A checklist of the benthic and epiphytic marine dinoflagellates of New Zealand, including Rangitāhua/Kermadec Islands

ABSTRACT

New Zealand benthic and epiphytic dinoflagellate records increased in recent years as harmful algal bloom research increased. This was largely due to risk assessments of micro-algal biotoxins for the seafood industry and concerns regarding ciguatera fish poisoning in humans from toxic finfish. High-throughput sequencing enabled the detection of dinoflagellate species that were previously overlooked by microscopy, particularly where diatoms or sediments obscured visual identification. This checklist includes new species records for New Zealand and species usually considered planktonic, but which have benthic life stages. Thirty-one dinoflagellate genera were recorded from isolations and descriptions of living cells: Alexandrium, Amphidinium, Archaeperidinium, Azadinium, Biecheleria, Blastodinium, Bysmatrum, Coolia, Crypthecodinium, Cryptoperidiniopsis, Durinskia, Ensiculifera, Fukuyoa, Gambierdiscus, Gymnodinium, Gyrodinium, Heterocapsa, Karenia, Kryptoperidinium, Ostreopsis, Pelagodinium, Pentapharsodinium, Pfiesteria, Polarella, Prorocentrum, Protodinium, Protoperidinium, Pseudopfiesteria, Scrippsiella, Togula and Vulcanodinium. A further nine genera were detected by high-throughput sequencing: Cachonina, Dinophysis, Fragilidium, Gonyaulax, Karlodinium, Lepidodinium, Protodinium, Symbiodinium and Woloszynskia.

KEYWORDS:

• Dinoflagellates
• marine epiphytic
• benthic
• New Zealand
• Rangitāhua
• Kermadec Islands
• metabarcoding

Introduction

Research on the unicellular eukaryotic dinoflagellates has evolved globally in recent years. In the last decades of the twentieth century, research was driven by the harmful effects of toxic and noxious dinoflagellate blooms on fisheries and aquaculture. The New Zealand shellfish industry and the New Zealand government biotoxin regulators responded to harmful algal blooms (HABs) around New Zealand's coastline by implementing internationally recognised biotoxin and phytoplankton monitoring programmes in 1993 and 1994, respectively (Rhodes et al. 2013b). These programmes are on-going and the samples monitored are shellfish flesh, to test for biotoxins, and seawater, to determine the presence and concentration of planktonic HAB species. The micro-algal identification is via light microscopy and is supported by molecular tests. Risk assessments are provided to seafood managers within 24 hours of sample receipt to help reduce impacts on commercial fisheries and public health (Rhodes & Munday 2016). Blooms of potentially toxin-producing dinoflagellate genera, such as Alexandrium (MacKenzie 2014), Dinophysis (MacKenzie et al. 2004), Karenia (de Salas et al. 2005; Haywood et al. 2004; Smith et al. 2007), Protoceratium (Satake et al. 1997) and Gymnodinium (for example, the paralytic shellfish toxin (PST) producer, G. catenatum; MacKenzie 2014), occur regularly around New Zealand's coastline.

Benthic and epiphytic dinoflagellates, however, occur mainly attached to surfaces, including sediments, coral, macroalgae and seagrasses, and motile forms or re-suspended cells (as may occur following storm events) are only occasionally detected in the sea water samples for phytoplankton monitoring. Even when the epiphytic dinoflagellate Ostreopsis siamensis covers macroalgae in dense concentrations in sub-tropical Northland, during the austral summers (Shears and Ross 2009; Rhodes 2011), only a few cells are detected in the phytoplankton monitoring samples collected from those areas (Cawthron Institute Microalgae Laboratory, unpublished data).

Benthic and epiphytic harmful algal bloom (BHABs) species produce diverse toxins and cause different issues to planktonic HAB species. For example, ciguatera fish poisoning (CFP) in humans is caused when the toxin producers, epiphytic species in the genus Gambierdiscus, are consumed along with macroalgae and coral by herbivorous fish. The toxins (ciguatoxins) bioaccumulate in fish tissue, which are then consumed by humans. BHAB species therefore need a specialised approach for their detection and quantification (GEOHAB 2012). One method which has been used by the authors in Northland, New Zealand, utilises an artificial substrate, which standardises sampling techniques and enables comparisons between locations, including international BHAB data (Tester et al. 2014; Berdalet et al. 2017; Smith et al. 2017a). This approach has also proved particularly useful for obtaining live dinoflagellate isolates free from debris, as well as for determining species presence using molecular techniques (e.g. quantitative PCR, high-throughput sequencing). Parsons et al. (2017), however, have advised caution in using the artificial substrates for determining Gambierdiscus abundance in some habitats as they found a lack of consistent correlation between cell concentrations from their deployments compared with those from the natural macrophyte hosts at the sites they investigated. The method was particularly useful, however, in New Zealand's protected northern embayments (Smith et al. 2017b).

The benthic life stages of some mainly planktonic dinoflagellate species may be found in the sediments or attached to macroalgae and eel grasses, but will also be detected in motile form during phytoplankton monitoring. Any study of the meroplankton, i.e. plankton spending at least one life stage in the benthos, must include dormant forms. Ten per cent of dinoflagellates have been estimated to produce resting cysts (Dale 1983), and these increase the chances of survival during unfavourable environmental conditions. There are also temporary or ecdysal (non-dormant) cysts which may also be a response to adverse conditions. The cycling between benthic and planktonic stages may be rapid, for example, dinoflagellates that inhabit shallow pools and are vulnerable to tidal cycles (Hoppenrath et al. 2014).

