599 Supplementary material

28 (A) Jaccard (B) Bray Curtis (O) 2 2 1 0 0 NMDS 2 NMDS 2 −1 −2 −2 −4 −3

−4 −2 0 2 4 −3 −2 −1 0 1 2 3 (SS) NMDS 1 NMDS1 0.4 2 0.2 1 Seawater 0 0.0 Adults NMDS 2 NMDS 2 −1 Larvae −0.2 −2 −0.4 −3

−0.6 −0.4 −0.2 0.0 0.2 0.4 −3 −2 −1 0 1 2 3 (PVT) NMDS 1 NMDS 1 6 2 4 1 2 0 0 NMDS 2 NMDS 2 −2 −1 −4 −6 −2

−4 −3 −2 −1 0 −10 −5 0 NMDS 1 NMDS 1

Figure S1: Non metric multidimensional (NMDS) scaling for Jaccard (left hand side) and Bray Curtis (right hand side) distances calculated on the top level of each bipartite network (i.e., the microbial communities). As expected, the microbial communities (i.e., the top level in each network) across host species increased in community similarity with increasing host-microbe specificity (Figure S1) .

29 B

PARENT 3 PARENT 2 PARENT 1 I.fasciculata A

PARENT 3 PARENT 2 PARENT 1 I.oros ADULTS ADULTS NO SIBLINGS OVERALL OVERALL SPONGE-SPECIFIC SPONGE-SPECIFIC iueS:Sefiuelgn below. legend figure See S2: Figure NO OFFSPRING NO OFFSPRING POTENTIALLY VERTICALLY POTENTIALLY VERTICALLY TRANSMITTED TRANSMITTED 30 TRANSMITTED VERTICALLY TRANSMITTED VERTICALLY P h P ylum h Gammaproteobacte Fi Epsilonproteobacte Deltaproteobacte C Chlorofl Chla Betaproteobacte ARKDMS−49 Alphaproteobacte AEGEAN−245 Actinobacte Acidobacte ylum y r Gammaproteobacte Fusobacte Fi Epsilonproteobacte El Deltaproteobacte −The C Chlorofl Chla Betaproteobacte Bacteroidetes ARKDMS−49 Aminicenantes Alphaproteobacte Actinobacte Acidobacte micutes anobacte y r e micutes anobacte m v−16S−509 m ydiae ydiae e e xi xi r r r r ia ia r r ia r ia ia ia ia r r r ia r ia r ia r r ia ia m ia r r r ia r ia us ia ia Unclassified V TM6_(Dependentiae) T Spirochaetae SBR1093 Proteobacte Proteobacte Saccha P Plancto P Nitrospi Nitrospinae Ma Latescibacte Ign Gemmatimonadetes ectomicrobia A er o Proteobacte Proteobacte Unclassified V T SP SBR1093 P Plancto pItb−vmat−80 P Nitrospi Nitrospinae Ma Lentisphae UC34f r r a r ibacte ectomicrobia A er o inimicrobia_(SAR406_clade) ucomicrobia vibacte UC34f r O r r ibacte inimicrobia_(SAR406_clade) ucomicrobia r m TSOCT00m83 r ibacte ae ycetes r ia m r r ae r r iae r ia_Ince ia ycetes r ia ia r r ia ae r r ia_Ince ia r tae_Sedis r tae_Sedis C C. crambe POTENTIALLY VERTICALLY VERTICALLY ADULTS OVERALL SPONGE-SPECIFIC TRANSMITTED TRANSMITTED

Phylum Lentisphaerae Marinimicrobia_(SAR406_clade) AEGEAN−245 Nitrospinae PARENT 1 PARENT ARKDMS−49 Omnitrophica Bacteroidetes Parcubacteria PAUC34f

NO SIBLINGS Deferribacteres Proteobacteria_Incertae_Sedis PARENT 2 PARENT Elev−16S−509 SBR1093 Spirochaetae Fibrobacteres SPOTSOCT00m83 Tectomicrobia TM6_(Dependentiae) Gemmatimonadetes Unclassified

PARENT 3 PARENT

D C. viridis POTENTIALLY VERTICALLY VERTICALLY ADULTS OVERALL SPONGE-SPECIFIC TRANSMITTED TRANSMITTED

Phylum Acidobacteria Latescibacteria

PARENT 1 PARENT Actinobacteria Marinimicrobia_(SAR406_clade) Alphaproteobacteria Nitrospinae ARKDMS−49 Nitrospirae Bacteroidetes Parcubacteria Betaproteobacteria PAUC34f Chlamydiae Planctomycetes Chloroflexi Poribacteria Cyanobacteria Proteobacteria

