BACKGROUND
reproductive proteins are essential for success of sexually reproducing species and indeed for the emergence of new species.
in the past it has been observed that reproductive proteins tend to be under positive selective pressure to change, i.e.
adaptive evolution, a trend found in a variety of animal species from abalone to primates  <cit> .
adaptive evolution or positive selection is a selective pressure placed on a protein by a change in environment in order to improve the fitness of the organism in that environment.
with changes in environment, that can include mating system, there is a subsequent selective pressure on the protein sequences related to those functions to adapt accordingly.
this variation can be detected using the well-known measurements of the rate of non-synonymous substitutions per non-synonymous site  and synonymous substitutions per synonymous site  and their ratio ω = dn/ds.
the detection of adaptive evolution, where the ratio exceeds unity, is referred to as positive darwinian selection.
detecting positive darwinian selection in a region of a protein, or indeed in a lineage of a phylogeny, indicates that there is a selective advantage in changing the amino acid sequence in this region.
these signals are essential for our understanding of functionally important residues in a protein sequence and protein functional shift.
in general, the rate of mutation that a gene undergoes is contingent on a number of factors including; protein structure, presence of gene duplicates, location in the genome, effective population size, germ line generation time, and composition of the sequence .
it has recently been shown that the number of physical interactions of a particular protein also influences the intrinsic rate of evolution  <cit> .
evidence for the generation time effect has come from studies on various proteins and species including analyses of substitution rates in higher primates and rodents  <cit> , substitution rates in higher grasses and in palms  <cit> , in mammalian genomes  <cit>  and in chloroplast and sex mutation rate ratios  <cit> .
with recent advances in sequencing we have an opportunity to examine these effects using a wider selection of proteins and species.
documented selective pressures associated with positive selection in reproductive proteins include:  intense sperm competition whereby sperm from numerous males, ejaculated into the female reproductive tract, compete with one another for the prized fertilization of the egg  <cit> ;  evasion of the immune system, whereby surface layer reproductive proteins evolve to evade destruction by the host's immune system  <cit> ; and finally  selective pressures enforced by mating system, related of course to point  above.
species that are more promiscuous have increased levels of selective pressure acting on reproductive proteins than species that are monogamous.
this later point is illustrated in the study of semg <dig>  where adaptive evolution was found to correlate with mating system in primates  <cit> .
in order to determine the variation in selective pressure in these proteins, there are a number of criteria that the data must meet.
firstly, the data must have a robust phylogenetic signal.
secondly, systematic biases that may exist in the data must be minimized, these include but are not limited to: long branch attraction , amino acid composition bias, base composition bias and unqualified ortholog predictions, all of which may lead to inaccurate estimates of phylogeny.
thirdly, sensitivity to taxa number is a known limitation of methods for detecting positive selection, therefore more than  <dig> taxa are needed to gain accurate estimations of selective pressure using the maximum likelihood  method applied here  <cit> .
in this study we have selected a subset of proteins that have roles to play in reproduction.
our dataset was composed of three major datatypes,  previously published reproductive proteins,  interacting proteins, here we identified proteins shown to interact with , and finally , genes identified from microarray experiments as being highly expressed in reproductive tissues.
for group  we assume that those proteins highly expressed in reproductive tissues are important for the function of that tissue.
the previously untested reproductive proteins analysed here are from data types  and  outlined above.
these novel proteins are sp <dig>  porimin and col1a <dig>  sp <dig> is sperm binding protein number  <dig>  this protein is a representative of the interacting protein subset of sequences analysed.
sp <dig> has been shown to interact with zp <dig> - a well-studied reproductive protein.
both porimin and col1a <dig> have been identified from published microarray experiments on normal human tissue  <cit> , and were selected for analysis due to their high levels of expression in reproductive tissues in that study.
porimin is a transmembrane protein that is highly expressed in the uterus, prostate and placenta and col1a <dig> is highly expressed in the uterus.
further evidence for the link between porimin and reproduction was not available in the literature and therefore results from this particular gene are taken with caution until this protein is further characterized.
col1a <dig> plays an important role during spermatogenesis where it mediates the detachment and migration of germ cells, thus adding further support for its role in reproduction  <cit> .
we have analyzed these data with an approach sensitive to all the systematic biases and limitations of methods given above.
a number of genes in our dataset have been analyzed previously but have not taken these limitations and considerations into account.
we have expanded these datasets to include a greater number of taxa, we have analyzed all of these genes for evidence of systematic biases and we have used improved models of codon evolution.
in this paper we have included models that allow for rate variation across the sequence and across the phylogeny.
RESULTS
we performed phylogenetic analyses on all  <dig> datasets.
the resultant gene trees were found to conflict with the canonical phylogeny species  amino acid and/or base composition bias,  lack of phylogenetic signal in the data, and finally , lba caused by mixtures of long and short germ line generation times .
what follows is a summary of the results of the tests of data quality and bias we performed, see table  <dig> for synopsis.
we carried out these tests to determine in each case whether these conflicting phylogenies are accurate descriptions of history or whether the data are subject to these known issues listed 1- <dig> above.
subsequent statistical comparison of the gene trees and species phylogeny using the shimodaira hasegawa  test  <cit>  revealed that there is no statistical difference between the gene and species trees in each case, see table  <dig> for results of sh tests.
the only exceptions were prkar2a and zp <dig> where the presence of polytomies in the gene trees caused the preference of the unresolved nodes over the resolved nodes.
genes with significant signal are given in the likelihood mapping, or, l.m.
category column, see text for explanation of the category  <dig> and  <dig> in this column.
results of the amino acid composition and nucleotide base composition bias tests, are shown in the a.a. comp bias and base comp bias columns respectively.
the phylogenetic trees for each gene are drawn using the substitution model described where g = gamma distributed rates across sites, i = invariable sites, f = frequency of amino acids, jtt = jones taylor thornton model.
in the case of lba analysis, no = no evidence of lba in the gene analysed, yes = evidence of lba in the gene analysed.
for each gene, the likelihood of estimated bayesian phylogeny  and corresponding ideal species tree  to fit the dataset were determined with the sh test at a 5% significance level.
values equal to  <dig>  represent the tree with the lowest log likelihood, values less than  <dig>  refer to those cases where there is a significant difference between the two topologies, and the gene tree is a significantly better fit to the data.
ns = no statistical significance between gene and species tree, in these cases the species tree was used.
