Copyright  1998 by the Genetics Society of America

Molecular Population Genetics of the Southern Mirounga leonina

Robert W. Slade,*,†,1 Craig Moritz,* A. Rus Hoelzel‡,2 and Harry R. Burton§ *Department of , University of Queensland, 4072, Australia, †Centre for Molecular and Cellular Biology, University of Queensland, 4072, Australia, ‡National Cancer Institute, Frederick, Maryland 21702 and §Australian Antarctic Division, Kingston, , 7002, Australia Manuscript received August 19, 1997 Accepted for publication May 11, 1998

ABSTRACT Southern elephant seals breed on sub-Antarctic islands and have a circumpolar distribution. We assayed mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) variation in the three main populations in the south Atlantic, south Indian, and south Pacific oceans, and a smaller continental population in South America. Population structure of mtDNA was strong and not consistent with isolation by distance. The nDNA loci, although less informative, were consistent with the mtDNA results. Geographic structure appears to be dominated by historical processes, not contemporary gene flow. Uncorrected levels of nucleotide diversity for mtDNA control region I (2.86%) and nDNA (0.09%) were similar to those in humans and mice. Mutation rates for control region I (75 ϫ 10Ϫ9 substitutions per site per year) and nDNA (1.23 ϫ 10Ϫ9) were similar to those in other . Female effective population size and total ϫ 104, indicating a twofold greater rate of drift for 4ف effective population size were roughly equal at mtDNA. Effective breeding sex ratio of four to five females per male was estimated from nucleotide diversity and mutation rates for mtDNA and nDNA, and was much less than behavioral observations would suggest. There was no evidence for selection at any of the assayed loci.

HE Mirounga leonina has a tern differences were environmentally determined. Al- Tcircumpolar distribution, and breeding colonies ternatively, the growth pattern differences could be ge- are concentrated on sub-Antarctic islands near the Ant- netically determined, in which case we might expect a arctic convergence (Figure 1). The three main popula- closer genetic relationship between the MQ and HD tions are centered on South Georgia (SG) in the south populations than either is to SG. Atlantic Ocean, the geographically close Heard (HD) Recent studies on population size change have also and in the south Indian Ocean, and indicated similarities between HD and MQ compared (MQ) in the south Pacific Ocean with SG. Population size was estimated as 350,000 for (Ling and Bryden 1992). There are several small breed- SG, 80,000 for HD, 157,000 for Kerguelen, and 136,000 ing populations on other sub-Antarctic islands, and for MQ, and the three main populations account for there is a continental breeding population in the south 96% of total population size (McCann 1985). These Atlantic ocean at Penı´nsula Valde´s (PV) in Argentina figures represent population size during the early 1950s (Campagna and Lewis 1992). On the basis of skull for SG, HD, and MQ, and during the 1970s for Ker- characters, Lydekker (1909) proposed that three sub- guelen. Although the SG population has been stable be recognized, falclandicus, crosetensis, and mac- since that time (McCann and Rothery 1988), popula- quariensis, corresponding to type localities in the south tions in the south Indian and south Pacific oceans have Atlantic, south Indian, and south Pacific oceanic re- declined markedly (Hindell and Burton 1987). The gions, respectively. There are different growth patterns MQ population, for example, has declined by 50% in between populations, and these probably underlie the 30 yr at a rate of Ϫ2% per year. One possible explanation skull character differences. Studies during the 1950s for these changes is that they are due to large-scale and 1960s showed that individuals from MQ and HD movement of individuals between populations (Hin- grew at a slower rate and had a smaller ultimate size dell and Burton 1987). Carrick Bryden than individuals from SG ( et al. 1962a; One of the aims of this study was to assess the extent Bryden 1968). (1968) suggested that the growth pat- of geographic structure in the southern elephant seal and to determine if the genetic relationship between populations corresponded to the relationship indicated Corresponding author: Rob Slade, Queensland Institute of Medical by the morphometric and demographic studies. A previ- Research, Post Office, Royal Brisbane Hospital, Queensland 4029, Australia. E-mail: [email protected] ous study analyzed the genetic relationship between 1Present address:Queensland Institute of Medical Research, Australia. only the MQ and HD populations, between which there 2Present address: Biological Sciences, University of Durham, England. were significant differences in allozyme frequency

Genetics 149: 1945–1957 (August 1998) 1946 R. W. Slade et al.

Figure 1.—Past and present distribu- tion of the southern elephant seal (taken from Ling and Bryden 1992 with permis- sion).

