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.

Last call: Passive acoustic monitoring shows continued rapid decline of critically endangered vaquita.

The vaquita is a critically endangered species of porpoise. It produces echolocation clicks, making it a good candidate for passive acoustic monitoring. A systematic grid of sensors has been deployed for 3 months annually since 2011; results from 2016 are reported here. Statistical models (to compensate for non-uniform data loss) show an overall decline in the acoustic detection rate between 2015 and 2016 of 49% (95% credible interval 82% decline to 8% increase), and total decline between 2011 and 2016 of over 90%. Assuming the acoustic detection rate is proportional to population size, approximately 30 vaquita (95% credible interval 8-96) remained in November 2016.

The myodural bridge existing in the Nephocaena phocaenoides.

Recent studies have identified that the myodural bridge (MDB) between the rectus capitis posterior minor (RCPmi) and the cervical spinal dura mater in the posterior atlanto-occipital interspace in humans. And it was supposed that the MDB may play essential physiological roles. As a result, the MDB is possibly a highly conserved structure in the evolution of mammals. However, there is little confirmative description about the existence of the MDB in marine mammals. The objective of this study was to explore the existence and the fiber property of the MDB in the Neophocaena phocaenoides. Six cadavers of the Neophocaena phocaenoides with formalin fixation were used in this study. One was used for head and neck CT scanning and three-dimensional (3D) reconstruction and suboccipital region dissection, two were for sectional observation by P45 plastinated sheets of head and neck, and three were for histological analysis of suboccipial structures. This is the first study to demonstrate the existence of the MDB in the aquatic mammals. The rectus capitis dorsal minor (RCDmi) originated from the inferior border of the occiput and inserted into the cervical spinal dura mater. At the ventral aspect of the RCDmi, the MDB directly extended through the posterior atlanto-occipital interspace and connected with the cervical spinal dura mater which was consisted of type Ⅰ collagen. In addition, the dorsal atlanto-occipital membrane was not found in the Neophocaena phocaenoides. The tendinous myodural bridge extended from the RCDmi to the spinal dura mater through the posterior atlanto-occipital interspace in the Neophocaena phocaenoides.

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.

Dual instrument passive acoustic monitoring of belugas in Cook Inlet, Alaska.

As part of a long-term research program, Cook Inlet beluga (Delphinapterus leucas) presence was acoustically monitored with two types of acoustic sensors utilized in tandem in moorings deployed year-round: an ecological acoustic recorder (EAR) and a cetacean and porpoise detector (C-POD). The EAR was used primarily to record the calls, whistles, and buzzes produced by belugas and killer whales (Orcinus orca). The C-POD was used to log and classify echolocation clicks from belugas, killer whales, and porpoises. This paper describes mooring packages that maximized the chances of successful long-term data collection in the particularly challenging Cook Inlet environment, and presents an analytical comparison of odontocete detections obtained by the collocated EAR and C-POD instruments from two mooring locations in the upper inlet. Results from this study illustrate a significant improvement in detecting beluga and killer whale presence when the different acoustic signals detected by EARs and C-PODs are considered together. Further, results from concurrent porpoise detections indicating prey competition and feeding interference with beluga, and porpoise displacement due to ice formation are described.

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).

Acoustic capture-recapture method for towed acoustic surveys of echolocating porpoises.

Passive acoustic monitoring for cetaceans mainly employ fixed-location methods or point transect samplings; an acoustic survey from a moving platform to conduct line transects is less common. In this study, acoustic capture-recapture by combining a double-observer method with line transect sampling was performed to observe Yangtze finless porpoises. Two acoustic devices were towed with the distance between them varying 0.5 to 89.5 m. The conditional probabilities that both devices would detect the porpoises within the same time window were calculated. In a 1-s time window, it became smaller as the distance between the devices increased, approaching zero when the distance between them was more than 50 m. It was considered that the devices with less than 50 m distance detected the same signals from the same animals, which means the identical detection. When the distance between them is too great, the recapture rate is reduced and the incidence of false matching may increase. Thus, a separation distance of around 50 m between two devices in acoustic capture-recapture of Yangtze finless porpoises was recommended. Note that the performance of the double detections can change depending on the particular device used and on animal behaviors such as vocalizing interval, ship avoidance.

Unique feeding morphology in a new prognathous extinct porpoise from the Pliocene of California.

