1
Supplementary Information
2
Supplemetary Table 1. Collection and voucher information for samples used in this study. All host genera are Tsuga.
Voucher
Accession No.
Sample
name
CNC HEM053427
NH04-85
Host species
Collection information
TAIWAN, Taichung Co., Tayuling; 23 Dec. 2004; Coll. H.Y. Wang
YPM ENT 764423 Ad03-01
YPM ENT 674511 Ad02-05
T. chinensis
var. formosana
T. chinensis
T. chinensis
CNC HEM053081
Ad02-10
T. chinensis
YPM ENT 764508 Ad02-34
T. chinensis
CNC HEM053368
NH04-35
T. diversifolia
CNC HEM053369
NH04-36
T. diversifolia
CNC HEM053370
NH04-37
T. diversifolia
CNC HEM053390
NH04-44
T. diversifolia
CNC HEM056237
Ad06-20
T. diversifolia
CNC HEM056235
Ad06-22
T. diversifolia
CNC HEM053366
NH04-33
T. seiboldii
CNC HEM053367
NH04-34
T. seiboldii
CNC HEM053364
NH04-31
T. sieboldii
CNC HEM053432
NH05-04
T. sieboldii
CHINA, Qioji, Boaxing Co., Sichuan; 28 Sep. 2003; Coll. M.E. Montgomery
CHINA, Shaanxi, Ningshan Co.; 13 Oct. 2002; Coll.: N.P. Havill, M.E. Montgomery, G.
Yu
CHINA, Shaanxi, Ningshan Co.; 13 Oct. 2002; Coll. N.P. Havill, M.E. Montgomery, G.
Yu
CHINA, Yunnan, Lijiang Co.; 25 Oct. 2002; Coll. N.P. Havill
JAPAN, Nagano Prefecture, Tashirobashi, Kamikochi, Azumi-mura; 10 June 2004; Coll.
N. Havill, S. Shiyake, G. Yu
JAPAN, Gifu Prefecture, Norikura skyline, Kamitakara-mura; 10 June 2004; Coll. N.
Havill, S. Shiyake, G. Yu
JAPAN, Gifu Pref., Norikura Skyline, Kamitakara-mura; 10 June 2004; Coll. N. Havill,
S. Shiyake, G. Yu
JAPAN, Yamanashi Prefecture, Mt. Fuji; 12 June 2004; Coll. N. Havill, S. Shiyake, G.
Yu
JAPAN, Nagano Prefecture; Ina; Mibu Valley; 10 May 2006; Coll. M.E. Montgomery,
S. Shiyake
JAPAN, JAPAN; Nara Pref.; Nakamichi; Odaigahara; 8 May 2006; Coll.: Y. Miyataki
JAPAN, Osaka Prefecture, Yoshikawa, Toyono-cho; 9 June 2004; Coll. N. Havill, S.
Shiyake, G. Yu
JAPAN, Osaka Prefecture, Nakahata, Takatsuki; 9 June 2004; Coll. N. Havill, S.
Shiyake, G. Yu
JAPAN, Wakayama Prefecture, Kongo-buji Temple, Koya-san; 8 June 2004; Coll. N.
Havill, S. Shiyake, G. Yu
JAPAN, Shizuoka Prefecture, Yugashima, Izu-shi; 19 March 2005; Coll. S. Shiyake
YPM ENT 764514 Ad06-04
T. sieboldii
YPM ENT 764528 Ad04-13
YPM ENT 764529 Ad04-14
CNC HEM053412 Ad04-18
T. canadensis
T. canadensis
T. canadensis
YPM ENT 764530 Ad05-13
YPM ENT 764498 AdNANY1
T. canadensis
T. canadensis
JAPAN, Osaka Prefecture, Takatsuki, Fuji shrine; 24 March 2006; Coll. A. Lamb, S.
Shiyake, T. McAvoy
JAPAN, Kyoto Prefecture, Kameoka, Uketa shrine; 28 March 2006; Coll. A. Lamb, S.
