Major rDNA and histone H3 clusters detected by rDNA FISH and TSA-FISH, respectively, on pachytene nuclei of representatives of the caddisfly superfamily Limnephiloidea (Trichoptera) and lepidopteran superfamilies Hepialoidea and Tischeroidea. a, c, e, g -FISH with 18S rDNA probe (red signals marked by arrowheads); b, d, f, h -TSA-FISH with histone H3 probe (red 

signals marked by arrowheads). Chromosomes are counterstained with DAPI (blue). a, b -pachytene nuclei of the caddisfly Glyphotaelius pellucidus (sex not determined); c, d -male pachytene nuclei of Hepialus humuli, note that the rDNA signals cover approximately half of one of the chromosomal bivalents and colocalize with heterochromatin (C and inset); e -female pachytene nucleus of Phymatopus californicus, f -male pachytene nucleus of P. californicus; g, h -male pachytene nuclei of Tischeria ekebladella, note that the cluster of rDNA signals is adjacent to a pair of heterochromatin blocks (g and inset). * DAPI positive heterochromatin. Scale bar = 10 µm. 

Species

Gene 



  




  




  




  




  




  




  




  




  




  




  




  




  




  


table 2 :

 2 Overview of results from this study and from literature. . Lukhtanov, V. A. Sex chromatin and sex chromosome systems in nonditrysian Lepidoptera (Insecta). J. Zool. Syst. Evol. Res. 38, 73-79 (2000). 3. Seiler, J. Research on the sex-chromosomes of Psychidae (Lepidoptera). Biol. Bull. 36, 399-404 (1919). 4. Hejníčková, M. et al. Absence of W chromosome in Psychidae moths and implications for the theory of sex chromosome evolution in Lepidoptera. Genes 10, 1016; 10.3390/genes10121016 (2019). 5. Seiler, J. Geschlechtschromosomen-Untersuchungen an Psychiden. Z. Indukt. Abstamm. Vererbungsl. 31, 1-99 (1922). 6. Dalíková, M. et al. New insights into the evolution of the W chromosome in Lepidoptera. J. Hered. 108, 709-719 (2017). 7. Prins, J. D., Prins, W. D. & Dall'Asta, U. The karyotype of Cameraria ohridella (Lepidoptera: Gracillariidae). Phegea 301, 5-10 (2002). 8. Yasukochi, Y. et al. Extensive conserved synteny of genes between the karyotypes of Manduca sexta and Bombyx mori revealed by BAC-FISH mapping. PLoS ONE 4, 


	NCBI AN

d 18S rDNA -ref. d Histone H3 -ref. d a preliminary diploid chromosome number determined only in male, female unknown b variable c chromosome numbers differ in subspecies (2n = 25/26 in S. c. sp. indet.; 2n = 26 in S. c. walkeri; 2n = 27/28 in S. c. ricini) d 1. Kiauta, B. & Lankhorst, L. The chromosomes of the caddis-fly, Glyphotaelius pellucidus (Retzius, 1783) (Trichoptera: Limnephilidae, limnephilinae). Genetica 40, 1-6 (1969). 2e7465; 10.1371/journal.pone.0007465 (2009). 
table S4 :

 S4 List of primers used for PCR amplification of marker genes. 1. Fuková, I., Nguyen, P. & Marec, F. Codling moth cytogenetics: karyotype, chromosomal location of rDNA, and molecular differentiation of sex chromosomes. Genome 48, 1083-1092; (2005). 2. Šíchová, J., Nguyen, P., Dalíková, M. & Marec, F. Chromosomal evolution in tortricid moths: Conserved karyotypes with diverged features. PLoS One 8, e64520; 10.1371/journal.pone.0064520 (2013). 