<dig>  tests of data quality and bias
test for amino acid and base composition biases
we tested all multiple sequence alignments  for evidence of significant levels of amino acid composition bias and base composition bias in each lineage using the treepuzzle software  <cit> .
we found that all alignments passed the significance test with p-values <  <dig> , see table  <dig> for summary.
for full set of amino acid and base composition bias test results, see additional files  <dig> and  <dig> respectively.
in summary the discordance between each of the gene trees and the canonical species phylogeny is not a result of amino acid or base composition biases providing evidence of false relationships.
test for phylogenetic signal
we performed the likelihood mapping procedure implemented in the treepuzzle software  <cit>  to determine the level of phylogenetic signal/conflict present in each alignment, for more detail see the methods section.
our initial dataset consisted of  <dig> genes, we used this filtering step to reduce our dataset to contain only those genes with phylogenetic signal.
we categorized the results from the likelihood mapping analysis into  <dig> main categories of signal: category  <dig> had strong phylogenetic signal , category  <dig> had medium level of phylogenetic signal  and category  <dig> had low/no levels of phylogenetic signal .
the results of the test for phylogenetic signal are summarized in table  <dig> and in total  <dig> out of the  <dig> genes had strong phylogenetic signal , with an additional  <dig> genes with moderate levels of phylogenetic signal .
the complete set of results for the likelihood mapping process is given in additional file  <dig>  the remaining  <dig> genes failed the test .
the category  <dig> genes  were subsequently removed from the analysis, only  <dig> genes were retained for further analysis.
long branch attraction  analysis
we assessed the data for evidence of lba which would manifest itself in the data by drawing species with a greater number of mutations in the gene of interest together erroneously on the phylogenetic tree.
the method applied uses the msa and the corresponding phylogeny to categorise rates amongst sites, using an approach we have previsouly published for mammalian data  <cit> , as described in detail the methods section.
in this method of site-stripping we apply the phylogenetic tree  and the msa to classify all sites in the alignment into one of eight categories of mutation rate.
these are arbitrary categories from 1-8; with  <dig> being the most highly conserved sites and  <dig> being the most highly variable.
essentially, these estimates allow us to select only the most conserved sites for phylogeny reconstruction.
sites are sequentially stripped from the alignments based on their rate of evolution and phylogenies are created based on slower evolving sites.
these site-stripped phylogenies are then compared to the species tree.
using two independent methods of comparison we determined whether the resultant stripped trees had topologies significantly similar to the species phylogeny.
the "root mean squared deviation", or rmsd, method is restricted to binary trees  <cit> , see additional file  <dig> for full set of results.
therefore we also employed the sh method of comparing phylogenies  <cit> , see additional file  <dig> for full set of results.
for a full description of the rmsd statistic used here  <cit> , see the corresponding section in the methods.
using this approach we could identify the signature of lba in the ph <dig> dataset alone, see table  <dig> for summary.
<dig>  analysis of selective pressures using codon models of evolution
following analysis of the phylogenies of these reproductive genes, we determined the selective forces at work on these  <dig> genes .
only those genes passing the data quality tests were analyzed here , see table  <dig>  in the case of catsper <dig>  we have analyzed the gene at two different evolutionary distances because it contains high levels of insertion and deletion events.
the two datasets for catsper <dig> are: exon  <dig> from the primates only, and, the entire gene from only distant mammalian groups.
hence the number of datasets is  <dig>  and the number of genes tested is  <dig>  the alignments in all cases reached significant levels following randomization tests .
in those cases where the genes had already been analyzed in previous studies, we expand upon the data in these studies and use more sophisticated models of evolution.
ml methods are sensitive to sample size with a minimum of  <dig> taxa recommended from simulation studies  <cit> .
for a summary of the site-specific and lineage-specific results, see table  <dig> and table  <dig> respectively.
for a summary of all likelihood ratio tests  performed in the analyses of these genes see table a <dig>  in general the lineages tested in the lineage specific analysis for each gene were as follows: modern human; the primate ancestor; modern mouse, and the rodent ancestor, these are indicated in figure  <dig>  for certain datasets the species tested varied depending on those species for which high quality sequence data existed for that gene, these are discussed on a gene-by-gene basis below.
in summary, for each of the  <dig> datasets tested, positive selection was detected.
in the site-specific test between  <dig> and  <dig> sites per gene were identified as positively selected.
in the lineage-specific analyses there were up to  <dig> lineages per gene identified as having evidence of positive selection.
below is a brief description of the results on a gene-by-gene basis, the complete set of all parameters, likelihood values and lrts are given in additional file  <dig>
q =  <dig>
15> <dig>
q =  <dig>
7> <dig>
catsper1_mammals
q =  <dig>
30> <dig>
q =  <dig>
21> <dig>
q =  <dig>
3> <dig>
q =  <dig>
13> <dig>
q =  <dig>
4> <dig>
q =  <dig>
5> <dig>
q =  <dig>
2> <dig>
q =  <dig>
9> <dig>
q =  <dig>
0> <dig>
following lrt analysis m <dig> was chosen in each case as the most significant model.
n refers to the number of taxa in each dataset.
the proportion of sites , evolving under each corresponding selective pressures  are shown.
for example, p <dig> refers to the proportion of the protein evolving under the selective pressure value given by ω <dig>  the parameters p and q describe the beta distribution.
the final column gives the number of sites with posterior probability  of  <dig> ,  <dig>  and  <dig>  that belong in the positively selected category or sites.
the number before the ">" refers to the number of sites with a specific pp value.
summary table of significant results for lineages specific analyses following lrt analyses.
lineages tested as foreground  are shown in the first column.
only those lineages with significant lrt values for model b or model a and ω > <dig> are shown here.
parameter estimates are given for the lrt values highlighted in bold.
p is the proportion of sites under selection the corresponding selective pressure as measured by ω. fwd ω and bck ω scores for the foreground species and background species respectively are given in the final column.
col1a1
possibly the most intriguing result from our entire analysis is that from the col1a <dig> protein.
according to the microarray study employed here  <cit> , col1a <dig> is highly expressed in the uterus tissue.
it is also found in most structural tissues including cartilage, bone, tendon, skin and part of the eye .
it is a member of the group  <dig> collagen proteins involved in the development of the uterine fibroids  <cit> .
there are two propeptide regions to the col1a <dig> gene, denoted n- and c-terminal propeptides.
according to studies on col1a <dig> function, a role has been established for col1a <dig> in spermatogenesis  <cit> .
our site-specific analysis shows  <dig> sites evolving with an ω value of  <dig> , see table  <dig>  in summary 35/ <dig> of our positively selected sites fall in the n-terminal propeptide region  and 9/ <dig> positively selected sites fall in the c-terminal propeptide region , this can be seen clearly in figure 5a.
position  <dig> in col1a <dig> is cleaved and modified by an endopeptidase, position  <dig> is also modified by pyrrolidone carboxcylic acid .
a positively selected site at position  <dig> is neighboring this multifunctional site, suggesting that there has been an evolutionary effort to improve cleavage and/or modification in this protein.
variations in col1a <dig> are linked with osteogenesis imperfecta , an autosomal dominant disease, resulting in an inability to make the correct collagen protein.
there are a spectrum of oi conditions, the most severe is oi type  <dig>  leading to death in the perinatal period.
a recent extensive study of the single nucleotide polymorphisms  associated with oi has revealed a number of substitutions of glycine residues within the triple helical domains of the col1a <dig> protein  <cit> .
the total number of disease implicated sites in the swiss-prot entry p <dig> for col1a <dig> is 99:  <dig> of these are oi non-specific,  <dig> are oi-i,  <dig> are oi_ii,  <dig> are oi-iv and  <dig> are snps .
one third of the mutations that result in substitutions for glycine in col1a <dig> are lethal whereas those between the start codon and position  <dig> are non-lethal.
only  <dig> of the sites we have identified as positively selected is in the non-lethal domain from position 1- <dig>  this is site  <dig>  this positively selected site is neighboring the snp position  <dig> that causes a mild oi phenotype.
in table  <dig> we show a list of  <dig> positively selected sites that fall in close proximity to sites associated with disease and are located between  <dig> and  <dig>  spanning the important triple helix region.
these positions are all within  <dig> to  <dig> amino acid residues of known disease variants,  <dig> of these disease variants are the severe/lethal oi-ii disease form.
two exclusively lethal regions, helix positions 691- <dig> and 910- <dig> aligned with major binding regions  <cit>  and we find a positively selected site in this region.
following a randomization test for the positively selected sites and disease implicated sites , we have found that the pattern we observe, i.e.
finding positively selected sites in close proximity to disease implicated sites is significant in  <dig> out of the  <dig> cases examined here .
from nearest
the sites under positive selection in the col1a <dig> protein and their associated posterior probabilities  are shown.
the third column shows variant positions  as determined using swiss-prot human  sequence.
the fourth and fifth columns show the residue distance "d" of the positively selected site from its nearest genetic variant, and the probability of being located "d" residues from any disease implicated site by random chance alone.
the sixth column uses single-letter amino acid symbols to show the genetic code distances between all observed character states at each positively selected site.