Salgado et al. (2018) also discuss the flattened cells of the marine dinoflagellates Protoceratium reticulatum and Ceratocorys mariaovidiorum (Order Gonyaulacales). (The name C. mariaovidiorum is a synonym for Pentaplacodinium saltonense (Mertens et al. 2017).) The flattened cells differ morphologically from the planktonic form of each species and, whilst mostly benthic, they may become motile if disturbed. Benthic life stages are also included in this study.

The research focus on biotoxin-producing dinoflagellates continues globally and toxicity testing (e.g. the mouse bioassay) is invaluable as it indicates when a toxic compound is present that has not been detected by current chemical tests (Munday et al. 2017). There has been a slight shift in dinoflagellate toxin production research recently, from sampling improvements and the development of new chemical tests, to the production of valuable compounds. For example, the cyclic imine portimine, which was isolated from the non-motile benthic cells of Vulcanodinium rugosum, has potential as an agent of apoptosis (Selwood et al. 2013). Portimine has very low acute toxicity to mice but is highly cytotoxic to cultured cells and is one of the most potent naturally derived inducers of apoptosis to be discovered (Cuddihy et al. 2016). Compounds are sought from other dinoflagellates for their nutritional value (for example, non-meat proteins) and for potential pharmacological applications (Assunção et al. 2017).

There is also a need to determine the microbial biodiversity of New Zealand in view of expected changes in the communities due to climate change. It has recently been discovered through novel metabarcoding approaches that, in the marine environment, dinoflagellates represent up to 50% of the planktonic species richness, representing a major source of primary production and nutrient cycling in the world's oceans. Of the approximately 5000 living microalgae species known (including the dinoflagellates), approximately 6% multiply to form HABs (Kohli et al. 2014, 2015). Less than 2% of the described species produce potent biotoxins (Hallegraeff 2014), which may be directly toxic to shellfish or fish, or toxic to animals or humans consuming contaminated seafood. Dinoflagellate distributions are already expanding, with species from tropical regions being detected in temperate to sub-tropical regions. For example, a previously considered tropical species, Gambierdiscus carpenteri, now blooms regularly in temperate southern New South Wales, Australia (Kohli et al. 2014). A combination of factors is probably responsible, including an increase in average coastal seawater temperatures along the south east Australian coastline and associated changes in the direction of the main currents (http://climatechange.environment.nsw.gov.au/About-climate-change-in-NSW/Evidence-of-climate-change/Observed-Australian-climate-change: accessed 5 March 2018). There is also the added complicating factor of adaptation of tropical species to cooler temperatures. Increases in average annual coastal seawater temperatures are also occurring in northern New Zealand waters (Ministry for the Environment 2016) and while the risk of CFP occurring due to local Gambierdiscus blooms is currently low, there is a potential risk of CFP occurring in New Zealand in the future (Rhodes et al. 2017a).

The identification of benthic dinoflagellates, historically based on light microscope morphological descriptions of motile cells and their resting cysts, now often requires electron microscope examination for a confident identification and DNA sequence data can be critical in deciding on the differentiation of morphologically similar species (for example, Gambierdiscus species, Smith et al. 2017a, 2017b). Differences in pigments and toxin production strengthen the characterisation of a species. For example, Gambierdiscus species have been observed in high concentrations in samples collected from macroalgae in Rangitāhua/Kermadec Islands (a New Zealand territory approximately 1000 km north-east of the mainland; Rhodes et al. 2017b). The genus is readily recognised under the light microscope, but identifications to species level in environmental samples have required molecular approaches, for example quantitative Polymerase Chain Reaction (qPCR) assays or high-throughput sequencing metabarcoding (Smith et al. 2017a). Genera that commonly co-occur with Gambierdiscus in New Zealand waters are Amphidinium, Coolia, Ostreopsis and Prorocentrum. These are readily recognised at the genus level, but may also require molecular approaches for species determination (Smith et al. 2017b).

Benthic dinoflagellate species from sub-tropical New Zealand

Sub-tropical New Zealand includes the Rangitāhua/Kermadec Islands as well as the northern coastal waters of New Zealand (Figures 1 and 2). For this study, the subtropics refers to geographic and climate zones located approximately between the tropics at latitude 23.5° and temperate zones (normally referring to latitudes 35°–66.5°) to the south of the Equator. Average annual sea surface temperatures (SSTs) at Raoul Island ranges from approximately 17°C–23°C (a 6°C range; Sutton et al. 2012, Trnski & de Lange 2015).

Figure 1. Map of Rangitāhua/Kermadec Islands highlighting the sampling sites where epiphytic dinoflagellates referred to in this study were collected from macroalgae.

Figure 2. Map of New Zealand highlighting sites where benthic and epiphytic dinoflagellates referred to in this study were collected.

For Northland, New Zealand, average annual SSTs range from approximately 13°C–23°C (a 10°C range; https://terra.nasa.gov/data/modis-data). Sampling for dinoflagellates in the Kermadec Islands has been carried out during four Auckland War Memorial Museum expeditions to determine the biodiversity of the region (Rhodes et al. 2014a, 2015, 2017b, 2017d; Smith et al. 2017a). Sampling of surface sediments in Northland has also been carried out on numerous occasions (Rhodes et al. 2010a, 2010b, 2013a).