(Gales et al. 1989). A second aim of this study was MATERIALS AND METHODS to obtain more detailed information on other popula- Samples and molecular methods: The southern elephant tion genetic parameters for both mitochondrial DNA seal blood tissue samples from Macquarie Island (MQ) in the (mtDNA) and nuclear DNA (nDNA). These other pa- south Pacific Ocean and Heard Island (HD) in the south rameters include mutation rate, effective population Indian Ocean were collected by the Australian Antarctic Divi- size, and selection for both mtDNA and nDNA. The sion. Samples from South Georgia (SG) in the south Atlantic Ocean were collected by the British Antarctic Survey using g of skin from the webbing in the 2–1ف framework for estimating these parameters relies upon ronguers to clip sequence data information on the pattern of variation rear flippers. The MQ and HD samples were from beaches within and between species. Our previous studies have representing the geographic extremes of each island. The Slade described the general approach for detecting variation samples from other species were described in et al. (1994). For initial sequencing, to determine levels of in orthologous nDNA genes within the southern ele- variation and to search for diagnostic markers, one individual phant seal and other organisms (Slade et al. 1993), and was sequenced from each of MQ, HD, and SG. Several genes between the southern elephant seal and other pinniped were then selected for further sequencing of a total of five species (Slade et al. 1994), and have also compared individuals from each population (Table 1). For large-scale individuals from each 30ف ,levels of mtDNA variation in southern and northern screening of diagnostic markers Hoelzel of MQ, HD, and SG were selected. The gene fragments ampli- elephant seals ( et al. 1993). In this article, we fied and sequenced are shown in Table 1. Here we detail only investigate several areas of the molecular population those protocols and primer sequences that have not been and evolutionary genetics of the southern elephant seal, described previously (Slade et al. 1993, 1994). including level of , rate of molecular For mtDNA, amplification with the THR and TDKD primers from 15 individuals (5 from each major population) resulted evolution, effective population size and effective breed- in 444 bp of sequence data, and analysis focused on a highly ing ratio, geographic distribution of genetic variation, variable 299-bp subset comprising all 264 bp of control region rate of gene flow, and population divergence time. I (CRI) and 35 bp of flanking sequence. This CRI subset Elephant Seal Population Genetics 1947 d — n I, Yes Rsa I I site Yes I, Bsl Marker spp. Nsp Fok @ n c position microsatellite at 146 is with respect to G/C @ 274 G/C @ 335 C/T @ 75 C/T @ 78 T/C @ 109 123–177 G/A @ 273 (1993, 1994). -globin ␤ b et al. 4 G/T @1 72 (1) A/T @ 1460 (0) — (GGAAA) — — — Slade e e e 38 38 See Figure 2 (1993); (4) f No. No. Mutations Applebaum and (bp) analyzed alleles sites a TABLE 1 Lessa Genes sequenced (1992); (3) et al. Moore ); (2) H2A4 H2A4 ALD2 ALD2 G/C @ 202 ALD2DQA2 ALD2 DQA2 DQA4 DQA2 DQA5 Reverse @ 201–206 OSCRFA2 OSCRFA2 TDKD TDKD BETA5 BETA5 (GGAAA) (1993). is that in the reference sequence in GenBank. The position of the A/T mutation in the et al. N/N loci amplified simultaneously, and therefore the number of alleles could not be determined (see text). materials and methods Hoelzel UTR U91906 OSCRFA1 OSCRFA1 2 209 6 1 0 — — — intron 4 H2A5 H2A5 exon2 LYSO2 exon 4 H2A5 intron 1 LYSO2 intron 2 H2A5 intron 2exon 3 BETA4 BETA4 globin - Ј Exon 5–6 L17497 ALD1 ALD1 3 478 30 5 4 (2) C/T @ 133 Exon 2–5 L17502 H2A6 H2A6 4 274 6 1 0 — — Yes Exon 2Exon 3 U91907Intron 2 U91907 DQA1 U91907 DQA1 SQA1 DQA1 DQA2 Universal 1, 4 169 4 4 230 447 30 30 4 2 2 1 2 (0) G/A @ 1 70 (1) 0 C/T @ 828 6 bp indel — — indel Yes Yes Exon 5–6 L17498 ALD1 ALD1 3 272 6 1 0 — — Yes Exon 2/ U91910 BETA3 BETA3 3 168 6 ? 5 Exon 4/ L17501 H2A8 H2A2 4 338 6 2 1 C/A @ 259 — Yes Intron 1/ U91909 LYSO1 LYSO2 1 311 6 2 2 C/T @ 239 — — Intron 3/ L17500 H2A8 H2A5 4 293 6 1 0 — — — Exon 1/ U91908 LYSO1 LYSO1 1 303 6 1 0 — — — Exon 2/ L17499 H2A8 H2A8 4 270 6 1 0 — — — Intron 2 U91911 BETA6 BETA6 1 215 30 ? Intron2/ U91911 BETA3 BETA4 3 351 30 ? ␤ . (1994) for details of sequence data. et al Slade end of the sequence and may vary between different size repeat units. Ј (1) This study (see See For those nDNA loci for which 30 genes were sequenced, the values in parentheses are the number of variant sites observed in the initial screen of six genes. The first mutation given in There were two Includes data from releasing factor a b c d e f H2AF -Globin -Globin -Globin ALD-A ⌿ Mhc-DQA Mhc-DQA Mhc-DQA mtDNA CRI U03591 THR THR 4 299 60 ALD-C ␤ Corticotropin- H2AF Lysozyme H2AF the 3 Lysozyme Genefragment Region No. Accession primers PCR primers Sequencing Ref. Size haplotypes No. variant and Other H2AF ␤ ␤ 1948 R. W. Slade et al. corresponds to sites 67–365 in the GenBank sequence. (All site numbers refer to the sequences in GenBank; accession numbers in Table 1.) This enabled us to make direct use of published mtDNA sequence data (Hoelzel et al. 1993) for the southern elephant seal populations at SG and PV. There were a total of 60 CRI individual sequences available for analy- sis comprising 28 from SG, 21 from PV, 6 from HD (one individual was heteroplasmic; see results), and 5 from MQ. ,bp gene fragment of the ␤-globin gene-800ف For nDNA, an spanning exons 2–3, was amplified with primers BETA3/ BETA4 using cycling parameters of 35 ϫ 94Њ for 1 min, 65Њ for 1 min, 72Њ for 3 min; 168 bp at the 5Ј end was sequenced with BETA3, and 351 bp at the 3Ј end was sequenced with BETA4. Sequencing with the BETA4 primer revealed a pen- tameric microsatellite (GGAAA)n, the 3Ј end of which was located 297 bp upstream from the beginning of exon 3. Prim- ers BETA6 and BETA5 were designed to regions flanking the microsatellite. The BETA6/BETA5 amplified product con- sisted of 149 bp of flanking sequence and from 6 to 20 repeats. The cycling parameters for BETA6/BETA5 were 35 ϫ 94Њ for bp fragment of the-260ف min, 65Њ for 1 min, 72Њ for 1 min.An 1 Corticotropin-releasing factor gene was amplified and sequenced with primers OSCRFA1/OSCRFA2 using cycling parameters of 35 ϫ 94Њ for 1 min, 50Њ for 1 min, and 72Њ for 1 min. kb fragment of the lysozyme gene was amplified with-1ف An primers LYSO1/LYSO2 using cycling parameters of 35 ϫ 94Њ for 1 min, 55Њ for 1 min, 72Њ for 3 min, and 303 bp at the 5Ј Figure 2.—(A) The 16 different sequence haplotypes of end was sequenced using LYSO1, and 311 bp at the 3Ј end CRI from 15 individuals. (B) The 5 different sequence haplo- was sequenced with LYSO2. The PCR primers not described types of from 15 individuals. The site numbers corre- previously are (5Ј 3Ј) LYSO1 (AGGTCTTTGRACGDTG ALD-A → spond to the sequences in GenBank, and the reference se- TGA), LYSO2 (GGGGTTTTGCCATCATTACA), DQA4 (ACA quences in GenBank are SG1 and haplotype a for CRI and CATACCATTGGTAG), BETA6 (AATTGGGCATGTGATGT ALD-A, respectively. Diagnostic RFLP sites are in bold, and ATGAG), and BETA5 (AATTAGTATGATGCTGGGCTGTC). for CRI these are FokI @ 196 and 326, RsaI @ 169, and BslI For microsatellite PCR, the BETA5 primer was end-labeled @ 305. For CRI, the heteroplasmic individual contained haplo- with[␥-33P]dATP accordingto a standard protocol (Sambrook types HD3 and HD4. See Hoelzel et al. (1993) for CRI haplo- 1989). This was used in a regular PCR reaction with the et al. types of the PV and other SG sequences. BETA6 primer except that the cycling parameters involved denaturation at 94Њ for 30 sec, variableannealing temperatures (see below), and extension at 72Њ for 2 min. The annealing temperatures followed a “touchdown” procedure to reduce distribution, to use in a large-scale survey (Figure 2A). For Don ALD-A (Figure 2B), there was a variant site at position 202 nonspecific amplification ( et al. 1991) and were 67Њ,30 Slade sec for the first 2 cycles, then 66Њ, 30 sec for 2 cycles, and then that was part of a NspI restriction site ( et al. 1993). The presence/absence of the 6-bp indel in intron 2 of 65Њ, 30 sec for 25 cycles. Formamide stop solution was added Mhc-DQA was assayed by double digestion of the PCR fragment with to the product, which was heated to 95Њ for 2 min, and 4 ␮l was 96I and IV and subsequent PAGE. The restriction di- loaded onto a 4.5% sequencing gel. The bands were visualized Sau Nla gests were conducted as in Slade et al. (1993). after autoradiography. Electrophoresis revealed up to four Data analysis: For those nDNA sequences with multiple vari- bands per individual, indicating that at least two microsatellite ant sites (i.e., ALD-A and Lysozyme), the haplotypes were deter- loci were being amplified. Direct sequencing of the BETA6/ mined. For ALD-A, the method of phase determination was BETA5 product from several individuals revealed a clean se- described in Slade et al. (1993). Briefly, the haplotypes of all quence with one variant site, located within the repeat unit. double