BACKGROUND
ants are a successful and diverse group of insects, playing a role in various ecosystem functions.
these roles include facilitating soil aeration, preying on herbivore insects, farming aphids for their honeydew, and cutting leaves to farm fungi for foraging.
ant eusociality is of great scientific interest and numerous works have addressed issues in its division of labour and cast-specific gene expression.
most ant genomes were sequenced due to their status as an invasive species , for their unusual cast division of labour , or for their negative impact on the economy .
the black garden ant lasius niger is a usual ant of the northern hemisphere and dwells in nearly all landscapes in europe, ranging from forests to big cities.
the last review of l. niger was in  <cit> , where the new species lasius platythorax was established.
workers in l. niger are small , while queens can reach 16 mm.
colonies are monogynous and have only one egg-producing queen, though polyandry does occurs  <cit> .
l. niger nests are usually underground or in sand mounds, but are also found in wall cracks and rotting wood.
l. niger feeds primarily on honeydew and other sugar-rich substances; small invertebrates are also part of their diet.
farming aphids for honeydew often occur in gardens, thus effectively making itself a pest.
these ants are also known to infest human housing, causing noticeable disturbances  <cit> .
l. niger dwells in holarctic region, inhabiting all of europe and parts of asia and north america.
l. niger is the most widespread of all sequenced ants.
in natural environments lasius niger occupy a subdominant position in ant communities, while species of the genera myrmica and formica play the main role as generalist predators.
when forest ecosystems are eroded, however, dominant ant species are the first to disappear, creating possible niches and opportunities for other ant species.
lasius niger has a very flexible ecological strategy and can inhabit different spatial tiers from soil to trees.
due to its ecological plasticity lasius niger has grown widespread in urbanised areas: the black garden ant had even increased its abundance in highly affected territories  <cit> .
here we report the draft genome of l. niger as a means of studying the genetic mechanisms underlying insect adaptation to urban environments.
we focus on gene families that could facilitate behavioural or physiological means of coping with specific urban stresses.
methods
source material
population samples for sequencing were obtained from two locations: near the campus of moscow state university and from the istra district ca.
70 km from moscow.
in early september  <dig> worker ants from three sites  at both locations were collected using aspirators and stored separately in 98% ethanol.
approximately 50– <dig> ants from each of six sites were treated as different samples.
for exact sample sizes see additional file 1: table s <dig>  samples were dry-freezed and transported to the university of southern california.
genomic procedures
genomic dna for each of the six samples were prepared into six libraries  and sequenced as paired-ended 100 bp reads on an illumina hiseq  <dig>  each library was individually barcoded and sequenced as a pooled sample of barcoded dna, generating  <dig> million  total reads.
since de novo assembly of complete genomes can be sensitive to sequencing errors, it is critical that the reads used to generate contigs have the highest sequencing quality.
reads were removed from consideration in the de novo assemblyif the read had a terminal “phred”  <cit>  , or if the read contained more than two unknown nucleotides.
reads were also filtered out with trimmomatic  <cit>  if they had similarity to known polymerase chain reaction primer and illumina adapter sequences.
the 272 m retained reads from all samples were assembled with spades version  <dig> .
<dig>  <cit> .
the data are deposited to bioproject prjna <dig>
genome assembly completeness was assessed using cegma  <cit>  and busco  <cit> .
cegma score was  <dig> % compared to  <dig> – <dig> % range for other ant genomes  <cit> .
the raw illumina data and the draft assembly have been deposited in ncbi under the bioproject prjna <dig> and will be released upon publication.
gene prediction and gene ontology  content
for gene prediction augustus  <cit>  was used with the nasonia vitripennis genome as a training set.
the prediction was done on both strands, including partial genes.
predicted genes were searched against the nr protein database .
contigs with bacterial and fungal genes were removed from the assembly.
bacterial 16 s rna were identified using blast searches against the bacterial 16 s rna database; selected sequences were then searched against the nr database for genus and species identification.
for more accurate searches of genes encoding cytochromes, odorant receptors, odorant-binding proteins and desaturases, hymenopterans protein sequences were used to build alignments using hmm model with hmmbuild.
we built separate models for each major cyp superfamily .
hmmsearch output was checked by blast  and delta-blast for the presence of specific domains.
genes with hmmersearch hits were aligned using blastp against the nr database, and genes with the majority of hits from other families were removed from further analysis.
we used delta blast to check for gene-specific domains.
we also searched for sequences from other examined species in the hymenoptera genomes portal  and in genbank .
predicted proteins of l. niger and other ant species were then annotated using blastp  and blast2go  <cit> .
we performed go mapping, blast-based annotation, interproscan annotation and joined blast and interproscan annotation data.
go terms were assigned for each protein, go category numbers for each species were normalized by total protein number.
dixon q-test for outliers was used to detect overrepresented and underrepresented go groups in the l. niger genome, assuming it as an outlier among all ants.
go distribution was normalized with arcsine transformation  <cit>  l. niger was treated as outlier in both cases - in over and under represented go ids.
proteins and genes of species with draft genomes that were not published yet  were not included in go-mapping and intersproscan annotation and was used only for or and cyp comparison and selection detection.
alignment and detection of selection
to eliminate potential pseudogenes, any sequence with predicted start and stop codons was discarded if it was shorter than its hypothetical product length by 25%.
we assumed  <dig> amino acid residues for desaturases and  <dig> for odorant receptors and p <dig> cytochromes, based on rounded off mean length of respective proteins in databases.
for p <dig> we kept only sequences containing phe-x-x-gly-x-arg-x-cys-x-gly motif, which is believed to be crucial to stabilizing the iron ion in the heme group.
the selected sequence predictions for p <dig>  odorant receptors and desaturases were than aligned for each family using the muscle software .
for the p <dig> cytochrome superfamily we aligned sequences in cyp <dig>  cyp <dig> and cyp <dig> separately for each family.
search for best substitution models were performed in mega <dig> .
tamura-nei substitution modelwas found best for our dataset and was used for phylogenetic analysis.
a maximum-likelihood phylogenetic tree was generated for each alignment using mega <dig>  .
to identify genes under selection, we applied a z-test for selection  <cit>  to nucleotide alignments of these genes.
the positive selection hypothesis was rejected in none of the cases.
a sequence was tagged as being under positive selection if both the neutrality and the purifying selection hypotheses were rejected.
for the cyp <dig> subfamily of p <dig> cytochromes we also searched for sites under selection.
for this analysis we used only complete coding sequences with start and stop codons in the length range of 1425– <dig> nucleotides due to limitations of selection search methods .
upon removal of stop codons, sequences were codon-aligned using clustalw software.
maximum-likelihood phylogenetic trees were then generated using mega <dig>  .
based on these trees, a selection search with fubar  and meme  methods was performed for each codon in a given alignment using hyphy <dig>  software.
the number of amino acid substitution events were then calculated based upon ancestor sequence reconstructed with slac.
RESULTS
genome assembly and annotation
we sequenced and annotated the draft genome of lasius niger and compared it with the draft genomes of other ants.
the 272 m reads were assembled with spades  <cit>  into  <dig>  contigs of more than 500 bp in length with a total length 245 mb and an n <dig> of  <dig>  bp.
these figures are similar to other available ant draft genomes and enable comparative genomic analysis  <cit> .
the assembly also included  <dig>  contigs of more than 1000 bp in length with a total length 237 mb.
cg content was found to be 38%, similar to that found in camponotus floridanus   <cit> .
median kmer coverage is  <dig>  exhibiting a bimodal distribution of kmer coverage with peaks at  <dig> and  <dig> cpg dinucleotides are overrepresented in the l. niger genome: observed/expected cpg ratio is  <dig> , similar to other ants :
we identified transposable elements throughout the l. niger genome using repeatmasker  <dig> .
