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Janssenswillenetal. BMC Ecol Evo (2021) 21:182
https://doi.org/10.1186/s12862-021-01910-w
RESEARCH ARTICLE
Odorant-binding proteins incanine anal sac
glands indicate anevolutionarily conserved role
inmammalian chemical communication
Sunita Janssenswillen1†, Kim Roelants1*† , Sebastien Carpentier2, Hilde de Rooster3, Mieke Metzemaekers4,
Bram Vanschoenwinkel5,6, Paul Proost4 and Franky Bossuyt1
Abstract
Background: Chemical communication is an important aspect of the behavioural ecology of a wide range of mam-
mals. In dogs and other carnivores, anal sac glands are thought to convey information to conspecifics by secreting a
pallet of small volatile molecules produced by symbiotic bacteria. Because these glands are unique to carnivores, it is
unclear how their secretions relate to those of other placental mammals that make use of different tissues and secre-
tions for chemical communication. Here we analyse the anal sac glands of domestic dogs to verify the secretion of
proteins and infer their evolutionary relationship to those involved in the chemical communication of non-carnivoran
mammals.
Results: Proteomic analysis of anal sac gland secretions of 17 dogs revealed the consistently abundant presence
of three related proteins. Homology searches against online databases indicate that these proteins are evolutionary
related to ‘odorant binding proteins’ (OBPs) found in a wide range of mammalian secretions and known to contrib-
ute to chemical communication. Screening of the dog’s genome sequence show that the newly discovered OBPs
are encoded by a single cluster of three genes in the pseudoautosomal region of the X-chromosome. Comparative
genomic screening indicates that the same locus is shared by a wide range of placental mammals and that it origi-
nated at least before the radiation of extant placental orders. Phylogenetic analyses suggest a dynamic evolution of
gene duplication and loss, resulting in large gene clusters in some placental taxa and recurrent loss of this locus in
others. The homology of OBPs in canid anal sac glands and those found in other mammalian secretions implies that
these proteins maintained a function in chemical communication throughout mammalian evolutionary history by
multiple shifts in expression between secretory tissues involved in signal release and nasal mucosa involved in signal
reception.
Conclusions: Our study elucidates a poorly understood part of the biology of a species that lives in close association
with humans. In addition, it shows that the protein repertoire underlying chemical communication in mammals is
more evolutionarily stable than the variation of involved glands and tissues would suggest.
Keywords: Odorant binding proteins, Chemical communication, Anal sac glands, Placental mammals, Carnivores,
Dogs
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Background
Carnivores use anal sac gland secretions (ASGS) to com-
municate by smell, whereby each individual is believed
to produce a distinct chemical profile, composed of a
Open Access
BMC Ecology and Evolution
*Correspondence: kim.roelants@vub.be
†Sunita Janssenswillen and Kim Roelants contributed equally to this work
1 Amphibian Evolution Lab, Biology Department, Vrije Universiteit Brussel,
Pleinlaan 2, 1050 Brussels, Belgium
Full list of author information is available at the end of the article
Page 2 of 16
Janssenswillenetal. BMC Ecol Evo (2021) 21:182
unique blend of scent molecules [1]. e exchange of
chemical profiles between individuals of the same spe-
cies can be a key determinant of their subsequent social
interaction. Behavioural experiments have shown that
ASGSs are used among conspecifics for territorial mark-
ing (wolf, hyena) [2–4], and for identification of gender
(ferret, brown bear) [5, 6], individuality (domestic cat,
honey badger, hyena, mongoose) [7–10], hierarchic sta-
tus (hyena) [8, 11], familiarity (ferret, hyena) [5, 11], and
kin recognition (meerkat) [12].
Dogs (Canis lupus familiaris), of all carnivores, argu-
ably live in closest association with humans and the
importance of anal scent assessment in their social
behaviour is known well beyond the scientific commu-
nity. Indeed, when dogs meet, their sniffing behaviour
reveals an obvious focus on the perianal zone that con-
tains the anal sac glands (Fig.1A, B) [13, 14]. Such scent
assessment not only takes place during a first encounter
but also on a daily basis among pack members. ASGSs
may also be used by dogs for scent marking when
released together with faeces, to advertise their presence
and/or territory, as shown in wolves [2, 3]. Finally, during
fear-induced responses, the entire content inside the anal
sacs can be released as a spray, resulting in an intense foul
odour and suggesting a common alarm or defence scent
signal [13, 15, 16]. Despite many indications of the mul-
tifunctionality of anal sac glands in dogs, investigation of
their molecular contents has remained limited to early
gas chromatography studies of volatile compounds [17,
18].
Anal sac glands are modified sweat and sebaceous apo-
crine glands that are a unique trait of carnivores [1]. e
sacs are often hypothesized to be fermentation chambers
in which small volatile metabolites produced by gland-
inhabiting bacteria are stored and secreted to be used
as scent signals [19–25]. Consistent with this hypothesis
(and because volatile molecules are most effective in cre-
ating a scent) most studies on carnivores have focused on
the identification of small volatile molecules [5, 8, 10, 18,
21, 26–30]. However, proteins endogenously produced
by the animal (and thus encoded by genes in its genome)
could be similarly important for chemical communica-
tion. Indeed, endogenously produced proteins have been
shown to play an important role in chemical signalling in
noncarnivoran mammals like rodents [31–37], primates
[38] and pigs [39, 40]. Despite early predictions of the
presence of proteins in carnivoran anal sac glands [41],
only one protein has ever been identified from the ASGS
of a carnivore [42].
To investigate the importance of proteins in carnivore
chemical communication, we conducted a comprehen-
sive proteomic study of the ASGS of dogs using pub-
lished genome sequences as a reference. To reconstruct
the evolutionary history of the newly found proteins,
we used comparative genomics and phylogenetic analy-
ses on related genes of a wide taxonomic range of mam-
mals. Our findings, besides providing new insights in the
functioning of canine scent glands, further elucidate the
evolution of chemical signalling in carnivores and, by
extension, in mammals.
Results
Identication ofproteins incanine ASGS
We sampled the anal sac secretion of 17 dogs of differ-
ent ages, including six males and 11 females (Additional
file 1). Four females were sampled twice, once during
anoestrus and once in oestrus during their fertility peak.
Bicinchoninic acid (BCA) protein assays indicated vari-
able protein concentration across canine anal sac samples
and animals, ranging between 5.44 and 868.68mg/ml (21
samples from 17 individuals; Additional file1). Despite
this variation in concentration, SDS-PAGE and RP-HPLC
indicated a similar protein composition across all individ-
uals. For three samples, the highest chromatogram peak
was analysed using a combination of Edman sequencing
and electrospray ionization mass spectrometry. It repre-
sented a protein of 17,468.28Da with the following 24
N-terminal amino acids: HLPLPNVLTQIxGPxKTLYVS-
SNN. A BLAST search against the Uniprot database [43]
identified this sequence as part of an Odorant Binding
Protein (OBP), a subclass of structurally related proteins
within the lipocalin family that can bind a wide range of
volatile molecules [44, 45].
To obtain a general overview of proteins secreted by
dog anal sacs, we conducted liquid-chromatography–
tandem mass spectrometry (LC–MS/MS) on all 21 sam-
ples. is analysis showed the existence of not one, but
three OBP isoforms (Fig.1C), all of which were present
in all samples, albeit at varying abundances (Additional
files 2 and 3). No apparent differences were observed
between age classes, genders, or oestrus states, but OBP
abundances were invariably higher in mixed breeds (stray
dogs) than in crossbreds and purebreds (Additional
file2).
Besides OBP, LC–MS/MS revealed a large diversity
of secretory proteins, many of which have previously
been shown to play a role in the mammalian immune
system. In total, peptide fragments of 57 unique pro-
teins at 0.01 false discovery rate (FDR; Additional file3)
were sequenced. Four of these represent antimicrobial
proteins: lactotransferrin (LTF), cathelicidin, prolactin-
induced protein (PIP) and C-type lysozyme. In various
mammals, each of these proteins has been shown to kill
bacteria or control their growth through different mecha-
nisms [46–49]. We also found six immunoproteins: poly-
meric immunoglobulin receptor (PIGR), joining chain
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Janssenswillenetal. BMC Ecol Evo (2021) 21:182
of multimeric IgA and IgM (JCHAIN), Immunoglobu-
lin Heavy Chain-like (IGH), Zinc-Alpha-2-glycoprotein
(AZGP1), and two Ig domain-like containing proteins. In
humans and other mammals these proteins are involved
in antigen processing and antigen presentation as part of
the humoral immune system [50–53]. In addition, sev-
eral types of protease inhibitors (WAP-type, KAZAL-
type protease inhibitors, serpins, cystatins and alpha-2
macroglobulins) were identified in the canine anal sac
content. e exact function of these proteins in ASGSs is
unclear but by controlling the activity of proteases, they
may contribute to a wide range of biological processes
[54]. Serum Albumin (SA) represents a final consistent
constituent of ASGS. Besides being a transport protein in
blood, SA has also been described as part of the phero-
mone signalling complex in Asian elephants (Elephas
maximus) [55].
Structural features ofodorant binding proteins
Canine OBPs are small (173 or 174 amino acids) extra-
cellular proteins that share a sequence similarity of 67%
(Fig.1C). Tertiary structure prediction using the online
tool PHYRE2 [56] confirmed that all three OBPs form
an eight-stranded antiparallel beta barrel, a structure
that has been previously determined for OBP3 (iden-
tified as the allergen Can f 4 in dog dander) [57] and is
shared among members of the lipocalin family [58]. ey
can reversibly bind a broad range of small organic com-
pounds in their hydrophobic pocket structures [44, 45,
59, 60]. As part of the lipocalin family, OBPs contain a
GxW motif (positions 29–31), a glycine residue (position
136), and two cysteines (positions 78 and 170; forming a
disulphide bridge), as characteristic sequence signatures
shared among most lipocalins [45]. An additional lipoca-
lin-specific motif, YxxxYxG (positions 93–99) was found
to be only partially conserved in canine OBPs (Fig.1C).
