1 Supplementary Material

2 Genomic and phylogenomic insights into the family lead

3 to proposal of Charcoactinosporaceae fam. nov. and 8 novel genera with

4 emended descriptions of calvus

5 Munusamy Madhaiyan1, †, *, Venkatakrishnan Sivaraj Saravanan2, †, Wah-Seng See-Too3, †

6 1Temasek Life Sciences Laboratory, 1 Research Link, National University of Singapore,

7 Singapore 117604; 2Department of Microbiology, Indira Gandhi College of Arts and Science,

8 Kathirkamam 605009, Pondicherry, India; 3Division of Genetics and Molecular Biology,

9 Institute of Biological Sciences, Faculty of Science, University of Malaya, Kuala Lumpur,

10 Malaysia

1

11 Table S3. List of the core genes in the genome used for phylogenomic analysis.

NCBI Protein Accession Gene WP_074993204.1 NUDIX hydrolase WP_070028582.1 YggS family pyridoxal phosphate-dependent enzyme WP_074992763.1 ParB/RepB/Spo0J family partition protein WP_070022023.1 lipoyl(octanoyl) transferase LipB WP_070025151.1 FABP family protein WP_070027039.1 heat-inducible transcriptional repressor HrcA WP_074992865.1 folate-binding protein YgfZ WP_074992658.1 recombination protein RecR WP_074991826.1 HIT domain-containing protein WP_070024163.1 adenylosuccinate synthase WP_009190566.1 anti-sigma regulatory factor WP_071828679.1 preprotein translocase subunit SecG WP_070026304.1 50S ribosomal protein L13 WP_009190144.1 30S ribosomal protein S5 WP_014674378.1 30S ribosomal protein S8 WP_070026314.1 50S ribosomal protein L5 WP_009300593.1 30S ribosomal protein S13 WP_003998809.1 50S ribosomal protein L36 WP_070026864.1 WhiB family transcriptional regulator WP_023550764.1 50S ribosomal protein L27 WP_070027822.1 ATP synthase F0 subcomplex C subunit WP_039656223.1 30S ribosomal protein S17 WP_070024148.1 DUF5063 domain-containing protein WP_009190136.1 50S ribosomal protein L16 WP_070024021.1 MoaD/ThiS family protein WP_070028831.1 30S ribosomal protein S6 WP_003956500.1 50S ribosomal protein L34 WP_070025179.1 glycine--tRNA ligase WP_018544333.1 DUF3117 domain-containing protein WP_003974250.1 50S ribosomal protein L30 WP_070022292.1 30S ribosomal protein S16 WP_070023573.1 2-phospho-L-lactate transferase WP_074993184.1 PHP domain-containing protein

2

12 Table S4. Similarity in polyphasic data observed between S. asterosporus DSM 41452T and S.

13 calvus CECT 3271T

Streptomyces asterosporus Characteristic† DSM 41452T CECT 3271T Morphological characters: Spore color Gray Gray Spore chain morphology Spira Spira Melanoid pigment production − − Spore wall ornamentation Spiny Hairy Physiological tests: D-Glucose + + D-Xylose − + L-Arbinose + + D-Rhamnose + + L-Rhamnose + + D-Galactose + + Raffinose − + D-Mannitol + + i-Inositol n + Salicin n + Sucrose n + G+C content (mol%) 72.5 72.5 Genome size (Mbp) 7.77 7.94 14 +, positive; −, negative; n-not determined. 15 †Data from Kämpfer et al. (2014).