PARENT 2 PARENT Deinococcus−Thermus Proteobacteria_Incertae_Sedis Deltaproteobacteria SBR1093 Elev−16S−509 Spirochaetae Epsilonproteobacteria SPOTSOCT00m83 Firmicutes Tectomicrobia Fusobacteria TM6_(Dependentiae) Gammaproteobacteria Verrucomicrobia Gemmatimonadetes Unclassified

PARENT 3 PARENT Hydrogenedentes

Figure S2: See figure legend below.

31 E D. avara POTENTIALLY VERTICALLY VERTICALLY ADULTS OVERALL SPONGE-SPECIFIC TRANSMITTED TRANSMITTED

Phylum Acidobacteria Latescibacteria Actinobacteria Lentisphaerae AEGEAN−245 Marinimicrobia_(SAR406_clade) PARENT 1 PARENT Alphaproteobacteria Nitrospinae Aminicenantes Nitrospirae ARKDMS−49 Omnitrophica Bacteroidetes Parcubacteria Betaproteobacteria PAUC34f Chlamydiae Peregrinibacteria Chloroflexi Planctomycetes Cyanobacteria Poribacteria PARENT 2 PARENT Deferribacteres Proteobacteria Deltaproteobacteria Proteobacteria_Incertae_Sedis Elev−16S−509 Saccharibacteria Epsilonproteobacteria SBR1093 Fibrobacteres Spirochaetae Firmicutes SPOTSOCT00m83 Fusobacteria Tectomicrobia Gammaproteobacteria

PARENT 3 PARENT Gemmatimonadetes Verrucomicrobia Gracilibacteria Unclassified

F H. columella POTENTIALLY VERTICALLY VERTICALLY ADULTS OVERALL SPONGE-SPECIFIC TRANSMITTED TRANSMITTED

Phylum Acidobacteria Marinimicrobia_(SAR406_clade) Actinobacteria Nitrospinae PARENT 1 PARENT AEGEAN−245 Nitrospirae Alphaproteobacteria Omnitrophica ARKDMS−49 Parcubacteria Bacteroidetes PAUC34f Betaproteobacteria Planctomycetes Chlamydiae Poribacteria

NO SIBLINGS Chloroflexi Proteobacteria Cyanobacteria Proteobacteria_Incertae_Sedis PARENT 2 PARENT Deferribacteres SBR1093 Deltaproteobacteria Spirochaetae Elev−16S−509 SPOTSOCT00m83 Epsilonproteobacteria Tectomicrobia Firmicutes Tenericutes Fusobacteria TM6_(Dependentiae) Gammaproteobacteria Verrucomicrobia NO OFFSPRING Gemmatimonadetes Unclassified PARENT 3 PARENT Lentisphaerae

Figure S2: See figure legend below.

32 G O. lobularis POTENTIALLY VERTICALLY VERTICALLY ADULTS OVERALL SPONGE-SPECIFIC TRANSMITTED TRANSMITTED

Phylum Acidobacteria Lentisphaerae Actinobacteria Marinimicrobia_(SAR406_clade)

PARENT 1 PARENT AEGEAN−245 Nitrospinae Alphaproteobacteria Nitrospirae ARKDMS−49 PAUC34f Bacteroidetes Peregrinibacteria Betaproteobacteria Planctomycetes Chlamydiae Poribacteria Chloroflexi Proteobacteria Cyanobacteria Proteobacteria_Incertae_Sedis Deferribacteres SBR1093 PARENT 2 PARENT Deltaproteobacteria Spirochaetae Elev−16S−509 SPOTSOCT00m83 Epsilonproteobacteria Tectomicrobia Firmicutes Tenericutes Fusobacteria TM6_(Dependentiae) Gammaproteobacteria Verrucomicrobia Gemmatimonadetes Unclassified Hydrogenedentes PARENT 3 PARENT

Figure S2: Donut charts showing the relative contribution of ASVs classifying to different microbial phyla (classes for Proteobacteria) in sponge parents and their offspring across the different networks depicted in Figure 2. The left-hand column of donuts corresponds to the three adult specimens, while the remaining donuts depict microbial communities in their offspring across the different networks. For the adult donut plots, the inner and outer donuts represent the overall and sponge-specific network, respectively. For the offspring donut plots, concentric donuts correspond to different offspring from the same parent (i.e. siblings). White donuts with a solid outline indicate a community where all the ASVs were unclassified. White donut with a dashed outline indicate a community where the focal offspring did not contain any ASVs found in the focal network. Colors represent different microbial phyla (classes for Proteobacteria). Plot A-G corresponds to host species: I. oros; I. fasciculata; C. crambe; C. viridis; D. avara; H. columella; and O. lobularis.

