DOI: 10.5061/dryad.9zw3r22j7
title: Humpback pregnancy rates and environmental variation: Supporting datasets

description: The krill surplus hypothesis of unlimited prey resources available for
 Antarctic predators due to commercial whaling in the 20th century has
 remained largely untested since the 1970s. Rapid warming of the Western
 Antarctic Peninsula (WAP) over the past 50 years has resulted in decreased
 seasonal ice cover and a reduction of krill. The latter is being
 exacerbated by a commercial krill fishery in the region. Despite this,
 humpback whale populations have increased but may be at a threshold for
 growth based on these human-induced changes. Understanding how
 climate-mediated variation in prey availability influences humpback whale
 population dynamics is critical for focused management and conservation
 actions. Using an eight-year dataset (2013–2020), we show that
 inter-annual humpback whale pregnancy rates, as determined from
 skin-blubber biopsy samples (n = 616), are positively correlated with
 krill availability and fluctuations in ice cover in the previous year.
 Pregnancy rates showed significant inter-annual variability, between 29%
 and 86%. Our results indicate that krill availability is in fact limiting
 and affecting reproductive rates, in contrast to the krill surplus
 hypothesis. This suggests that this population of humpback whales may be
 at a threshold for population growth due to prey limitations. As a result,
 continued warming and increased fishing along the WAP, which continue to
 reduce krill stocks, will likely impact this humpback whale population and
 other krill predators in the region. Humpback whales are sentinel species
 of ecosystem health, and changes in pregnancy rates can provide
 quantifiable signals of the impact of environmental change at the
 population level. Our findings must be considered paramount in developing
 new and more restrictive conservation and management plans for the
 Antarctic marine ecosystem and minimizing the negative impacts of human
 activities in the region.
description: <em>Biopsy collection</em> We
 collected skin and blubber samples from female humpback whales during the
 2013–2020 austral summers (December-March). This was done in the nearshore
 waters of the Western Antarctic Peninsula (WAP) using standard biopsy
 techniques (Fig. 1). We used a crossbow to project modified bolts and 40mm
 stainless steel biopsy tips (CetaDart) to obtain samples from a distance
 of 10–30 meters, targeting the area of the body below the dorsal fin.  
 Samples were collected opportunistically when whales were encountered
 during prey or visual surveys conducted within ~10 nautical miles of
 scientific research stations (i.e., Palmer Station, Anvers Island, USA, or
 Akademik Vernadsky Station, Galindez Island, Ukraine). Dedicated research
 cruises or platforms of opportunity, including ecotour vessels, were also
 used. Dependent calves were not sampled during seasons 2013–2019, but all
 age and sex classes of humpback whales were sampled during 2020. Because
 of this change in protocol, samples from calves were not included in any
 analysis. However, the presence of a calf was recorded and identified, as
 evident by its smaller size (less than half of the presumed mother's
 length) and close association with an adult, presumed to be the mother. 
 Supplementary data (including location and group size) were recorded at
 every biopsy event. Samples were stored frozen whole at -20°C until used
 for analysis. <em>DNA
 profiling</em> A standard DNA profile, including
 sex-specific markers and microsatellite genotypes, was used to identify
 individual whales. DNA was extracted from the skin-blubber interface using
 a commercially available kit (DNeasy 96 Blood & Tissue Kit,
 Qiagen, Hilden, Germany). The sex of each sampled whale was determined by
 amplification of sex-specific markers following the protocols of Gilson et
 al. Results were compared to controls for a known male and female using
 gel electrophoresis. Samples were genotyped using 10
 previously published microsatellite loci to resolve the individual
 identity of each sampled whale and remove potential duplicates (Table S1).
 Alleles were sized and binned using the software program Genemapper v3.7
 (Applied Biosystems). The total number of amplified loci for a given
 sample was considered as an added quality control threshold, with samples
 amplifying for less than 7 loci considered poor quality and repeated or
 removed from final dataset. Given the estimated probability of identity
 for these loci from previous studies, we assumed that samples matching at
 a minimum of seven loci to be recaptures of the same individual.
