Streaked horned lark <Emphasis Type="Italic">Eremophila alpestris strigata</Emphasis> has distinct mitochondrial DNA

The Streaked Horned Lark (STHL; Eremophila alpestris strigata) is a federal candidate for listing under the Endangered Species Act. We evaluated the conservation status and level of genetic diversity of the STHL using the complete mitochondrial ND2 gene. We sampled 32 STHLs from the southern Puget Sound region, the Pacific coast, and Whites Island in the Columbia River of Washington, and additional 68 horned larks from Alaska, alpine and eastern Washington, Oregon, California, and Asia (outgroups). Our Maximum Likelihood analysis of 32 haplotypes identified three geographically concordant clades in Pacific coast states: Pacific Northwest (alpine and eastern Washington, Alaska), Pacific Coast (western Washington, California), and Great Basin (eastern Oregon). Each of the three clades was supported by bootstrap values ≥86%. The distance among them varied from 0.72 to 0.79% nucleotide divergence excluding intraclade variation. The relationship among the clades was not resolved. AMOVA also showed significant structuring of haplotypes among the three clades. Differences among clades accounted for 75.7% of sequence variation, differences among localities within clades accounted for 12.1%, and differences among individuals within localities accounted for the remaining 12.2%. Although STHL populations were closely related to the Californian sample, they appeared unique and isolated. All pairwise F _st values involving the STHL samples were significant (except between themselves). STHLs appear to have remarkably low genetic diversity; all 32 STHLs shared the same haplotype. Even with small sample sizes, all other localities had multiple haplotypes. Because the STHL appears to be unique and isolated, and to have little genetic diversity our data suggest it should be a conservation priority.     

GENETIC ANALYSIS OF KILLER WHALE (ORCINUS ORCA) HISTORICAL BONE AND TOOTH SAMPLES TO IDENTIFY WESTERN U.S. ECOTYPES

Little is known about the historical range of killer whale ecotypes in the eastern North Pacific (ENP). It is possible that ranges have shifted in the last few decades because of changes in availability of food. In particular, the southern resident ecotype, currently found primarily in the inland waters of Washington State, is known to prey extensively on salmon, which have declined in recent decades along the outer coasts of Washington, Oregon, and California. To investigate historical distributions of this and the other ENP ecotypes, samples of teeth and bones were obtained from NMFS and museum collections. We amplified a short section of the mitochondrial DNA control region that contains four diagnostic sites that differentiate between haplotypes found in ecotypes of ENP killer whales. Results did not show any southern resident haplotypes in samples from south of the Washington State inland waterways. One whale genetically identified as a northern resident extends the known southernmost distribution of the population from Oregon to California. Items of diet identified from stomach contents of six of the whales genetically identified to ecotype conformed with what is known of the feeding habits of the various ecotypes.     

Characterization of Otostrongylus circumlitus from Pacific harbor and northern elephant seals

Otostrongylus circumlitus (Railliet, 1899) from Pacific harbor seals (Phoca vitulina richardsi) and northern elephant seals (Mirounga angustirostris) were examined using morphological and molecular methods to determine whether northern elephant seals along the central California coast are infected by the same species of Otostrongylus as are Pacific harbor seals in the same area. Fixed nematodes were examined and measured using light microscopy. The polymerase chain reaction (PCR) was used to amplify and sequence the second internal transcribed spacer (ITS-2) and D3 expansion (26S) regions of ribosomal DNA of O. circumlitus from Pacific harbor and northern elephant seals. The ITS-2 region was also amplified from Parafilaroides sp. from the Pacific harbor seal, northern elephant seal, and California sea lion (Zalophus californianus) and used for restriction fragment length polymorphism (RFLP) analysis. Morphologically, it was not possible to distinguish O. circumlitus from Pacific harbor and northern elephant seals, and over a consensus length of 443 base pairs (bp) for ITS-2 and 321 bp for D3 the sequences of O. circumlitus from both hosts were identical. With the PCR-RFLP assay, it was possible to distinguish O. circumlitus from Parafilaroides sp. The results suggest that O. circumlitus is the same species in Pacific harbor and northern elephant seals, and molecular methods make it possible to distinguish this nematode from related nematodes.     

