Multilocus DNA metabarcoding diet analyses of small cetaceans: a case study on highly vulnerable humpback dolphins and finless porpoises from the Pearl River Estuary, China.

Accurate diet identification of top predators is crucial to fully understand their ecological roles. Compared to terrestrial animals, gathering dietary information from cetaceans is notoriously difficult. Here, we applied a multilocus metabarcoding approach to investigate the diet of vulnerable Indo-Pacific humpback dolphins and Indo-Pacific finless porpoises from the Pearl River Estuary (PRE), China. Our analyses identified 21 prey fish species from the 42 humpback dolphin stomachs, as well as 10 species of fish and 1 species of cephalopod from the 13 finless porpoise stomachs. All of the taxa were assigned to the species level, highlighting that the multimarker approach could facilitate species identification. Most of the prey species were small- and medium-sized fishes that primarily fed on zooplankton. The calculated similarity index revealed a moderated dietary overlap between the 2 cetaceans, presumably due to the feeding of the 2 predators in association with fishing vessels in the PRE. A more diverse diet was observed in humpback dolphins in the closed fishing season compared to the fishing season, implying the influence on the dolphin diet due to the availability of commercial fishery resources. However, according to the results of species rarefaction curves, our findings on the feeding habits of the 2 cetaceans are still limited by insufficient sample size and therefore should be interpreted with caution. This study represents a first attempt to apply the multilocus DNA metabarcoding technique in the diet analysis of small cetaceans, although more efforts are needed to improve this type of analysis.

Gapless genome assembly of East Asian finless porpoise.

In recent years, conservation efforts have increased for rare and endangered aquatic wildlife, especially cetaceans. However, the East Asian finless porpoise (Neophocaena asiaeorientalis sunameri), which has a wide distribution in China, has received far less attention and protection. As an endangered small cetacean, the lack of a chromosomal-level reference for the East Asian finless porpoise limits our understanding of its population genetics and conservation biology. To address this issue, we combined PacBio HiFi long reads and Hi-C sequencing data to generate a gapless genome of the East Asian finless porpoise that is approximately 2.5 Gb in size over its 21 autosomes and two sex chromosomes (X and Y). A total of 22,814 protein-coding genes were predicted where ~97.31% were functionally annotated. This high-quality genome assembly of East Asian finless porpoise will not only provide new resources for the comparative genomics of cetaceans and conservation biology of threatened species, but also lay a foundation for more speciation, ecology, and evolutionary studies. Measurement(s) Neophocaena asiaeorientalis sunameri • Gapless genome assembly • sequence annotation Technology Type(s) MGISEQ. 2000 • PacBio HiFi Sequencing • Hi-C Sample Characteristic - Organism Neophocaena asiaeorientalis sunameri Sample Characteristic - Environment seawater Sample Characteristic - Location Yellow Sea near Lianyungang City, Jiangsu Province, China.

Population Structure of Finless Porpoise (Neophocaena phocaenoides) Discovered off Coastal Waters, Republic of Korea.

The finless porpoise (Neophocaena phocaenoides Cuvier, 1829) is distributed in the coastal waters of Asia, throughout Indonesia to the east, and as far north as the Taiwan Strait. The finless porpoise has been declared critically endangered by the WWF (World Wide Fund for Nature), and in 2017 was rated vulnerable on the IUCN Red Threatened Species List. Since this species is distributed near the coast and has many interactions with humans, effective conservation of the species requires further studies into their genetic diversity and population. In this study, 45 samples were obtained from bycatch or stranded individuals in the East, South, and West Seas, where Korean porpoises were mainly distributed from 2017-2021. We compared 473 bp mtDNA sequences from the control region. Pairwise fixation indices (FST) revealed that the two populations differed significantly (FST = 0.4557, p = 0.000). In contrast to high levels of genetic differentiation, gene flow was identified as medium levels (Nm of 0.04-0.71). Our data suggest that finless porpoises may have undergone a historic differentiation event, and that finless porpoises in the three regions could be divided into two populations: West and East/South.