"clinical association" show the replacement substitution at the human variant position and its clinical association with that human variant.
oi = osteolysis imperfecta, oi-i to -iv.
the final entry for dbsnp is database entry number rs <dig> and as yet has not been associated with oi although it is in the same domain as the other disease-causing snps.
lineage-specific analysis shows evidence for positive selection in this protein in the rodent ancestor.
in total,  <dig> % of the sites in the rodent ancestor have ω =  <dig> , while the rest of the species are evolving under purifying selection, ω =  <dig> .
for a summary of site and lineage specific results for col1a <dig>  see table  <dig> and  <dig>  for complete set of results see additional file  <dig>
prkar2a
prkar2a is a camp dependent protein kinase that is attached to the sperm flagella via regulatory subunit   <cit> .
protein tyrosine phosphorylation has been linked with successful fertilization due to hyper-activated sperm motility  <cit> .
this increase in phosphorylation is part of a camp dependent pathway that activates protein kinase a  <cit> .
the prka families were previously tested for positive selection using  <dig> to  <dig> taxa and site-specific model m <dig> with no significant results for positive selection reported.
with our  <dig> taxa dataset, we were able to detect that  <dig> % of sites were evolving at a rate of ω =  <dig> , see table  <dig> for summary of details.
positively selected sites detected in the site-specific analysis of prkar2a were compared to the human swiss-prot sequence .
in total  <dig> sites were predicted to be positively selected,  <dig> of these sites occur in the region of the protein associated with dimerization and phosphorylation , see figure  <dig>  in the swiss-prot entry there are a number of residues listed as being modified by phosphoserine.
these are positions  <dig>   <dig>   <dig>   <dig> and phosphothreonine at position  <dig>  the sites estimated to be positively selected from our analysis are:  <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>  these sites are at or in close proximity to these modified residues.
the regulatory subunit alpha  <dig> of prkar2a has been shown in vitro to interact with semg <dig>  the phosphorylation of semg <dig> may lead to its activation into forming a gel matrix in the female reproductive tract.
from our analysis it is shown that while semg <dig> has positively selected sites dispersed throughout its sequence, whereas the positively selected sites for prkar2a are localized to the n-terminus region, and the remainder of the gene is under strong purifying selection.
literature has so far not specified an exact phosphorylation site for semg <dig>  which prevents us from commenting further on its interactions with prkar2a.
lineage-specific analysis shows that prkar2a in the macaque has undergone a greater selective pressure to change when compared with other mammalia in the dataset, with  <dig> % of sites evolving at ω =  <dig> , see table  <dig> for summary of results.
for complete set of results for prkar2a, see additional file  <dig>
ph <dig>
ph <dig> is expressed in the testis and found in the acrosome of the sperm.
it is also codes for a receptor that is involved in the sperm to zona pellucida  adhesion  <cit> .
previous analysis conducted on this protein involved  <dig> taxa  <cit> .
here we have increased the number of taxa to  <dig>  we have omitted the carnivores from our analysis of ph <dig> as the sequences were spurious.
we found evidence for lba in the ph <dig> dataset.
by removing fast evolving sites a fully resolved gene phylogeny is obtained.
this gene tree now is in agreement with the ideal species phylogeny , see figure  <dig> for results.
catalytically important resides  <dig>   <dig>   <dig>  <dig> and  <dig> when mutated result in a reduction in, or loss of, activity  <cit> .
it has been shown experimentally that mutations in the region of this active site significantly reduce or completely block the function of this protein  <cit> .
our results show that  <dig> of the positively selected sites,  <dig>   <dig>   <dig>  are in close proximity to these regions.
another  <dig> positively selected sites:  <dig>   <dig>   <dig>   <dig> and  <dig> are close to glycosylation sites, see figure  <dig>  these sites when modified are known to change the structure and function of the ph <dig> protein.
for complete set of results for ph <dig> see additional file  <dig>  these results are of significance as the ph <dig> protein changes position in the sperm during the different stages of the fertilization process.
in guinea pig ph <dig> protein is known to migrate from the post acrosomal membrane to the inner acrosomal membrane  <cit> .
thus finding these positively selected sites in close proximity to these glycosylation sites in guinea pig suggests that these sites have been selected to modify the ph <dig> structure more effectively thus increasing the chance of capacitation.
sp <dig>
the binding of sperm to the zona pellucida  is crucial for gamete formation to take place.
the exact mechanisms of this process are still to be uncovered therefore any predictions on important residues will greatly improve knowledge by directing mutational studies.
sp <dig> has been shown through photoaffinity cross-linking experiments to have a specific binding affinity for zp <dig>  <cit> .
therefore it is believed to play an important role in the binding of sperm to the zp matrix.
experiments have shown that during capacitation sp <dig> is released from the acrosomal matrix and becomes situated in the sperm head membrane, enabling it to act as a zp <dig> binding protein  <cit> .
here we have found  <dig> positions in the sp <dig> protein that are under positive selection  following site-specific analysis.
these sites were compared to the human sp <dig> entry in swiss-prot  to determine possible links to function.
one of these  <dig> positively selected sites is position  <dig>  regarded as a snp number  in dbsnp database  <cit> .
although further experimental work needs to be conducted to decipher the clinical association of this position, it is extremely interesting that our most significant positively selected site also displays variation in the population, especially given the overall high level of conservation in this gene.
for summary of results see tables  <dig> and  <dig>  and for full set of results for this gene see additional file  <dig>
zp2
zona pellucida  proteins form the complex glycoprotein coat that surrounds the oocyte  <cit> .
these zp proteins have been shown to be under strong pressure to change, and results have been published on both site and lineage analyses  <cit> .
here we have expanded the analysis of zp <dig> to include  <dig> taxa .
we have also applied more complex models of evolution and have sampled deeper branches on the phylogeny including a representative of the afrotheria - elephant.
in this case, the results of our larger dataset and more complex models show that the values of ω determined here vary slightly when compared to previous analyses  <cit> .
this previous test showed  <dig> % of sites to have ω =  <dig> , increasing the size of the dataset in this study results in  <dig> sites in zp <dig> that have an ω value of  <dig> .
see additional file  <dig> for complete results.
positively selected sites were compared to the human swiss-prot entry for zp <dig>  to identify possible function for these sites, see figure  <dig>  zp <dig> contains  <dig> carbohydrate chains situated between sites 87- <dig>  these are important for the sperm to bind to the zp of the egg coat  <cit> .