 Recaptures of the same individual were removed from the analysis. The
 expected probability of identity (P<sub>ID</sub>; the
 probability that two individuals drawn at random from a population will
 have the same genotype by chance) for each locus was calculated in GenAlEx
 v6.5. Cervus 3.0.7 was used to compute the number of alleles (K), observed
 and expected heterozygosity (HO and HE), and the probability of identity
 for all individual matches. <em>Hormone
 extraction and quantification</em> We extracted
 steroid hormones from the blubber portion of the biopsy samples following
 standard methods. Briefly, to quantify hormone biomarkers (i.e.,
 progesterone), we sub-sectioned a cross-sectional sub-sample (~0.15g)
 spanning from the epidermis-blubber interface to the most internal layer
 of the biopsy. These sub-samples were then homogenized multiple times
 using an automated bead mill homogenizer (Bead Ruptor Elite, Omni
 International). Following the completion of the homogenization process, we
 isolated progesterone using a series of chemical washes, evaporations, and
 separations. The final hormone residue was stored at -20°C until
 analysis. We quantified the amount of hormone in each
 extract using a commercially available enzyme immunoassay used extensively
 in similar studies. Our progesterone EIA kit (EIA kit 900-011, ENZO Life
 Sciences, Farmingdale, NY) had a 100% reactivity with progesterone and an
 assay detection limit between 15 and 500 pg/mL. Two additional standard
 dilutions were added to allow for a lower detection limit of the standard
 curve to 3.81 pg/mL. We determined extraction efficiency by spiking
 subsamples of blubber from a dead, stranded animal of known pregnancy
 status, with 150ng of progesterone and including these with every
 extraction. We calculated the percentage of progesterone recovered after
 each extraction and adjusted each sample concentration to this efficiency
 prior to statistical analyses. An extraction efficiency greater than 60%
 was adequate and is based on the reported range of efficiencies seen using
 these methods.  If the efficiency of an extraction set was less than 60%,
 the sample extracts were discarded, and the blubber samples were
 re-extracted and re-analyzed. Each assay was evaluated for color
 development using a Biotek plate reader Epoch
 (Gen5<sup>TM</sup> software [Biotek, USA]) with reading and
 correction wavelengths of 405 nm and 630 nm. Blubber hormone
 concentrations were then transformed into nanograms of progesterone per
 gram of blubber (wet weight). <em>Pregnancy
 classification</em> We assigned pregnancy of
 female humpback whales following previously published methods. Biopsy
 samples (n = 29) were collected from individuals of a known life-history
 stage from the Gulf of Maine feeding aggregation by the Center for Coastal
 Studies in Provincetown, MA. Using these control samples from the Gulf of
 Maine, the pregnancy state relative to blubber progesterone concentrations
 was modeled using a standard logistic regression model. Each WAP humpback
 sample of unknown pregnancy status was entered into the model, and the
 model returned a probability of being pregnant for each female sampled. If
 the probability of being pregnant was greater than 99.9%, that female was
 given an assignment of pregnant. If the probability of being pregnant was
 less than 0.1%, that female was assigned as not pregnant. If a biopsied
 female's probability of being pregnant was between those two bounds,
 that female was set as undetermined pregnancy. 
description: Please see the README document
 ("README-HumpbackPregnancyEnvironmentalChange.rmd") and the
 accompanying published article: L.J. Pallin, N.M. Kellar, D. Steel, N.
 Botero-Acosta, C.S. Baker, J. A. Conroy, D.P. Costa,  C.M. Johnson, D.W.
 Johnston, R. C. Nichols, D.P. Nowacek, A.J. Read, O. Savenko11,12, O.M.
 Schofield, S.E. Stammerjohn, D.K. Steinberg, A.S. Friedlaender. A surplus
 no more? Variation in krill availability impacts reproductive rates of
 Antarctic baleen whales. Global Change Biology.