heterozygotes were separated by digestion with NspI, ␤ It is likely, therefore, that there are at least two -globin genes and the separate fragments sequenced. For Lysozyme, phase in the southern elephant seal and that these are the result of a was determined by reference to the unambiguous haplotypes very recent duplication or possibly a very recent homogenizing observed after sequencing homozygotes. There was also one event between two preexisting ␤-globin genes. The presence of individual that was heteroplasmic for mtDNA, and the two two ␤-globin genes in the southern elephant seal was previously haplotypes were determined by reference to the unambiguous Seal suggested by isozyme studies ( et al. 1971). Although it haplotypes. Phase can be inferred from the unambiguous hap- was not possible to allocate alleles to separate microsatellite lotypes assuming that there has been no recombination that loci, the clear and reproducible dosage differences did allow is reasonable for short PCR fragments (Clark 1990). scoring of the combined loci allele frequencies. For example, The pairwise genetic distances between sequences were cal- for individuals with three bands there was always one band culated with the “MEGA” program (Kumar et al. 1993) using with increased intensity, indicating the presence of two alleles pairwise-deletion to account for gaps. The genetic distances for that number of repeats. were the uncorrected number or proportion of differences, For each sequence with a variant nucleotide site, a computer andfor some analyses ofthe CRI data, distances were corrected search was made using the “MacVector” program (IBI-Kodak) using the Tamura-Nei gamma model (Tamura and Nei 1993). for corresponding variant restriction sites. For mtDNA CRI For the latter, the scale parameter for the negative-binomial there were several variant restriction sites, and three enzymes distribution was estimated by maximum-likelihood using the (RsaI, FokI, BslI) were chosen, on the basis of their geographic macro “kfit” (Crawley 1993) for the program “GLIM” (Re- Elephant Seal Population Genetics 1949 lease 3.772; Royal Statistical Society, London). The input data the polymorphism within the mtDNA was corrected down by were the number of substitutions that occurred at each site, one-half. and this was estimated from a parsimony tree (Wakeley 1993) The total effective population size (N) and the female effec- Felsenstein constructed in “PHYLIP” ( 1993). The frequency tive population size (Nf) were estimated from the formulae distribution of the inferred number of substitutions per site ␪ϭ4Nug and ␪ϭ2Nfug, respectively. The neutral parameter was compared with expected poisson (Sokal and Rohlf 1969, ␪ was taken to be equivalent to nucleotide diversity (␲). The p. 86) and negative binomial (Bliss and 1953) distribu- mutation rate per site per generation (u g) was calculated tions. as the mutation rate per year multiplied by the generation Nucleotide diversity within populations was calculated as in time. The generation time for the southern elephant seal equation 10.6 in Nei (1987). For nDNA data, this was based of 8 yr was calculated from female life tables of the stable on the uncorrected proportional distance, and for CRI data population at South Georgia (McCann 1985) using the for- it was based on both the uncorrected proportional distance mula Rxlxmx/Rlxmx, where x is age in years, lx is the proportion and the Tamura-Nei gamma corrected distance. The variance of females surviving to age x, and mx is the number of females of nucleotide diversity was that because of estimation errors produced per female at age x (Begon et al. 1986). The effective of nucleotide substitutions. For the uncorrected proportional breeding ratio was estimated from data on mutation rates (␮n Nei Jin distance, the variances were calculated as in and (1989) and ␮mt) and levels of diversity (␲n and ␲mt) for nDNA and using the program “SEND.” For the Tamura-Nei corrected mtDNA, respectively. The mutation rates do not depend on distance, the maximum variance was calculated as in Taka- effective population size, although diversity does, and so for hata and Tajima (1991) using a BASIC program modified neutrally evolving sequences, and assuming mutation-drift by R. W. Slade from one kindly provided by Y. Satta. equilibrium, the following should be true: The distribution of genetic variation between populations N ␲ ␮ was estimated in several ways. For CRI data only, we utilized a f ϭ mt n. (1) procedure analogous to analysis of variance with the “AMOVA” 2N ␲n␮mt program (Excoffier et al. 1992). As recommended, the dis- tance matrix used was the number of nucleotide differences N is composed of male and female components such that Wright between pairs of sequences, and the significance levels were N ϭ 4NmNf/(Nm ϩ Nf)( 1931). Substitution of the determined after 1000 permutations. Also, for CRI data only, right-hand side into Equation 1 above results in the following: we calculated gross and net nucleotide divergence between N 8␲ ␮ populations as in equations 10.20 and 10.21, respectively, in f ϭ ΂ mt n Ϫ 1΃. (2) Nei (1987). For CRI haplotype and nDNA allele data, ␹2 tests Nm ␲n␮mt of independence were conducted using “Monte” (Roff and Bentzen 1989) in the “REAP” suite of programs (McElroy et al. 1992), and significance levels were determined after 1000 permutations. RESULTS To estimate levels of long-term gene flow, we calculated analogs of G ST between pairs of populations from the CRI From mtDNA, we analyzed 299 bp of CRI from 60 sequence data and from the nDNA allele frequency data. individuals and assayed three diagnostic restriction sites For the CRI sequence data, estimates of G ST were calculated from 115 individuals. From nDNA we sequenced 3594 ϭ Ϫ Hudson between pairs of populations as K ST 1 K S/K T ( bp of mostly noncoding regions from seven single-copy et al. 1992), where K is the average nucleotide diversity S loci from 6 haplotypes (three individuals) from the geo- within the two populations, and KT is the total nucleotide diversity among all sequences from the populations. The de- graphic extremes of the range, and 877 bp of this was gree of maternal gene flow (Nfm f) between each pair of popula- sequenced from 30 haplotypes. Three nDNA diagnostic tions was then estimated from KST ϭ 1/(2Nfm f ␣ϩ1), markers (restriction site, indel, microsatellite) were as- ␣ϭ Ϫ 2 .haplotypes 186ف where (n/(n 1)) and n is the number of populations sayed from exchanging migrants (Crow and Aoki 1984), in this case Sequence variation: nDNA: The pilot analysis of one four. For the nDNA data, estimates of G ST between pairs of populations were obtained from allele frequency data from individual per main population of SG, HD, and MQ several loci using the program “BIOSYS-11.7”(Swofford and (i.e., a total of 6 haplotypes) detected variation in 7 of Selander 1989), and total gene flow (Nm) between pairs of the 15 nDNA gene segments (Table 1). From this initial ϭ ␣ϩ Crow populations was estimated from K ST 1/(4Nm 1) ( screening, several segments were selected to increase and Aoki 1984). The CRI sequences were analyzed for any pattern of selec- the sample size from one individual per population to tion among haplotypes using the methods of Tajima (1989) five individuals per population (i.e., a total of 30 haplo- and Fu and Li (1993). For the latter, a northern elephant types). The segments chosen for further analysis were seal sequence was used as the outgroup. All sequences were ALD-A, exons 2 and 3 of Mhc-DQA, and two segments of analyzed for selection using 2 ϫ 2 tables of within-species ␤-globin. Subsequently, it was discovered that two ␤-globin polymorphism and between-species fixed differences for two loci were being amplified simultaneously and that it loci as in Kreitman and Aguade´ (1986). For this test, a sig- nificant departure from a neutral model could be because of was not possible to distinguish alleles from loci (see linkage and stochastic factors rather than selection (Hudson materials and methods). Therefore, the 734 bp of et al. 1987). However, the ease of the ␹2 test means that it is an ␤-globin sequence was not included in further analyses appropriate first choice and that, subsequently, any significant of sequence variation. The 3594 bp of nDNA analyzed associations can be revisited with the more conservative HKA (excluding the 734 bp of ␤-globin) comprised 877 bp test. For comparisons between mtDNA and nDNA sequences, the difference in effective population size needs to be ac- sequenced from 30 haplotypes and 2717 bp sequenced counted for (Nachman et al. 1994). All nDNA was assumed from 6 haplotypes. Of the total 3594 bp, there were to be autosomal, and because Nf was found to be equal to Ne, 2838 bp of silent sites comprised of 467 bp sequenced 1950 R. W. Slade et al. from 30 haplotypes and 2371 bp sequenced from 6 TABLE 2 haplotypes. The silent sites were assumed to be the syn- Total (Ne) and female (Nf) effective population sizes onymous sites in exons, all intron sites except for the

