BACKGROUND
regarding adaptation to infectious diseases, genes of the major histocompatibility complex  are the most commonly studied in mammals.
mhc genes play a significant role in the functionality and effectiveness of the immune response of vertebrates and are directly involved in fitness and adaptation of hosts to their pathogens  <cit> .
therefore, studying mhc gene diversity in non-model animals, known to be reservoirs of pathogens, is a critical tool to assess its evolutionary process and implication in  the variation of individual fitness,  population viability and  evolutionary potential in a changing environment  <cit> .
studies have essentially focused on the genetic variability of exon  <dig> of the mhc class ii dr beta  gene given that this exon encodes the peptide-binding region   <cit> .
hence, most of the variability displayed is recorded on this exon.
variations within the pbr define the repertoire of antigenic determinants and subsequently the ability to recognize a variable number of circulating pathogens  <cit> .
optimum resistance to pathogens is, therefore, the result of all intrinsic and extrinsic factors that influence the genetic variability of individuals’ immune system  <cit> .
most of the vertebrate populations studied so far exhibited high levels of mhc diversity both in the number of alleles and in their heterozygosities and allelic variation .
pathogen-driven balancing selection and sexual selection seem to play a fundamental role in the preservation of this high level of polymorphism  <cit> .
pathogen-driven balancing selection acts through antagonistic host–parasite coevolution via several mechanisms: overdominant and frequency-dependent selection  <cit> , as well as spatial and temporal variation in host pathogens  <cit> .
sexual selection pressures encompass mechanisms such as maternal–fetal interactions  <cit>  and mate selection  <cit> .
however, when assessing the genetic variability of both neutral markers and mhc genes, studies have highlighted the role of past demographic processes  in shaping the pattern of mhc variability that can sometimes surpass that of natural selection .
local immunogenetic adaptation of hosts that live in different environments was associated with different parasite and pathogen pressures .
indeed, differences in the diversity of pathogens  are directly linked to environmental components.
these latter  modulate parasite and pathogen species richness, their survival and adaptability, as well as their distribution, transmission, developmental success and their ability to induce diseases  <cit> .
environmental components likewise impact the richness, population dynamics, immunocompetence and nutritional status of host species, all of which subsequently determine resistance or susceptibility to disease  <cit> .
a strong correlation was also shown between host and parasite species richness, their life history and ecological traits .
moreover, anthropogenic alterations of habitats induce changes in host–pathogen–environment interactions and are consequently linked to the emergence of infectious zoonotic diseases .
therefore, considering the role of the environment is critical for the assessment of mhc gene variability.
bats  are the second largest species-rich mammalian order, accounting for 20 % of all mammal species in the world.
compared to other mammals, their uncommon biological features  and their long-term evolutionary history with pathogens make them excellent reservoirs and spreaders of viruses , which have a significant impact on both human and animal health  <cit> .
however, bats rarely exhibit clinical or pathological signs of diseases and appear to coexist with their pathogens in a disease-free state, characteristic of reservoir hosts  <cit> .
at the order level, the monophyly of chiroptera was evidenced via phylogenetic relationships inferred from intron sequences of drb  <cit> .
the monophyletic origin of drb genes was also evidenced at the family level via phylogenetic relationships inferred from drb sequences from saccopteryx bilineata, myotis spp., noctilio spp.
and carollia perspicillata  <cit> .
other studies, investigating the diversity of mhc drb, showed significant differences in the polymorphism of mhc genes between species.
this polymorphism was essentially influenced by  a pathogen-driven selection  <cit> ,  a social structure driven by mhc-mediated post-copulatory mechanisms  <cit> ,  diversifying selection and recombination events  <cit>  and  geographical constraints resulting in spatial variation of pathogen-mediated selection and enhanced susceptibility to environmental changes  <cit> .
the amazon, a major biodiversity hotspot in south america, possesses a large diversity of bat species and pathogens in a wide variety of climates and vegetation formations  <cit> .
french guiana, a tropical amazonian region near the equatorial zone, harbors a great diversity of bats, with  <dig> species registered  <cit> .
in this region, the impact of deforestation on the composition and dynamics of bat communities was assessed  <cit>  as well as, the role of habitats on rabies virus circulation and maintenance  <cit> .
in this study, we analyzed the genetic variability of the expressed mhc class ii drb exon  <dig> in three bat species: two phyllostomidae, carollia perspicillata and desmodus rotundus, and one molossidae, molossus molossus.
these three widely distributed species have ecological plasticity and tolerance to disturbance.
in french guiana, these species are sympatric and use the same habitats .
the key hypothesis of this study was that composition and distribution of mhc drb alleles were specific to the environments , rather than randomly distributed in space.
consequently, we should observe local immunogenetic adaptation to the contrasting pathogen pressures or equally adapted alleles.
to assess which are the best factors that predict the mhc diversity, pathogen-mediated selection, recombination, gene conversion, demographic history and population structure were investigated.
there is a higher diversity of microorganisms in forest environments, compared to disturbed environnments, due to greater host species richness and better transmission-promoting parameters  <cit> .
for this reason, we expect higher levels of mhc diversity in forest environments facing lower disturbance pressures, where higher parasite and pathogen diversities imply a higher selection pressure.
furthermore, assuming that bats using the same roosting area and/or the same foraging areas would be subjected to similar pathogen pressures, we should observe similar trends in intra- and inter-specific mhc diversity.
in contrast, once the demographic neutral genetic histories—which may also influence mhc diversity—are controlled, we should observe differences in selective histories between bats inhabiting different environments.
to identify different signatures of selection in the drb exon  <dig>  which would imply area-specific recognition capabilities, conformation of the hla-drb <dig> was used as a reference to detect putative sites directly involved in antigen binding  and consequently subjected to selection.
additionally, to reduce the misidentification bias of abs, the selection signature within the pbr was investigated without a priori identifying species-specific abs for further comparisons.
mhc genes exhibit high levels of allele similarity within species as well as between related species and the occurrence of identical mhc alleles in related species is frequent.
convergence and trans-species polymorphism are thought to be responsible for this trans-species evolution.
to highlight which of these two mechanisms acts predominantly on the evolutionary history of the drb gene in the three species investigated, phylogenetic relationships were inferred from the drb sequences obtained here and with other available chiropteran sequences.
finally, mhc spatial diversity was compared to that of neutral markers  to highlight the impact of demographic processes and population structure on the diversity pattern in the three bat species investigated.
methods
ethics statement
animals were captured, handled, sampled and, whenever necessary, euthanized following asm guidelines  <cit>  under the supervision of researchers who had been granted the french “expérimentation animale niveau 1” diploma.
bats are not protected by law in french guiana; however, the project was submitted to the conseil scientifique régional pour le patrimoine naturel de la guyane and approved.
captures that occurred within protected areas  received approval by the conseil scientifique régional du patrimoine naturel on  <dig> january  <dig> and ad-hoc authorizations .
study areas, sample collection, dna and rna extraction
the three bat species  were sampled at  <dig> sites.
three sites were located in pristine lowland forests  with low disturbance pressure, four sites were located in edge habitats , five in anthropized areas , and two in urban and periurban areas  .fig.
<dig> map of sampling sites of all individuals used in this study across four environments.
for clarity, nearby collecting sites within 15 km were grouped.
sites are numbered and labeled according to the type of environment to which they correspond: edge habitats , anthropized areas , pristine primary lowland forests , urban and periurban areas .
pie charts indicate the proportion of the most frequent alleles ; dero-drb* <dig> and  <dig> for d. rotundus ; momo-drb* <dig> for m. molossus ), other shared alleles and private alleles  in each collecting sites/environments, for each species: c. perspicillata , d. rotundus  and m. molossus
bats were trapped with mist nets erected near breeding sites, roosts, at forest edges, around livestock, or through putative foraging courses.
bat species were identified in the field using external morphology and, prior to release, a brachial vein puncture and wing biopsies were performed.
a few individuals captured were euthanized at the laboratory to collect organs.
a total of  <dig> c. perspicillata,  <dig> d. rotundus and 42 m. molossus were sampled.
nucleic acids were extracted from blood, liver or spleen depending on sampling.
extractions were performed using the nuclisens easymag bio-robot .
amplification and genotyping of the mhc class ii drb gene
all amplifications of mhc drb genes for the three species were performed using cdna obtained from liver and spleen rnas.
all cdnas were synthesized using superscript® iii reverse transcriptase  and random hexamers  following the manufacturer’s recommendations.
expressed mhc drb genes of the three bat species were amplified by a minimum of two independent polymerase chain reactions  using a standard procedure with the amplitaq gold dna polymerase pcr kit .
to optimize the detection of different alleles, a minimum of two distinct pcrs per individual were performed.
species-specific primers were designed for c. perspicillata and d. rotundus to avoid occurrence of non-amplifying alleles .
to amplify the m. molossus drb gene, primers designed by richman et al.
<cit>  were modified.
primers located in conserved parts of exon  <dig> , exon  <dig> , exon  <dig>  and exon  <dig>  were used to amplify functional mhc class ii drb alleles from the cdna of three bats from each species, c. perspicillata, d. rotundus and m. molossus.
the sequences obtained were used to design specific primers complementary to conserved parts of exon  <dig>  and batcmhiif.
<dig> , table 1) and exon  <dig>  and batcmhiir.
<dig> , table 1).
the primer combinations f-r and f1-r <dig> were used as the first pcr for d. rotundus and c. perspicillata .
for the second pcr, all of the newly designed primers were used together with jsex <dig> , ex1rd, ex3rd and l <dig> in different combinations to screen the cdna of a total of  <dig> c. perspicillata and  <dig> d. rotundus .
two different primer combinations with js2cape, ex3rd, r and r <dig> were used to amplify the drb alleles from the cdna of 40 m. molossus .
the pcr products were cloned using the pcr™4-topo® ta cloning® kit .
ten clones per pcr amplification were sent for sequencing to beckman coulter genomics  using t <dig> and t <dig> primers.table  <dig> pcr primers used to amplify mhc class ii dr beta loci in the three species investigated: c. perspicillata, d. rotundus and m. molossus
d. rotundus
ex1fdb
jsex <dig> -drb
js2cape-drb
c. perspicillata
ex3rdb
d. rotundus
asee additional file: figure s <dig>  bprimer modified from richman et al.