Canine anal sac gland OBPs are encoded byagene cluster
ontheX‑chromosome
Screening of the dog genome (UCSC, NCBI and Ensembl
genome browsers) revealed that the anal sac OBP iso-
forms are encoded by three different genes organised in
a single cluster on the X chromosome (Fig.1D). None
of the isoforms showed evidence of alternative splic-
ing or the combined transcription of exons of multiple
genes (yielding chimeric proteins), as confirmed by our
LC–MS/MS protein data (not shown). For obp3, encod-
ing the dander allergen Can f 4, a transcript was previ-
ously cloned [61]. Mapping of this transcript and the
protein sequences inferred here indicated that the three
genes share the same structure composed of seven
exons (Fig. 1E) with coding regions spanning exons 1
through 6. Peptide abundance data inferred from our
LC–MS/MS analysis shows that the first 16 amino-acids
Fig. 1 Odorant-binding proteins (OBP) in anal sac gland secretions of dogs. A Picture of dog behaviour that shows the investigation of anal zones.
B Canine anal sacs are situated at both sides of the anus. C Sequence alignment of OBP isoforms of canine ASGS. The signal peptide and the
functional protein are marked in grey and black, respectively. Stars show identical amino acids in all proteins. Signature residues and motifs that
are conserved among most mammalian lipocalins are included below the amino acid sequences. D Obp gene organisation in the dog genome.
The obp gene cluster is situated on the X chromosome and obtains three genes with nucleotide positions of obp1: 2,893,438–2,899,738, obp2:
2,905,438–2,911,062, and obp3: 2,918,198–2,923,699. E Obp gene structure: every dog obp gene contains seven exons of which exons 1 to 6 span
the coding region. UTR = untranslated regions at the 5ʹ and 3ʹ ends; SP = signal peptide
Page 4 of 16
Janssenswillenetal. BMC Ecol Evo (2021) 21:182
encoded by these regions are not included in the
secreted proteins, indicating the posttranslational exci-
sion of a conserved N-terminal fragment. is fragment
(MKILLLCLILVLACSDA) is confirmed to be a signal
peptide, characterising secretory proteins.
Conserved synteny oftheobp gene repertoire inplacental
mammals
To investigate the evolutionary history of the dog’s obp
repertoire, we used the sequences of the newly discovered
dog proteins to screen 98 genomes, including those of 19
other carnivores as well as representatives of the major
mammalian lineages. Homologous genes sharing syn-
teny with the dog’s obp genes (as evidenced by the same
order of flanking genes) were found in 35 placental spe-
cies representing a wide phylogenetic range (Additional
file4). In 15 of those, obp homologues were confirmed
to be situated on the X-chromosome (Fig. 2). In the
majority of these species, the obp locus is situated in a
relatively distal position on the X-chromosome, at dis-
tances below 4Mb from one of the chromosome’s ends.
Such distal position is likely within the pseudoautosomal
region (PAR) of the X-chromosome, a short region that
maintains high sequence similarity with a corresponding
region on the Y-chromosome, allowing recombination
during meiosis in males [62]. Based on inferred pseudo-
autosomal boundaries in the genes grp143 and shroom2
[63–65], our genome mapping confirms the position of
the obp locus within the PAR for cattle (Bos taurus), pig
(Sus scrofa), cat (Felis catus) and dog (Fig.2). In horse
(Equus caballus) however, the obp gene cluster lies out-
side the PAR due the presence of a more distal bound-
ary, within a horse-specific xkrp3-like gene [62, 66]. In
house mouse (Mus musculus), the obp gene cluster lies in
a far more central position of the X-chromosome, at over
75Mb distance from a very short and modified PAR [67].
e mapping of obp genes within the PAR of the X-chro-
mosome implies that identical copies or alleles are very
likely present in the corresponding PAR of the Y-chro-
mosome. Indeed, BLAST screening revealed the pres-
ence of identical obp gene copies on the Y-chromosome
in pig and common bottlenose dolphin (Tursiops trunca-
tus), two of only few species for which separate sequence
data for the Y-chromosome are available. In pig, one gene
copy was additionally found in the nonautosomal region
of the Y-chromosome, at approximately 8.73Mb from
the chromosome’s end (Fig.2). If correct, this observa-
tion implies the origin of a male-specific obp gene copy.
Screening of five marsupial and two monotreme genomes
reveals the strong synteny of genes that flank obp genes
in placental mammals. However, in all cases, this synteny
is restricted to autosomal chromosomes, suggesting that
their position on the X-chromosome is unique to placen-
tal mammals. Similarly, no obp genes were found in any
marsupial or monotreme genome.
Dynamic evolution oftheobp gene repertoire inplacental
mammals
Placental mammals show substantial variation in the
number of obp genes, suggesting differential rates of
gene diversification across taxa. Among carnivores, can-
ids (dogs and foxes) share the largest number (three) of
obp genes. Remarkably however, the dingo (Canis lupus
dingo, Australia’s feral dog) only has two obp genes
despite its very recent (and incomplete) divergence
from domesticated dogs [68]. e majority of carnivores
as well as other placental species share one or two obp
genes. Absence of obp genes was observed for meerkat
(Suricata suricatta), all bats (19 genomes screened), Chi-
nese treeshrew (Tupaia chinensis), Sunda flying lemur
(Galeopterus variegatus) and all primates (29 genomes
screened). For some species (most eulipotyphlans and
all lagomorphs), the presence, number and organisation
of obp genes could not be established due to the frag-
mentary nature of the available draft genomes. e larg-
est obp gene repertoires are found in rodents, pig and
pecorans (horned mammals including cattle). While this
repertoire has been well characterised in house mouse
[69–73] and golden hamster [74–76], only few obp genes
of pig and cattle encode previously identified proteins
[77–79]. In several species, identified obp homologues
are probably pseudogenes (Additional file 4). All obp
homologues found in pinniped species (walrus, sea lions
and seals) share premature stop codons, while in ceta-
ceans (whales and dolphins), both obp homologues are
apparently missing exons. In other species, additional
Fig. 2 Variation in size and genomic organisation of obp gene repertoires in placental mammals. Each line represents a schematic map of the
genomic organisation of obp genes (in red) and flanking genes (in blue) for the species and chromosome/scaffold indicated on the left. Species
sharing the same organisation are listed on the right. Individual genes are depicted as arrows indicating their orientation on the chromosome.
To visualise the shared synteny, orthologous genes are vertically aligned across species when feasible. Shades of blue of flanking genes indicate
whether they typically lie distal (dark blue), proximal (medium blue) or at a large distance (light blue) from the obp locus. Genes on a different
genome scaffold (in Iberian mole) are coloured ochre. Numbers above obp genes indicate their distance (in Mb) from the nearest chromosome
end. Known chromosome ends are indicated by a vertical cap typically on the left of a map. Yellow windows in delineate known pseudoautosomal
regions
(See figure on next page.)
Page 5 of 16
Janssenswillenetal. BMC Ecol Evo (2021) 21:182
Fig. 2 (See legend on previous page.)
Page 6 of 16
Janssenswillenetal. BMC Ecol Evo (2021) 21:182
exon and gene fragments were found in the vicinity of
identified obp genes, indicating duplication and/or loss of
incomplete genes.
Phylogenetic analyses of 102 sequences retrieved from
three dog breeds, dingo and 36 other placental species
further elucidate the evolutionary history of the obp
gene repertoire (Additional files 4 and 5). Because high
sequence divergence between obp and other lipocalins
complicates sequence alignment and may lead to spuri-
ous rooting and poor branch support, we conducted
phylogenetic analyses without outgroup sequences.
e resulting unrooted obp gene tree is relatively well
resolved, with high Bayesian posterior probabilities
(BBP > 0.95) and nonparametric bootstrap percentages
(NPBS > 75%) for major clades grouping obp sequences
from the same mammalian taxa, like Carnivora, Pecora,
Rodentia and Afrotheria. To reconstruct obp gene diver-
sification throughout the placental mammalian evolu-
tionary history, we performed a gene-tree/species-tree
reconciliation (GTSTR) analysis, which superimposed
the inferred gene tree on the phylogeny of placental
mammals [81–84] while minimizing the inferred num-
ber of gene duplication events and losses. A GTSTR
analysis using a fixed gene tree (prioritising confidence
in the gene tree including weakly supported branches
over minimising the number of events) reconstructed 42
gene duplication events and 30 gene losses (Additional
file6). Instead, when rearrangement of weakly supported
branches (BPP < 0.9 and NPBS < 70%) was allowed in
favour of an improved reconciliation, a far more parsimo-
nious reconstruction was obtained, with 38 duplications
events and 15 losses (Fig.3). is difference reflects an
effect of phylogenetic uncertainty in the gene tree and
is mostly restricted to basal branches in the mammalian
tree (Additional file 6). Despite their differences, both
reconciliations indicate that obp gene duplication started
during the early placental radiation, creating at least two
basal paralogues before the divergence between Laura-
siatheria (including carnivores, artiodactyls and relatives)
and Euarchontoglires (including rodents, primates and
relatives). One obp paralogue (indicated by blue line-
ages in Fig.3) was subsequently lost in the ancestors of
Euarchontoglires (incl. rodents, primates and relatives)
and Chiroptera (bats) and along the branches leading
to meerkat and Iberian mole (Talpa occidentalis). How-
ever, the same paralogue duplicated further in canids,
felids, horse, pig and pecorans. A second paralogue (indi-
cated by red lineages in Fig.3) was lost in the ancestor
of Euarchonta (primates and relatives), bats, carnivores,
white rhinoceros (Ceratotherium simum) and pig but
diversified in rodents and pecorans. e shared presence
of three obp paralogues in dogs and foxes is explained
by two gene duplication along the canid stem lineage.