Rangitāhua / Kermadec Islands

The Kermadec Islands are at the north-eastern limit of New Zealand's territories (Figure 1), approximately 1000 km from mainland New Zealand. They currently include a 747,000-hectare marine reserve with the only human habitation being Raoul Island Station.

Collection of samples for the isolation of epiphytic dinoflagellates from macroalgae was carried out at Rangitāhua/Raoul Island in November 2013 (Rhodes et al. 2014a, 2014b), North Meyer Island in November 2015 (Rhodes et al. 2015) and at Macauley Island in 2016 and 2017 (Rhodes et al. 2017b). Samples were transported back to Cawthron Institute, Nelson, New Zealand, for dinoflagellate identification. Isolates of Gambierdiscus (Family Ostreopsidaceae), the causative organism of CFP, were of interest as CFP afflicts many people in the Pacific region, including Australia (Kohli et al. 2017), following consumption of contaminated fish, even causing deaths (Rongo & van Woesik 2012). The genus Gambierdiscus is common throughout tropical and sub-tropical regions (Litaker et al. 2010; Parsons et al. 2012) and species identified from the Kermadec expeditions were G. australes (the dominant species in samples, from all sites and a producer of maitotoxins), G. honu (a previously undescribed species; Rhodes et al. 2017c) and G. polynesiensis, a known ciguatoxin (CTX) producer, from Macauley Island (Rhodes et al. 2017b). Two G. polynesiensis isolates from Macauley Island were cultured and one produced trace levels of CTX-3 as well as isomers of CTX-3B/C and -4A/B (isolate CAWD254), but the second isolate produced no known CTXs (CAWD259).

Associated dinoflagellates from the same family included Ostreopsis sp. 3 (Sato et al. 2011), O. ovata and Coolia malayensis (Rhodes et al. 2014b). Isolates from the family Gymnodiniaceae included Amphidinium massartii and A. carterae and from the family Prorocentraceae included Prorocentrum emarginatum, P. hoffmannianum and P. lima. All were identified by analysis of DNA sequencing data (large subunit ribosomal RNA gene region) of clonal cultures as well as by their morphology (Rhodes et al. 2014a, 2014b, 2015, 2017a, 2017b, 2017d).

Northland (Rangaunu and Whangaroa Harbours and the Bay of Islands)

The far north of New Zealand has a sub-tropical climate and the coastline varies from muddy bottomed mangrove (Avicennia marina) dominated harbours surrounded by eel grass (Zostera muelleri) to rocky tidal pools (providing habitat for macroalgae) and sandy beaches (Figure 2). Comprehensive field studies have been carried out over the last two decades in Rangaunu Harbour, where Pacific oyster (Crassostrea gigas) farmers suffered economic losses through regulatory closures of their leases. The closures were for extended periods due to positive toxin results for the oysters using the mouse bioassay. The biotoxins proved to be pinnatoxins, produced by the dinoflagellate Vulcanodinium rugosum (Family Peridiniales incertae sedis; Rhodes et al. 2011), which formed dense mats of non-motile cells on the surface sediments near the edge of mangrove and eel grass habitat around the harbour. At times, the cells become motile and are taken up by the farmed oysters grown on racks further out in the harbour. A morphologically similar and genetically related species, Bysmatrum sp. (Dr Lincoln MacKenzie, Cawthron Institute, unpublished data), were also been found in the same habitat as V. rugosum.

Another dinoflagellate found as mats on surface sediments in Rangaunu Harbour and on oyster farm infrastructure was Prorocentrum lima. Cells were also detected in sea water samples when the mats were disturbed by storm events (Rhodes & Syhre 1995). Okadaic acid (OA), the cause of diarrhetic shellfish poisoning, and OA-diol esters were found at approximately equal levels in P. lima strains from Rangaunu Harbour. Some strains produced the diarrhetic shellfish toxin DTX1 at low concentrations and a novel nitrogen-containing polyether compound was also detected (Rhodes et al. 2006a). Other Prorocentrum species isolated from Rangaunu Harbour surface sediments included P. maculosum and P. hoffmanianum, and from coralline turfs at Te Uenga Bay in the Bay of Islands, P. rhathymum, P. thermaeum and P. triestinum (the latter usually planktonic). The related species P. balticum, P. compressum and P. gracile have only been found as planktonic species.

Amphidinium species were found in the same sediment and macroalgae samples as P. lima. They included A. boggayum, A. carterae, A. massartii, A. mootonorum and A. thermaeum. The type specimen of A. trulla was isolated from seawater in Rangaunu Harbour, but the cells have been described as benthic in Australia (Murray et al. 2004). Many antifungal and haemolytic amphidinol analogues have been isolated from an A. carterae isolate from New Zealand and their structures characterised (Houdai et al. 2001; Echigoya et al. 2005).