Shiyake, T. McAvoy
JAPAN, Nara Prefecture, Nara, Wakakusa-Yama; 17 April 2006; Coll. A. Lamb, S.
Shiyake, T. McAvoy
JAPAN, Wakayama Prefecture, Koya, Ojiyama san; 17 April 1996; Coll. A. Lamb, S.
Shiyake, T. McAvoy
JAPAN, Wakayama Prefecture, Koya-san town; 23 Sep. 2003; Coll. M.E. Montgomery;
S. Shiyake
USA, CT, New Haven Co., Hamden; 6 Nov. 2003; Coll. K. Shields
USA, CT, New Haven Co., Hamden; 3 Dec. 2003; Coll. K. Shields
USA, CT, Litchfield Co., Bridgewater; 15 June 2002; Coll.: N.P. Havill
USA, CT, Middlesex Co., Killingworth, Chatfield Hollow State Park; 26 June 2003; Coll.
D. Mikus
USA, CT, Middlesex Co., Killingworth, Chatfield Hollow State Park; 26 June 2003; Coll.
D. Mikus
USA, CT, Middlesex Co., Killingworth, Chatfield Hollow State Park; 26 June 2003; Coll.
D. Mikus
USA, CT, New Haven Co., Hamden; 23 July 2003; Coll. K. Shields
USA, CT, New Haven Co., Hamden; 6 Feb. 2004; Coll. K. Shields
USA, CT, New Haven Co., Hamden; 9 Feb. 2005; Coll. K. Shields
USA, CT, Middlesex Co., Killingworth; 27 Oct. 2005; Coll. K. Shields
USA, CT, New Haven Co., Hamden; 26 April 2006; Coll. K. Shields
USA, GA, Clayton Co., Chattahoochee Nat. For.; Reed Creek Gap; 11 May 2004; Coll.
M.E. Montgomery
USA, MA, Suffolk Co., Jamaica Plain; Arnold Arboretum; 23 June 2003; Coll. N.P.
Havill
USA, MD, Allegheny Co., Flintstone; 18 March 2004
USA, NC; 18 March 2004; Coll. R. Rhea
USA, NC, Otto Co., Hurricane Gap, Coweta Hydrologic Lab; 12 May 2004; Coll. M.E.
Montgomery
USA, NC; 26 Oct. 2005; Coll. R. Rhea
USA, NY, Ulster Co., Ashokan; 11 Dec. 2001; Coll. L. Jones
YPM ENT 764515 Ad06-07
T. sieboldii
YPM ENT 764516 Ad06-14
T. sieboldii
YPM ENT 764517 Ad06-15
T. sieboldii
CNC HEM053336
AdJ1
T. sieboldii
YPM ENT 764518
YPM ENT 764519
YPM ENT 764496
YPM ENT 764520
AdCT03
AdCT04
AdNACT25
Ad03-01
T. canadensis
T. canadensis
T. canadensis
T. canadensis
CNC HEM053347
T. heterophylla
USA, OR, Clackamas County, Sandy; 17 Dec. 2003; Coll. B. Wilhite
YPM ENT 764521 Ad03-03
T. canadensis
YPM ENT 764522 Ad03-04
T. canadensis
YPM ENT 764523
YPM ENT 764524
YPM ENT 764525
YPM ENT 764526
YPM ENT 764527
CNC HEM053410
Ad03-06
Ad04-03
Ad05-01
Ad05-11
Ad06-18
Ad04-17
T. canadensis
T. canadensis
T. canadensis
T. canadensis
T. canadensis
T. canadensis
CNC HEM053181
AdNAAA
T. seiboldii
Ad04-04
YPM ENT 764531
YPM ENT 764532
YPM ENT 764503
YPM ENT 764533
YPM ENT 764534
YPM ENT 764535
CNC HEM053161
YPM ENT 764536
YPM ENT 764537
YPM ENT 764538
CNC HEM053405
YPM ENT 764539
CNC HEM053422
YPM ENT 764540
YPM ENT 764541
YPM ENT 764542
YPM ENT 764543
Ad04-05
Ad04-06
Ad04-07
Ad05-12
Ad06-12
AdOR2
AdNAWA1
Ad04-08
Ad04-09
Ad04-10
Ad04-11