Detection of Cetacean Poxvirus in Peruvian Common Bottlenose Dolphins (Tursiops truncatus) Using a Pan-Poxvirus PCR.

Cetacean poxviruses (CePVs) cause 'tattoo' skin lesions in small and large cetaceans worldwide. Although the disease has been known for decades, genomic data for these poxviruses are very limited, with the exception of CePV-Tursiops aduncus, which was completely sequenced in 2020. Using a newly developed pan-pox real-time PCR system targeting a conserved nucleotide sequence located within the Monkeypox virus D6R gene, we rapidly detected the CePV genome in typical skin lesions collected from two Peruvian common bottlenose dolphins (Tursiops truncatus) by-caught off Peru in 1993. Phylogenetic analyses based on the sequencing of the DNA polymerase and DNA topoisomerase genes showed that the two viruses are very closely related to each other, although the dolphins they infected pertained to different ecotypes. The poxviruses described in this study belong to CePV-1, a heterogeneous clade that infects many species of dolphins (Delphinidae) and porpoises (Phocoenidae). Among this clade, the T. truncatus CePVs from Peru were more related to the viruses infecting Delphinidae than to those detected in Phocoenidae. This is the first time that CePVs were identified in free-ranging odontocetes from the Eastern Pacific, surprisingly in 30-year-old samples. These data further suggest a close and long-standing pathogen-host co-evolution, resulting in different lineages of CePVs.

Biofilm formation, antimicrobial assay, and toxin-genotypes of Clostridium perfringens type C isolates cultured from a neonatal Yangtze finless porpoise.

This is a culture-dependent study with the objective of pure culturing and characterizing pathogenic bacteria from the blowhole, lung, stomach and fecal samples of a neonatal crucially endangered Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis) that died 27 days after birth. Bacteria were inoculated using a swab onto blood and MacConkey agar plates and representative isolates were identified through 16S rRNA gene sequence analysis. A total of three Clostridium perfringens type C strains from the fecal samples were isolated. Toxin genes, including cpa, cpb and cpb2, were detected by PCR amplification, whereas the etx, iap and cpe genes were not detected. Biofilm formation of the three strains was then examined. Only one strain was capable of biofilm formation. In addition, isolates showed strong resistance against the antibiotics amikacin (3/3), erythromycin (1/3), gentamicin (3/3), streptomycin (3/3), and trimethoprim (3/3), while sensitivity to ampicillin (3/3), bacitracin (3/3), erythromycin (2/3), penicillin G (3/3), and tetracycline (3/3). The results suggested C. perfringens type C could have contributed to the death of this neonatal porpoise.

Relationship of stranded cetaceans in Thai territorial waters to global populations: Mitochondrial DNA diversity of Cuvier's beaked whale, Indo Pacific finless porpoise, pygmy sperm whale, and dwarf sperm whale.

Cetaceans inhabit oceans throughout the world. Four specific odontocetes, namely Cuvier's beaked whale (Ziphius cavirostris), Indo Pacific finless porpoise (Neophocaena phocaenoides), pygmy sperm whale (Kogia breviceps), and dwarf sperm whale (Kogia sima), have occasionally been found stranded along Thailand's coastal waters (the Andaman Sea and the Gulf of Thailand). Although shared haplotypes of each species for many locations have been found, and some species have revealed genetic structure through haplotype networks, cetaceans in Thai waters have never been investigated in terms of comparing haplotypes to those that have existed before. Herein, we have illustrated the matrilineally phylogeographic relationships among worldwide populations through Bayesian Phylogenetic tree computations using Markov Chain Monte Carlo (MCMC) and Median-Joining Networks (MJNs). Unique haplotypes of the control region mitochondrial DNA of Thai odontocetes were found for all species. Moreover, a high degree of worldwide haplotype diversity (hd) above 0.8 among the four species was detected, while the lowest degree of nucleotide diversity (π) was observed in the Indo Pacific finless porpoise (1.12% ± 0.184%). An expansion of the effective female population size worldwide of three odontocete species was detected using Bayesian Skyline Reconstruction, but this did not include the Indo Pacific finless porpoise. Because Thai seas are located within the Indo Polynesian province, where this biodiversity hotspot exists, we speculate that these odontocetes may also inhabit specific habitats within the Malay Peninsula and Thailand's territorial waters. Therefore, closer attention and monitoring of these cetacean populations will be necessary for future conservation efforts.