Total mercury in stranded marine mammals from the Oregon and southern Washington coasts

Muscle samples from 105 marine mammals stranded along the Oregon and Washington coasts (2002–2009) were tested for levels of total mercury (THg) by Cold Vapor Atomic Fluorescence Spectrometry. The THg present is in the form of the highly toxic methylmercury. After normalizing tissue to 75% water weight, Steller sea lions and northern elephant seals exhibited the highest mean concentrations of THg followed by harbor seals, harbor porpoises, and California sea lions, 0.34 ± 0.278, 0.34 ± 0.485, 0.21 ± 0.216, 0.17 ± 0.169, and 0.15 ± 0.126 mg/kg normalized wet weight (ww), respectively. The mean normalized values demonstrate limited muscle methylmercury accumulation in these species in the Pacific Northwest. However, actual ww concentrations in some of the stranded carcasses may pose a risk to scavengers. Normalizing muscle mercury concentrations eliminated the variability from desiccation, and allowed for a clearer indication of the amount of mercury the animal accumulated before stranding.     

Novel and canine genotypes of Giardia duodenalis in harbor seals ( Phoca vitulina richardsi)

Feces of harbor seals (Phoca vitulina richardsi) and hybrid glaucous-winged/western gulls (Larus glaucescens / occidentalis) from Washington State's inland marine waters were examined for Giardia and Cryptosporidium spp. to determine if genotypes carried by these wildlife species were the same genotypes that commonly infect humans and domestic animals. Using immunomagnetic separation followed by direct fluorescent antibody detection, Giardia spp. cysts were detected in 42% of seal fecal samples (41/97). Giardia-positive samples came from 90% of the sites (9/10) and the prevalence of positive seal fecal samples differed significantly among study sites. Fecal samples collected from seal haulout sites with over 400 animals were 4.7 times more likely to have Giardia spp. cysts than samples collected at smaller haulout sites. In gulls, a single Giardia sp. cyst was detected in 4% of fecal samples (3/78). Cryptosporidium spp. oocysts were not detected in any of the seals or gulls tested. Sequence analysis of a 398 bp segment of G. duodenalis DNA at the glutamate dehydrogenase locus suggested that 11 isolates originating from seals throughout the region were a novel genotype and 3 isolates obtained from a single site in south Puget Sound were the G. duodenalis canine genotype D. Real-time TaqMan PCR amplification and subsequent sequencing of a 52 bp small subunit ribosomal DNA region from novel harbor seal genotype isolates showed sequence homology to canine genotypes C and D. Sequence analysis of the 52 bp small subunit ribosomal DNA products from the 3 canine genotype isolates from seals produced mixed sequences at could not be evaluated.     

INTERCHANGE AND ISOLATION OF HUMPBACK WHALES OFF CALIFORNIA AND OTHER NORTH PACIFIC FEEDING GROUNDS

Humpback whales feed in several high-latitude areas of the North Pacific. We examined the interchange of humpback whales between one of these areas, off California, and those in other feeding grounds in the eastern North Pacific:. Fluke photographs of 597 humpback whales identified off California between 1986 and 1992 were compared with those off Oregon and Washington (29); British Columbia (81); southeastern Alaska (343); Prince William Sound, Alaska (141); Kodiak Island, Alaska (104); Shumagin Islands, Alaska (22); and in the Bering Sea (7). A high degree of interchange, both inter-and intrayear, was found among humpback whales seen off California, Oregon, and Washington., A low rate of interchange was found between British Columbia and California.: two whales seen near the British Columbia/Washington border were photographed off California in a different year, No interchange was found between California and the three feeding areas in Alaska. Humpback whales off California, Oregon, and Washington form a single intermixing feeding aggregation with only limited interchange with areas farther north. These findings are consistent with photographic identification studies in the North Atlantic and with genetic studies in both the North Atlantic and North Pacific.     

Seroprevalence of Toxoplasma gondii in harbor seals (Phoca vitulina) in southern Puget Sound, Washington.