Within the dingo, the lost gene turns out to be obp3 (Can
f 4).
Absence of tissue expression data for the majority of
presently identified obp genes precludes a detailed recon-
struction of the evolutionary changes in OBP secretion.
However, our finding of OBP in canid anal sac glands
combined with previously published proteomic data on
OBP in elephant, rodents, pigs and cattle (Additional
file 7) implies the expansion of expression sites across
multiple mammalian taxa to include their presence in
nasal mucosa, skin, saliva, tears, vaginal secretion and
anal sac gland secretion. Noteworthy, these sites the evo-
lution of functions at the receiving end of chemical com-
munication (nasal mucosa) as well as at the signalling end
(all others). Mapping of these secretion data on a pruned
version of the inferred obp gene tree using the parsimony
principle yields a first tentative image of OBP expression
evolution (Fig. 4). e most parsimonious reconstruc-
tion (assuming a single ancestral origin for each observed
secretion site and not counting losses) suggests that nasal
mucosa was the original expression site in the last com-
mon placental ancestor, with skin expression originating
no later than the basal obp gene duplication during basal
placental diversification (see also Fig. 3). e origin of
lacrimal, salivary and vaginal secretion in rodents must
have happened no later than the last ancestor of golden
hamster and house mouse. Finally, as anal sac glands are
(See figure on next page.)
Fig. 3 Evolutionary history of the obp gene repertoire in placental mammals. The taxonomic diversification of placental mammals is depicted
here as a timetree (thick light grey branches) with divergence times inferred from ref. 84 (see “Methods”). Obp gene lineages are drawn as thin
branches superimposed on the timetree with gene duplication events and gene losses along branches depicted as vertical bars (I) and crosses
(X), respectively. Ancestral and extant gene repertoires are schematically shown at key internal nodes and terminal nodes respectively. Red and
blue branches and genes represent two paralogous gene lineages that descended from a gene duplication inferred at the base of the placental
radiation, in the ancestor of Euarchontoglires and Laurasiatheria. Probable pseudogenes are coloured in lighter shades. The reconstruction was
obtained by reconciling an estimated gene tree with the mammalian timetree while inferring the lowest possible number of gene duplication
events and losses. As a gene tree, we used a consensus phylogram from Bayesian and nonparametric bootstrapping analyses in which weakly
supported branches (posterior probabilities < 0.9 and bootstrap percentages < 70%) were allowed to be rearranged if this reduced the number of
inferred gene duplication events and losses. An alternative reconstruction in which such rearrangements were not allowed is shown in Additional
file 6
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Janssenswillenetal. BMC Ecol Evo (2021) 21:182
Fig. 3 (See legend on previous page.)
Page 8 of 16
Janssenswillenetal. BMC Ecol Evo (2021) 21:182
a carnivore synapomorphy, they must have originated
along the stem branch of Carnivora, after its divergence
from Pholidota (pangolins) and before the last common
ancestor of all carnivores (ochre time window in Fig.4).
Consequently, OPB secretion in these glands must have
similarly originated after the carnivore–pangolin diver-
gence and before the duplication events that created
three canid paralogues in an ancestor of dogs and foxes.
Discussion
Research of various mammal species has demonstrated
that OBP proteins are involved in two key phases of
chemical communication. In vaginal secretions, saliva,
tears and urine of rodents, they are an essential part of
signalling complexes that are exchanged during sexual
and other social interactions [32, 35, 59, 69, 85–89]. In
nasal mucosa of elephants, rodents, and some ungu-
lates (pig, cow, goat, sheep), OBP proteins assist with the
perception of molecules inhaled from the environment
[77, 78, 90–94]. e function of OBP in other mam-
mals and the role of anal sac glands in carnivore scent
signalling together provide a compelling indication that
canine OBP proteins contribute to conspecific chemical
communication. Fuelled by the fermentation chamber
hypothesis, anal sac research has predominantly pur-
sued the identification of microbiomes and associated
volatiles [5, 8, 10, 18, 21, 26–30]. However, our finding of
OBP proteins as major components in ASGSs indicates
that endogenously produced proteins are a crucial part
of this signalling system as well. Since canids share anal
sac glands with other carnivores (felids, hyenas, mon-
gooses, bears and musteloids), the involvement of OBP in
anal scent communication may be more widespread than
currently known. Only one protein-related study of car-
nivore anal sac glands has been performed. In the domes-
tic cat, the protein Fel d 1, a secretoglobin described as
a human allergen and unrelated to lipocalins, was iden-
tified [42]. However, as this study targeted this protein
using immunochemistry, no broader information on pro-
tein content of feline ASGSs is known.
OBP proteins may perform a range of biological func-
tions in ASGSs. First, these proteins could transport and
store odorants (the actual scent signals) by the formation
of protein–ligand complexes [45]. Second, OBP proteins
could extend the preservation of a signal in faecal scent
marks, by delaying the release of their ligands into the
environment [95, 96]. ird, selective binding of specific
ligands could control the information that is presented
to the receiving animal. is observation was made
for SAL1 in pigs [40] and aphrodisin in hamsters [59].
Fourth, OBP proteins could act as a trigger to release
volatile ligands during investigation by another animal.
Dogs tend to press their nose against another individual’s
anal zone or scent mark, sometimes even licking it. is
contact could change the physicochemical environment
of the transport protein–ligand complexes, causing their
dissociation. Such mechanism has been postulated for
elephants [55]: pre-ovulating females release urine marks
which contain complexes of SA proteins and volatile sex
pheromones. e trunk of a male inspecting a female
urine mark changes the acidic environment while touch-
ing the urine, which triggers the release of the SA-pher-
omone complex, creating a burst of pheromone scent.
Finally, OBP proteins could act as signals themselves.
is function was previously shown in mice for major
urinary proteins (MUPs), distantly related lipocalins that
are involved in sexual attraction and individual recogni-
tion [34, 37].
Scent in dogs is thought to convey information regard-
ing age, gender, heat cycle, social status, health and fit-
ness [97, 98]. One question that arises from our findings
Fig. 4 Tentative reconstruction of the evolution of new OBP
secretion sites. Information on expression sites of extant obp genes
is inferred from the present study (dog obp) and previous proteomic
studies of nasal mucosa (N), saliva (S), skin (Sk), tears (T ) and vaginal
secretion (V; Additional file 7). The depicted tree is based on the
previously inferred gene tree pruned to include only genes for
which expression information are available and modified to fit
placental mammal divergence times retrieved from Ref. [84]. Nodes
representing gene duplication events are labelled with ‘d’. For each
expression site, the history is traced back (coloured branches) to a
single origin (a labelled vertical bar). Origins were mapped on the
tree by ancestral state reconstruction using the parsimony principle
(see “Methods”). Uncertainty in the origin of OBP secretion in anal sac
glands is indicated by a dashed line. The time window in which anal
sac glands most likely originated is shown as a yellow window
Page 9 of 16
Janssenswillenetal. BMC Ecol Evo (2021) 21:182
is whether OBP variation across dog individuals reflects
such information. Although we investigated only a lim-
ited number of individuals, OBP abundances seemed
consistently higher in mixed breeds than in crossbreds
and purebreds (Additional files 2 and 3). A possible expla-
nation for this observation could be different social envi-
ronments in which the dogs were born. Purebred dogs
typically descend from pedigrees characterised by limited
social structure and non-competitive mate selection con-
trolled by humans. In contrast, all mixed-bred dogs of the
current study were captured as stray dogs by animal shel-
ter organisations on the streets of Romanian and Spanish
towns, where they were part of large packs. Such packs
are characterised by a well-developed hierarchic struc-
ture and mate selection controlled by highly competitive
social interaction [99]. In such a complex social environ-
ment, a well-functioning communication gland is essen-
tial. An expanded comparative study including additional
breeds and integrating observations of mate choice in
feral conditions could substantiate this hypothesis.
Besides a general role in social interaction, OBPs of
several mammalian taxa have been shown to underlie
chemical communication related to sexual reproduc-
tion. In this perspective, the position of obp genes on the
X-chromosome enables a possible genetic mechanism for
the evolution of new sex-specific signals. Our genomic
analyses show that obp genes of dog, cat, cattle and pig lie
within the pseudoautosomal region (PAR). Similar distal
positions in other taxa (Fig.2) suggest that this pattern
may be common in placental mammals. e PAR is a seg-
ment of typically less then 10Mb that shares 96–100%
sequence identity with a corresponding segment on the
Y-chromosome [62]. Consequently, obp genes in many
species may have identical copies or closely related alleles
on the Y-chromosome. In house mouse and horse how-
ever, the obp loci lie outside the PAR, due to their relo-
cation to a far more proximal locus (mouse), or through
the origin of a new pseudoautosomal boundary slightly
more distal than the obp gene cluster (horse [66]; Fig.2).