16

3

Streptomyces lincolnensis NRRL 2936T (CP016438) Streptomyces caeruleatus NRRL B-24802T (KQ948975) DSM 40281T (LIQS01000280) Streptomyces acidiscabies NRRL B-16524T (D63865) Streptomyces curacoi DSM 40107T (KQ948008) Streptomyces humi MUSC 119T (LBMU02000042) Streptomyces hyaluromycini NBRC 110483T (BCFL01000051) NRRL B-2286T (AB184155) 100 Streptomyces griseofuscus NRRL B-5429T (AB184206) Streptomyces corchorusii DSM 40340T (KQ948396) DSM 41781T (KQ948892) 73 Streptomyces longwoodensis DSM 41677T (KQ948572) Streptomyces griseochromogenes ATCC 14511T (CP016279) Streptomyces cellostaticus DSM 40189T (KQ948097) Streptomyces achromogenes subsp. achromogenes NRRL B-2120T (AB184109) Streptomyces durhamensis NRRL B-3309T (JNXR01000068) 93 Streptomyces puniciscabiei NRRL B-24456T (AF361785) MA-4680T (BA000030) Streptomyces atriruber NRRL B-24165T (LIPN01000196) Streptomyces davaonensis JCM 4913T (HE971709) Streptomyces cinnabarinus NRRL B-12382T (JNXQ01000585) Streptomyces yokosukanensis DSM 40224T (KQ948269) 92 Streptomyces hokutonensis R1-NS-10T (BARG01000157) NBRC 13075T (BARF01000082) Streptomyces aureocirculatus NRRL ISP-5386T (JOAP01000131) Streptomyces bobili NRRL B-1338T (MUBA01000087) Streptomyces phaeoluteigriseus DSM 41896T (MPOH01000466) Streptomyces populi A249T (MF100127) Streptomyces resistomycificus DSM 40133T (JOBA01000220) DSM 40017T (KQ948708) Streptomyces pseudovenezuelae DSM 40212T (KQ948163) NRRL B-1832T (LIQN01000245) 98 NRRL B-3811T (AB184165) NRRL B-24310T (JNXP01000045) Streptomyces olivochromogenes DSM 40451T (KQ948511) 84 NRRL B-2808T (AB249976) Streptomyces kanamyceticus NRRL B-2535T (AB184388) NRRL ISP-5412T (AB184259) 67 Streptomyces phaeochromogenes NRRL B-1248T (AB184738) Streptomyces bottropensis ATCC 25435T (KB911581) 90 Streptomyces stelliscabiei NRRL B-24447T (LBNW01000338) Streptomyces capitiformicae 1H-SSA4T (KX777629) Streptomyces graminilatus NRRL B-59124T (LIQQ01000213) Streptomyces dengpaensis XZHG99T (CP026652) Streptomyces rishiriensis NBRC 13407T (AB184383) Streptomyces reticuliscabiei NRRL B-24446T (MUNI01000325) 76 Streptomyces geranii A301T (MF100124) 99 Streptomyces flavovariabilis NRRL B-16367T (JNXD01000071) Streptomyces variegatus NRRL B-16380T (JYJH01000110) Streptomyces griseorubiginosus DSM 40469T (KQ948757) Streptomyces yerevanensis NRRL B-16943T (JNWJ01000157) Streptomyces kebangsaanensis SUK12T (HM449824) Streptomyces hirsutus NRRL B-2713T (LIQT01000254) 100 Streptomyces prasinus NRRL B-2712T (LIRH01000162) Streptomyces indicus CGMCC 4.5727T (EF157833) 99 Streptomyces amritsarensis MTCC 11845T (MQUR01000179) Streptomyces yangpuensis fd2-tbT (LBMK01000002) 91 NRRL ISP-5094T (JOAK01000082) Streptomyces xanthophaeus NRRL B-5414T (JOFT01000080) subsp. lavendulae NRRL B-2774T (JOEW01000098) 86 Streptomyces vietnamensis GIM4.0001T (DQ311081) NRRL B-1049T (JNWL01000107) ATCC 31255T (AP017424) ATCC 10712T (FR845719) NRRL B-2924T (JNZP01000081) NRRL 3426T (JNWK01000124) Streptomyces purpureus NBRC 13927T (AB184547) Streptomyces flavidovirens DSM 40150T (AB184270) Streptomyces kanasensis ZX01T (JN572690) Streptomyces lunaelactis MM109T (KM207217) NRRL ISP-5083T (AB184163) ATCC 15855T (AB184439) 74 99 Streptomyces mutomycini NRRL B-65393T (MAPV01000102) Streptomyces atroolivaceus NRRL ISP-5137T (JNXG01000049) 75 NRRL ISP-5068T (AB184142) Streptomyces cyaneofuscatus NRRL B-2570T (JOEM01000050) subsp. griseus DSM 40236T (M76388) Streptomyces