As part of the Puget Sound Ambient Monitoring Program of the Washington Department of Fish and Wildlife, serum samples from 380 harbor seals (Phoca vitulina) were tested for antibodies to Toxoplasma gondii in the modified agglutination test (MAT) incorporating formalin-fixed tachyzoites and mercaptoethanol. Antibodies to T. gondii were found in 29 of 380 (7.6%) seals with titers of 1:25 in 13, 1:50 in 14, and > or = 1:500 in 2 seals. Results indicate natural exposure of these wild marine mammals to T. gondii oocysts.     

Limited realized dispersal and introgressive hybridization influence genetic structure and conservation strategies for brown rockfish, <Emphasis Type="Italic">Sebastes auriculatus</Emphasis>

Understanding patterns of connectivity among marine fish populations with demersal adults and pelagic larvae is critical for effective conservation of west coast rockfishes. The brown rockfish (Sebastes auriculatus) occurs in nearshore habitat and is common from northern Baja California, Mexico to northern California, rare off the outer coast of Oregon and Washington and again common in the inland waters of Puget Sound, Washington. Here we examine patterns of microsatellite DNA diversity from throughout the species’ range as an indirect measure of long-term trends in larval dispersal. Genetic divergence was large and highly significant over all populations (F _ST=0.056, P<0.0001), and was significantly correlated with geographic distance when considering coastal populations. The best estimates of mean coastal dispersal distance were on the order of 10 km or less per generation. Diversity was relatively low in the Puget Sound, suggesting that Puget Sound rockfish populations experienced a post-glacial founder effect followed by genetic isolation and low effective population size. Puget Sound individuals appeared to have recent mixed ancestry as a result of introgression with S. maliger and S. caurinus. Genetic isolation of Puget Sound fish provides a basis for consideration as a Distinct Population Segment (DPS) under the provisions of the Endangered Species Act. We recommend that coastal brown rockfish fisheries be managed at regional rather than coast-wide scales, and that design of marine reserve networks considers the surprisingly low realized dispersal distance of some species with high dispersal potential.     

Biochemical polymorphism of starry flounder, Platichthys stellatus, from the northwestern and northeastern Pacific Ocean.

Three enzyme systems, MDH, PGM and AGPDH, were investigated in starry flounder collected from off northwestern Hokkaido Island, Japan; western Kamchatka; USSR; Puget Sound, Washington, USA; and Yaquina Bay, Oregon, USA. Significant differences in MDH allele distribution were found between the Asian-Puget Sound and Yaquina Bay-Puget Sound samples suggesting that this system may be useful to separate different population units. The allele frequencies of MDH and PGM did not show significant variation with body length and handedness in starry flounder collected from Puget Sound, Washington, and did not support the hypothesis of differential viability between dextral and sinistrial forms of starry flounder.     

Organochlorine exposure and health effects in stranded Steller sea lions (Eumetopias jubatus) and Pacific harbor seals (Phoca vitulina richardii) from Oregon and southern Washington coasts

Organochlorine (OC) pesticides and polychlorinated biphenyls (PCBs) have been detected in a variety of marine mammal species at levels associated with adverse health effects. Little is known about OC levels and impacts on health in pinnipeds with different life histories. We determined the health and levels of 18 OC pesticides and 16 PCB congeners in blubber samples from 20 Steller sea lions and 39 Pacific harbor seals stranded from Oregon and Southern Washington. The most commonly detected OC at the highest concentration was p,p′- dichlorodiphenyldichloroethylene (DDE). PCBs were detected in all samples as well. Hypothesis testing indicated that diseased Steller sea lions (males and females combined) had higher contaminant concentrations than healthy Steller sea lions, and diseased Pacific harbor seals had higher concentrations of total OCs than healthy animals. Differences were also noted between diseased and healthy animals when looking at individual sexes of each species. Diseased Steller sea lions had higher mean contaminant levels than diseased harbor seals and healthy Steller sea lions had higher mean contaminant concentrations than healthy Pacific harbor seals. These results show that species differences exist in both contaminant loads and sensitivity to contaminants, which may be due to differences in life histories and physiology.     

