Biogeography of Leptospira in wild animal communities inhabiting the insular ecosystem of the western Indian Ocean islands and neighboring Africa

Figures & data

Details of the Leptospira-positive samples from terrestrial small mammals and bats used in the analyses, the number of genetic sequences obtained, and the associated Leptospira spp

Order: family	Species	No. of samples	Nb of sequences			Leptospira sp
			rrs2	secY	MLST5
Madagascar
Rodentia: Nesomyidae	Eliurus minor	15	15	13	13	Lb
Afrotheria: Tenrecidae	Hemicentetes nigriceps	1	1	1	1	Lk
	Hemicentetes semispinosus	1	1	1	0	Lk
	Microgale cowani	2	2	2	1	Lb, Lm
	Microgale dobsoni	5	5	4	4	Lm
	Microgale longicaudata	1	1	1	1	Lb
	Microgale majori	2	2	2	2	Lb
	Microgale principula	2	2	2	2	Lb
Chiroptera: Vespertilionidae	Myotis goudoti	1	1	1	1	Lb
Chiroptera: Miniopteridae	Miniopterus gleni	1	1	1	1	Lb
	Miniopterus griffithsi	1	1	1	1	Lb
	Miniopterus mahafaliensis	3	3	3	3	Lb
	Miniopterus majori	1	1	1	0	Lb
	Miniopterus sororculus	1	1	1	0	Lb
Chiroptera: Molossidae	Otomops madagascariensis	1	1	1	1	Lb*
	Mormopterus jugularis	2	2	2	1	Lb*
Chiroptera: Pteropodidae	Pteropus rufus	1	1	0	0	Lsp
Chiroptera: Rhinonycteridae	Triaenops menamena	5	5	5	3	Li, Lb*, Lsp
Tanzania
Rodentia: Nesomyidae	Cricetomys sp.	7	7	7	7	Lb, Lk
Rodentia: Muridae	Mastomys sp.	5	5	5	5	Li, Lb
Soricomorpha: Soricidae	Crocidura sp.	11	11	11	11	Li, Lb
Mayotte
Rodentia: Muridae	*Rattus rattus	8	8	8	8	Li, Lb
Afrotheria: Tenrecidae	*Tenrec ecaudatus	8	8	8	8	Lm
Seychelles
Rodentia: Muridae	*Rattus rattus	1	1	1	1	Li
Chiroptera: Pteropodidae	Pteropus seychellensis	1	1	0	0	Lsp
La Réunion
Rodentia: Muridae	*Rattus rattus	3	3	2	2	Li
Chiroptera: Molossidae	Mormopterus francoismoutoui	4	4	4	4	Lb*
Union of the Comoros
Chiroptera: Miniopteridae	Miniopterus griveaudi	1	1	1	1	Lb
Chiroptera: Pteropodidae	Rousettus obliviosus	2	2	1	1	Li, Lb
Mauritius
Chiroptera: Molossidae	Mormopterus acetabulosus	8	8	8	7	Lb*
South Africa
Chiroptera: Vespertilionidae	Glauconycteris variegata	1	1	0	0	Lk
	Scotophilus dinganii	1	1	1	1	Lb
Chiroptera: Miniopteridae	Miniopterus natalensis	8	8	7	6	Lb, Lk
Chiroptera: Nycteridae	Nycteris thebaica	2	2	2	2	Lb
Chiroptera: Pteropodidae	Rousettus aegyptiacus	8	8	5	1	Li, Lb*
Swaziland
Chiroptera: Nycteridae	Nycteris thebaica	1	0	1	0	Li
Mozambique
Chiroptera: Miniopteridae	Miniopterus mossambicus	1	1	1	1	Lb
Total		127	126	115	101

MLST5 includes the rrs2, secY, adk, lipL41, and icdA genes. The identification of the different Leptospira species is based on single (Figure S2 and S3) and concatenated gene phylogenies (Fig. 3). Introduced species are designated with an asterisk. Two endemic lineages of terrestrial small mammals occur on Madagascar, which include the family Tenrecidae and the subfamily Nesomyinae of the family Nesomyidae. The genus Cricetomys belongs to a separate subfamily, the Cricetomyinae, of the Nesomyidae

Li=L. interrogans, Lb = L. borgpetersenii, Lb* = sequences closely related to L. borgpetersenii, Lm = L. mayottensis, Lk = L. kirschneri. Lsp corresponds to unidentified Leptospira species.