<dig>  <cit>  against the repbase te library v2016-08- <dig>  <cit> .
we found  <dig>  mb of known transposable elements .
however, there are many intact orfs of transposon proteins compared to other ants , which suggest high recent transposon activity in the l.niger lineage.
transposons detected by repeatmasker include  <dig> lines, mostly from r1/loa/jockey family, and  <dig> ltr elements .
application of the gene prediction tool augustus  <cit>  revealed  <dig>  genes in l. niger contigs, with  <dig>  having both start and stop codons.
annotated genes were shorter than in other ant species and contained fewer exons, but total exon number and length is similar to values observed in other ants .
therefore we conclude that the gene space of l. niger annotation is as complete as other ants, although the genome is more fragmented.
to distinguish between genes in the ant genome and those originating from microbes, we used blast search against the non-redundant protein database.
analysis of 16s and 18s ribosomal rna genes revealed the following non-ant species:  <dig> bacteria , one enthomopathogenic fungus ophiocordyceps unilateralis, an unidentified spider and a plant from family fabiaceae.
high protein blast-hit similarity additionally identified enterobacteriacea , propionobacter, nematode, lactobacillus, actinomycete , and, potentially, fusarium .
after removing hits to bacterial and fungal contigs we obtained a total of  <dig>  ant genes;  <dig>  potentially complete and  <dig> partial sequences.
all these contigs were also removed from the bioproject.
among non-bacterial annotated contigs the vast majority  are annotated as proteins, common with camponotus floridanus .
c. floridanus belongs to the same family formicinae as l. niger and their similarity in proteins corresponds to morphological likeness.
three other ant species from the fig.
<dig> – solenopsis invicta, acromyrmex echinator and harpegnatos saltator are highly specialized and diverged from formicinae lineage in late cretaceous  <cit> .fig.
<dig> top blast hits of l. niger metagenome  and genome  assembly.
most hits are from c. floridanus, closest sequenced relative of l. niger, and other ants
go distribution
the predicted proteome of l. niger is significantly  different from other ant genomes in terms of abundance of several go categories.
the proteome is enriched in domains involved in nucleic acid binding , dna repair , nucleotidyl transferase activity , heterocyclic compound binding  and contain fewer domains involved in neuropeptide signaling .
l. niger might also contain fewer odorant binding domains than other ants , but this difference was not statistically significant in a q-test.
overrepresentation of nucleic acid binding domains is due to high abundance of retrotransposon genes coding gag-pol polyproteins or separate reverse transcriptases.
we detected  <dig> pol and gag-pol genes in the l. niger genome compared to  <dig> in camponotus and between  <dig> and  <dig> in other ant species.
no env genes were found to match the abundance of pol and gag-pol.
we conclude that the pol and gag-pol genes belong to retrotransposomes and are not from ongoing retroviral infections.
dna repair domains are overrepresented in l. niger mostly because of duplications of pif1-like helicases.
the l. niger genome encodes  <dig> pif1-like helicases compared to  <dig> in camponotus and  <dig> in most other ants.
other dna repair proteins duplicated in l. niger include chromosome structure maintenance proteins 1a and  <dig>  dna damage-binding protein  <dig> and tyrosyl-phosphodiesterase.
detoxification system genes
as our focus is on potential genomic features enabling success in urban environments, we paid special attention to the gene families involved in detoxification.
glutathione-s-transferases and cytochrome p <dig> families are the most common detoxification genes among hymenoptera  <cit>  and we examined these two families in detail.
we expected to find multiple duplications in these families, and in glutathione-s-transferase family we found  <dig> genes, , though most had incomplete and short sequences.
we found no evidence of species-specific duplications of glutathione-s-transferase in the l. niger genome.
we did find cyp genes being highly duplicated in l. niger, with  <dig> sequences matching cyp genes across  <dig> families .
this is the second largest value among ants, second only to  <dig> cyp <dig> genes in c. floridanus from the same family .
compared to a. mellifera we see fewer cyp families, but more genes per family in those present .
interestingly, there are  <dig> tandem clusters of cyp <dig> genes found in the l. niger genome assembly, most of which are members of cyp9e subfamily.
most cyp <dig> genes in ant genomes are members of cytochrome p <dig> 9th, 6th and 4th families .
the cyp <dig> family is highly expanded in l. niger, with  <dig> complete and  <dig> nearly complete members - over twice as much as any other ant.
the number of cyp <dig> and cyp <dig> gene families in l. niger genome is within the diversity range of other ants.
genes of the 9e <dig> family are organized in  <dig> duplicated clusters, with genes of 6 k, 6a and 4g families also exhibiting tandem duplications.fig.
<dig> comparative abundance of cyp <dig> subfamilies in hymenopteran genomes.
various subfamilies are expanded in different species.
even closely related c. floridanus and l. niger differ significantly in relative abundance of cyp <dig> and cyp <dig> subfamilies
positive selection in cytochrome p <dig> genes
for phylogenetic analyses and selection tests we selected only complete and nearly complete  p <dig> genes where the conservative heme-binding motif phe-x-x-gly-x-arg-x-cys-x-gly was present .
in the phylogenetic tree, most l. niger genes form  <dig> clusters near those of c. floridanus, another ant in formicinae subfamily .
most of the cyp <dig> genes in the examined species are under positive selection according to pairwise z test .
except for recently duplicated genes phylogenetic trees of cyp <dig> and cyp <dig> always show a. mellifera and n. vitripennis as outgroups.
l. niger contain two duplications of cyp <dig> genes after the divergence of lasius and camponotus lineages.
five l. niger genes of cyp <dig> family sit on the phylogenetic tree separately, but form clusters with c. floridanus genes.
two are putative pseudogenes.
most ant genes from the cyp <dig> and cyp <dig> families were under positive selection since their divergence with other hymenoptera according to a pairwise z-test for selectionfig.
<dig> phylogenetic tree of hymenopteran cyp <dig> genes.
branches where statistically significant positive selection was found by z-test are in red.
positive selection was found in most of the terminal branches of the phylogenetic tree.
many lineage-specific duplications are evident in l. niger, a. echinator and c. biroi
fig.
<dig> positive  and negative  selected amino acids of cyp <dig> l. niger.
ligand-binding pocket is located in the upper right part of the protein molecule, with heme group in the bottom of the pocket.
most sites under positive selection are in the ligand-binding pocket, while structural alpha spiral domains are under purifing selection
as noted previously, the cyp <dig> and cyp <dig> gene families are not amplified in lasius niger.
nearly all ants with sequenced genomes have between  <dig> and  <dig> fragments of cyp <dig> genes, coding for more than half of the protein .
camponotus has  <dig> full-length genes and  <dig> partial genes, with the partial genes coding for more than half of the protein.
non-ant hymenopteran species a.mellifera and n.vitripennis have  <dig> and  <dig> predicted cyp <dig> family proteins.
despite the  <dig> found fragments of genes, similar to the number of cyp <dig> found in other species, only  <dig> code for more than half of the protein and only  <dig> have the sequence of the gem-iron stabilizing motif.
other examined species have between  <dig> and  <dig> fragments of cyp <dig> genes, with only  <dig> to  <dig> containing coding sequences of more than 750 bp.
every ant species except for c. obscurior has lineage specific duplication of cyp <dig> genes .
the most abundant cyp <dig> genes  are in invasive species , but in half of the genes the sequence of gem stabilizing motif is absent.
the poorest representation is in c. biroi – .
overall, the cyp <dig> tree has many species-specific duplications for all ants, except l. niger and a. cephalotes and c. obscurior .
the most amplified are cyp <dig> in c. floridanus and s. invicta, both having  <dig> groups of 3– <dig> genes and  <dig> separately lying genes.
positive selection in cyp <dig> family, as well as in the abovementioned families, was not significant for recent duplications; most of them are in c. floridanus.fig.
