Supplemental Table 1.Taxon names, MCZbase codes, original MCZ DNA database codes, GenBank codes, and collection locality information for all specimens used.

Species / MCZbase code (Invert. Zoo.) / Old MCZ DNA code (DNA-) / Population code / H3 / 18S / 28S / COI / 12S / 16S / Location / Lat., Long.
Metasiroamericanus / 134557 / 101532 / A / KJ405955–964 / KT200610-9 / KT200839-48 / Torreya State Park, Liberty Co., Florida, USA / 30.56472, -84.95139
“ / 133791, 133792 , 133797 / 103805, 103806, 103811 / B / KT201076 / KT201064 / KT201067 / KJ405965–76, KJ405986–992 / KT200620-31, KT200641-7 / KT200849-60, KT200870-6 / Torreya State Park, Liberty Co., Florida, USA / 30.568, -84.95167
“ / 133793–5 / 103807–9 / C / KJ405977–85 / KT200632-40 / KT200861-9 / Upper Sweetwater Creek, Liberty Co., Florida, USA / 30.52558, -84.93677
“ / 134493 / 103813 / D / KJ405993–999 / KT200648-54 / KT200877-83 / Flat Creek, Gadsden Co., Florida, USA / 30.62793, -84.83528
“ / 134492 / 103814 / E / KJ406000–009 / KT200655-64 / KT200884-93 / Chattahoochee, Gadsden Co., Florida, USA / 30.70232, -84.82556
“ / 133808 / 105654 / P / KT201078 / KT201061 / KT201068 / KJ406107–26 / KT200762-80 / KT200991-1010 / Florida Caverns State Park, Jackson Co., Florida, USA / 30.82119, -85.24639
“ / 133809 / 105655 / Q / KJ406127–36 / KT200782-91 / KT201011-20 / “ / “
“ / 133810 / 105656 / R / KJ406137–43 / KT200792-8 / KT201021-27 / “ / “
“ / 133811 / 105657 / S / KT201073 / KJ406144–53 / KT200799-808 / KT201028-37 / “ / “
“ / 133812 / 105662 / T / KT201074 / KJ406154–163 / KT200809-18 / KT201038-47 / “ / 30.81433, -85.24667
“ / 133813 / 105663 / U / KT201079 / KT201060 / KJ406164–175 / KT200819-30 / KT201048-60 / “ / 30.81408, -85.24722
M. sassafrasensis / 134534–5 / 105643–4 / F / KT201075, KT201080 / KT201063 / KT201070 / KJ406010–21 / KT200665-76 / KT200894-905 / Jocosse Gorge, Sassafras Mt., Pickens Co., South Carolina, USA / 35.06228, -82.795
M. savannahensis / 133799 / 105645 / G / KJ406022–30 / KT200677-85 / KT200906-14 / Kingfisher Pond, Savannah National Wildlife Refuge, Jasper Co., South Carolina, USA / 32.18923, -81.08
“ / 133800 / 105646 / H / KT201077 / KT201062 / KT201069 / KJ406031–40 / KT200686-95 / KT200915-24 / “ / “
“ / 133801 / 105647 / I / KT201066 / KJ406041–9 / KT200696-704 / KT200925-33 / “ / “
“ / 133802 / 105648 / J / KT201071 / KJ406050–8 / KT200705-13 / KT200934-42 / “ / “
“ / 133803 / 105649 / K / KT201065 / KJ406059–67 / KT200714-22 / KT200943-51 / “ / “
“ / 133804 / 105650 / L / KT201072 / KJ406068–77 / KT200723-32 / KT200952-61 / “ / “
“ / 133805 / 105651 / M / KJ406078–87 / KT200733-42 / KT200962-71 / “ / “
“ / 133806 / 105652 / N / KJ406088–96 / KT200743-51 / KT200972-80 / “ / “
“ / 133807 / 105653 / O / KT201081 / KJ406097–106 / KT200752-61 / KT200981-90 / “ / “
Aorakidenticulata major / 134644 / 100959 / EU673700 / EU673620 / EU673656 / DQ992203 / EU673585 / New Zealand / -43.03408, 171.76464
