Ecological Archives E096-258-A1

Ecological Archives E096-258-A1

Sophie Lardy, Dominique Allainé, Christophe Bonenfant, and Aurélie Cohas. 2015. Sex-specific determinants of fitness in a social mammal. Ecology 96:2947–2959. http://dx.doi.org/10.1890/15-0425.1

Appendix A. Molecular and parentage analyses.

For genetic analyses, hairs were collected from 1992 to 1997, and tissue biopsies thereafter from all trapped individuals. All captured individuals were typed at 16 microsatellite loci: SSBibl1, SS-Bibl18, SS-Bibl20, SS-Bibl31, SS-Bibl4 (Klinkicht 1993); MS41, MS45, MS47, MS53, MS56, MS6, ST10 (Hanslik and Kruckenhauser 2000); Ma002, Ma018, Ma066, Ma091 (Da Silva et al. 2003). Microsatellite characteristics are given in Table A1. Tests of Hardy-Weinberg carried with the R library ‘genetics’ (Warnes, 2011) indicated that none of the loci showed deviation from Hardy–Weinberg equilibrium (1>P>0.05), except for MS53 (χ²=43.19, P=0.02, 10000 replicates). These tests were performed on dominant adults only to avoid potential bias caused by family structure and on all cohorts pooled to ensure sufficient sample size (n=230). Parental relationships were then confirmed through genetic exclusion and parentage analyses.

Parentage analyses were performed in two ways. First, the genotypes of each pup and of the dominant pair were compared to check maternity. For 16x674 mother-pup comparisons, only 1 mismatch at one locus (SS-Bibl20) between the putative mother (in 671 cases the dominant female and in 3 cases a subordinate female) and one of its pups was found. We then defined a pup as fathered by the dominant male if no mismatch was observed with the dominant male genotype (630 of 674 pups) and as not fathered by the dominant male if at least one mismatch was observed with the dominant male genotype (44 pups: one to eight mismatches). For eight pups, exclusions of paternity were based on only one mismatch with the dominant male. We nevertheless consider it unlikely that these pups were fathered by the dominant male, because (1) genotyping error rate was low (probability of finding an error for one allele should not exceed 0.0003, for details see Cohas et al. 2008), (2) all these pups and their parents were retyped and their genotypes confirmed, (3) the average mutation rate for microsatellites is low (1.67x10-4 per generation in M. marmota, Rassmann et al. 1994) and (4) only one mismatch with the putative mother has been found (see above). The genotypes of pups not fathered by the dominant male were then compared to the genotypes of all known sexually mature males in the family group. Among the 44 pups not fathered by the dominant male, 20 had genotypes compatible with that of a subordinate male in their family and 24 had a genotype incompatible with all subordinate males of their family.

Second, parentage analyses was repeated using the software CERVUS 3.0.3 (Kalinowski et al. 2007), with the following simulation settings: two candidate fathers per pup, 97% of candidate parents sampled, error rate of 1% to allow for mistyping and for mutations or null alleles, and assignment at a 98% confidence level. We then ran the parentage analyses with the mother identity known and with all the sexually matures males present for a given year in the population as putative fathers. The previous results were confirmed except for four pups where paternity could be assigned to both the dominant and a subordinate male. However, MHC markers (Ferrandiz et al. In prep) confirmed the dominant male to be the father of two and a subordinate male to be the father of another one. In the last case, even when considering these additional markers, the pup could still be assigned to both the dominant and a subordinate male. However, the sexual organs of the putative subordinate father showed no sign of development at capture, and all the other pups of the litter were assigned with no ambiguity to the dominant male. Thus, we parsimoniously considered this last pup to be fathered by the dominant male. Among the 24 pups that were neither fathered by the dominant male nor by a subordinate of the groups, seven were found to be fathered by an individual born in our study population in dispersal while the other 17 were fathered by unknown males.

In total, the 52 males for whom we estimated fitness produced 297 pups that survived to the age of one. Among these 297 pups, 288 (97%) were mothered by the dominant female and nine (3%) were mothered by three subordinate females. The 39 females for whom we estimated the LRS produced 269 pups that survived to the age of one. Among these 269 pups, 254 (94.4%) were fathered by the dominant male, 11 (4.10%) were fathered by a subordinate of the group unrelated to the dominant female and 13 (4.83%) were fathered by a transient male.

Table A1. Characteristics of the 16 microsatellites.