Temporary and resting dinoflagellate cysts were found associated with beached macroalgae and sediments from Ninety Mile Beach, which is situated in north-west New Zealand adjacent to Rangaunu Harbour (Figure 2). At times, the beach wrack is covered with Greenshell^TM mussel spat (Perna canaliculus), which is desirable for on-growing on marine farms in more southern, temperate waters. Monitoring of the macroalgae bearing the spat, prior to transport by truck and ferry to the Marlborough Sounds and Stewart Island aquaculture regions, was carried out to prevent transference of toxin dinoflagellates to other parts of New Zealand (Rhodes et al. 1994). Temporary cysts of Karenia mikimotoi (Family Brachidiniaceae) bearing a mucilaginous outer coat and resting cysts of Alexandrium ostenfeldii (Family Ostreopsidaceae), a spirolide producer, were found during monitoring of the macroalgae. Cysts of Gymnodinium catenatum were also found in sediments collected from Kaipara and Aotea Harbours (north-west Northland) as well as in Whangarei Harbour to the north-east (Mackenzie et al. 1996; MacKenzie 2009; Taylor and MacKenzie 2001).

In the Bay of Islands, Fukuyoa paulensis, previously classified as Gambierdiscus cf. yasumotoi (Rhodes et al. 2013a), was discovered at Te Uenga Bay in 2013. This raised the concern that, with increasing coastal seawater temperatures, toxic Gambierdiscus populations could become established in Northland, leading to a risk of CFP from New Zealand fish populations. Gambierdiscus was reported in Northland, New Zealand, nearly two decades ago as G. toxicus (Chang 1996). The morphological plasticity of the species in the genus made microscopic identification difficult in the past and the classification of G. toxicus before 1999 is uncertain, but molecular markers now allow rapid determination to species level (Parsons et al. 2012).

The producer of palytoxin (PLTX)-like compounds, Ostreopsis siamensis, has been a bloom former down the eastern coastline of Northland for many years (Shears and Ross 2009; Rhodes 2011) and occurred with F. paulensis in the Bay of Islands. Despite dense blooms covering macroalgae in Northland during 2014, and the fact that cultured isolates were positive for PLTXs (0.2 pg PLTX equiv./cell), no PLTX was found in herbivorous fish or other marine grazers at that time (Rhodes et al. 2015).

A study carried out in the Bay of Islands in 2014 resulted in new taxonomic records for New Zealand based on high-throughput sequencing metabarcoding analyses. Many of the species reported were not detected by microscopic investigation of surface sediment or macro-algal samples, which highlighted the strength of this high-throughput sequencing technique (Smith et al. 2017b; Tables 1–3).

Table 1. Sub-tropical benthic/epiphytic dinoflagellates, including species with benthic life stages, isolated and identified from New Zealand and its territories.