Integrated analysis of blood mRNAs and microRNAs reveals immune changes with age in the Yangtze finless porpoise (Neophocaena asiaeorientalis).

Populations of Yangtze finless porpoises (YFPs) have rapidly declined in recent decades, raising the specter of extinction. In order to protect YFPs, a greater understanding of their biology is needed, including studying how their immune functioning changes with age. Here, we systematically studied the hematologic and biochemical parameters, as well as mRNAs and miRNAs profiles of old, adult, and young YFPs. The lymphocyte (LYMPH), neutrophils (NEUT) and eosinophils (EOS) counts in old YFPs were lower than those in young or adult YFPs. When comparing old to adult YFPs, the latter showed higher expression of genes associated with the innate and adaptive immune systems, including complement components, major histocompatibility complex, interleukins, TNF receptors, and chemokines/cytokines. When comparing old to young YFPs, the most striking difference was in higher toll-like receptor signaling in the latter. When comparing adult to young YFPs, the former exhibited higher expression of genes related to adaptive immunity and the FoxO signaling pathway, but lower expression of genes associated with the PI3K-Akt signaling pathway. Negative miRNA-mRNA interactions were predicted in comparisons of the old and adult (326), old and young (316), adult and young (211) groups. Overall, these results delineate a progression from early innate immune function dominance to adaptive immune function enhancement (young to adult) and deterioration (adult to old), and the changes in miRNAs profile correlate with the effects of age on immune functions. This study is the first to observe the changes of immune function of Yangtze finless porpoise with age using transcriptome method, and the study's findings are of great significance for protecting this endangered species.

Investigating the distribution of the Yangtze finless porpoise in the Yangtze River using environmental DNA.

Determining the distribution of the Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis, YFP) in the Yangtze River has to date relied on traditional visual and counting methods, but such field surveys are time-consuming and expensive. Analyses using environmental DNA (eDNA) to investigate the presence and range of endangered aquatic species have proven to be more economical and effective detection methods, and are a non-invasive approach to sampling. A challenge of relying on eDNA for YFP monitoring is that the Yangtze River is characterized by high turbidity and a strong current. Here, we used an eDNA-based approach to estimate the presence of YFP at 18 sites in the Yangtze River in August 2017 and at an additional 11 sites in January 2018. At each sampling site, we filtered six 1 L water samples with 5 µm pore size filter paper and quantified the amount of YFP eDNA in each water sample using quantitative real-time polymerase chain reaction (qPCR). In addition, YFP eDNA was successfully detected in locations where we visually observed YFP, as well as in locations where YFP were not observed directly. We found that our eDNA-based method had higher detection rates than traditional field survey methods. Although YFP was visually observed in the Yangtze River in winter, water samples collected during the summer contained significantly higher YFP eDNA than winter water samples. Our results demonstrate the potential effectiveness of eDNA detection methods in determining the distribution of YFP in the Yangtze River.

Evaluating monitoring options for conservation: comparing traditional and environmental DNA tools for a critically endangered mammal.