	Bibl1		Bibl18	Bibl20			Bibl31		Bibl4		Ma002		Ma018		Ma066		Ma091		MS41		MS45		MS47		MS53		MS56		MS6		ST10
	Alleles	Freq	Alleles	Freq	Alleles	Freq	Alleles	Freq	Alleles	Freq	Alleles	Freq	Alleles	Freq	Alleles	Freq	Alleles	Freq	Alleles	Freq	Alleles	Freq	Alleles	Freq	Alleles	Freq	Alleles	Freq	Alleles	Freq	Alleles	Freq
	95	0.15	132	<0.01	206	0.01	157	0.50	175	0.13	265	<0.01	296	0.25	231	0.62	159	0.13	184	0.17	107	0.40	176	0.03	132	0.13	104	0.02	142	0.05	116	0.14
	97	0.22	133	<0.01	208	0.19	159	0.27	178	<0.01	271	0.19	298	0.75	233	0.02	167	0.09	186	0.83	109	0.49	180	0.26	140	0.43	106	0.30	158	0.87	118	0.26
	101	0.43	137	0.01	216	0.38	161	0.18	188	0.17	279	0.50			241	0.36	169	0.04			111	0.10	182	0.16	142	0.42	108	0.68	160	0.07	120	0.21
	103	<0.01	143	0.36	218	0.33	163	0.05	190	0.66	281	0.30					171	0.01					184	0.18	144	<0.01	110	<0.01			130	0.05
	107	0.15	145	0.11	220	0.08			192	0.04	283	<0.01					173	0.17					186	0.32	148	<0.01					132	0.15
	109	0.04	147	0.38	222	<0.01											175	0.44					188	0.02							134	0.14
			149	0.10													177	0.02					190	0.01							136	0.03
																	179	0.09
																	188	<0.01															Total
N	1045		1042	1011			1044		1042		1015		1026		1035		1038		1032		1041		1033		1038		1037		1033		1031
Nb of Alleles	6		7	6			4		5		5		2		3		9		2		3		7		5		4		3		7		4.88
PIC	0.67		0.63	0.64			0.58		0.47		0.55		0.30		0.38		0.71		0.24		0.50		0.72		0.53		0.36		0.21		0.79		0.519
NE-1P	0.69		0.74	0.72			0.78		0.86		0.80		0.93		0.88		0.64		0.96		0.83		0.63		0.81		0.90		0.97		0.54		0.020
NE-2P	0.52		0.57	0.56			0.62		0.71		0.66		0.85		0.80		0.46		0.88		0.71		0.46		0.68		0.81		0.88		0.36		0.0007
NE-PP	0.33		0.40	0.38			0.45		0.54		0.51		0.76		0.69		0.26		0.80		0.57		0.28		0.53		0.70		0.79		0.19		<0.0001

Literature cited

Klinkicht, M. 1993. Untersuchungen zum Paarungssystem des Alpenmurmeltiers, Marmota M. marmota mittels DAN Fingerprinting. Ph.D. thesis, University of Munich.

Hanslik, S., and L. Kruckenhauser. 2000. Microsatellite loci for two European sciurid species (Marmota marmota, Spermophilus citellus). Molecular Ecology 9:2163–2165.

Da Silva, A., G. Luikart, D. Allainé, P. Gautier, P. Taberlet, and F. Pompanon. 2003. Isolation and characterization of microsatellites in European alpine marmots (Marmota marmota). Molecular Ecology Notes3:189–190.

Warnes, G., G. Gorjanc, F. Leisch, and M. Man. 2011. genetics: Population Genetics. R package version 1.3.6. http://CRAN.R-project.org/package=genetics.

Cohas, A., N. Yoccoz, C. Bonenfant, B. Goossens, C. Genton, M. Galan, B. Kempenaers, and D. Allainé. 2008. The genetic similarity between pair members influences the frequency of extrapair paternity in alpine marmots. Animal Behaviour 76:87–95

Rassmann, K., W. Arnold, and D. Tautz. 1994. Low genetic variability in a natural alpine marmot population (Marmota marmota, Sciuridae) revealed by DNA fingerprinting. Molecular Ecology 3:347–353.

Kalinowski, S. T., M. L. Taper, and T. C. Marshall. 2007. Revising how the computer program CERVUS accommodates genotyping error increases success in paternity assignment. Molecular Ecology 16:1099–1106.

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