Kostovcik, Bateman, Kolarik, Stelinski, Jordal, Hulcr

The ambrosia symbiosis is specific in some species and promiscuous in others: evidence from high-throughput community sequencing

Kostovcik, Bateman, Kolarik, Stelinski, Jordal, Hulcr

Supplementary materials

Supplementary Table 1. Samples included in the analysis, with location information and barcode sequence used for labeling sequencing reads.

Samplenumber / Species / Location / Barcode
1 / X. affinis / Colt Creek / GAGAGTACTA
2 / X. affinis / Colt Creek / CGTAGTATAC
3 / X. affinis / Colt Creek / CATTCCTCTA
4 / X. affinis / Colt Creek / GCACTAGATA
5 / X. affinis / Colt Creek / GACCTATTCT
6 / X. affinis / Colt Creek / CTATGCTAAG
7 / X. affinis / Colt Creek / GTTCGGTATA
8 / X. affinis / Colt Creek / CTTCTACCAA
9 / X. affinis / Colt Creek / CTTCCATAGT
10 / X. affinis / Colt Creek / GAGGAATAAC
11 / X. affinis / Colt Creek / CTTATTGGTC
12 / X. affinis / Colt Creek / GATGATCAGT
13 / X. affinis / Colt Creek / GTGAGATGAT
14 / X. affinis / Colt Creek / GATCACATCA
15 / X. affinis / Colt Creek / CCATATGAAC
16 / X. affinis / Colt Creek / CAACCTAACA
17 / X. affinis / Colt Creek / CTACGTGATT
18 / X. affinis / Colt Creek / CGGAACTTAT
19 / X. affinis / Colt Creek / GTAACTCGTT
20 / X. affinis / Colt Creek / CAAGAAGTTC
21 / X. affinis / Colt Creek / ATCTCGATAC
22 / X. affinis / Colt Creek / CATTGAGTGT
23 / X. affinis / Colt Creek / CAAGGACAAT
24 / X. affinis / Colt Creek / TGTTACCTAC
25 / X. affinis / Colt Creek / ACTTAGAAGG
26 / X. affinis / Colt Creek / CCACAATCAT
27 / X. affinis / Colt Creek / AAGATCTGAG
28 / X. affinis / Colt Creek / TCCAATACTC
29 / X. affinis / Colt Creek / AGGAGAATCA
30 / X. affinis / Colt Creek / AATACCAGGA
31 / X. affinis / Colt Creek / AGAATCGGTA
32 / X. affinis / Colt Creek / AATAGGTAGC
33 / X. affinis / Lake Wales / TTACAGTACG
34 / X. affinis / Lake Wales / AGTCCTTAAC
35 / X. affinis / Lake Wales / TTCTCCTTCA
36 / X. affinis / Lake Wales / CGCTTAATTG
37 / X. affinis / Lake Wales / AGCTGTCATA
38 / X. affinis / Lake Wales / AACAGTCTCT
39 / X. crassiusculus / Colt Creek / ACAGAAGACT
40 / X. crassiusculus / Colt Creek / ACCTTACCTT
41 / X. crassiusculus / Colt Creek / ACCTCTTGAA
42 / X. crassiusculus / Colt Creek / AACCGAGTTA
43 / X. crassiusculus / Colt Creek / ACGGTTCTAA
44 / X. crassiusculus / Colt Creek / ACACGTATGA
45 / X. crassiusculus / Colt Creek / AACCAATAGG
46 / X. crassiusculus / Colt Creek / AATACTTCCG