Species name	CICCM^a code	GenBank code	Toxins	Sites	Comments	References
Alexandrium ostenfeldii (Paulsen) Balech & Tangen	CAWD135,136	NA	Spirolides	Macroalgae (beach wrack): Ninety Mile Beach, Northland	Found as hypnozygotes	Rhodes et al. (1994); MacKenzie et al. (1996)
Amphidinium boggayum Murray & Patterson	CAWD164	NA	N	Sediments: Rangaunu Harbour, Northland	Unpublished finding	Identification by M Hoppenrath, Senckenberg am Meer, Germany, pers. comm.
A. carterae Hulburt	Various isolates maintained from temperate and sub-tropical regions; cryopreserved.	KY069056	Amphidinols	Sediments: Rangaunu Harbour, Northland. North Meyer Island, Kermadec Islands	Also found in the water column and in temperate coastal waters with A. operculatum	Rhodes et al. (2017d, personal observation)
A. massartii Biecheler A. cf. massartii	CAWD132 CAWD156 CAWD122	KM259619	NT	Sediments: Whangaroa and Rangaunu Harbours, Northland; Macroalgae, Kermadec Islands Raoul Island, Kermadec Islands		Rhodes et al. (2010a, 2014a)
A. mootonorum Murray & Patterson	CAWD161	JX262532 and JX262523	NT	Sediments: Rangaunu Harbour, Northland	Unpublished finding	Identification by Prof Shauna Murray, University of Technology Sydney, Australia, pers. comm.
A. thermaeum Dolapsakis et Economou-Amilli	Isolate died	NA	NT	Coralline turfs: Bay of Islands, Northland		Rhodes et al. (2015)
A. trulla Murray, Rhodes et Flo Jorgensen	CAWD68 (cryopreserved)	AY460594	NT	Sediments: Rangaunu Harbour, Northland	The type species was isolated from seawater samples from Rangaunu Harbour	Murray et al. (2004); Karafas et al. (2017)
Archaeperidinium minutum (Kofoid) Jørgensen		NA	NT	Coralline turfs: Bay of Islands, Northland	MB	Smith et al. (2017b)
Azadinium dalianense (Kofoid) Jørgensen		NA	NT	Coralline turfs: Bay of Islands, Northland	MB	Smith et al. (2017b)
Biecheleria cincta (Siano, Montresor & Zingone) Siano		NA		Coralline turfs: Bay of Islands, Northland	MB; planktonic isolate CAWD76 held in CICCM from Hauraki Gulf waters	Smith et al. (2017b)
Blastodinium crissum Chatton		NA	NT	Coralline turfs: Bay of Islands, Northland	MB	Smith et al. (2017b)
Bysmatrum sp.^b Faust & Steidinger		NA	N	Coralline turfs: Bay of Islands, Northland; Sediments: Rangaunu Harbour, Northland	MB; Rangaunu isolate held by AL MacKenzie, Cawthron Institute, Nelson, NZ, pers. comm.	Smith et al. (2017b)
Coolia malayensis Leaw, Lim & Usup	CAWD77 CAWD154 CAWD175 CAWD214	KR608283 KJ422852 KR605285 KJ422855 KR605286 KJ422856 KJ422858	T	Sediments and coralline turfs: Rangaunu Harbour and Bay of Islands, Northland	Also found as far south as Nelson; previously published as C. monotis	Leaw et al. (2016); Rhodes et al. (2014b)
Crypthecodinium sp.^b Biecheler		NA	NT	Coralline turfs: Bay of Islands, Northland	MB	Smith et al. (2017b)
Durinskia sp.^b Carty & Cox		NA	NT	Coralline turfs: Bay of Islands, Northland	MB	Smith et al. (2017b)
Ensiculifera cf. loeblichii Cox & Arnott		NA	NT	Coralline turfs: Bay of Islands, Northland	MB	Smith et al. (2017b)
Fukuyoa paulensis Gómez, Qiu, Lopes & Lin	CAWD210 CAWD238	AB859986 and AB859987 NA	MTX-3	Coralline turfs: Bay of Islands, Northland	Previously published as Gambierdiscus cf. yasumotoi	Rhodes et al. (2013a, 2015)
Gambierdiscus australes Chinian & Faust	CAWD244 CAWD245 CAWD246 CAWD255 CAWD256	KY069059 NA NA MF109033 MF109034	MTX-1, MTX-3	Macroalgae: Kermadec Islands		Rhodes et al. (2017b, 2017d); Smith et al. (2017a)
G. honu Rhodes, Smith & Murray	CAWD242	KY062662	MTX-3	Macroalgae: Kermadec Islands		Rhodes et al. (2017d); Smith et al. (2017a)
G. polynesiensis Chinian Faust	CAWD254^b CAWD259	MF109032 NA	CTX, MTX-3		^bNon-toxic isolate: MTX-3 but no CTX	Rhodes et al. (2017d); Smith et al. (2017a)
G. cf. toxicus		NA		Macroalgae: Northland	Uncertain classification	Chang (1996)
Gymnodinium catenatum Graham	CAWD101 CAWD102	AY036128 NA	STX and analogues	Sediments: Ninety Mile Beach, Northland	Usually motile but found as resting cysts	Taylor and MacKenzie (2001); MacKenzie (2009)
G. dorsalisulcum (Hulbert, McLaughlin & Zahl) Murray, de Salas & Hallegraeff	CAWD225	NA	N	Coralline turfs: Bay of Islands, Northland		Rhodes et al. (2015)
Gyrodinium fissum (Levander) Kofoid & Swezy		NA	NT	Coralline turfs: Bay of Islands, Northland	MB	Smith et al. (2017b)
Heterocapsa rotundata (Lohmann) Hansen		NA	NT	Coralline turfs: Bay of Islands, Northland	MB	Smith et al. (2017b)
Karenia mikimotoi (Miyake & Kominami ex Oda) Hansen & Moestrup		NA	NT	Macroalgae (beach wrack): Ninety Mile Beach, Northland	Found as temporary cysts	Rhodes et al. (1994)
Kryptoperidinium sp.^b Lindemann		NA	NT	Coralline turfs: Bay of Islands, Northland	MB	Smith et al. (2017b)
Ostreopsis siamensis Schmidt	CAWD206 CAWD208 CAWD243	KJ422860 NANA	PlTx	Macroalgae: throughout Northland	Also found as far south as the Wellington coast and Marlborough Sounds	Rhodes (2011, pers. observation)
Ostreopsis sp. 3	CAWD221	KM360088 KY197854	PlTx (low conc.)	Macroalgae: Kermadec Islands	Refer Sato et al. (2011)	Rhodes et al. (2014a)
Pelagodinium sp.^b Siano, Montresor, Probert & Vargas		NA	NT	Coralline turfs: Bay of Islands, Northland	MB	Smith et al. (2017b)
Pentapharsodinium tyrrhenicum (Balech) Montresor, Zingone & Marino		NA	NT	Coralline turfs: Bay of Islands, Northland	MB	Smith et al. (2017b)
Polarella sp.^b Montresor, Procaccini & Stoecker		NA	NT	Coralline turfs: Bay of Islands, Northland	MB	Smith et al. (2017b)
Prorocentrum cf. emarginatum Fukuyo	CAWD228	KM360089	N	Macroalgae: Kermadec Islands		Rhodes et al. (2014a)
P. fukuyoi Murray & Nagahama		NA	NT	Coralline turfs: Bay of Islands, Northland	MB	Smith et al. (2017b)
P. hoffmanianum Faust		NA	NT	Macroalgae: North Meyer Is., Kermadec Islands		Rhodes et al. (2017d)
P. lima (Ehrenberg) Stein	CAWD33 CAWD157 CAWD176	NA NA NA	OA and OA-diol esters, DTX1	Sediments and macroalgae: throughout New Zealand, including Kermadec Islands		Rhodes et al. (2006a, 2015); Mackenzie et al. (2011)
P. maculosum Faust	CAWD158	NA	N	Sediments: Rangaunu Harbour, Northland		Rhodes et al. (2015)
P. rhathymum Loeblich III, Sherley & Schmidt	CAWD266	NA	N	Coralline turfs: Bay of Islands, Northland		Rhodes et al. (2015)
P. triestinum Schiller		NA	N	Coralline turfs: Bay of Islands, Northland		Rhodes et al. (2015)
Protodinium simplex Lohmann		NA	NT	Coralline turfs: Bay of Islands, Northland	MB	Smith et al. (2017b)
Protoperidinium bipes (Paulsen) Balech		NA	NT	Coralline turfs: Bay of Islands, Northland	MB	Smith et al. (2017b)
P. parthenopes Zingone & Montresor		NA	NT	Coralline turfs: Bay of Islands, Northland	MB	Smith et al. (2017b)
Scrippsiella lachrymose Lewis		NA	NT	Coralline turfs: Bay of Islands, Northland	MB	Smith et al. (2017b)
Vulcanodinium rugosum Nézan & Chomérat	CAWD163 CAWD166 CAWD167 CAWD168 CAWD170 CAWD171 CAWD178	NA JF267779 JF267773 JF267777 JF267774 JF267778 JF267780 JF267775 JF267781 JF267776	Pinnatoxins	Sediments: Rangaunu Harbour, Northland		Rhodes et al. (2010a, 2010b, 2011)