<cit>  to amplify the exon  <dig> of mhc class ii drb gene
amplification and sequencing of the mtdna d-loop
all amplifications of mitochondrial dna  control region  for the three species were performed using genomic dna.
the primers f and p  <cit>  were used to amplify the hypervariable domain hvi of mtdna d-loop.
pcrs were performed using a standard procedure with biotaq™ dna polymerase pcr kit .
sequencing was carried out by beckman coulter genomics  using f and p primers.
sampling analysis
to assess the environmental impact on the mhc class ii drb diversity, the data were analyzed according to the location of the capture sites and the type of environment at these sites.
for d. rotundus and m. molossus, each capture site corresponded to one environment type.
d. rotundus sampling sites corresponded to two different roots  and cave m ), considered as pf sites, and one foraging area ), an eh site .
m. molossus individuals were captured in one foraging area ), an aa site, and two different roosts located at the eh site with different disturbance levels: paracou  with a high level of disturbance and the sa site with a low level of disturbance .
c. perspicillata individuals were captured in the four types of environments pf, eh, aa and ur .
to explore the potential bias induced by the spatial structure of collecting sites in c. perspicillata samples, spatial structure was compared with environment structure.
data analysis
sequences were edited using geneious r <dig>  <cit> .
dna sequences were aligned using the mafft alignment tool  <cit>  included in the software.
sequences were confirmed as mhc class ii drb and mtdna d-loop by homology analysis using the ncbi blast search  <cit> .
a drb clone sequence was regarded as valid if the following criteria were met:  incidence in at least two independent pcrs either from the same individual or different ones, or amplification by two different primer pairs, and  identified by at least three identical clones  <cit> .
haplotypes were reconstructed for each bat species using the dnasp  <dig>  <cit> .
as proposed by  <cit> , the nomenclature of mhc alleles for non-human species was used: to each allele a prefix  was given, with the serial number attached as follows: cape-drb for c. perspicillata, dero-drb for d. rotundus and momo-drb for m. molossus.
cape-drb alleles identified in this study were named after the  <dig> alleles previously described by  <cit> .
sequence diversity
allele frequencies , proportion of segregating sites , nucleotide diversity , observed  and expected  heterozygosities, and gene diversity  were estimated using arlequin  <dig>   <cit> .
allelic richness , with a correction for sample size using a rarefaction method and inbreeding coefficients  were calculated using fstat  <dig> .
<cit> .
the mean number of differences between nucleotide and amino acid alleles were counted using mega  <dig>   <cit> .
analyses were performed per species for the entire dataset, and for each environment subgroup separately.
gene conversion and recombination events
evidence of genetic recombination or gene conversion events between drb sequences was assessed using geneconv  <dig> a  <cit> .
this program detects lengthy patterns shared between sequences despite the existence of a pronounced polymorphism.
p-values were determined from both global and pairwise permutation tests, with  <dig>  replicates.
no mismatches between fragments were accepted, and global p-values were corrected for multiple comparisons .
recombination breakpoints were identified using the gard and sbp algorithms  <cit>  implemented in the hyphy package  <cit> , available in the standard datamonkey analysis library .
codon-based analysis of positive selection
putative antigen-binding sites  were identified by comparison with the human abs of the hla-drb <dig>  <cit> .
the relative rates of dn and ds substitutions were calculated for putative abs, non-abs and all codons following the method of nei and gojobori with the jukes and cantor correction for multiple hits  <cit> .
a z-test was performed to assess the deviation of the dn/ds  ratio from  <dig> .
these tests were performed using mega  <dig> .
codons potentially subjected to positive selection were further identified with no a priori assumption of abs.
for this purpose, two maximum-likelihood  frameworks were carried out as proposed by  <cit> .
first, two alternative models comprised in the codeml program included in the paml package version  <dig>  were executed  <cit> .
the first one  allows codons to evolve either neutrally  or under purifying selection , while the second one  considers a class of sites under positive selection .
these models were compared with a likelihood ratio test  with two degrees of freedom   <cit> .
as previously described, each analysis was run twice, with starting ω values of  <dig>  and  <dig> , to ensure convergence  <cit> .
the f3 ×  <dig> model of codon frequencies was assumed for all analyses.
the amino acids positively selected were identified using the bayes empirical bayes approach , as recommended by yang  <cit> , with the cutoff posterior probability set at 90 %.
secondly, five ml methods proposed in the hyphy package were used.
for all analyses, the best fitting nucleotide substitution model was assessed with the automatic model selection tool available on the server.
a single likelihood ancestor counting  model was used to detect evidence of the non-neutral evolution of the drb gene  <cit> .
based on ancestral reconstruction, this model counts dn and ds changes at each codon position in a phylogeny.
fixed  and random effects likelihood  models were performed to estimate the ω ratio on a site-by-site basis  <cit> .
for the fel model, ω ratios were estimated with an a priori distribution of rates across sites, while this distribution was determined by the present data for the rel model.
an internal fel  model was carried out to highlight selection pressure at the population level .
finally, a mixed effects model of evolution  was performed to highlight both diversifying and importantly episodic selection at individual sites  <cit> .
codon positions were regarded as candidates for positive selection if the following two criteria were met:  p-values <  <dig>  for slac, fel, ifel and meme and  bayes factor >  <dig> for rel.
accordingly, only sites identified as being under positive selection by at least two approaches were considered  <cit> .
analyses were performed per species for the entire dataset and for each environment subgroup separately.
genetic differentiation and past population dynamics
for both neutral and functional markers, the genetic differentiation  among environment subgroups and collecting sites was calculated through an implementation of analysis of molecular variance  using arlequin  <dig> .
isolation-by-distance  was examined with the web service idbws v. <dig>   by assessing the statistical significance of the correlation in a mantel test with  <dig>  permutations.
the quantity f
st/ was used as a genetic distance according to rousset  <cit> .
geographic distances between populations were determined from gps coordinates recorded at each collecting site.
to add a measurement of relationships between alleles at the intraspecific level, haplotype networks were constructed for each species using the parsimony statistical algorithm tcs implemented in popart  <dig>   <cit> .
a bayesian skyline plot was used to investigate changes in effective population size  over time with beast  <dig> .
<dig>  <cit> , using mtdna d-loop data only.
markov chain monte carlo samples were based on 2 ×  <dig> generations, logging every  <dig> steps, with the first  <dig> , <dig> generations discarded as the burn-in.
the best-fitted model hky + g was used with a relaxed molecular clock.
the number of stepwise changes in ne was set at five.
mhc class ii drb phylogeny
phylogenetic relationships and trans-species polymorphism between drb alleles were inferred using a bayesian inference approach implemented in mrbayes  <dig>  <cit> .
the hla-drb <dig> sequence  was used as outgroup.
the gtr + g + i model was selected as the best-fitted model of nucleotide substitution using jmodeltest  <dig>  <cit>  under corrected akaike information criteria .
the program was run 10 ×  <dig> generations, with a sampling frequency of  <dig> and a 25 % burn-in.
validation of the inference was assessed based on the standard deviation of split frequencies, less than the expected threshold value of  <dig> .
RESULTS
drb sequence characterization
one hundred and three bats  were tested for the amplification of mhc drb using cdna.
pcr product sizes ranged from 357 bp to 600 bp, for both c. perspicillata and d. rotundus and covered all of exon  <dig>  .
for m. molossus, we successfully amplified 244 bp, excluding primers, of exon  <dig>  we successfully obtained  <dig> mhc drb-like fragments from  <dig> bats .
based on blast searches, sequences showed homologies to only drb loci.
considering validation criteria,  <dig> sequences were validated, corresponding to  <dig> bats .
one hundred and ten sequences were validated for  <dig> c. perspicillata, corresponding to  <dig> cape-drb alleles .
two hundred and thirty-three sequences were validated for  <dig> d. rotundus, corresponding to  <dig> dero-drb alleles .
one hundred and forty-seven sequences were validated for 28 m. molossus, corresponding to  <dig> momo-drb alleles .
all amino acid sequences were unique.
a maximum of three transcribed alleles per individual was detected in one c. perspicillata and two d. rotundus individuals .
no more than two transcribed alleles were identified in m. molossus samples .
all sequences showed blast homology with the hla-drb <dig> locus , with a maximum of 85 % nucleotide identity and 75 % amino acid identity.
the percentage of nucleotide and amino acid identity with other bat species  was above 87 % and 75 % for all transcripts, respectively.
detection of indels
two alleles  presented a 3-bp deletion at position 217– <dig> of the nucleotide alignment .
these single-codon indels did not induce a frame-shifting mutation or a stop codon.
bats carrying these alleles were heterozygotes and found in eh and ur environments.
two other alleles  presented a 6-bp insertion at position 115– <dig> of the alignment.
these two-codon indels – one serine  and one asparagine  – did not alter the reading frame.
dero-drb* <dig> was carried by four bats, all captured in sa, with one homozygote.
dero-drb* <dig> was carried by one heterozygote bat captured in cf.
no indel event was detected for m. molossus.fig.
<dig> amino acid sequence variation and overall frequency of mhc class ii dr beta exon  <dig> alleles of c. perspicillata, d. rotundus and m. molossus.
drb sequences of homo sapiens , c. perspicillata , n. albiventris  and a. jamaicensis  are given for comparison.
antigen-binding sites  of the hla-drb <dig> molecule are shadowed  <cit> .
dots mark identity with the top sequence.
numbers in italics indicate the amino acid positions within the beta  <dig> domain of the hla-drb <dig> molecule.
asterisks indicate the positively selected sites  identified in each species, according to acceptance criteria described in the methods section  <cit> .
† indicates pss identified by  <cit> , and ‡ indicates pss identified by  <cit>
mtdna d-loop sequence characterization
we amplified the first hypervariable segment  of the mitochondrial dna  control region  for the  <dig> bats tested.
we obtained a 291-bp alignment for m. molossus, a 340-bp alignment for c. perspicillata and a 359-bp alignment for d. rotundus.
thirty-nine mtdna d-loop haplotypes were identified for c. perspicillata ,  <dig> for m. molossus  and only five for d. rotundus despite the high number of sequenced individuals  .table  <dig> the mhc class ii dr beta  allelic diversity in  <dig> c. perspicillata,  <dig> d. rotundus and 28 m. molossus
c. perspicillata
d. rotundus
m. molossus
the sample size , number of alleles , allelic richness , number of segregating sites , nucleotide diversity , number of nucleotide and amino acid differences  per sequence from averaging overall sequence pairs, observed  and expected  heterozygosities, gene diversity , inbreeding coefficients  and their p-values are given for each species and defined subgroups.