AG/GT splice sites, and all of the 5Ј UTR and pseu- Population/species Ne Nf (n) dogene sites. The variants detected included 9-point substitutions, MQ — 18,000 (5) HD — 22,000 (6) a 6-bp indel, and a pentameric microsatellite. Of the SG — 30,000 (28) nine variant sites, five were found in introns, two were PV — 3,000 (21) silent polymorphisms in exons, and there were two non- Southern elephant seal 40,000a 40,000 (60) synonymous polymorphisms, glycine/arginine in exon Northern elephant sealb — 5,000 (41)

2ofMhc-DQA and glycine/arginine in exon 2 of Lyso- a See text for sample size n for Ne. zyme. There were five transitions and four transversions b Sequence data taken from Hoelzel et al. (1993). resulting in a roughly twofold bias of transitions over transversions. The total diversity (uncorrected) for nDNA of 0.09 Ϯ 0.03% was calculated from 877 bp from 30 method of Fu and Li (1993) on the 60 CRI sequences, haplotypes and 2717 bp from 6 haplotypes. The level with the as the outgroup, the of silent site diversity of 0.08 Ϯ 0.03% was calculated test statistic D was equal to 0.55, which is not significant from 467 bp from 30 haplotypes and 2371 bp from 6 (P Ͼ 0.05). The Kreitman and Aguade´ ␹2 test of inde- haplotypes. The diversity calculated from the samples pendence on all the variant nDNA sequences and the from the initial screening was the same, and therefore 60 CRI sequences showed no significant deviations we assume that no systematic bias was introduced by from the neutral expectation of levels of polymorphism selecting some gene fragments for further sampling. within species, given the number of fixed differences Phase was determined for those nDNA genes with more between species. than one polymorphic site, ALD-A and Lysozyme. For Rate of molecular evolution, Ne and Nf:Nm: The rate of -bp of silent sites in pinni 1200ف ALD-A, the phase of the variant sites is shown in Figure neutral evolution for 2B. for Lysozyme, the T/C polymorphism at site 239 was ped nDNA (see Table 1 for genes sequenced) was pre- in phase with the A/G polymorphism at site 273. viously estimated with a Kimura two-parameter model mtDNA: The level of polymorphism in the 299 bp of (Slade et al. 1994) from three fossil-record calibrations mtDNA CRI sequence was considerably higher with 16 (divergence times of 4.5, 14.5, and 23 mya) as being different haplotypes observed from 15 individuals, 5 1.23 Ϯ 0.24 substitutions per nucleotide site per 109 yr from each of the three main populations (Table 1, Fig- (i.e., 1.23 Ϯ 0.24 ϫ 10Ϫ9). For the 299 bp of CRI, and ure 2A). One HD individual was heteroplasmic. All 26 using the Tamura-Nei gamma model with one fossil- variant sites were transitions. All further analyses of CRI record calibration (the 4.5 mya divergence between Mir- sequence variation combined this data set of 16 se- ounga spp. in one and Hydrurga leptonyx and Lep- quences with the data set of Hoelzel et al. (1993), giv- tonychotes weddelli in the other clade; Ray 1976; Slade ing a total of 60 individual sequences with 38 variant et al. 1994), the rate of CRI evolution was estimated as sites (five transversions) and 38 different haplotypes. 75 Ϯ 46 ϫ 10Ϫ9.