Note: Results from metabarcoding analyses are also presented (Smith et al. 2017b).

^aCICCM is Cawthron Culture Collection of Microalgae; NA: not available; MTX: maitotoxin; PlTx: palytoxin; N: no toxin detected by LCMS/MS analysis, T: toxic in bioassays, NT: not tested, MB: metabarcoding results only.

^bWhere the species name has not been determined the genus authority is given.

Table 2. Temperate benthic/epiphytic dinoflagellates, including species with benthic life stages, isolated and identified from New Zealand and its territories.

Species name	CICCM^a code	GenBank code	Toxins	Sites	Comments	References
Alexandrium ostenfeldii (Paulsen) Balech et Tangen	CAWD135 CAWD136	AB753841 AB753842	GTXs, STXs, Spirolides	Sediments: from sub-tropical to temperate coastal waters around New Zealand	Found as hypnozygotes	MacKenzie et al. (1996); Munday et al. (2012)
A. pacificum Litaker in John et al.	CAWD260 CAWD261 CAWD262	NA		Sediments: Whangakoko, Port Underwood	Previously named A. catenella	Knight and MacKenzie (2015); MacKenzie (2014)
Azadinium poporum Tillmann & Elbrächter	NA	NA	NT	Sediments: Whangakoko, Port Underwood	Detected by species-specific quantitative qPCR	Smith, unpublished data
Coolia malaysensis Leaw, Lim & Usup in Leaw et al.	CAWD98	KR605284 and KJ422853	T	Sediments in intake pond	Glenhaven Hatchery, Nelson; previously published as C. monotis	Leaw et al. (2016); Rhodes et al. (2014b)
Cryptoperidiniopsis brodyi Steidinger, Landsberg, Mason, Vogelbein, Tester & Litaker	NA	NA	NT	Waiwera/Lake Forsyth, Canterbury	By species-specific qPCR assay	Smith, unpublished data
Pfiesteria piscicida Steidinger & Burkholder	NA	NA	NT	Moutere Inlet, Tasman Bay and Te Waihora/Lake Ellesmere, Canterbury	By species-specific PCR assay	Rhodes et al. (2006b)
Prorocentrum lima (Ehrenberg) Stein	CAWD95	NA	OA	Croissilles Harbour, Tasman Bay	Found associated with macroalgae on mussel growing lines; OA detected by PP-2A	Rhodes et al. (2001)
Pseudopfiesteria shumwayae (Glasgow & Burkholder) Litaker, Steidinger, Mason, Shields & Tester	NA	NA	NT	North and South Island estuaries and Waiwera/Lake Forsyth and Te Waihora/Lake Ellesmere, Canterbury	Previously known as Pfiesteria shumwayae Glasgow & Burkholder By species-specific PCR assay?	Litaker et al. (2005); Rhodes et al. (2002, 2006b)
Togula jolla Flø Jørgensen, Murray & Daugbjerg	CAWD41 CAWD58	NA AY568561	NT	Awaroa, Tasman Bay; Napier	Found in estuary associated with sand ripples; harbour sediments	Jørgensen et al. (2004)

Note: Benthic life stages of Gymnodinium catenatum and Karenia mikimotoi have been recorded in sub-tropical waters (Table 1), but only planktonic forms have been detected in temperate waters.

^aCICCM is Cawthron Culture Collection of Microalgae; NA: not available; GTXs: sulpho-carbamate derivatives of STX; STX: saxitoxin; MTX: maitotoxin; PlTx: palytoxin; OA: okadaic acid; N: no toxin detected by LCMS/MS analysis, T: toxic in bioassays, PP-2A: Protein phosphatase inhibition assay; NT: not tested.

Table 3. List of epiphytic and benthic families and genera in the Class Dinophyceae F.E. Fritsch reported in New Zealand, including records from metabarcoding analyses (Smith et al. 2017a, 2017b).