Huitacaboyacaensis / 132276 / 101407 / DQ518167 / DQ518090 / DQ825596 / DQ518129 / DQ518050 / Colombia / 5.77956, -73.45377
Metagovea sp. / 132282 / 101409 / JF786426 / DQ825544 / DQ825599 / DQ825646 / DQ825619 / Colombia / 1.28500, -78.07367
Metagovea sp. / 132283 / 101410 / DQ518168 / DQ518091 / DQ518048 / GQ912860 / JF935034 / Colombia / 1.28500, -78.07367
Ogoveacameroonensis / 132315 / 104617 / JF786417 / JF934960 / JF934994 / JF786392 / JF935026 / Cameroon / 3.64621, 11.29079
Parogovia sp. / 132305 / 101052 / DQ825515 / DQ825546 / DQ825601 / DQ825648 / DQ825620 / Equatorial Guinea / 1.65815, 10.31143
Parogoviasironoides / 132302 / 101059 / DQ518169 / DQ518092 / DQ825606 / DQ518131 / DQ518051 / Bioko, Equatorial Guinea / 3.72570, 8.83828
Parogoviasironoides / 132304 / 101061 / DQ825519 / DQ825550 / DQ825607 / DQ825650 / DQ825624 / Bioko, Equatorial Guinea / 3.70284, 8.87520
Parogovia sp. / 132299 / 104615 / JF786427 / JF934966 / JF935021 / JF786410 / KT200831 / JF935044 / Cameroon / 4.80084, 9.66326
Parogovia cf. sironoides / 132293 / 104619 / JF786428 / JF934967 / JF935022 / JF786409 / KT200832 / JF935045 / Cameroon / 2.74108, 9.88181
Parogoviagabonica / 132297 / 104620 / JF934969 / JF935019 / JF786411 / KT200833 / JF935047 / Gabon / 0.50448, 12.79525
Pettalusthwaitesi / 78875 / 101227 / DQ518138 / DQ517975 / DQ518018 / DQ518101 / DQ518057 / Sri Lanka / 7.27251, 80.59333
Siro rubens / 132391 / 100457 / AY428834 / AY428818 / DQ133735 / DQ513111 / France / 44.08338, 3.58140
Leptopsalis sp. / 70646 / 101934 / GQ488131 / GQ488296 / GQ488358 / GQ488165 / KT200834 / GQ488208 / Sulawesi, Indonesia / 0.759167, 124.421667
Miopsalis sp. / 134360 / 103259 / GQ488116 / GQ488260 / GQ488375 / GQ488142 / KT200835 / GQ488193 / Borneo, Malaysia / 5.81, 116.24
Suzukielussauteri / 132256 / 101543 / DQ518166 / DQ513138 / DQ513116 / DQ513108 / DQ518086 / Japan / 35.63440, 139.24122
Troglosiroaelleni / 134764 / 100345 / DQ518164 / AY639497 / AY639525 / AY639584 / AY639555 / New Caledonia / -21.1833, 165.3059
Troglosirolongifossa / 134765 / 100867 / DQ518165 / DQ518089 / DQ825582 / DQ518127 / DQ518084 / New Caledonia / -22.3527, 166.9736
Troglosiromonteithi / 51948 / 101580 / EU887101 / EU887116 / EU887043 / KT200836 / EU887074 / New Caledonia / -21.6000, 165.7167
Troglosiro cf. juberthiei / 133875 / 101703 / EU887045 / KT200837 / EU887082 / New Caledonia / -22.25, 166.816667
Troglosirourbanus / 134780 / 101710 / JF786340 / EU887102 / EU887119 / EU887040 / EU887073 / New Caledonia / -22.1945, 166.5017
Troglosirowilsoni / 134787 / 102324 / EU887107 / EU887125 / EU887061 / KT200838 / EU887075 / New Caledonia / -22.1770, 166.5106
Forsteropsalisturneri / 134832 / 100783 / KJ871492 / KJ871558 / New Zealand / -46.01975, 167.74095

Supplemental Table 2.Primers and temperature profiles used in this study. All primers are written 5’–3’.