of the  <dig> sites identified to be under positive selection,  <dig> fall between positions 66- <dig>  this region contains  <dig> of the binding domains of the carbohydrate chains.
the clustering can be seen more clearly in figure  <dig>  another cluster of positively selected sites  occurs in the propeptide region .
it has been suggested that upon the cleavage of the propeptide region, the mature zp <dig> protein plays a role in the prevention of polyspermy  <cit> .
zp3
analysis of site-specific evolution in zp <dig> identified  <dig> positively selected sites.
of specific interest are positively selected positions  <dig>   <dig>   <dig>   <dig>   <dig>   <dig>  as these sites were in close proximity to identified sperm binding sites   <cit> , see table  <dig>  the furin cleavage site is identified at position , and the propeptide domain at position .
when cleavage takes place the zp <dig> undergoes a conformational change that inhibits any further sperm binding to the coat thus preventing polyspermy  <cit> .
of the  <dig> positively selected sites identified,  <dig> fall within the propeptide domain, with an additional  <dig> occurring close to the vicinity of the furin cleavage and sperm binding sites, thus suggesting that there is a pressure to improve binding and prevent polyspermy.
for complete set of results for zp <dig>  see additional file  <dig>
adam <dig>
adam <dig> is a cell adhesion molecule that plays a fundamental role in the final binding of sperm to the oocyte membrane  <cit> .
indirect interactions have been shown with female proteins cd <dig>  <cit> .
.
previous results have been published reporting positive selection using site-specific analysis on  <dig> taxa  <cit> .
here we have included  <dig> taxa for adam <dig> and we have investigated the possible functional implications of positively selected sites found.
in the site-specific analysis we find  <dig> % of sites with ω =  <dig> , this corresponds to  <dig> sites in total, see table  <dig>  comparison of these positions to human swiss-prot adam <dig> sequence , we determine that 39/ <dig> positively selected sites are situated in the c-terminus region.
on closer investigation of these sites we find that 12/ <dig> positively selected sites occur in the disintegrin domain .
the disintegrin domain has been shown to be involved in the binding of adam <dig> to the oocyte  <cit> .
a cysteine-rich domain occurs between , 16/ <dig> positively selected sites fall in this region.
it has been suggested for adam <dig>  , that the cysteine-rich domain plays a role in mediating the cellular interactions via syndecans and integrin  <cit> , a similar role for this domain in adam <dig> can be postulated.
overall the results for adam <dig> suggest a selective pressure for increased binding of adam <dig> to the oocyte regardless of species of origin.
for a complete set of results and lrts for adam <dig>  see additional file  <dig>
catsper1
catsper <dig> is involved in regulating the calcium cation channel in sperm flagella, the result of which is movement of sperm  <cit> .
previous studies on catsper <dig> exon  <dig> have been performed  <cit> .
we intended to expand our analysis to span all exons and expand the data set to include a variety of mammalia.
however, the exon  <dig> of non-primate mammalia is so highly variable that an accurate alignment cannot be constructed.
the remaining exons were highly conserved across all species.
we therefore split our catsper <dig> dataset into two sections each of which produced a good quality alignment for analysis,  exon <dig> of catsper <dig> for the primates, and  entire catsper <dig> gene for non-primate mammalia.
catsper <dig> exon  <dig> primates
site-specific analysis of this protein identified 17% of the protein under positive selection with ω =  <dig> .
previous analysis of this exon showed positive selection on indel substitutions in this gene  <cit> .
the positively selected sites are situated throughout exon <dig>  little is known about the functional significance of these sites.
however, it is known that exon  <dig> has a significant role to play in altering the rate of calcium ion channel inactivation.
different lengths in the n-terminus result in different rates of channel inactivation, where a long terminus results in a longer time to activation than the shorter terminus.
this is described most effectively by the ball and chain mechanism described in  <cit> .
see additional file  <dig> for complete results.
these results show the importance of this protein, and specifically the first exon, for reproductive success.
catsper <dig> entire gene non-primate mammals
our site-specific analysis identified  <dig> % of the sites under positive selection with an ω =  <dig> , see table  <dig>  these sites all cluster in exon  <dig>  while the rodent ancestor appears to be under positive selection with  <dig> % of its sites evolving at ω =  <dig>  see additional file  <dig> for complete set of results.
a previous study of  <dig> rodent species, including mus musculus individuals from  <dig> different populations, has shown that within the rodent order there has been a continued pressure to evolve, with positive selection for indel substitutions in exon <dig> of the catsper <dig> gene  <cit> .
semg2
a member of the family of semenogelin genes, semg <dig> is involved in the formation of a postcopulatory plug  <cit> .
previously, positive selection has been reported for both site-specific and lineage-specific analysis for semg <dig>  <cit> .
we have expanded the data set from previous analyses to incorporate more species.
in our site-specific analysis, we found that  <dig> % of our sites had an ω value of  <dig> , see table  <dig>
we have performed a novel functional analysis of these positively selected sites by comparing them to the human semg <dig> sequence  in the swiss-prot database.
this is a step not previously taken by other studies of semg <dig>  a striking pattern emerged - all known domains of this protein have several positively selected sites.
there is a probable glycosylation site at position  <dig>  which is located close to a large stretch of positively selected sites .
it is so far unknown how significant this glycosylation site is in semg <dig> and whether it plays a role in modifying the protein to form a copulatory plug.
however, the results indicate that this protein, and in particular the region around the glycosylation site, has been under significant pressure to change.
a complete set of results for semg <dig> is given in additional file  <dig>  the lineage-specific results are not described here in detail as lineage analyses have been carried out previously on the primate semg <dig> gene  <cit> .
it has been shown recently that the rate of evolution for this protein varies depending on the level of sperm competition  <cit> .
our results are in agreement with this finding, thus further verifying our approach.
porimin
two isoforms of this protein have been identified; we have focused on isoform  <dig> in the mammalia, as isoform  <dig> contains an additional human specific region between residues 34- <dig>  to date the exact mechanisms of this transmembrane receptor are unknown.
this protein is not well characterized biochemically and its function cannot be verified as reproduction related, therefore we only discuss the results briefly below.
on site-specific analysis of this protein we determined that  <dig> of the sites are under positive selection , see table  <dig>  from analysis of the sites on the swiss-prot entry for human porimin , we could determine that two positively selected sites , were found in a highly conserved region and fall in close proximity to the n-linked glycosylation site.
for complete set of results for porimin, see additional file  <dig>
CONCLUSIONS
testing for phylogenetic signal and biases, such as amino acid composition bias and lba, indicated that there was adequate phylogenetic signal for  <dig> of the genes and in general no evidence of systematic biases.
on testing for lba, ph <dig> was the only protein in this dataset that displayed the typical signature of this bias with gene and species tree agreement being maximized with the removal of the fastest evolving categories.
this would suggest that while germ line generation times vary greatly in the dataset, the effect of the resultant lba does not impact on the sequence data to any great extent .
selective pressures for the reproductive proteins studied here are heterogeneous.
all proteins exhibited regions of strong conservation proving the importance of maintaining structural stability and overall function in these proteins.
all but  <dig> protein  exhibited evidence of positive selection in specific lineages, and all proteins without exception exhibited positive selection in regions of catalytic/functional importance.