47 / X. crassiusculus / Colt Creek / TAGTGATGCA
48 / X. crassiusculus / Colt Creek / ATGGAAGGAA
49 / X. crassiusculus / Colt Creek / AAGCTTATCC
50 / X. crassiusculus / Colt Creek / TCGATCAACT
51 / X. crassiusculus / Gainesville / TGTCATTCCT
52 / X. crassiusculus / Gainesville / TGTGGTTATG
53 / X. crassiusculus / Gainesville / AGAACCACAT
54 / X. crassiusculus / Gainesville / TTAAGCCTTG
55 / X. crassiusculus / Gainesville / AATCGAACCT
56 / X. crassiusculus / Gainesville / AATGCACTAC
57 / X. crassiusculus / Gainesville / GAGTTGGATC
58 / X. crassiusculus / Gainesville / GTAAGCGAGA
59 / X. crassiusculus / Gainesville / CTAACGATCG
60 / X. crassiusculus / Gainesville / AATTAAGGCC
61 / X. crassiusculus / Gainesville / GGCATCCTAA
62 / Mock_Raffaelea / Raffaelea reference community / ATTGTGTTCC
63 / X. crassiusculus / Lake Wales / GCTAAGTCAC
64 / X. crassiusculus / Lake Wales / CCTGTTAGCT
65 / X. crassiusculus / Lake Wales / GTGTACCATG
66 / X. crassiusculus / Lake Wales / GGATACAAGC
67 / X. crassiusculus / Lake Wales / AAGTCGTTGT
68 / X. crassiusculus / Lake Wales / ACAACATGGT
69 / X. crassiusculus / Lake Wales / CGTCAAGAAG
70 / X. crassiusculus / Lake Wales / ACCATTGTTG
71 / X. crassiusculus / Lake Wales / AACACGGAAT
72 / X. crassiusculus / Lake Wales / CAAGGTTGGA
73 / X. crassiusculus / Lake Wales / GTTCATGACC
74 / Mock_all / Ophiostomatales & Microascales reference / CTTAGCTGCA
75 / X. crassiusculus / Venus / AACAACAACC
76 / X. ferrugineus / Colt Creek / AATGCTGGTT
77 / X. ferrugineus / Colt Creek / TCCAGGAAGA
78 / X. ferrugineus / Jefferson Ct. / CGGTCATGTT
79 / X. ferrugineus / Jefferson Ct. / AGAGGAAGTG
80 / X. ferrugineus / Jefferson Ct. / GGCGTTACAT
81 / X. ferrugineus / Jefferson Ct. / GCAATTACCG
82 / X. ferrugineus / Jefferson Ct. / AGCTACTGTC
83 / X. ferrugineus / Jefferson Ct. / ATATCCGTGG
84 / X. ferrugineus / Jefferson Ct. / AGAAGTGACC
85 / X. ferrugineus / Jefferson Ct. / CTGGACTCTC
86 / X. ferrugineus / Venus / AGTATAGCGC
87 / X. ferrugineus / Venus / TTGTCTCGAC
88 / X. ferrugineus / Venus / ACACTCCAAG
89 / X. ferrugineus / Venus / GGAAGACCTC
90 / X. ferrugineus / Venus / ACATGGTCCA
91 / X. ferrugineus / Venus / CGGATGAGGA
92 / X. ferrugineus / Venus / TCTCGACGTT
93 / X. ferrugineus / Venus / GGAGCTATGG
94 / X. ferrugineus / Venus / ACAAGAGCAG
95 / X. ferrugineus / Venus / AGCTCAACGA
96 / neg.c. / potential contaminants / ACGTAGCGAT

