Testing the efficacy of different molecular tools for parasite conservation genetics: a case study using horsehair worms (Phylum: Nematomorpha).

In recent years, parasite conservation has become a globally significant issue. Because of this, there is a need for standardized methods for inferring population status and possible cryptic diversity. However, given the lack of molecular data for some groups, it is challenging to establish procedures for genetic diversity estimation. Therefore, universal tools, such as double-digest restriction-site-associated DNA sequencing (ddRADseq), could be useful when conducting conservation genetic studies on rarely studied parasites. Here, we generated a ddRADseq dataset that includes all 3 described Taiwanese horsehair worms (Phylum: Nematomorpha), possibly one of the most understudied animal groups. Additionally, we produced data for a fragment of the cytochrome c oxidase subunit I (COXI) for the said species. We used the COXI dataset in combination with previously published sequences of the same locus for inferring the effective population size (Ne) trends and possible population genetic structure.We found that a larger and geographically broader sample size combined with more sequenced loci resulted in a better estimation of changes in Ne. We were able to detect demographic changes associated with Pleistocene events in all the species. Furthermore, the ddRADseq dataset for Chordodes formosanus did not reveal a genetic structure based on geography, implying a great dispersal ability, possibly due to its hosts. We showed that different molecular tools can be used to reveal genetic structure and demographic history at different historical times and geographical scales, which can help with conservation genetic studies in rarely studied parasites.

Potential PCR amplification bias in identifying complex ecological patterns: Higher species compositional homogeneity revealed in smaller-size coral reef zooplankton by metatranscriptomics.

PCR-based high-throughput sequencing has permitted comprehensive resolution analyses of zooplankton diversity dynamics. However, significant methodological issues still surround analyses of complex bulk community samples, not least as in prevailing PCR-based approaches. Marine drifting animals-zooplankton-play essential ecological roles in the pelagic ecosystem, transferring energy and elements to higher trophic levels, such as fishes, cetaceans and others. In the present study, we collected 48 size-fractionated zooplankton samples in the vicinity of a coral reef island with environmental gradients. To investigate the spatiotemporal dynamics of zooplankton diversity patterns and the effect of PCR amplification biases across these complex communities, we first took metatranscriptomics approach. Comprehensive computational analyses revealed a clear pattern of higher/lower homogeneity in smaller/larger zooplankton compositions across samples respectively. Our study thus suggests changes in the role of dispersal across the sizes. Next, we applied in silico PCR to the metatranscriptomics datasets, in order to estimate the extent of PCR amplification bias. Irrespective of stringency criteria, we observed clear separations of size fraction sample clusters in both metatranscriptomics and in silico datasets. In contrast, the pattern-smaller-fractioned communities had higher compositional homogeneity than larger ones-was observed in the metatranscriptomics data but not in the in silico datasets. To investigate this discrepancy further, we analysed the mismatches of widely used mitochondrial CO1 primers and identified priming site mismatches likely driving PCR-based biases. Our results suggest the use of metatranscriptomics or, although less ideal, redesigning the CO1 primers is necessary to circumvent these issues.

Modeling the geographical distributions of Chordodes formosanus and its mantis hosts in Taiwan, with considerations for their niche overlaps.

Species distribution models (SDMs) have conventionally been used for evaluating the distribution of individual species, but they can also be used, through comparing different SDMs, to evaluate the geographic similarity between taxa. In this study, we used a parasite and host system to infer the geographic overlaps between species with tight biological interaction, for example, parasites and their obligate host. Specifically, we used the horsehair worm Chordodes formosanus and its three mantis hosts to study the extent of niche overlap. We retrieved presence points for the host species and the parasite, and then we built SDMs with MaxEnt implemented in ENMeval using selected bioclim variables (based on variance inflation factor values) at 30s scale. The models showed that the hosts and parasite do not occur in the high elevation areas in Taiwan, which is expected based on their biology. Interestingly, the predicted parasite distribution included areas without collection records, implying local extinction or sampling bias. We subsequently evaluated niche overlap between hosts and the parasite according to five similarity indices (Schoener's D, I statistic, relative rank, Pearson correlation coefficient, and the rank correlation coefficient rho). Our models showed a high similarity of SDM predictions between hosts and the parasite. There were differences among metrics for which host shared the highest similarity with the parasite, but the majority of the results indicated that the Japanese boxing mantis had the highest niche similarity with the horsehair worm. The choice of the niche overlap metric to use can uncover information on the parasite's ecology, which can be important for endangered species. SDMs are reliable tools for host and parasite conservation management and could help improve our understanding of parasite biology and ecology.

Utilisation of Oxford Nanopore sequencing to generate six complete gastropod mitochondrial genomes as part of a biodiversity curriculum.

High-throughput sequencing has enabled genome skimming approaches to produce complete mitochondrial genomes (mitogenomes) for species identification and phylogenomics purposes. In particular, the portable sequencing device from Oxford Nanopore Technologies (ONT) has the potential to facilitate hands-on training from sampling to sequencing and interpretation of mitogenomes. In this study, we present the results from sampling and sequencing of six gastropod mitogenomes (Aplysia argus, Cellana orientalis, Cellana toreuma, Conus ebraeus, Conus miles and Tylothais aculeata) from a graduate level biodiversity course. The students were able to produce mitogenomes from sampling to annotation using existing protocols and programs. Approximately 4 Gb of sequence was produced from 16 Flongle and one MinION flow cells, averaging 235 Mb and N50 = 4.4 kb per flow cell. Five of the six 14.1-18 kb mitogenomes were circlised containing all 13 core protein coding genes. Additional Illumina sequencing revealed that the ONT assemblies spanned over highly AT rich sequences in the control region that were otherwise missing in Illumina-assembled mitogenomes, but still contained a base error of one every 70.8-346.7 bp under the fast mode basecalling with the majority occurring at homopolymer regions. Our findings suggest that the portable MinION device can be used to rapidly produce low-cost mitogenomes onsite and tailored to genomics-based training in biodiversity research.

First detection of Colletotrichumfructicola (Ascomycota) on horsehair worms (Nematomorpha).

Fungal members of Colletotrichum (Ascomycota) were found to be associated with Chordodesformosanus, one of the three currently known horsehair worm (Nematomorpha) species in Taiwan. The fungi were identified as Colletotrichumfructicola, which is mostly known as a plant pathogen, through the use of the nuclear ribosomal internal transcribed spacer and partial large subunit (nrITS + nrLSU) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) DNA sequences. To our knowledge, this report represents both the first records for Colletotrichum associated with hairworms and for fungi on Nematomorpha. These findings expand the knowledge on the ecological relationships of both clades.