Ad04-19
Ad04-25
Ad03-08
Ad03-09
Ad03-10
Ad03-11
T. heterophylla
T. heterophylla
T. heterophylla
T. heterophylla
T. heterophylla
T. heterophylla
T. heterophylla
T. heterophylla
T. heterophylla
T. heterophylla
T. heterophylla
T. heterophylla
T. heterophylla
T. heterophylla
T. heterophylla
T. heterophylla
T. heterophylla
USA, OR Clackamas County, Sandy; 3 Feb. 2004; Coll. B. Wilhite
USA, OR Clackamas County, Sandy; 3 March 2004; Coll. B. Wilhite
USA, OR, Clackamas Co., Sandy; 5 April 2004; Coll. B. Wilhite
USA, OR, Benton Co., Corvallis; 27 Oct. 2005
USA, OR, Eola Hills; 11 April 2006
USA, OR, Clackamas Co., Sandy; 18 Nov. 2003; Coll. B. Wilhite
USA, WA, Clark Co., Vancouver, Ridgefield; 5 May 2003; Coll.: N.P. Havill
USA, WA; 15 April 2004
USA, WA, King County; 28 April 2004
USA, WA, Thurston County; 28 April 2004
USA, WA, Clallam Co., Sequim, Dungeness NWR; 27 April 2004; Coll. K. Ripley
USA, WA; 15 April 2004
USA, WA, Snohomish Co., Edmonds; 30 Sep. 2004; Coll. K. Saltonstall
CANADA, Pacific Forestry Centre, Victoria; 14 Nov. 2003
CANADA, Pacific Forestry Centre, Victoria; 14 Nov. 2003
CANADA, British Columbia, Victoria; 3 Dec. 2003
CANADA, Pacific Forestry Centre, Victoria; 14 Nov. 2003
Supplementary Table 2. Primers and probes used in this study.
Primer/Probe Name
10f
27f
340f
341f
570r
766f
810r (ER15)
1495r
1507r
AT Beta 70Ar
AT Beta 70Br
AT Beta 191Ar
AT Beta 191Br
AT Beta 440Ar
AT Beta 440Br
AT Beta 844Ar
AT Beta 844Br
AT Beta 1032Ar
AT Beta 1032Br
AT Beta 1256Ar
AT Beta 1256Br
AT BetaA 1009r
AT BetaB 1009r
AT BetaA 460f
AT BetaB 460f
AT BetaA 460r
AT BetaB 460r
BetaA 74r
BetaA 454r
BetaA 836r
BetaA 1003r
BetaA 1021r
BetaB 74r
Sequence (5' - 3')
AGTTTGATCATGGCTCAGATTG
GAGAGTTTGATCCTGGCTCAG
TTCTAAGGAAGGCAGCAGTG
CCTACGGGAGGCAGCAGTGG
CGCCCTGTAATTCCGATTAACGC
AAAGCGTGGGGAGCAAACAG
GCGTGGACTACCAGGGTATC
CTACGGCTACCTTGTTACGA
TACCTTGTTACGACTTCACCCCAG
CAGCGCGGGGCAACCTGGC
CAGCACGGGGGCAACCCTGGT
GATGAAAGTGGGGGACCTTC
GAAGAAAGCGGGGGATCTTT
TGTCAGGGAAGAAACGGCCGA
TGTCCGGAAAGAAAACCTTTT
AATTAACTTGGTAACGCAGC
CATTGACTTAGTAACGTAGC
CCGAAAGGGAACCTGAAC
TCGAAAGAGAACCGATAC
CCGCCAACCCGCGAGGGG
CTGCCAACCCGTGAGGGG
CCTTTCAGCCAGGTTCCGACC
GATCTCTCCGGACTTCCTGAC
ACCTTTTGGTTAATACCCGAGGGG
CGGCCGAGGATAATACCTTTGGCT
CCCCTCGGGTATTAACCAAAAGGT
AGCCAAAGGTATTATCCTCGGCCG
ACGGGGGCAACCCTGGT
ACCTTTTGGTTAATACCCGAGG
TGGGTCTTCATTGACTTAGTAACGT
GTCGGAGCCTGGCTGAAA
GCTGGGGTGCTCGAAAGAG
GCGGGGCAACCTGGC
Reference/Target taxon
(Munson et al., 1991)/general eubacterial
(Lane, 1991)/general eubacterial
(Spaulding and von Dohlen, 2001)/general eubacterial
(Spaulding and von Dohlen, 1998)/general eubacterial
This study/general eubacterial