Modern porpoises (Odontoceti: Phocoenidae) are some of the smallest cetaceans and usually feed near the seafloor on small fish and cephalopods [1-3]. Within both extinct and extant phocoenids, no evidence for specialized mandibular morphology has been documented [4-7]. Here we describe a new species of extinct porpoise, Semirostrum ceruttii, from the marine Pliocene San Diego (4.2-1.6 mega-annum, Ma) and Purisima (5-2.5 Ma) formations of California. The mandibles comprise a long, fused, and nearly edentulous prognathous symphysis, extending farther beyond the rostrum than in any known mammal. Phylogenetic analyses based on morphology reconstruct Semirostrum ceruttii as sister to extant (crown) porpoise species with moderate support. We describe the spectacularly preserved holotype specimen based on computed tomography (CT) scans, which allowed visualization of the elongate mental and accessory canals within the symphysis. The elongate canals are similar to those found in Rynchops birds [8] and were likely involved in sensory function. Oblique labial wear facets present on numerous small conical mandibular teeth posterior to the symphysis suggest regular contact with benthic substrate. The unique mandibular and dental characteristics, along with robust scapulae, sternum, and unfused cervical vertebrae, support the interpretation that this species employed a form of benthic skim feeding by using its mandible to probe for and obtain prey.

Genome-wide SNP and population divergence of finless porpoises.

Single nucleotide polymorphisms (SNPs) are rapidly becoming the population genomic markers in addressing ecology, evolution, and conservation issues for their high capacity to access variability across the genome. We isolated a total of 140 ideal SNPs from the finless porpoise and used 78 (under Hardy-Weinberg equilibrium) of them to conduct those issues especially for addressing population genetic differentiation. Bayesian clustering and principal component analyses all suggested that finless porpoises in Chinese waters could be divided into three distinct genetic groupings. Low levels of within-population genetic variation (mean HE = 0.3405, standard deviation = 0.1188) and significant differentiation among populations (FST = 0.1050-0.1628, P < 0.01) were confirmed. Limited gene flow was found especially between the freshwater Yangtze River porpoise and the oceanic Yellow Sea and South China Sea populations, which strongly suggested that some barriers might have restricted their genetic exchange. These evidences not only support a recent subdivision of the finless porpoise into two species but also suggest a full species status for the Yangtze finless porpoise, especially considering the significant genetic divergence between freshwater and marine porpoises, in combination with the unique distribution of Yangtze finless porpoises in freshwater and their distinctness in physiological and morphological features.

Nested clade phylogeographical analysis of the finless porpoise (Neophocaena phocaenoides) inhabiting Chinese and Japanese coasts.

The finless porpoise (Neophocaena phocaenoides) is a small cetacean whose survival is largely affected by human activity. The characteristics and structures of 3 populations in China and 4 to 5 populations in Japan have been well documented, although their history and origins remain poorly understood. In this study, nested clade phylogeographical analysis was applied to mtDNA sequences from finless porpoises to delineate the historical factors shaping the divergence pattern of this species. The sampling range covered most habitats of the finless porpoise, including the Chinese and Japanese coasts and the Yangtze River. A nested cladogram was constructed, and all 3 (2-step) clades were linked together without interior or tip status. Our data indicated that haplotype C was the most common among most individuals and populations, which could be the most ancient haplotype among all of the sampled types. As populations expanded to reside in different areas, ancient types with haplotype C mutated to other haplotypes, which were kept endemic by geological barriers that changed during glacial cycles that retarded gene flow. Populations in Japanese Pacific coasts and inland sea and post-arc marginal seacoasts and the Yangtze River were formed by the residual individuals left when allopatric fragmentation occurred in the ancestral population. The existence of highly endemic haplotypes and high genetic diversity from the Ariake Sound, Tachibana Bay, and the Yellow Sea indicated a relatively isolated state in these areas during the evolutionary history of the finless porpoise. The phylogenetic pattern revealed in the present study provided a better understanding of the biogeographical events that affected the finless porpoise within this region.

Comparative morphology of porpoise (Cetacea: Phocoenidae) pterygoid sinuses: phylogenetic and functional implications.

High-resolution X-ray computed tomographic scans were used to examine pterygoid sinus morphology within extant porpoise species and one delphinid (Tursiops truncatus), in order to consider: 1) intraspecific and interspecific variation among the studied species; 2) the most parsimonious sequence of character acquisition; and 3) the potential functional roles of the preorbital lobes of the sinuses in sound reflection. Scans revealed that the pterygoid/palatine regions are mediolaterally broader in the earliest diverging phocoenid (Neophocaena phocaenoides) and Tursiops truncatus than the dorsoventrally elongated sinuses observed in other species. Rostrocaudal lengths of the sphenoidal regions of the sinuses in all individuals studied are proportionally similar, indicating conservatism in this region across species. The neonate Phocoena phocoena has shorter preorbital lobes than adults, but they are still proportionally longer than Neophocaena phocaenoides and Phocoena spinipinnis. The preorbital lobes broaden mediolaterally to varying degrees across species; in particular, Phocoenoides dalli has the largest dorsal and lateral expansion of this region. Assuming the highest pulse frequency produced by porpoises is 150 kHz, all regions of the preorbital lobes are thick enough to reflect the wavelengths produced. In addition, the neonate preorbital lobes are not as elongated as they are in adults, and the dorsal third of this region may not reflect sound to the same extent. This study reinforces the importance of using nondestructive methods to quantify variation in endocranial anatomy and the value of CT data for recovering phylogenetically useful information, as well as functional roles sinuses play in concert with the soft tissue head anatomy for biosonar.