Coxiella burnetii infection of marine mammals in the Pacific Northwest, 1997-2010

Q fever is a zoonotic disease caused by the bacterium Coxiella burnetii. Humans are commonly exposed via inhalation of aerosolized bacteria derived from the waste products of domesticated sheep and goats, and particularly from products generated during parturition. However, many other species can be infected with C. burnetii, and the host range and full zoonotic potential of C. burnetii is unknown. Two cases of C. burnetii infection in marine mammal placenta have been reported, but it is not known if this infection is common in marine mammals. To address this issue, placenta samples were collected from Pacific harbor seals (Phoca vitulina richardsi), harbor porpoises (Phocoena phocoena), and Steller sea lions (Eumetopias jubatus). Coxiella burnetii was detected by polymerase chain reaction (PCR) in the placentas of Pacific harbor seals (17/27), harbor porpoises (2/6), and Steller sea lions (1/2) collected in the Pacific Northwest. A serosurvey of 215 Pacific harbor seals sampled in inland and outer coastal areas of the Pacific Northwest showed that 34.0% (73/215) had antibodies against either Phase 1 or Phase 2 C. burnetii. These results suggest that C. burnetii infection is common among marine mammals in this region.     

Biochemical polymorphism of starry flounder, Platichthys stellatus, from the northwestern and northeastern Pacific Ocean

Three enzyme systems, MDH, PGM and AGPDH, were investigated in starry flounder collected from off northwestern Hokkaido Island, Japan; western Kamchatka; USSR; Puget Sound, Washington, USA; and Yaquina Bay, Oregon, USA. Significant differences in MDH allele distribution were found between the Asian-Puget Sound and Yaquina Bay-Puget Sound samples suggesting that this system may be useful to separate different population units.
The allele frequencies of MDH and PGM did not show significant variation with body length and handedness in starry flounder collected from Puget Sound, Washington, and did not support the hypothesis of differential viability between dextral and sinistrial forms of starry flounder.     

Antibodies to phocine herpesvirus-1 are common in North American harbor seals (Phoca vitulina)

Phocine herpesvirus-1 (PhHV-1) has been associated with morbidity and high mortality in neonatal harbor seals (Phoca vitulina) along the Pacific coast of California (USA) and in northern Europe. Seals dying with PhHV-1 associated disease in California primarily have histopathologic evidence of adrenal necrosis or adrenalitis with herpesviral inclusion bodies. Little is known about prevalence of exposure to PhHV-1, modes of disease transmission, and viral pathogenesis in free-ranging harbor seal populations. To evaluate the prevalence in North America, 866 serum samples collected between 1994 and 2002 from harbor seals captured or stranded on the Pacific and Atlantic coasts of North America were assayed by enzyme linked immunosorbent assay (ELISA) for evidence of PhHV-1 exposure. Samples from three harbor seal age classes (pre-weaned, weaned, and subadults/adults) were obtained from each of four regions to compare exposure among sex, age class, and region. We found increasing prevalence with age as 37.5% of pre-weaned pups, 87.6% of weaned pups, and 99.0% of subadults and adults were seropositive. When accounting for age, no associations between seropositivity and sex or location of harbor seals were detected. These data indicate that PhHV-1 is endemic in the harbor seal populations of North America.     

Genetic Evidence of Postglacial Population Expansion in Puget Sound Rockfish (Sebastes emphaeus)

Several rockfish species (genus Sebastes) along the northeastern Pacific Ocean have rapidly declined in abundance owing in part to overfishing. A striking exception is the dwarf-like Puget Sound rockfish Sebastes emphaeus, whose densities have increased by several orders of magnitude over the last several decades. To describe their genetic structure, we sequenced 395 bp from the mitochondrial control region of 128 S. emphaeus adults from 5 locations spanning approximately 120 km of the Northwest Straits of Washington state. We detected no significant genetic differentiation among these populations and substantial genetic variation within populations, a pattern that may indicate high levels of ongoing gene flow. Preliminary data from 2 microsatellite loci are also consistent with panmixia. The mtDNA sequences also suggest that Puget Sound rockfish populations have expanded substantially since the retreat of Pleistocene glaciers made habitat in Puget Sound region available approximately 12,000 years ago.     

