Madagascar is one of the most active plague regions in the world. However, few studies have investigated the molecular epidemiology of Y. pestis
from Madagascar and none have done so using very high resolution genomic methodologies. Here, we investigated the phylogeography and molecular epidemiology of Y. pestis
in Madagascar by using a combination of SNPs and MLVA to analyze 262 Malagasy isolates from 25 districts from 1939–2005. In contrast with previous analyses that utilized ribotyping or SNPs alone 
, we identified a very high level of genetic diversity with 226 MLVA genotypes among the 262 isolates. These genotypes were distributed amongst 15 subclades that displayed significant geographic separation (), leading to insights into the maintenance and spread of plague in Madagascar.
The use of MLVA was particularly effective at identifying genetic groups in Madagascar. SNPs, though useful, mostly provided confidence in genetic groups that were already apparent via MLVA. This is somewhat counter to the conventional hierarchical approach wherein SNPs are used first to identify major genetic groups followed by MLVA to provide resolution within those groups, thus minimizing the problems of mutational saturation/homoplasy that can occur with highly variable markers such as VNTRs 
. In this study, only SNP Mad-43 (Table S2
), which differentiated Groups I and II, was useful in this conventional sense to identify “major genetic groups” that were obscured in the MLVA phylogeny (data not shown). All of the other subclades identified by SNPs were also identified by MLVA, suggesting that at this regional scale, MLVA alone may be effective at identifying robust genetic groups. Importantly, though MLVA was excellent at identifying these genetic groups, the relationships among those groups, such as the division between Groups I and II, remained unclear using MLVA alone (data not shown) whereas they were very clearly depicted as a star phylogeny in the SNP phylogeny (). Where knowledge of deeper genetic relationships or fine-scale phylogenetic analysis of specific lineages (e.g., the strain MG05-1020 lineage here) is desired, SNPs will remain the preferred methodology for clonal pathogens such as Y. pestis
. However, until whole genome sequencing for entire isolate collections becomes feasible, MLVA will continue to be a useful tool for examining genetic diversity whether used in conjunction with SNPs or alone.
Our analyses suggest that plague is being maintained in Madagascar in multiple geographically separated subpopulations. We revealed significant geographic separation among the identified subclades (), suggesting that these subclades are undergoing local cycling with limited gene flow from other subclades. This is consistent with the population genetics and ecology of the black rat (Rattus rattus
), the primary plague host in rural Madagascar 
. The black rat in Madagascar exhibits limited gene flow between subpopulations 
as well as limited geographic ranges 
. This limited mobility, a high reproduction rate 
and the development of some resistance to plague 
are all likely important factors that allow the black rat to maintain plague in these genetically distinct, geographically separated subpopulations. The two flea vectors, X. cheopis
and S. fonquerniei 
, may also play a role in maintaining genetically distinct subpopulations (i.e., Groups I and II), though more data would be needed to confirm this hypothesis.
In contrast, transport of Y. pestis
across longer distances in Madagascar is likely human-mediated. Historically, there is ample evidence for the influence of human traffic on the spread of plague, including transport along trade routes such as the Silk Road in the early pandemics and transport via steam ship to numerous new locations during the “third” pandemic 
. The SNP phylogeny determined by Morelli et al. 
suggests the progression of plague from Israel to Madagascar to Turkey (), a series of transfer events that were almost certainly human-mediated, though the details remain unknown. In Madagascar, plague was most likely transported from its introduction point on the coast to the central highlands, where it became permanently established, via the railroad linking Toamasina and Antananarivo 
. More recently, plague was most likely reintroduced to Mahajanga via the transport of infected rats and fleas together with foodstuffs from the central highlands. Indeed, our data suggest multiple transfers between Mahajanga and the central highlands, all likely human-mediated. Additional long distance transfers of Y. pestis
in Madagascar are suggested by the multiple subclades identified in cities/communes such as Antananarivo and Andina Firaisana (, S1
, Table S1
Though long distance transfers of Y. pestis
undoubtedly occur, it is unclear how often such transfers result in the successful establishment of the transferred genotypes in new locations. At least one transfer to Mahajanga became successfully established and underwent local cycling as evidenced by the Mahajanga I.A subcluster described here (). However, many of the other examples of long distance transfers where multiple subclades were found in a single location are not as clear regarding the establishment of the transferred subclade(s). Antananarivo, for example, is clearly dominated by subclade I.A with only 1–2 representatives of each of the other five subclades identified there (, S1
, Table S1
), suggesting that the presence of these alternative subclades may have been only transitory.
Successful establishment of subclades in new locations following a long distance transfer may be related to adaptive advantages possessed by some genotypes 
. For instance, subclade I.A appears to be particularly successful in our analysis. The earliest subclade I.A isolate in our dataset was collected in 1974 from the Ambositra district (Table S1
), one of the most active plague districts in Madagascar 
. Subsequent isolates indicate that this subclade continued to exist in a small area of the Ambositra district but also became well established over a large geographic area including and surrounding the capital, Antananarivo. This subclade was also successfully introduced to and established in Mahajanga and appears to have been transferred to the Fianarantsoa district, though it is unclear whether or not it became established there (, S1
, Table S1
). This widespread geographical success may indicate that this subclade possesses an adaptive advantage that enhances its ability to be transferred long distances and become established in new locations 
. Alternatively, the particular success of this subclade may simply be due to chance.
The central highlands focus remains the most active plague focus in Madagascar 
and is, consequently, a likely place for new genotypes to emerge. This is particularly true for those central highlands districts with the highest plague activity. For instance, the three unique ribotypes identified in a previous study belonged to isolates from two highly active districts, Ambositra and Ambohimahasoa 
. Here, isolates belonging to Group II and its subclades were found in three highly active districts, Betafo, Manandriana and Ambositra (, S1
). As discussed above, Ambositra may also have been the district of origin for the highly successful subclade I.A. Overall, the Ambositra district was one of the two most diverse districts in our analysis, containing representatives from six different subclades (, Table S1
). This diversity is consistent with the Ambositra district's status as one of the three most important plague districts in Madagascar 
The maintenance and spread of Y. pestis in Madagascar is a dynamic and highly active process, depending on the natural cycle between the black rat and its flea vectors as well as human activity. Y. pestis in Madagascar is maintained in multiple, genetically distinct, geographically separated subpopulations, likely via the black rat. The exact geographic landscape of these subpopulations is probably ever changing, with some subclades going extinct or decreasing in frequency (e.g., subclade I.K), new subclades emerging and becoming established and some subclades being transferred to new locations, where they may become established either temporarily or more long-term. Much of the long distance spread of Y. pestis in Madagascar is likely due to human activities that allow for the transport of plague infected rats and fleas from one location to another.