Supplementary Table 2:Significance of diversity differences between beetle species, assessed by a non-parametric two-sample t-test. The test wasbased on 999 Monte Carlo permutations. Significant difference(α = 0.05) levels are marked with asterisks. All three species have comparable species richness of their mycangial fungal communities (observed species p-value). The Dominance index is significantly higher for X. crassiusculus,suggesting lower evenness in species distribution. Similarly, the Shannon index reflecting species richness,as well as,evennessis also significantly lower for X. crassiusculus.

Species1 / Species2 / Dominance / Chao1 / Shannon / Observed species
ferrugineus / affinis / 0.399 / 0.099 / 1.00 / 0.12
crassiusculus / affinis / 0.003* / 0.498 / 0.01* / 1.00
crassiusculus / ferrugineus / 0.003* / 0.021* / 0.01* / 0.09

Supplementary Table 3: OTUs that statistically contribute to the differentiation of fungal communities between beetle species when analyzed by G-test (based on OTU presence/absence). Significant p-values (α = 0.05) are marked with asterisks. Left columns indicate the number of samples of a beetle species positive for a given OTU (observed, “obs”) and the number of expected observations if the OTU was randomly distributed throughout samples (expected, “exp”). Right columns report data in the same manner except for negative observations (=absences).

OTU / Consensus Lineage / g_value / g_prob / Bonferroni corrected / FDR corrected / X. affinis / X. ferrugineus / X. crassiusculus
[obs, exp] presence / [obs, exp] absence / [obs, exp] presence / [obs, exp] absence / [obs, exp] presence / [obs, exp] absence
New.Ref OTU0 / unknown fungus / 34.1310 / 2.24E-06* / 2.69E-05* / 2.69E-05* / [20, 9.39] / [18, 28.60] / [3, 4.94] / [17, 15.05] / [0, 8.65] / [35, 26.34]
New.Ref OTU1 / family Pichiaceae / 26.0199 / 8.84E-05* / 0.0011* / 0.0005* / [31, 20.83] / [7, 17.16] / [3, 10.96] / [17, 9.03] / [17, 19.19] / [18, 15.80]
JN882303 / Cryptococcus sp / 11.8579 / 0.0368* / 0.4415 / 0.1472 / [26, 31.46] / [12, 6.53] / [17, 16.55] / [3, 3.44] / [34, 28.97] / [1, 6.02]

Supplementary Table 4: Identification details for core members of fungal communities of the three beetle species (identified from persistence/abundance plots). Identification is based on the RDP classifier algorithm searching against the hand-curated UNITE+INSDC database (release August 27, 2013), and on GenBank BLASTn (accessed November 18, 2013). Frequency refers to fraction of samples in which fungus was recovered. Reads count and relative count are reporting absolute number of reads and their relative portions, respectively, for a given fungal species within aparticular beetle species.