Family	Genus
Amphidiniopsidaceae	Archaeperidinium JØrgensen^a
Amphidomataceae	Azadinium Elbrächter & Tillmann^a
Blastodiniaceae	Blastodinium Chatton^a
Brachidiniaceae	Karenia Gert Hansen & Moestrup Karlodinium J. Larsen^b
Crypthecodiniaceae	Crypthecodinium Biecheler^a
Dinophysaceae	Dinophysis Ehrenberg^b
Dinothrichaceae	Durinskia S. Carty & E.R. Cox^a Kryptoperidinium Lindemann^a
Gonyaulacaceae Subfamily Gonyaulacoideae	Gonyaulax Diesing^b
Gymnodiniaceae	Amphidinium É. Claperède & J. Lachmann Gymnodinium F. Stein Gyrodinium Kofoid & Swezy^a Lepidodinium Watanabe, Suda, Inouye, Sawaguchi & Chihara^b Togula Flø Jorgensen, Shauna Murray & N. Daugbjerg
Heterocapsaceae	Heterocapsa F. Stein^a
Oodiniaceae	Protodinium Lohmann^b
Ostreopsidaceae	Alexandrium Halim Coolia A. Meunier Gambierdiscus Adachi & Fukuyo Ostreopsis Johs. Schmidt
Peridiniaceae	Cachonina A.R. Loeblich III^b
Peridiniales incertae sedis	Bysmatrum M.A. Faust & K.A. Steidinger Pentapharsodinium Indelicato & Loeblich III^a Vulcanodinium E. Nézan & N. Chomérat
Prorocentraceae	Prorocentrum Ehrenberg^a
Protoperidiniaceae	Protoperidinium R.S. Bergh^a
Pyrophacaceae	Fragilidium Balech ex Loeblich III^b
Suessiaceae	Biecheleria Lindberg & Daugbjerg^a Pelagodinium Siano, Montresor, Probert & Vargas^a Polarella Montresor, Procaccini & D.K .Stoecker^a Protodinium Lohmann^a Symbiodinium Freudenthal ex H.J. Jeong et al.^b
Thoracosphaeraceae	Cryptoperidiniopsis Steidinger, Landsberg, Mason, Vogelbein, Tester & Litaker Ensiculifera Balech ex K.Matsuoka, S. Kobayashi & G. Gains^a Fukuyoa F. Gómez, D.X. Qiu, R.M. Lopes & Senjie Lin Pfiesteria K.A. Steidinger & J.M. Burkholder Pseudopfiesteria R.W. Litaker, K.A. Steidinger, P.L. Mason, J.D. Shields & P.A. Tester Scrippsiella Balech ex A.R. Loeblich III
Woloszynskiaceae	Woloszynskia sp. RH Thompson^b

^aWhere the species name has not been determined the genus authority is given.

^bBenthic and epiphytic forms detected only by high-throughput sequencing.

Benthic dinoflagellate species from temperate New Zealand

The temperate epiphytic/benthic dinoflagellate microflora of New Zealand differs from the sub-tropical microflora, particularly from those species found in Rangitāhua/Kermadec Islands. The annual average range in SSTs for the temperate region is approximately 9°C–20°C (cooler in Canterbury where the range is 8°C–12°C; https://terra.nasa.gov/data/modis-data).). There are several species, however, that have been found in both the sub-tropical northern and the cooler southern waters of New Zealand's mainland (Figure 2). Coolia malayensis, for example, was found in unlined hatchery pond sediments at Glenhaven, Nelson (Rhodes et al. 2014b). Ostreopsis siamensis has been reported in low cell concentrations from as far south as temperate Wellington coastal waters (New Zealand's National Marine Biotoxin monitoring programme records; Rhodes 2011). The chain-forming dinoflagellate G. catenatum, found in cyst form in northern sediments, was probably introduced to southern waters during transfer of drift macroalgae bearing mussel spat collected by the truckload from Ninety Mile Beach. This species has been detected in its motile form in phytoplankton samples collected from the outer Marlborough Sounds (New Zealand's National Marine Biotoxin monitoring programme records). One species found throughout both sub-tropical and temperate regions, in water samples, surface sediments and associated with macroalgae, is P. lima.

The success of New Zealand's phytoplankton monitoring programmes is exemplified by the excellent management of blooms of the PST producing HAB species G. catenatum, A. pacificum (previously A. catenella), A. minutum and A. ostenfeldii in recent years (MacKenzie 2014). However, benthic cyst forms of these species are not included in micro-algal monitoring programmes and are only known to be a vital component of the microflora because of on-going research efforts (Knight and MacKenzie 2015). In fact, resting cysts (hypnozygotes) of A. ostenfeldii have been reported in sediments collected from all around the New Zealand coastline (MacKenzie et al. 1996). Vegetative cell cultures were established by hatching cysts and the toxicity of these cultures was ascertained using high-performance liquid chromatography, with considerable differences detected in the toxin profiles of strains isolated from different sites (MacKenzie et al. 1996). Cysts of A. pacificum have been found in sediments following bloom events in the temperate waters of Queen Charlotte Sound, Marlborough Sounds, with a heavily impacted area being Opua Bay. QPCR assays have been developed to rapidly quantify the cysts to determine the future risk to Greenshell^TM mussel farms in the vicinity.