for sp <dig> and col1a <dig> the site-specific results are entirely novel.
the lineage-specific results described here for prkar2a and catsper <dig> exon  <dig> in primates, are also novel.
we have shown that, in the case of catsper <dig>  there is a fundamental protein functional shift between new world monkeys and old world monkeys.
the dn/ds measurement applied here assumes that neutral substitution rate is akin to ds, therefore no selection on silent sites.
there have been many publications of late to the contrary therefore we are mindful of examining the rate of silent substitution in all our analyses  <cit> .
for the reproductive genes in our dataset, we show that lineages evolve at unique rates and at functionally crucial sites, specifically those involved in phosphorylation.
we have also shown that a number of these proteins  show positive selection for example in the ancestral rodent lineage and evidence of purifying selection in the subsequent divergent species.
overall our analyses of these reproductive proteins show how important it is to carefully examine data for systematic biases prior to testing for lineage and/or site specific positive selection.
we have also demonstrated the importance of including large numbers of taxa/lineages in these analyses.
this finding was highlighted in our analysis of prkar2a where previous analysis of this protein had included only  <dig> taxa and therefore reported a negative result.
we do not observe any large-scale effect of germ line generation time in our dataset, with only  <dig> protein  with evidence of long branch attraction.
the results of col1a <dig> indicate that the positively selected sites may have been of such importance for this protein that neighboring mutated sites may have been maintained in the population despite their propensity for causing disease.
the location of positively selected sites determined using this approach and in regions of functional importance in the proteins in this dataset, provides us with further evidence of the link between functional shift and positive selection.

 reproductive proteins are essential for success of sexually reproducing species and indeed for the emergence of new species. in the past it has been observed that reproductive proteins tend to be under positive selective pressure to change, i.e. adaptive evolution, a trend found in a variety of animal species from abalone to primates. adaptive evolution or positive selection is a selective pressure placed on a protein by a change in environment in order to improve the fitness of the organism in that environment. with changes in environment, that can include mating system, there is a subsequent selective pressure on the protein sequences related to those functions to adapt accordingly. this variation can be detected using the well-known measurements of the rate of non-synonymous substitutions per non-synonymous site and synonymous substitutions per synonymous site and their ratio ω = dn/ds. the detection of adaptive evolution, where the ratio exceeds unity, is referred to as positive darwinian selection. detecting positive darwinian selection in a region of a protein, or indeed in a lineage of a phylogeny, indicates that there is a selective advantage in changing the amino acid sequence in this region. these signals are essential for our understanding of functionally important residues in a protein sequence and protein functional shift. in general, the rate of mutation that a gene undergoes is contingent on a number of factors including; protein structure, presence of gene duplicates, location in the genome, effective population size, germ line generation time, and composition of the sequence. it has recently been shown that the number of physical interactions of a particular protein also influences the intrinsic rate of evolution. evidence for the generation time effect has come from studies on various proteins and species including analyses of substitution rates in higher primates and rodents, substitution rates in higher grasses and in palms, in mammalian genomes and in chloroplast and sex mutation rate ratios. with recent advances in sequencing we have an opportunity to examine these effects using a wider selection of proteins and species. documented selective pressures associated with positive selection in reproductive proteins include: intense sperm competition whereby sperm from numerous males, ejaculated into the female reproductive tract, compete with one another for the prized fertilization of the egg; evasion of the immune system, whereby surface layer reproductive proteins evolve to evade destruction by the host's immune system; and finally selective pressures enforced by mating system, related of course to point above. species that are more promiscuous have increased levels of selective pressure acting on reproductive proteins than species that are monogamous. this later point is illustrated in the study of semg where adaptive evolution was found to correlate with mating system in primates. in order to determine the variation in selective pressure in these proteins, there are a number of criteria that the data must meet. firstly, the data must have a robust phylogenetic signal. secondly, systematic biases that may exist in the data must be minimized, these include but are not limited to: long branch attraction, amino acid composition bias, base composition bias and unqualified ortholog predictions, all of which may lead to inaccurate estimates of phylogeny. thirdly, sensitivity to taxa number is a known limitation of methods for detecting positive selection, therefore more than taxa are needed to gain accurate estimations of selective pressure using the maximum likelihood method applied here. in this study we have selected a subset of proteins that have roles to play in reproduction. our dataset was composed of three major datatypes, previously published reproductive proteins, interacting proteins, here we identified proteins shown to interact with, and finally, genes identified from microarray experiments as being highly expressed in reproductive tissues. for group we assume that those proteins highly expressed in reproductive tissues are important for the function of that tissue. the previously untested reproductive proteins analysed here are from data types and outlined above. these novel proteins are sp porimin and col1a sp is sperm binding protein number this protein is a representative of the interacting protein subset of sequences analysed. sp has been shown to interact with zp - a well-studied reproductive protein. both porimin and col1a have been identified from published microarray experiments on normal human tissue, and were selected for analysis due to their high levels of expression in reproductive tissues in that study. porimin is a transmembrane protein that is highly expressed in the uterus, prostate and placenta and col1a is highly expressed in the uterus. further evidence for the link between porimin and reproduction was not available in the literature and therefore results from this particular gene are taken with caution until this protein is further characterized. col1a plays an important role during spermatogenesis where it mediates the detachment and migration of germ cells, thus adding further support for its role in reproduction. we have analyzed these data with an approach sensitive to all the systematic biases and limitations of methods given above. a number of genes in our dataset have been analyzed previously but have not taken these limitations and considerations into account. we have expanded these datasets to include a greater number of taxa, we have analyzed all of these genes for evidence of systematic biases and we have used improved models of codon evolution. in this paper we have included models that allow for rate variation across the sequence and across the phylogeny.  we performed phylogenetic analyses on all datasets. the resultant gene trees were found to conflict with the canonical phylogeny species amino acid and/or base composition bias, lack of phylogenetic signal in the data, and finally, lba caused by mixtures of long and short germ line generation times. what follows is a summary of the results of the tests of data quality and bias we performed, see table for synopsis. we carried out these tests to determine in each case whether these conflicting phylogenies are accurate descriptions of history or whether the data are subject to these known issues listed 1- above. subsequent statistical comparison of the gene trees and species phylogeny using the shimodaira hasegawa test revealed that there is no statistical difference between the gene and species trees in each case, see table for results of sh tests. the only exceptions were prkar2a and zp where the presence of polytomies in the gene trees caused the preference of the unresolved nodes over the resolved nodes. genes with significant signal are given in the likelihood mapping, or, l.m. category column, see text for explanation of the category and in this column. results of the amino acid composition and nucleotide base composition bias tests, are shown in the a.a. comp bias and base comp bias columns respectively. the phylogenetic trees for each gene are drawn using the substitution model described where g = gamma distributed rates across sites, i = invariable sites, f = frequency of amino acids, jtt = jones taylor thornton model. in the case of lba analysis, no = no evidence of lba in the gene analysed, yes = evidence of lba in the gene analysed. for each gene, the likelihood of estimated bayesian phylogeny and corresponding ideal species tree to fit the dataset were determined with the sh test at a 5% significance level. values equal to represent the tree with the lowest log likelihood, values less than refer to those cases where there is a significant difference between the two topologies, and the gene tree is a significantly better fit to the data. ns = no statistical significance between gene and species tree, in these cases the species tree was used. tests of data quality and bias