Vector-borne pathogens have evolved molecular mechanisms of vector-pathogen interactions that involve genetic traits of both the vector and the pathogen [5, 35]. For the tick-borne pathogenA. marginale, recent studies have characterized tick and pathogen-derived genes that are involved in tick-pathogen interactions [2, 3, 6–9, 15]. Phylogenetic studies using tick and/or pathogen-derived genetic markers have contributed to our understanding of the evolution ofA. marginale strains and tick-pathogen relationships [5, 6, 20]. However, the impact of these studies has been limited by the genetic diversity of genes involved in tick-pathogen interactions and thus are likely to reflect pathogen evolution and tick-pathogen relationships .
In this study we took a different approach to characterize the evolution ofA. marginale strains. This is the first study to use remotely sensed vegetation features as a surrogate of an environmental envelope to which genetic variability and structure of a single pathogen is associated. Biogeographic research seeks to identify the processes structuring organism diversity at a variety of geographic and taxonomic scales . Remote sensing is being used increasingly as a tool to discover ecological traits through definite signatures. NDVI is a measure of the vegetation stress, thus a time series of NDVI values over a region reflects the seasonal cycle of vegetation as a surrogate of the seasonal variation in climate. NDVI and other climate features are commonly used to detect ecologically suitable areas for some pathogens and their vectors [37–39]. For example, Randolph and Rogers  indicated that climate has directed and constrained the evolution of flaviviruses of the TBE group. Six flaviviruses (SSEV, WTBEV, Russian TBEV, OHFV, and Kyasanur forest virus) fall within a distinct eco-climatic space defined by factors derived from thermal and moisture conditions. Herein we showed thatA. marginale MSP1a R1 repeats evolved under positive selection, were associated with specific ecoregion clusters and were not arranged according to geographical features. The different evolutionary pressures operating over different MSP1a repeats was demonstrated previously , but the possibility of using the MSP1a R1 repeat as a biogeographical marker has only been suggested . In contrast, MSP1a RL repeat sequences, while still linked to a similar set of ecoregion clusters, did not evolve under positive selection. Consequently, RL repeat sequences were not good genetic markers for the characterization ofA. marginale biogeography and evolution.
Analysis of MSP1a microsatellite sequences demonstrated thatA. marginale strains are associated with specific ecoregion clusters, thus corroborating the results obtained with repeat sequences. These results may have a functional significance. It has been shown that the SD-ATG distance between the ribosome binding site (Shine-Dalgarno sequence) and the translation initiation codon affects gene expression in prokaryotes . Little is known about the regulation of gene expression inA. marginale . However, as shown here inE. coli, the length of the MSP1a microsatellite could affect the expression of MSP1a, which varies duringA. marginale multiplication in both tick cells and bovine erythrocytes, thus affecting pathogen infection and transmission . Since MSP1a repeats and microsatellites are unambiguously associated to ecoregion clusters, these results suggested a new factor that may affect the efficiency by which differentA. marginale strains are transmitted under different environmental conditions.
A. marginale is an obligate intracellular parasite, which alternates between the tick vector and the vertebrate host. Our hypothesis was that the link between pathogen strains and definite portions of the environmental envelope could reflect the effects of climate on the tick vector. Temperature and rainfall, which are indirectly captured by the specific signatures of NDVI, are the main factors affecting the ecology and population dynamics of tick species  and these operate at critical levels of selection of tick populations, selecting also specific strains of the pathogen. This framework is further obscured by the 'noise' produced by invasive events of the pathogen (by cattle movement or other factors), or by selection of strains transmitted mechanically in areas where ticks are eradicated by acaricide application, contributing to the absence of total consistence between ecoregion clusters and strains.
Adequate reports exist about distribution, seasonal dynamics and abundance ofR. microplus populations in the study area, allowing a direct comparison with results presented herein. Ecoregion cluster 1 contained the R1 repeats with the lowest percentage of conserved amino acids and the highest positive selection pressure, in areas with high temperature and medium rainfall.R. microplus ticks are common in these areas and a strict seasonality in tick population dynamics has been reported, allowing for a high selection of tick populations due to winter mortality . Ecoregion cluster 2 contained sites with constant high temperature and rainfall. In these sites,R. microplus ticks are abundant throughout the year without marked seasonality and climate is not a limiting factor in tick mortality [44, 45]. In ecoregion cluster 3, tick populations suffer drastic limitations in effectiveness because of low and inadequate rainfall , and this high selection pressure on tick populations might be adverse for pathogen transmission and selection. The R1 repeat sequences in ecoregion clusters 2 and 3 had higher number of conserved amino acids and lower positive selection pressure when compared with R1 sequences in ecoregion cluster 1. Finally, the R1 repeat sequences ascribed to ecoregion cluster 4 had the highest percentage of conserved amino acids and the lowest positive selection pressure, recorded only in sites whereR. microplus ticks are absent because of the low yearly temperature and thus other tick species act as vectors ofA. marginale . The analysis of MSP1a microsatellite sequences also supported differences among all ecoregion clusters, except for ecoregion clusters 3 and 4 whereR. microplus has low prevalence or is absent.
Some R1 repeat sequences such as A, B, D and alpha as well as microsatellite genotypes C-D, G and H were present across several ecoregion clusters. These sequences appeared inA. marginale strains collected in zones whereR. microplus ticks are common (ecoregion clusters 1 and 2) and in sites, such as central Argentina and southern parts of the USA, whereR. microplus has been prevalent in the past but has been eradicated . Additionally, these sequences were also found in sites where other tick vectors such asDermacentor spp. are prevalent . These R1 repeats and microsatellite sequences could have evolved from ancestor pathogen strains transmitted byR. microplus as the main vector, and then evolved under lower selection pressure, due to pathogen transmission by other tick species or mechanically. The presence of these sequences in sites whereR. microplus has been historically absent (that is, north-western USA) and now adapted to transmission byDermacentor spp. ticks, could be interpreted as invasive events. The results reported here showed that lowest selection pressure exist in sites whereDermacentor spp. ticks are the main biological vectors or where mechanical transmission is predominant because of eradication ofR. microplus. Therefore, R1 repeats are evolving under high selection pressure only in sites whereR. microplus is the main vector and is subjected to selection because of climate constraints. This hypothesis did not explain the absence of MSP1a genetic diversity in Australia. Analysis of fourA. marginale strains in Australia revealed the presence of a single repeat type 8 . We would expect evolution ofA. marginale MSP1a towards different repeat sequences, sharing the consensus sequence found in ecoregion cluster 1, into which R1 type 8 is ascribed, even in the case of a single invasive event. Reasons accounting for such a lack of diversity are currently unknown, but the combined pressure exerted by tick population structure [47, 48] theA. centrale vaccine, acaricide treatments and cattle movement for pathogen and tick control may have impacted onA. marginale genetic diversity in Australia .
A. marginale exclusively infects cattle and wild ruminants . Such high host specificity may results in a relatively low impact of vertebrate host factors on the evolution ofA. marginale strains, thus leaving tick-pathogen interactions as the main contributing factor affecting its biogeography and evolutionary history. However, as previously discussed, cattle movement may have contributed to the genetic diversity ofA. marginale strains worldwide . Nevertheless, the results reported herein may be relevant in studying the evolution of other vector-borne pathogens. Many vector-borne pathogens, such as someBabesia,Theileria,Rickettsia,Ehrlichia andPlasmodium species, are also highly host-specific  and vector-pathogen interactions may play a crucial role in their evolution and biogeography .