Interspecific variation of ontogeny and skull shape among porpoises (Phocoenidae).

All extant members of Phocoenidae (porpoises) have been characterized as pedomorphic based on skeletal characters. To investigate the ontogenetic background for pedomorphosis and assess interspecific differences in ontogeny among phocoenids, samples of the six extant species were compared in terms of development of both epiphyseal and cranial suture fusion. Across all species, full maturity of the vertebral column was rare. Vertebral epiphyseal development did not progress so far in most Phocoena phocoena as in Phocoenoides dalli and Phocoena dioptrica. P. phocoena, Phocoena spinipinnis, Ph. dalli, and P. dioptrica, for which large series were available, were further compared in terms of ontogeny of cranial shape by three-dimensional geometric morphometrics. Ph. dalli and P. dioptrica generally showed further development of cranial sutures than the other species. Postnatal skull shape development was similar for all species studied; the majority of interspecific shape differences are present at parturition. Smaller species had a higher rate of shape development relative to growth in size than Ph. dalli and P. dioptrica, but they still showed less allometric development due to less postnatal growth. Interspecific shape differences indicate phylogenetic relationships similar to that proposed based on morphology or convergent evolution of the two pelagic species, Ph. dalli and P. dioptrica, under the scenarios suggested by recent molecular studies. A shape trend coinciding with habitat preference was detected; in species with pelagic preference the position and orientation of the foramen magnum aligned the skull with the vertebral column; the rostrum showed less ventral inclination, and the facial region was larger and more concave in lateral aspect.

Sequence polymorphism and geographical variation at a positively selected MHC-DRB gene in the finless porpoise (Neophocaena phocaenoides): implication for recent differentiation of the Yangtze finless porpoise?

Sequence polymorphism at the MHC class II DRB locus was investigated in three finless porpoise (Neophocaena phocaenoides) populations in Chinese waters. Intragenic recombination and strong positive selection were the main forces in generating sequence diversity in the DRB gene. MHC sequence diversity changed significantly along the study period. Significant decrease in heterozygosity and lost alleles have been detected in the Yangtze River population and South China Sea population since 1990. Furthermore, there is a trend of increasing population differentiation over time. Especially, the genetic differentiation between the Yangtze River population and the Yellow Sea population was very low prior to 1990 (F (ST) = 0.036, P = 0.009), but became very significant after 1990 (F (ST) = 0.134, P < 0.001), suggesting a recent augmentation of genetic differentiation between both populations probably in a relatively short-term period. Porpoises from the Yangtze River displayed divergent frequencies of shared and private alleles from those displayed by two marine populations, which suggest that the former riverine population has been under a different selection regime (characteristic of a fresh water environment) than that of its marine counterparts.

Detecting recent speciation events: the case of the finless porpoise (genus Neophocaena).

Recent speciation events provide important insights into the understanding and conservation of Earth's biodiversity, representing recent adaptations to a changing environment and an important source of future evolutionary potential. However, the most frequently applied criterion for molecular-based speciation investigations, that of reciprocal monophyly of mitochondrial sequences, overlooks recent speciation events where insufficient time has passed for fixed molecular differences to develop between putative species. Two morphologically distinguishable forms of finless porpoise (genus Neophocaena) exist in sympatry in the strait of Taiwan, however the taxonomic relationship of these different forms is controversial. To test the hypothesis that the two forms represent different species, a study was conducted based on morphological characters and microsatellite and mitochondrial markers. The data suggest that the two forms are highly differentiated in terms of both morphology and genetic characteristics, despite being sympatric, and therefore represent different species as defined by the biological species concept. Moreover, the two forms appear to have been reproductively isolated since sharing a common ancestor prior to the last major glaciation event approximately 18 000 years ago. However, this represents an insufficient amount of time for reciprocal monophyly to have developed, and thus previous studies based on this criterion have overlooked this speciation event and resulted in incorrect taxonomic classification of these forms.

Natriuretic peptides in cetaceans: identification, molecular characterization and changes in plasma concentration after landing.