(Spaulding and von Dohlen, 1998)/general eubacterial
(Widjojoatmodjo et al., 1995)/general eubacterial
(Adolph, 1996)/general eubacterial
(Munson et al., 1991)/general eubacterial
This study/BS1
This study/BS2
This study/BS1
This study/BS2
This study/BS1
This study/BS2
This study/BS1
This study/BS2
This study/BS1
This study/BS2
This study/BS1
This study/BS2
This study/BS1
This study/BS2
This study/BS1
This study/BS2
This study/BS1
This study/BS2
This study/BS1
This study/BS1
This study/BS1
This study/BS1
This study/BS1
This study/BS2
BetaB 443r
BetaB 454r
BetaB 836r
BetaB 1003r
GamA 805r
GamB 1119r
GamB 437f
GamB 807r
GamC 440r
GamC 476f
GamC 476r
GamC 847r
GamC 1256
GANA 192r
GANA 1118r
CAGGGAAGAAACGGCCGA
CGGCCGAGGATAATACCTTT
CGGGTTTTAATTAACTTGGTAACGC
ATCTCTCCGGACTTCCTGA
CTTCTAACGGCTAGTTGA
GAGTTCCCGACTTTATCG
GCAATGTCTTATTAACACATTACC
GGGCACAACCTCCAAATC
CAATAGCATAAGAAGGTAATGAAA
ATTTTGTTATCAGACGTTAGCTAT
ATAGCTAACGTCTGATAACAAAAT
ATTATAAGTCAAAGCTTT
ATGCAATATTGTAAAATA
TGATAGTGCAAGGCCTTG
GAGTGCCCACCATGATGT
This study/BS2
This study/BS2
This study/BS2
This study/BS2
This study/GS-A
This study/GS-B
This study/GS-B
This study/GS-B
This study/GS-C
This study/GS-C
This study/GS-C
This study/GS-C
This study/GS-C
This study/GS-A
This study/GS-A
Supplementary Figure Legends
Figure S1. Maximum-parsimony phylogram of Gammaproteobacteria GS-B (‘Ca. Serratia
symbiotica’) sequences amplified from Japanese T. sieboldii populations from the island of
Honshu and from eastern North American populations; all other samples yielded no
amplifications of that phylotype. Numbers above branches indicate inferred substitutions along
the branch. GenBank accessions are indicated in brackets.
Figure S2. Laser-scanning confocal images from FISH with the general eubacterial probe 1507r
(labeled with A-568). A. Japanese sample from T. sieboldii first-instar stage, showing probed
bacteria (in red) in both the body cavity and salivary glands. Inset: high magnification of a
probed salivary gland. B. Same sample as in A, but different insect; no differentiated bacteriome
is visible. C. Japanese sample from T. sieboldii, fourth-instar or adult stage, showing probed
bacteria in the hemocoel and the central region of a bacteriome; insets: high magnification of
bacteriome-dwelling bacteria and hemocoel bacteria. D. Western North America sample from T.
heterophylla, third-instar stage cross-section, showing probed rod-shaped bacteria (in red) in the
hemocoel and completely filling several bacteriocytes. Green = autofluorescence from insect
tissue. b = bacteriome, s = salivary gland, h = hemocoel, n = bacteriocyte nucleus.