fulvissimus DSM 40593T (CP005080) Streptomyces luridiscabiei NRRL B-24455T (LIQV01000394) Streptomyces setonii NRRL ISP-5322T (MUNB01000146) papulosa NRRL B-16504T (JNYQ01000038) Streptomyces niveus NCIMB 11891T (DQ442532) Streptomyces clavuligerus ATCC 27064T (CM000913) 76 Streptomyces tsukubensis NRRL18488T (AJSZ01000237) Streptomyces alni CGMCC 4.3510T (DQ460470) 99 Streptomyces lushanensis NRRL B-24994T (MAUD01000416) Streptomyces odonnellii NRRL B-24891T (LATD01000204) Streptomyces scopuliridis NRRL B-24574T (JOEI01000056) Streptomyces eurocidicus ATCC 27428T (AY999790) 80 Streptacidiphilus jiangxiensis NBRC 100920T (BBPN01000092) Streptacidiphilus anmyonensis NBRC 103185T (BBPQ01000139) 74 Streptacidiphilus melanogenes NBRC 103184T (BBPP01000099) 84 Streptacidiphilus rugosus AM-16T (JQMJ01000004) 74 Streptacidiphilus pinicola MMS16-CNU450T (MG189375) Streptacidiphilus carbonis DSM 41754T (AF074412) Streptacidiphilus T 87 Streptacidiphilus albus NBRC 100918 (BBPL01000138) Streptacidiphilus neutrinimicus DSM 41755T (AF074410) 97 Streptacidiphilus bronchialis DSM 106435T (CP031264) 70 Streptacidiphilus griseoplanus NRRL B-3064T (LIQR01000258) Streptacidiphilus oryzae TH49T (JQMQ01000005) 95 Kitasatospora cineracea DSM 44780T (RJVJ01000002) 88 Kitasatospora niigatensis DSM 44781T (RKQG01000001) Kitasatospora setae KM-6054T (AP010968) 96 Kitasatospora cheerisanensis KCTC 2395T (KK853997) Kitasatospora phosalacinea NRRL B-16230T (JNWZ01000148) 87 Kitasatospora viridis DSM 44826T (VIWT01000001) Kitasatospora Kitasatospora aureofaciens ATCC 10762T (JPRF02000033) Kitasatospora atroaurantiaca NRRL B-24282T (DQ026645) Streptomyces novaecaesareae NRRL B-1267T (JNWQ01000181) Kitasatospora azatica KCTC 9699T (JQMO01000002) 98 Kitasatospora purpeofusca NRRL B-1817T (JODS01000077) Streptomyces inhibens NEAU-D10T (QUAC01000041) 95 BCRC 12349T (NDXL01000004) Streptomyces celluloflavus NRRL B-2493T (JOEL01000102) Streptomyces decoyicus NRRL 2666T (LGUU01000106) DSM 40041T (AB045882) Streptomyces monomycini NRRL B-24309T (JNYL01000005) 76 NRRL B-3857T (AB184352) Streptomyces ochraceiscleroticus NRRL ISP-5594T (JOAX01000062) 92 Streptomyces violens NRRL ISP-5597T (JOBH01000039) subsp. rimosus NRRL ISP-5260T (ANSJ01000404) Streptomyces sclerotialus NRRL ISP-5269T (JOBC01000056) Streptomyces albofaciens JCM 4342T (AB045880) 77 Streptomyces chrestomyceticus NBRC 13444T (BHZC01000001) Streptomyces chattanoogensis NRRL ISP-5002T (LGKG01000206) ATCC 11455T (CP011533) NRRL ISP-5461T (RDTD01000009) Streptomyces angustmyceticus NRRL B-2347T (MUAY01000275) Streptomyces catenulae NRRL B-2342T (JODY01000075) 97 Streptomyces viridosporus NRRL 2414T (MSGP01000087) Streptomyces triticisoli NEAU-DSCPA1-4-4T (MF993053) Streptomyces thermovulgaris NRRL B-12375T (MUME01000064) Streptomyces gilvigriseus MUSC 26T (KJ632660) DSM 40063T (MIFZ01000280) Streptomyces speibonae NRRL B-24240T (JNXM01000266) Streptomyces iakyrus NRRL ISP-5482T (JNXI01000062) NRRL B-2373T (JNXT01000131) NBRC 13193T (AB184326) 94 NRRL B-3009T (JOFH01000101) Streptomyces leeuwenhoekii C34T (LN831790) MUSC 135T (CP021080) Streptomyces cadmiisoli ZFG47T (CP030073) VK-A60T CP031742 87 Streptomyces albidoflavus NRRL B-1271T (Z76676) NRRL B-2867T (KL569104) Streptomyces carpinensis NRRL B-16921T (MUBM01000291) Streptomyces pseudogriseolus NRRL B-3288T (MUNG01000290) ATCC 14921T (DF968281) Streptomyces africanus NRRL B-24243T (MUKA01000156) Streptomyces