– computations not performed
drb allelic diversity
overall, c. perspicillata presented the highest values of allelic richness , number of segregating sites , nucleotide diversity , gene diversity  and mean nucleotide and amino acid differences , while m. molossus exhibited the lowest values .
the highest observed heterozygosity  was recorded in d. rotundus and, as for the two other species, h
o was lower than expected heterozygosities .
considering environment subgroups, the highest value of r was detected in ur for c. perspicillata, , in cf for d. rotundus , and in pa for m. molossus .
s ranged from  <dig> in cc for m. molossus to  <dig> in eh for c. perspicillata.
the highest values of π were found in pf for c. perspicillata, cf for d. rotundus and cc for m. molossus .
fis values were high in all species as well as in all the subgroups per species, with values ranging from  <dig>   for d. rotundus to  <dig>   for m. molossus.
recombination tests did not reveal fragments involved in gene conversion, recombination events or breakpoints among individuals from the same species or within a given environment or between species.
mtdna d-loop allelic diversity
c. perspicillata had the highest allelic richness values , number of segregating sites , gene diversity  and mean nucleotide differences:  <dig>  ±  <dig>  .
nucleotide diversity, for each species, was low .
considering environment subgroups, π values ranged from  <dig>  ±  <dig>  in cf  to  <dig>  ±  <dig>  in aa .
the highest r values were detected in sa , aa and eh .
s ranged from one in cf  to  <dig> in aa , and h
d ranged from  <dig>  in cf  to  <dig>  in aa and eh .
distribution of drb alleles across the different environments
c. perspicillata
three alleles  were shared either between bats trapped in the same environment  or between bats trapped in distinct environments .
these three alleles were the most common, with an overall frequency  of  <dig>  ±  <dig>  for cape-drb* <dig> and  <dig>  ±  <dig>  for both cape-drb* <dig> and cape-drb* <dig> .
cape-drb* <dig> was shared between one bat trapped in pf  and two bats trapped in eh.
cape-drb* <dig> was shared between two bats trapped in eh .
nine individuals in this region were homozygotes .
cape-drb* <dig> was shared between two bats trapped in pf and eh.
alleles found in ur were private alleles and three of the five trapped bats were heterozygotes.
d. rotundus
up to  <dig> alleles were shared either between bats roosting in the same area or between bats foraging in the same area.
out of these alleles, one  was shared between six bats from the three sampling sites  .
five alleles  were shared between bats from sa and cm sites, and two alleles  between the sa and cf sites.
dero-drb* <dig> and  <dig> had the highest overall frequency of  <dig>  ±  <dig>  .
four alleles  were exclusively shared between individuals trapped in sa.
two alleles  were solely shared between individuals trapped in cm.
we did not find shared alleles between bats trapped in cf .
sixteen of the  <dig> bats captured in sa were heterozygotes, five in cm  and three in cf .
m. molossus
up to ten shared alleles were identified but none was shared between the three sampling sites .
three alleles  were shared between bats captured in the sa and pa sites.
two alleles  were shared between individuals from the sa and cc sites.
one allele  was shared between two bats captured in the cc and pa sites.
three alleles  were exclusively shared within sa.
momo-drb* <dig> had the highest overall frequency:  <dig>  ±  <dig>  .
four of the five bats trapped in cc were homozygotes, seven in pa , and  <dig> in sa .
detection of selection signature
an excess of dn substitutions was detected for the three species .
more specifically, in putative antigen binding sites , dn/ds values were highly significant  and ranged from  <dig>  for d. rotundus to  <dig>  for c. perspicillata.
we found significant dn substitutions also occurring in putative non-abs for c. perspicillata  but none in both d. rotundus and m. molossus samples .table  <dig> non-synonymous  and synonymous  substitutions  for c. perspicillata, d. rotundus and m. molossus
p-value
c. perspicillata
d. rotundus
m. molossus
putative abs and non-abs were identified assuming functional homology to the human abs of the hla-dr <dig> molecule brown et al.
<cit> .
n is the number of codons in each sequence region.
z-test values and p-values are shown
paml analyses  showed significant support for m <dig> and m <dig> , 2Δl = , df =  <dig>  p <  <dig> ), indicating an effect of positive selection acting on the drb gene.
overall,  <dig> codons appeared to be under positive selection for c. perspicillata, eight for d. rotundus, and six for m. molossus .
three positively selected sites  were common to the three species investigated.
these codons were the only common between d. rotundus and m. molossus.
six of the eight pss identified in d. rotundus were also found in c. perspicillata, and all of the pss identified in m. molossus were identified in c. perspicillata.table  <dig> identification of species-specific positively selected sites  by maximum likelihood  analysis
sample size  considered for each analysis is shown.
sites identified as positively selected by at least two ml analyses are underlined.
codons are given with a posterior probability ≥  <dig> % in the bayes empirical bayes  analysis for paml m8; p-values <  <dig>  for slac, fel, ifel and meme; bayes factor >  <dig> for rel.
† indicates pss found only in a specific region for each species.
validated pss also found with human antigen-binding sites  are shadowed
regarding environment subgroups for all species, a maximum of  <dig> pss was identified in ur for c. perspicillata, eight in cf for d. rotundus and  <dig> in sa for m. molossus.
three pss were found in all environment subgroups for d. rotundus, four in c. perspicillata and four in m. molossus.
a maximum of six unique pss  was observed for c. perspicillata, four for m. molossus and two unique pss were observed in the two roosts  for d. rotundus .
none of the pss detected in c. perspicillata was shared between ur and pf.
the same result was obtained between sa and cm for d. rotundus and between pa and cc or sa for m. molossus.
thirteen of the  <dig> pss identified were located within the human abs region for c. perspicillata, seven for d. rotundus and five for m. molossus .
all the pss identified in m. molossus were described as pss for n. albiventris,  <dig> for c. perspicillata and six for d. rotundus.
six pss in c. perspicillata were also found in a. jamaicensis, three for m. molossus and one for d. rotundus.
the sites where pss differed were located within one to six amino acid residues  of the human abs or other bat species pss, depending on the bat species investigated.
drb population structure
we found no significant genetic differentiation between drb alleles of the environment subgroups defined for each species  to  <dig> ]; d. rotundus: fst range: ; m. molossus: fst range: , additional file 1: table s6 − 8).
taking into account the collecting sites, pairwise differentiation tests did not reveal significant genetic differentiation between drb alleles  to  <dig> ]; d. rotundus: fst range: ; m. molossus: fst range: ,  − 8).
minimum spanning trees showed complex connections between the haplotypes, marked by numerous nucleotide mutations, without clustering of haplotypes either by site or by environment .
we did not find relevant isolation-by-distance in our samples .fig.
<dig> haplotype networks of mhc class ii dr beta exon  <dig> alleles and of the mitochondrial dna  control region  at the nucleotide level for c. perspicillata.
nodes are proportional to the number of bats carrying each haplotype, colored by the environments where the bats were trapped .
hatch marks represent mutations.
interruptions in lines indicate the presence of more than ten mutations
fig.
<dig> haplotype networks of mhc class ii dr beta exon  <dig> alleles and of the mitochondrial dna  control region  at the nucleotide level for c. perspicillata based on collecting sites.
nodes are proportional to the number of bats carrying each haplotype, colored by the capture site where the bats were trapped .
hatch marks represent mutations
fig.
<dig> haplotype networks of mhc class ii dr beta exon  <dig> alleles and the mitochondrial dna  control region  at the nucleotide level for d. rotundus.
nodes are proportional to the number of bats carrying each haplotype, colored by the capture site where the bats were trapped .
hatch marks represent mutations.
interruptions in lines indicate the presence of more than ten mutations
fig.
<dig> haplotype networks of mhc class ii dr beta exon  <dig> alleles and of the mitochondrial dna  control region  at the nucleotide level for m. molossus.
nodes are proportional to the number of bats carrying each haplotype, colored by the capture site where the bats were trapped .
hatch marks represent mutations.
interruptions in lines indicate the presence of more than ten mutations
mtdna d-loop population structure
we found significant genetic differentiation between some populations of d. rotundus .
no significant genetic differentiation was observed between populations for the two other species  to ]; m. molossus fst range: , additional file 1: table s6–8).
pairwise computations performed between collecting sites did not reveal significant genetic differentiation  to  <dig> ], additional file 1: table s6–8).
the haplotype networks for c. perspicillata revealed two main groups of haplotypes that possessed all the haplotypes found in the different environments or collecting sites .
the same result was obtained for m. molossus, with a maximum of four mutations between haplotypes .
the lowest number of mutations was found for d. rotundus for which the five haplotypes were linked by one connection, marked by only one or two mutations .
no population expansion or collapse was observed for the three species.
we did not find relevant isolation-by-distance in our samples .
drb phylogenetic relationships
the analysis included the  <dig> drb sequences identified in this study , as well as the  <dig> published chiropteran drb sequences selected randomly from a. jamaicensis  <cit> ,  <dig> from c. perspicillata  <cit> ,  <dig> from myotis spp.
,  <dig> from noctilio spp.
and  <dig> from s. bilineata  <cit> .
the phylogenetic tree showed six major clades .
we observed a clustering by species or genus supported by medium and high posterior probabilities – c. perspicillata , d. rotundus , m. molossus , noctilio spp.
– with an intermingled clustering of n. albiventris and n. leporinus drb alleles.
within the c. perspicillata clade, drb sequences clustered with the previously published sequences, with a high posterior probability .
one drb sequence of c. perspicillata was related to a d. rotundus clade with a posterior probability of  <dig> .
one drb sequence from s. bilineata clustered with the clade noctilio spp., with a low posterior probability value.
the  <dig> drb sequences of the a. jamaicensis species clustered with five drb sequences of d. rotundus and two drb sequences of c. perspicillata, with a posterior probability of  <dig> .
within this phyllostomid clade, we observed a species-dependent clustering with high posterior probabilities.
two drb sequences of c. perspicillata clustered with a low posterior probability with two sequences from a. jamaicensis.
we did not observe a characteristic clustering for s. bilineata and myotis spp.
however, one drb sequence from m. velifer clustered together with six drb sequences from s. bilineata with a low posterior probability.fig.