The uncorrected nucleotide diversity in these 60 CRI The total effective population size of roughly Ne ϭ sequences was 2.86 Ϯ 0.49%. Several studies have shown 4 ϫ 104 (Table 2) was calculated from the silent site substitution rate variation among sites in the mamma- nucleotide diversity given above. We considered that lian control region as evidenced by departure from a there was not enough nDNA variation to properly esti- Poisson distribution of the number of substitutions per mate Ne within populations. The female effective popu- site (e.g., Kocher and Wilson 1991). The mean of the lation size calculated from the 60 CRI sequences was 4 inferred number of substitutions per site from the 60 roughly Nf ϭ 4 ϫ 10 , and each of the three main 4 CRI sequences was less than the variance (0.2843 and populations has an Nf of roughly 2–3 ϫ 10 (Table 2). 0.8820, respectively), indicating a non-Poisson distribu- The PV population is an of magnitude smaller tion. The frequency distribution of the inferred number than the other southern elephant seal populations, and of substitutions per site did not fit a Poisson distribution the effective number of northern elephant seals is an (␹2 ϭ 161.3, P Ͻ 0.001) but did fit a negative-binomial order of magnitude smaller than that for the southern distribution (␹2 ϭ 0.98, P Ͼ 0.25). The scale parameter elephant seal. The effective breeding ratio was estimated for the negative-binomial distribution was estimated by from Equation 2 to be four to five females to one male. maximum-likelihood as 0.1042, and therefore we sup- Geographic structure: Distribution of CRI sequence varia- plied this to the Tamura-Nei gamma model of pairwise tion: The levels of CRI nucleotide diversity and diver- distances giving a nucleotide diversity of 4.75 Ϯ 2.67%. gence within and between populations are shown in Selection: Using the method of Tajima (1989) on the Table 3. Diversity was highest within the SG and HD 60 CRI sequences, the test statistic D was equal to populations and very low by comparison in the PV popu- Ϫ0.427, which is not significant (P Ͼ 0.1). Using the lation. The greatest divergence was between the PV and Elephant Seal Population Genetics 1951

TABLE 3 Percent diversity (uncorrected) within and between populations for CRI

PV (n ϭ 21) SG (n ϭ 28) HD (n ϭ 6) MQ (n ϭ 5) PV 0.297 3.365 3.071 5.224 SG 2.073 2.289 2.394 3.872 HD 1.908 0.235 2.029 3.801 MQ 4.273 1.926 1.985 1.605 Diversity within populations in italics on diagonal. Gross divergence between populations above diagonal, and net divergence (i.e., gross divergence minus within-population diversity) between populations below diagonal.

MQ populations, and the least was between the SG and single multiallelic locus in a tetraploid. This approach HD populations. Although PV and SG are in the same (Utter et al. 1992), or a similar one (Gharrett et al. oceanic region, there is almost an order of magnitude 1987; Bartley and Gall 1990; Gall et al. 1992), has of greater divergence between those two populations been used previously for duplicated loci in salmonid than between the separate oceanic populations of SG fish (see Waples 1988; Waples and Aebersold 1990 and HD. The AMOVA results showed that 57% of CRI for discussion). This is a conservative approach for de- variation was distributed between populations. This dis- tecting population structure because for any one micro- tribution of mtDNA variation was significantly different satellite allele, the frequency differences between popu- from random (P Ͻ 0.001). lations could only be the same or greater by partitioning For a hierarchical AMOVA, we compared oceanic into two loci. The genotype of individuals with fewer regions, with SG and PV representing the south Atlantic than four bands was deduced from the relative band ocean, HD the south Indian ocean, and MQ the south intensitites. Of the 93 individuals assayed, there were Pacific ocean. This resulted in a decomposition of varia- 0 single-band, 7 double-band, 31 triple-band, and 55 tion of Ϫ5% among the three oceanic regions, 61% quadruplex-band genotypes, indicating a high degree among SG and PV within the south Atlantic oceanic of heterozygosity at both loci. That is, 59% (55/93) region, and 44% within populations. The breakdown of individuals were heterozygous at both loci, and a into oceanic regions thus explained none of the varia- minimum of 92% (55ϩ31/93) were heterozygous for tion. This is because of heterogeneity within the south at least one locus. There was significant heterogeneity Atlantic ocean between SG and PV and the close rela- of allele frequencies for the microsatellite loci among tionship between haplotypes from SG and HD repre- senting different regions. Distribution of nDNA allele and CRI haplotype frequencies: individuals 30ف Diagnostic markers were screened in from each of the three main populations. For CRI, the combined RFLP haplotypes and their population fre- quencies are shown in Figure 3. Most haplotypes were found in more than one population; however, none of the MQ haplotypes were shared with HD, SG, or PV. Haplotype frequency differences were analyzed by ␹2, and significant heterogeneity was found among all pop- ulations (␹2 ϭ 224.76, P Ͻ 0.001). For each of the pair- wise comparisons, there was significant heterogeneity (P Ͻ 0.001 for each comparison). For the nDNA loci, the markers analyzed were the presence/absence of the Mhc-DQA 6-bp deletion, the ALD-A NspI restriction site, and the two ␤-globin pentam- eric microsatellites (Table 4). There were no departures Figure 3.—The CRI RFLP haplotypes and their frequencies from the Hardy-Weinberg equilibrium (HWE) for geno- in four populations. Only the polymorphic sites are shown. types at the Mhc-DQA or ALD-A loci. The ␤-globin micro- Presence of a restriction site ϭ 1 and absence ϭ 0. For each satellite data were not tested for HWE because single- of the 21 individuals from PV (Hoelzel et al. 1993), the 299 locus genotypes could not be determined. There was bp of CRI sequence data was converted to a single RFLP haplotype. An extra polymorphic restriction site was observed no significant heterogeneity among the three popula- for the RsaI restriction site @ 251–254 in the large-scale screen- 2 tions for alleles at the Mhc-DQA and ALD-A loci. The ␹ ing that was not observed in the smaller sequence sample analysis of the two microsatellite loci treated them as a shown in Figure 2. 1952 R. W. Slade et al.

