Electronic Supplementary Material ESM
Tables S1-S3
Reference genome sequencing, paternity assignment methods and putative sire haplogroup confirmation
figure S1
Padua, Zeh, Bonilla and Zeh
“Sisters’ curse: sexually antagonistic effects constrain the spread of a mitochondrial haplogroup superior in sperm competition”
Table S1. Primers and PCR conditions for next generation sequencing of the Cordylochernes scorpioides mitochondrial genome.
Long PCR was used to amplify the genome in three adjacently overlapping fragments.
Position in Ahaplogroup
Amplicon
genome
length (bp)
1,405-1,432
9,011
10,443-10,416
Mitochondrial
region amplified
COX1CYTB
Fragment 1
Primer names
CscCOX1F1F
CscCYTBF1R
Primer sequence
CTATAATAATTGGGGGTTTTGGDAACTG
CCCCTCCTATTTTGTTAGGGACACAACG
CYTBrRNA
Fragment 2
CscCYTBF2F
Cscl-rRNAF2R
TRCTGACAATAAATTACTGCCCATCYCC
AGAGAAAAATTACGCCAGGGATAACAGG
9,721-9,748
14,093-14,120
4,399
ND1COX1
Fragment 3
CscND1F3F
CscCOX1F3R
TTGCTGAGGTTAAAGAGTTTACAACTGG
GATAAATTGTTCATCCAGCTCCACAYC
13,085-13,112
1,552-1,578
4,434
PCR was performed in a 35 µL reaction volume containing ~10 ng of genomic DNA, 3.5 µL of 10X Advantage Genomic LA Buffer
(25 mM MgCl2), 350 mM dNTPs, 750 nM primers, and 1.5U of Advantage Genomic LA Polymerase Mix (Clontech, Mountain
View, CA, USA). The PCR amplification conditions involved a 1 min hot start at 95°C, followed by 34 cycles of the following
conditions: 93°C melting for 25 s, 54 to 620C melting for 40 s, and a 68°C extension for 10-12 min.
Table S2. Mean number of nucleotide substitutions and mean uncorrected divergence within
and between the A, B1 and B2 haplogroups of the Cordylochernes scorpioides mitochondrial
genome. Comparisons were restricted to the 13 protein-coding genes of the oxidative
phosphorylation pathway, the 22 tRNAs, and the large and small subunit ribosomal RNA genes.
Total genome length, excluding the control region, varied from 13,704 to 13,736 bp across the
15 sequenced genomes.
mean number of nucleotide
mean uncorrected
comparison
substitutions
divergence (%)
Within A haplogroup
33
0.20
Within B1 haplogroup
47
0.29
Within B2 haplogroup
11
0.04
A versus B1 haplogroup
915
8.36
A versus B2 haplogroup
949
8.89
B1 versus B2 haplogroup
495
4.06
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Table S3. Sequence lengths and nucleotide substitution properties of the 13 protein-coding
genes of the Cordylochernes scorpioides mitochondrial oxidative phosphorylation system.
Genes are listed in the order in which they are arranged in the genome. pi(-), pi(N) and pi(+) are
the proportion of sites under negative selection, neutrality and positive selection, respectively.
proportion of sites under
number or percentage of
negative selection,
sequence length by
codons with nonneutrality or positive
haplogroup (bp)
synonymous substitutions selection
gene
ND2
COX1
COX2
ATP8
ATP6
COX3
ND3
ND5
ND4
ND4L
ND6
CYTB
ND1
A
B1
930
1536
705
183
672
807
345
1662
1356
270
465
1098
885
894
1536
705
183
675
807
345
1662
1257
270
465
1101
885
B2
894
1536
705
183
675
807
345
1662
1287
270
465
1101
885
number percentage
19
6.44
10
1.97
4
1.72
9
15.00
14
6.28
9
3.37
20
17.70
46
8.35
15
3.69
9
10.23
13
8.50
20
5.49
18
6.32
3
pi(-)
0.933
0.971
0.932
0.424
0.936
0.952
0.791
0.904
0.931
0.461
0.883
0.941
0.753
pi(N)
0.045
0.020
0.032
0.334
0.015
0.025
0.118
0.049
0.033
0.257
0.059
0.032
0.157
pi(+)
0.020
0.009
0.037
0.241
0.048
0.022
0.091
0.046
0.036
0.282
0.059
0.027
0.090
Reference genome sequencing, paternity assignment methods and putative sire haplogroup
confirmation
Sequencing of the reference C. scorpioides mitochondrial genome
An entire C. scorpioides mitochondrial genome was PCR amplified in two segments, using the
Expand Long Template PCR kit (Roche). High-Tm, C. scorpioides-specific primers were
designed by first amplifying an ~ 660-bp segment of the COX1 gene, using the highly conserved
chelicerate forward1 (5'-TACTCTACTAATCATAAAGACATTGG – 3’) and reverse2 (5’ –
GGATGGCCAAAAAATCAAAATAAATG – 3’) primers [1], and an ~ 450 bp region of the
CYTB gene Cytb424-449 (5’ – GGWTAYGTWYTWCCWTGRGGWCARAT – 3’ and
Cytb876-847 (5’ – GCRTAWGCRAAWARRAARTAYCAYTCWGG – 3’) [2]. COXI and
CYTB are located approximately opposite one another in the circular mitochondrial genome, and
primers from these two genes can be used to amplify the entire genome in two fragments of
similar length. In order to span the entire genome, 35 primer pairs were designed, with ~ 100-bp
overlap between contiguous segments
TreeSAAP Methods
For each gene, the phylogeny of the 15 sequences was reconstructed using Bayesian methods and
imported into TreeSAAP, which was then used to estimate the magnitude of change across the
entire gene sequence for 31 physicochemical properties. TreeSAAP rates substitutions on a scale
of 1 (most conservative) to 8 (most radical), with a significant positive z-score indicating more
non-synonymous substitutions than expected under neutrality. Following author
recommendations, only the most radical amino acid substitutions (categories 7-8), with z-scores
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statistically significant at the 0.001 level, were considered biologically significant and further
analyzed, using a sliding window of 15 codons to pinpoint specific gene regions under positive
selection.
