Taxonomy, barcoding and phylogeny of Hydroides (Serpulidae, Annelida), the largest genus of notorious fouling and invading calcareous tubeworms

Hydroides Gunnerus, 1768 is the most speciose genus of the calcareous tubeworm familySerpulidae (Polychaeta, Annelida). It is one of the most economically important group of marine invertebrates as it includes notorious foulers and common bioinvaders, yet the biodiversity of the genus is poorly known. Molecular tools, especially cytochrome c oxidase subunit I (COI) barcoding that have proven useful for species delimitation and discovery in eukaryotes, have not been widely used in Hydroides due to the high amplification failure rate of the COI gene. The aim of this study was to revise this genus using both morphological and molecular approaches and to provide reliable identification tools for biosecurity purposes. The first detailed regional morphological taxonomic revision of Hydroides of Australia was conducted, based both on collections from Australian museums and newly collected material from New South Wales, Victoria, Queensland, Northern Territory, and Western Australia. As a result, 25 species are currently considered valid in Australia, including three newly described species. Furthermore, based on collections of 21 museums in 15 countries, taxonomic identification keys accompanied by diagnoses and original high-quality photographs were developed for 96 species word-wide. To develop molecular identification tools for Hydroides, the entire mitochondrial genomes of eight Hydroides species were sequenced by next-generation sequencing and the resultant reference sequences were used to design new taxon-specific COI primers. Analyses of mitochondrial genomes of Hydroides showed a substantial rearrangement in gene order and numerous rarely reported large intergenic regions when compared with genomes of other known polychaetes. Using the newly designed primers, a COI barcoding database including 45 species of Hydroides was established and further used to explore cryptic speciation and invasion pathways in Hydroides. In particular, COI sequence data revealed the existence of two cryptic species in the common global invader Hydroides dianthus and suggested that the native range of H. dianthus sensu stricto is the Mediterranean rather than the Atlantic coast of the USA as historically assumed. In addition to COI barcoding, another four gene markers (18S, 28S, ITS, cytochrome b) were also used to explore the taxonomic status and phylogenetic relationship of Hydroides. Combined data of three gene markers (18S, ITS, cytochrome b) and morphological data were used to resolve the taxonomic status of another widely distributed species, Hydroides brachyacantha. The study showed that H. brachyacantha is a complex of species with regionally restricted distribution ranges. A new Australian species H. amri and its cryptic lineage H. nikae were separated from the complex. A large-scale study of phylogenetic relationships of Hydroides world-wide, based on 45 species using the integrated data of all five genetic markers, indicated closer phylogenetic relationships among species that occur in close biogeographic regions. While the current study resolved the taxonomic status of most morphologically distinct Hydroides species, it also revealed the presence of a large number of cryptic species in the genus, which casts doubts on the status of other widespread species of Hydroides. Further molecular studies of widely dispersed species are needed to fully understand whether such wide distributions are caused by forces such as anthropogenic transport or are an indication of unresolved species complexes.