TABLE 4 Frequency of nDNA alleles

Frequency Locus Allele SG HD MQ Significance test results Mhc-DQA 6-bp indel ϩ 57 61 60 Overall ␹2 ϭ 1.99 Ϫ 358(Pϭ0.4130 Ϯ 0.0156) Total 60 66 68 (n ϭ 30) (n ϭ 33) (n ϭ 34) ALD-A NspI site ϩ 19 23 30 Overall ␹2 ϭ 2.00 Ϫ 35 39 34 (P ϭ 0.3360 Ϯ 0.0149) Total 54 62 64 (n ϭ 27) (n ϭ 31) (n ϭ 32) ␤-Globin No. repeats 6 4 15 23 Overall ␹2 ϭ 58.87 7036(Pϭ0.0010 Ϯ 0.0010) 8 7 11 7 9 8 7 6 SG/HD ␹2 ϭ 20.75 10 26 15 11 (P ϭ 0.1000 Ϯ 0.0095) 11 32 36 38 12 12 15 14 SG/MQ ␹2 ϭ 42.82 13 5 7 0 (P Ͻ 0.001) 14 2 2 1 15 6 6 4 HD/MQ ␹2 ϭ 23.42 16 3 6 6 (P ϭ 0.0320 Ϯ 0.0056) 17 1 3 1 18 7 2 1 19 2 0 10 20 1 0 0 Total 116 128 128 (n ϭ 29) (n ϭ 32) (n ϭ 32)

the three populations. Pairwise comparisons showed sig- changed per generation (m f) were estimated from the nificant differences in microsatellite allele frequency uncorrected and Tamura-Nei corrected distances (Ta- between MQ and the other two populations, but not ble 5). To estimate m f from N fm f, N f was assumed to be between HD and SG. the arithmetic mean of the N f estimates from the two Gene flow: For the 60 CRI sequences, the spatial vari- populations (Chakraborty and Nei 1974). There was ance of gene frequency (K ST), the number of female little difference in the estimates between the uncor- migrants exchanged between populations per genera- rected and Tamura-Nei corrected distances, because tion (N fm f), and the proportion of female migrants ex- the relative levels of diversity and divergence are similar

TABLE 5

Estimates of K ST, N fm f, and m f from CRI variation

Uncorrected diversities Tamura-Nei diversities

Mean Nf Distance m f m f 3 Ϫ5 Ϫ5 Comparison (n) between: (ϫ 10 ) (km) K ST N fm f (ϫ 10 ) K ST N fm f (ϫ 10 ) SG (28) and PV (21) 16.5 2,400 0.348 0.53 3.21 0.416 0.39 2.36 HD (6) and SG (28) 26 8,400 0.035 7.74 29.77 0.035 7.74 29.77 HD (6) and PV (21) 12.5 10,800 0.354 0.51 4.08 0.404 0.41 3.28 MQ (5) and SG (28) 24 11,500 0.248 0.85 3.54 0.31 0.63 2.63 MQ (5) and HD (6) 20 7,500 0.267 0.77 3.85 0.348 0.53 2.65 MQ (5) and PV (21) 10.5 11,000 0.6 0.19 1.81 0.705 0.12 1.14 Elephant Seal Population Genetics 1953