test for amino acid and base composition biases we tested all multiple sequence alignments for evidence of significant levels of amino acid composition bias and base composition bias in each lineage using the treepuzzle software. we found that all alignments passed the significance test with p-values <, see table for summary. for full set of amino acid and base composition bias test results, see additional files and respectively. in summary the discordance between each of the gene trees and the canonical species phylogeny is not a result of amino acid or base composition biases providing evidence of false relationships. test for phylogenetic signal we performed the likelihood mapping procedure implemented in the treepuzzle software to determine the level of phylogenetic signal/conflict present in each alignment, for more detail see the methods section. our initial dataset consisted of genes, we used this filtering step to reduce our dataset to contain only those genes with phylogenetic signal. we categorized the results from the likelihood mapping analysis into main categories of signal: category had strong phylogenetic signal, category had medium level of phylogenetic signal and category had low/no levels of phylogenetic signal. the results of the test for phylogenetic signal are summarized in table and in total out of the genes had strong phylogenetic signal, with an additional genes with moderate levels of phylogenetic signal. the complete set of results for the likelihood mapping process is given in additional file the remaining genes failed the test. the category genes were subsequently removed from the analysis, only genes were retained for further analysis. long branch attraction analysis we assessed the data for evidence of lba which would manifest itself in the data by drawing species with a greater number of mutations in the gene of interest together erroneously on the phylogenetic tree. the method applied uses the msa and the corresponding phylogeny to categorise rates amongst sites, using an approach we have previsouly published for mammalian data, as described in detail the methods section. in this method of site-stripping we apply the phylogenetic tree and the msa to classify all sites in the alignment into one of eight categories of mutation rate. these are arbitrary categories from 1-8; with being the most highly conserved sites and being the most highly variable. essentially, these estimates allow us to select only the most conserved sites for phylogeny reconstruction. sites are sequentially stripped from the alignments based on their rate of evolution and phylogenies are created based on slower evolving sites. these site-stripped phylogenies are then compared to the species tree. using two independent methods of comparison we determined whether the resultant stripped trees had topologies significantly similar to the species phylogeny. the "root mean squared deviation", or rmsd, method is restricted to binary trees, see additional file for full set of results. therefore we also employed the sh method of comparing phylogenies, see additional file for full set of results. for a full description of the rmsd statistic used here, see the corresponding section in the methods. using this approach we could identify the signature of lba in the ph dataset alone, see table for summary. analysis of selective pressures using codon models of evolution following analysis of the phylogenies of these reproductive genes, we determined the selective forces at work on these genes. only those genes passing the data quality tests were analyzed here, see table in the case of catsper we have analyzed the gene at two different evolutionary distances because it contains high levels of insertion and deletion events. the two datasets for catsper are: exon from the primates only, and, the entire gene from only distant mammalian groups. hence the number of datasets is and the number of genes tested is the alignments in all cases reached significant levels following randomization tests. in those cases where the genes had already been analyzed in previous studies, we expand upon the data in these studies and use more sophisticated models of evolution. ml methods are sensitive to sample size with a minimum of taxa recommended from simulation studies. for a summary of the site-specific and lineage-specific results, see table and table respectively. for a summary of all likelihood ratio tests performed in the analyses of these genes see table a in general the lineages tested in the lineage specific analysis for each gene were as follows: modern human; the primate ancestor; modern mouse, and the rodent ancestor, these are indicated in figure for certain datasets the species tested varied depending on those species for which high quality sequence data existed for that gene, these are discussed on a gene-by-gene basis below. in summary, for each of the datasets tested, positive selection was detected. in the site-specific test between and sites per gene were identified as positively selected. in the lineage-specific analyses there were up to lineages per gene identified as having evidence of positive selection. below is a brief description of the results on a gene-by-gene basis, the complete set of all parameters, likelihood values and lrts are given in additional file q = 15> q = 7> catsper1_mammals q = 30> q = 21> q = 3> q = 13> q = 4> q = 5> q = 2> q = 9> q = 0> following lrt analysis m was chosen in each case as the most significant model. n refers to the number of taxa in each dataset. the proportion of sites, evolving under each corresponding selective pressures are shown. for example, p refers to the proportion of the protein evolving under the selective pressure value given by ω the parameters p and q describe the beta distribution. the final column gives the number of sites with posterior probability of, and that belong in the positively selected category or sites. the number before the ">" refers to the number of sites with a specific pp value. summary table of significant results for lineages specific analyses following lrt analyses. lineages tested as foreground are shown in the first column. only those lineages with significant lrt values for model b or model a and ω > are shown here. parameter estimates are given for the lrt values highlighted in bold. p is the proportion of sites under selection the corresponding selective pressure as measured by ω. fwd ω and bck ω scores for the foreground species and background species respectively are given in the final column. col1a1 possibly the most intriguing result from our entire analysis is that from the col1a protein. according to the microarray study employed here, col1a is highly expressed in the uterus tissue. it is also found in most structural tissues including cartilage, bone, tendon, skin and part of the eye. it is a member of the group collagen proteins involved in the development of the uterine fibroids. there are two propeptide regions to the col1a gene, denoted n- and c-terminal propeptides. according to studies on col1a function, a role has been established for col1a in spermatogenesis. our site-specific analysis shows sites evolving with an ω value of, see table in summary 35/ of our positively selected sites fall in the n-terminal propeptide region and 9/ positively selected sites fall in the c-terminal propeptide region, this can be seen clearly in figure 5a. position in col1a is cleaved and modified by an endopeptidase, position is also modified by pyrrolidone carboxcylic acid. a positively selected site at position is neighboring this multifunctional site, suggesting that there has been an evolutionary effort to improve cleavage and/or modification in this protein. variations in col1a are linked with osteogenesis imperfecta, an autosomal dominant disease, resulting in an inability to make the correct collagen protein. there are a spectrum of oi conditions, the most severe is oi type leading to death in the perinatal period. a recent extensive study of the single nucleotide polymorphisms associated with oi has revealed a number of substitutions of glycine residues within the triple helical domains of the col1a protein. the total number of disease implicated sites in the swiss-prot entry p for col1a is 99: of these are oi non-specific, are oi-i, are oi_ii, are oi-iv and are snps. one third of the mutations that result in substitutions for glycine in col1a are lethal whereas those between the start codon and position are non-lethal. only of the sites we have identified as positively selected is in the non-lethal domain from position 1- this is site this positively selected site is neighboring the snp position that causes a mild oi phenotype. in table we show a list of positively selected sites that fall in close proximity to sites associated with disease and are located between and spanning the important triple helix region. these positions are all within to amino acid residues of known disease variants, of these disease variants are the severe/lethal oi-ii disease form. two exclusively lethal regions, helix positions 691- and 910- aligned with major binding regions and we find a positively selected site in this region. following a randomization test for the positively selected sites and disease implicated sites, we have found that the pattern we observe, i.e. finding positively selected sites in close proximity to disease implicated sites is significant in out of the cases examined here. from nearest the sites under positive selection in the col1a protein and their associated posterior probabilities are shown. the third column shows variant positions as determined using swiss-prot human sequence. the fourth and fifth columns show the residue distance "d" of the positively selected site from its nearest genetic variant, and the probability of being located "d" residues from any disease implicated site by random chance alone. the sixth column uses single-letter amino acid symbols to show the genetic code distances between all observed character states at each positively selected site. "clinical