Kinship influences sperm whale social organization within, but generally not among, social units

Sperm whales have a multi-level social structure based upon long-term, cooperative social units. What role kinship plays in structuring this society is poorly understood. We combined extensive association data (518 days, during 2005–2016) and genetic data (18 microsatellites and 346 bp mitochondrial DNA (mtDNA) control region sequences) for 65 individuals from 12 social units from the Eastern Caribbean to examine patterns of kinship and social behaviour. Social units were clearly matrilineally based, evidenced by greater relatedness within social units (mean r = 0.14) than between them (mean r = 0.00) and uniform mtDNA haplotypes within social units. Additionally, most individuals (82.5%) had a first-degree relative in their social unit, while we found no first-degree relatives between social units. Generally and within social units, individuals associated more with their closer relatives (matrix correlations: 0.18–0.25). However, excepting a highly related pair of social units that merged over the study period, associations between social units were not correlated with kinship (p > 0.1). These results are the first to robustly demonstrate kinship's contribution to social unit composition and association preferences, though they also reveal variability in association preferences that is unexplained by kinship. Comparisons with other matrilineal species highlight the range of possible matrilineal societies and how they can vary between and even within species.

To examine association preferences across different time scales, we used a variety of sampling periods, chosen depending on the definition of association and the association index used. The shortest period used was two hours, which corresponds to approximately two dive cycles in sperm whales and has been applied in other studies of this species [3,4]. With this sampling period, we aimed to maximize the number of samples while allowing ample opportunity for clusters to disband and clusters with new compositions to form. The longest period used was 'year', which has also been previously applied in this species [4] to highlight longterm associations, removing potential autocorrelation across sequential days.

Microsatellite Genotyping
All PCRs for microsatellite loci were carried out in 20 µl reactions in 1x PCR buffer, with 1.5 mM MgCl2, 0.2mM of each dNTP, 0.3 µM of each primer, 0.05 U/µl of GoTaq Flexi DNA polymerase (Promega, Madison, WI) and 10 ng of template DNA (based on functional concentration, determined based on ZFX/ZFY gene fragment brightness). Reactions were run on an ABI Veriti 96 well thermal cycler (Applied Biosystems, Foster City, CA) with the following parameters: initial denaturing for 5 min at 94°C, then cycles of denaturation for 30 s at 94°C, annealing for 1 min, and extension for 1 min at 72°C, followed by a final elongation step, of either 10 min at 72°C or 45 min at 60°C. For locus-specific annealing temperatures and numbers of cycle, see Table S2. We included a no-template negative control with all reactions.
For four loci that did not amplify well with this standard procedure, a biphasic touchdown (TD) PCR protocol was used, to maximize amplification of low quality DNA while minimizing spurious amplification. This protocol consisted of a phase of TD-PCR [5], where annealing temperature (Ta) was started at 10°C above the final Ta and dropped by 0.5°C with each cycle, for 20 cycles, followed by 10 cycles at the final Ta. In a second phase of PCR, 2 µl of this first PCR product was used as template DNA, and the same cycle parameters were used as for the standard procedure.
To genotype the samples, we performed capillary electrophoresis to size separate and visualize the PCR product, using an ABI 3500xl Genetic Analyzer (Applied Biosystems, Foster City, CA). Before loading samples for genotyping, PCR products for some loci were diluted in distilled water (see Table S2 for dilution ratios), and up to three loci (that were labelled with different fluorescent molecules and had been amplified in separate PCRs) were combined. We used the program GeneMarker (SoftGenetics, State College, PA) to automatically score fluorescence peaks, and all allele calls were confirmed manually by eye and then manually re-inspected a second time.

mtDNA haplotype sequencing
For the majority (80%) of sequencing reactions, initial PCRs were carried out in 20 µl reactions in 1x PCR buffer, with 1.5 mM MgCl2, 0.2mM of each dNTP, 0.3 µM of each primer, 0.05 U/µl of GoTaq Flexi DNA polymerase and 10 ng of template DNA (based on functional concentration). Reactions were run on an ABI Veriti 96 well thermal cycler with the following parameters: initial denaturing for 5 min at 94°C, then cycles of denaturation for 30 s at 94°C, annealing for 1 min at 55°C, and extension for 1 min at 72°C, followed by a final elongation step, of 45 min at 60°C. Excess dNTPs and primers were digested in an enzymatic reaction containing 5 μl PCR product, 0.65μl Antarctic phosphatase buffer (50 mM Bis-Tris-Propane-HCl, 1 mM MgCl2, 0.1 mM ZnCl2, pH 6.0), 0.1μl Antarctic phosphatase (New England Biolabs, Ipswich, MA), and 0.03μl exonuclease I (New England Biolabs, Ipswich, MA). For this reaction, samples were incubated for 15 min at 37°C, followed by 15 min at 80°C. Sequencing reactions, using the product from the preceding reaction, were then carried out in 15μl reactions using the BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, Foster City, CA), containing 1.5μl of Reaction Mix, 3μl of Sequencing Buffer, and 1μl (at 10 μM) of the primer t-Pro [6]. Reactions were run on an ABI Veriti 96 well thermal cycler with the following parameters: initial denaturing for 2 min at 96°C, then cycles of denaturation for 20 s at 96°C, annealing for 20 sec at 50°C, and extension for 4 min at 60°C.
The remaining 20% of reactions were carried out using a BigDye® Direct Cycle Sequencing Kit and the accompanying protocol (Applied Biosystems, Foster City, CA), which used M13 tailed primers.
After the sequencing reaction, salts, nucleotides and primers were removed via ethanol precipitation [7] and resuspended in 10μl of HiDi formamide (Applied Biosystems, Foster City, CA). We included a no-template negative control with all reactions, and 14 samples were duplicated as blind replicates to estimate the consistency of haplotype sequencing. The PCR products were visualized using an ABI 3500xl Genetic Analyzer. Sequences were manually trimmed and edited using 4Peaks (nucleoytes.com) and were manually aligned using BioEdit 7.2.5 [8].

Supplemental Discussion of Social Context and Social Structure
In social unit A, variation in association rates between the two matrilineal families did not have a clear relationship with changes in unit composition, but the year with the highest rate of association did correspond with the presence of two new calves (Table S3).
Notable increases in association rates between social units U and F correspond with the first observations of a new calf in social unit F in 2008, the loss of two adult members from social unit F in 2011, and the departure of a juvenile male from each social unit in 2012 (Table S4).