	Supplementary			
	Gene Superfamily Limnephiloidea	Forward primer Species Glyphotaelius pellucidus	Reverse primer Origin Znojmo, CZ	Product length [bp] fresh water, 15°C, Fagus sp. Annealing temp. [°C] Rearing conditions, diet Reference a
	18 S rDNA (Trichoptera) Hepialoidea	CGATACCGCGAATGGCTCAA Phymatopus californicus	ACAAAGGGCAGGGACG Bodega Marine Reserve,	1850 15°C, Daucus carota 58	[1]
	Histone H3 5S rDNA Tischerioidea Tineoidea	ATGGCNCGTACNAARCARAC Hepialus humuli GCCAACGTCCATACCAYGYTGA Tischeria ekebladella a Taleporia tubulosa a	TANGCACGYTCNCCNCGGAT California, USA Bochov, Czech Republic AAGCCAACGNCACGYGGT České Budějovice, CZ České Budějovice, CZ	300-500 outdoor container with planted Daucus carota 62 [2] 120 58 ambient conditions, Quercus sp. This study ambient conditions
	U1 snRNA	CAAAAATTACGCGTCCGAG Proutia betulina a Psyche crassiorella a	CGTAGGGGACACCGTGAT České Budějovice, CZ České Budějovice, CZ	120 ambient conditions 58 ambient conditions	This study
	U2 snRNA AGATCAAAGTGTAGTATCTGTTC Tineola bisselliella	CTGCAATGCCGGGCCRAC České Budějovice, CZ	140 25 ± 1°C, 16/8 h (light/dark) regime, wool 58 This study
	Gracillarioidea	Cameraria ohridella a	České Budějovice, CZ	ambient conditions, Aesculus hippocastanum
	Yponomeutoidea Plutella xylostella	Laboratory strain	25 ± 1°C, 16/8 h (light/dark) regime, artificial
					diet [1]
			Yponomeuta evonymella	Amsterdam, NL	ambient conditions, Prunus padus
	Tortricoidea		Cydia pomonella	Laboratory strain Krym-	27±1 °C ; 16/8 h (light/dark) regime, artificial
				61	diet [2]
	Cossoidea		Cossus cossus a	České Budějovice, CZ	ambient conditions
	Papilionoidea		Pieris brassicae	Levín, CZ	ambient conditions, Brassica napus
			Pieris rapae	Levín, CZ	ambient conditions, Brassica oleracea
			Gonepteryx rhamni a	Levín and České	ambient conditions
				Budějovice, CZ	
			Inachis io	Vrábče, CZ	ambient conditions, Urtica dioica
			Aglais urticae	Vrábče, CZ	ambient conditions, Urtica dioica
	Gelechioidea		Depressaria daucella a	Tábor, CZ	ambient conditions, Carum sp.
			Limnaecia phragmitella a	České Budějovice, CZ	ambient conditions, Typha sp.
			Tuta absoluta	Laboratory strain	25 ± 1°C, 16/8 h (light/dark) regime, Solanum
					lycopersicum [3]
	Pyraloidea		Ephestia kuehniella	Laboratory strain WT-C02 25 ± 1°C, 16/8 h (light/dark) regime, artificial
					diet [4]
	Noctuoidea		Cerura vinula	Chrudim, CZ	ambient conditions, Salix alba
			Phalera bucephala a	České Budějovice, CZ	
			Spodoptera frugiperda	Laboratory of Heckel D,	25 ± 1°C, 16/8 h (light/dark) regime, artificial
				Jena, DE	diet [5]
	Geometroidea	Biston betularia	Laboratory of Saccheri I,	ambient conditions, Betula pendula and Malus
				Liverpool,UK	domestica
			Abraxas grossulariata	Near Hrabětice, CZ	Ambient conditions, Ribes rubrum [6]
	Bombycoidea		Euthrix potatoria	Sedlec, CZ	ambient conditions, Poaceae
			Bombyx mori	Laboratory strain p50	ambient conditions, Morus sp.
			Hyalophora cecropia	Connecticut, USA	ambient conditions, Acer platanoides

a 
Table 6 :

 6 Primers and their concentrations used in the qPCR experiments.1. Dalíková, M. et al.New Insights into the Evolution of the W Chromosome in Lepidoptera.J. Hered.108, 709-719(2017).Voleníková, A. Karyotype and sex chromosomes analysis of two species from basal lepidopteran family Hepialidae (Lepidoptera: Hepialidae). Master thesis (in Czech), University of South Bohemia, České Budějovice, Czech Republic (2014). 