<dig> phylogenetic tree of hymenopteran cyp <dig> genes.
branches where statistically significant positive selection was found by z-test are in red.
positive selection was found in most of the terminal branches of the phylogenetic tree.
many lineage-specific duplications are evident in n. vitripennis, c. floridanus and c. biroi
fig.
<dig> phylogenetic tree of hymenopteran cyp <dig> genes.
branches where statistically significant positive selection was found by z-test are in red.
positive selection was found in most of the terminal branches of the phylogenetic tree.
many lineage-specific duplications are evident in n. vitripennis and a. mellifera
sites under selection in cyp9
to test whether an expansion of the cyp <dig> family was concordant with strong evolution on gene sequences, we executed a more detailed analysis.
an alignment of full-length cyp <dig> coding sequences between  <dig> and 1800 bp with start and stop codons was used to search for sites under positive selection.
these criteria identified  <dig> coding cyp <dig> sequences in a. cephalotes,  <dig> sequences in a. echinator,  <dig> in a. mellifera,  <dig> in c. biroi,  <dig> in c. floridanus,  <dig> in h. saltator,  <dig> in l. niger,  <dig> in n. vitripennis,  <dig> in m.pharaonis,  <dig> in c.obscurior,  <dig> in v. emeryi,  <dig> in w.auropunctata and  <dig> in s. invicta.
meme detected  <dig> alignment positions under positive selection  .
three positions were confirmed by other selection search methods , one was significant in all four methods.
negative selection was detected in  <dig> sites with statistical significance for fubar with aposteriori probability >  <dig> ).
eleven positively selected positions are in ligand-binding pockets of the enzyme .
an additional four are at the c-terminus, but this part of alignment was not present in all sequences.
most of negatively selected sites lie in structural domains of the enzyme.
the most frequent non-synonymous substitutions in cyp <dig> family did not change the charge and polarity of amino-acid residues  .
nevertheless, some substitutions changed charge and polarity – lys to glu, asn to asp, ala to thr and leu and ile to met.
the rarest were changes of cys and trp.
the meme analysis found different preferred substitutions in different subtrees of the l.niger cyp9e tree.
in one subtree, arg was changed to hys and asn, while another arg was changed to ala. this trending of changing arg to other amino-acids occurred only in l. niger alignments – in other ants not only arg was changed, but also ala and hys.
fubar analysis detects negative selection in most positions, where meme detects positive.
these sites are conservative in the majority of lineages but in other leading to l. niger diversifying selection did occur.
olfactory receptors and odorant-binding proteins
olfaction related genes frequently exhibited signatures of directional selection or selection relaxation.
hhmsearch and reciprocal blastp of l. niger predicted proteins revealed  <dig> putative odorant receptors and  <dig> odorant-binding proteins.
one putative odorant-binding protein, having high similarity with other ant obp, is  <dig> amino acids in length and is not recognized by blastp in the refseq protein database.
other ants have additional obps, from  <dig> in c. floridanus and a. echinator to  <dig> in v. emeryi.
only  <dig> of 74 l. niger predicted or genes had coding sequence longer than 750 bp and were used for phylogenetic analysis and selection detection.
other ants have between  <dig> and  <dig> fragments of odorant receptor genes, from  <dig> to  <dig> of this manifold were used for the phylogenetic analysis.
apis mellifera has  <dig> fragments of genes and  <dig> fragments were used to construct the phylogenetic tree.
.
genes of odorant receptors in l. niger have  <dig> amplified clusters –  <dig> duplications and  <dig> 5-genes repeats.
most tandem duplications have related sequences in other ants.
we did not find any l. niger duplication that was species-specific .
not only did l. niger have only  <dig> genes of suitable quality for odorant receptors, more than half were putative pseudogenes.
despite the poor repertoire of l. niger odorant receptors, half were under positive selection.
in contrast to the cyp genes, odorant receptors of l. niger do not predominantly form clusters with c. floridanus genes, but often cluster with genes of more distant ant species.
the phylogenetic tree of odorant receptors indicates that there are many amplifications and deletions of odorant receptor genes, providing an explantation as to why closely related a. cephalotes and a. echinator have very different odorant receptor genes, perhaps due to pseudogenisation and lineage sorting in their putative ancestor’s gene set.
z-test found positive selection in average in all sequence pairs.
fatty acid desaturases
cuticular hydrocarbons are important molecules in ant chemocommunication.
these compounds are produced by fatty acid desaturases enzymes.
ants with sequenced genomes have from  <dig> in c.biroi to  <dig> in h. saltator.
l. niger and c. floridanus both have  <dig> fatty acid desaturase genes, incomplete genes not included .
desaturases of hymenoptera are grouped into six subfamilies, with one being unique for this order and other subfamilies orthologous to five drosophila melanogaster desaturases .
no orthologs of d. melanogaster cg <dig> were found in l. niger and p. barbatus; other ants have one.
hymenopteran specific desaturases are absent from c. biroi.
other subfamilies of desaturases are present in all ant genomes.
in all ant species, orthologs to d.melanogaster cg <dig> are under positive selection, while there was no selection within c. floridanus duplications.
almost all orthologs to d. melanogaster cg <dig> and desat <dig> are positively selected as well.
in hymenopteran specific desaturase group, the selection was significant predominantly in myrmicinae and formicinae subfamilies.
no significant differences between l. niger and other ants can be found in desaturase gene content and selection mode .
telomerases
ants are known for their great intraspecific differences in life spans and l. niger has the greatest: from several months in males to decades in queens  <cit> .
in contrast to drosophila in which telomere length is maintained through retrotransposition  <cit>  and is consistent with other studied animals species, ants’ life span is based on telomerase activity.
jemielty et al.
<cit>  showed caste-specific differences in expression level of telomerases.
our annotation contains several genes related to telomerase activity.
one copy of telomerase reverse transcriptase was found.
thirteen copies of another important component of telomerase ribonucleoprotein  complex – telomerase binding protein est1a  <cit>  – were found .
four genes similar to telomerase cajal body protein were found.
future functional analysis of telomerase sequences in l. niger could shed light on life span regulation in this species.
vitellogenins
vitellogenins were duplicated in many formicoid lineages.
caste-specific expression is characteristic for vitellogenin paralogs in ants.
however, the c. floridanus genome contains only one vitellogenin gene.
we found only one vitellogenin in the lasius genome, with high similarity to the camponotus vitellogenin.
hmm search for hymenopteran vitellogenins also revealed only one vitellogenin in harpegnatos, cerapachys and pogonomyrmex.
other ant species had more vitellogenins; we found  <dig> vitellogenins in solenopsis,  <dig> in linepithema and wasmania and  <dig> vitellogenins in volenhovia and monomorium.
one more sole vitellogenin in formicinae can not, of course, reject a hypothesis of all-formicidae initial vitellogenin duplication, but add some more doubts to it.
all mentioned vitellogenins were vgg, we did not search for vgf.
discussion
high loads of fungal parasites and retroelements
here, we assembled the draft genome of l. niger and compared it with the genomes of other ant species.
surprisingly, retrotransposons are an order of magnitude more abundant in this ant compared to others.
discovered retrotransposon genes do not contain internal stop codons and expected to be active.
we also discovered the expansion of dna repair proteins.
here, we hypothesize that expansion of dna repair proteins could be a genome response to the expansion of retroelements.
dna repair system takes part in transposon integration and is required to restore dna damage caused by incorrect integration.
tes are known to interfere with host repair, therefore the diversification of dna repair system may represent the host’s response to retrotransposon expansion  <cit> .
we further hypothesize that the diversification of dna repair system might be an exaptation pre-adapting l. niger to explore urban environments.