TABLE 6 of DNA sequence variation using several recently devel- Kreitman Aguade´ Tajima Time of population and species divergence oped methods ( and 1986; from CRI data 1989; Fu and Li 1993), although the power of at least some of these tests is limited, given the sample size in Comparison (n) between: Time (yr) this study (Braverman et al. 1995; Simonsen et al. 1995). For all these methods, the distributions of the mtDNA MQ (5) and HD (6) 270,000 and nDNA variants in the southern elephant seal (with MQ (5) and SG (28) 260,000 HD (6) and SG (28) 23,000 the exception of the ␤-globin microsatellites that were MQ (5) and PV (21) 600,000 not tested) were compatible with neutrality. Second, it HD (6) and PV (21) 215,000 is reasonable to assume that selection is not a factor if SG (28) and PV (21) 270,000 there are concordant geographic patterns among sev- Southern (60) and northern (41) elephant seals 800,000 eral unlinked loci. Although not as informative, the available data from nDNA loci are concordant with the mtDNA data. Three allozyme loci (Ada, Pgam, Pgm) despite a large difference in the absolute amounts of showed significant differences in allele frequency be- estimated variation. There was limited maternal gene tween HD and MQ (Gales et al. 1989) as did the ␤-globin flow between all populations, except between South microsatellites, and there are nonsignificant indications Georgia and Heard Island. For the latter, maternal gene from the latter of differences between HD and SG. flow was roughly an order of magnitude greater than Mutation rate, genetic diversity, and effective popula- among other populations. For the nDNA data, the level tion size: The rate of silent substitution in pinniped of sequence variation and the number of loci assayed nDNA was previously estimated as 1.23 Ϯ 0.24 substitu- were too low to use the sequence-based approach. How- tions per site per 109 yr (Slade et al. 1994). This was ever, it was possible to obtain an estimate of total gene based on three different divergences and independent flow between Heard and Macquarie Islands using allele fossil-record dates of 4.5, 14.5, and 23 mya, each of frequency data from three polymorphic allozyme loci, which gave very similar rate estimates of 1.37, 0.93, and Acp-1, Ada-1, and Pgm-1 (Gales et al. 1989), and the 1.40 ϫ 10Ϫ9, respectively. As discussed previously (Slade two polymorphic nDNA loci from this study (Mhc-DQA, et al. 1994), this is at the low end of rates among mam- ALD-A). This resulted in an estimate of Nm between mals, that is, 6.5 ϫ 10Ϫ9 in rodents, 3 ϫ 10Ϫ9 in primates Heard and Macquarie Islands of 4.11. and artiodactyls, and 1 ϫ 10Ϫ9 in humans (Li et al. Time since divergence: An alternative approach is to 1987). A slower rate of nDNA evolution in carnivores assume that there has been no gene flow between popu- compared with other mammals has also been indicated lations and that the level of nucleotide divergence be- by immunological distances (Sarich 1985). However, tween populations reflects only time since population these estimates should be treated with caution because divergence. The time of divergence between southern they are based on the fossil record, and it is clear that elephant seal populations and between the southern reinterpretation of parts of the mammalian fossil record and northern elephant seal populations was estimated is necessary (Wilson et al. 1987; Easteal 1990). from CRI data using the Tamura-Nei net divergences The rate of mtDNA evolution was estimated for 299 and the estimated mutation rate of 75 ϫ 10Ϫ9 (Table bp of CRI and was calibrated against only the 4.5 mya 6). In this historical association model, MQ and PV last fossil-record date. Other calibrations were not possible shared a common ancestral population some 600,000 because of alignment difficulties between more diver- yr ago, SG and HD separated as recently as 20,000 yr gent sequences. The rate of evolution in CRI was esti- ago, whereas all other population divergences occurred mated as 75 Ϯ 46 ϫ 10Ϫ9. It is clear that the rate is at some 200,000–300,000 yr ago. The northern and south- least an order of magnitude higher than that for nDNA, ern elephant seals were estimated to have diverged and it may be close to two orders of magnitude higher. 800,000 yr ago. In terms of percent change, the nDNA is evolving at roughly 0.1%/million years (myr) along a lineage, and the mtDNA CRI is evolving at a rate of 5–10%/myr. DISCUSSION The rate of substitution averaged over the entire mtDNA In this study, we assessed the contribution of mutation molecule in other was estimated as roughly 1%/ rate, effective population size, gene flow, and selection myr along a lineage (Wilson et al. 1985), and the silent to the amount and distribution of genetic variation in rate for the ND4 and ND5 mtDNA genes in primates .(myr (Brown et al. 1982/%5ف some regions of mtDNA and nDNA of the southern was estimated to be elephant seal. Estimates of the rate of control region evolution include Selection: Two approaches were used to infer that 103 ϫ 10Ϫ9 and 74 ϫ 10Ϫ9 for CRI and CRII, respectively, selection had not played a significant role in the level in great apes (Horai et al. 1995), 75 ϫ 10Ϫ9 for the or distribution of genetic variation in the gene regions entire control region in great apes (Tamura and Nei analyzed. First, selection can be detected from patterns 1993), 114 ϫ 10Ϫ9 for the entire control region in hu- 1954 R. W. Slade et al. mans (Stoneking et al. 1992), and 20–40 ϫ 10Ϫ9 for Considering this, the above data suggest that the average CRI in the horse (Ishida et al. 1995). Therefore, the breeding male would sire 20 offspring in his lifetime, estimate presented here of 75 ϫ 10Ϫ9 for CRI in the although there would likely be a much larger variance southern elephant seal is consistent with estimates for associated with this figure than that for the average other mammals. female. We should be cautious when interpreting the The uncorrected level of nucleotide diversity in the estimated effective breeding ratio for two reasons. One, 299 bp of CRI was 2.86 Ϯ 0.49%. This is among the there is a large variance associated with each of the higher levels so far observed among vertebrate species mutation rates and diversities that would probably be (see Table 2 in Slade 1998) and is similar to that ob- amplified in the estimated ratio. Second, the ratio may served in control regions I and II in African humans of reflect the presence of population structure and sex- 2.08% (Vigilant et al. 1991). Consistent with its lower biased gene flow, rather than a sex-biased breeding mutation rate, nucleotide diversity in nDNA was much ratio. lower at 0.09%. Less comparative data are available for Geographic structure: This is the first global survey nDNA compared to mtDNA. Diversity in southern ele- of genetic variation in the southern elephant seal. For phant seal nDNA is similar to the 0.11% found for hu- mtDNA, there was significant geographic structure Li Sadler mans ( and 1991) and to the 0.08% found for among the four populations of the southern elephant Nachman mice ( 1997) but lower than that in Drosophila seal for distribution of sequence variation and distribu- pseudoobscura, D. simulans, D. ananassae,orD. melano- tion of haplotype frequencies. A previous phylogeo- gaster (2.2%, 1.4%, 1.0%, and 0.4%, respectively; sum- graphic analysis had also shown geographic structure Aquadro marized in 1992). The heterozygosity of 3.2% for distribution of lineages (Slade 1998). Most of the Gales in the protein products of 35 nDNA genes ( et al. geographic structure was because of the divergent MQ 1989) is similar to the mammalian average of 4.1% Nevo and PV populations, the lineages of which formed dis- ( et al. 1984). crete monophyletic groups. The SG and HD popula- Given that the neutral mutation rate and nucleotide tions shared haplotype lineages, but nonetheless there diversity in southern elephant seal nDNA is similar to was significant geographic structure for the distribution that found in humans, it is not surprising, then, that of those lineages as well as for the distribution of genetic )of4ϫ104 is the total effective population size (Ne variation and haplotype frequencies. The limited varia- also similiar to that in humans, which is 104 to 105 tion at the nDNA loci allowed only an analysis of allele ف depending on the time scale over which the estimates frequency differences, which, among the populations of are calculated (Takahata 1993). If the worldwide SG, HD, and MQ, did not exhibit as strong a geographic population size of southern elephant seals is 750,000 structure as the mtDNA data. There were no significant (McCann 1985), then the ratio of effective to current differences in allele frequency markers at the ALD-A and population size (N /N) would be 0.05. N /N ratios are e e Mhc-DQA loci, whereas for the ␤-globin microsatellites a highly variable among species; in a summary of ratios, significant difference was found between MQ and the those of nine species of ranged from 0.08 to other two populations. The difference in the microsatel- 0.76 (Frankham 1994). The low ratio for the southern lite allele frequencies between SG and HD was signifi- elephant seal would reflect, in part, its highly polygynous cant at the 10%, but not the 5%, level and suggests that breeding system. Such a breeding system would also Birky further analysis of the distribution of mDNA variation explain the similarity between Nf and Ne ( et al. 1983). between these two populations is warranted, preferably We estimated the effective breeding ratio as four to with highly variable markers such as microsatellites. The five females to one male. This ratio is perhaps lower than genetic discreteness of the HD and MQ populations has expected considering that, in one study, the average also been shown in a previous study of allozyme variation Gales number of cows per bull in a varied between 28 among those two populations ( et al. 1989). It is and 53 (Carrick et al. 1962a). However, not only are clear, therefore, that there is strong geographic struc- estimates of copulatory success often overestimated by ture for the female (i.e., mtDNA) component among behavioral observations (e.g., Pemberton et al. 1992; the four populations, but for the biparental (i.e., nDNA) Amos et al. 1993; Lambert et al. 1994) but also, although component this is clear only between MQ and both HD males may sire a large number of offspring in the one or SG. The genetic relationship between populations or two years they are a beachmaster, their lifetime repro- does not correlate with the relationship determined by ductive success would be much lower than observation growth patterns, thus supporting the hypothesis that during beachmaster years would indicate. A female at the growth pattern differences are environmentally de- South Georgia that survives to breeding age produces, termined (Bryden 1968). The degree of genetic struc- on average, five offspring in her lifetime. [This figure ture also did not support the hypothesis that the demo- was derived from the female life tables in McCann graphic changes within populations were because of (1985) and from the estimated fecundity rate of 0.391 differences in movement of individuals between popula- females born per female per year (Hindell 1991).] tions (Slade 1998). Elephant Seal Population Genetics 1955