Either way, an X-specific position may preclude recombi-
nation during meiosis in males and render obp genes sub-
ject to dosage compensation in females by inactivation
of one X-chromosome. However, if they stay close to the
pseudoautosomal boundary (as is the case in horse), they
may escape X-inactivation, leading to sex-specific differ-
ences in expression [100]. is observation been made
for human X-specific genes like nlgn4x [101], which in
most mammals flanks the obp genes (Fig.2). More pro-
foundly, a similar relocation of genes from the PAR to the
Y-specific region would effectively create male-specific
gene copies. In the case of obp, such relocation could
lead to the origin of male-specific pheromone-binding
proteins and thus a sexually dimorphic chemical signal.
In pig, such relocation may have already happened, as a
single obp gene copy was found at 8.73Mb from the end
of the Y-chromosome, outside the PAR (Fig.2). In the
expanding field of mammalian genomics, sequencing of
the Y-chromosome has lagged behind due to sequenc-
ing difficulties caused by the high abundance of repeat
segments [65]. Targeted sequencing efforts focusing
on Y-chromosomes may reveal additional examples of
Y-specific obp genes.
e history of the obp repertoire in mammals as recon-
structed here matches a birth-and-death model of gene
family evolution [102] with frequent gene duplication
and loss. is process has created substantial variation
in obp gene repertoires across placental mammals. e
loss of obp genes in several lineages could either indi-
cate a reduced importance of chemical communication
or the functional replacement of obp by other proteins.
e apparent pseudogenization of obp genes in pinnipeds
and cetaceans could be linked to their shift to a marine
life where chemical communication may be replaced by
acoustic and tactile signals. In contrast, the presence of
expanded repertories in canids, horse, horned mam-
mals and rodents could be interpreted as reflecting an
increased importance of OBP in chemical communica-
tion. Yet, this hypothesis is difficult to substantiate: the
dingo for example has only two obp genes but there is
no indication that scent communication has become less
important in these feral dogs compared to their domes-
ticated relatives. A large repertoire of OBP proteins may
not even be required for effective chemical communica-
tion. Indeed, a small number of ligand-specific OBP may
suffice if only a limited number of ligands is involved in
chemical communication. In addition, some proteins
may be capable of binding a multitude of ligands, as has
been demonstrated for aphrodisin in the golden hamster
(Mesocricetus auratus) [59]. Finally, a single gene may
suffice to produce multiple odorant-binding isoforms.
In pig, proteins encoded by the same lipocalin genes can
undergo different posttranslational modifications, creat-
ing isoforms with different ligand affinities [92, 103].
Given the multitude of secretions and tissues in which
various OBP proteins have been found across species,
gene diversification may have been paralleled by an
equally dynamic evolution of gene expression. Expression
variation that includes both signal sending and receiv-
ing secretions/tissues can even be found among closely
related genes within a single species, as evidenced by
OBP in the mouse (Fig.4) [89]. Use of the same proteins
at signaling and perceiving ends might facilitate the adap-
tive evolution of chemical communication, as it does not
require coevolution of different genes or protein families
on both sides. Instead, mutation of a single protein family
may allow for simultaneous adaptation of both processes.
Page 10 of 16
Janssenswillenetal. BMC Ecol Evo (2021) 21:182
With the current knowledge of OBP expression, it is too
early to reliably infer the timing and order in which new
expression sites emerged during mammalian evolution
and the reconstruction in Fig. 4 should be interpreted
very cautiously. However, OBP expression in anal sac
glands very likely originated in an ancestral carnivore
after its divergence from pangolins and before the diver-
gence of dogs and foxes, since (1) these glands do not
exist in non-carnivores and (2), the three OBP isoforms
found here duplicated before the dog-fox divergence. In
contrast, expression of obp genes in nasal mucosa may
have evolved once in an early placental ancestor (as
shown in Fig.4) or may have originated in parallel in in
several taxa. Detailed comparative analyses of multiple
tissues across a wide range of mammals will be required
to further resolve the evolution of expression sites and
related functions.
Conclusions
For decades, carnivoran anal sacs have been mostly con-
sidered as fermentation chambers from which volatile
molecules produced by symbiotic bacteria are secreted
as scent signals to communicate with conspecific ani-
mals. For the first time, we identified endogenously pro-
duced proteins as an abundant component of canine anal
sac glands, which are likely to play an important role in
chemical communication, probably by enabling efficient
transfer of volatile signals or possibly by being part of
the signal themselves. ese odorant binding proteins
are encoded by a gene cluster that originated on the
X-chromosome early in placental mammal evolution
and maintained a role in chemical communication across
mammalian taxa by shifting expression to various tissues
involved in both signalling and receiving scent molecules.
Methods
Animals
Anal sac secretions were obtained from six male and 11
female dogs of different breeds and age categories (Addi-
tional file1). Four of the female dogs were sampled two
times, once in oestrus and once in anoestrus. Oestrus
samples were taken when male housemates mounted the
female, or around the time that blood progesterone lev-
els reached 2ng/ml (in dogs where blood was taken for
breeding purposes).
Protein analysis
Collection ofanal sac gland secretion
Dogs were handled by their owners while one of the two
anal sacs was emptied by gently squeezing the surround-
ing tissue. Secretion was collected in a 10ml falcon tube,
and immediately transported on ice to the lab. Samples
were vortexed for 30 s and centrifuged for 15 min at
21,000g at 4 °C. Twenty microliter of supernatant was
detained for total protein concentration measurement by
using the Pierce BCA Protein Assay Kit (Sigma-Aldrich).
e remaining supernatant was divided into aliquots and
stored at −80°C until further handling.
RP‑HPLC/MS andEdman sequencing
To conduct reversed-phase-high-performance liquid
chromatography (RP-HPLC) of OBP proteins, 100 µl
supernatant per sample was thawed on ice, diluted
20× in 2% (v/v) acetonitrile (ACN) − 0.05% (v/v) trif-
luoroacetic acid (TFA) solution (4 °C), and loaded on
an activated reversed-phase adsorbent cartridge RP-C8
(Sep-Pak plus cartridge, Waters) to prepare the samples
for further protein analysis. Cartridges were washed 3
times by applying 10ml of 20% (v/v) ACN in 0.05% (v/v)
TFA. Molecules were eluted with 6ml 70% (v/v) ACN
in 0.05% (v/v) TFA, and ACN was removed by 90min of
lyophilization (Speedvac SCV-100H, Savant instruments,
Farmingdale, NY). e remaining volume was filled up
to 2ml with 1% (v/v) ACN in 0.1% (v/v) TFA and loaded
onto a 150 × 4.6mm, 5µm Proto300 C4 HPLC column
(Higgins Analytical Inc., Mountain View, CA). Proteins
were eluted using a gradient of ACN in 0.1% (v/v) TFA at
1ml/min, increasing from 0 to 80% (v/v) ACN in 80min.
Absorbance was measured at a wavelength of 214 nm.
Mass spectra of all fractions were measured in parallel
on an AmaZon SL ion trap mass spectrometer (Bruker
Daltonics, Bremen, Germany). To visualize the protein
content of both unfractionated samples and HPLC peak
fractions, aliquots were adjusted to 10mM TRIS, loaded
on SDS-PAGE precast gels (Any kD Mini-PROTEAN
TGX, Bio-Rad), and silver-stained (Silverquest Silver
Staining kit, Invitrogen). Protein gel bands were trans-
ferred onto a polyvinylidene difluoride membrane by
semi-dry blotting (Trans Blot Turbo System, Bio-Rad)
and stained with 0.1% Coomassie brilliant blue R-250
(Sigma-Aldrich). Bands were excised and destained with
methanol, for N-terminal amino-acid sequencing using
Edman degradation (491 Procise cLC protein sequencer,
Applied Biosystems).
Proteins in 100µl aliquots of OBP peak fractions were
concentrated to 50μl and 5μl was used for analysis with
nano-scale RP-HPLC (Ultimate 3000 RSLCnano system,
ermo Scientific). Purification of proteins was accom-
plished using a 5 × 0.3mm PepMap 300 C4 pre-column
(ermo Scientific) combined with a 50 × 0.15 mm Proto
300 C4 column (Higgins Analytical Inc.). Samples were
loaded in 4% (v/v) ACN in 0.1% (v/v) TFA and elution
was performed with an ACN gradient in 0.08% (v/v)
formic acid (flow rate of 0.5μl/min). e column efflu-
ent was directly injected into an AmaZon speed electron
transfer dissociation (ETD) ion trap mass spectrometer
Page 11 of 16
Janssenswillenetal. BMC Ecol Evo (2021) 21:182
(Bruker Daltonics) with target mass at 2200m/z. Aver-
aged profile spectra of proteins were obtained using
Bruker Daltonics deconvolution software (data analy-
sis 4.1). e experimentally obtained relative molecular
weight (Mr) of the proteins was compared to the theo-
retical Mr. To calculate the theoretical Mrs, predicted
RNA precursor sequences were obtained from the dog
genome (CanFam 3.1, NCBI Genome Browser), manually
adjusted if needed, and translated into proteins using the
ExPASy Translate tool [104]. Signal Peptides were pre-
dicted using SignalP 5.0 [105] and determined by peptide
abundance data inferred from our LC–MS/MS analyses,
and removed before the theoretical Mr of mature pro-
teins were calculated on the Genscript website [106].
Tandem mass spectrometry
Forty microliter supernatant per sample was thawed on
ice, diluted 20×, and subjected to RP-C8 cartridge pro-
tein purification as described above, but in this case
cartridges were washed with a 2% ACN–0.05% TFA
solution, molecules were eluted with 90% (v/v) ACN in
0.05% (v/v) TFA, and lyophilisation lasted for 1–5h. Lyo-
philized RP-C8 eluates were dissolved in lysis buffer (8M
urea, 5mM DTT, 30mM TRIS). Twenty microgram of
proteins were reduced and alkylated, diluted in 50mM
ammonium bicarbonate (Fluka) to 2 M of urea and
digested overnight at 37°C with 0.2μg of trypsin (Pierce
MS grade, ermo Scientific). e peptide samples were
desalted using Pierce C18 spin columns (ermo Sci-
entific) according to the manufacturer’s instructions.