Figure S3. Laser-scanning confocal images of A. tsugae after FISH with the GS-A-specific probe
GANA 1118r (labeled with A-568), indicating GS-A (Pseudomonas adelgestsugas; in red) are
found only extracellularly in the hemocoel. A. Japanese sample from T. sieboldii (first instar)
showing rod-shaped bacteria in the posterior body cavity. B. Western North America sample
from T. heterophylla (second instar). C. Japanese sample from T. sieboldii egg showing bacteria
free in the posterior cavity and clustered around developing bacteriome (arrows). D. Japanese
sample from T. diversifolia egg, showing bacteria in the posterior region. Green =
autofluorescence from insect tissue; b = bacteriome; h = hemocoel.
Figure S4. Laser-scanning confocal images of A. tsugae after FISH with the GS-C-specific probe
GamC440r (labeled with A-568), indicating GS-C (‘Ca. Annandia adelgestsuga’; in red) are
harbored in bacteriocytes, are transmitted to eggs, and invade salivary glands in early instars. AC, samples from Japan. A. Sample from T. sieboldii, egg stage, showing ‘Ca. A. adelgestsuga’
clustered in two regions of one pole; not all bacteria appear to be contained within discrete
bacteriocytes at this stage (arrow). B. Sample from T. diversifolia, first-instar stage showing ‘Ca.
A. adelgestsuga’ in both bacteriome and salivary gland. C. Sample from T. diversifolia, firstinstar stage, showing ‘Ca. A. adelgestsuga’ in bacteriocytes. D-F, samples from North America.
D. Sample from eastern North America, second-instar stage, showing ‘Ca. A. adelgestsuga’ in
most areas of the bacteriome; some areas are unprobed (arrow). E. Sample from eastern North
America, third-instar stage showing ‘Ca. A. adelgestsuga’ in most areas of the bacteriome; other
areas are unprobed (arrows). F. Sample from western North America, first-instar stage, showing
‘Ca. A. adelgestsuga’ in both bacteriome and salivary gland (arrow). Green = autofluorescence
from insect tissue; b= bacteriocyte/bacteriome, s = salivary gland.
References
Adolph, K.W. (ed) (1996) Microbial Genome Methods: CRC Press.
Lane, D.J. (1991) 16S/23S rRNA sequencing. In Nucleic Acid Techniques in Bacterial
Systematics. Stackebrandt, E., and Goodfellow, M. (eds). New York, NY: John Wiley and
Sons, pp. 115-175.
Munson, M.A., Baumann, P., Clark, M.A., Baumann, L., Moran, N.A., Voegtlin, D.J., and
Campbell, B.C. (1991) Evidence for the establishment of aphid-eubacterium endosymbiosis
in an ancestor of four aphid families. Journal of Bacteriology 173: 6321-6324.
Spaulding, A.W., and von Dohlen, C.D. (1998) Phylogenetic characterization and molecular
evolution of bacterial endosymbionts in psyllids (Hemiptera: Sternorrhyncha). Molecular
Biology and Evolution 15: 1506-1513.
Spaulding, A.W., and von Dohlen, C.D. (2001) Psyllid endosymbionts exhibit patterns of cospeciation with hosts and destabilizing substitutions in ribosomal RNA. Insect Molecular
Biology 10: 57-67.
Widjojoatmodjo, M.N., Fluit, A.C., and Verhoef, J. (1995) Molecular identification of bacteria
by fluorescence-based PCR-single-strand conformation polymorphism analysis of the 16S
rRNA gene. Journal of Clinical Microbiology 33: 2601-2606.