extrafloral nectaries ( efns ) are nectarsecreting glands located outside of flowers and have been observed on a huge diversity of species , spanning over 93 families and 332 genera ( koptur 1992a , b ; marazzi et  al .
1978 ; becerra and venable 1989 ; wagner and kay 2002 ; heil 2011 ) , but primarily they are known to provide indirect defense against herbivores by attracting predatory insects , predominantly ants ( bentley 1977 ; koptur 1992a , b ; rosumek et  al .
2009 ; heil 2015 ) . despite their unquestionable importance , relatively little is known about the factors that regulate efn production .
uncovering these factors can help us understand how plants regulate their investment in defense , and how they manage and maintain interactions with beneficial insects .
light conditions are likely to be particularly influential in controlling the expression of plant defensive traits , as light not only represents a crucial aspect of resource availability , but may also serve as an indicator of insect activity ( karban et  al .
plants are well known to sense changes in spectral signals ( e.g. , weller et  al . 1997 ; adams et  al .
farred light , for example , is a component of the solar spectrum ( 710850  nm ) that is heavily reflected by plant tissues ( izaguirre et  al .
increases in farred radiation , relative to the red portion of sunlight ( 620750  nm ) , can be detected through the photoreceptor , phytochrome b ( ballare et  al . 1990 ; ballare 2014 ) .
a low red / farred ( r / fr ) light ratio is known to indicate the close proximity of competitors for many plants ( ballare 1999 , 2014 ) .
plants exposed to low r / fr light conditions often express a suite of competitionfocused traits collectively known as the shadeavoidance syndrome ( ballare 1999 , 2014 ; pierik et  al .
responses associated with the shadeavoidance syndrome include increased stem elongation , reduced lateral branching , and a reduction in resource allocation to defensive traits ( izaguirre et  al .
the expression of several direct plant defenses such as phenolic compounds ( moreno et  al .
2009 ) , and latex ( rasmann et  al . 2009 ; agrawal et  al . 2012 ) , is known to be reduced in low r / fr light .
far less is known about the effects of light conditions on the expression of indirect plant defenses such as efn .
light intensity is known to affect tradeoffs between indirect defenses in mallotus japonicus ( yamawo and hada 2010 ) ; however , only the effects of light intensity , and not light quality , were observed and so we know little about how these plants may respond to informational light signals . in lima beans ( phaseolus lunatus ) , the induction of efn production with jasmonic acid ( ja ) has been shown to be dependent on light intensity , and on the ratio of r / fr wavelengths ( radhika et  al .
( 2013 ) also observed efn production in passion fruit ( passiflora edulis ) in carefully manipulated light conditions .
plants ( and plant parts ) exposed to low r / fr light conditions exhibited reduced efn production , compared to those exposed to higher r / fr light ratios , particularly in response to simulated herbivory .
pine rockland habitats contain a high proportion of efnbearing plants ( 27% ) ( koptur 1992a , b ) , but the dynamics of efn production in these species have rarely been studied ( but see rutter and rausher 2004 ; jones and koptur 2015 ) .
chapmanii ( hereafter referred to as senna chapmanii ) is an herbaceous legume native to the pine rocklands of south florida and the caribbean .
we have already shown that s.  chapmanii plants produce more efn in response to leaf damage ( jones and koptur 2015 ) . we have also observed that plants in shady conditions are less well defended by ants than those in direct sunlight ( i. m. jones , s. koptur , j. tardanico , h. gallegos and p. trainer , unpubl .
data ) . in this study , we investigated the effects of light intensity and r / fr light ratio on efn production in s.  chapmanii . both artificially defoliated and undamaged plants were tested .
we expected efn production would be increased in response to leaf damage and high light intensity , but reduced in response to low r / fr light ratios .
understanding the factors that control efn production is important , because such variations affect the number and identity of insect visitors , and the effectiveness of plant defense ( fig .  1 ) .
to control s.  chapmanii light environments , film cylinders ( 50  cm in circumference and 60  cm in height ) were constructed using three calibrated light filtration films .
