Molecular phylogenetics of the Clupeiformes based on exon-capture data and a new classification of the order.

The Clupeiformes, including among others herrings, anchovies, shads and menhadens are ecologically and commercially important, yet their phylogenetic relationships are still controversial. Previous classification of Clupeiformes were based on morphological characters or lack of synapomorphic characters. More recent studies based on molecular data as well as new morphological evidence are keeping challenging their phylogenetic relations and there is still no consensus on many interrelationships within the Clupeiformes. In this study, we collected nuclear sequence data from 4,434 single-copy protein coding loci using a gene-capture method. We obtained a robust phylogeny based on 1,165 filtered loci with less than 30 % missing data. Our major findings include: 1) reconfirmation of monophyly of the Clupeiformes, that is, Denticipitidae is sister to all other clupeiforms; 2) the polyphyletic nature of dussumieriids and early branching of Spratelloididae from all other clupeoids were confirmed using datasets curated for less missing data and more balanced base composition in the respective taxa. The next branching clade is the monophyletic Engraulidae. Pristigasteridae also is monophyletic, but it was nested in the previously defined "Clupeidae". Within Pristigasteridae there is no support for monophyletic Pelloninae. Chirocentrus is close to Dussumieria and not to engraulids. The miniaturized Sundasalanx is placed close to the ehiravine Clupeonella, however, with a relatively deep split. The genus Clupea, is not part of the diverse "Clupeidae", but part of a clade containing additionally Sprattus and Etrumeus. Within the crown group clades, Alosidae and Dorosomatidae are retrieved as sister clades. Based on new fossil calibration points, we found that major lineages of the clupeiforms diverged in the late Cretaceous and early Paleogene. The extinction event at the end of the Cretaceous may have created ecological niches, which could have fueled the diversification of clupeiform fishes. Based on the strong evidence of the present study, we propose an updated classification of Clupeiformes consisting of ten families: Denticipitidae; Spratelloididae; Engraulidae (Engraulinae + Coiliinae); Clupeidae; Chirocentridae; Dussumieriidae; Pristigasteridae; Ehiravidae; Alosidae, Dorosomatidae.

A modified protocol with less clean-up steps increased efficiency and product yield of sequencing library preparation.

Library preparation is an essential step for the next-generation sequencing, such as whole-genome sequencing, reduced-representation genome sequencing, exome sequencing and transcriptome sequencing. The library preparation often involves many steps, including DNA fragmentation, end repair, ligation and amplification. Each step involves different enzymes and buffer systems, so many washing steps are implemented in between to clean-up the enzymes and solutes from the previous step. Those extra washing steps not only are tedious and costly, but more importantly may introduce cross-contamination and reduce the final library yield. Here, we modified the common protocol of Illumina library prep to reduce the washing steps by deactivating the enzymes with high temperature. The modified protocol has two less washing steps than the original one, which can save more than 40 min of hands-on time and reduce potential risk of cross-contamination. We compared our protocol with the original one by constructing libraries using 200 ng DNA of Tetraodon nigroviridis. The results showed that libraries prepared with the modified protocol had higher yields than that using the original protocol (53.4 ± 16.8 ng/ml vs. 8 ± 0.7 ng/ml), whereas the coverage and PCR duplication rate were similar. Furthermore, we eliminated the very first washing step after DNA shearing to preserve short DNA fragments, which increased proportion of fragments less than 100 bp DNA from 0.82 to 2.99%. In conclusion, using the modified protocols not only can save time and money, but also can generate higher yield and keep more short DNA fragments. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03168-5.

First report of de novo assembly and annotation from brain and blood transcriptome of an anadromous shad, Alosa sapidissima.

OBJECTIVES: American shad (Alosa sapidissima) is an important migratory fish under Alosinae and has long been valued for its economic, nutritional and cultural attributes. Overfishing and barriers across the passage made it vulnerable to sustain. To protect this valuable species, aquaculture action plans have been taken though there are no published genetic resources prevailing yet. Here, we reported the first de novo assembled and annotated transcriptome of A. sapidissima using blood and brain tissues. DATA DESCRIPTION: We generated 160,481 and 129,040 non-redundant transcripts from brain and blood tissues. The entire work strategy involved RNA extraction, library preparation, sequencing, de novo assembly, filtering, annotation and validation. Both coding and non-coding transcripts were annotated against Swissprot and Pfam datasets. Nearly, 83% coding transcripts were functionally assigned. Protein clustering with clupeiform and non-clupeiform taxa revealed ~ 82% coding transcripts retained the orthologue relationship which improved confidence over annotation procedure. This study will serve as a useful resource in future for the research community to elucidate molecular mechanisms for several key traits like migration which is fascinating in clupeiform shads.

