources. The genetic relationships of C. taxifolia on a global scale remain unclear despite the inclusion of sequences from a wide geographic range in our analysis. Although we could not conclusively demonstrate the most likely source regions for some of these populations, it is equally important for management to exclude source populations, such as the Mediterranean/ aquarium ‘strain’. This study therefore demonstrated the usefulness of genetic techniques for management decisions on introduced species. A future approach could include targeted analyses of populations in potential source regions, identified by a vector risk analysis that includes information about infections of donor ports and length of voyage (K. Hayes, CRIMP, pers. comm.). Although useful additional information, genetic laboratory techniques alone cannot provide definite answers to environmental managers and, indeed, ecological and physiological studies, and analyses of strengths and pathways of introduction vector remain essential.

Acknowledgements We are grateful to Julie Phillips (University of Queensland) and staff of the Queensland EPA, and Marcus Miller (NSW Fisheries) for the collection of C. taxifolia samples. We also thank Bob Ward and Ron Thresher for comments on the manuscript.

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