33 ADULT 1 ADULT 2 ADULT 3

O SS A. aerophoba PVT

O SEC

O SS NO OFFSPRING I. oros PVT

O SEC

O SS NO OFFSPRING I. fasciculata PVT

O SEC

O SS C. crambe PVT

O SEC

O SS C. viridis PVT

O SEC

O SS D. avara PVT

O SEC

O SS H. columella NO OFFSPRING PVT

O SEC

O O. lobularis SS PVT

O SEC 0 25 50 75 100 0 25 50 75 100 0 25 50 75 100

Proportion of vertically transmitted ASV

parent-offspring non-parent adults-larvae

Figure S3: The average proportion of vertically transmitted ASVs shared between sponge larvae and either (i) their known parents (orange bars), or (ii) two non-parental conspecific adults (green bars) for each sponge species and network. The three columns represent the three adults from each sponge species. The abbreviations O, SS, and PVT on the Y-axis correspond to the overall, sponge-specific and potentially vertically transmitted network, respectively. Finally, the abbreviation O SEC corresponds to the overall network but for sponge-enriched clusters. Note that in 12 sets of plots, offspring shared more ASVs with their parents, in 9 sets of plots, offspring shared more ASVs with nearby conspecifics who were not their parents (3 plots show no sharing with either parents or conspecific adults).

34 C. crambe

A B C Logit transformed proportions Logit transformed proportions Logit transformed proportions 35

Adults-larvae n=16 Parent-ofspring Non-parent Non-parent n=8 Non-parent adults-larvae adults-larvae adults-larvae n=16 Parent-ofspring n=16 Parent-ofspring n=16 Parent-ofspring n=8 n=8 n=8 (A) C. crambe

Figure S4: Gardner-Altman comparison plots of the logit transformed proportions of vertically transmitted ASVs shared between sponge larvae and either (i) their known parents (dark gray dots), or (ii) two non-parental conspecific adults (light gray dots) for the sponge species C. crambe that showed significant signatures of vertical transmission between parents and offspring. As in Figure 4, each dot represents one comparison; either a larva and its parent or a larva and one of the non-parental conspecific adults. Plots A-C correspond to the overall, sponge-specific, and potentially vertically transmitted network. The axis on the right-hand side shows the mean difference between groups (the maximum value of the mean difference distribution), and the narrowness of the confidence interval reflects the precision of the effect size. 36 itiuin,adtenrons ftecndneitra ie la mrsino fetsz precision. the size effect to of a correspond impression either A-C clear comparison; Plots a gives one interval adults. represents confidence conspecific dot the non-parental each of narrowness the 4, species the of Figure sponge and one two distribution), in the and As for larva dots) offspring. a and transmitted gray and larvae or vertically (light parents sponge parent adults between between conspecific its shared non-parental transmission and two ASVs vertical larva (ii) transmitted of or vertically signatures dots), of gray significant proportions (dark showed transformed parents logit known the their of (i) either plots comparison Gardner-Altman S4: Figure

Logit transformed proportions O. lobularis A

Non-parent adults-larvae n=16 ewr.Teai ntergthn iesostema ifrnebtengop temxmmvleo h endifference mean the of value maximum (the groups between difference mean the shows side right-hand the on axis The network. Parent-ofspring n=8

Logit transformed proportions B

Non-parent adults-larvae n=16 (B) .lobularis O.

Parent-ofspring n=8

Logit transformed proportions overall C

Non-parent adults-larvae n=16 , sponge-specific

Parent-ofspring n=8 and , lobularis O potentially that A

B ∆

Overall Potentially

Sponge-specific vertically transmitted

Figure S5: Gardner-Altman comparison plots for Normalized Mutual Information (NMI) across the three different networks (blue, overall network; green, sponge-specific network; and red, potentially vertically transmitted network). The top panel (A) shows the mean std for each network (the gray lines represent the std while the gap corresponds to the mean). Dots represent each sponge± species. The lower panel (B) shows the difference in NMI scores relative to the prior expectation of perfect parent-offspring modules (i.e., NMI=1).