Phylogeography of the intertidal copepod Tigriopus californicus reveals substantially reduced population differentiation at northern latitudes

Previous studies of the intertidal copepod Tigriopus californicus revealed one of the highest levels of mitochondrial DNA differentiation ever reported among conspecific populations. The present study extends the geographical sampling northward, adding populations from northern California to south-east Alaska. The mitochondrial phylogeny for the entire species range, based on cytochrome oxidase I sequences for a total of 49 individuals from 27 populations, again shows extreme differentiation among populations (up to 23%). However, populations from Oregon northwards appear to be derived and have interpopulation divergences five times lower than those between southern populations. Furthermore, although few individuals were sequenced from each locality, populations from Puget Sound northward had significantly reduced levels of within-population variation. These patterns are hypothesized to result from the contraction and expansion of populations driven by recent ice ages.     

Deep mitochondrial divergence in Baja California populations of an aquilopelagic elasmobranch: the golden cownose ray

Assessing the realized effect of dispersal in the genetic makeup of a species has significant evolutionary, ecological, and economical consequences. Here, we investigate the genetic diversity and population differentiation in the aquilopelagic golden cownose ray Rhinoptera steindachneri from the Gulf of California (GC) and the Pacific coast of Baja California (PCBC) using the mitochondrial NADH2 gene. Low levels of genetic diversity were found with only 4 polymerase chain reaction-restriction fragment length polymorphism haplotypes among 76 specimens. Pacific coast organisms were fixed for a unique haplotype not shared with rays from the gulf; 92% of GC rays possessed a single NADH2 haplotype not found in the Pacific. This produced significant differentiation between the GC and the PCBC (Φ(CT) = 0.972, P < 0.001). A pronounced phylogeographic pattern was found in which GC haplotypes were reciprocally monophyletic relative to a very divergent Pacific lineage (d = 10%). Our results indicate that despite high dispersal potential, GC and PCBC golden cownose ray populations are characterized by highly divergent mitochondrial lineages. Although more evidence is needed to corroborate the genetic isolation and systematic status of PCBC and GC golden cownose rays, our results suggest a possible cryptic species in the region.     

GROWTH AND DEVELOPMENT IN FREE-RANGING HARBOR SEAL (PHOCA VITULINA) PUPS FROM SOUTHERN BRITISH COLUMBIA, CANADA

Harbor seals ( Phoca vitulina ) are small pinnipeds that are widely distributed throughout the temperate coastal regions of the Atlantic and Pacific oceans. We determined birth mass, neonatal growth rates, weaning age, and weaning mass of NE Pacific harbor seals ( P. v. richardsi ) during a capture-recapture study that spanned the nursing period (Sidney Island, British Columbia, Canada). Of 46 harbor seal pups initially captured, 28 were classified as newborns ( i. e. , < 24 h old). Mean body mass of newborns was 11.2 ± SE 0.31 kg. Pups were individually tagged and recaptured throughout the nursing period. Average daily mass gain during the nursing period was 394 ± 26 g. Mean birth mass of males did not differ significantly from females, although pups found with fetal pelage (lanugo) (21.4% of all newborns) were smaller at birth (9.8 ± 0.44 kg) than non-lanugo pups (11.6 ± 0.33 kg). Mean weaning mass was estimated at 23.6 ± 1.2 kg at a mean weaning age of 32 d ± 1.5 d. While birth and weaning masses differed little from the published data for offshore Sable Island harbor seals ( P. v. concolor ), British Columbia harbor seals are characterized by half the daily mass gain and a longer nursing period.     

Clines revisited: The timing of pupping in the harbour seal (Phoca vitulina)

The regional variation in the pupping season of the harbour seal ( Phoca vitulina ) was reviewed using the birth periods reported for 65 colony sites distributed over a range from 30.4 to 78.5 North latitude. The birth timing of P. v. vitulina was not related to latitude, but birthing in P. v. concolor along eastern North America exhibited a latitudinal cline. The timing of birth in P. v. richardsi varied in three distinct patterns: (1) a significant unidirectional latitudinal cline extending between Baja California and the west coast of Washington; (2) a cluster comprised of Puget Sound, Washington and Vancouver Island, British Columbia colonies in which birthing occurred an average of 65 days later than on the Washington coast; and (3) a cluster from northern British Columbia and Alaska which did not demonstrate a latitudinal cline. Insufficient data were available for the analysis of P. v. mellonae or P. v. stejnegeri .
We found great regional variation in the timing of birth among all colonies, with mean birth dates occurring as early as 15 March and as late as 3 September. Little variation existed north of 50. To the south of 50, however, most of the variation could be attributed to correlation with latitude or to affiliation with the Puget Sound, Washington-Vancouver Island, British Columbia geographic area.
Clinal variation in pupping could result from: (1) geographic variations in a selective factor with perhaps gene exchange between contiguous populations playing a role in smoothing the variation; or (2) for populations between Mexico and the west coast of Washington, regional variation in a non-selective environmental variable, such as photoperiod.     