<dig> phylogenetic relationships of the mhc class ii dr beta sequences based on part of exon  <dig>  with values of posterior probabilities for nodes.
species designation follows the genbank accession numbers for previously described drb sequences.
arja a. jamaicensis, cape c. perspicillata, dero d. rotundus, momo m. molossus, myve m. velifer, myvi m. vivesi, noal n. albiventris, nole n. leporinus, sabi s. bilineata.
hla-drb <dig> was used as the outgroup.
tsp indicates trans-species polymorphism of the newly described drb alleles with previously described drb alleles
discussion
in this study, we explored the genetic variability of the expressed mhc drb genes of three sympatric neotropical bat species, looking for selection signatures within the pbr and investigating the role of the environment and the population structure on mhc diversity.
diversity patterns in the mhc class ii drb gene
in the three species, identified alleles  were unique in amino acid sequences, suggesting a non-redundant nucleotide polymorphism.
the number of alleles detected per species and per environment subgroup within each species indicated a relatively high level of mhc drb variation.
these results are in agreement with previously reported allelic copy number variation between or within bat species .
this high drb polymorphism was observed in the three species whatever the degree of habitat disturbance .
in addition, no loss of polymorphism induced by low host and/or pathogen diversity, expected to occur in disturbed environments  <cit>  was observed.
furthermore, mtdna d-loop sequences, considered as neutral, revealed high levels of genetic diversity for c. perspicillata and m. molossus.
high levels of neutral diversity have been associated with either multiple geographic origin and/or population admixture  <cit> , suggesting that c. perspicillata and m. molossus populations originated from one of these two phenomena or from both.
the high level of drb polymorphism can be related to population history for these two species with the effects of additional selection factors such as pathogen-mediated selection.
a high number of drb homozygotes was also found in m. molossus in all habitats as well as in c. perspicillata but in edge habitats only.
for these two species, the presence of a specific allele  ensuring an adequate immunity can be hypothesized  <cit> .
considering d. rotundus, a high number of heterozygotes for drb was observed in all habitats.
this result suggests a heterozygote advantage related to the forest  environment known to encompass higher species and pathogen richness  <cit> .
the most frequent mhc drb alleles encountered in each bat species  were all identified in forest environments.
these alleles are likely ancient and play a significant role in the immune response, conferring a selective advantage to the bats in sympatry in these habitats.
indeed, given that forest environments are characterized by a high level of pathogen diversity, these alleles most likely possess enhanced recognition capabilities and might be able to recognize a broader range of pathogens.
they thus allow a more efficient immune response in line with the ecological diversity.
moreover, the geographical structure highlighted in d. rotundus, using mtdna d-loop results, did not affect the allelic distribution.
indeed, most of the alleles identified in d. rotundus were shared between sites, and one allele  was present at all sites.
in contrast, no geographical structure was found in c. perspicillata even if the alleles identified in urban and periurban areas were rare and not shared with other groups.
these results suggest that environment rather than population structure drives the allelic distribution, corroborating a local adaptation hypothesis.
although edge and forest habitats may differ in pathogenic pressures , the results observed for c. perspicillata may be related to the high tolerance of this species to habitat disturbance and to its dominance in bat communities in edge habitats  <cit> , likely allowing continuous exposure to the communities of pathogens of both pristine and disturbed forests, and pathogen transfer between these habitats.
duplication detected in phyllostomidae
gene conversion and recombination events were not detected for any species.
the identification of three expressed drb alleles in individuals from c. perspicillata and d. rotundus supported the existence of at least two functional drb loci.
however, underestimation of the number of alleles and expressed loci can be suspected, considering the limited number of recombinant clones per primer combinations and the use of cdna only allowing the detection of the most frequently expressed alleles.
the occurrence of multiple mhc drb loci varying in the number of loci between individuals and species was reported in neotropical bat species such as c. perspicillata , s. bilineata   <cit> , a. jamaicensis   <cit> , as well as in other animals  <cit> .
together with environmental and biological patterns, gene conversion and recombination processes are known to drive the extensive mhc polymorphism  <cit> .
variable gene duplication between closely related taxa and among individuals from the same species is characteristic of mhc genes and plays a critical role in the adaptive evolution of the host  <cit> .
gene duplication can occur over different time scales and therefore predates or follows speciation events.
furthermore, the absence of recombination between alleles of different species was reported.
this result was regarded as evidence of recent duplication events between loci that occurred after speciation  <cit> .
our findings support this hypothesis since some recent radiation events occurred in neotropical bat species during the pleistocene era  .
despite the indels detected in some c. perspicillata and d. rotundus sequences, the reading frames remained unaltered, suggesting that these sequences encode functional proteins .
nevertheless, the heterozygote-dominant character of individuals with these alleles suggests a turnover of these loci generated by evolutionary processes such as the silencing of duplicate genes by mutation or deletion  <cit> .
under the birth-and-death model, genes created by duplication can be maintained for long periods, deleted or transformed as pseudogenes and, therefore, contribute to host fitness and adaptability  <cit> .
the phenomenon is especially significant among heterozygote d. rotundus, for which one of the two alleles is among the most widespread in the population studied , and supports a shift towards an allele with better recognition capabilities.
evidence of historical positive selection
higher rates of non-synonymous  vs. synonymous  nucleotide substitutions were observed, especially in sites supposedly involved in antigen-binding.
this excess of dn over ds in abs is consistent with balancing selection acting on drb loci, as a sign of historical positive selection in polymorphic mhc genes  <cit> .
furthermore, an unexpected excess of dn in putative non-abs for c. perspicillata suggests strong positive selection acting on these sites and highlights species-specific divergence on sites involved in antigen recognition.
a high congruence was observed between species-specific positively selected sites  in the three species and human abs demonstrating the functional homology of these sites.
in contrast, pss not identified as human abs might play a role in mhc drb recognition capability or in the molecule stability for the species studied.
here, nine of the pss were detected in c. perspicillata, while only one was detected in both d. rotundus and m. molossus.
therefore, the observed excess of dn in c. perspicillata was attributed to pss identified as non-abs in humans.
this result demonstrates the critical role of positive selection in shaping mhc diversity and corroborates that positive selection is driven by a species-specific immune response.
focusing on c. perspicillata, comparisons of pss identified here with those previously described revealed high congruence, even among pss not identified as human abs  <cit> .
a similar result was observed between c. perspicillata and a. jamaicensis, two phyllostomidae with the same diet  <cit> .
taken together, these findings reveal family and species-specific selection pressures acting on mhc genes.
moreover, the high similarity of pss between a. jamaicensis and c. perspicillata highlights that bat species sharing similar feeding strategies might be subjected – independently of their habitats – to similar pathogenic pressures, mainly from their microbiota  <cit> .
comparisons between each environment subgroup, for each present case, revealed the existence of private pss depending on the area, suggesting a habitat-specific selection pressure process.
indeed, while six private pss were counted in urban and periurban areas for c. perspicillata, only two were detected in edge habitats and one in pristine forest.
phylogeny, trans-species polymorphism and demographic process
two mechanisms are thought to be responsible for the trans-specific similarity of mhc genes: convergent evolution  <cit>  and trans-species polymorphism  <cit> .
evidence of trans-species polymorphism in bats was reported in both myotis spp.
and noctilio spp.
<cit>  and at the family level between the two phyllostomids a. jamaicensis and c. perspicillata  <cit> .
phylogenetic analyses in our study did not reveal any characteristic clustering of mhc alleles depending on the habitat, but rather clades at the family, genus and species levels .
these results suggest either similar pathogenic pressures or that these alleles possess a larger antigenic recognition capability that constitutes a major asset to the immune response of these species.
intermingled clusterings observed in this survey  are in agreement with trans-species polymorphism reported in bats  <cit> .
the observed trans-species polymorphism for myotis spp.
<cit>  was not detected in the present study.
the results highlighted clustering between s. bilineata and m. velifer.
however, this clustering was no longer observed by analyzing full-length exon  <dig>  observation of trans-species polymorphism within bat families  strengthens the scenario of independent modes of evolution of mhc drb alleles, allowing balancing selection to retain substantial allelic lineages before speciation events  <cit> .
extended periods of host–pathogen coevolution is thought to contribute to trans-species polymorphism given that host-sharing pathogens and exposure to similar pathogen pressures are believed to induce similar changes in different host species  <cit> .
although pathogen-driven selection is known to be crucial in mhc diversity  <cit> , demographic processes such as genetic drift, bottlenecks, expansions, fragmentation and geographic isolation have been demonstrated to also participate in the shaping of this extensive polymorphism  <cit> .
evolutionary analyses of mhc drb alleles for the three species did not reveal any area-dependent clustering effect.
furthermore, analyses of partial sequences of the mtdna d-loop revealed no structuration for c. perspicillata and m. molossus, whereas it was observed for d. rotundus.
demographic analysis performed for the three species did not detect any substantial bottleneck or expansion.
while the patterns observed for the first two species corroborate the hypothesis of a similarity between the intrinsic genetic diversity of the species and that of mhc, the lack of similarity between these patterns of diversity for d. rotundus contradicts this statement.
these results suggest that the signature of an area-limited pathogen-driven selection, supported by the presence of private pss rather than by demographic processes such as distance isolation, is observed in this case.
CONCLUSIONS
this study was the first to investigate mhc drb polymorphism in three sympatric bat species, m. molossus, c. perspicillata and d. rotundus, from the amazonian region.
our results revealed a high genetic variability in the mhc drb gene.
natural selection, as well as local adaptation driven by different parasite and pathogen exposures across environments, contribute to the maintenance of an extensive mhc polymorphism.
the richness of pathogens in the different habitats should be investigated to strengthen our assumptions on potential local adaptation.
further studies using finer neutral markers such as microsatellites will be necessary to detect possible confounding effects, such as hidden structuring and demographic history, and thus to better understand the drivers of mhc allelic diversity.
additional files
additional file 1: table s <dig>  supplementary methods, including the characteristics of all capture sites ﻿, the list of samples successfully amplified for the mhc class ii drb loci , the distribution of drb alleles per species , the pairwise differentiation computations per species , as well as the correlations of genetics and geographic distance .
additional file 2: figure s <dig>  map of french guiana showing the capture sites of c. perspicillata, d. rotundus and m. molossus.
for clarity, nearby sites within 15 km were grouped.
sites are numbered and labeled according to the type of environment to which they correspond: edge habitats , anthropized areas , pristine primary lowland forests , urban and periurban areas .
pie chart indicates the proportion of bat species sampled, with c. perspicillata in light grey, d. rotundus in black and m. molossus in dark grey.
small charts indicate a total number of individuals caught ≤  <dig>  medium-sized charts  , large charts  .
characteristics of the different sites are given in additional file 1: table s <dig>
additional file 3: figure s <dig>  positions of pcr primers used to amplify the indicated fragments of the mhc class ii loci in c. perspicillata, d. rotundus and m. molossus, based on cdna.
the structure of the cdna of the mhc class ii drb gene is based on  <cit> .
according to each species, boxes indicate the amplified region using each couple of primers.
the shaded region represents the region of interest, namely exon  <dig>  sequences and references of all primers used are given in table  <dig>
abbreviations
absantigen-binding sites
aicccorrected akaike information criteria
bebbayes empirical bayes
drbexon  <dig> of the mhc class ii dr beta
felfixed effects likelihood
ifelinternal fixed effects likelihood
mememixed effects model of evolution
mhcmajor histocompatibility complex
mlmaximum-likelihood
mtdna d-loopmitochondrial dna control region
pbrpeptide-binding region
pcrpolymerase chain reaction
pssspecies-specific positively selected sites
relrandom effects likelihood
slacsingle likelihood ancestor counting
we are grateful to françois catzeflis and marguerite delaval for useful discussions on the biology, ecology and dynamics of bats.
all field volunteers and owners and/or managers of capture sites are warmly acknowledged for their assistance in captures.
funding
a. salmier was supported by a grant from european funds  and "investissement d’avenir" managed by agence nationale de la recherche .
this study was conducted within the carolia program supported by european funds  and assistance from région guyane and direction régionale pour la recherche et la technologie.
it also received a european commission "regpot-ct-2011-285837- stronger" grant within the fp <dig> and "investissement d’avenir" grants managed by the agence nationale de la recherche .
the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
availability of data and materials
nucleotide sequences for the  <dig> drb alleles and for mtdna d-loop are available on genbank .
authors’ contribution
conceived and designed the experiments: as al.
analyzed the data: as al.
contributed reagents/materials/analysis tools: as bdt vl al.
wrote the paper: as bdt bcr vl al.
field work and acquisition data: as al bdt.
all authors read and approved the final manuscript.
competing interests
the authors declare that they have no competing interests.