specifically, enhanced dna repair enzyme repertoire may play a role in resistance against dna-damaging xenobiotics such as polycyclic aromatic hydrocarbons.
furthermore, both protein and 18s rrna blast results show high level of fungal dna from genus ophiocordyceps.
these fungi are well-known entomopathogens  <cit> , infecting various insects including ants.
fungal 18 s rdna from l. niger assembly is most similar  to sequences from o. unilateralis infecting camponotus carpenter ants, suggesting high rate of fungal infection in l. niger.
however, characteristic fruit bodies protruding from the dead ant’s head have never been seen in l. niger.
it was shown that laboratory settings that ophiocordyceps infects and kills many ant species besides its natural host  <cit>  however, reproduction and spore dispersal of ophiocordyceps requires complex host manipulation , which is successful only in its natural host.
two species of camponotus  live in the moscow region  <cit>  and they may be natural hosts of ophiocordyceps found in l. niger.
alternatively, l. niger might be a natural host for this fungus but its transmission does not involve typical fruit body formation.
nevertheless, the observed fungal dna level suggests that ophiocordyceps infection may be an important cause of mortality among l. niger and there may be the selective pressure toward resistance to this pathogen.
cyp <dig> expansion
insects can cope with pollution in two ways - physiological or behavioral.
metabolic detoxification of xenobiotics is an example of physiological adaptation.
we studied the cytochrome p <dig> superfamily in detail, as it is known for its crucial role in resistance to xenobiotics  <cit> .
compared to other ants, l. niger has a highly amplified cyp <dig> subfamily.
phylogenetic analysis and comparison of cyp <dig> genes of l. niger with a. mellifera and n. vitripennis allocated all genes of this subfamily in ants into subfamilies cyp9e and cyp9p.
functions of these subfamilies are unknown, but closely related subfamilies  are known for their roles in insecticide resistance  <cit> .
entomopathogenic cordycepitales are known to synthesize wide range of toxic compounds  <cit> .
we have previously shown using molecular docking and virtual screening  <cit>  that l. niger cyp <dig> cytochromes form complexes with low free energy with fusarin c, fumonisin, ergot-alkaloids, enniatin and beauvericin.
these compounds are produced by ophiocordyceps and are important pathogenicity factors  <cit> .
for some of l. niger cyp <dig> free energy of complexes with fusarin are below −480 kj/mol that suggests high affinity of enzymes to this substrate.
we hypothesize, therefore, that the expansion of cyp <dig> in l. niger genome may result from coevolution with enthomopathogenic fungi.
cytochrome p <dig> superfamily evolution in formicidae was extensively studied  <cit> .
differential expansions and contractions of p <dig> subfamilies were observed in different genera.
these changes are linked with adaptations to novel ecological niches and changes in colony lifestyle.
trophic specialization was found to promote the contraction of p <dig> family in p. barbatus  <cit> .
l. niger and c. floridanus, which belong to the subfamily formicinae, contain approximately the same number of cytochrome p <dig> genes, and this number is higher than that in other species of ants.
like l. niger, c. floridanus dwells in an urban environment and similarly omnivorous  <cit> .
its diet includes living and dead insects and the secretions of aphids and scale insects  <cit> .
cytochrome p <dig> genes have wide range of substrates and single amino acid substitution can drastically change substrate specificity  <cit> .
it is possible that the large set of cytochrome p <dig> genes provide for polyphagia by enabling specific and nonspecific neutralization of various phyto and mycotoxins .
cytochrome p <dig> genes may also be important in resistance to urban pollutants of different chemical composition .
some cytochrome p <dig> genes can oxidize some of these chemicals, e.g., benzopyren  <cit> .
the importance of detoxification system in l. niger xenobiotic resistance may be further increased due to scarcity of anti-fungal protective behaviour.
for example, l. niger does not remove aphids infected with parasitic fungi from symbiotic aphid colonies, while this behaviour is common in other aphid-herding ants such as formica s. str.
.
odorant communication
genes of odorant receptors and odorant-binding proteins, including partial sequences, appear to be less abundant in the assembly of l. niger than in other ant genomes.
we hypothesize that the lasius genus lost some odorant receptor and odorant-binding genes in the course of their evolution.
our reanalysis of previously published data enables unbiased comparison between species.
in s. invicta, there are only  <dig> fragments of odorant receptors and odorant-binding proteins, instead of  <dig> complete genes and more than  <dig> fragments that were previously reported  <cit> .
this observation could be explained by the fact that the insect odorant receptors were considered to be homologous to odorant receptors of vertebrates prior to  <dig>  <cit> .
accordingly, g-protein coupled receptors were treated as hypothetically odor detection related, while now we know that all insect odorant receptors are ion channels.
another published prediction that was not confirmed in our work, is an increase in amounts of odorant receptors in ants as compared to a. mellifera and n. vitripennis.
on the contrary, we observed that, in ants, odorant receptors and odorant-binding protein families underwent gene loss.
this loss may be caused by transition of ants to terrestrial lifestyle and, therefore, increase in the role of antennal contacts in species communication .
l. niger was shown to prefer visual information over pheromone trails when these stimuli were in conflict, contrary to other ant species  <cit> .
specifically in respect to urban environments, odorant receptor loss can be very useful, as it is increasing the resistance of the species to repellents.
CONCLUSIONS
in conclusion, we hypothesize that a number of seemingly unrelated events – expansion of dna repair, olfactory system genes loss and cyp <dig> gene amplification - can be viewed as preadaptations required for the successful colonization of urban areas.
among these preadaptations, the changes in olfactory system genes and cyp genes provide two different ways to fight against xenobiotics.
the latter is achieved rather by avoiding poisonous chemicals versus metabolizing them with the help of detoxication system, thus, opening the potential for trade-off between these two systems.
moreover, this hypothesis is experimentally supported  <cit>  by a negative correlation between cyp and odorant-binding proteins expression levels in insects.
in our future work, we will test whether urban-life associated selection sweeps are enriched in the genes that comprise xenobiotic avoidance and metabolizing systems.
additional files
additional file 1: tables s1- <dig>  samples, number of cyp, or, obp and desturases genes, results of selection detection, neuropeptides.
additional file 2: phylogenetic tree of ants fatty acid desaturases.
<dig> - hymenoptera-specific desaturases,  <dig> d. melanogaster cg <dig> orthologs,  <dig> — d. melanogaster cg <dig> orthologs,  <dig> — d. melanogaster desat <dig> orthologs,  <dig> — d. melanogaster cg <dig> orthologs,  <dig> — d. melanogaster cg <dig> orthologs.
authors thank n. yu.
oparina and t. s. putyatina for useful discussion.
declarations
this article has been published as part of bmc evolutionary biology vol  <dig> suppl  <dig>  2017: selected articles from bgrs\sb-2016: evolutionary biology.
the full contents of the supplement are available online at https://bmcevolbiol.biomedcentral.com/articles/supplements/volume-17-supplement- <dig>
funding
this work was supported by the russian scientific foundation, project no.
14-14- <dig> — specimen collecting, dna extraction, library preparation, sequencing, genome assembly and publication fee and project no.
16-16-00007— data analysis and discussion.
availability of data and materials
the genome assembly and predicted protein sequences have been deposited in the genbank under the bioproject id prjna <dig>
authors’ contributions
vas conceived and designed the experiment, eak and snl collected the animal samples, extracted dna and rna and prepared illumina libraries, all authors participated in sequence data analysis, eak, man, svn and vas wrote the manuscript.
all authors read and approved the final manuscript.
competing interests
the authors declare that they have no competing interests.
consent for publication
not applicable.
ethics approval and consent to participate
the animal use protocol was performed in accordance with bioethics committee of lomonosov moscow state university.
at present, the committee do not provide a specific approval numbers for invertebrate animal research.
animals used in this study do not belong to regulated or endangered species.

