Table 1:

Museum specimen metadata.

Sequencing Based Species ID	Date Collected	Location (1)	Donating Institution	BioSample (2)	Sample ID (3)	Museum Accession	Storage Type	Extraction Method	Concentration (ng/μl) (4)
D. melanogaster	1896	Urbana, IL	Illinois Natural History Survey	SAM N38071661	Illinois_1896	1004284	Dried	Non-Destructive	4
D. melanogaster	1908	Albany, NY	New York State Museum	SAM N38071662	NewYork_1908	-	Dried	Destructive	152
D. melanogaster	1908	Urbana, IL	Illinois Natural History Survey	SAMN38071654	Illinois_1908	1004277	Dried	Non-Destructive	54.6
D. melanogaster	1915	Urbana, IL	Illinois Natural History Survey	SAMN38071656	Illinois_1915_1	1004279	Dried	Non-Destructive	84
D. putrida	1915	Urbana, IL	Illinois Natural History Survey	SAMN38071657	Illinois_1915_2	1004280	Dried	Non-Destructive	U
D. melanogaster	1915	Urbana, IL	Illinois Natural Historv Survey	SAMN38071658	Illinois_1915_3	1004281	Dried	Non-Destructive	12.7
D. melanogaster	1915	Urbana, IL	Illinois Natural Historv Survey	SAM N38071659	Illinois_1915_4	1004282	Dried	Non-Destructive	16.9
D. melanogaster	1919	St. Paul, MN	University of Minnesota, Insect Collection	SAMN38090828	Minnesota_1919_1	-	Dried	Destructive	0
D. melanogaster	1919	St. Paul, MN	University of Minnesota, Insect Collection	SAM N38071687	Minnesota_1919_2	-	Dried	Destructive	21.6
D. melanogaster	1919	St. Paul, MN	University of Minnesota, Insect Collection	SAM N38071688	Minnesota_1919_3	-	Dried	Destructive	18.9
D. melanogaster	1927	Albany, NY	New York State Museum	SAMN38071663	NewYork_1927	-	Dried	Destructive	21
D. melanogaster	1930	Urbana, IL	Illinois Natural History Survey	SAM N38071660	Illinois_1930	1004283	Dried	Non-Destructive	13.6
D. melanogaster	1942	Glassboro, NJ	Illinois Natural Historv Survey	SAMN38071655	NewJersev_1942	1004278	Dried	Non-Destructive	10.2
D. melanogaster	1950	Oahu, HI	University of Hawaii, Insect Museum	-	Hawaii_1950_1	-	Dried	Destructive	0
D. melanogaster	1950	Oahu, HI	University of Hawaii, Insect Museum	-	Hawaii_1950_2	-	Dried	Destructive	0
D. melanogaster	1951	Honolulu, HI	University of Hawaii, Insect Museum	-	Hawaii_1951_1	-	Dried	Destructive	0
D. melanogaster	1951	Oahu, HI	University of Hawaii, Insect Museum	-	Hawaii_1951_2	-	Dried	Destructive	0
D. melanogaster	1952	Kauai, HI	University of Hawaii, Insect Museum	-	Hawaii_1952_1	-	Dried	Destructive	0
D. melanogaster	1953	Kauai, HI	University of Hawaii, Insect Museum	SAM N38071677	Hawaii_1953_1	-	Dried	Destructive	0
D. melanogaster	1963	Lebanon Co, PA	Frost Entomological Museum	SAMN38071668	Pennsylvania_1963_1	-	Dried	Non-Destructive	66.4
D. melanogaster	1963	Lebanon Co, PA	Frost Entomological Museum	SAM N38071669	Pennsylvania_1963_2	-	Dried	Non-Destructive	24.4
D. melanogaster	1968	St Cloud, MN	University of Minnesota, Insect Collection	SAM N38071684	Minnesota_1968	-	Dried	Destructive	13
D. melanogaster	1969	St Cloud, MN	University of Minnesota, Insect Collection	SAMN38071685	Minnesota_1969	-	Dried	Destructive	38.6
D. melanogaster	1991	St Cloud, MN	University of Minnesota, Insect Collection	SAM N38071686	Minnesota_1991	-	Dried	Destructive	24.4
