Cathryn L. Abbott

New primers for DNA barcoding of digeneans and cestodes (Platyhelminthes)

Digeneans and cestodes are species‐rich taxa and can seriously impact human health, fisheries, aqua‐ and agriculture, and wildlife conservation and management. DNA barcoding using the COI Folmer region could be applied for species detection and identification, but both ‘universal’ and taxon‐specific COI primers fail to amplify in many flatworm taxa. We found that high levels of nucleotide variation at priming sites made it unrealistic to design primers targeting all flatworms. We developed new degenerate primers that enabled acquisition of the COI barcode region from 100% of specimens tested (n = 46), representing 23 families of digeneans and 6 orders of cestodes. This high success rate represents an improvement over existing methods. Primers and methods provided here are critical pieces towards redressing the current paucity of COI barcodes for these taxa in public databases.

Reproducibility of pyrosequencing data for biodiversity assessment in complex communities

High-throughput sequencing is rapidly becoming a popular method to profile complex communities and has generated deep insights into community biodiversity. However, the reproducibility of this method for biodiversity assessment remains largely unexplored. Here we evaluated reproducibility by analysing 454 pyrosequenced biological replicates of two complex plankton communities collected from one freshwater port and one marine port. We also tested whether reproducibility potentially influences biodiversity estimates, notably - and -diversity. Our evaluation of reproducibility revealed a complex scenario, having both technical and biological significance. At the Operational Taxonomic Unit (OTU) level, reproducibility was 100% for high-abundance OTUs (>100 sequences), although it was lower for low-abundance OTUs, and sometimes 88% of irreproducible OTUs had high sequence similarity to existing records, suggesting that some singletons may reflect rare lineages/genotypes in communities. However, spurious amplification of distantly related taxonomic groups generated mainly low-abundance OTUs that were characterized by low reproducibility. At a broad taxonomic level (i.e. order level), reproducibility decreased as the abundance of OTUs decreased and was particularly low for distantly related taxonomic groups such as algae and protists that were not the targets of our zooplankton biodiversity survey. At a lower taxonomical level (i.e. family-level), overall reproducibility was high (>80%) for crustaceans, the dominant group in zooplankton samples. Therefore, we suggest that random variation during both sample collection and sequencing processes can be responsible for low reproducibility. Our analyses also suggest that random sampling processes may influence both - and -diversity estimates. Our results add to growing evidence that caution needs to be applied when designing and interpreting experiments utilizing high-throughput sequencing data for biodiversity assessments. Technical replicates are needed to statistically correct intra-sample variation, while field-based replicate samples are desirable to substantiate results. An overestimation of species diversity can occur when OTUs are uniquely characterized by spuriously amplified sequences and errors/artifacts. Therefore, careful management of low-abundance OTUs is required to reveal unique/rare lineages. Our results suggest that further studies are needed to determine the ecological significance of low-abundance OTUs in complex communities.

Sequence homogeneity of internal transcribed spacer rDNA in Mikrocytos mackini and detection of Mikrocytos sp. in a new location.

Mikrocytos mackini is a microcell parasite of Pacific oysters only known to occur on the Pacific coast of North America. It is the only described species in the genus, although a genetically divergent Mikrocytos sp. organism has been reported once in both the Atlantic Ocean and China. We developed methods for sequencing the internal transcribed spacer (ITS) of rDNA for the purpose of characterizing extant diversity within M. mackini throughout its known geographic range, and surveying for other evidence of Mikrocytos sp. organisms. Our specific aims were to examine relatedness of M. mackini among sites to make inferences about its recent evolutionary history, and to provide baseline data for future development of a species-specific molecular detection method. We found a total lack of genetic variation within M. mackini across the complete ITS1-5.8S-ITS2 array in over 70 samples collected throughout its range. We hypothesize that this could be a result of a founder effect if the parasite had been introduced into its known range alongside its host, which was imported from Asia beginning around 1914 to about 1961. We detected a single divergent sequence at a short stretch of 18S that was identical to the Mikrocytos sp. detected elsewhere, which adds to the recent and growing body of evidence that Mikrocytos is much more broadly distributed than the limited range of M. mackini suggests. A 1903 bp section of rDNA from Mikrocytos sp. was generated that contained regions of high divergence from M. mackini (in ITS1 and ITS2) that could be exploited for molecular diagnostics.