Dolphins are aquatic animals free from gravity, and this may have imposed significant changes in their cardiovascular status and its hormonal regulation compared with terrestrial animals. This study molecularly characterized two major cardiovascular hormones, atrial and B-type natriuretic peptides (ANP and BNP) and measured their changes in dolphin plasma concentrations in relation to the cardiovascular status of the animal. We initially identified ANP and BNP in three species of dolphins (Lagenorhynchus obliquidens, Phocoenoides dalli and Tursiops truncatus). ANP precursors are highly conserved in most mammals, but dolphin BNP precursors were more variable. In molecular phylogenetic analyses, dolphin ANP and BNP precursors grouped with those of artiodactyls, particularly to the camel peptides. The chromatographic characterization of tissue and plasma molecular forms using specific radioimmunoassays showed that the predominant ANP and BNP in the atrium are prohormone and mature peptide, respectively, whereas mature ANP and BNP are circulating in the dolphin blood. A mass spectrometric analysis showed that atrial BNP consists of 26 amino acids, rather than the 32-amino-acid form detected in other mammals. Finally, changes in plasma ANP and BNP concentrations were examined in captive bottlenose dolphins (Tursiops truncatus) after their pool was drained. Plasma ANP and BNP concentrations did not change after landing, unlike terrestrial mammals. Plasma angiotensin II and cortisol concentrations did not change either, showing minor stress after landing. Since landed dolphins show a different cardiovascular status on land than terrestrial mammals, plasma ANP and BNP concentrations seem to reflect the cardiovascular status characteristic of dolphins.

Genetic variation of the MHC DQB locus in the finless porpoise (Neophocaena phocaenoides).

The Major Histocompatibility Complex (MHC) is a large multigene coding for glycoproteins that play a key role in the initiation of immune responses in vertebrates. The exon 2 region of the MHC DQB locus was analyzed using 160 finless porpoises from 5 populations in Japanese waters. The 5 populations were based on a previous mitochondrial DNA control region analysis, which showed distinct geographical separation. Eight DQB alleles were detected, and the geographical distribution of the alleles indicated that most of them are shared among the populations. Heterozygosity of the DQB alleles in each population ranged from 0.55 to 0.78, and for all 5 populations was 0.78. Low MHC variability is not a common feature in marine mammals, but the finless porpoise populations inhabiting coastal waters had a relatively high MHC heterozygosity. Balancing selection in the MHC DQB alleles of the finless porpoise was indicated by the higher rate of nonsynonymous than synonymous substitutions for PBR; however, an excess of hetrozygotes compared to expectation was not observed. This suggests that the MHC DQB locus in the finless porpoise may have been under balancing selection for a long evolutionary time period, and is influenced by genetic drift beyond the effect of balancing selection for short time periods in small local populations.

DNA surveillance: web-based molecular identification of whales, dolphins, and porpoises.

DNA Surveillance is a Web-based application that assists in the identification of the species and population of unknown specimens by aligning user-submitted DNA sequences with a validated and curated data set of reference sequences. Phylogenetic analyses are performed and results are returned in tree and table format summarizing the evolutionary distances between the query and reference sequences. DNA Surveillance is implemented with mitochondrial DNA (mtDNA) control region sequences representing the majority of recognized cetacean species. Extensions of the system to include other gene loci and taxa are planned. The service, including instructions and sample data, is available at http://www.dna-surveillance.auckland.ac.nz.

Phylogeography, intraspecific structure and sex-biased dispersal of Dall's porpoise, Phocoenoides dalli, revealed by mitochondrial and microsatellite DNA analyses.

Mitochondrial DNA (mtDNA) control-region sequences and microsatellite loci length polymorphisms were used to estimate phylogeographical patterns (historical patterns underlying contemporary distribution), intraspecific population structure and gender-biased dispersal of Phocoenoides dalli dalli across its entire range. One-hundred and thirteen animals from several geographical strata were sequenced over 379 bp of mtDNA, resulting in 58 mtDNA haplotypes. Analysis using F(ST) values (based on haplotype frequencies) and phi(ST) values (based on frequencies and genetic distances between haplotypes) yielded statistically significant separation (bootstrap values P < 0.05) among most of the stocks currently used for management purposes. A minimum spanning network of haplotypes showed two very distinctive clusters, differentially occupied by western and eastern populations, with some common widespread haplotypes. This suggests some degree of phyletic radiation from west to east, superimposed on gene flow. Highly male-biased migration was detected for several population comparisons. Nuclear microsatellite DNA markers (119 individuals and six loci) provided additional support for population subdivision and gender-biased dispersal detected in the mtDNA sequences. Analysis using F(ST) values (based on allelic frequencies) yielded statistically significant separation between some, but not all, populations distinguished by mtDNA analysis. R(ST) values (based on frequencies of and genetic distance between alleles) showed no statistically significant subdivision. Again, highly male-biased dispersal was detected for all population comparisons, suggesting, together with morphological and reproductive data, the existence of sexual selection. Our molecular results argue for nine distinct dalli-type populations that should be treated as separate units for management purposes.