37 A. aerophoba I. oros C. viridis D. avara 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.9 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.150.17 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.8

0.53 0 0 0.01 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.01 0 0 0 0.7 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.6

0.5 I. faciculata C. crambe H. columella O. lobularis

0 0 0 0 0 0 0 0 0 0 0 0 0 0.045 0.61 0 0.4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.3 Jaccard index (binary similarity) 0 0 0 0 0 0 0 0 0 0.2 0 0 0 0 0.05 0 0 0.1

0

Figure S6: Siblings almost never inherit the same vertically transmitted taxa assigning to sponge-enriched clusters as show by pairwise Jaccard coefficients calculated for assemblages of vertically transmitted ASVs between larvae that shared the same parent. Each cell represents a larva and sets of siblings from the same parent are indicated by cells bordered by the same color (green, purple, or red). In cases where parents only had one offspring, the diagonal is bordered by a dashed line. Cells with gray boarders correspond to Jaccard coefficients calculated for assemblages of vertically transmitted ASVs assigning to sponge-enriched clusters between conspecific larvae that did not share the same parent. Gray cells represent the comparison with self. The Jaccard index ranges between 0 (no ASVs shared) and 1 (all ASVs shared)

38 Figure S7: Sketch of the constructed traps that were used to capture dispersing larvae from adult sponges.

39 Table S1: Shows the number of sequence reads, and the number ASVs those reads assigned to for the overall and sponge-specific network. Each row corresponds to an adult sponge.

reads ASV s ASV s Sponge species N NO NSS 144148 1581 588 Aplysina aerophoba 194322 1539 668 150522 1334 581 283401 2153 906 Ircinia oros 287524 1981 910 299987 2217 867 89896 1027 356 Ircinia fasciculata 182657 1319 439 256141 1557 563 48893 1130 200 Crambe crambe 180038 1099 149 190642 810 104 176885 490 70 Cliona viridis 147933 654 103 140974 748 86 70402 1512 164 Dysidea avara 127357 1921 224 4232 324 40 536732 2247 337 Hemimycale columella 114731 879 114 1797 270 34 149832 1333 248 Oscarella lobularis 330103 1779 293 521058 2558 489

40 Table S2: Shows the number of sequence reads, and the number ASVs those reads assigned to for the different networks, including the subset of vertically transmitted microbes. Each row corresponds to a larval offspring.

reads ASV s ASV s ASV s ASV s Sponge species Source adult N NO NSS NPVT NVT 5227 68 9 3 1 Adult 1 1849 59 5 1 1

27846 206 35 9 5

Adult 2 3058 52 1 0 0

169456 954 198 114 63 A. aerophoba 439760 1456 480 314 159

267481 1337 438 304 175

Adult 3 18199 154 20 5 1

5801 78 6 2 0

29836 161 16 6 2

10397 85 8 3 1

11260 77 6 4 3

Adult 1 221306 790 125 40 23

17141 185 30 13 5 I. oros 32123 211 28 10 3

55825 299 46 21 12

Adult 2 16737 82 7 2 1

15971 145 17 9 6

6008 103 9 5 1 Adult 1 I. fasciculata 6899 87 12 5 2

Adult 2 5479 62 10 2 0

5272 114 8 2 1

2709 45 6 5 1 Adult 1 17264 186 26 11 4

C. crambe Continued on next page

41 Table S2 – Continued from previous page

reads ASV s ASV s ASV s ASV s Sponge species Source adult N NO NSS NPVT NVT 2976 134 7 1 1

Adult 2 3054 97 8 1 1

18918 220 32 12 1 Adult 3 54956 419 59 18 2

88167 284 40 11 2

2083 126 12 4 0 Adult 1 6669 96 7 3 0

6631 113 12 3 0

193439 443 53 19 5 C. viridis Adult 2 77960 315 55 10 2

2961 145 14 4 1

42482 129 12 2 1 Adult 3 3517 61 8 2 0

5187 90 14 4 0

12651 66 11 1 0

Adult 1 30050 244 36 16 1

8630 67 4 1 0

78829 307 25 13 7

49906 234 34 10 4 Adult 2 44314 235 19 6 2 D. avara 16470 225 38 8 1

37901 229 24 12 1

9215 113 13 4 1

Adult 3 27264 228 21 6 0

6937 91 13 7 0

Continued on next page

42 Table S2 – Continued from previous page

reads ASV s ASV s ASV s ASV s Sponge species Source adult N NO NSS NPVT NVT 372660 1111 258 68 3