	Species	Gene	Forward	Reverse	Reference a	Final conc.
	Glyphotaelius pelucidus Acetylcholinesterase 2	CCCCGGATTCGAAGGAGAAG	ATCCGCCGCCATAAATCCAA	this study	0,8 μM
		U1 snRNA	TAGGGAAAATCGCAAGGGTCAG	GTAGGGGACACCGTGATCAAT	this study	0,8 μM
		U2 snRNA	GTGGAGCAAGCCCCGAACATC	GCAATGCGGGACCAGTATATT	this study	0,8 μM
		5S rDNA	GCCAACGTCCATACCATGCTGA	ACCGCGTGTCGTTGGCTT	this study	0,8 μM
	Hepialus humuli	paralytic	ATTCCCTTGGCTCCCTTAAC	CCCACATTGCTGAGAGTGG	[1]	1,2 μM
		U1 snRNA	TAGGGAAAATCGCAAGGGTCAG	CCGTGATCATGAAGGCGGTA	this study	0,8 μM
		U2 snRNA	GTGGAGCAAGCCCCGAACATC	ATCTGATAGTTCCCGCATTGC	this study	0,8 μM
		5S rDNA	TGAAAACACCGGTTCTCGTCC	CACCCATCCAAGTACTGACCG	this study	0,4 μM
	Tischeria ekebladella	Acetylcholinesterase 2	GACACTATTGTGGCCTCGATG	CCAGGAGTCTCTTCACTTTGC	[2]	0,8 μM
		U1 snRNA	ATTACGCGTCCGAGATATCCA	TTGCACTAGGGTTGGGTTGAC	this study	0,8 μM
		U2 snRNA	AGCCCCTATCTCCGCATCAG	GCTTAATATCTGAAAGTTCCCGCAA	this study	0,8 μM
		5S rDNA	CCCATCCAAGTACTGACCACG	TGAATACACCGGTTCTCGTCC	this study	0,8 μM
	Tineola bisselliella	Acetylcholinesterase 2	GAGGCACCCAAATATTCAAATACAA	ATACGAATATTTTCCAGGATTTGAGG	[2]	1 μM
		U1 snRNA	GGGTAATCACAGGGGTCAACC	ATCAAGAAGGCGGTTCCCC	this study	0,4 μM
		U2 snRNA	GTGGAGCAAGCCCCGAACATC	ATCTGATAGTTCCCGCATTGC	this study	0,8 μM
		5S rDNA	ACCCATCCAAGTACTGACCAC	TACACCGGTTCTCGTCCGAT	this study	0,8 μM
	Taleporia tubulosa	Acetylcholinesterase 2	TGGCGGAAAGACCTAAGGTTC	TACAAGTCTAGGGTCGCAGTG	this study	0,4 μM
		U1 snRNA	CCCACATTAGGGGTAATCGCA	CGTGATCACAAAGGCGGTTC	this study	0,4 μM
		U2 snRNA	TCAGCTTAATATCTGAAAGTTCCAG	TGGAGCGAGCCCCGAACAT	this study	0,4 μM
		5S rDNA	GCGGTCACCCATCCAAGTAC	TGAATACACCGGTTCTCGTCC	this study	0,4 μM
	Cameraria ohridella	Acetylcholinesterase 2	TACACGGACTGGGAGGAGATAAC	TAGTTTGTAGGGCAGATGAAGAAG	[2]	0,8 μM
		U1 snRNA	CCGTGATCAAGTAGGCGGTT	TAGGGATAATCGCAGGGGTCA	this study	0,8 μM
		U2 snRNA	CAGCTTAATATCTGAAAGTTCCCTCAC	AGCCCCAACTACCCATCTAGA	this study	0,8 μM
		5S rDNA	TGAAAACACCGGTTCTCGTCC	CACCCATCCAAGTACTGACCG	this study	0,8 μM
	Yponomeuta evonymella Acetylcholinesterase 2	AGCTGGAGCTGTTTCTGTCTC	CGCATAATGCTCTCTTCTCTTG	this study	0,8 μM
		U1 snRNA	CCGTGATCAAGTAGGCGGTT	TAGGGATAATCGCAGGGGTCA	this study	0,8 μM
		U2 snRNA	GTAACGGGAGTGGAGCGAG	CAGCTTAATATCTGAAAGTTCCCCC	this study	0,8 μM
		5S rDNA	AGCCAACGACACGTGGTG	TGAATACACCGGTTCTCGTCC	this study	0,8 μM
	Cydia pomonella	Acetylcholinesterase 2	CTGCCACATTCATGCGTTCA	ACCCAAAGCATAACAGCTGC	this study	0,4 μM
		U1 snRNA	CAAAAATTACGCGTCCGAG	CGTAGGGGACACCGTGAT	this study	0,4 μM
		U2 snRNA	AGTGTAGTATCTGTTCTTTTCAGC	CCGTGACGGGAGTGGAG	this study	0,4 μM
		5S rDNA	CCAACGTCCATACCATGCTGAAT	TCACCCATCCAAGTACTGACCT	this study	0,4 μM