Difference between mtDNA and nDNA: Differences be- females is such that movement between oceanic regions tween mtDNA and nDNA in the level of observed geo- is certainly possible. The longest interisland movement graphic structure may reflect either differences in rates recorded was some 3000 km between Heard and Marion of gene flow between males and females, or differences Islands in the south Indian ocean (Carrick et al. 1962b), in characteristics of the genes, such as mutation rate and males and females regularly make long-distance and/or rate of . If males and females have round trips of up to 6000 km from breeding sites an equal probability of migrating, then Nm is expected to feeding areas (Hindell et al. 1991; McConnell Fedal to be twice that of Nfm f. The observed result showing and 1996). However, because only very small Nm between HD and MQ to be five to eight times greater amounts of gene flow are required to generate panmixia Wright than Nfm f suggests that the rate of male gene flow may ( 1931), it is reasonable to suggest that geo- be up to four times greater than female gene flow. The graphic structure in the southern elephant seal, espe- result must be viewed cautiously, because the estimate cially with respect to females, is not dominated by any of Nfm f was derived from one locus, and because the substantial amounts of contemporary gene flow. This estimate of Nm was derived from only one pairwise popu- suggestion is supported by the observation that mtDNA lation comparison. Nonetheless, male-biased gene flow gene flow (or genetic distance) between populations is consistent with ecological studies on dispersal of male does not correspond very well with geographic distance and female southern elephant seals (Slade 1998) and (Table 5), particularly when comparing SG and PV with sex-biased dispersal during the pelagic feeding (Nfm f ϭ 0.53, distance ϭ 2400 km) with SG and HD phase of the annual cycle in which males generally travel (Nfm f ϭ 7.74, distance ϭ 8000 km), and that resight individuals tagged mostly in the 20,000ف farther than females to reach their respective feeding records of grounds (Hindell et al. 1991). south Indian and south Pacific ocean regions between However, we suggest that most of the difference be- 1950 and 1980 showed no movement between regions tween mtDNA and nDNA in the observed level of geo- but some movement, mostly by immatures, within re- graphic structure is accounted for by differences in the gions (Slade 1998). characteristics of the genes. The greater sensitivity of If historical association, rather than contemporary mtDNA compared with nDNA is usually explained by gene flow, dominates the observed geographic structure two factors: its smaller effective population size and its of the southern elephant seal, then how do we explain higher mutation rate (Birky et al. 1983; Wilson et al. the close genetic relationship between HD and SG, de-

1985). For the southern elephant seal, Nf is roughly spite their being as geographically separate as HD and equal to Ne, and so the rate of drift of mtDNA is expected MQ? In the historical model, SG and HD separated to be roughly twice that of the nDNA, thereby contribut- as recently as 20,000 yr ago, and all other population ing to the increased resolution of the mtDNA variation. divergences occurred over 200,000 yr ago. We suggest However, most of the increased sensitivity for detecting that the estimated relatively recent separation time be- subdivision from mtDNA point mutations compared tween SG and HD is linked with the last ice age 18,000 with nDNA point mutations would come from the 50– yr ago (CLIMAP 1976). It is likely that the distribution 100 times greater mutation rate. The higher mutation of southern elephant seals during that period would rate would decrease the frequency of shared alleles have been different from the current distribution, be- when one population separates into two and would in- cause of the unsuitability of current breeding beaches crease the chance that isolated populations contain that were iced over at that time (R. W. Slade, unpub- unique alleles that arose within each population after lished results). Rather than being distributed on sub- separation (Slade et al. 1994). That differences in muta- Antarctic islands, the southern elephant seal was proba- tion rate account for a significant part of the difference bly distributed primarily on the southern edge of the in detecting subdivision is supported by the results from continental land masses of Australia, Africa, and South the highly variable microsatellites, which alone among America, and we suggest that at that time HD and SG the nDNA loci in this study showed evidence of geo- were a single breeding population on the coast of South graphic structure. Microsatellite loci have mutation Africa. Records of southern elephant seals in South Ϫ Ϫ rates of the order of 10 2–10 4 per locus per generation Africa indicate that it is currently used as a haul-out (Weber and Wong 1993). This is similar to the mutation for immature seals to moult, although several births rate per locus per generation for CRI, which is 1.8 ϫ have occurred there also (Oosthuizen et al. 1988). It Ϫ Ϫ 10 4 (i.e.,75ϫ10 9 ϫ 299 bp ϫ 8 yr). Conversely, the is known that Australia was a former breeding colony per locus mutation rate of the other 250–450-bp nDNA (Pemberton and Skira 1989), and PV may be the rem- Ϫ6 gene fragments is 2.5–4.5 ϫ 10 , some two orders of nant of the population breeding on the coast of South magnitude lower. America. Gene flow vs. historical association: Geographic structure Thanks to Hamish McCallum, Yuki Takahata, and Monty Slat- reflects some combination of contemporary levels of kin for advice; Tom Arnbom and Ian Boyd for sample collection at gene flow and recent historical association. Considering South Georgia; Chris Schneider, Dick Hudson, and two reviewers the former, the dispersal capacity of adult males and for comments on the manuscript; and especially Anita Heideman 1956 R. W. Slade et al. for assistance in the lab. This work was funded principally by grant Gall, G. A. E., D. Bartley, B. Bentley, J. Brodziak, R. 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