Haplogroup effects on sperm competitive ability
DNA was extracted from the dams, putative sires and protonymphs using Invitrogen
Chargeswitch gDNA Micro Tissue Kit (Life Technologies, Grand Island, NY, USA). PCR was
performed in a 30 µL reaction volume containing ~10 ng of genomic DNA, 3 µL of 10X
Advantage Genomic LA Buffer (25 mM MgCl2), 350 mM dNTPs, 750 nM primers, and 1.5U of
Advantage Genomic LA Polymerase Mix (Clontech, Mountain View, CA, USA). The PCR
amplification conditions involved a 1 min hot start at 95°C, followed by 34 cycles of the
following conditions: 93°C melting for 25 s, and 68°C annealing/extension for 8 min For each
replication, PCR products from the mother, the two putative sires and an average of 22 offspring
were run on a 1.5% agarose gel and stained with ethidium bromide to visualize alleles. Several
measures were taken to ensure amplification of both alleles from heterozygous individuals. First,
a strict consensus primer pair (cCscMS23 2+: CAGTGTTGCCGGGTGGATTATTAACC and
cCscMS23 2-: ATGGCGTGKGAAGCAGAGAACTCTAGG) was previously designed from a
large sample (N=22) of cCscMS23 alleles. Second, the high primer-pair Tm enabled a robust,
two-step PCR reaction. Finally, PCR was performed using a mixture of DNA polymerases
capable of amplifying high molecular weight alleles (to 22 kb) from femptogram quantities of
DNA.
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Putative sire haplogroup confirmation in sperm competition experiment
DNA sequencing of the mitochondrial ND2 locus from our C. scorpioides laboratory matrilines
has established that haplotypes in the A but not the B2 haplogroup possess a ClaI restriction site.
ClaI digested ND2 amplicons were therefore used to confirm the mitochondrial haplotype of all
putative sires from successful replicates of the sperm competition experiment. DNA from frozen
adults was extracted as described above and PCR was conducted using the ND2 mitochondrial
DNA locus forward (5’ – TGTAAGTCTTAAAAYAAAGAAAACC – 3’) and reverse primers
(5’ – AAGTCATCGAATAGARACRTTAGC – 3’). PCR reactions were performed, as
described above, except that the conditions of the 34 cycles were modified to: 93°C for 25 s,
48C for 50 s, and 68C for 90 s. Digestion reactions were carried out using 20 μL mixtures of
1X buffer, 1 μg (approximately 12 μL) of PCR product and 1μL of Cla I restriction enzyme.
Products were incubated for 60 min at 37C and the enzyme inactivated at 65C for 20 min.
Products were visualized on 1.5% agarose gels run for approximately 80 volt-hr to determine
presence (two fragments) or absence (one fragment) of the restriction site in the ND2 sequence
from each male.
References
1. Barrett, R. D. H. & Hebert, P. D. N. Identifying spiders through DNA barcodes. Can. J. Zool.
83, 481-491 (2005).
2. Cook, C. E. The complete mitochondrial genome of the stomatopod crustacean Squilla
mantis. BMC Genomics 6, 105 (2005).
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figure S1. Female mitochondrial haplotype and sexual receptivity at first and second mating. Ahaplogroup females (striped bars) and B2-haplogroup females (black bars) were equally likely to
accept a sperm packet in their first mating. However, the propensity of B2-haplogroup females to
engage in polyandry was significantly lower (*P = 0.05) than that of A-haplogroup females.
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