D. putrida	2003	Lebanon Co, PA	Frost Entomological Museum	SAM N38071673	Pennsylvania_2003_1	4908	Dried	Non-Destructive	71.2
D. putrida	2003	Lebanon Co, PA	Frost Entomological Museum	SAMN38071672	Pennsylvania_2003_2	4887	Dried	Non-Destructive	13.9
S. pallida	2003	Lebanon Co, PA	Frost Entomological Museum	SAMN38071675	Pennsytvania_2003_3	5867	Dried	Non-Destructive	-
S. pallida	2003	Lebanon Co, PA	Frost Entomological Museum	SAM N38071676	Pennsylvania_2003_4	5873	Dried	Non-Destructive	120
S. pallida	2003	Lebanon Co, PA	Frost Entomological Museum	SAM N38071674	Pennsytvania_2003_5	5863	Dried	Non-Destructive	25.6
D. melanogaster	2006	Davidson, NC	The Davidson College Entomology Collection	SAM N38071666	NorthCaroBna_2006_1	-	Dried	Destructive	19.6
D. melanogaster	2006	Davidson, NC	The Davidson College Entomology Collection	SAMN38071667	NorthCarolina_2006_2	-	Dried	Destructive	56.8
D. melanogaster	2006	Davidson, NC	The Davidson College Entomology Collection	-	NorthCarolina_2006_3	-	Dried	Destructive	0
D. melanogaster	2010	Puslinch Township, Ontario, Canada	University of Guelph, Centre for Biodiversity Genomics	SAMN38071680	Canada_2010_11	PHDIP1072-11	Ethanol	Destructive	0
D. simulans	1952	Kauai, HI	University of Hawaii, Insect Museum	-	Hawaii_1952_2	-	Dried	Destructive	0
D. simulans	1952	Kauai, HI	University of Hawaii, Insect Museum	-	Hawaii_1952_3	-	Dried	Destructive	0
D. simulans	1953	Kilauea, HI	University of Hawaii, Insect Museum	SAMN38071678	Hawaii_1953_2	-	Dried	Destructive	0
D. simulans	1958	Maui, HI	University of Hawaii, Insect Museum	-	Hawaii_1958	-	Dried	Destructive	0
D. simulans	1960	Oahu, HI	University of Hawaii, Insect Museum	-	Hawaii_1960	-	Dried	Destructive	0
D. simulans	1963	Lebanon Co, PA	Frost Entomological Museum	SAM N38071670	Pennsytvania_1963_3	-	Dried	Non-Destructive	84.6
D. simulans	19G3	Lebanon Co, PA	rrost Entomological Museum	SAMN30O71671	Pennsytvania_1963_4	-	Dried	Non-Destructive	40
D. simulans	1965	Oahu, HI	University of Hawaii, Insect Museum	SAM N38071679	Hawaii_1965	-	Dried	Destructive	0
D. simulans	2000	Santa Barbara, CA	Santa Barbara Museum of Natural History	SAM N38071683	California_2000_1	-	Dried	Destructive	17.8
D. simulans	2000	Santa Barbara, CA	Santa Barbara Museum of Natural Historv	SAMN38071682	California_2000_2	-	Dried	Destructive	55.4
D. simulans	2010	Puslinch Township, Ontario, Canada	University of Guelph, Centre for Biodiversity Genomics	SAM N38071681	Canada_2010_2	PHDIP946-11	Ethanol	Destructive	17.8
D. simulans	2011	Ventura Co, CA	University of Guelph, Centre for Biodiversity Genomics	SAMN38071664	California_2011_1	BBDIV1549-12	Ethanol	Destructive	74.2
D. simulans	2011	Ventura Co, CA	University of Guelph, Centre for Biodiversity Genomics	SAM N38071665	California_2011_2	BBDIV1550-12	Ethanol	Destructive	15.9

⁽¹⁾All specimens from USA unless otherwise noted

⁽²⁾Specimens that did not yield sufficient sequencing library were not sequenced and thus have no BioSample ID

⁽³⁾Our study sample ID

⁽⁴⁾RNA concentrations below the limit of detection on the Qubit were assigned a concentration of 0