polyandry , multiple mating by females , occurs frequently in animals . by mating with several males , females gain benefits such as reduced harassment ( wilcox 1984 ) , nuptial gifts ( zeh and smith 1985 ) , fertilization insurance ( ridley 1983 ) , or genetic variety among offspring ( keller and reeve 1994 ) .
however , it is generally believed that the proportion of eggs fertilized due to polyandry , is not in the in the interests of a male .
indeed , males have evolved numerous tactics preventing females from re - mating , from mate guarding ( birkhead and parker 1997 ) , to mating plugs ( devine 1977 ) .
hymenopteran social insects ( ants , bees and wasps ) are a notable exception to the general prevalence of polyandry , with most females mating just once and using sperm from that copulation for the rest of their lives .
to date polyandry has been documented in only about a half - dozen genera of ants , bees and wasps ( boomsma and sundstrm 1998 , strassmann 2001 ) .
although their mates do not provide nuptial gifts or parental care , as they die shortly after mating , social insect females are nonetheless expected to benefit from polyandry . in particular
, females may either acquire a potentially advantageous mixture of genes for their offspring ( page and metcalff 1982 ; crozier and page 1985 ; sherman et al .
1988 ; keller and reeve 1994 ) , obtain a quantity of sperm necessary for long term production of workers and large colony sizes ( cole 1983 ) or reduce kin conflict with daughters over sex ratios and male parentage ( starr 1984 ; moritz 1985 ) .
indeed , genetic advantages of polyandry have been demonstrated both in naturally polyandrous harvester ants ( cole and wiernasz 1999 ) and , by artificial insemination , in singly mating bumblebees ( baer and schmid - hempel 1999 ) .
mating frequency in social insects determines the relatedness of workers and thus the genetic structure of the colony , which affects numerous aspects of behavior .
for example , changes in relatedness brought upon by polyandry may be involved in resolving conflicts over male parentage and offspring sex ratios ( ratnieks 1988 ; boomsma and grafen 1990 ) .
accordingly , knowledge of the forces underlying the evolution of multiple mating by queens is fundamental to our understanding of social evolution .
the interests of males have been largely ignored when considering social insect polyandry , probably because of the secondary roles males appear to play in these societies , but see hlldobler and bartz ( 1985 ) and boomsma and sundstrm ( 2000 ) , for alternative perspectives .
however , secretions of male accessory glands ( mags ) , which attenuate re - mating by females , have been documented in numerous non - social insects ( chen 1984 ) .
these plugs , which consist principally of a mixture of four fatty acids , were found to inhibit re - mating by females ( baer et al .
male fire ants transfer at least two of the same four fatty acids to females during mating ( mikheyev , 2003 ) suggesting that re - mating suppression of polyandry may occur in many hymenopteran social insects ( sauter et al .
2001 ) , and may involve the same chemical mechanism as the bumblebees . the fungus gardening ants have been the focus of several phylogenetic ( schultz and meier 1995 , wetterer et al .
1998 ) and paternity studies ( kerr 1961 , moser 1967 , corso and serzedello 1981 , villesen et al .
while the basal species are singly mating , leaf cutters in the genera atta and acromyrmex , made a single transition to polyandry ( villesen et al .
thus , these ants provide a means of examining the possible association between mags and the evolution of polyandry . specifically ,
if mag secretions in some way affect female mating frequency , then the evolutionary transition from monandry to polyandry would likely be correlated with morphological changes in the glands themselves .
males from seven attine genera ( seventeen species ) were dissected under magnification , their internal reproductive systems were photographed and the volumes of their male accessory glands ( mags ) and seminal vesicles measured by approximating them as either cylinders or spheroids .
in addition , a photograph of the male reproductive system ( hung and vinson 1975 ) of the polyandrous ( moser 1967 ) at .
males of the fire ant solenopsis invicta , a known singly mated species ( ross and fletcher 1985 ) with a well - studied reproductive system ( ball and vinson 1984 ) , were dissected and included as an outgroup .
s. invicta , which belongs in the same subfamily as the attines ( myrmicinae ) , illustrates the internal reproductive morphology common to most ants studied previously ( beck 1972 ; hung and vinson 1975 ; wheeler and krutzsch 1992 ) , which appears to be the primitive condition in ants . the seminal vesicles and mags of the multiply mating genera acromyrmex and atta , and a few singly mates species ,
the mags and seminal vesicles were dissected out , fixed for 1624 h with either bouins solution or z-5 , then dehydrated with a succession of 95% ethanol ( 15 min ) , 100% ethanol ( 15 min ) , methyl salicylate ( 30 min ) and two changes of xylene .
the tissues were then infiltrated with two changes of paraplast plus ( fisher scientific , www.fishersci.com/ ) mixed with a few drops of honeybee wax ( 1 hour each infiltration ) .
tissues were sectioned at 10 m , mounted on slides and dried overnight . after being deparaffinized and hydrated with distilled water ,
the slides were stained with hematoxylin ( 30 sec ) and eosin - y ( 15 sec ) .
the estimates of mag volumes for acromyrmex and atta species were made from stained sections mounted on slides . in this manner sperm
could be clearly visible in the seminal vesicles , while mag contents stained a solid red .
a male 's gaster contains the bare minimum of life support organs with the rest being occupied by the reproductive structures ; mags and the seminal vesicles .
as there appeared to be a trade - off between allocating space to mags versus seminal vesicles , the relative changes in their morphology were expressed as a ratio of mag volume over seminal vesicle volume . to eliminate any potential problems caused by phylogenetic non - independence due to unequal genetic distance between species , phylogenetic information derived from ( wetterer et al .