Beetle species / OTU code / UNITE+INSDC / RDP classifier confidence / GenBank / E value / Identity / Frequency / Relative count
X. crassiusculus / New.ReferenceOTU2 / Ceratocystis sp CspXger3 / 0.97 / Ceratocystis sp. / 1E-68 / 96% / 1.00 / 0.648
JN882303 / Cryptococcus sp JLH 2011 / 1 / Cryptococcus sp. / 0 / 100% / 0.97 / 0.088
Bandoniozyma glucofermentans / 0 / 100%
GU117066 / Malassezia restricta / 1 / Malassezia restricta / 7E-138 / 100% / 0.91 / 0.017
GU721625 / order Capnodiales / 1 / Cladosporium sp. / 0 / 100% / 0.86 / 0.037
HM211210 / unknown fungus / 0.94 / Fusarium solani / 0 / 100% / 0.71 / 0.016
GU721291 / Epicoccum nigrum / 0.81 / Epicoccum nigrum / 1E-170 / 93% / 0.60 / 0.016
New.ReferenceOTU4 / Tricladium patulum / 0.93 / Xylariales sp. / 5E-73 / 99% / 0.51 / 0.007
New.CleanUpReferenceOTU146 / Ceratocystis sp. / 0.88 / Ceratocystis sp. / 2E-41 / 83% / 0.49 / 0.005
JN693502 / Diplodia corticola / 0.98 / Diplodia corticola / 0 / 100% / 0.46 / 0.006
New.ReferenceOTU1 / family Pichiaceae / 0.81 / Candida sp. / 3E-45 / 85% / 0.46 / 0.003
New.CleanUpReferenceOTU206 / Ceratocystis sp CspXger3 / 0.89 / Ceratocystis sp. / 8E-52 / 89% / 0.43 / 0.001
New.CleanUpReferenceOTU49 / unknown fungus / 1 / Sugiyamaella smithiae / 0.001 / 73% / 0.40 / 0.013
X. ferrugineus / New.ReferenceOTU2 / Ceratocystis sp CspXger3 / 0.97 / Ceratocystis sp. / 1E-68 / 96% / 1.00 / 0.144
GU721625 / order Capnodiales / 1 / Cladosporium sp. / 0 / 100% / 0.90 / 0.157
JN882303 / Cryptococcus sp JLH 2011 / 1 / Cryptococcus sp. / 0 / 100% / 0.85 / 0.169
Bandoniozyma glucofermentans / 0 / 100%
GU117066 / Malassezia restricta / 1 / Malassezia restricta / 7E-138 / 100% / 0.60 / 0.018
GQ850385 / Strelitziana africana / 1 / Strelitziana africana / 0 / 100% / 0.60 / 0.061
New.ReferenceOTU4 / Tricladium patulum / 0.93 / Xylariales sp. / 5E-73 / 99% / 0.55 / 0.030
HM211210 / unknown fungus / 0.94 / Fusarium solani / 0 / 100% / 0.50 / 0.065
X. affinis / GU721625 / order Capnodiales / 1 / Cladosporium sp. / 0 / 100% / 1.00 / 0.249
New.ReferenceOTU2 / Ceratocystis sp CspXger3 / 0.97 / Ceratocystis sp. / 1E-68 / 96% / 0.97 / 0.077
New.ReferenceOTU1 / family Pichiaceae / 0.81 / Candida sp. / 3E-45 / 85% / 0.82 / 0.098
GU117066 / Malassezia restricta / 1 / Malassezia restricta / 7E-138 / 100% / 0.71 / 0.018
JN882303 / Cryptococcus sp JLH 2011 / 1 / Cryptococcus sp. / 0 / 100% / 0.68 / 0.044
Bandoniozyma glucofermentans / 0 / 100%
New.ReferenceOTU4 / Tricladium patulum / 0.93 / Xylariales sp. / 5E-73 / 99% / 0.61 / 0.025
HM211210 / unknown fungus / 0.94 / Fusarium solani / 0 / 100% / 0.58 / 0.023
New.ReferenceOTU0 / unknown fungus / 1 / Candida laemsonensis / 3E-70 / 98% / 0.53 / 0.177
JN693502 / Diplodia corticola / 0.98 / Diplodia corticola / 0 / 100% / 0.45 / 0.016
GU721291 / Epicoccum nigrum / 0.81 / Epicoccum nigrum / 1E-170 / 93% / 0.42 / 0.023
EU568927 / Pichia jadinii / 1 / Pichia jadinii / 0 / 100% / 0.39 / 0.021
FJ381698 / Candida mycetangii / 1 / Candida mycetangii / 0 / 100% / 0.34 / 0.008

Supplementary Table 5:Shapiro-Wilk and Anderson-Darling tests of fit to log-normal distribution of the whole mycangia fungal communities, the core member sets and the transient member sets.

Beetle species community / Goodness-of-fit tests for log-normality
Shapiro-Wilk / Anderson-Darling
Crassiusculus
Whole community / W = 0.9257, p-value < 0.001 / A = 2.4837, p-value < 0.001
Core members / W = 0.9437, p-value = 0.5476 / A = 0.3353, p-value = 0.4451
Transient members / W = 0.9646, p-value < 0.001 / A = 1.3992, p-value < 0.001
Affinis
Whole community / W = 0.9551, p-value < 0.001 / A = 1.634, p-value < 0.001
Core members / W = 0.9266, p-value = 0.3451 / A = 0.4643, p-value = 0.208
Transient members / W = 0.9711, p-value < 0.001 / A = 1.083, p-value < 0.001
Ferrugineus
Whole community / W = 0.9727, p-value = 0.01393 / A = 0.6075, p-value = 0.112
Core members / W = 0.8979, p-value = 0.3183 / N/A
Transient members / W = 0.9632, p-value = 0.003013 / A = 1.0924, p-value = 0.007

Supplementary Table 6: Reference cultures. Identification of Ambrosiella and Raffaelea morphotypes was confirmed using LSU rDNA sequencing and GenBank BLAST.