 regarding adaptation to infectious diseases, genes of the major histocompatibility complex are the most commonly studied in mammals. mhc genes play a significant role in the functionality and effectiveness of the immune response of vertebrates and are directly involved in fitness and adaptation of hosts to their pathogens. therefore, studying mhc gene diversity in non-model animals, known to be reservoirs of pathogens, is a critical tool to assess its evolutionary process and implication in the variation of individual fitness, population viability and evolutionary potential in a changing environment. studies have essentially focused on the genetic variability of exon of the mhc class ii dr beta gene given that this exon encodes the peptide-binding region. hence, most of the variability displayed is recorded on this exon. variations within the pbr define the repertoire of antigenic determinants and subsequently the ability to recognize a variable number of circulating pathogens. optimum resistance to pathogens is, therefore, the result of all intrinsic and extrinsic factors that influence the genetic variability of individuals’ immune system. most of the vertebrate populations studied so far exhibited high levels of mhc diversity both in the number of alleles and in their heterozygosities and allelic variation. pathogen-driven balancing selection and sexual selection seem to play a fundamental role in the preservation of this high level of polymorphism. pathogen-driven balancing selection acts through antagonistic host–parasite coevolution via several mechanisms: overdominant and frequency-dependent selection, as well as spatial and temporal variation in host pathogens. sexual selection pressures encompass mechanisms such as maternal–fetal interactions and mate selection. however, when assessing the genetic variability of both neutral markers and mhc genes, studies have highlighted the role of past demographic processes in shaping the pattern of mhc variability that can sometimes surpass that of natural selection. local immunogenetic adaptation of hosts that live in different environments was associated with different parasite and pathogen pressures. indeed, differences in the diversity of pathogens are directly linked to environmental components. these latter modulate parasite and pathogen species richness, their survival and adaptability, as well as their distribution, transmission, developmental success and their ability to induce diseases. environmental components likewise impact the richness, population dynamics, immunocompetence and nutritional status of host species, all of which subsequently determine resistance or susceptibility to disease. a strong correlation was also shown between host and parasite species richness, their life history and ecological traits. moreover, anthropogenic alterations of habitats induce changes in host–pathogen–environment interactions and are consequently linked to the emergence of infectious zoonotic diseases. therefore, considering the role of the environment is critical for the assessment of mhc gene variability. bats are the second largest species-rich mammalian order, accounting for 20 % of all mammal species in the world. compared to other mammals, their uncommon biological features and their long-term evolutionary history with pathogens make them excellent reservoirs and spreaders of viruses, which have a significant impact on both human and animal health. however, bats rarely exhibit clinical or pathological signs of diseases and appear to coexist with their pathogens in a disease-free state, characteristic of reservoir hosts. at the order level, the monophyly of chiroptera was evidenced via phylogenetic relationships inferred from intron sequences of drb. the monophyletic origin of drb genes was also evidenced at the family level via phylogenetic relationships inferred from drb sequences from saccopteryx bilineata, myotis spp., noctilio spp. and carollia perspicillata. other studies, investigating the diversity of mhc drb, showed significant differences in the polymorphism of mhc genes between species. this polymorphism was essentially influenced by a pathogen-driven selection, a social structure driven by mhc-mediated post-copulatory mechanisms, diversifying selection and recombination events and geographical constraints resulting in spatial variation of pathogen-mediated selection and enhanced susceptibility to environmental changes. the amazon, a major biodiversity hotspot in south america, possesses a large diversity of bat species and pathogens in a wide variety of climates and vegetation formations. french guiana, a tropical amazonian region near the equatorial zone, harbors a great diversity of bats, with species registered. in this region, the impact of deforestation on the composition and dynamics of bat communities was assessed as well as, the role of habitats on rabies virus circulation and maintenance. in this study, we analyzed the genetic variability of the expressed mhc class ii drb exon in three bat species: two phyllostomidae, carollia perspicillata and desmodus rotundus, and one molossidae, molossus molossus. these three widely distributed species have ecological plasticity and tolerance to disturbance. in french guiana, these species are sympatric and use the same habitats. the key hypothesis of this study was that composition and distribution of mhc drb alleles were specific to the environments, rather than randomly distributed in space. consequently, we should observe local immunogenetic adaptation to the contrasting pathogen pressures or equally adapted alleles. to assess which are the best factors that predict the mhc diversity, pathogen-mediated selection, recombination, gene conversion, demographic history and population structure were investigated. there is a higher diversity of microorganisms in forest environments, compared to disturbed environnments, due to greater host species richness and better transmission-promoting parameters. for this reason, we expect higher levels of mhc diversity in forest environments facing lower disturbance pressures, where higher parasite and pathogen diversities imply a higher selection pressure. furthermore, assuming that bats using the same roosting area and/or the same foraging areas would be subjected to similar pathogen pressures, we should observe similar trends in intra- and inter-specific mhc diversity. in contrast, once the demographic neutral genetic histories—which may also influence mhc diversity—are controlled, we should observe differences in selective histories between bats inhabiting different environments. to identify different signatures of selection in the drb exon which would imply area-specific recognition capabilities, conformation of the hla-drb was used as a reference to detect putative sites directly involved in antigen binding and consequently subjected to selection. additionally, to reduce the misidentification bias of abs, the selection signature within the pbr was investigated without a priori identifying species-specific abs for further comparisons. mhc genes exhibit high levels of allele similarity within species as well as between related species and the occurrence of identical mhc alleles in related species is frequent. convergence and trans-species polymorphism are thought to be responsible for this trans-species evolution. to highlight which of these two mechanisms acts predominantly on the evolutionary history of the drb gene in the three species investigated, phylogenetic relationships were inferred from the drb sequences obtained here and with other available chiropteran sequences. finally, mhc spatial diversity was compared to that of neutral markers to highlight the impact of demographic processes and population structure on the diversity pattern in the three bat species investigated. methods ethics statement animals were captured, handled, sampled and, whenever necessary, euthanized following asm guidelines under the supervision of researchers who had been granted the french “expérimentation animale niveau 1” diploma. bats are not protected by law in french guiana; however, the project was submitted to the conseil scientifique régional pour le patrimoine naturel de la guyane and approved. captures that occurred within protected areas received approval by the conseil scientifique régional du patrimoine naturel on january and ad-hoc authorizations. study areas, sample collection, dna and rna extraction the three bat species were sampled at sites. three sites were located in pristine lowland forests with low disturbance pressure, four sites were located in edge habitats, five in anthropized areas, and two in urban and periurban areas.fig. map of sampling sites of all individuals used in this study across four environments. for clarity, nearby collecting sites within 15 km were grouped. sites are numbered and labeled according to the type of environment to which they correspond: edge habitats, anthropized areas, pristine primary lowland forests, urban and periurban areas. pie charts indicate the proportion of the most frequent alleles; dero-drb* and for d. rotundus; momo-drb* for m. molossus ), other shared alleles and private alleles in each collecting sites/environments, for each species: c. perspicillata, d. rotundus and m. molossus bats were trapped with mist nets erected near breeding sites, roosts, at forest edges, around livestock, or through putative foraging courses. bat species were identified in the field using external morphology and, prior to release, a brachial vein puncture and wing biopsies were performed. a few individuals captured were euthanized at the laboratory to collect organs. a total of c. perspicillata, d. rotundus and 42 m. molossus were sampled. nucleic acids were extracted from blood, liver or spleen depending on sampling. extractions were performed using the nuclisens easymag bio-robot. amplification and genotyping of the mhc class ii drb gene all amplifications of mhc drb genes for the three species were performed using cdna obtained from liver and spleen rnas. all cdnas were synthesized using superscript® iii reverse transcriptase and random hexamers following the manufacturer’s recommendations. expressed mhc drb genes of the three bat species were amplified by a minimum of two independent polymerase chain reactions using a standard procedure with the amplitaq gold dna polymerase pcr kit. to optimize the detection of different alleles, a minimum of two distinct pcrs per individual were performed. species-specific primers were designed for c. perspicillata and d. rotundus to avoid occurrence of non-amplifying alleles. to amplify the m. molossus drb gene, primers designed by richman et al. were modified. primers located in conserved parts of exon, exon, exon and exon were used to amplify functional mhc class ii drb alleles from the cdna of three bats from each species, c. perspicillata, d. rotundus and m. molossus. the sequences obtained were used to design specific primers complementary to conserved parts of exon and batcmhiif., table 1) and exon and batcmhiir., table 1). the primer combinations f-r and f1-r were used as the first pcr for d. rotundus and c. perspicillata. for the second pcr, all of the newly designed primers were used together with jsex, ex1rd, ex3rd and l in different combinations to screen the cdna of a total of c. perspicillata and d. rotundus. two different primer combinations with js2cape, ex3rd, r and r were used to amplify the drb alleles from the cdna of 40 m. molossus. the pcr products were cloned using the pcr™4-topo® ta cloning® kit. ten clones per pcr amplification were sent for sequencing to beckman coulter genomics using t and t primers.table pcr primers used to amplify mhc class ii dr beta loci in the three species investigated: c. perspicillata, d. rotundus and m. molossus d. rotundus ex1fdb jsex -drb js2cape-drb c. perspicillata ex3rdb d. rotundus asee additional file: figure s bprimer modified from richman et al. to amplify the exon of mhc class ii drb gene amplification and sequencing of the mtdna d-loop all amplifications of mitochondrial dna control region for the three species were performed using genomic dna. the primers f and p were used to amplify the hypervariable domain hvi of mtdna d-loop. pcrs were performed using a standard procedure with biotaq™ dna polymerase pcr kit. sequencing was carried out by beckman coulter genomics using f and p primers. sampling analysis to assess the environmental impact on the mhc class ii drb diversity, the data were analyzed according to the location of the capture sites and the type of environment at these sites. for d. rotundus and m. molossus, each capture site corresponded to one environment type. d. rotundus sampling sites corresponded to two different roots and cave m ), considered as pf sites, and one foraging area ), an eh site. m. molossus individuals were captured in one foraging area ), an aa site, and two different roosts located at the eh site with different disturbance levels: paracou with a high level of disturbance and the sa site with a low level of disturbance. c. perspicillata individuals were captured in the four types of environments pf, eh, aa and ur. to explore the potential bias induced by the spatial structure of collecting sites in c. perspicillata samples, spatial structure was compared with environment structure. data analysis sequences were edited using geneious r. dna sequences were aligned using the mafft alignment tool included in the software. sequences were confirmed as mhc class ii drb and mtdna d-loop by homology analysis using the ncbi blast search. a drb clone sequence was