Samples (0.5 μg/5 μl) were separated in an Ultimate
3000 (ermo Scientific) UPLC system, followed by a Q
Exactive Orbitrap mass spectrometer (ermo Scien-
tific). e Ultimate 3000 UPLC system (Dionex, ermo
Scientific) was equipped with an Acclaim PepMap100
pre-column (C18 particle size 3 μm pore size–100 Å,
diameter 0.075 mm, length 20 mm, ermo Scientific)
and a C18 PepMap RSLC analytical column (particle size
2μm, pore size–100Å, diameter 50μm, length-150mm,
ermo Scientific) using a linear gradient (0.300μl/min).
e composition of buffer A is pure water containing
0.1% formic acid. e composition of buffer B is pure
water containing 0.08% formic acid and 80% acetoni-
trile. e fraction of buffer B increased from 0–4% in
3min, from 4–10% in 12min, from 10–35% in 20min,
from 35–65% in 5min, from 65–95% in 1min, stayed
at 95% for 10min. e fraction of buffer B decreased
from 95–5% in 1min and stayed at 5% for 10min. e Q
Exactive Orbitrap mass spectrometer (ermoScientific)
was operated in positive ion mode with a nanospray volt-
age of 2.1kV and a source temperature of 250°C. Pierce
LTQ Velos ESI positive ion calibration mix (ermo Sci-
entific) was used as an external calibrant. e instrument
was operated in data-dependent acquisition mode with a
survey MS scan at a resolution of 70,000 (fwhm at m/z
200) for the mass range of m/z 400–1600 for precur-
sor ions, followed by MS/MS scans of the top ten most
intense peaks with + 2, + 3, + 4, and + 5 charged ions
above a threshold ion count of 1e+6 at 17,500 resolu-
tion using normalized collision energy of 25eV with an
isolation window of 3.0m/z, apex trigger of 5–15s and
dynamic exclusion of 10s. All data were acquired with
Xcalibur 3.1.66.10 software (ermoScientific). For pro-
tein identification, we used MASCOT version 2.2.06
(Matrix Science) against Uniprot Canis lupus familiaris
protein databases to which we added all possible exon
splice variants of the three obp genes, yielding 29,672
sequences. e parameters used to search at MASCOT
were: parent tolerance of 10 PPM, fragment tolerance of
20mmu, variable modification deamidation of NQ and
oxidation of M, fixed modification with carbamidomethyl
C and up to two missed cleavages for trypsin. To calcu-
late the FDR [107] and judge the protein inference, the
sample-specific mgf files were loaded into Scaffold 3.6.5.
e proteins were quantified in Progenesis version 4.0
(Nonlinear dynamics) based on the normalized abun-
dance of all matching features. All compound ion abun-
dances yi have been multiplied by a scalar factor αk to
give a normalised abundance
To implement the normalisation, the most suitable ref-
erence sample was determined and the scalar factor ratio
between each sample being normalised and the reference
sample was calculated.
Genome screening forobp genes
A total of 98 mammalian genomes was screened to inves-
tigate the diversity and organisation of obp genes and
retrieve their sequences for phylogenetic analyses. We
used two screening strategies. First, a homology-based
strategy involved genome-wide BLAT, BLASTn and
tBLASTn searches against the UCSC genome browser
Databases, the NCBI Genome Data Viewer, and Ensembl
Genome Browser using previously retrieved obp tran-
scripts and proteins as query sequences. Second, a
synteny-based strategy involved detailed screening of
intergenic regions between genes that flank obp genes
previously identified in other genomes. At first instance,
our screening effort focused on the genomes of three
dog breeds and dingo as well as genomes representing
major mammalian clades. Eventually, 102 obp genes were
retrieved from 40 genomes (Additional file4) to compile
a data set for phylogenetic analyses (see below). If no obp
homologues were found in the selected representative
of a specific clade, additional genomes of the same clade
y′
i
:
y′
i
=α
kyi.
Page 12 of 16
Janssenswillenetal. BMC Ecol Evo (2021) 21:182
were screened to confirm the absence of obp genes in the
entire clade. is approach resulted in the screening of
19 bat genomes, 29 primate genomes, two monotreme
genomes and five marsupial genomes.
Phylogenetic reconstruction ofobp gene evolution
e coding sequences of the 102 retrieved obp genes
(Additional file4) were aligned using the E-INS-I algo-
rithm with default parameters implemented in Mafft v7
[108]. A general time-reversible model with a gamma
distribution to accommodate among-site rate hetero-
geneity and an estimated proportion of invariable sites
(GTR + G + I) was identified as the best-fitting DNA
substitution model using Akaike and Bayesian informa-
tion criteria in ModelTest-NG 0.1.6 [109]. Consequently,
this model was applied for Bayesian phylogeny inference
with using MrBayes 3.2.7a [110] and maximum likeli-
hood bootstrap analyses, sing RAXML 8.2.12 [111], both
accessed through the CIPRES Science Gateway [112].
For the Bayesian analyses, two runs of four Markov
chain Monte Carlo chains each were run in parallel for
12 million generations, with a sampling interval of 1000
generations and a burnin of two million generations.
Convergence of the parallel runs was verified by split
frequency standard deviations (< 0.01) and potential
scale reduction factors (approximating 1.0) for all model
parameters. Maximum Likelihood bootstrap support val-
ues were obtained by performing 1000 “rapid” bootstrap
replicates in RAXML. e consensus trees resulting from
the Bayesian and bootstrapping analyses were nearly
identical in topology and the few conflicting branches
received weak support by either analysis. In the absence
of closely related outgroup sequences, phylogenetic anal-
yses were performed on ingroup (obp) sequences only,
producing unrooted gene trees.
e resulting Bayesian consensus phylogram was used
to reconstruct gene duplication events and gene losses on
the mammalian taxon tree using a gene tree/species tree
reconciliation (GTSTR) analysis under the parsimony
principle as implemented in Notung 2.9 [113]. A taxon
tree that matches our gene tree was retrieved from the
timetree.org database [84] as a chronogram with diver-
gence times as averages calculated from a database of
published molecular clock estimates. e cost of gene
duplications and losses were both set to 1.0. We made
two reconstructions that represent contrasting prior-
itisation strategies. A first GTSTR was based on the
unchanged Bayesian gene tree without allowing rear-
rangements of weakly supported branches even if this
would improve the reconciliation (yield lower estimated
number of duplications and losses). is reconstruc-
tion prioritises confidence in the gene tree over opti-
mizing tree reconciliation. A second GTSTR allowed
the rearrangement of weakly supported branches if this
results in lower estimated number of duplications and
losses. is reconstruction prioritises minimization
of the number of events over maintaining unreliable
branches. Branches were considered weakly supported
if they received Bayesian posterior probabilities < 0.9 and
bootstrap percentages < 70%. As expected, application of
the unchanged tree implicated a higher number of gene
duplications and losses than a partially rearranged tree
(Additional file6). Both reconstructions also allowed us
to tentatively root the gene tree, by defining the branch
on which the root position would implicate the least
number of gene duplication events and losses. Under
both reconstruction strategies, the most optimal root
was located on the branch separating the xenarthran and
afrotherian sequences from all other sequences (Addi-
tional file5).
In the current absence of comprehensive expression
information for the majority of species, the evolution of
OBP secretion sites was reconstructed on a pruned tree
including the 13 genes for which expression informa-
tion could be inferred from the present study or previ-
ous proteomic studies (Additional file7). e origin of
expression sites was mapped on this gene tree using the
maximum parsimony principle as implemented in Mes-
quite 3.61 [114].
Abbreviations
ASGS: Anal sac gland secretions; GTSTR: Gene tree/species tree reconstruction;
OBP: Odorant binding protein; PAR: Pseudoautosomal region.
Supplementary Information
The online version contains supplementary material available at https:// doi.
org/ 10. 1186/ s12862- 021- 01910-w.
Additional le1. Information on individual dogs and total protein con-
centrations of ASGS samples.
Additional le2. Normalized abundances of OBP isoform in dog ASGS of
pure breeds, crossbreeds, and mixed breeds. Although OBP abundances
are higher in mixed breeds, more samples of different breeds are required
to validate this pattern.
Additional le3. Proteins identified by LC-MS/MS in the anal sac gland
secretions of 21 samples (17 dogs). All 56 proteins are listed in major
functional classes. For each protein, domain families, Uniprot accession
numbers and normalised abundance per sample are provided.
Additional le4. List of identified obp genes per species, their accession
numbers, and the genomes from which they were retrieved. All of these
genes were included in the phylogenetic analyses.
Additional le5. Bayesian consensus phylogram for 102 obp genes,
retrieved from 40 placental mammal genomes. Numbers near branches
indicate Bayesian posterior probabilities (BPP; left) and nonparametric
bootstrap percentages (NPBS; right). Branches supported by BBP > 0.95
and NPBS > 75% are drawn as thick lines. Branches supported by
BBP < 0.90 and NPBS < 70% (indicated in red) are drawn as dashed lines
and were allowed to be rearranged in a gene tree/species tree reconcili-
ation analysis (see Fig. 3). Branch colours delineate obp sequence clades
unique to one of the following mammalian taxa: Carnivora (yellow),
Page 13 of 16
Janssenswillenetal. BMC Ecol Evo (2021) 21:182
Pecora (light blue), Cetacea (teal), Pig (pink), Perissodactyla (light brown),
Pholidota (green), Rodentia (orange) and Eulipotyphla (dark brown),
Afrotheria (light green), Xenarthra (grey).