Fig. 1 Diversity and geographic distribution of Leptospira in terrestrial small mammals and bats of the western Indian Ocean islands and neighboring Africa.

a Map of the sample locations. b Schematic representation of the Leptospira phylogenetic relationships based on the maximum-likelihood secY and rrs2 phylogenetic trees (details in Figures S1 and S2). Circle sizes are proportional to the number of samples included for each branch. Colors within the pie charts refer to the different countries as shown on the map. Samples denoted by an asterisk (*) refer to the sequences that could not be assigned to any clade

Fig. 2 Comparison of Leptospira haplotype diversity (± standard error, se) among the host groups and locations in the western Indian Ocean islands and neighboring Africa.

“tsm” refers to terrestrial small mammals. “MAU”=Mauritius, “RUN”=La Réunion, “MAY”=Mayotte, “SA” = South Africa, “TANZ” = Tanzania, “MADA” = Madagascar, “COM” = Union of the Comoros. Letters a–c above the bars refer to significantly different averages based upon a Tukey HSD test and “ns” = non-significant. Bars can have more than one letter to reflect the “overlap” between them

Results of the hierarchical AMOVA analysis

	d.f.	Φ Statistics	% Total variation
Geographic structure
Among islands/countries (South Africa, Madagascar, Comoros, La Réunion, Tanzania and Mayotte)	5	ΦCT = 0.035	3.45
Among host species within islands/countries	15	ΦSC = 0.600***	57.50
Within populations	71	ΦST = 0.610***	39.05
Structure by host groups (bats vs. terrestrial small mammals)
Among host groups	1	ΦCT = 0.074*	7.42
Among host species within host groups	22	ΦSC = 0.613***	56.76
Within populations	77	ΦST = 0.642***	35.83

*p < 0.05

***p < 0.001

Fig. 3 Leptospira–host associations in the western Indian Ocean islands and neighboring Africa based on a Bayesian multilocus phylogenetic analysis.

The genetic clades identified in Fig. 1 are shown in the gray circle, and the major genetic groups within these clades (squares, numbers 1 to 12) are highlighted by dashed boxes. Animal silhouettes represent host groups. Sequences from the samples specific to this study are in black and coded with the sample ID, geographic location, and host species/genus. Colored circles at the tip of the branches correspond to the geographic locations as shown in Fig. 1. Reference samples are in gray and are coded as follow: Li = L. interrogans, Lk = L. kirschneri, and Lb = L. borgpetersenii (see Table S1 for details). Posterior probabilities higher than 80% are represented by white circles at the nodes. Clade C was not included (refer to Results section for more information)

Pairwise distance (F_ST) among the different genetic Leptospira clades in the western Indian Ocean islands and neighboring Africa

		L. borgpetersenii-related		L. mayottensis	L. interrogans	L. kirschneri
		Clade B	Clade G	Clade D	Clade E	Clade F
L. borgpetersenii	Clade A	0.761***	0.794**	0.845***	0.888***	0.887***
L. borgpetersenii-related	Clade B		0.736**	0.853***	0.871***	0.870***
	Clade G			0.917**	0.857**	0.818**
L. mayottensis	Clade D				0.971***	0.947***
L. interrogans	Clade E					0.858***

Clade C was not included because of the full genotyping failure or suspected co-infections

**p < 0.05

***p  < 0.001