A New Genus and Species of Gyrodactylid (Monogenea) From Silver Hake, Merluccius Bilinearis, in the Bay of Fundy, New Brunswick, Canada

Abstract Gyrocerviceanseris passamaquoddyensis n. gen., n. sp. is described from the gills of silver hake, Merluccius bilinearis, in Passamaquoddy Bay, southwestern New Brunswick, Canada. Diagnostic features include an oval haptor with asymmetrically distributed marginal hooks (3 pairs anterior, 5 pairs posterior) and a prominent muscular flange on the lateral margins, between the anterior and posterior groups of hooks. The hamuli are well developed, with a straight point, a short shaft, and an elongate, blade-like ventral root having a ventrally directed, gooseneck-shaped tip. The ventral bar is rectangular, with a small, hourglass-shaped posterior membrane. There is no dorsal bar. The marginal hook sickle is sharply curved, with a long fine point, an upturned toe, a small heel, and a straight handle. A partial DNA sequence from 18S rDNA was generated and found to be over 11% divergent from its closest matches in GenBank. However, it was less divergent (7.8%) from a previously unpublished sequence of Gyrodactylus emembranatus Malmberg, 1970, suggesting this species is its closest known relative. Interestingly, both species have almost identical marginal hook sickles and a male copulatory organ (MCO) with small spines in multiple arched rows. Gyrocerviceanseris n. gen. is the sixth genus of viviparous monogeneans to be described from marine and estuarine fishes of the northwestern Atlantic Ocean.

Phylogenetic species delimitation for crayfishes of the genus Pacifastacus.

Molecular genetic approaches are playing an increasing role in conservation science by identifying biodiversity that may not be evident by morphology-based taxonomy and systematics. So-called cryptic species are particularly prevalent in freshwater environments, where isolation of dispersal-limited species, such as crayfishes, within dendritic river networks often gives rise to high intra- and inter-specific genetic divergence. We apply here a multi-gene molecular approach to investigate relationships among extant species of the crayfish genus Pacifastacus, representing the first comprehensive phylogenetic study of this taxonomic group. Importantly, Pacifastacus includes both the widely invasive signal crayfish Pacifastacus leniusculus, as well as several species of conservation concern like the Shasta crayfish Pacifastacus fortis. Our analysis used 83 individuals sampled across the four extant Pacifastacus species (omitting the extinct Pacifastacus nigrescens), representing the known taxonomic diversity and geographic distributions within this genus as comprehensively as possible. We reconstructed phylogenetic trees from mitochondrial (16S, COI) and nuclear genes (GAPDH), both separately and using a combined or concatenated dataset, and performed several species delimitation analyses (PTP, ABGD, GMYC) on the COI phylogeny to propose Primary Species Hypotheses (PSHs) within the genus. All phylogenies recovered the genus Pacifastacus as monophyletic, within which we identified a range of six to 21 PSHs; more abundant PSHs delimitations from GMYC and ABGD were always nested within PSHs delimited by the more conservative PTP method. Pacifastacus leniusculus included the majority of PSHs and was not monophyletic relative to the other Pacifastacus species considered. Several of these highly distinct P. leniusculus PSHs likely require urgent conservation attention. Our results identify research needs and conservation priorities for Pacifastacus crayfishes in western North America, and may inform better understanding and management of P. leniusculus in regions where it is invasive, such as Europe and Japan.

Molecular taxonomy of Mikrocytos boweri sp. nov. from Olympia oysters Ostrea lurida in British Columbia, Canada.

Mikrocytos mackini is a microcell parasite that usually infects Crassostrea gigas distributed along the Pacific Northwest coast of North America. For many years, M. mackini was the only known species in the genus, but there have been multiple recent findings of genetically divergent forms of Mikrocytos in different hosts and in distantly located geographic locations. This note describes M. boweri sp. nov. found in Olympia oysters Ostrea lurida collected from and native to British Columbia, Canada, primarily using a molecular taxonomic approach.