regarded as valid if the following criteria were met: incidence in at least two independent pcrs either from the same individual or different ones, or amplification by two different primer pairs, and identified by at least three identical clones. haplotypes were reconstructed for each bat species using the dnasp. as proposed by, the nomenclature of mhc alleles for non-human species was used: to each allele a prefix was given, with the serial number attached as follows: cape-drb for c. perspicillata, dero-drb for d. rotundus and momo-drb for m. molossus. cape-drb alleles identified in this study were named after the alleles previously described by. sequence diversity allele frequencies, proportion of segregating sites, nucleotide diversity, observed and expected heterozygosities, and gene diversity were estimated using arlequin. allelic richness, with a correction for sample size using a rarefaction method and inbreeding coefficients were calculated using fstat.. the mean number of differences between nucleotide and amino acid alleles were counted using mega. analyses were performed per species for the entire dataset, and for each environment subgroup separately. gene conversion and recombination events evidence of genetic recombination or gene conversion events between drb sequences was assessed using geneconv a. this program detects lengthy patterns shared between sequences despite the existence of a pronounced polymorphism. p-values were determined from both global and pairwise permutation tests, with replicates. no mismatches between fragments were accepted, and global p-values were corrected for multiple comparisons. recombination breakpoints were identified using the gard and sbp algorithms implemented in the hyphy package, available in the standard datamonkey analysis library. codon-based analysis of positive selection putative antigen-binding sites were identified by comparison with the human abs of the hla-drb. the relative rates of dn and ds substitutions were calculated for putative abs, non-abs and all codons following the method of nei and gojobori with the jukes and cantor correction for multiple hits. a z-test was performed to assess the deviation of the dn/ds ratio from. these tests were performed using mega. codons potentially subjected to positive selection were further identified with no a priori assumption of abs. for this purpose, two maximum-likelihood frameworks were carried out as proposed by. first, two alternative models comprised in the codeml program included in the paml package version were executed. the first one allows codons to evolve either neutrally or under purifying selection, while the second one considers a class of sites under positive selection. these models were compared with a likelihood ratio test with two degrees of freedom. as previously described, each analysis was run twice, with starting ω values of and, to ensure convergence. the f3 ×  model of codon frequencies was assumed for all analyses. the amino acids positively selected were identified using the bayes empirical bayes approach, as recommended by yang, with the cutoff posterior probability set at 90 %. secondly, five ml methods proposed in the hyphy package were used. for all analyses, the best fitting nucleotide substitution model was assessed with the automatic model selection tool available on the server. a single likelihood ancestor counting model was used to detect evidence of the non-neutral evolution of the drb gene. based on ancestral reconstruction, this model counts dn and ds changes at each codon position in a phylogeny. fixed and random effects likelihood models were performed to estimate the ω ratio on a site-by-site basis. for the fel model, ω ratios were estimated with an a priori distribution of rates across sites, while this distribution was determined by the present data for the rel model. an internal fel model was carried out to highlight selection pressure at the population level. finally, a mixed effects model of evolution was performed to highlight both diversifying and importantly episodic selection at individual sites. codon positions were regarded as candidates for positive selection if the following two criteria were met: p-values <  for slac, fel, ifel and meme and bayes factor >  for rel. accordingly, only sites identified as being under positive selection by at least two approaches were considered. analyses were performed per species for the entire dataset and for each environment subgroup separately. genetic differentiation and past population dynamics for both neutral and functional markers, the genetic differentiation among environment subgroups and collecting sites was calculated through an implementation of analysis of molecular variance using arlequin. isolation-by-distance was examined with the web service idbws v. by assessing the statistical significance of the correlation in a mantel test with permutations. the quantity f st/ was used as a genetic distance according to rousset. geographic distances between populations were determined from gps coordinates recorded at each collecting site. to add a measurement of relationships between alleles at the intraspecific level, haplotype networks were constructed for each species using the parsimony statistical algorithm tcs implemented in popart. a bayesian skyline plot was used to investigate changes in effective population size over time with beast., using mtdna d-loop data only. markov chain monte carlo samples were based on 2 ×  generations, logging every steps, with the first, generations discarded as the burn-in. the best-fitted model hky + g was used with a relaxed molecular clock. the number of stepwise changes in ne was set at five. mhc class ii drb phylogeny phylogenetic relationships and trans-species polymorphism between drb alleles were inferred using a bayesian inference approach implemented in mrbayes. the hla-drb sequence was used as outgroup. the gtr + g + i model was selected as the best-fitted model of nucleotide substitution using jmodeltest under corrected akaike information criteria. the program was run 10 ×  generations, with a sampling frequency of and a 25 % burn-in. validation of the inference was assessed based on the standard deviation of split frequencies, less than the expected threshold value of.  drb sequence characterization one hundred and three bats were tested for the amplification of mhc drb using cdna. pcr product sizes ranged from 357 bp to 600 bp, for both c. perspicillata and d. rotundus and covered all of exon. for m. molossus, we successfully amplified 244 bp, excluding primers, of exon we successfully obtained mhc drb-like fragments from bats. based on blast searches, sequences showed homologies to only drb loci. considering validation criteria, sequences were validated, corresponding to bats. one hundred and ten sequences were validated for c. perspicillata, corresponding to cape-drb alleles. two hundred and thirty-three sequences were validated for d. rotundus, corresponding to dero-drb alleles. one hundred and forty-seven sequences were validated for 28 m. molossus, corresponding to momo-drb alleles. all amino acid sequences were unique. a maximum of three transcribed alleles per individual was detected in one c. perspicillata and two d. rotundus individuals. no more than two transcribed alleles were identified in m. molossus samples. all sequences showed blast homology with the hla-drb locus, with a maximum of 85 % nucleotide identity and 75 % amino acid identity. the percentage of nucleotide and amino acid identity with other bat species was above 87 % and 75 % for all transcripts, respectively. detection of indels two alleles presented a 3-bp deletion at position 217– of the nucleotide alignment. these single-codon indels did not induce a frame-shifting mutation or a stop codon. bats carrying these alleles were heterozygotes and found in eh and ur environments. two other alleles presented a 6-bp insertion at position 115– of the alignment. these two-codon indels – one serine and one asparagine – did not alter the reading frame. dero-drb* was carried by four bats, all captured in sa, with one homozygote. dero-drb* was carried by one heterozygote bat captured in cf. no indel event was detected for m. molossus.fig. amino acid sequence variation and overall frequency of mhc class ii dr beta exon alleles of c. perspicillata, d. rotundus and m. molossus. drb sequences of homo sapiens, c. perspicillata, n. albiventris and a. jamaicensis are given for comparison. antigen-binding sites of the hla-drb molecule are shadowed. dots mark identity with the top sequence. numbers in italics indicate the amino acid positions within the beta domain of the hla-drb molecule. asterisks indicate the positively selected sites identified in each species, according to acceptance criteria described in the methods section. † indicates pss identified by, and ‡ indicates pss identified by mtdna d-loop sequence characterization we amplified the first hypervariable segment of the mitochondrial dna control region for the bats tested. we obtained a 291-bp alignment for m. molossus, a 340-bp alignment for c. perspicillata and a 359-bp alignment for d. rotundus. thirty-nine mtdna d-loop haplotypes were identified for c. perspicillata, for m. molossus and only five for d. rotundus despite the high number of sequenced individuals.table the mhc class ii dr beta allelic diversity in c. perspicillata, d. rotundus and 28 m. molossus c. perspicillata d. rotundus m. molossus the sample size, number of alleles, allelic richness, number of segregating sites, nucleotide diversity, number of nucleotide and amino acid differences per sequence from averaging overall sequence pairs, observed and expected heterozygosities, gene diversity, inbreeding coefficients and their p-values are given for each species and defined subgroups. – computations not performed drb allelic diversity overall, c. perspicillata presented the highest values of allelic richness, number of segregating sites, nucleotide diversity, gene diversity and mean nucleotide and amino acid differences, while m. molossus exhibited the lowest values. the highest observed heterozygosity was recorded in d. rotundus and, as for the two other species, h o was lower than expected heterozygosities. considering environment subgroups, the highest value of r was detected in ur for c. perspicillata,, in cf for d. rotundus, and in pa for m. molossus. s ranged from in cc for m. molossus to in eh for c. perspicillata. the highest values of π were found in pf for c. perspicillata, cf for d. rotundus and cc for m. molossus. fis values were high in all species as well as in all the subgroups per species, with values ranging from for d. rotundus to for m. molossus. recombination tests did not reveal fragments involved in gene conversion, recombination events or breakpoints among individuals from the same species or within a given environment or between species. mtdna d-loop allelic diversity c. perspicillata had the highest allelic richness values, number of segregating sites, gene diversity and mean nucleotide differences:  ± . nucleotide diversity, for each species, was low. considering environment subgroups, π values ranged from  ±  in cf to  ±  in aa. the highest r values were detected in sa, aa and eh. s ranged from one in cf to in aa, and h d ranged from in cf to in aa and eh. distribution of drb alleles across the different environments c. perspicillata three alleles were shared either between bats trapped in the same environment or between bats trapped in distinct environments. these three alleles were the most common, with an overall frequency of  ±  for cape-drb* and  ±  for both cape-drb* and cape-drb*. cape-drb* was shared between one bat trapped in pf and two bats trapped in eh. cape-drb* was shared between two bats trapped in eh. nine individuals in this region were homozygotes. cape-drb* was shared between two bats trapped in pf and eh. alleles found in ur were private alleles and three of the five trapped bats were heterozygotes. d. rotundus up to alleles were shared either between bats roosting in the same area or between bats foraging in the same area. out of these alleles, one was shared between six bats from the three sampling sites. five alleles were shared between bats from sa and cm sites, and two alleles between the sa and cf sites. dero-drb* and had the highest overall frequency of  ± . four alleles were exclusively shared between individuals trapped in sa. two alleles were solely shared between individuals trapped in cm. we did not find shared alleles between bats trapped in cf. sixteen of the bats captured in sa were heterozygotes, five in cm and three in cf. m. molossus up to ten shared alleles were identified but none was shared between the three sampling sites. three alleles were shared between bats captured in the sa and pa sites. two alleles were shared between individuals from the sa and cc sites. one allele was shared between two bats captured in the cc and pa sites. three alleles were exclusively shared within sa. momo-drb* had the highest overall frequency:  ± . four of the five bats trapped in cc were homozygotes, seven in pa, and in sa. detection of selection signature an excess of dn substitutions was detected for the three species. more specifically, in putative antigen binding sites, dn/ds values were highly significant and ranged from for d. rotundus to for c. perspicillata. we found significant dn substitutions also occurring in putative non-abs for c. perspicillata but none in both d. rotundus and m. molossus samples.table non-synonymous and synonymous substitutions for c. perspicillata, d. rotundus