While conservation management has made tremendous strides to date, deciding where, when and how to invest limited monitoring budgets is a central concern for impactful decision-making. New analytical tools, such as environmental DNA (eDNA), are now facilitating broader biodiversity monitoring at unprecedented scales, in part, due to time, and presumably cost, of methodological efficiency. Genetic approaches vary from conventional PCR (cPCR; species presence), to metabarcoding (community structure), and qPCR (relative DNA abundance, detection sensitivity). Knowing when to employ these techniques over traditional protocols could enable practitioners to make more informed choices concerning data collection. Using 12 species-specific primers designed for cPCR, eDNA analysis of the Yangtze finless porpoise (YFP; Neophocaena asiaeorientalis asiaeorientalis), a critically endangered aquatic mammal within the Yangtze River, we validated and optimized these primers for use in qPCR. We tested repeatability and sensitivity to detect YFP eDNA and subsequently compared the cost of traditional (visual and capture) sampling to eDNA tools. Our results suggest cPCR as the least expensive sampling option but the lack of PCR sensitivity suggests it may not be the most robust method for this taxon, predominately useful as a supplementary tool or with large expected populations. Alternatively, qPCR remained less expensive than traditional surveys, representing a highly repeatable and sensitive method for this behaviorally elusive species. Cost comparisons of surveying practices have scarcely been discussed; however, given budgetary constraints particularly for developing countries with limited local oversight but high endemism, we encourage managers to carefully consider the trade-offs among accuracy, cost, coverage, and speed for biodiversity monitoring.

Population genomics of finless porpoises reveal an incipient cetacean species adapted to freshwater.

Cetaceans (whales, dolphins, and porpoises) are a group of mammals adapted to various aquatic habitats, from oceans to freshwater rivers. We report the sequencing, de novo assembly and analysis of a finless porpoise genome, and the re-sequencing of an additional 48 finless porpoise individuals. We use these data to reconstruct the demographic history of finless porpoises from their origin to the occupation into the Yangtze River. Analyses of selection between marine and freshwater porpoises identify genes associated with renal water homeostasis and urea cycle, such as urea transporter 2 and angiotensin I-converting enzyme 2, which are likely adaptations associated with the difference in osmotic stress between ocean and rivers. Our results strongly suggest that the critically endangered Yangtze finless porpoises are reproductively isolated from other porpoise populations and harbor unique genetic adaptations, supporting that they should be considered a unique incipient species.

Genetic footprint of population fragmentation and contemporary collapse in a freshwater cetacean.

Understanding demographic trends and patterns of gene flow in an endangered species is crucial for devising conservation strategies. Here, we examined the extent of population structure and recent evolution of the critically endangered Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis). By analysing genetic variation at the mitochondrial and nuclear microsatellite loci for 148 individuals, we identified three populations along the Yangtze River, each one connected to a group of admixed ancestry. Each population displayed extremely low genetic diversity, consistent with extremely small effective size (≤92 individuals). Habitat degradation and distribution gaps correlated with highly asymmetric gene-flow that was inefficient in maintaining connectivity between populations. Genetic inferences of historical demography revealed that the populations in the Yangtze descended from a small number of founders colonizing the river from the sea during the last Ice Age. The colonization was followed by a rapid population split during the last millennium predating the Chinese Modern Economy Development. However, genetic diversity showed a clear footprint of population contraction over the last 50 years leaving only ~2% of the pre-collapsed size, consistent with the population collapses reported from field studies. This genetic perspective provides background information for devising mitigation strategies to prevent this species from extinction.

Loop-mediated isothermal amplification (LAMP) assay-A rapid detection tool for identifying red fox (Vulpes vulpes) DNA in the carcasses of harbour porpoises (Phocoena phocoena).

Carcasses of wild animals are often visited by different scavengers. However, determining which scavenger caused certain types of bite marks is particularly difficult and knowledge thereof is lacking. Therefore, a loop-mediated isothermal amplification (LAMP) assay (target sequence cytochrome b) was developed to detect red fox DNA in carcasses of harbour porpoises. The MSwab™ method for direct testing without prior DNA isolation was validated. As a detection device, the portable real-time fluorometer Genie® II was used, which yields rapid results and can be used in field studies without huge laboratory equipment. In addition to in vitro evaluation and validation, a stranded and scavenged harbour porpoise carcass was successfully examined for red fox DNA residues. The developed LAMP method is a valuable diagnostic tool for confirming presumable red fox bite wounds in harbour porpoises without further DNA isolation steps.