459628 2245 769 326 72

71813 316 48 14 9 Adult 1 H. columella 20188 139 17 5 2

349906 1468 519 267 45

Adult 2 9300 74 22 5 0

1218 79 6 4 1 Adult 1 6691 78 11 4 3

2872 79 5 4 0 Adult 2 110912 537 64 27 12 O. lobularis 437458 1798 629 297 75

1116 52 8 4 1 Adult 3 2948 59 9 3 1

26825 151 13 4 1

600

43 Table S3: Weighted modularity and NMI across host species and networks

Sponge species Network Number of modules Normalized modularity NMI O 6 0.558 0.433 A. aerophoba SS 9 0.778 0.529 PVT 5 0.690 0.361 O 8 0.978 0.427 I. oros SS 7 0.995 0.447 PVT 4 0.997 0.304 O 4 0.246 0.615 I. fasciculata SS 5 0.559 0.607 PVT 2 0.995 0.106 O 7 0.536 0.489 C. crambe SS 8 0.981 0.493 PVT 3 0.995 0.237 O 8 0.948 0.349 C. viridis SS 8 0.985 0.406 PVT 5 0.577 0.233 O 7 0.907 0.191 D. avara SS 8 0.973 0.306 PVT 2 0.944 0.0521 O 4 0.992 0.284 H. columella SS 7 0.876 0.518 PVT 6 0.863 0.552 O 6 0.826 0.323 O. lobularis SS 8 0.549 0.465 PVT 8 0.438 0.493

Table S4: Weighted (normalized) modularity (Qnorm) and NMI across networks

Network Number of modules Normalized modularity NMI O 21 0.906 0.500 SS 22 0.894 0.396 PVT 18 0.865 0.390

44 Table S5: For each species and network: the top left cell corresponds to the number of unique sponge-enriched clusters found; the top right cell corresponds to the number of ASVs those clusters assigned to; the bottom left cell corresponds to contribution (%) of those sponge-enriched clusters to total ASV richeness; and the bottom right cell corresponds to the contribution (%) of those clusters to the total sequence count.

Potentially vertically Sponge species Overall Sponge-specific Vertically transmitted transmitted 85 809 67 480 44 181 38 122 A. aerophoba 20.564 50.44 31.979 43.723 42.191 62.851 47.843 62.245 83 836 63 508 17 22 14 18 I. oros 20.232 39.27 31.553 27.887 28.571 24.356 36.735 33.516 72 492 47 248 2211 I. fasciculata 19.984 32.519 31.353 38.676 28.571 30.993 33.333 88.876 72 299 35 70 4511 C. crambe 11.262 59.398 14.17 17.335 27.778 0.514 10 0.13 65 257 31 53 4523 C. viridis 12.561 15.053 13.66 1.731 26.316 6.977 33.333 12.903 77 327 35 84 6745 D. avara 12.561 3.258 12.227 0.238 15.556 0.658 25 1.701 84 718 63 386 22 36 18 29 H. columella 15.801 52.262 28.382 29.664 30.508 32.02 33.333 35.277 81 601 61 286 23 38 21 32 O. lobularis 15.801 12.939 22.68 8.72 31.405 8.709 35.165 7.019 94 507 Seawater 5.172 0.23

Table S6: The percentage of unique clusters found in each host species and network from the total number of unique clusters identified in the focal network.

Sponge species Overall Sponge-specific Potentially vertically Vertically transmitted (n=105) (n=77) transmitted (n=51) (n=47) A. aerophoba 80.952 87.013 86.275 80.851 I. oros 79.048 81.818 33.333 29.787 I. fasciculata 68.571 61.039 3.922 2.128 C. crambe 68.571 45.455 7.843 2.128 C. viridis 61.905 40.260 7.843 4.255 D. avara 73.333 45.455 11.765 8.511 H. columella 80.000 81.818 43.137 38.298 O. lobularis 77.143 79.221 45.098 44.681 Seawater 89.524

45