CORRECTING AERIAL SURVEY COUNTS OF HARBOR SEALS (PHOCA VITULINA RICHARDSI) IN WASHINGTON AND OREGON

Aerial surveys of harbor seals on land produce only a minimum assessment of the population; a correction factor to account for the missing animals is necessary to estimate total abundance. In 1991 and 1992, VHF radio tags were deployed on harbor seals ( n = 124) at six sites in Washington and Oregon. During aerial surveys a correction factor to account for seals in the water was determined from the proportion of radio-tagged seals on shore during the pupping season. This proportion ranged from 0.54 to 0.74. Among the six sites there was no significant difference in the proportion of animals on shore nor was there a difference in age/sex categories of seals on shore between sites. The pooled correction factor for determining total population abundance was 1.53. An additional 32 seals were radio tagged in 1993 at one of the sites used in 1991. Comparing data from the two years, we found no interannual variation. Aerial surveys of all known harbor seal haul-out sites in Washington ( n = 319) and Oregon ( n = 68) were flown during the peak of the pupping season, 1991–1993. The Washington and Oregon harbor seal population was divided into two stocks based on pupping phenology, morphometics, and genetics. Mean counts for the Washington inland stock were 8,710 in 1991, 9,018 in 1992, and 10,092 in 1993. Oregon and Washington coastal stock mean counts were 18,363 in 1991, 18,556 in 1992, and 17,762 in 1993. Multiplying the annual count by the correction factor yielded estimates of harbor seal abundance in the Washington inland stock of 13,326 (95% CI = 11,637–15,259) for 1991, 13,798 (95% CI = 11,980–15,890) for 1992, and 15,440 (95% CI = 13,382–17,814) for 1993. In the Oregon and Washington coastal stock the corrected estimate of harbor seal abundance was 28,094 (95% CI = 24,697–31,960) in 1991, 28,391 (95% CI = 24,847–32,440) for 1992, and 27,175 (95% CI = 23,879–30,926) for 1993.     

Cytochrome P450 1A expression and organochlorine contaminants in harbour seals (Phoca vitulina): evaluating a biopsy approach

We previously reported in vivo induction of cytochrome P450 1A (CYP1A) by beta-naphthoflavone in skin and liver biopsies of captive harbour seals (Phoca vitulina richardsi). The present study evaluated CYP1A expression (immunoblot analysis and ethoxyresorufin O-deethylase activity-EROD) in harbour seals using two study designs: i) skin and liver biopsies from 20 harbour seal pups captured from coastal British Columbia (BC, Canada) and temporarily housed in captivity; and ii) skin biopsies from 42 free-ranging harbour seals captured and sampled on-site in multiple locations in BC and Washington State (USA). Toxic Equivalency Quotients (TEQs) were calculated for polychlorinated biphenyl, polychlorinated dibenzo-p-dioxin, and polychlorinated dibenzofuran residues measured in blubber from a subset of study animals (n=30). CYP1A data from the seal pups held temporarily in captivity show that CYP1A protein levels were greater in liver than skin and that CYP1A protein and EROD activity were correlated in skin and liver. However, analysis of free-ranging seals from different sites revealed that blubber organochlorine TEQ values did not correlate with skin CYP1A levels. CYP1A protein levels and EROD activities in skin of seal pups from the BC locations and from Puget Sound were relatively low, possibly reflecting contaminant levels that were not high enough to elicit a response, a small sample size, or methodological limitations. Our results show that CYP1A measurements in skin show promise as a biomarker of contaminant exposure, but that refinements to techniques and a larger sample size are needed.