swartbergensis HMC13T (GU220476) Streptomyces zinciresistens K42T (AGBF01000432) 84 Streptomyces luteus TRM 45540T (KN039946) NRRL B-2410T (MUMD01000370) 99 NRRL B-2706T (AB184843) Streptomyces pharetrae CZA14T (MRYD01000560) ATCC 23877T (CP012382) DSM 40306T (FNTD01000004) Streptomyces jietaisiensis CGMCC 4.1859T (AY314783) Streptomyces silaceus NRRL B-24166T (LIRJ01000287) ATCC 14899T (CP009313) Streptomyces lavenduligriseus NRRL ISP-5487T (JOBD01000085) Streptomyces coelicoflavus NBRC 15399T (AB184650) NBRC 12862T (AY999917) subsp. ardesiacus NBRC 15402T (DQ026631) Streptomyces uncialis DCA2648T (LFBV01000015) Streptomyces rubrogriseus NBRC 15455T (AJ781373) Streptomyces violaceorubidus NRRL B-16381T (AJ781374) Streptomyces tricolor NRRL B-16925T (AB184687) 72 Streptomyces humiferus DSM 43030T (AF503491) Streptomyces himastatinicus ATCC 53653T (EF408736) Streptomyces hygroscopicus subsp. hygroscopicus NBRC 13472T (BBOX01000593) Streptomyces rapamycinicus NRRL 5491T (EF408733) Streptomyces rhizosphaericus NRRL B-24304T (AB249941) Streptomyces castelarensis NRRL B-24289T (AY508511) 89 Streptomyces melanosporofaciens DSM 40318T (FNST01000002) Streptomyces orinoci NRRL B-3379T (AB184866) Streptomyces griseocarneus NRRL B-24281T (MUFU01000078) Streptomyces lilacinus NRRL B-1968T (JNXU01001020) Streptomyces varsoviensis NRRL ISP-5346T (JOBF01000056) NBRC 13819T (AORZ01000256) Streptomyces roseifaciens MBT76T (MG947385) Streptomyces bingchenggensis BCW-1T (CP002047) 93 Streptomyces sparsogenes ATCC 25498T (MAXF01000077) Streptomyces pathocidini NRRL B-24287T (AB184501) Streptomyces spongiicola HNM0071T (KT327868) 89 Streptomyces wuyuanensis CGMCC 4.7042T (jgi.1085054) Streptomyces xinghaiensis S187T (CP023202) Streptomyces glauciniger CGMCC 4.1858T (jgi.1076272) Streptomyces guanduensis CGMCC 4.2022T (AY876942) CGMCC 4.2026T (AY876941) Streptomyces yeochonensis CN732T (JQNR01000005) Streptomyces paucisporeus CGMCC 4.2025T (jgi.1076282) Streptomyces yanglinensis CGMCC 4.2023T (AY876940) subsp. cacaoi NRRL B-1220T (MUBL01000215) Streptomyces qinglanensis 172205T (HQ660227) Streptomyces diacarni LHW51701T (KX347890) Streptomyces reniochalinae LHW50302T (QOIM01000066) Streptomyces flocculus NRRL B-2465T (LIQO01000098) Streptomyces almquistii NRRL B-1685T (LIPO01000052) 100 Streptomyces gibsonii NRRL B-1335T (LIQP01000245) NRRL B-1811T (BBQG01000088) 100 DSM 40104T (DQ442546) Streptomyces xiaopingdaonensis L180T (EF577241) Streptomyces nanshensis SCSIO 01066T (EU589334) Streptomyces oceani SCSIO 02100T (LJGU01000141) Streptomyces armeniacus ATCC 15676T (AB018092) Streptomyces pini PL19T (FOSG01000050) Streptomyces barkulensis RC 1831T (JQ862602) 88 Streptomyces radiopugnans CGMCC 4.3519T (DQ912930) NRRL B-16372T (AB184617) NRRL 8057T (FQ859185) 318T (EF012099) Streptomyces harbinensis CGMCC 4.7047T (JQ750974) 100 Streptomyces thermoautotrophicus UBT1T (JYIJ01000013) Allostreptomyces psammosilenae YIM DR4008T (KX689228) Streptomyces vitaminophilus ATCC 31673T (ARCK01000107) Streptomyces aidingensis CGMCC 4.5739T (HQ286045) Allostreptomyces Streptomyces klenkii KCTC 29202T (KF656772) Streptomyces hoynatensis KCTC 29097T (RBAL01000049) 96 Streptomyces avicenniae NRRL B-24776T (JOEK01000028) Streptomyces specialis GW41-1564T (LN929789) Streptomyces zhaozhouensis CGMCC 4.7095T (OCNE01000042) 0.01 17 Fig. S1. Maximum likelihood tree based on 16S rRNA gene sequences showing the phylogenetic