 ants are a successful and diverse group of insects, playing a role in various ecosystem functions. these roles include facilitating soil aeration, preying on herbivore insects, farming aphids for their honeydew, and cutting leaves to farm fungi for foraging. ant eusociality is of great scientific interest and numerous works have addressed issues in its division of labour and cast-specific gene expression. most ant genomes were sequenced due to their status as an invasive species, for their unusual cast division of labour, or for their negative impact on the economy. the black garden ant lasius niger is a usual ant of the northern hemisphere and dwells in nearly all landscapes in europe, ranging from forests to big cities. the last review of l. niger was in, where the new species lasius platythorax was established. workers in l. niger are small, while queens can reach 16 mm. colonies are monogynous and have only one egg-producing queen, though polyandry does occurs. l. niger nests are usually underground or in sand mounds, but are also found in wall cracks and rotting wood. l. niger feeds primarily on honeydew and other sugar-rich substances; small invertebrates are also part of their diet. farming aphids for honeydew often occur in gardens, thus effectively making itself a pest. these ants are also known to infest human housing, causing noticeable disturbances. l. niger dwells in holarctic region, inhabiting all of europe and parts of asia and north america. l. niger is the most widespread of all sequenced ants. in natural environments lasius niger occupy a subdominant position in ant communities, while species of the genera myrmica and formica play the main role as generalist predators. when forest ecosystems are eroded, however, dominant ant species are the first to disappear, creating possible niches and opportunities for other ant species. lasius niger has a very flexible ecological strategy and can inhabit different spatial tiers from soil to trees. due to its ecological plasticity lasius niger has grown widespread in urbanised areas: the black garden ant had even increased its abundance in highly affected territories. here we report the draft genome of l. niger as a means of studying the genetic mechanisms underlying insect adaptation to urban environments. we focus on gene families that could facilitate behavioural or physiological means of coping with specific urban stresses. methods source material population samples for sequencing were obtained from two locations: near the campus of moscow state university and from the istra district ca. 70 km from moscow. in early september worker ants from three sites at both locations were collected using aspirators and stored separately in 98% ethanol. approximately 50– ants from each of six sites were treated as different samples. for exact sample sizes see additional file 1: table s samples were dry-freezed and transported to the university of southern california. genomic procedures genomic dna for each of the six samples were prepared into six libraries and sequenced as paired-ended 100 bp reads on an illumina hiseq each library was individually barcoded and sequenced as a pooled sample of barcoded dna, generating million total reads. since de novo assembly of complete genomes can be sensitive to sequencing errors, it is critical that the reads used to generate contigs have the highest sequencing quality. reads were removed from consideration in the de novo assemblyif the read had a terminal “phred”, or if the read contained more than two unknown nucleotides. reads were also filtered out with trimmomatic if they had similarity to known polymerase chain reaction primer and illumina adapter sequences. the 272 m retained reads from all samples were assembled with spades version.. the data are deposited to bioproject prjna genome assembly completeness was assessed using cegma and busco. cegma score was % compared to – % range for other ant genomes. the raw illumina data and the draft assembly have been deposited in ncbi under the bioproject prjna and will be released upon publication. gene prediction and gene ontology content for gene prediction augustus was used with the nasonia vitripennis genome as a training set. the prediction was done on both strands, including partial genes. predicted genes were searched against the nr protein database. contigs with bacterial and fungal genes were removed from the assembly. bacterial 16 s rna were identified using blast searches against the bacterial 16 s rna database; selected sequences were then searched against the nr database for genus and species identification. for more accurate searches of genes encoding cytochromes, odorant receptors, odorant-binding proteins and desaturases, hymenopterans protein sequences were used to build alignments using hmm model with hmmbuild. we built separate models for each major cyp superfamily. hmmsearch output was checked by blast and delta-blast for the presence of specific domains. genes with hmmersearch hits were aligned using blastp against the nr database, and genes with the majority of hits from other families were removed from further analysis. we used delta blast to check for gene-specific domains. we also searched for sequences from other examined species in the hymenoptera genomes portal and in genbank. predicted proteins of l. niger and other ant species were then annotated using blastp and blast2go. we performed go mapping, blast-based annotation, interproscan annotation and joined blast and interproscan annotation data. go terms were assigned for each protein, go category numbers for each species were normalized by total protein number. dixon q-test for outliers was used to detect overrepresented and underrepresented go groups in the l. niger genome, assuming it as an outlier among all ants. go distribution was normalized with arcsine transformation l. niger was treated as outlier in both cases - in over and under represented go ids. proteins and genes of species with draft genomes that were not published yet were not included in go-mapping and intersproscan annotation and was used only for or and cyp comparison and selection detection. alignment and detection of selection to eliminate potential pseudogenes, any sequence with predicted start and stop codons was discarded if it was shorter than its hypothetical product length by 25%. we assumed amino acid residues for desaturases and for odorant receptors and p cytochromes, based on rounded off mean length of respective proteins in databases. for p we kept only sequences containing phe-x-x-gly-x-arg-x-cys-x-gly motif, which is believed to be crucial to stabilizing the iron ion in the heme group. the selected sequence predictions for p odorant receptors and desaturases were than aligned for each family using the muscle software. for the p cytochrome superfamily we aligned sequences in cyp cyp and cyp separately for each family. search for best substitution models were performed in mega. tamura-nei substitution modelwas found best for our dataset and was used for phylogenetic analysis. a maximum-likelihood phylogenetic tree was generated for each alignment using mega. to identify genes under selection, we applied a z-test for selection to nucleotide alignments of these genes. the positive selection hypothesis was rejected in none of the cases. a sequence was tagged as being under positive selection if both the neutrality and the purifying selection hypotheses were rejected. for the cyp subfamily of p cytochromes we also searched for sites under selection. for this analysis we used only complete coding sequences with start and stop codons in the length range of 1425– nucleotides due to limitations of selection search methods. upon removal of stop codons, sequences were codon-aligned using clustalw software. maximum-likelihood phylogenetic trees were then generated using mega. based on these trees, a selection search with fubar and meme methods was performed for each codon in a given alignment using hyphy software. the number of amino acid substitution events were then calculated based upon ancestor sequence reconstructed with slac.  genome assembly and annotation we sequenced and annotated the draft genome of lasius niger and compared it with the draft genomes of other ants. the 272 m reads were assembled with spades into contigs of more than 500 bp in length with a total length 245 mb and an n of  bp. these figures are similar to other available ant draft genomes and enable comparative genomic analysis. the assembly also included contigs of more than 1000 bp in length with a total length 237 mb. cg content was found to be 38%, similar to that found in camponotus floridanus. median kmer coverage is exhibiting a bimodal distribution of kmer coverage with peaks at and cpg dinucleotides are overrepresented in the l. niger genome: observed/expected cpg ratio is, similar to other ants: we identified transposable elements throughout the l. niger genome using repeatmasker. against the repbase te library v2016-08-. we found  mb of known transposable elements. however, there are many intact orfs of transposon proteins compared to other ants, which suggest high recent transposon activity in the l.niger lineage. transposons detected by repeatmasker include lines, mostly from r1/loa/jockey family, and ltr elements. application of the gene prediction tool augustus revealed genes in l. niger contigs, with having both start and stop codons. annotated genes were shorter than in other ant species and contained fewer exons, but total exon number and length is similar to values observed in other ants. therefore we conclude that the gene space of l. niger annotation is as complete as other ants, although the genome is more fragmented. to distinguish between genes in the ant genome and those originating from microbes, we used blast search against the non-redundant protein database. analysis of 16s and 18s ribosomal rna genes revealed the following non-ant species: bacteria, one enthomopathogenic fungus ophiocordyceps unilateralis, an unidentified spider and a plant from family fabiaceae. high protein blast-hit similarity additionally identified enterobacteriacea, propionobacter, nematode, lactobacillus, actinomycete, and, potentially, fusarium. after removing hits to bacterial and fungal contigs we obtained a total of ant genes; potentially complete and partial sequences. all these contigs were also removed from the bioproject. among non-bacterial annotated contigs the vast majority are annotated as proteins, common with camponotus floridanus. c. floridanus belongs to the same family formicinae as l. niger and their similarity in proteins corresponds to morphological likeness. three other ant species from the fig.  – solenopsis invicta, acromyrmex echinator and harpegnatos saltator are highly specialized and diverged from formicinae lineage in late cretaceous.fig. top blast hits of l. niger metagenome and genome assembly. most hits are from c. floridanus, closest sequenced relative of l. niger, and other ants go distribution the predicted proteome of l. niger is significantly different from other ant genomes in terms of abundance of several go categories. the proteome is enriched in domains involved in nucleic acid binding, dna repair, nucleotidyl transferase activity, heterocyclic compound binding and contain fewer domains involved in neuropeptide signaling. l. niger might also contain fewer odorant binding domains than other ants, but this difference was not statistically significant in a q-test. overrepresentation of nucleic acid binding domains is due to high abundance of retrotransposon genes coding gag-pol polyproteins or separate reverse transcriptases. we detected pol and gag-pol genes in the l. niger genome compared to in camponotus and between and in other ant species. no env genes were found to match the abundance of pol and gag-pol. we conclude that the pol and gag-pol genes belong to retrotransposomes and are not from ongoing retroviral infections. dna repair domains are overrepresented in l. niger mostly because of duplications of pif1-like helicases. the l. niger genome encodes pif1-like helicases compared to in camponotus and in most other ants. other dna repair proteins duplicated in l. niger include chromosome structure maintenance proteins 1a and dna damage-binding protein and tyrosyl-phosphodiesterase. detoxification system genes as our focus is on potential genomic features enabling success in urban environments, we paid special attention to the gene families involved in detoxification. glutathione-s-transferases and cytochrome p families are the most common detoxification genes among hymenoptera and we examined these two families in detail. we expected to find multiple duplications in these families, and in glutathione-s-transferase family we found genes,, though most had incomplete and short sequences. we found no evidence of species-specific duplications of glutathione-s-transferase in the l. niger genome. we did find cyp genes being highly duplicated in l. niger, with sequences matching cyp genes across families. this is the second largest value among ants, second only to cyp genes in c. floridanus from the same family. compared to a. mellifera we see fewer cyp families, but more genes per family in those present. interestingly, there are tandem clusters of cyp genes found in the l. niger genome assembly, most of which are members of cyp9e subfamily. most cyp genes in ant genomes are members of cytochrome p 9th, 6th and 4th families. the cyp family is highly expanded in l. niger, with complete and nearly complete members - over twice as much as any other ant. the number of cyp and cyp gene families in l. niger genome is within the diversity range of other ants. genes of the 9e family are organized in duplicated clusters, with genes of 6 k, 6a and 4g families also exhibiting tandem duplications.fig. comparative abundance of cyp subfamilies in hymenopteran genomes. various subfamilies are expanded in different species. even closely related c. floridanus and l. niger differ significantly in relative abundance of cyp and cyp subfamilies positive selection in cytochrome p genes for phylogenetic analyses and selection tests we selected only complete and nearly complete p genes where the conservative heme-binding motif phe-x-x-gly-x-arg-x-cys-x-gly was present. in the phylogenetic tree, most l. niger genes form clusters near those of c. floridanus, another ant in formicinae subfamily. most of the cyp genes in the examined species are under positive selection according to pairwise z test. except for recently duplicated genes phylogenetic trees of cyp and cyp always show a. mellifera and n. vitripennis as outgroups. l. niger contain two duplications of cyp genes after the divergence of lasius and camponotus lineages. five l. niger genes of cyp family sit on the phylogenetic tree separately, but form clusters with c. floridanus genes. two are putative pseudogenes. most ant genes from the cyp and cyp families were under positive selection since their divergence with other hymenoptera according to a pairwise z-test for selectionfig. phylogenetic tree of hymenopteran cyp genes. branches where statistically significant positive selection was found by z-test are in red. positive selection was found in most of the terminal branches of the phylogenetic tree. many lineage-specific duplications are evident in l. niger, a. echinator and c. biroi fig. positive and negative selected amino acids of cyp l. niger. ligand-binding pocket is located in the upper right part of the protein molecule, with heme group in the bottom of the pocket. most sites under positive selection are in the ligand-binding pocket, while structural alpha spiral domains are under purifing selection as noted previously, the cyp and cyp gene families are not amplified in lasius niger. nearly all ants with sequenced genomes have between and fragments of cyp genes, coding for more than half of the protein. camponotus has full-length genes and partial genes, with the partial genes coding for more than half of the protein. non-ant hymenopteran species a.mellifera and n.vitripennis have and predicted cyp family proteins. despite the found fragments of genes, similar to the number of cyp found in other species, only code for more than half of the protein and only have the sequence of the gem-iron stabilizing motif. other examined species have between and fragments of cyp genes, with only to containing coding sequences of more than 750 bp. every ant species except for c. obscurior has lineage specific duplication of cyp genes. the most abundant cyp genes are in invasive species, but in half of the genes the sequence of gem stabilizing motif is absent. the poorest representation is in c. biroi –. overall, the cyp tree has many species-specific duplications for all ants, except l. niger and a. cephalotes and c. obscurior. the most amplified are cyp in c. floridanus and s. invicta, both having groups of 3– genes and separately lying genes. positive selection in cyp family, as well as in the abovementioned families, was not significant for recent duplications; most of them are in c. floridanus.fig. phylogenetic tree of hymenopteran cyp genes. branches where statistically significant positive selection was found by z-test are in red. positive selection was found in most of the terminal branches of the phylogenetic tree. many lineage-specific duplications are evident in n. vitripennis, c. floridanus and c. biroi fig. phylogenetic tree of hymenopteran cyp genes. branches where statistically significant positive selection was found by z-test are in red. positive selection was found in most of the terminal branches of the phylogenetic tree. many lineage-specific duplications are evident in n. vitripennis and a. mellifera sites under selection in cyp9 to test whether an expansion of the cyp family was concordant with strong evolution on gene sequences, we executed a more detailed analysis. an alignment of full-length cyp coding sequences between and 1800 bp with start and stop codons was used to search for sites under positive selection. these criteria identified coding cyp sequences in a. cephalotes, sequences in a. echinator, in a. mellifera, in c. biroi, in c. floridanus, in h. saltator, in l. niger, in n. vitripennis, in m.pharaonis, in c.obscurior, in v. emeryi, in w.auropunctata and in s. invicta. meme detected alignment positions under positive selection. three positions were confirmed by other selection search methods, one was significant in all four methods. negative selection was detected in sites with statistical significance for fubar with aposteriori probability >  ). eleven positively selected positions are in ligand-binding pockets of the enzyme. an additional four are at the c-terminus, but this part of alignment was not present in all sequences. most of negatively selected sites lie in structural domains of the enzyme. the most frequent non-synonymous substitutions in cyp family did not change the charge and polarity of amino-acid residues. nevertheless, some substitutions changed charge and polarity – lys to glu, asn to asp, ala to thr and leu and ile to met. the rarest were changes of cys and trp. the meme analysis found different preferred substitutions in different subtrees of the l.niger cyp9e tree. in one subtree, arg was changed to hys and asn, while another arg was changed to ala. this trending of changing arg to other amino-acids occurred only in l. niger alignments – in other ants not only arg was changed, but also ala and hys. fubar analysis detects