Additional le6. Alternative reconstruction of the evolutionary history
of the obp gene repertoire in placental mammals. Similar to Fig. 3, obp
gene lineages are drawn as thin branches superimposed on a timetree of
placental mammals, with gene duplication events and gene losses along
branches depicted as blue vertical bars (I) and red crosses (X), respectively.
Ancestral and extant gene repertoires are schematically shown at key
internal nodes and terminal nodes, respectively. This reconstruction was
obtained by gene tree/species tree reconciliation with the consensus
phylogram from the Bayesian analyses (Additional file 5) as a fixed gene
tree. Unlike the reconstruction in Fig. 3, arrangement of weakly supported
branches to minimise the inferred number of gene duplication events and
losses was not allowed prioritising confidence in the inferred gene tree of
maximising the reconciliation. As a result, this reconstruction implicates
substantially more duplication events (indicated by light blue vertical
bars), losses (indicated by light red crosses) and ancestral genes for basal
branches (coloured light blue).
Additional le7. Overview of obp genes with previously identified secre-
tion sites of their corresponding proteins.
Acknowledgements
We would like to thank all dog owners for their interest and involvement in
this research, and Kusay Arat for his technical contribution. Special thanks to
Elleke Verschoren (vanhoght.be) for her valuable insights.
Authors’ contributions
SJ conceived this research. HdR and SJ collected the samples. SJ, SC, MM,
and PP performed, analysed and interpreted the protein data. SJ, KR and FB
performed the genome and phylogenetic analyses. BVS did the statistical
assessments. SJ and KR interpreted the phylogenetic data, wrote the manu-
script and designed the figures. All authors reviewed, edited. All authors read
and approved the final manuscript.
Funding
This work was supported by the Strategic Research Program SRP-Growth, Vrije
Universiteit Brussel (Grant no. SRP30, received by FB); and Research Founda-
tion – Flanders (FWO, Fonds voor Wetenschappelijk Onderzoek—Vlaanderen)
(Grant no. 12O1517N, received by SJ). The funding bodies played no role in
the design of the study and collection, analysis, and interpretation of data and
in writing the manuscript.
Availability of data and materials
All protein and DNA sequences analysed in this study are available in the NCBI
and Ensembl databases and can be accessed using the accession numbers
provided in Additional file 3. A fasta files of the DNA sequences and HPLC
chromatograms are available from the corresponding author upon reasonable
request.
Declarations
Ethics approval and consent to participate
For all sampled dogs, written owner consents were obtained. This study does
not include any animal experiments according to Belgian (Art. 2.6 of the
Belgian Law of May 4th 1995; Annex VII, Belgian Law of May 29th 2013) and
European legislation (European Convention for the protection of Vertebrate
animals used for experimental and other scientific purposes). Sampling has
been conducted in agreement with the Ethical Committee of Animal Experi-
ments of Vrije Universiteit Brussel (Project 16-634-3).
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Author details
1 Amphibian Evolution Lab, Biology Department, Vrije Universiteit Brussel,
Pleinlaan 2, 1050 Brussels, Belgium. 2 Proteomics Core - SyBioMa, Katholieke
Universiteit Leuven, Herestraat 49 - 03.313, 3000 Leuven, Belgium. 3 Small Ani-
mal Department, Faculty of Veterinary Medicine, Ghent University, Salisbury-
laan 133, 9820 Merelbeke, Belgium. 4 Rega Institute, Molecular Immunology,
Katholieke Universiteit Leuven, Herestraat 49 - Bus1042, 3000 Leuven, Belgium.
5 Community Ecology Lab, Biology Department, Vrije Universiteit Brussel,
Pleinlaan 2, 1050 Brussels, Belgium. 6 Center for Environmental Management,
University of the Free State, Bloemfontein 9030, South Africa.
Received: 16 November 2020 Accepted: 10 September 2021
References
1. Gittleman JL. Carnivore behavior, ecology, and evolution. 1st ed. New
York: Cornell University Press; 1996.
2. Asa CS, Peterson EK, Seal US, Mech LD. Deposition of anal-sac secre-
tions by captive wolves (Canis lupus). J Mammal. 1985;66(1):89–93.
3. Asa CS, Mech LD, Seal US. The use of urine, faeces, and anal-gland
secretions in scent-marking by a captive wolf (Canis lupus) pack. Anim
Behav. 1985;33(3):1034–6.
4. Drea CM, Vignieri SN, Kim HS, Weldele ML, Glickman SE. Responses to
olfactory stimuli in spotted hyenas (Crocuta crocuta): II. Discrimination
of conspecific scent. J Comp Psychol. 2002;116(4):342.
5. Clapper ton BK, Minot O, Crump DR. An olfactory recognition system
in the ferret Mustela furo L. (Carnivora: Mustelidae). Anim Behav.
1988;36(2):541–53.
6. Jojola SM, Rosell F, Warrington I, Swenson JE, Zedrosser A. Subadult
brown bears (Ursus arctos) discriminate between unfamiliar adult male
and female anal gland secretion. Mammal Biol. 2012;77(5):363–8.
7. Begg CM, Begg KS, Du Toit JT, Mills MGL. Scent-marking behaviour
of the honey badger, Mellivora capensis (Mustelidae), in the southern
Kalahari. Anim Behav. 2003;66(5):917–29.
8. Burgener N, Dehnhard M, Hofer H, East ML. Does anal gland scent
signal identity in the spotted hyaena? Anim Behav. 2009;77(3):707–15.
9. Gorman ML. A mechanism for individual recognition by odour
in Herpestes auropunctatus (Carnivora: Viverridae). Anim Behav.
1976;24(1):141–5.
10. Miyazaki T, Nishimura T, Yamashita T, Miyazaki M. Olfactory discrimina-
tion of anal sac secretions in the domestic cat and the chemical profiles
of the volatile compounds. J Ethol. 2018;36(1):99–105.
11. Theis KR. Scent marking in a highly social mammalian species, the
spotted hyena, Crocuta crocuta. Dissertation for the degree of Ph.D.,
Michigan State University; 2008.
12. Leclaire S, Nielsen JF, Thavarajah NK, Manser M, Clutton-Brock TH.
Odour-based kin discrimination in the cooperatively breeding meerkat.
Biol Lett. 2012;9(1):20121054.
13. Pageat P, Gaultier E. Current research in canine and feline pheromones.
Vet Clin N Am Small Anim Pract. 2003;33(2):187–211.
14. Spotte S. Societies of wolves and free-ranging dogs. 1st ed. Cambridge:
Cambridge University Press; 2012.
15. Donovan CA. Canine anal glands and chemical signals (pheromones). J
Am Vet Med Assoc. 1969;155:1995–6.
16. Doty RL, Dunbar I. Attraction of beagles to conspecific urine, vaginal
and anal sac secretion odors. Physiol Behav. 1974;12(5):825–33.
17. Natynczuk S, Bradshaw JWS, McDonald DW. Chemical constituents of
the anal sacs of domestic dogs. Biochem Syst Ecol. 1989;17(1):83–7.
18. Preti G, Meutterties EL, Furman J, Kennelly JJ, Johns BE. Volatile con-
stituents of dog (Canis familiaris) and coyote (Canis latrans) anal sacs. J
Chem Ecol. 1976;2:177–87.
19. Albone ES, Eglinton G, Walker JM, Ware GC. The anal sac secretion of
the red fox (Vulpes vulpes); its chemistry and microbiology. A com-
parison with the anal sac secretion of the lion (Panthera leo). Life Sci.
1974;14(2):387–400.
20. Archie EA, Theis KR. Animal behaviour meets microbial ecology. Anim
Behav. 2011;82:425–36.
Page 14 of 16
Janssenswillenetal. BMC Ecol Evo (2021) 21:182
21. Gorman ML, Nedwell DB, Smith RM. An analysis of the contents of the
anal scent pockets of Herpestes auropunctatus (Carnivora: Viverridae). J
Zool. 1974;172:389–99.
22. Svendsen GE, Jollick JD. Bacterial contents of the anal and castor glands
of beaver (Castor canadensis). J Chem Ecol. 1978;4(5):563–9.
23. Leclaire S, Jacob S, Greene LK, Dubay GR, Drea CM. Social odours covary
with bacterial community in the anal secretions of wild meerkats. Sci
Rep. 2017;7(1):1–13.
24. Theis KR, Schmidt TM, Holekamp KE. Evidence for a bacterial mecha-
nism for group-specific social odors among hyenas. Sci Rep. 2012;2:615.
25. Theis KR, Venkataraman A, Dycus A, Koonter KD, Schmitt-Matzen EN,
Wagner AP, et al. Symbiotic bacteria appear to mediate hyena social
odors. Proc Natl Acad Sci USA. 2013;110(49):19832–7.
26. Albone ES, Perry GC. Anal sac secretion of the red fox, Vulpes vulpes;
volatile fatty acids and diamines: implications for a fermentation
hypothesis of chemical recognition. J Chem Ecol. 1976;2(1):101–11.
27. Raymer J, Wiesler D, Novotny M, Asa C, Seal US, Mech LD. Chemical
investigations of wolf (Canis lupus) anal-sac secretion in relation to
breeding season. J Chem Ecol. 1985;11(5):593–608.
28. Rosell F, Jojola SM, Ingdal K, Lassen BA, Swenson JE, Arnemo JM,
Zedrosser A. Brown bears possess anal sacs and secretions may code
for sex. J Zool. 2011;283(2):143–52.