and m. molossus p-value c. perspicillata d. rotundus m. molossus putative abs and non-abs were identified assuming functional homology to the human abs of the hla-dr molecule brown et al.. n is the number of codons in each sequence region. z-test values and p-values are shown paml analyses showed significant support for m and m, 2Δl =, df =  p <  ), indicating an effect of positive selection acting on the drb gene. overall, codons appeared to be under positive selection for c. perspicillata, eight for d. rotundus, and six for m. molossus. three positively selected sites were common to the three species investigated. these codons were the only common between d. rotundus and m. molossus. six of the eight pss identified in d. rotundus were also found in c. perspicillata, and all of the pss identified in m. molossus were identified in c. perspicillata.table identification of species-specific positively selected sites by maximum likelihood analysis sample size considered for each analysis is shown. sites identified as positively selected by at least two ml analyses are underlined. codons are given with a posterior probability ≥ % in the bayes empirical bayes analysis for paml m8; p-values < for slac, fel, ifel and meme; bayes factor > for rel. † indicates pss found only in a specific region for each species. validated pss also found with human antigen-binding sites are shadowed regarding environment subgroups for all species, a maximum of pss was identified in ur for c. perspicillata, eight in cf for d. rotundus and in sa for m. molossus. three pss were found in all environment subgroups for d. rotundus, four in c. perspicillata and four in m. molossus. a maximum of six unique pss was observed for c. perspicillata, four for m. molossus and two unique pss were observed in the two roosts for d. rotundus. none of the pss detected in c. perspicillata was shared between ur and pf. the same result was obtained between sa and cm for d. rotundus and between pa and cc or sa for m. molossus. thirteen of the pss identified were located within the human abs region for c. perspicillata, seven for d. rotundus and five for m. molossus. all the pss identified in m. molossus were described as pss for n. albiventris, for c. perspicillata and six for d. rotundus. six pss in c. perspicillata were also found in a. jamaicensis, three for m. molossus and one for d. rotundus. the sites where pss differed were located within one to six amino acid residues of the human abs or other bat species pss, depending on the bat species investigated. drb population structure we found no significant genetic differentiation between drb alleles of the environment subgroups defined for each species to ]; d. rotundus: fst range:; m. molossus: fst range:, additional file 1: table s6 − 8). taking into account the collecting sites, pairwise differentiation tests did not reveal significant genetic differentiation between drb alleles to ]; d. rotundus: fst range:; m. molossus: fst range:,  − 8). minimum spanning trees showed complex connections between the haplotypes, marked by numerous nucleotide mutations, without clustering of haplotypes either by site or by environment. we did not find relevant isolation-by-distance in our samples.fig. haplotype networks of mhc class ii dr beta exon alleles and of the mitochondrial dna control region at the nucleotide level for c. perspicillata. nodes are proportional to the number of bats carrying each haplotype, colored by the environments where the bats were trapped. hatch marks represent mutations. interruptions in lines indicate the presence of more than ten mutations fig. haplotype networks of mhc class ii dr beta exon alleles and of the mitochondrial dna control region at the nucleotide level for c. perspicillata based on collecting sites. nodes are proportional to the number of bats carrying each haplotype, colored by the capture site where the bats were trapped. hatch marks represent mutations fig. haplotype networks of mhc class ii dr beta exon alleles and the mitochondrial dna control region at the nucleotide level for d. rotundus. nodes are proportional to the number of bats carrying each haplotype, colored by the capture site where the bats were trapped. hatch marks represent mutations. interruptions in lines indicate the presence of more than ten mutations fig. haplotype networks of mhc class ii dr beta exon alleles and of the mitochondrial dna control region at the nucleotide level for m. molossus. nodes are proportional to the number of bats carrying each haplotype, colored by the capture site where the bats were trapped. hatch marks represent mutations. interruptions in lines indicate the presence of more than ten mutations mtdna d-loop population structure we found significant genetic differentiation between some populations of d. rotundus. no significant genetic differentiation was observed between populations for the two other species to ]; m. molossus fst range:, additional file 1: table s6–8). pairwise computations performed between collecting sites did not reveal significant genetic differentiation to ], additional file 1: table s6–8). the haplotype networks for c. perspicillata revealed two main groups of haplotypes that possessed all the haplotypes found in the different environments or collecting sites. the same result was obtained for m. molossus, with a maximum of four mutations between haplotypes. the lowest number of mutations was found for d. rotundus for which the five haplotypes were linked by one connection, marked by only one or two mutations. no population expansion or collapse was observed for the three species. we did not find relevant isolation-by-distance in our samples. drb phylogenetic relationships the analysis included the drb sequences identified in this study, as well as the published chiropteran drb sequences selected randomly from a. jamaicensis, from c. perspicillata, from myotis spp., from noctilio spp. and from s. bilineata. the phylogenetic tree showed six major clades. we observed a clustering by species or genus supported by medium and high posterior probabilities – c. perspicillata, d. rotundus, m. molossus, noctilio spp. – with an intermingled clustering of n. albiventris and n. leporinus drb alleles. within the c. perspicillata clade, drb sequences clustered with the previously published sequences, with a high posterior probability. one drb sequence of c. perspicillata was related to a d. rotundus clade with a posterior probability of. one drb sequence from s. bilineata clustered with the clade noctilio spp., with a low posterior probability value. the drb sequences of the a. jamaicensis species clustered with five drb sequences of d. rotundus and two drb sequences of c. perspicillata, with a posterior probability of. within this phyllostomid clade, we observed a species-dependent clustering with high posterior probabilities. two drb sequences of c. perspicillata clustered with a low posterior probability with two sequences from a. jamaicensis. we did not observe a characteristic clustering for s. bilineata and myotis spp. however, one drb sequence from m. velifer clustered together with six drb sequences from s. bilineata with a low posterior probability.fig. phylogenetic relationships of the mhc class ii dr beta sequences based on part of exon with values of posterior probabilities for nodes. species designation follows the genbank accession numbers for previously described drb sequences. arja a. jamaicensis, cape c. perspicillata, dero d. rotundus, momo m. molossus, myve m. velifer, myvi m. vivesi, noal n. albiventris, nole n. leporinus, sabi s. bilineata. hla-drb was used as the outgroup. tsp indicates trans-species polymorphism of the newly described drb alleles with previously described drb alleles discussion in this study, we explored the genetic variability of the expressed mhc drb genes of three sympatric neotropical bat species, looking for selection signatures within the pbr and investigating the role of the environment and the population structure on mhc diversity. diversity patterns in the mhc class ii drb gene in the three species, identified alleles were unique in amino acid sequences, suggesting a non-redundant nucleotide polymorphism. the number of alleles detected per species and per environment subgroup within each species indicated a relatively high level of mhc drb variation. these results are in agreement with previously reported allelic copy number variation between or within bat species. this high drb polymorphism was observed in the three species whatever the degree of habitat disturbance. in addition, no loss of polymorphism induced by low host and/or pathogen diversity, expected to occur in disturbed environments was observed. furthermore, mtdna d-loop sequences, considered as neutral, revealed high levels of genetic diversity for c. perspicillata and m. molossus. high levels of neutral diversity have been associated with either multiple geographic origin and/or population admixture, suggesting that c. perspicillata and m. molossus populations originated from one of these two phenomena or from both. the high level of drb polymorphism can be related to population history for these two species with the effects of additional selection factors such as pathogen-mediated selection. a high number of drb homozygotes was also found in m. molossus in all habitats as well as in c. perspicillata but in edge habitats only. for these two species, the presence of a specific allele ensuring an adequate immunity can be hypothesized. considering d. rotundus, a high number of heterozygotes for drb was observed in all habitats. this result suggests a heterozygote advantage related to the forest environment known to encompass higher species and pathogen richness. the most frequent mhc drb alleles encountered in each bat species were all identified in forest environments. these alleles are likely ancient and play a significant role in the immune response, conferring a selective advantage to the bats in sympatry in these habitats. indeed, given that forest environments are characterized by a high level of pathogen diversity, these alleles most likely possess enhanced recognition capabilities and might be able to recognize a broader range of pathogens. they thus allow a more efficient immune response in line with the ecological diversity. moreover, the geographical structure highlighted in d. rotundus, using mtdna d-loop results, did not affect the allelic distribution. indeed, most of the alleles identified in d. rotundus were shared between sites, and one allele was present at all sites. in contrast, no geographical structure was found in c. perspicillata even if the alleles identified in urban and periurban areas were rare and not shared with other groups. these results suggest that environment rather than population structure drives the allelic distribution, corroborating a local adaptation hypothesis. although edge and forest habitats may differ in pathogenic pressures, the results observed for c. perspicillata may be related to the high tolerance of this species to habitat disturbance and to its dominance in bat communities in edge habitats, likely allowing continuous exposure to the communities of pathogens of both pristine and disturbed forests, and pathogen transfer between these habitats. duplication detected in phyllostomidae gene conversion and recombination events were not detected for any species. the identification of three expressed drb alleles in individuals from c. perspicillata and d. rotundus supported the existence of at least two functional drb loci. however, underestimation of the number of alleles and expressed loci can be suspected, considering the limited number of recombinant clones per primer combinations and the use of cdna only allowing the detection of the most frequently expressed alleles. the occurrence of multiple mhc drb loci varying in the number of loci between individuals and species was reported in neotropical bat species such as c. perspicillata, s. bilineata, a. jamaicensis, as well as in other animals. together with environmental and biological patterns, gene conversion and recombination processes are known to drive the extensive mhc polymorphism. variable gene duplication between closely related taxa and among individuals from the same species is characteristic of mhc genes and plays a critical role in the adaptive evolution of the host. gene duplication can occur over different time scales and therefore predates or follows speciation events. furthermore, the absence of recombination between alleles of different species was reported. this result was regarded as evidence of recent duplication events between loci that occurred after speciation. our findings support this hypothesis since some recent radiation events occurred in neotropical bat species during the pleistocene era. despite the indels detected in some c. perspicillata and d. rotundus sequences, the reading frames remained unaltered, suggesting that these sequences encode functional proteins. nevertheless, the heterozygote-dominant character of individuals with these alleles suggests a turnover of these loci generated by evolutionary processes such as the silencing of duplicate genes by mutation or deletion. under the birth-and-death model, genes created by duplication can be maintained for long periods, deleted or transformed as pseudogenes and, therefore, contribute to host fitness and adaptability. the phenomenon is especially significant among heterozygote d. rotundus, for which one of the two alleles is among the most widespread in the population studied, and supports a shift towards an allele with