negative selection in most positions, where meme detects positive. these sites are conservative in the majority of lineages but in other leading to l. niger diversifying selection did occur. olfactory receptors and odorant-binding proteins olfaction related genes frequently exhibited signatures of directional selection or selection relaxation. hhmsearch and reciprocal blastp of l. niger predicted proteins revealed putative odorant receptors and odorant-binding proteins. one putative odorant-binding protein, having high similarity with other ant obp, is amino acids in length and is not recognized by blastp in the refseq protein database. other ants have additional obps, from in c. floridanus and a. echinator to in v. emeryi. only of 74 l. niger predicted or genes had coding sequence longer than 750 bp and were used for phylogenetic analysis and selection detection. other ants have between and fragments of odorant receptor genes, from to of this manifold were used for the phylogenetic analysis. apis mellifera has fragments of genes and fragments were used to construct the phylogenetic tree.. genes of odorant receptors in l. niger have amplified clusters – duplications and 5-genes repeats. most tandem duplications have related sequences in other ants. we did not find any l. niger duplication that was species-specific. not only did l. niger have only genes of suitable quality for odorant receptors, more than half were putative pseudogenes. despite the poor repertoire of l. niger odorant receptors, half were under positive selection. in contrast to the cyp genes, odorant receptors of l. niger do not predominantly form clusters with c. floridanus genes, but often cluster with genes of more distant ant species. the phylogenetic tree of odorant receptors indicates that there are many amplifications and deletions of odorant receptor genes, providing an explantation as to why closely related a. cephalotes and a. echinator have very different odorant receptor genes, perhaps due to pseudogenisation and lineage sorting in their putative ancestor’s gene set. z-test found positive selection in average in all sequence pairs. fatty acid desaturases cuticular hydrocarbons are important molecules in ant chemocommunication. these compounds are produced by fatty acid desaturases enzymes. ants with sequenced genomes have from in c.biroi to in h. saltator. l. niger and c. floridanus both have fatty acid desaturase genes, incomplete genes not included. desaturases of hymenoptera are grouped into six subfamilies, with one being unique for this order and other subfamilies orthologous to five drosophila melanogaster desaturases. no orthologs of d. melanogaster cg were found in l. niger and p. barbatus; other ants have one. hymenopteran specific desaturases are absent from c. biroi. other subfamilies of desaturases are present in all ant genomes. in all ant species, orthologs to d.melanogaster cg are under positive selection, while there was no selection within c. floridanus duplications. almost all orthologs to d. melanogaster cg and desat are positively selected as well. in hymenopteran specific desaturase group, the selection was significant predominantly in myrmicinae and formicinae subfamilies. no significant differences between l. niger and other ants can be found in desaturase gene content and selection mode. telomerases ants are known for their great intraspecific differences in life spans and l. niger has the greatest: from several months in males to decades in queens. in contrast to drosophila in which telomere length is maintained through retrotransposition and is consistent with other studied animals species, ants’ life span is based on telomerase activity. jemielty et al. showed caste-specific differences in expression level of telomerases. our annotation contains several genes related to telomerase activity. one copy of telomerase reverse transcriptase was found. thirteen copies of another important component of telomerase ribonucleoprotein complex – telomerase binding protein est1a – were found. four genes similar to telomerase cajal body protein were found. future functional analysis of telomerase sequences in l. niger could shed light on life span regulation in this species. vitellogenins vitellogenins were duplicated in many formicoid lineages. caste-specific expression is characteristic for vitellogenin paralogs in ants. however, the c. floridanus genome contains only one vitellogenin gene. we found only one vitellogenin in the lasius genome, with high similarity to the camponotus vitellogenin. hmm search for hymenopteran vitellogenins also revealed only one vitellogenin in harpegnatos, cerapachys and pogonomyrmex. other ant species had more vitellogenins; we found vitellogenins in solenopsis, in linepithema and wasmania and vitellogenins in volenhovia and monomorium. one more sole vitellogenin in formicinae can not, of course, reject a hypothesis of all-formicidae initial vitellogenin duplication, but add some more doubts to it. all mentioned vitellogenins were vgg, we did not search for vgf. discussion high loads of fungal parasites and retroelements here, we assembled the draft genome of l. niger and compared it with the genomes of other ant species. surprisingly, retrotransposons are an order of magnitude more abundant in this ant compared to others. discovered retrotransposon genes do not contain internal stop codons and expected to be active. we also discovered the expansion of dna repair proteins. here, we hypothesize that expansion of dna repair proteins could be a genome response to the expansion of retroelements. dna repair system takes part in transposon integration and is required to restore dna damage caused by incorrect integration. tes are known to interfere with host repair, therefore the diversification of dna repair system may represent the host’s response to retrotransposon expansion. we further hypothesize that the diversification of dna repair system might be an exaptation pre-adapting l. niger to explore urban environments. specifically, enhanced dna repair enzyme repertoire may play a role in resistance against dna-damaging xenobiotics such as polycyclic aromatic hydrocarbons. furthermore, both protein and 18s rrna blast results show high level of fungal dna from genus ophiocordyceps. these fungi are well-known entomopathogens, infecting various insects including ants. fungal 18 s rdna from l. niger assembly is most similar to sequences from o. unilateralis infecting camponotus carpenter ants, suggesting high rate of fungal infection in l. niger. however, characteristic fruit bodies protruding from the dead ant’s head have never been seen in l. niger. it was shown that laboratory settings that ophiocordyceps infects and kills many ant species besides its natural host however, reproduction and spore dispersal of ophiocordyceps requires complex host manipulation, which is successful only in its natural host. two species of camponotus live in the moscow region and they may be natural hosts of ophiocordyceps found in l. niger. alternatively, l. niger might be a natural host for this fungus but its transmission does not involve typical fruit body formation. nevertheless, the observed fungal dna level suggests that ophiocordyceps infection may be an important cause of mortality among l. niger and there may be the selective pressure toward resistance to this pathogen. cyp expansion insects can cope with pollution in two ways - physiological or behavioral. metabolic detoxification of xenobiotics is an example of physiological adaptation. we studied the cytochrome p superfamily in detail, as it is known for its crucial role in resistance to xenobiotics. compared to other ants, l. niger has a highly amplified cyp subfamily. phylogenetic analysis and comparison of cyp genes of l. niger with a. mellifera and n. vitripennis allocated all genes of this subfamily in ants into subfamilies cyp9e and cyp9p. functions of these subfamilies are unknown, but closely related subfamilies are known for their roles in insecticide resistance. entomopathogenic cordycepitales are known to synthesize wide range of toxic compounds. we have previously shown using molecular docking and virtual screening that l. niger cyp cytochromes form complexes with low free energy with fusarin c, fumonisin, ergot-alkaloids, enniatin and beauvericin. these compounds are produced by ophiocordyceps and are important pathogenicity factors. for some of l. niger cyp free energy of complexes with fusarin are below −480 kj/mol that suggests high affinity of enzymes to this substrate. we hypothesize, therefore, that the expansion of cyp in l. niger genome may result from coevolution with enthomopathogenic fungi. cytochrome p superfamily evolution in formicidae was extensively studied. differential expansions and contractions of p subfamilies were observed in different genera. these changes are linked with adaptations to novel ecological niches and changes in colony lifestyle. trophic specialization was found to promote the contraction of p family in p. barbatus. l. niger and c. floridanus, which belong to the subfamily formicinae, contain approximately the same number of cytochrome p genes, and this number is higher than that in other species of ants. like l. niger, c. floridanus dwells in an urban environment and similarly omnivorous. its diet includes living and dead insects and the secretions of aphids and scale insects. cytochrome p genes have wide range of substrates and single amino acid substitution can drastically change substrate specificity. it is possible that the large set of cytochrome p genes provide for polyphagia by enabling specific and nonspecific neutralization of various phyto and mycotoxins. cytochrome p genes may also be important in resistance to urban pollutants of