The family Gymnodiniaceae includes the genera Togula as well as Amphidinium and both have been found in temperate New Zealand waters associated with surface sediments and on marine structures. The species T. jolla was found on sand ripples in Awaroa Inlet, Tasman Bay, in the north-east of the South Island of New Zealand. The inlet is known for its golden sands and the golden-brown Togula blended with its surroundings. The same species has also been isolated from surface sediments near Napier in the east of the North Island. Amphidinium spp. are common in sub-tropical habitats associated with macroalgae and with sediments (especially in the non-motile form), but A. carterae is also regularly reported in sea water monitoring samples collected from temperate waters, often associated with A. operculatum.

The heterotrophic dinoflagellate family Thoracosphaeraceae includes Pfiesteria piscicida, caused major fish kills along the eastern USA seaboard in the northern summer of 1997 (Burkholder and Glasgow 2001). Such fish kills had previously been associated with neurological health symptoms in local fishers (Burkholder et al. 1992). The economic ramifications were considerable as consumers panicked and sales of seafood went down with a calculated loss of US$43M for the state. The ‘halo effect’ also led to the avoidance of Maryland for boat charters with the recreational fishing industry also losing several million dollars. An overall economic loss of US$50M was estimated for just a four-month period (CENR 2000). Since then, Pfiesteria species have been reported from at least 15 countries and the genus is considered to have a global distribution (Rublee et al. 2004; 2005). In 2000, Pseudopfiesteria shumwayae (Litaker et al. 2005) (synonym: Pfiesteria shumwayae; Guiry & Guiry 2017) was reported in surface sediments from estuaries in the Kaipara Harbour in the North Island of New Zealand. In the South Island, it was detected, using molecular methods, in sediment samples from Tasman Bay, Golden Bay, Marlborough and Southland. It was also detected in Canterbury in Wairewa/Lake Forsyth and Te Waihora/Lake Ellesmere (Rhodes et al. 2002). PCR-based assays also determined the presence of P. piscicida DNA in Tasman Bay and the Canterbury Lakes (Rhodes et al. 2006b). A research study in Tasmania, Australia, showed cross-reactivity of the related species, Cryptoperidiniopsis brodyi, with the PCR assay for P. piscicida and, while there has been scanning electron microscope confirmation of the presence of P. piscicida in New Zealand, there may have been an over-estimation of occurrence in the earliest studies (Park 2006; Park et al. 2007). Using a specifically designed PCR assay, C. brodyi was detected in 2007 in Wairewa/Lake Forsyth (Rhodes and Smith, unpublished data).

The azaspiracid-producing dinoflagellate genus, Azadinium (Family Amphidomataceae), has been detected in cyst form in surface sediments from the Marlborough Sounds (Rhodes and Smith, unpublished data). The Irish shellfish aquaculture industry, with a value of >US$65M per annum, suffered economic losses from the closure of export markets due to azaspiracids in 1995. The organism responsible for toxin production was not identified until more than a decade later. It was classified as Azadinium spinosum (Tillmann et al. 2009). Many other Azadinium species, both toxic and non-toxic, have been identified since 2007 (Tillmann et al. 2014). Motile cells of Azadinium poporum were isolated from seawater in the Marlborough Sounds, but the isolate proved non-toxic (Smith et al. 2016) and Azadinium dalianense was detected in benthic dinoflagellate communities from Northland, New Zealand (Smith et al. 2017b).

Metabarcoding results

Nineteen new species records of dinoflagellate species for New Zealand were detected in samples from Northland, collected from macroalgae and on artificial substrates by high-throughput sequencing, using metabarcoding techniques, and the results were published in 2017 (Smith et al. 2017b, refer Table 4 in that publication). Metabarcoding has also been used to characterise benthic dinoflagellates communities in the Kermadec Islands. The analyses demonstrated a dominance by Gambierdiscus species and the first confirmed identification of G. polynesiensis, a known CTX producer, in New Zealand waters. Other known toxin-producing genera were also detected, including Alexandrium, Amphidinium, Azadinium, Dinophysis, Ostreopsis and Prorocentrum. It is expected that these novel next-generation sequencing methods, including metabarcoding, will reveal many more cryptic species in New Zealand's epiphytic and benthic marine habitats.

Conclusions

The epiphytic and benthic dinoflagellates of New Zealand have been the subject of research in recent years, but despite that the group have been barely touched on. Further studies are planned, particularly in shellfish growing areas where there are perceived health risks from dinoflagellate cyst ‘seed’ beds. In northern New Zealand, where a potential risk of CFP has been noted, a continuation of monitoring using high-throughput molecular tools is planned. It is likely that in just a few years this checklist will be increased substantially as sites are reinvestigated and new sites explored. In the interim, this study provides a baseline for future work with 30 genera reported.

Acknowledgements

Samples for identification of dinoflagellates from Rangitāhua/Kermadec Islands were collected by Tom Trnski and colleagues during expeditions carried out by the Auckland War Memorial Museum. Thanks also for technical help to Janet Adamson, Laura Biessy and Kendall Morman, including obtaining DNA sequence data. Thanks to Krystyna Ponikla and Sarah Challenger for maintaining the unique Cawthron Institute Culture Collection of Microalgae.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

Funding is acknowledged from the New Zealand Ministry for Business, Innovation and Employment, Contracts CAWX1317 and CAWX 0902.