better recognition capabilities. evidence of historical positive selection higher rates of non-synonymous vs. synonymous nucleotide substitutions were observed, especially in sites supposedly involved in antigen-binding. this excess of dn over ds in abs is consistent with balancing selection acting on drb loci, as a sign of historical positive selection in polymorphic mhc genes. furthermore, an unexpected excess of dn in putative non-abs for c. perspicillata suggests strong positive selection acting on these sites and highlights species-specific divergence on sites involved in antigen recognition. a high congruence was observed between species-specific positively selected sites in the three species and human abs demonstrating the functional homology of these sites. in contrast, pss not identified as human abs might play a role in mhc drb recognition capability or in the molecule stability for the species studied. here, nine of the pss were detected in c. perspicillata, while only one was detected in both d. rotundus and m. molossus. therefore, the observed excess of dn in c. perspicillata was attributed to pss identified as non-abs in humans. this result demonstrates the critical role of positive selection in shaping mhc diversity and corroborates that positive selection is driven by a species-specific immune response. focusing on c. perspicillata, comparisons of pss identified here with those previously described revealed high congruence, even among pss not identified as human abs. a similar result was observed between c. perspicillata and a. jamaicensis, two phyllostomidae with the same diet. taken together, these findings reveal family and species-specific selection pressures acting on mhc genes. moreover, the high similarity of pss between a. jamaicensis and c. perspicillata highlights that bat species sharing similar feeding strategies might be subjected – independently of their habitats – to similar pathogenic pressures, mainly from their microbiota. comparisons between each environment subgroup, for each present case, revealed the existence of private pss depending on the area, suggesting a habitat-specific selection pressure process. indeed, while six private pss were counted in urban and periurban areas for c. perspicillata, only two were detected in edge habitats and one in pristine forest. phylogeny, trans-species polymorphism and demographic process two mechanisms are thought to be responsible for the trans-specific similarity of mhc genes: convergent evolution and trans-species polymorphism. evidence of trans-species polymorphism in bats was reported in both myotis spp. and noctilio spp. and at the family level between the two phyllostomids a. jamaicensis and c. perspicillata. phylogenetic analyses in our study did not reveal any characteristic clustering of mhc alleles depending on the habitat, but rather clades at the family, genus and species levels. these results suggest either similar pathogenic pressures or that these alleles possess a larger antigenic recognition capability that constitutes a major asset to the immune response of these species. intermingled clusterings observed in this survey are in agreement with trans-species polymorphism reported in bats. the observed trans-species polymorphism for myotis spp. was not detected in the present study. the results highlighted clustering between s. bilineata and m. velifer. however, this clustering was no longer observed by analyzing full-length exon observation of trans-species polymorphism within bat families strengthens the scenario of independent modes of evolution of mhc drb alleles, allowing balancing selection to retain substantial allelic lineages before speciation events. extended periods of host–pathogen coevolution is thought to contribute to trans-species polymorphism given that host-sharing pathogens and exposure to similar pathogen pressures are believed to induce similar changes in different host species. although pathogen-driven selection is known to be crucial in mhc diversity, demographic processes such as genetic drift, bottlenecks, expansions, fragmentation and geographic isolation have been demonstrated to also participate in the shaping of this extensive polymorphism. evolutionary analyses of mhc drb alleles for the three species did not reveal any area-dependent clustering effect. furthermore, analyses of partial sequences of the mtdna d-loop revealed no structuration for c. perspicillata and m. molossus, whereas it was observed for d. rotundus. demographic analysis performed for the three species did not detect any substantial bottleneck or expansion. while the patterns observed for the first two species corroborate the hypothesis of a similarity between the intrinsic genetic diversity of the species and that of mhc, the lack of similarity between these patterns of diversity for d. rotundus contradicts this statement. these results suggest that the signature of an area-limited pathogen-driven selection, supported by the presence of private pss rather than by demographic processes such as distance isolation, is observed in this case.  this study was the first to investigate mhc drb polymorphism in three sympatric bat species, m. molossus, c. perspicillata and d. rotundus, from the amazonian region. our results revealed a high genetic variability in the mhc drb gene. natural selection, as well as local adaptation driven by different parasite and pathogen exposures across environments, contribute to the maintenance of an extensive mhc polymorphism. the richness of pathogens in the different habitats should be investigated to strengthen our assumptions on potential local adaptation. further studies using finer neutral markers such as microsatellites will be necessary to detect possible confounding effects, such as hidden structuring and demographic history, and thus to better understand the drivers of mhc allelic diversity. additional files additional file 1: table s supplementary methods, including the characteristics of all capture sites ﻿, the list of samples successfully amplified for the mhc class ii drb loci, the distribution of drb alleles per species, the pairwise differentiation computations per species, as well as the correlations of genetics and geographic distance. additional file 2: figure s map of french guiana showing the capture sites of c. perspicillata, d. rotundus and m. molossus. for clarity, nearby sites within 15 km were grouped. sites are numbered and labeled according to the type of environment to which they correspond: edge habitats, anthropized areas, pristine primary lowland forests, urban and periurban areas. pie chart indicates the proportion of bat species sampled, with c. perspicillata in light grey, d. rotundus in black and m. molossus in dark grey. small charts indicate a total number of individuals caught ≤  medium-sized charts, large charts . characteristics of the different sites are given in additional file 1: table s additional file 3: figure s positions of pcr primers used to amplify the indicated fragments of the mhc class ii loci in c. perspicillata, d. rotundus and m. molossus, based on cdna. the structure of the cdna of the mhc class ii drb gene is based on. according to each species, boxes indicate the amplified region using each couple of primers. the shaded region represents the region of interest, namely exon sequences and references of all primers used are given in table  abbreviations absantigen-binding sites aicccorrected akaike information criteria bebbayes empirical bayes drbexon of the mhc class ii dr beta felfixed effects likelihood ifelinternal fixed effects likelihood mememixed effects model of evolution mhcmajor histocompatibility complex mlmaximum-likelihood mtdna d-loopmitochondrial dna control region pbrpeptide-binding region pcrpolymerase chain reaction pssspecies-specific positively selected sites relrandom effects likelihood slacsingle likelihood ancestor counting we are grateful to françois catzeflis and marguerite delaval for useful discussions on the biology, ecology and dynamics of bats. all field volunteers and owners and/or managers of capture sites are warmly acknowledged for their assistance in captures. funding a. salmier was supported by a grant from european funds and "investissement d’avenir" managed by agence nationale de la recherche. this study was conducted within the carolia program supported by european funds and assistance from région guyane and direction régionale pour la recherche et la technologie. it also received a european commission "regpot-ct-2011-285837- stronger" grant within the fp and "investissement d’avenir" grants managed by the agence nationale de la recherche. the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. availability of data and materials nucleotide sequences for the drb alleles and for mtdna d-loop are available on genbank. authors’ contribution conceived and designed the experiments: as al. analyzed the data: as al. contributed reagents/materials/analysis tools: as bdt vl al. wrote the paper: as bdt bcr vl al. field work and acquisition data: as al bdt. all authors read and approved the final manuscript. competing interests the authors declare that they have no competing interests.
here we examined spatial patterns of diversity of the expressed mhc class ii drb gene of three sympatric neotropical bats, carollia perspicillata and desmodus rotundus, and molossus molossus, all of which use the same environments. mhcdrb polymorphismselectioncarollia perspicillatadesmodus rotundusmolossus molossuseuropepo fse 2007-2013regpot-ct-2011-285837- strongersalmier arielle lacoste vincent http://dx.doi.org/ /501100001665agence nationale de la rechercheanr-10- labex-25-01anr-10- labex-25-01salmier arielle lavergne anne erdf/federcarollialavergne anne issue-copyright-statement© the author 2016 in contrast, drb diversity of d. rotundus was shaped by environment rather than demographic history. the drb polymorphism was high in all sampling sites and different signatures of positive selection were detected depending on the environment. twenty-three drb alleles were identified in c. perspicillata, alleles in d. rotundus and alleles in 28 m. molossus. the patterns of drb diversity were similar to those of neutral markers for c. perspicillata and m. molossus. furthermore, multiple geographic origins and/or population admixtures observed in c. perspicillata and m. molossus populations acted as an additional force in shaping drb diversity. this study of mhc diversity demonstrated the strength of the environment and contrasting pathogen pressures in shaping drb diversity. in contrast, these patterns were different for d. rotundus for which a geographical structure was highlighted. a heterozygote advantage was also identified for this species. no recombination or gene conversion event was found and phylogenetic relationships showed a trans-species mode of evolution in the phyllostomids. differences between positively selected sites identified in bat species highlighted the potential role of gut microbiota in shaping immune responses. although bats are natural reservoirs of many pathogens, few studies have been conducted on the genetic variation and detection of selection in major histocompatibility complex genes.
 although bats are natural reservoirs of many pathogens, few studies have been conducted on the genetic variation and detection of selection in major histocompatibility complex genes. these genes are critical for resistance and susceptibility to diseases, and host–pathogen interactions are major determinants of their extensive polymorphism. here we examined spatial patterns of diversity of the expressed mhc class ii drb gene of three sympatric neotropical bats, carollia perspicillata and desmodus rotundus, and molossus molossus, all of which use the same environments. comparison with neutral marker diversity was performed at the same time.  twenty-three drb alleles were identified in c. perspicillata, alleles in d. rotundus and alleles in 28 m. molossus. the occurrence of multiple drb loci was found for the two phyllostomidae species. the drb polymorphism was high in all sampling sites and different signatures of positive selection were detected depending on the environment. the patterns of drb diversity were similar to those of neutral markers for c. perspicillata and m. molossus. in contrast, these patterns were different for d. rotundus for which a geographical structure was highlighted. a heterozygote advantage was also identified for this species. no recombination or gene conversion event was found and phylogenetic relationships showed a trans-species mode of evolution in the phyllostomids.  this study of mhc diversity demonstrated the strength of the environment and contrasting pathogen pressures in shaping drb diversity. differences between positively selected sites identified in bat species highlighted the potential role of gut microbiota in shaping immune responses. furthermore, multiple geographic origins and/or population admixtures observed in c. perspicillata and m. molossus populations acted as an additional force in shaping drb diversity. in contrast, drb diversity of d. rotundus was shaped by environment rather than demographic history. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords mhcdrb polymorphismselectioncarollia perspicillatadesmodus rotundusmolossus molossuseuropepo fse 2007-2013regpot-ct-2011-285837- strongersalmier arielle lacoste vincent http://dx.doi.org/ /501100001665agence nationale de la rechercheanr-10- labex-25-01anr-10- labex-25-01salmier arielle lavergne anne erdf/federcarollialavergne anne issue-copyright-statement© the author 2016 