2002 ) was incorporated to compute a set of phylogenetically independent contrasts ( felsenstein 1985 ) using the contrast module of phylip ( version 3.5 ) .
the loss or extreme reduction of mags exactly corresponds with the appearance of polyandry in the fungus gardening ants .
while the mags of the basal attines and fire ants are prominent , with conspicuous lobes protruding laterally off the ejaculatory duct , atta possess minute accessory glands , relative to the size off their seminal vesicles , and acromyrmex appear to lack mags entirely .
figure 1 shows the presence or absence of large mags mapped onto the attine phylogeny ( schultz and meier 1995 , wetterer et al .
the reproductive system of attine males , excluding the leaf cutter clade , is remarkably similar to that of fire ants and to that of most ants studied to date in that they all possess slender seminal vesicles and prominent lobed mags . in the singly mating species ,
however , a major transition in internal morphology occurs in the leaf cutting genera with the extreme reduction of mags in atta and their complete disappearance in acromyrmex .
figure 2 illustrates the inverse relationship between the volumes of mags relative to seminal vesicle volume and a species ' effective paternity , which is a measure of the genetic diversity within a colony due to multiple matings .
the effective mating frequency controls for unequal sperm usage by weighing the relative contribution to fertilizations of the different mates of the queen .
the phylogenetically independent contrasts of relative mag volume and effective paternity were negatively correlated ( r=0.77 , n=6 ) for all the species listed in figure 2 , except ac .
the change in mag volume , which decreases in multiply mating species , suggests that these glands may be involved in regulating multiple mating by females .
ideally , behavioral assays using instrumentally inseminated females should be used to test the function of mags , as has been done in bumblebees ( baer et al .
however , to the best of my knowledge , no attine ant has ever been instrumentally inseminated or mated in a laboratory setting .
thus , for now , morphological correlations , somewhat corroborated by evidence for fatty acid transfer from fire ant mags ( mikheyev , 2003 ) , provide the best , though inconclusive evidence that mags regulate multiple mating in attine ants
. however , as is the case for all correlative evidence , these observations are consistent with a number of other alternative hypotheses .
for instance , mags could be the source of nuptial gifts , which are no longer offered by males of acromyrmex and atta due to some unknown change in the direction of sexual selection accompanying polyandry .
( 2001 ) argued that the bumblebees ' mating plug is too small to provide measurable nutritional support and the fire ants secretion also appears miniscule ( mikheyev , 2003 ) , there are no data on size or composition of mag secretions in the attine ants and this hypothesis can not be excluded .
likewise , mag secretions may be used for intersexual signaling that happens differently in singly and multiply mated species .
additionally , the evolution polyandry and subsequent sperm competition may have reduced the adaptive value of large mags relative to the need for large seminal vesicles .
assuming that mag secretions do enforce monandry , we can speculate about forces causing the evolution of polyandry .
for instance , polyandry may have evolved when higher attine females , through an unknown mechanism , were able to overcome the male 's manipulative strategy .
if so , males would not be expected reduce mag size , but to pursue a coevolutionary arms race , perhaps even leading to mag hypertrophy .
more likely , the evolution of polyandry in the fungus gardeners was driven not solely by female preference for multiple mating but by selection on the males themselves . in theory ,
polyandry ( i.e. increased genetic diversity ) amplifies the fitness of a multiply mated colony , relative to a singly mated colony ( page and metcalff 1982 ; sherman et al . 1988 ) .
if a queen 's multiple mating confers a colony - level fitness boost sufficient to overcompensate her mates for effects of genetic dilution due to polyandry , males will be selected to stop monopolizing females .
the beneficial effects of polyandry in harvester ants described by cole and wiernasz ( 1999 ) appear sufficient to give the necessary fitness amplification , especially considering the high rates of mortality among the more genetically homogeneous colonies . given the relative rarity of polyandry in the social insects ,
the inhibition of female re - mating via mag secretions may be a widespread phenomenon in insects ( chen 1984 ) .
the high relatedness within social insect colonies , which has been proposed for the evolution of eusociality through kin selection ( hamilton 1964 ) , presupposes monandry .
thus , mag - enforced single mating by females in some insect groups may be yet another factor facilitating the evolution of complex kin - based social systems .
the transition indicated on the phylogeny corresponds to both the evolution of polyandry , as well as to the reduction of mags in the monophyletic leafcutter clade including acromyrmex and atta .
representative dissections of males , illustrating the primitive and derived morphologies , are shown alongside the phylogeny .
an outgroup , the fire ants ( solenopsis ) has been included for an informal comparison .
relationship between relative mag volume and the number of matings by queens of attine ants ( strassmann 2001 , villesen et al .
the polyandrous species ( acromyrmex and atta ) have much smaller mags relative to their seminal vesicles than the monandrous genera ( apterostigma , cyphomyrmex and sericomyrmex ) .
 polyandry in hymenopteran social insects is surprisingly rare , despite its likely colony - level fitness benefits . 
 ordinarily , a male 's fitness will be at odds with that of a colony when the genetic representation within it is diluted by multiple mating by the queen . 
 consequently , males are expected to be under selection to limit female re - mating , for example via secretions of their accessory glands . 
 i hypothesized that if accessory glands in some way regulate female mating frequency , an evolutionary transition from single to multiple mating would likely be accompanied with a change in the morphology of the accessory glands . 
 the accessory gland morphology was examined in the fungus - gardening ants , which have made a single transition from single to multiple mating . 
 the evolution of polyandry within this clade corresponds to the loss of male accessory glands , lending tentative support to the idea that they may be involved in regulating mating frequency.abbreviation:magmale accessory gland 
Introduction
Materials and Methods
Statistical analysis
Results
Discussion
Figures and Tables