different chemical composition. some cytochrome p genes can oxidize some of these chemicals, e.g., benzopyren. the importance of detoxification system in l. niger xenobiotic resistance may be further increased due to scarcity of anti-fungal protective behaviour. for example, l. niger does not remove aphids infected with parasitic fungi from symbiotic aphid colonies, while this behaviour is common in other aphid-herding ants such as formica s. str.. odorant communication genes of odorant receptors and odorant-binding proteins, including partial sequences, appear to be less abundant in the assembly of l. niger than in other ant genomes. we hypothesize that the lasius genus lost some odorant receptor and odorant-binding genes in the course of their evolution. our reanalysis of previously published data enables unbiased comparison between species. in s. invicta, there are only fragments of odorant receptors and odorant-binding proteins, instead of complete genes and more than fragments that were previously reported. this observation could be explained by the fact that the insect odorant receptors were considered to be homologous to odorant receptors of vertebrates prior to. accordingly, g-protein coupled receptors were treated as hypothetically odor detection related, while now we know that all insect odorant receptors are ion channels. another published prediction that was not confirmed in our work, is an increase in amounts of odorant receptors in ants as compared to a. mellifera and n. vitripennis. on the contrary, we observed that, in ants, odorant receptors and odorant-binding protein families underwent gene loss. this loss may be caused by transition of ants to terrestrial lifestyle and, therefore, increase in the role of antennal contacts in species communication. l. niger was shown to prefer visual information over pheromone trails when these stimuli were in conflict, contrary to other ant species. specifically in respect to urban environments, odorant receptor loss can be very useful, as it is increasing the resistance of the species to repellents.  in conclusion, we hypothesize that a number of seemingly unrelated events – expansion of dna repair, olfactory system genes loss and cyp gene amplification - can be viewed as preadaptations required for the successful colonization of urban areas. among these preadaptations, the changes in olfactory system genes and cyp genes provide two different ways to fight against xenobiotics. the latter is achieved rather by avoiding poisonous chemicals versus metabolizing them with the help of detoxication system, thus, opening the potential for trade-off between these two systems. moreover, this hypothesis is experimentally supported by a negative correlation between cyp and odorant-binding proteins expression levels in insects. in our future work, we will test whether urban-life associated selection sweeps are enriched in the genes that comprise xenobiotic avoidance and metabolizing systems. additional files additional file 1: tables s1- samples, number of cyp, or, obp and desturases genes, results of selection detection, neuropeptides. additional file 2: phylogenetic tree of ants fatty acid desaturases. - hymenoptera-specific desaturases, d. melanogaster cg orthologs, — d. melanogaster cg orthologs, — d. melanogaster desat orthologs, — d. melanogaster cg orthologs, — d. melanogaster cg orthologs. authors thank n. yu. oparina and t. s. putyatina for useful discussion. declarations this article has been published as part of bmc evolutionary biology vol suppl 2017: selected articles from bgrs\sb-2016: evolutionary biology. the full contents of the supplement are available online at https://bmcevolbiol.biomedcentral.com/articles/supplements/volume-17-supplement- funding this work was supported by the russian scientific foundation, project no. 14-14- — specimen collecting, dna extraction, library preparation, sequencing, genome assembly and publication fee and project no. 16-16-00007— data analysis and discussion. availability of data and materials the genome assembly and predicted protein sequences have been deposited in the genbank under the bioproject id prjna authors’ contributions vas conceived and designed the experiment, eak and snl collected the animal samples, extracted dna and rna and prepared illumina libraries, all authors participated in sequence data analysis, eak, man, svn and vas wrote the manuscript. all authors read and approved the final manuscript. competing interests the authors declare that they have no competing interests. consent for publication not applicable. ethics approval and consent to participate the animal use protocol was performed in accordance with bioethics committee of lomonosov moscow state university. at present, the committee do not provide a specific approval numbers for invertebrate animal research. animals used in this study do not belong to regulated or endangered species.
notably, the cyp family is highly expanded with complete and nearly complete members - over twice as many compared to other ants. sequences from 272 m illumina reads were assembled into contigs with total length of 245 mb, and n of  bp, similar to other ant genome assemblies enabling comparative genomic analysis. here, we present the draft genome assembly of the black garden ant lasius niger – the most successful urban inhabitant of all ants – and we compare it with the genomes of other ant species, including the closely related camponotus floridanus. remarkably, the predicted proteome of l. niger is significantly enriched relative to other ant genomes in terms of abundance of domains involved in nucleic acid binding, dna repair, and nucleotidyl transferase activity, reflecting transposable element proliferation and a likely genomic response. our results suggest that the success of l. niger in urbanized areas may be the result of fortuitous coincidence of several factors, including the expansion of the cyp cytochrome family due to coevolution with parasitic fungi, the diversification of dna repair systems as an answer to proliferation of retroelements, and the reduction of olfactory system and behavioral preadaptations from non-territorial subdominant life strategies found in natural environments. this family exhibits the signatures of strong directional selection, with eleven positively selected positions in ligand-binding pockets of enzymes. gene family contraction was detected for several components of the olfactory system, accompanied by instances of both directional selection and relaxation. lasius nigerurban environmentdraft genometransposable elementdetoxificationdirectional selectionthe international conference on bioinformatics of genome regulation and structure\systems biology novosibirsk, russia august- september issue-copyright-statement© the author 2017 diversification of cytochromes and dna repair systems along with reduced odorant communication are the basis of l. niger pollutant resistance and polyphagy, while non-territorial and mobilization strategies allows more efficient exploitation of large but patchy food sources.
 the world is rapidly urbanizing, and only a subset of species are able to succeed in stressful city environments. efficient genome-enabled stress response appears to be a likely prerequisite for urban adaptation. despite the important role ants play in the ecosytem, only the genomes of ~ have been sequenced so far. here, we present the draft genome assembly of the black garden ant lasius niger – the most successful urban inhabitant of all ants – and we compare it with the genomes of other ant species, including the closely related camponotus floridanus.  sequences from 272 m illumina reads were assembled into contigs with total length of 245 mb, and n of  bp, similar to other ant genome assemblies enabling comparative genomic analysis. remarkably, the predicted proteome of l. niger is significantly enriched relative to other ant genomes in terms of abundance of domains involved in nucleic acid binding, dna repair, and nucleotidyl transferase activity, reflecting transposable element proliferation and a likely genomic response. with respect to environmental stress, we note a proliferation of various detoxification genes, including glutatione-s-transferases and those in the cytochrome p families. notably, the cyp family is highly expanded with complete and nearly complete members - over twice as many compared to other ants. this family exhibits the signatures of strong directional selection, with eleven positively selected positions in ligand-binding pockets of enzymes. gene family contraction was detected for several components of the olfactory system, accompanied by instances of both directional selection and relaxation.  our results suggest that the success of l. niger in urbanized areas may be the result of fortuitous coincidence of several factors, including the expansion of the cyp cytochrome family due to coevolution with parasitic fungi, the diversification of dna repair systems as an answer to proliferation of retroelements, and the reduction of olfactory system and behavioral preadaptations from non-territorial subdominant life strategies found in natural environments. diversification of cytochromes and dna repair systems along with reduced odorant communication are the basis of l. niger pollutant resistance and polyphagy, while non-territorial and mobilization strategies allows more efficient exploitation of large but patchy food sources. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords lasius nigerurban environmentdraft genometransposable elementdetoxificationdirectional selectionthe international conference on bioinformatics of genome regulation and structure\systems biology novosibirsk, russia august- september issue-copyright-statement© the author 2017 