association" show the replacement substitution at the human variant position and its clinical association with that human variant. oi = osteolysis imperfecta, oi-i to -iv. the final entry for dbsnp is database entry number rs and as yet has not been associated with oi although it is in the same domain as the other disease-causing snps. lineage-specific analysis shows evidence for positive selection in this protein in the rodent ancestor. in total, % of the sites in the rodent ancestor have ω =, while the rest of the species are evolving under purifying selection, ω =. for a summary of site and lineage specific results for col1a see table and for complete set of results see additional file prkar2a prkar2a is a camp dependent protein kinase that is attached to the sperm flagella via regulatory subunit. protein tyrosine phosphorylation has been linked with successful fertilization due to hyper-activated sperm motility. this increase in phosphorylation is part of a camp dependent pathway that activates protein kinase a. the prka families were previously tested for positive selection using to taxa and site-specific model m with no significant results for positive selection reported. with our taxa dataset, we were able to detect that % of sites were evolving at a rate of ω =, see table for summary of details. positively selected sites detected in the site-specific analysis of prkar2a were compared to the human swiss-prot sequence. in total sites were predicted to be positively selected, of these sites occur in the region of the protein associated with dimerization and phosphorylation, see figure in the swiss-prot entry there are a number of residues listed as being modified by phosphoserine. these are positions and phosphothreonine at position the sites estimated to be positively selected from our analysis are: these sites are at or in close proximity to these modified residues. the regulatory subunit alpha of prkar2a has been shown in vitro to interact with semg the phosphorylation of semg may lead to its activation into forming a gel matrix in the female reproductive tract. from our analysis it is shown that while semg has positively selected sites dispersed throughout its sequence, whereas the positively selected sites for prkar2a are localized to the n-terminus region, and the remainder of the gene is under strong purifying selection. literature has so far not specified an exact phosphorylation site for semg which prevents us from commenting further on its interactions with prkar2a. lineage-specific analysis shows that prkar2a in the macaque has undergone a greater selective pressure to change when compared with other mammalia in the dataset, with % of sites evolving at ω =, see table for summary of results. for complete set of results for prkar2a, see additional file ph ph is expressed in the testis and found in the acrosome of the sperm. it is also codes for a receptor that is involved in the sperm to zona pellucida adhesion. previous analysis conducted on this protein involved taxa. here we have increased the number of taxa to we have omitted the carnivores from our analysis of ph as the sequences were spurious. we found evidence for lba in the ph dataset. by removing fast evolving sites a fully resolved gene phylogeny is obtained. this gene tree now is in agreement with the ideal species phylogeny, see figure for results. catalytically important resides and when mutated result in a reduction in, or loss of, activity. it has been shown experimentally that mutations in the region of this active site significantly reduce or completely block the function of this protein. our results show that of the positively selected sites, are in close proximity to these regions. another positively selected sites: and are close to glycosylation sites, see figure these sites when modified are known to change the structure and function of the ph protein. for complete set of results for ph see additional file these results are of significance as the ph protein changes position in the sperm during the different stages of the fertilization process. in guinea pig ph protein is known to migrate from the post acrosomal membrane to the inner acrosomal membrane. thus finding these positively selected sites in close proximity to these glycosylation sites in guinea pig suggests that these sites have been selected to modify the ph structure more effectively thus increasing the chance of capacitation. sp the binding of sperm to the zona pellucida is crucial for gamete formation to take place. the exact mechanisms of this process are still to be uncovered therefore any predictions on important residues will greatly improve knowledge by directing mutational studies. sp has been shown through photoaffinity cross-linking experiments to have a specific binding affinity for zp. therefore it is believed to play an important role in the binding of sperm to the zp matrix. experiments have shown that during capacitation sp is released from the acrosomal matrix and becomes situated in the sperm head membrane, enabling it to act as a zp binding protein. here we have found positions in the sp protein that are under positive selection following site-specific analysis. these sites were compared to the human sp entry in swiss-prot to determine possible links to function. one of these positively selected sites is position regarded as a snp number in dbsnp database. although further experimental work needs to be conducted to decipher the clinical association of this position, it is extremely interesting that our most significant positively selected site also displays variation in the population, especially given the overall high level of conservation in this gene. for summary of results see tables and and for full set of results for this gene see additional file zp2 zona pellucida proteins form the complex glycoprotein coat that surrounds the oocyte. these zp proteins have been shown to be under strong pressure to change, and results have been published on both site and lineage analyses. here we have expanded the analysis of zp to include taxa. we have also applied more complex models of evolution and have sampled deeper branches on the phylogeny including a representative of the afrotheria - elephant. in this case, the results of our larger dataset and more complex models show that the values of ω determined here vary slightly when compared to previous analyses. this previous test showed % of sites to have ω =, increasing the size of the dataset in this study results in sites in zp that have an ω value of. see additional file for complete results. positively selected sites were compared to the human swiss-prot entry for zp to identify possible function for these sites, see figure zp contains carbohydrate chains situated between sites 87- these are important for the sperm to bind to the zp of the egg coat. of the sites identified to be under positive selection, fall between positions 66- this region contains of the binding domains of the carbohydrate chains. the clustering can be seen more clearly in figure another cluster of positively selected sites occurs in the propeptide region. it has been suggested that upon the cleavage of the propeptide region, the mature zp protein plays a role in the prevention of polyspermy. zp3 analysis of site-specific evolution in zp identified positively selected sites. of specific interest are positively selected positions as these sites were in close proximity to identified sperm binding sites, see table the furin cleavage site is identified at position, and the propeptide domain at position. when cleavage takes place the zp undergoes a conformational change that inhibits any further sperm binding to the coat thus preventing polyspermy. of the positively selected sites identified, fall within the propeptide domain, with an additional occurring close to the vicinity of the furin cleavage and sperm binding sites, thus suggesting that there is a pressure to improve binding and prevent polyspermy. for complete set of results for zp see additional file adam adam is a cell adhesion molecule that plays a fundamental role in the final binding of sperm to the oocyte membrane. indirect interactions have been shown with female proteins cd.. previous results have been published reporting positive selection using site-specific analysis on taxa. here we have included taxa for adam and we have investigated the possible functional implications of positively selected sites found. in the site-specific analysis we find % of sites with ω =, this corresponds to sites in total, see table comparison of these positions to human swiss-prot adam sequence, we determine that 39/ positively selected sites are situated in the c-terminus region. on closer investigation of these sites we find that 12/ positively selected sites occur in the disintegrin domain. the disintegrin domain has been shown to be involved in the binding of adam to the oocyte. a cysteine-rich domain occurs between, 16/ positively selected sites fall in this region. it has been suggested for adam, that the cysteine-rich domain plays a role in mediating the cellular interactions via syndecans and integrin, a similar role for this domain in adam can be postulated. overall the results for adam suggest a selective pressure for increased binding of adam to the oocyte regardless of species of origin. for a complete set of results and lrts for adam see additional file catsper1 catsper is involved in regulating the calcium cation channel in sperm flagella, the result of which is movement of sperm. previous studies on catsper exon have been performed. we intended to expand our analysis to span all exons and expand the data set to include a variety of mammalia. however, the exon of non-primate mammalia is so highly variable that an accurate alignment cannot be constructed. the remaining exons were highly conserved across all species. we therefore split our catsper dataset into two sections each of which produced a good quality alignment for analysis, exon of catsper for the primates, and entire catsper gene for non-primate mammalia. catsper