a 
table S7 :

 S7 Information about digestion reactions used for Southern hybridization. 


	Species	Gene	Restriction enzymes	Restriction enzymes	Restriction enzymes
			I	II	III
	Glyphotaelius pellucidus	U1 snRNA 5S rDNA	AfeI + XbaI a AfeI + BamHI a	AfeI + BamHI a PstI + SalI a	PstI + SalI a AfeI + XbaI a
	Hepialus humuli	U1 snRNA 5S rDNA	AfeI + XbaI a AfeI + XbaI a	AfeI + BamHI a AfeI + BamHI a	PstI + SalI a PstI + SalI a
	Tischeria ekebladella	U1 snRNA 5S rDNA	-PstI + BspHI a	-XbaI + BamHI a	-XbaI + NspI a
	Taleporia tubulosa	U1 snRNA 5S rDNA	-AfeI + XbaI a	-AfeI + BamHI a	-PstI + SalI a
	Tineola bisselliella	U1 snRNA 5S rDNA	AfeI + XbaI a AfeI + XbaI a	AfeI + BamHI a AfeI + BamHI a	PstI + SalI a PstI + SalI a
	Cameraria ohridella	U1 snRNA 5S rDNA	AfeI + XbaI a AfeI + XbaI a	AfeI + BamHI a AfeI + BamHI a	PstI + SalI a PstI + SalI a
	Yponomeuta evonymella	U1 snRNA 5S rDNA	NotI + XhoI b PstI + BspHI a	AfeI + BamHI a BspHI + BamHI a	BamHI + SalI a XbaI + NspI a
	Ephestia kuehniella	U1 snRNA 5S rDNA	PstI + BspHI a PstI + BspHI a	XbaI + BamHI a XbaI + BamHI a	XbaI + NspI a XbaI + NspI a
	Cydia pomonella	U1 snRNA 5S rDNA	NotI + PstI b NotI + PstI b	BamHI + SalI a BamHI + SalI a	AfeI + BamHI a AfeI + BamHI a
	Bombyx mori	U1 snRNA 5S rDNA	PstI + BspHI a PstI + BspHI	XbaI + BamHI a	XbaI + NspI a

a BspHI + BamHI a XbaI + NspI a a CutSmart buffer, NEB (New England Biolabs, Ipswich, MA, USA) b Buffer H + BSA + Triton (ThermoFisher Scientific, Waltham, MA, USA) 
 S7. 

Supplementary