29. Yuan H, Liu D, Sun L, Wei R, Zhang G, Sun R. Anogenital gland secretions
code for sex and age in the giant panda, Ailuropoda melanoleuca. Can J
Zool. 2004;82(10):1596–604.
30. Zhang JX, Liu D, Sun L, Wei R, Zhang G, Wu H, Zhang H, Zhao C. Poten-
tial chemosignals in the anogenital gland secretion of giant pandas,
Ailuropoda melanoleuca, associated with sex and individual identity. J
Chem Ecol. 2008;34(3):398–407.
31. Beynon RJ, Hurst JL. Multiple roles of major urinary proteins in the
house mouse, Mus domesticus. Biochem Soc Trans. 2003;31(1):142–6.
32. Briand L, Trotier D, Pernollet JC. Aphrodisin, an aphrodisiac lipocalin
secreted in hamster vaginal secretions. Peptides. 2004;25(9):1545–52.
33. Haga S, Hattori T, Sato T, Sato K, Matsuda S, Kobayakawa R, Sakano H,
Yoshihara Y, Kikusui T, Touhara K. The male mouse pheromone ESP1
enhances female sexual receptive behaviour through a specific vome-
ronasal receptor. Nature. 2010;466(7302):118.
34. Roberts SA, Simpson DM, Armstrong SD, Davidson AJ, Robertson
DH, McLean L, Beynon RJ, Hurst JL. Darcin: a male pheromone that
stimulates female memory and sexual attraction to an individual male’s
odour. BMC Biol. 2010;8(1):75.
35. Srikantan S, De PK. Sex differences in expression and differential regula-
tion by androgen and estrogen of two odorant-binding tear lipocalins
in lacrimal glands of immature hamsters. Gen Comp Endocrinol.
2008;158(3):268–76.
36. Stopková R, Vinkler D, Kuntová B, Šedo O, Albrecht T, Suchan J,
Dvořáková-Hortová K, Zdráhal Z, Stopka P. Mouse lipocalins (MUP, OBP,
LCN) are co-expressed in tissues involved in chemical communication.
Front Ecol Evol. 2016;4:47.
37. Roberts SA, Prescott MC, Davidson AJ, McLean L, Beynon RJ, Hurst JL.
Individual odour signatures that mice learn are shaped by involatile
major urinary proteins (MUPs). BMC Biol. 2018;16(1):48.
38. Unsworth J, Loxley GM, Davidson A, Hurst JL, Gómez-Baena G, Mundy
NI, Beynon RJ, Zimmermann E, Radespiel U. Characterisation of urinary
WFDC12 in small nocturnal basal primates, mouse lemurs (Microcebus
spp.). Sci Rep. 2017;7:42940.
39. Perry GC, Patterson RLS, MacFie HJH, Stinson CG. Pig courtship behav-
iour: pheromonal property of androstene steroids in male submaxillary
secretion. Anim Sci. 1980;31(2):191–9.
40. Marchese S, Pes D, Scaloni A, Carbone V, Pelosi P. Lipocalins of boar
salivary glands binding odours and pheromones. Eur J Biochem.
1998;252(3):563–8.
41. Montagna W, Parks HF. A histochemical study of the glands of the anal
sac of the dog. Anatom Rec. 1948;100(3):297–317.
42. De Andrade AD, Birnbaum J, Magalon C, Magnol JP, Lanteaume A,
Charpin D, Vervloet D. Fel d I levels in cat anal glands. Clin Exp Allergy.
1996;26(2):178–80.
43. The UniProt Consortium. UniProt: a worldwide hub of protein knowl-
edge. Nucleic Acids Res. 2019;47(D1):D506–15.
44. Pevsner J, Snyder SH. Odorant-binding protein: odorant trans-
port function in the vertebrate nasal epithelium. Chem Senses.
1990;15(2):217–22.
45. Tegoni M, Pelosi P, Vincent F, Spinelli S, Campanacci V, Grolli S, et al.
Mammalian odorant binding proteins. Biochim Biophys Acta.
2000;1482(1–2):229–40.
46. Iacono VJ, MacKay BJ, DiRienzo S, Pollock JJ. Selective antibacte-
rial properties of lysozyme for oral microorganisms. Infect Immun.
1980;29(2):623–32.
47. Lee B, Bowden GH, Myal Y. Identification of mouse submaxillary gland
protein in mouse saliva and its binding to mouse oral bacteria. Arch
Oral Biol. 2002;47(4):327–32.
48. Sanchez L, Calvo M, Brock JH. Biological role of lactoferrin. Arch Dis
Child. 1992;67(5):657.
49. Sang Y, Ortega MT, Rune K, Xiau W, Zhang G, Soulages JL, et al. Canine
cathelicidin (K9CATH): gene cloning, expression, and biochemical activ-
ity of a novel pro-myeloid antimicrobial peptide. Dev Comp Immunol.
2007;31(12):1278–96.
50. Abbas AK, Lichtman AH, Pillai S. Cellular and molecular immunology
E-book. 8th ed. Amsterdam: Elsevier Health Sciences; 2014.
51. Baenziger JU. Structure of the oligosaccharide of human J chain. J Biol
Chem. 1979;254(10):4063–71.
52. Kaetzel CS. The polymeric immunoglobulin receptor: bridging innate
and adaptive immune responses at mucosal surfaces. Immunol Rev.
2005;206(1):83–99.
53. Rolli V, Radosavljevic M, Astier V, Macquin C, Castan-Laurell I, Visentin V,
et al. Lipolysis is altered in MHC class I zinc-α2-glycoprotein deficient
mice. FEBS Lett. 2007;581(3):394–400.
54. Acton A. Protease inhibitors: advances in research and application. 1st
ed. Atlanta: Scholarly Editions; 2012.
55. Lazar J, Rasmussen LEL, Greenwood DR, Bang IS, Prestwich GD.
Elephant albumin: a multipurpose pheromone shuttle. Chem Biol.
2004;11(8):1093–100.
56. Kelley LA, Mezulis S, Yates CM, Wass MN, Sternberg MJ. The Phyre2
web portal for protein modelling, prediction and analysis. Nat Protoc.
2015;10(6):845–58.
57. Niemi MH, Rytkönen-Nissinen M, Jänis J, Virtanen T, Rouvinen J.
Structural aspects of dog allergies: the crystal structure of a dog dander
allergen Can f 4. Mol Immunol. 2014;61(1):7–15.
58. Flower DR. The lipocalin protein family: structure and function. Biochem
J. 1996;318(1):1–14.
59. Briand L, Blon F, Trotier D, Pernollet JC. Natural ligands of hamster aph-
rodisin. Chem Senses. 2004;29(5):425–30.
60. Pelosi P. Odorant-binding proteins: structural aspects. Ann N Y Acad Sci.
1998;855(1):281–93.
61. Mattsson L, Lundgren T, Olsson P, Sundberg M, Lidholm J. Molecular
and immunological characterization of Can f 4: a dog dander allergen
cross-reactive with a 23 kDa odorant-binding protein in cow dander.
Clin Exp Allergy. 2010;40(8):1276–87.
62. Raudsepp T, Chowdhary BP. The eutherian pseudoautosomal region.
Cytogenet Genome Res. 2015;147(2–3):81–94.
63. Van Laere AS, Coppieters W, Georges M. Characterization of the bovine
pseudoautosomal boundary: documenting the evolutionary history of
mammalian sex chromosomes. Genome Res. 2008;18(12):1884–95.
64. Das PJ, Mishra DK, Ghosh S, Avila F, Johnson GA, Chowdhary BP,
Raudsepp T. Comparative organization and gene expression profiles
of the porcine pseudoautosomal region. Cytogenet Genome Res.
2013;141(1):26–36.
65. Li G, Davis BW, Raudsepp T, Pearks Wilkerson AJ, Mason VC, et al.
Comparative analysis of mammalian Y chromosomes illuminates
ancestral structure and lineage-specific evolution. Genome Res.
2013;23(9):1486–95.
66. Raudsepp T, Chowdhary BP. The horse pseudoautosomal region (PAR):
characterization and comparison with the human, chimp and mouse
PARs. Cytogenet Genome Res. 2008;121(2):102–9.
67. Soh YQ, Alföldi J, Pyntikova T, Brown LG, Graves T, Minx PJ, et al.
Sequencing the mouse Y chromosome reveals convergent gene
acquisition and amplification on both sex chromosomes. Cell.
2014;159(4):800–13.
Page 15 of 16
Janssenswillenetal. BMC Ecol Evo (2021) 21:182
68. Zhang SJ, Wang GD, Ma P, Zhang LL, Yin TT, Liu YH, et al. Genomic
regions under selection in the feralization of the dingoes. Nat Commun.
2020;11(1):671.
69. Černá M, Kuntová B, Talacko P, Stopková R, Stopka P. Differential regula-
tion of vaginal lipocalins (OBP, MUP) during the estrous cycle of the
house mouse. Sci Rep. 2017;7(1):1–10.
70. Kuntová B, Stopková R, Stopka P. Transcriptomic and proteomic profil-
ing revealed high proportions of odorant binding and antimicrobial
defense proteins in olfactory tissues of the house mouse. Front Genet.
2018;9:26.
71. Stopka P, Kuntová B, Klempt P, Havrdová L, Černá M, Stopková R. On the
saliva proteome of the Eastern European house mouse (Mus musculus
musculus) focusing on sexual signalling and immunity. Sci Rep.
2016;6:32481.
72. Stopková R, Dudková B, Hájková P, Stopka P. Complementary roles of
mouse lipocalins in chemical communication and immunity. Biochem
Soc Trans. 2014;42(4):893–8.