Primer name / Reference / Sequence (5’ to 3’) / Protocol
18SrRNA
/ Standard1, T=47
1F / (Giribet et al. 1996) / TAC CTG GTT GAT CCT GCC AGT AG
3F / “ / GTT CGA TTC CGG AGA GGG A
4F / “ / CCA GCA GCC GCG CTA ATT C
4R / “ / GAA TTA CCG CGG CTG CTG G
5R / “ / CTT GGC AAA TGC TTT CGC
9R / “ / GAT CCT TCC GCA GGT TCA CCT AC
18Sa2.0 / (Whiting et al. 1997) / ATG GTT GCA AAG CTG AAA C
18Sbi / “ / GAG TCT CGT TCG TTA TCG GA
28SrRNA
/ Standard1, T=47
28SD1f / (Park and Foighil 2000) / GGG ACT ACC CCC TGA ATT TAA GCA T
28Sa / (Whiting et al. 1997) / GAC CCG TCT TGA AAC ACG GA
28Sb / “ / TCG GAA GGA ACC AGC TAC
28SrD4b / (Whiting 2002) / CCT TGG TCC GTG TTT CAA GAC
28SrD5b / “ / CCA CAG CGC CAG TTC TGC TTA C
28Srd4.8a / (Schwendinger and Giribet 2005) / ACC TAT TCT CAA ACT TTA AAT GG
28Srd7b1 / “ / GAC TTC CCT TAC CTA CAT
16SrRNA / Standard1, T=46
16Sa / (Xiong and Kocher 1991) / CGC CTG TTT ATC AAA AAC AT
16SaPo / CGA CTG TTT ATC AAA AAC AT
16Sb / (Xiong and Kocher 1991) / CTC CGG TTT GAA CTC AGA TCA
16SbPo / CAC CGG TTT GAA CTC AAA TCA
Front loop of 12S / Standard1, T=46
12SFb1 / CGT GCC AGC CGC CGC GGT TAG AC
12SFb1-long / CGT GCC AGC CGC CGC GGT TAG ACC A
12SaiREV / ATA ATA GGG TAT CTA ATC CTA GTT T
12SaiREV3 / ATA ATA GGG TAT CTA ATC CTA GTT TAT C
12S / Standard1, T=46
12Sai / (O'Neill and Dobson 2008) / AAA CTA GGA TTA GAT ACC CTA TTA T
12SdegR / VAR AGY GAC GGG CRA TAT GTA CAT
COI / Standard1, T=46
LCO1490 / (Folmer et al. 1994) / GGT CAA CAA ATC ATA AAG ATA TTG G
LCOdeg1 / TCH ACD AAY CAY AAA GAT ATT GGW ACW AT
LCOMeta2 / KKT CAA CGA ATC ATA AAG ATA TTG G
LCOMeta8 / TKK TCA ACG AAT CAT AAA GAT ATT
HCOoutout / (Whiting 2002) / GTA AAT ATA TGR TGD GCT C
HCOooOpi2 / GTA AAT ATA TGR TGD GCT CAT AC
HCOoutMeta / ATA GTG ACA ATA AAA TTA ATA GCT CC
Histone H3 / TD62, TD6-483
H3aF / (Xiong and Kocher 1991) / ATG GCT CGT ACC AAG CAG ACV GC
H3aR / “ / ATA TCC TTR GGC ATR ATR GTG AC

1 Standard: 35x[94°C (0:30), T (0:30), 72°C (1:30)], [72°C (10:00)]

2 TD6: [94°C (1:00), 58–45°C (1:00), 72°C (1:00)], 25x[94°C (0:30), 58°C (0:45), 72°C (0:45)] , [72°C (3:00)]

3 TD6-48 [94°C (1:00), 58.5–48°C (1:00), 72°C (1:00)], 25x[94°C (0:30), 58°C (0:45), 72°C (0:45)] , [72°C (3:00)]

4 TD53: [94°C (1:00), 63–53°C (1:00), 72°C (1:00)], 4x[94°C (1:00), 53°C (1:00), 72°C (1:00)], 25x[94°C (0:30), 58°C (0:45), 72°C (0:45)] , [72°C (3:00)]

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