exon primates site-specific analysis of this protein identified 17% of the protein under positive selection with ω =. previous analysis of this exon showed positive selection on indel substitutions in this gene. the positively selected sites are situated throughout exon little is known about the functional significance of these sites. however, it is known that exon has a significant role to play in altering the rate of calcium ion channel inactivation. different lengths in the n-terminus result in different rates of channel inactivation, where a long terminus results in a longer time to activation than the shorter terminus. this is described most effectively by the ball and chain mechanism described in. see additional file for complete results. these results show the importance of this protein, and specifically the first exon, for reproductive success. catsper entire gene non-primate mammals our site-specific analysis identified % of the sites under positive selection with an ω =, see table these sites all cluster in exon while the rodent ancestor appears to be under positive selection with % of its sites evolving at ω = see additional file for complete set of results. a previous study of rodent species, including mus musculus individuals from different populations, has shown that within the rodent order there has been a continued pressure to evolve, with positive selection for indel substitutions in exon of the catsper gene. semg2 a member of the family of semenogelin genes, semg is involved in the formation of a postcopulatory plug. previously, positive selection has been reported for both site-specific and lineage-specific analysis for semg. we have expanded the data set from previous analyses to incorporate more species. in our site-specific analysis, we found that % of our sites had an ω value of, see table we have performed a novel functional analysis of these positively selected sites by comparing them to the human semg sequence in the swiss-prot database. this is a step not previously taken by other studies of semg a striking pattern emerged - all known domains of this protein have several positively selected sites. there is a probable glycosylation site at position which is located close to a large stretch of positively selected sites. it is so far unknown how significant this glycosylation site is in semg and whether it plays a role in modifying the protein to form a copulatory plug. however, the results indicate that this protein, and in particular the region around the glycosylation site, has been under significant pressure to change. a complete set of results for semg is given in additional file the lineage-specific results are not described here in detail as lineage analyses have been carried out previously on the primate semg gene. it has been shown recently that the rate of evolution for this protein varies depending on the level of sperm competition. our results are in agreement with this finding, thus further verifying our approach. porimin two isoforms of this protein have been identified; we have focused on isoform in the mammalia, as isoform contains an additional human specific region between residues 34- to date the exact mechanisms of this transmembrane receptor are unknown. this protein is not well characterized biochemically and its function cannot be verified as reproduction related, therefore we only discuss the results briefly below. on site-specific analysis of this protein we determined that of the sites are under positive selection, see table from analysis of the sites on the swiss-prot entry for human porimin, we could determine that two positively selected sites, were found in a highly conserved region and fall in close proximity to the n-linked glycosylation site. for complete set of results for porimin, see additional file  testing for phylogenetic signal and biases, such as amino acid composition bias and lba, indicated that there was adequate phylogenetic signal for of the genes and in general no evidence of systematic biases. on testing for lba, ph was the only protein in this dataset that displayed the typical signature of this bias with gene and species tree agreement being maximized with the removal of the fastest evolving categories. this would suggest that while germ line generation times vary greatly in the dataset, the effect of the resultant lba does not impact on the sequence data to any great extent. selective pressures for the reproductive proteins studied here are heterogeneous. all proteins exhibited regions of strong conservation proving the importance of maintaining structural stability and overall function in these proteins. all but protein exhibited evidence of positive selection in specific lineages, and all proteins without exception exhibited positive selection in regions of catalytic/functional importance. for sp and col1a the site-specific results are entirely novel. the lineage-specific results described here for prkar2a and catsper exon in primates, are also novel. we have shown that, in the case of catsper there is a fundamental protein functional shift between new world monkeys and old world monkeys. the dn/ds measurement applied here assumes that neutral substitution rate is akin to ds, therefore no selection on silent sites. there have been many publications of late to the contrary therefore we are mindful of examining the rate of silent substitution in all our analyses. for the reproductive genes in our dataset, we show that lineages evolve at unique rates and at functionally crucial sites, specifically those involved in phosphorylation. we have also shown that a number of these proteins show positive selection for example in the ancestral rodent lineage and evidence of purifying selection in the subsequent divergent species. overall our analyses of these reproductive proteins show how important it is to carefully examine data for systematic biases prior to testing for lineage and/or site specific positive selection. we have also demonstrated the importance of including large numbers of taxa/lineages in these analyses. this finding was highlighted in our analysis of prkar2a where previous analysis of this protein had included only taxa and therefore reported a negative result. we do not observe any large-scale effect of germ line generation time in our dataset, with only protein with evidence of long branch attraction. the results of col1a indicate that the positively selected sites may have been of such importance for this protein that neighboring mutated sites may have been maintained in the population despite their propensity for causing disease. the location of positively selected sites determined using this approach and in regions of functional importance in the proteins in this dataset, provides us with further evidence of the link between functional shift and positive selection. 
however, the mammalia are a diverse group in terms of mating habits, population sizes and germ line generation times. reproductive proteins are central to the continuation of all mammalian species. we have examined the selective pressures at work on a number of novel reproductive proteins across a wide variety of mammalia. we show that selective pressures on reproductive proteins are highly varied. this work has highlighted the widespread rate heterogeneity in mutational rates across the mammalia and specifically has shown that the evolution of reproductive proteins is highly varied depending on the species and interacting partners. our analysis of sp and col1a are entirely novel and the results show positively selected sites present in each gene. of the genes analyzed in detail, all contain signatures of positive selection either across specific sites or in specific lineages or a combination of both. positive selection has been demonstrated in many of these proteins with particular focus on primate lineages. our findings for the col1a gene are suggestive of a link between positive selection and severe disease type. we have shown that positive selection and disease are closely linked in the col1a gene. our in silico analyses show positively selected sites are occurring near catalytically important regions suggesting selective pressure to maximize efficient fertilization.
 reproductive proteins are central to the continuation of all mammalian species. the evolution of these proteins has been greatly influenced by environmental pressures induced by pathogens, rival sperm, sexual selection and sexual conflict. positive selection has been demonstrated in many of these proteins with particular focus on primate lineages. however, the mammalia are a diverse group in terms of mating habits, population sizes and germ line generation times. we have examined the selective pressures at work on a number of novel reproductive proteins across a wide variety of mammalia.  we show that selective pressures on reproductive proteins are highly varied. of the genes analyzed in detail, all contain signatures of positive selection either across specific sites or in specific lineages or a combination of both. our analysis of sp and col1a are entirely novel and the results show positively selected sites present in each gene. our findings for the col1a gene are suggestive of a link between positive selection and severe disease type. we find evidence in our dataset to suggest that interacting proteins are evolving in symphony: most likely to maintain interacting functionality.  our in silico analyses show positively selected sites are occurring near catalytically important regions suggesting selective pressure to maximize efficient fertilization. in those cases where a mechanism of protein function is not fully understood, the sites presented here represent ideal candidates for mutational study. this work has highlighted the widespread rate heterogeneity in mutational rates across the mammalia and specifically has shown that the evolution of reproductive proteins is highly varied depending on the species and interacting partners. we have shown that positive selection and disease are closely linked in the col1a gene. 