Using environmental DNA to assess population-wide spatiotemporal reserve use.

Scientists increasingly rely on protected areas to assist in biodiversity conservation, yet the efficacy of these areas is rarely systematically assessed, often because of underfunding. Still, adaptive management strategies to maximize conservation success often rely on understanding the temporal and spatial dynamism of populations therein. Examination of environmental DNA (eDNA) is a time and cost-effective way to monitor species' distribution, and quantitative polymerase chain reaction (qPCR) provides information on organismal abundance. To date, however, such techniques remain underused for population assessments in protected areas. We determined eDNA concentration of the critically endangered Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis) to describe its occurrence, range, and use of the Tian e-Zhou National Nature Reserve in Hubei, China, across seasons and hydrological depths. Despite the observation that total eDNA concentrations were highest in surface waters in summer, finless porpoise eDNA concentrations were significantly higher in deeper waters than in surface waters in summer. During the breeding season (spring), eDNA signals were site specific and restricted to the core area of the reserve. However, postbreeding eDNA concentrations were widespread across the reserve, encompassing sites previously thought to be unfrequented by the species. Our results suggest spatiotemporal idiosyncrasies in site, depth, and seasonal use of the reserve and a propensity for postbreeding population dispersal. With eDNA and qPCR we were able to assess an entire population's use of a protected area. Illuminating nuances in habitat use via eDNA could be valuable to set pragmatic conservation goals for this, and other, species.

The complete mitochondrial genome of the Neophocaena asiaeorientails sunamrei (Phocaenidae: Neophocaena).

The complete sequence of the mitochondrial DNA (mtDNA) of the East Asian finless porpoise (Neophocaena asiaeorientails sunamrei) has been completed and annotated in this study. The circular genome is 16 385 bp in length, consisting of 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA genes, and 2 non-coding areas. Ten protein-coding genes use ATG as the initiation codon, except that ND2 started with ATT, and ND3 and ND5 started with ATA. Canonical TAA and TAG termination codons are found in seven protein-coding genes, the remaining four (ND4, ND2, ND3, and COIII) have incomplete termination codons (T or TA), in addition, the COI ends with AGG, and the Cyt b ends with AGA. On the neighbor-joining phylogenetic tree, these results show that the relationship of Phocoenidae is closer with Delphinidae.

Trace element concentrations, risks and their correlation with metallothionein genes polymorphism: A case study of narrow-ridged finless porpoises (Neophocaena asiaeorientalis) in the East China Sea.

The concentration of trace elements (TEs) and their risk to narrow-ridged finless porpoises (Neophocaena asiaeorientalis) are still unclear. The present study determined the concentration of typical TEs in liver, kidney, and muscle tissues from porpoises in the East China Sea, assessed potential health risk of TEs to porpoises, and explored the relationship between TE concentration and metallothionein genes (MTs) polymorphism. It was found that Zn, Cu, Mn, Cd and Hg were highly accumulated in liver, and Cd was highly accumulated in kidney. The concentrations of Cr, As, Pb and Ni were very low in all three tissues. TE concentrations showed significant positive correlation with body length, and sexual variation. The levels of most TEs were higher in tissues of porpoises in Ningbo and Nantong than in Pingtan, which is likely related to the local environment pollution level. The risk assessment showed that porpoises from Nantong and Ningbo could face health risks due to Hg, As, Cd, Pb, and Cr exposure. Moreover, two polymorphic sites on the MT4 gene were found to be significantly associated with increased levels of Hg, Cd, Zn and Mn. Whether these two polymorphic sites are involved in expression of MTs, or other functional processes, needs further research.

Parentage-Based Group Composition and Dispersal Pattern Studies of the Yangtze Finless Porpoise Population in Poyang Lake.