Twelve microsatellite markers in the invasive tunicate, Didemnum vexillum, isolated from low genome coverage 454 pyrosequencing reads

Abstract454 pyrosequencing reads were used to isolate microsatellites in the global marine ascidian invader, Didemnum vexillum. This method allows simple and cost-effective isolation of new markers from organisms without existing genomic information and, to our knowledge, has not been used before to develop a polymorphic microsatellite marker set. Loci had between two and eleven alleles and overall mean observed and expected heterozygosities of 0.57 and 0.62, respectively. These markers will greatly facilitate research required to develop control and mitigation strategies for D. vexillum.

Review of Mikrocytos microcell parasites at the dawn of a new age of scientific discovery

The genus Mikrocytos is traditionally known for Mikrocytos mackini, the microcell parasite that typically infects Pacific oysters along the west coast of North America. Multiple factors have conspired to create difficulty for scientific research on Mikrocytos parasites. These include their tiny cell size, infections that are often of light intensity, lack of suitable cell lines and techniques for in vitro culture, and the seasonal nature of infections. The extreme rate of molecular evolution in Mikrocytos stymied new species discovery and confounded attempts to resolve its phylogenetic position for many years. Fortunately, 2 recent landmark studies have paved the way forward for future research by drastically changing our understanding of the evolution and diversity of these parasites. No longer an orphan eukaryotic lineage, the phylogenetic placement of Mikrocytos has been confidently resolved within Rhizaria and as sister taxon to Haplosporidia. The genus has also found a taxonomic home within the newly-discovered order, Mikrocytida - a globally distributed lineage of parasites infecting a wide range of invertebrate hosts. Here we review available scientific information on Mikrocytos parasites including their evolution and diversity, host and geographic ranges, epizootiology, and detection of the regulated pathogen, M. mackini. We also make recommendations towards a consistent taxonomic framework for this genus by minimally suggesting the use of 18S rDNA sequence, host species information, and histopathological presentation in new species descriptions. This is timely given that we are likely embarking on a new era of scientific advancements, including species discovery, in this genus and its relatives.

Denman Island disease (causative agent Mikrocytos mackini) in a new host, Kumamoto oysters Crassostrea sikamea.

Mikrocytos mackini, causative agent of Denman Island disease in Pacific oysters Crassostrea gigas and other oyster species, was found in 2011 in a previously unreported host, the Kumamoto oyster C. sikamea, in Humboldt Bay, California, USA. The detection was also the first reported finding of M. mackini in California. Prevalence was estimated as high as approximately 27% from pooled samples analyzed by PCR. Higher prevalence appeared related to longer residence time in the bay and somewhat colder than typical winter seawater temperatures. No M. mackini was detected in Humboldt Bay juvenile Kumamoto oysters or Pacific oyster brood or seed stock in 2011 or 2012.

Phylogeographic insights into the invasion history and secondary spread of the signal crayfish in Japan.

Abstract Successful invasion by nonindigenous species is often attributed to high propagule pressure, yet some foreign species become widespread despite showing reduced genetic variation due to founder effects. The signal crayfish (Pacifastacus leniusculus) is one such example, where rapid spread across Japan in recent decades is believed to be the result of only three founding populations. To infer the history and explore the success of this remarkable crayfish invasion, we combined detailed phylogeographical and morphological analyses conducted in both the introduced and native ranges. We sequenced 16S mitochondrial DNA of signal crayfish from across the introduced range in Japan (537 samples, 20 sites) and the native range in western North America (700 samples, 50 sites). Because chela size is often related to aggressive behavior in crayfish, and hence, their invasion success, we also measured chela size of a subset of specimens in both introduced and native ranges. Genetic diversity of introduced signal crayfish populations was as high as that of the dominant phylogeographic group in the native range, suggesting high propagule pressure during invasion. More recently established crayfish populations in Japan that originated through secondary spread from one of the founding populations exhibit reduced genetic diversity relative to older populations, probably as a result of founder effects. However, these newer populations also show larger chela size, consistent with expectations of rapid adaptations or phenotypic responses during the invasion process. Introduced signal crayfish populations in Japan originate from multiple source populations from a wide geographic range in the native range of western North America. A combination of high genetic diversity, especially for older populations in the invasive range, and rapid adaptation to colonization, manifested as larger chela in recent invasions, likely contribute to invasion success of signal crayfish in Japan.