film 1 ( treatment film ) was a metal sputtercoated film designed to mimic shading by other plants by reducing photosynthetically active radiation ( par ) by approximately 80% , and reducing the ratio of r / fr light .
film 2 ( control film ) , a dyeimpregnated film , was designed to reduce par by approximately 80% , but without impacting r : fr light ratio .
paul , mn , 55144 , usa ) and have been used previously to test the effects of irradiance and spectral quality on forest tree seedling development ( lee et  al .
film 3 was a clear acetate film which allowed approximately 90% par transmission and had no impact on r : fr light ratio .
film 3 was supplied by blick art materials ( galesburg , il , 61402 , usa ) , and controlled for the effects of the cylinders themselves .
twentyfour film cylinders ( 8 of each type ) were placed on a glasshouse bench .
the open bottom of each cylinder was placed around the plant pot , and the top end was sealed closed using clear tape .
cylinders composed of the three film types were placed alternately in three rows running east to west ( fig .  2 ) .
the letter d indicates plants subjected to leaf damage . to determine the actual light environments within the film cylinders ,
the intensity and spectral distribution of light within the glasshouse were measured using a radiospectrometer ( unispecdc , pp systems , amesbury , ma , usa ) .
these measurements were then compared with measurements taken inside the film cylinders 1 , 2 , and 3 .
percentage transmittance of light through each film type , at a range of wavelengths ( 3001000  nm ) , was then calculated .
mean percentage of light of different wavelengths ( 3001000  nm ) transmitted through the three filter types .
light gray bands indicate red and farred light wavelengths , while the dark gray band indicates crossover between the two .
the sharp rise in percentage light transmission in film 1 , starting at around 710  nm , indicates the desired increase in r : fr light ratio within film 1 cylinders . 

senna chapmanii was grown from seeds in the glasshouse on the modesto maidique campus at florida international university .
after 3  weeks , seedlings were transplanted into 0.6l pots , and 1.5  g of slow release fertilizer ( nutricote npk ; florikan , sarasota , fl , usa ) was added to each plant .
after 48  h inside the cylinders , 12 plants ( 4 from each cylinder type ) were subjected to 50% leaf damage .
the same damage treatments were used in a previous study ( jones and koptur 2015 ) and induced a highly significant increase in efn production in s.  chapmanii .
extrafloral nectar production , by each plant , was measured 12 and 24  h after leaf damage as the increase in efn production by s.  chapmanii in response to leaf damage has been shown to be greatest during this period ( jones and koptur 2015 ) .
leaf damage was inflicted at 7  am , so nectar measurements took place at 7  pm on the day of leaf damage , and at 7  am the following morning .
taking measurements in the morning , and at night , allowed us to calculate a mean efn production for each plant , controlling for natural diurnal variations in efn production .
nectar volume was measured using 1 , 2 , and 10  l micropipettes , and its concentration determined using a handheld refractometer .
total sugar production by each plant was then calculated as described by jones and koptur ( 2015 ) .
we report efn production as the mean mass of sugar ( mg ) produced by each plant , as this provides the best representation of defensive investment .
previous studies have often reported only nectar volume or concentration , both of which are affected by environmental factors such as temperature and humidity . where we refer to efn production in the discussion
wallis htests were used to compare efn production between the three light treatments in damaged and undamaged plants .
kruskal  wallis htests were used to compare efn production between the three light treatments in damaged and undamaged plants .
holm 's sequential bonferroni adjustments were applied to control for type 1 errors . damaged and undamaged plants within each light treatment
radiospectrometer readings confirmed that light conditions inside the cylinders were as expected ( fig .  3 ) .
film 1 admitted between 10% and 30% of light in photosynthetically active wavelengths , but transmittance rose to almost 90% in the farred wavelength band .
film 1 therefore adequately mimicked shading by other plants , as compared to film 2 , which admitted around 20% of light in photosynthetically active wavelengths , with transmittance rising only slightly in the farred band .