source species / morphotype / genus / Avg of CFU proportion / proportion within spp
affinis / ambrosia_morhp_tough / Raffaelea / 26.4 / 0.085904876
affinis / ambrosia_morph_mycelium / Raffaelea / 10 / 0.032539726
affinis / ambrosia_morph_spikey / Raffaelea / 53.75 / 0.174901025
affinis / ambrosia_morph_yeast / Raffaelea / 49.16666667 / 0.159986984
affinis / Ambrosiella / Ambrosiella / 20 / 0.065079451
affinis / white_fluff / other / 10 / 0.032539726
affinis / white_myce / other / 3 / 0.009761918
affinis / yeast_matt / yeast / 50 / 0.162698628
affinis / yeast_smooth / yeast / 60 / 0.195238353
affinis / yeast_w_slimy / yeast / 25 / 0.081349314
crassiusculus / fun_grey_fluff / Ambrosiella / 100 / 0.454545455
crassiusculus / yeast / yeast / 100 / 0.454545455
crassiusculus / yeast_red_appressed / yeast / 20 / 0.090909091
ferrugineus / ambrosia_morhp_tough / Raffaelea / 10 / 0.047619048
ferrugineus / ambrosia_morph_spikey / Raffaelea / 20 / 0.095238095
ferrugineus / ambrosia_morph_yeast / Raffaelea / 20 / 0.095238095
ferrugineus / white_fluff / other / 50 / 0.238095238
ferrugineus / yeast_beige / yeast / 10 / 0.047619048
ferrugineus / yeast_w_matt / yeast / 50 / 0.238095238
ferrugineus / yeast_w_smooth / yeast / 50 / 0.238095238

Supplementary Table 7: Mean morphotype frequencies in reference cultures.

Beetle species / Morphotype frequency, mean
Raffaelea / Ambrosiella / yeast / other
Xyleborus affinis / 0.453 / 0.065 / 0.439 / 0.042
Xyleborus ferrugineus / 0.238 / 0 / 0.524 / 0
Xylosandrus crassiusculus / 0 / 0.455 / 0.545 / 0.238

Supplementary Table8: OTUs that significantly contribute to the differentiation of fungal communities between beetle species when analyzed by ANOVA based on read abundances. ANOVA was performed on 100 iterations of de-novo rarefied OTU tables. Identification is a consensus of BLASTn, RDP classifier and reference sequence communities. Values in the beetle species columns represent averaged relative abundances of a given OTU.

OTU / ANOVA prob. / FDR corrected / X. affinis / X. crassiusculus / X. ferrugineus / Consensus identification
New.Reference OTU2 / 2.27E-41 / <0.001* / 0.0293 / 0.6375 / 0.0839 / Ambrosiella sp.
GU721625 / 1.23E-09 / <0.001* / 0.2065 / 0.0321 / 0.1026 / Cladosporium sp.
JN882303 / 6.40E-08 / <0.001* / 0.0407 / 0.0808 / 0.1651 / Cryptococcus sp./ Bandoniozyma glucofermentans
New.Reference OTU0 / 1.21E-07 / <0.001* / 0.1543 / 0.0000 / 0.0034 / Candida laemsonensis
New.Reference OTU1 / 4.71E-06 / <0.001* / 0.1090 / 0.0032 / 0.0041 / family Pichiaceae
JQ990198 / 6.24E-05 / <0.001* / 0.0262 / 0.0003 / 0.0144 / Cladosporium sp.
New.CleanUp.Reference OTU680 / 6.35E-05 / <0.001* / 0.0003 / 0.0056 / 0.0010 / Microascales/Ophiostomatales
HQ538467 / 3.08E-04 / <0.001* / 0.0461 / 0.0069 / 0.0642 / Raffaelea sp.
New.ReferenceOTU4 / 0.0131 / 0.02* / 0.0251 / 0.0060 / 0.0299 / Xylariales sp.

Supplementary Figure 1: Individual-based rarefaction curves of fungal OTUs for all three beetle species indicate sufficient sequencing coverage. OTUs are defined as sequence clusters with 97% similarity. Vertical lines indicate the average number of fungal species per beetle species and ± SD. Red – Xyleborus affinis, orange - X. ferrugineus, blue – Xylosandrus crassiusculus.