73. Stopková R, Klempt P, Kuntová B, Stopka P. On the tear proteome of the
house mouse (Mus musculus musculus) in relation to chemical signal-
ling. PeerJ. 2017;5:e3541.
74. Mägert HJ, Hadrys T, Cieslak A, Gröger A, Feller S, Forssmann WG. cDNA
sequence and expression pattern of the putative pheromone carrier
aphrodisin. Proc Natl Acad Sci USA. 1995;92(6):2091–5.
75. Thavathiru E, Jana NR, De PK. Abundant secretory lipocalins displaying
male and lactation-specific expression in adult hamster submandibular
gland: cDNA cloning and sex hormone-regulated repression. Eur J
Biochem. 1999;266(2):467–76.
76. Srikantan S, Parekh V, De PK. cDNA cloning and regulation of two
sex-hormone-repressed hamster tear lipocalins having homology
with odorant/pheromone-binding proteins. Biochim Biophys Acta.
2005;1729(3):154–65.
77. Bignetti E, Cavaggioni A, Pelosi P, Persaud KC, Sorbi RT, Tirindelli R. Puri-
fication and characterization of an odorant binding protein from cow
nasal tissue. Eur J Biochem. 1985;149:227–31.
78. Paolini S, Scaloni A, Amoresano A, Marchese S, Napolitano E, Pelosi
P. Amino acid sequence, post-translational modifications, binding
and labelling of porcine odorant-binding protein. Chem Senses.
1998;23(6):689–98.
79. Rautiainen J, Rytkönen M, Syrjänen K, Pentikäinen J, Zeiler T, Virtanen
T, Mäntyjärvi R. Tissue localization of bovine dander allergen Bos d2. J
Allergy Clin Immunol. 1998;101(3):349–53.
80. Stopková R, Hladovcová D, Kokavec J, Vyoral D, Stopka P. Multiple roles
of secretory lipocalins (MUP, OBP) in mice. Folia Zool. 2009;58(1):29–40.
81. Esselstyn JA, Oliveros CH, Swanson MT, Faircloth BC. Investigating
difficult nodes in the placental mammal tree with expanded taxon
sampling and thousands of ultraconserved elements. Genome Biol
Evol. 2017;9(9):2308–21.
82. Foley NM, Springer MS, Teeling EC. Mammal madness: is the mam-
mal tree of life not yet resolved? Philos Trans R Soc Lond B Biol Sci.
2016;371(1699):20150140.
83. Murphy WJ, Pringle TH, Crider TA, Springer MS, Miller W. Using genomic
data to unravel the root of the placental mammal phylogeny. Genome
Res. 2007;17(4):413–21.
84. Kumar S, Stecher G, Suleski M, Hedges SB. TimeTree: a resource for time-
lines, timetrees, and divergence times. Mol Biol Evol. 2017;34(7):1812–9.
85. Briand L, Huet JC, Perez V, Lenoir G, Nespoulous C, Boucher Y, et al.
Odorant and pheromone binding by aphrodisin, a hamster aphrodisiac
protein. FEBS Lett. 2000;476(3):179–85.
86. Hagemeyer P, Begall S, Janotova K, Todrank J, Heth G, Jedelsky PL, et al.
Searching for major urinary proteins (MUPs) as chemosignals in urine of
subterranean rodents. J Chem Ecol. 2011;37(7):687–94.
87. Macrides F, Clancy AN, Singer AG, Agosta WC. Male hamster investiga-
tory and copulatory responses to vaginal discharge: an attempt to
impart sexual significance to an arbitrary chemosensory stimulus.
Physiol Behav. 1984;33(4):627–32.
88. Ranganathan V, Jana NR, De PK. Hormonal effects on hamster lacrimal
gland female-specific major 20 kDa secretory protein and its immu-
nological similarity with submandibular gland major male-specific
proteins. J Steroid Biochem Mol Biol. 1999;70(4–6):151–8.
89. Stopková R, Vinkler D, Kuntová B, Šedo O, Albrecht T, Suchan J, et al.
Mouse lipocalins (MUP, OBP, LCN) are co-expressed in tissues involved
in chemical communication. Front Ecol Evol. 2016;4:47.
90. Cann P, Chabi M, Delsart A, Le Danvic C, Saliou JM, Chasles M, et al. The
olfactory secretome varies according to season in female sheep and
goat. BMC Genomics. 2019;20(1):794.
91. Lazar J, Greenwood DR, Rasmussen LEL, Prestwich GD. Molecular and
functional characterization of an odorant binding protein of the Asian
elephant, Elephas maximus: implications for the role of lipocalins in
mammalian olfaction. Biochemistry. 2002;41(39):11786–94.
92. Nagnan-Le Meillour P, Joly A, Le Danvic C, Marie A, Zirah S, Cornard J-P.
Binding specificity of native odorant-binding protein isoforms is driven
by phosphorylation and O-N-Acetylglucosaminylation in the pig Sus
scrofa. Front Endocrinol. 2019;9:816.
93. Pes D, Pelosi P. Odorant-binding proteins of the mouse. Comp Biochem
Physiol B Biochem Mol Biol. 1995;112(3):471–9.
94. Pes D, Mameli M, Andreini I, Krieger J, Weber M, Breer H, Pelosi P. Clon-
ing and expression of odorant-binding proteins Ia and Ib from mouse
nasal tissue. Gene. 1998;212(1):49–55.
95. Hurst JL, Robertson DH, Tolladay U, Beynon RJ. Proteins in urine scent
marks of male house mice extend the longevity of olfactory signals.
Anim Behav. 1998;55(5):1289–97.
96. Silva C, Matamá T, Azoia NG, Mansilha C, Casal M, Cavaco-Paulo A. Odor-
ant binding proteins: a biotechnological tool for odour control. Appl
Microbiol Biotechnol. 2014;98(8):3629–38.
97. Miklósi A. The perceptual world of the dog. In: Miklósi A, editor. Dog
behaviour, evolution, and cognition. 2nd ed. Oxford: Oxford University
Press; 2015. p. 183–200.
98. Wyatt T. Pheromones and animal behaviour: chemical signals and
signatures. 2nd ed. Cambridge: Cambridge University Press; 2014.
99. Beaver BV. Canine behavior: insights and answers. 2nd ed. Amsterdam:
Elsevier Health Sciences/Saunders; 2008.
100. Posynick BJ, Brown CJ. Escape from X-chromosome inactivation: an
evolutionary perspective. Front Cell Dev Biol. 2019;7:241.
101. Maxeiner S, Benseler F, Krasteva-Christ G, Brose N, Südhof TC. Evolution
of the Autism-associated neuroligin-4 gene reveals broad erosion of
pseudoautosomal regions in rodents. Mol Biol Evol. 2020;37(5):1243–58.
102. Nei M, Gu X, Sitnikova T. Evolution by the birth-and-death process in
multigene families of the vertebrate immune system. Proc Natl Acad Sci
USA. 1997;94(15):7799–806.
103. Nagnan-Le Meillour P, Vercoutter-Edouart A-S, Hilliou F, Le Danvic C,
Lévy F. Proteomic analysis of pig (Sus scrofa) olfactory soluble proteome
reveals O-linked-N-Acetylglucosaminylation of secreted odorant-bind-
ing proteins. Front Endocrinol. 2014;5:202.
104. Artimo P, Jonnalagedda M, Arnold K, Baratin D, Csardi G, de Castro E,
et al. ExPASy: SIB bioinformatics resource portal. Nucleic Acids Res.
2012;40(W1):W597–603.
105. Armenteros JJA, Tsirigos KD, Sonderby CK, Petersen TN, Winther O,
Brunak S, et al. SignalP 5.0 improves signal peptide predictions using
deep neurol networks. Nat Biotechnol. 2019;37:420–3.
106. Peptide Molecular Weight Calculator. http:// gensc ript. com/ tools/ pepti
de- molec ular- weight- calcu lator. Accessed Oct 2019.
107. Kall L, Storey JD, MacCoss MJ, Noble WS. Assigning significance to pep-
tides identified by tandem mass spectrometry using decoy databases. J
Proteome Res. 2007;7(1):29–34.
108. Katoh K, Standley D. MAFFT multiple sequence alignment software
version 7: improvements in performance and usability. Mol Biol Evol.
2013;30:772–80.
109. Darriba D, Posada D, Kozlov AM, Stamatakis A, Morel B, Flouri T.
ModelTest-NG: a new and scalable tool for the selection of DNA and
protein evolutionary models. Mol Biol Evol. 2020;37(1):291–4.
110. Ronquist F, Teslenko M, Van Der Mark P, Ayres DL, Darling A, Höhna S,
et al. MrBayes 3.2: efficient Bayesian phylogenetic inference and model
choice across a large model space. Syst Biol. 2012;61(3):539–42.
111. Stamatakis A. RAxML version 8: a tool for phylogenetic analysis and
post-analysis of large phylogenies. Bioinformatics. 2014;30(9):1312–3.
112. Miller MA, Pfeiffer W, Schwartz T. The CIPRES science gateway: a com-
munity resource for phylogenetic analyses. In: Proceedings of the 2011
TeraGrid conference: extreme digital discovery. 2012:1–8.
Page 16 of 16
Janssenswillenetal. BMC Ecol Evo (2021) 21:182
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113. Stolzer M, Lai H, Xu M, Sathaye D, Vernot B, Durand D. Inferring duplica-
tions, losses, transfers and incomplete lineage sorting with nonbinary
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114. Maddison WP, Maddison DR. Mesquite: a modular system for evolu-
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