Table 2. Summary of genetic variation across 151 isolates of the L. donovani complex for previously described loci involved in resistance or treatment failure of antimonial drugs and Miltefosine.

locus/complex	gene id			gene name	function prediction	involved in resistance (R)/treatment failure (TF) to drug:	reference	evidence from reference	gene copy number (gene CN)
	L. infantum, JPCM5, v41	L. infantum, JPCM5, v38	L. donovani ortholog, BPK282A1, v41
H-locus	LINF_230007700	LinJ.23.0280	LdBPK_230280	terbinafine resistance gene (HTBF), (YIP1)		Antimonials (R)	Callahan and Beverley, 1991; Dias et al., 2007	The Leishmania H region is frequently amplified in drug-resistant lines and is associated with metal resistance (genes YIP1, MRPA, PTR1).	Genes have an increased CN in 30% (CN +1 to +44), and reduced CN in 9% (CN −1). 37% of all samples have an insertion including at least three genes (always YIP1, MRPA and argininosuccinate synthase). These amplifications are in groups Ldon1 (42/45), Ldon3 (13/19) and Ldon5 (1/8). The insertion boundaries in isolates from groups Ldon1 and Ldon3 are shared (Figure 9—figure supplement 1A).
	LINF_230007800	LinJ.23.0290	LdBPK_230290	P-glycoprotein A (MRPA); pentamidine resistance protein 1	ATP-binding cassette (ABC) transporter, ABC-thiol transporter	Antimonials (R)	Callahan and Beverley, 1991; Dias et al., 2007; Leprohon et al., 2009	Increased expression of MRPA is often due to the amplification of its gene in antimony-resistant strains.
	LINF_230007900	LinJ.23.0300	LdBPK_230300		argininosuccinate synthase - putative	Antimonials	Grondin et al., 1993; Leprohon et al., 2009
	LINF_230008000	LinJ.23.0310	LdBPK_230310	Pteridine reductase 1 (PTR1)		Antimonials (R)	Callahan and Beverley, 1991; Dias et al., 2007	see above, evidence only for H-locus in general
						Antifolate (R)	Vickers and Beverley, 2011	Leishmania salvage folate from their hosts. Thereby folates are reduced by a DHFR (dihydrofolate reductase)-TS (thymidylate synthase) and PTR1. PTR1 can act as a metabolic bypass of DHFR inhibition, reducing the effectiveness of existing antifolate drugs.
Mitogen-activated protein kinase, MAPK1	LINF_360076200	LinJ.36.6760	LdBPK_366760	LMPK, mitogen-activated protein kinase	protein phosphorylation	Antimonials (R)	Singh et al., 2010; Ashutosh et al., 2012	Conflicting evidence between up- and down-regulation of Mitogen-Activated Protein Kinase one between different studies.	45% of all isolates showed an increased CN, with all isolates of Ldon1 andLdon3 being affected and smaller fractions in other L. donovani groups (Figure 9—figure supplement 2A).
Aqua-glyceroporin, AQP1	LINF_310005100	LinJ.31.0030	LdBPK_310030	Aquaglyceroporin 1, AQP1	drug transmembrane transport	Antimonials (R)	Gourbal et al., 2004; Uzcategui et al., 2008; Monte-Neto et al., 2015; Andrade et al., 2016; Imamura et al., 2016	A frequently resistant L. donovani population has a two base-pair insertion in AQP1 preventing antimonial transport. Increased resistance with decrease in gene CN or expression, while increase leads to higher drug sensitivity.	Gene CN deletions and insertions of small effect sizes (CN −2 to −1 and +1 to +3) are present in 6% and 35% of isolates but never leading to loss of the locus.
Miltefosine transporter and associated genes	LINF_130020800	LinJ.13.1590	LdBPK_131590	Miltefosine transporter, LdMT	phospholipid transport	Miltefosine (R)	Pérez-Victoria et al., 2006; Shaw et al., 2016	Gene deletion or different changes in two different strains evolved in promastigote culture for Miltefosine resistance. strain Sb-S: locus deletion and A691P; strain Sb-R: E197D	15 isolates: +1 gene CNV (CUK, Ldon1, Ldon2, Ldon3, Ldon5)
	LINF_130020900	LinJ.13.1600	LdBPK_131600	hypothetical protein	unknown function	Miltefosine (R)	Shaw et al., 2016	Deleted along with the Miltefosine transporter gene in a single line evolved for Miltefosine resistance in promastigote culture.	three isolates: +1 gene CNV (Ldon1, Linf1)
	LINF_320015500	LinJ.32.1040	LdBPK_321040	Ros3, LdRos3	Vps23 core domain containing protein - putative	Miltefosine (R)	Pérez-Victoria et al., 2006	Putative subunit of LdMT; LdMT and LdRos3 seem to form part of the same translocation machinery that determines flippase activity and Miltefosine sensitivity in Leishmania.	one isolate: +1 gene CNV (Ldon1)
Miltefosine sensitivity locus, MSL	LINF_310031200	LinJ.31.2370	LdBPK_312380		3'-nucleotidase/nuclease - putative	Miltefosine (TF)	Carnielli et al., 2018	MSL: a deletion of this locus was associated with Miltefosine treatment failure in Brazil. While the frequency of the MSL was still relatively high in the North-East it was almost absent in the South-East of Brazil, and it was absent in L.infantum/L.donovani in the Old World.	Genes have a reduced CN in 55% (CN −1 to −8) and increased in 4% (CN +1). Four isolates, show a complete loss of the MSL at identical boundaries: WC, Cha001, HN167 and HN336 (2/4 isolates from Brazil, 2/2 isolates from Honduras). Two isolates show a reduction of all four genes at this locus but with various deletion boundaries: IMT373cl1 (Portugal), CH35 (Cyprus) (Figure 9B).
	LINF_310031300	LinJ.31.2380	LdBPK_312380		3'-nucleotidase/nuclease - putative	Miltefosine (TF)	Carnielli et al., 2018
	LINF_310031400	LinJ.31.2390	LdBPK_312390		helicase-like protein	Miltefosine (TF)	Carnielli et al., 2018
	LINF_310031500	LinJ.31.2400	LdBPK_312320, LdBPK_312400		3–2-trans-enoyl-CoA isomerase - mitochondrial precursor - putative	Miltefosine (TF)	Carnielli et al., 2018