18 relationship among Streptomyces and closely related genera belongs to Streptomycetaceae family

19 members. The numbers at the nodes are given as percent and represent the levels of bootstrap

20 support (>70%) based on maximum likelihood analyses of 500 re-sampled data sets. Scale bar,

21 0.01 nt substitutions per position.

4

Micromonospora rosaria DSM 803T (LRQV01000286) Micromonospora chalcea DSM 43026T (X92594) Micromonospora inositola DSM 43819T (LT607754) Micromonospora costi CS1-12T (RBAN01000020) Micromonospora globbae WPS1-2T (LC177396) Micromonospora pallida DSM 43817T (FMHW01000002) Micromonospora echinospora DSM 43816T (LT607413) Micromonospora inyonensis DSM 46123T (FMHU01000001) Micromonospora soli SL3-70T (AB981051) Micromonospora echinaurantiaca DSM 43904T (LT607750) 71 Micromonospora fluostatini PWB-003T (LC033898) Micromonospora eburnea DSM 44814T (FMHY01000002) Micromonospora nigra DSM 43818T (FMHT01000003) Micromonospora rhizosphaerae DSM 45431T (FMHV01000002) Micromonosporales; Micromonospora radicis AZ1-13T (LC177517) Micromonospora deserti 13K206T (MG770841) Micromonospora sediminimaris CGMCC 4.3550T (jgi.1076312) 81 Micromonospora polyrhachis NEAU-ycm2T (KC139400) Micromonospora sonneratiae 274745T (JQ619535) Micromonospora ovatispora 2701SIM06T (JQ836686) Micromonospora pattaloongensis DSM 45245T (jgi.1107896) Polymorphospora rubra TT 97-42T (AB223089)