Social behaviors are poorly known for the critically endangered Yangtze finless porpoise (YFP, Neophocaena asiaeorientalis asiaeorientalis). Here, group composition and dispersal patterns of the YFP population living in the Poyang Lake were studied by parentage-based pedigree analyses using 21 microsatellite loci and a 597 bp segment of the mitochondrial DNA control region. In this study, 21 potential mother-offspring pairs and six potential father-offspring pairs (including two potential parents-offspring pairs) were determined, among which 12 natural mother-offspring groups and a maternal group of three generations were found. No genetically-determined fathers were found associated with their offspring. This study also found that maternally related porpoises at the reproductive state tend to group together. This suggest maternal relationship and reproductive state may be factors for grouping in the YFP population. In natural mother-offspring groups, male offspring were all younger than two years old, which suggest male offspring may leave their mothers at approximately two years of age, or at least they were not in tight association with their mothers as they may have been under two years old. However, female offspring can stay longer with their mothers and can reproduce in the natal group.

Organization and characteristics of the major histocompatibility complex class II region in the Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis).

Little is known about the major histocompatibility complex (MHC) in the genome of Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis) (YFP) or other cetaceans. In this study, a high-quality YFP bacterial artificial chromosome (BAC) library was constructed. We then determined the organization and characterization of YFP MHC class II region by screening the BAC library, followed by sequencing and assembly of positive BAC clones. The YFP MHC class II region consists of two segregated contigs (218,725 bp and 328,435 bp respectively) that include only eight expressed MHC class II genes, three pseudo MHC genes and twelve non-MHC genes. The YFP has fewer MHC class II genes than ruminants, showing locus reduction in DRB, DQA, DQB, and loss of DY. In addition, phylogenic and evolutionary analyses indicated that the DRB, DQA and DQB genes might have undergone birth-and-death evolution, whereas the DQB gene might have evolved under positive selection in cetaceans. These findings provide an essential foundation for future work, such as estimating MHC genetic variation in the YFP or other cetaceans. This work is the first report on the MHC class II region in cetaceans and offers valuable information for understanding the evolution of MHC genome in cetaceans.

Mitochondrial genome of the Neophocaena asiaeorientalis asiaeorientalis (Phocaenidae: Neophocaena).

Yangtze finless porpoise, Neophocaena asiaeorientalis asiaeorientalis, is a cetacea animal which has been reclassified as a Critically Endangered species by IUCN in 2013. In this study, we determined the complete mitochondrial genome of N. a. asiaeorientalis for use in future phylogenetic analyses. The complete mitochondrial genome of N. a. asiaeorientalis (16,385 bp in length) contained 37 genes (13 protein-coding genes, 2 rRNA genes, and 22 tRNA genes) and a non-coding region (D-loop). Additionally, a rep-origin (35 bp) exists which is located between tRNA(Asn) and tRNA(Cys).

Assembly and characterization of the MHC class I region of the Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis).

The Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis; YFP) is the sole freshwater subspecies of N. asiaeorientalis and is now critically endangered. Major histocompatibility complex (MHC) is a family of highly polymorphic genes that play an important immunological role in antigen presentation in the vertebrates. Currently, however, little is known about MHC region in the genome of the YFP, which hampers conservation genetics and evolutionary ecology study using MHC genes. In this work, a nucleotide sequence of 774,811 bp covering the YFP MHC class I region was obtained by screening a YFP bacterial artificial chromosome (BAC) library, followed by sequencing and assembly of positive BAC clones. A total of 45 genes were successfully annotated, of which four were MHC class I genes. There are high similarities among the four YFP MHC class I genes (>94%). Divergence in the coding region of the four YFP MHC class I genes is mainly localized to exons 2 and 3, which encode the antigen-binding sites of MHC class I genes. Additionally, comparison of the MHC structure in YFP to those of cattle, sheep, and pig showed that MHC class I genes are located in genome regions with regard to the conserved genes, and the YFP contains the fewest MHC class I genes among these species. This is the first report characterizing a cetacean MHC class I region and describing its organization, which would be valuable for further investigation of adaptation in natural populations of the YFP and other cetaceans.