DOI: 10.5061/dryad.k98sf7m3j
title: Prolonged maternal investment in northern bottlenose whales alters our understanding of beaked whale reproductive life history

description: Nursing and weaning periods are poorly understood in cetaceans due to the
 difficulty of assessing underwater behaviour in the wild. However, the
 onset and completion of weaning are critical turning points for individual
 development and survival, with implications for a species life history
 including reproductive potential. δ15N and δ13C deposited in odontocete
 teeth annuli provide a lifetime record of diet, offering an opportunity to
 investigate variation and trends in fundamental biology. While available
 reproductive parameters for beaked whales have largely been inferred from
 single records of stranded or hunted animals and extrapolated across
 species, here we examine the weaning strategy and duration of nursing in
 northern bottlenose whales (Hyperoodon ampullatus) by measuring stable
 isotopes deposited in teeth growth layer groups (GLGs). Using a collection
 of H. ampullatus teeth taken from whales killed during the whaling era (N
 = 48) and from two stranded specimens, we compared ontogenetic variation
 of δ15N and δ13C found in annual GLGs across all individuals, by sex and
 by region. We detected age-based trends in both δ15N and δ13C that are
 consistent across regions and males and females, and indicate that nursing
 is prolonged and weaning does not conclude until whales are 3-4 years old,
 substantially later than previous estimates of 1 year. Incorporating a
 prolonged period of maternal care into H. ampullatus life history
 significantly reduces their reproductive potential, with broad
 implications for models of beaked whale life history, energetics and the
 species’ recovery from whaling.
description: <strong>Materials and methods</strong>   Tooth collection and dentine sampling Teeth were taken from <i>H. ampullatus</i> killed by Norwegian whalers in the waters off northern Iceland in 1967 and northern Labrador in 1971 [27] (<b>Fig 1</b>). Northern bottlenose whales are usually found in groups of one to four, and whalers would take all the whales they encountered, regardless of sex or age class, so we assume our dataset has low demographic capture bias [21]. Individuals included in this analysis ranged from 4-27 years old (median age = 14). The teeth of two <i>H. ampullatus</i> that stranded in northeast Newfoundland in 2004 were also analyzed. The jaws of whaled specimens were originally boiled for two hours to facilitate tooth extraction [27]. Teeth were sectioned along the longitudinal midline and stored unpreserved at room temperature in individual sachets for over 40 years prior to this study. Genetic analysis of gum-tissue from the teeth used in this study confirmed the sex documented in the whaling records [28,29]. The teeth from Newfoundland animals were extracted from decomposed specimens, air dried and stored whole until being sectioned for this study. Similar to other odontocetes [30-32], <i>H. ampullatus</i> dentine is laminated, with one clear and one opaque layer defining each annual GLG within the cone of the tooth [23] (<b>Fig 2</b>). Only teeth with a clear neo-natal line and defined GLG structure across the first five years were retained for isotope analysis, reducing our sample size to 50 individuals (N = 6 from Iceland, N = 42 from Labrador, N = 2 from Newfoundland).  To improve GLG definition, tooth sections were initially polished using 30μm aluminum oxide lapping film [16] and then acid-etched using 10% formic acid [33]. GLGs were counted and aged assuming annual deposition, starting at the line that divides prenatal and postnatal dentine [16,23]. Using a single section of each tooth, GLGs 1-5 were sampled individually at a depth of 250-μm with a 300-μm-diameter drill bit, using a high-resolution micro-mill (New Wave Research, Freemont, California). When sufficient prenatal dentine was present it was sampled at a depth of 150 μm.  For mature individuals (&gt; 9 years old) [27], we also collected samples from older GLGs to estimate the range in δ<sup>15</sup>N in adult diet (N = 29).  However, as whales age their GLGs become compressed and are not wide enough to sample individually. Instead we collected samples representative of the mature age class by drilling across GLGs 8 -12 as a group with a 1 mm-diameter drill bit using a Dremel hand tool. Stable isotope analysis (δ<sup>15</sup>N / δ<sup>13</sup>C) Powdered dentine from each sampled GLG was weighed (~1 mg) into tin cups for isotopic analysis on a Vario EL Cube elemental analyzer (Elementar, Germany) connected to a DELTA Advantage isotope ratio mass spectrometer (Thermo, Germany). Isotope ratios are reported in Delta notation (δ) as per mil (‰) deviation from isotope ratios of atmospheric N<sub>2</sub> for nitrogen and Vienna Pee-Dee Belemnite (V-PDB) limestone for carbon. δ<sup>15</sup>N or δ<sup>13</sup>C are defined as δ = (R<sub>sample</sub> – R<sub>standard</sub>)/R<sub>standard</sub>), where R is the ratio of the abundance of the heavy to the light isotope. Values are normalized to internal standards nicotinamide, ammonium sulfate + sucrose, caffeine, and glutamic acid, whose isotopic compositions cover the natural range of samples (δ<sup>15</sup>N -16.61 to 16.58‰, δ<sup>13</sup>C -34.46 to ‒11.94‰) and are calibrated to international standards IAEA-N1(+0.4‰), IAEA-N2(+20.3‰), USGS-40(-4.52‰) and USGS-41(47.57‰) for δ<sup>15</sup>N, and IAEA-CH-6(-10.4‰), NBS-22(-29.91‰), USGS-40(-26.24‰) and USGS-41(37.76‰) for δ<sup>13</sup>C. Analytical precision based on repeated measures of laboratory reference materials not used in calibrations was ~0.1‰ for both δ<sup>15</sup>N and δ<sup>13</sup>C within multiple laboratory runs. Variation between duplicate measures of ~10% of samples had an absolute mean of 0.26 ‰ for δ<sup>15</sup>N and 0.21 ‰ for δ<sup>13</sup>C. The small size of some GLGs meant it was sometimes necessary to collect amounts less than 1 mg. A linearity study showed samples &lt;0.5 mg appeared to have a positive bias in d<sup>15</sup>N but not d<sup>13</sup>C, and further analysis was restricted to samples weighing &gt;0.5mg, reducing the number of GLG samples available for some individuals. Additionally, we omitted the smallest duplicate sample, so that only a single sample from an individual GLG was included in further analysis.   Data Analysis Following the screening for duplicates and sample weight described above, 50 individuals were included in summary statistics regardless of how many GLGs were available. However ontogenetic trend analysis was restricted to those individuals which had stable isotope data available from at least GLGs 1-3 (N = 37). Data structure, variables, and sample sizes are identified in <b>Table 1</b> and variable inclusion rationale and data sources are further described in <b>S2 Table. </b> Table 1. Data structure, variables and sample sizes. Dependent variables Independent variables Total individuals N = GLG samples GLG chronologies N = GLG samples δ15N δ13C Region GLG – Year, Age Class Sex Individual Age 50 IDs N = 244 GLGs (*288 including duplicate samples)   39 IDs N = 207 GLGs   For comparison with other published values and ecological studies, carbon isotope values were adjusted for the oceanic Suess effect, applying a factor of 0.0019‰ yr <sup>-1</sup> to δ<sup> 13</sup>C measured in GLGs; δ<sup> 13</sup>C<sub>cor</sub> values are approximately relative to the year 2000 [16,34,35]. The isotope values sampled from a cross section of mature GLGs (age 8-12) were assumed to represent the average isotopic profile of adult whales, and used as a benchmark for assessing when the weaning associated δ<sup>15</sup>N decline ended.                        
description: The dataset represents Nitrogen and Carbon stable isotope values
 from 50 individuals and 244 GLGs (&gt;0.5 mg), not including duplicate
 samples.