T 99 Micromonospora pisi DSM 45175 (RBKT01000001) Planosporangium thailandense HSS8-18T (AB560970) 99 Planosporangium flavigriseum YIM 46034T (AM232832) Actinoplanes octamycinicus NBRC 14524T (AB047494) 75 Actinoplanes philippinensis DSM 43019T (jgi.1085831) albida DSM 43870T (CP007155) Pseudonocardiales; 100 Kutzneria viridogrisea JCM 3282T (U58530) Jiangella mangrovi 3SM4-07T (AB976540) Jiangella rhizosphaerae NEAU-YY265T (MH144586) 73 Jiangella anatolica GTF31T (POTW01000167) 100 Jiangellales; Jiangellaceae Jiangella gansuensis DSM 44835T (KI911782) Jiangella endophytica KE2-3T (LC032128)

T 72 Jiangella muralis DSM 45357 (LFXL01000071) 97 Jiangella alba DSM 45237T (FNUC01000003) Streptomyces thermoautotrophicus UBT1T (JYIJ01000013) Charcoactinosporaceae Acidothermus cellulolyticus ATCC 43068T (CP000481) Acidothermales; Acidothermaceae coriariae BMG5.1T (JWIO01000013) 78 Frankia alni ACN14AT (CT573213) Frankia canadensis ARgP5T (FZMO01000249) 95 Frankia saprophytica CN3T (MF960832) 94 96 Frankia asymbiotica M16386T (KY744730) Frankiales; Frankiaceae Frankia inefficax EuI1cT (CP002299) Frankia discariae BCU110501T (KB891152) Frankia elaeagni BMG5.12T (KB893676) 89 Frankia irregularis DSM 45899T (jgi.1068000)

T 100 Cryptosporangium aurantiacum DSM 46144 (jgi.1107629) Cryptosporangiales; Cryptosporangiaceae Cryptosporangium arvum DSM 44712T (KK073874) 100 Motilibacter rhizosphaerae RS-16T (FM998018) Motilibacteraceae Motilibacter peucedani RP-AC37T (RBWV01000010) Streptomyces albus NBRC 13014T (BBQG01000088) Streptomyces reniochalinae LHW50302T (QOIM010000660) 100 Streptomycetales; Streptomycetaceae T 85 Streptomyces sclerotialus NRRL ISP-5269 (JOBC01000056) 95 Streptomyces rimosus subsp. rimosus ATCC 10970T (ANSJ01000404)

0.01 22 Fig. S2. Maximum likelihood tree of the class families showing the presence of 9

23 orders/families based on 16S rRNA gene sequence comparison. The phylogenetic relatedness of

24 the families of the class Actinobacteria is outlined. Taxa newly described in this paper are

25 highlighted in bold. The numbers at the nodes are given as percent and represent the levels of

26 bootstrap support (>70%) based on maximum likelihood analyses of 1000 re-sampled data sets.

27 Scale bar, 0.01 nt substitutions per position.

5

28 Supplementary references

29 Kämpfer P, Glaeser SP, Parks L, van Keulen G, Dyson P. The family Streptomycetaceae. In:

30 Rosenberg E, Delong EF, Lory S, Stackebrandt E, Thompson F. (editors) The Prokaryotes:

31 Actinobacteria. 4th Edn. Springer Reference. 2014; p.1065

32 Tajima K, Takahashi Y, Seino A, Iwai Y, Omura S. Description of two novel species of the

33 genus Kitasatospora Omura et al. 1982, Kitasatospora cineracea sp. nov. and Kitasatospora

34 niigatensis sp. nov. Int J Syst Evol Microbiol 2001; 51:1765-1771.

6