Taxonomy and toxicity of Prorocentrum from Perhentian Islands (Malaysia), with a description of a non-toxigenic species Prorocentrum malayense sp. nov. (Dinophyceae)

Highlights

• A new non toxigenic species Prorocentrum malayense sp. nov. is described herein based on morphological and molecular data.

• Other five benthic epiphytic Prorocentrum species were reported from Perhentian Islands Marine Park, Malaysia.

• Okadaic acid production of P. lima and P. caipirignum was confirmed by LC–MS/MS.

Abstract

Thirteen isolates of Prorocentrum species were established from the coral reefs of Perhentian Islands Marine Park, Malaysia and underwent morphological observations and molecular characterization. Six species were found: P. caipirignum, P. concavum, P. cf. emarginatum, P. lima, P. mexicanum and a new morphotype, herein designated as P. malayense sp. nov. Prorocentrum malayense, a species closely related to P. leve, P. cf. foraminosum, P. sp. aff. foraminossum, and P. concavum (Clade A sensu Chomérat et al. 2018), is distinguished from its congeners as having larger thecal pore size and a more deeply excavated V-shaped periflagellar area. Platelet arrangement in the periflagellar area of P. malayense is unique, with the presence of platelet 1a and 1b, platelet 2 being the most anterior platelet, and a broad calabash-shaped platelet 3. The species exhibits consistent genetic sequence divergences for the nuclear-encoded large subunit ribosomal RNA gene (LSU rDNA) and the second internal transcribed spacer (ITS2). The phylogenetic inferences further confirmed that it represents an independent lineage, closely related to species in Clade A sensu Chomérat et al. Pairwise comparison of ITS2 transcripts with its closest relatives revealed the presence of compensatory base changes (CBCs). Toxicity analysis showed detectable levels of okadaic acid in P. lima (1.0–1.6 pg cell^˗1) and P. caipirignum (3.1 pg cell^˗1); this is the first report of toxigenic P. caipirignum in the Southeast Asian region. Other Prorocentrum species tested, including the new species, however, were below the detection limit.

Introduction

The genus Prorocentrum Ehrenberg is a group of dinoflagellates that can be found in planktonic and benthic marine ecosystems (Faust, 1990; Chomérat et al., 2010; Hoppenrath et al., 2013), with 75 valid species hitherto (Guiry and Guiry, 2018), bisectioned into planktonic and benthic/epiphytic. At least ten species (P. borbonicum Ten-Hage, Turquet, Quod, Puiseux-Dao & Couté, P. caipirignum Fraga, Menezes & Nascimento, P. concavum Fukuyo, P. emarginatum Fukuyo, P. faustiae Morton, P. hoffmannianum Faust, P. lima (Ehrenberg) Stein, P. mexicanum Osorio-Tafall, P. texanum Henrichs, Steidinger, Scott & Campbell, and P. leve Faust) have been confirmed to produce a suite of diarrhetic toxins, okadaic acid (OA) and the methyl derivatives dinophysistoxins (DTXs) (reviewed in Hoppenrath et al., 2013), of which nine are benthic (Murakami et al., 1982; Holmes et al., 2001; Hoppenrath et al., 2013,2014; Nascimento et al., 2016; Luo et al., 2017). The toxins that accumulated in molluscan shellfish are responsible for diarrhetic shellfish poisoning (DSP) in humans. Some species of Prorocentrum also produce various bioactive compounds (Faust and Gulledge, 2002; reviewed in Hoppenrath et al., 2013). While DSP toxins-producing Prorocentrum species have been recorded, reports of DSP incidents related to benthic Prorocentrum species were scarce, to our knowledge, only one DSP event from Patagonian Gulfs, Argentina has been reported to attribute to P. lima (Gayoso et al., 2002). Documentation on the occurrence and outbreaks of this particular poisoning have been proven difficult owing to its relatively generic symptoms as compared to other forms of shellfish poisonings (e.g., paralytic and amnesic shellfish poisonings), it is even difficult to detect the outbreaks of toxic Prorocentrum species, particularly of those with the benthic nature.

Species of the genus are generally identified morphologically based on the features of lateral plates and the periflagellar platelets (reviewed in Hoppenrath et al., 2013). However, sometimes the identity of species is uncertain since it cannot be readily determined, partly due to plasticity in the defined morphological traits and ambiguity in the original descriptions (Herrera‐Sepúlveda et al., 2015; Chomérat et al., 2018). In this case, many species have been synonymized over the years: P. arabianum Morton & Faust (=P. concavum Fukuyo) (Mohammad-Noor et al., 2007a; Mohammad-Noor et al., 2007b) and P. maculosum Faust (=P. hoffmannianum Faust) (Rodríguez et al., 2018). But as yet, precise species identification is crucial in order to better understand the ecology of Prorocentrum species, as well as their toxicity. With the recent advancements of molecular phylogenetic, several new species of Prorocentrum have been erected (Murray et al., 2007; Chomérat et al., 2010; Henrichs et al., 2013; Han et al., 2016). Likewise, molecular data have been used in evaluating species crypticity of Prorocentrum, allowing better taxonomic decision, for example, the erection of P. caipirignum from the P. lima complex sensu lato (s.l.) (Nascimento et al., 2017).

Numerous studies have revealed high diversity of Prorocentrum in benthic reef ecosystems. In this study, a field survey was undertaken at the fringing coral reefs of Perhentian Islands Marine Park (Malaysia) to collect Prorocentrum isolates and to document their morphological characteristics. With that, a new morphotype was discovered. Phylogenetic reconstructions based on the nuclear-encoded ribosomal DNA in the large subunit (LSU) and the second internal transcribed spacer (ITS2) regions were used to infer phylogenetic relationships among Prorocentrum species found in this and other studies. The outcomes of the phylogenetic analyses supported our morphological observations that the morphotype is a distinct species, herein described as P. malayense sp. nov. Its ability to produce OA was assessed.