Inline index helped in cleaning up data contamination generated during library preparation and the subsequent steps.

BACKGROUND: High-throughput sequencing involves library preparation and amplification steps, which may induce contamination across samples or between samples and the environment. METHODS: We tested the effect of applying an inline-index strategy, in which DNA indices of 6 bp were added to both ends of the inserts at the ligation step of library prep for resolving the data contamination problem. RESULTS: Our results showed that the contamination ranged from 0.29 to 1.25% in one experiment and from 0.83 to 27.01% in the other. We also found that contamination could be environmental or from reagents besides cross-contamination between samples. CONCLUSIONS: Inline-index method is a useful experimental design to clean up the data and address the contamination problem which has been plaguing high-throughput sequencing data in many applications.

A high-resolution genome of an euryhaline and eurythermal rhinogoby (Rhinogobius similis Gill 1895).

Rhinogobius similis is distributed in East and Southeast Asia. It is an amphidromous species found mostly in freshwater and sometimes brackish waters. We have obtained a high-resolution assembly of the R. similis genome using nanopore sequencing, high-throughput chromosome conformation capture (Hi-C), and transcriptomic data. The assembled genome was 890.10 Mb in size and 40.15% in GC content. Including 1373 contigs with contig N50 is 1.54 Mb, and scaffold N50 is 41.51 Mb. All of the 1373 contigs were anchored on 22 pairs of chromosomes. The BUSCO evaluation score was 93.02% indicating high quality of genome assembly. The repeat sequences accounted for 34.92% of the whole genome, with retroelements (30.13%), DNA transposons (1.64%), simple repeats (2.34%), and so forth. A total of 31,089 protein-coding genes were predicted in the genome and functionally annotated using Maker, of those genes, 26,893 (86.50%) were found in InterProScan5. There were 1910 gene families expanded in R. similis, 1171 gene families contracted and 170 gene families rapidly evolving. We have compared one rapidly change gene family (PF05970) commonly found in four species (Boleophthalmus pectinirostris, Neogobius melanostomus, Periophthalmus magnuspinnatus, and R. similis), which was found probably related to the lifespan of those species. During 400-10 Ka, the period of the Guxiang Ice Age, the population of R. similis decreased drastically, and then increased gradually following the last interglacial period. A high-resolution genome of R. similis should be useful to study taxonomy, biogeography, comparative genomics, and adaptive evolution of the most speciose freshwater goby genus, Rhinogobius.

Cross-species gene enrichment revealed a single population of Hilsa shad (Tenualosa ilisha) with low genetic variation in Bangladesh waters.

Tenualosa ilisha is a popular anadromous and significant trans-boundary fish. For sustainable management and conservation of this fish, drawing an appropriate picture reflecting population status of this species is very essential based on their all-strategic habitats in Bangladesh. In this study, 139 samples from 18 sites were collected and cross-species gene enrichment method was applied. Like most of the Clupeiforms, nucleotide diversity of this shad was very low (0.001245-0.006612). Population differences between most of the locations were low and not significant (P > 0.05). However, P values of a few locations were significant (P < 0.05) but their pairwise FST values were very poor (0.0042-0.0993), which is inadequate to recognize any local populations. Our study revealed that the presence of a single population in the Bangladesh waters with some admixtured individuals, which may contain partial genes from other populations. Most of the individuals were admixed without showing any precise grouping in the ML IQtree and Network, which might due to their highly migratory nature. Fishes from haors and small coastal rivers were not unique and no genetic differences between migratory cohorts. The hilsa shad fishery should be managed considering it as a single panmictic population in Bangladesh with low genetic diversity.

Gene markers for exon capture and phylogenomics in ray-finned fishes.

Gene capture coupled with the next-generation sequencing has become one of the preferred methods of subsampling genomes for phylogenomic studies. Many exon markers have been developed in plants, sharks, frogs, reptiles, fishes, and others, but no universal exon markers have been tested in ray-finned fishes. Here, we identified a suite of "single-copy" protein-coding sequence (CDS) markers through comparing eight fish genomes, and tested them empirically in 83 species (33 families and nine orders or higher clades: Acipenseriformes, Lepisosteiformes, Elopomorpha, Osteoglossomorpha, Clupeiformes, Cypriniformes, Gobiaria, Carangaria, and Eupercaria; sensu Betancur et al. 2013). Sorting the markers according to their completeness and phylogenetic decisiveness in taxa tested resulted in a selection of 4,434 markers, which were proven to be useful in reconstructing phylogenies of the ray-finned fishes at different taxonomic levels. We also proposed a strategy of refining baits (probes) design a posteriori based on empirical data. The markers that we have developed may greatly enrich the batteries of exon markers for phylogenomic study in ray-finned fishes.