film 3 , the clear film , admitted around 90% of light across all wavelengths ( fig .  3 ) . in all three light treatments , damaged plants produced more efn than undamaged plants ( film 1 : z  =  2.492 , df  =  46 , p  =  0.013 ; film 2 : z  =  2.474 , df  =  46 , p  =  0.013 ; film 3 : z  =  2.062 , df  =  46 , p  =  0.039 ) .
light treatments significantly affected efn production in both damaged (   =  18.355 , df  =  2 , p  <  0.001 ) and undamaged plants (   =  23.014 , df  =  2 , p  <  0.001 ) ( fig .  4 ) . mean sugar production ( mg ) by damaged and undamaged plants subjected to three light treatments .
letters indicate significant differences . among damaged plants , those in clear tubes ( film 3 ) produced significantly more efn than those in 70% shade with reduced r / fr light ratio ( z  =  3.843 , df  =  46 , p  <  0.001 ) , and those in 70% shade ( z  =  3.350 , df  =  46 , p  =  0.001 ) .
there was no difference in sugar production between plants in 70% shade with reduced r / fr light ratio , and those in 70% shade ( z  =  1.012 , df  =  46 , p  =  0.311 ) ( fig .  4 ) . among undamaged plants , those in clear tubes ( film 3 ) produced significantly more efn than those
is 70% shade with reduced r / fr light ratio ( z  =  4.245 , df  =  46 , p  <  0.001 ) , and those in 70% shade ( z  =  3.343 , df  =  46 , p  =  0.001 ) .
there was no difference in sugar production between plants in 70% shade with reduced r / fr light ratio , and those in 70% shade ( z  =  1.343 , df  =  46 , p  =  0.179 ) ( fig . 
extrafloral nectar is an extremely widespread , often inducible trait that mediates foodforprotection interactions between plants and ants . although the ecological role of efn is well established ( bentley 1977 ; koptur 1992a , b ; rosumek et  al .
2009 ) , far less is known about how changes in environmental conditions , even over small scales , may affect its production and , therefore , the outcomes of the interactions it mediates . plants in all three light treatments produced more efn in response to leaf damage .
inducible efn nectar production has been reported in many species ( stephenson 1982 ; koptur 1989 ; agrawal and rutter 1998 ; engel et  al .
2001 ; heil et  al . 2001 ; mondor and addicott 2003 ; choh and takabayashi 2006 ; lach et  al . 2009
indeed , increased efn production in response to leaf damage has been observed previously in s.  chapmanii ( jones and koptur 2015 ) .
we observe for the first time , however , that this induced response is maintained in lightlimited conditions , albeit at a lower level .
it should be noted that , in the present study , we observed the effects of mechanical leaf damage and not true herbivory .
plants have been observed to respond to the oral secretions of specific herbivores ( kessler et  al . 2010 ) , and responses to damage have been shown to vary based on herbivore feeding guild ( schmidt et  al .
light intensity had a significant impact on efn production , as both damaged and undamaged plants produced more efn at high light intensities .
it might seem intuitive that a reduction in the availability of photosynthetically active light would reduce the level of resources available for defense .
indeed , some socalled green nectaries may be isolated from phloem tissue and produce nectar only at a rate that can be supported by their own photosynthesis ( lttge 2013 ) . the resource availability hypothesis ( rah ) , however , suggests that lownutrient environments may promote greater investment in defensive traits , compared to nutrientrich environments where plants must grow quickly in order to compete ( coley et  al .
the relationship between nutrient availability and defense is therefore complex , and assumptions that increased resources should lead to a greater investment in defense may be misplaced .
the carbon  nutrient balance hypothesis ( cnbh ) suggests that when a given resource limits plant growth , other resources , found in relative excess , may be allocated toward defenses ( bryant et  al .
for example , in lightrich and nutrientpoor conditions , plants are expected to invest in carbonbased defenses , while in shaded but nutrientrich conditions , plants should invest more heavily in nitrogenbased defenses ( lerdau and coley 2002 ) .
yamawo and hada ( 2010 ) found that light intensity affected the tradeoff between two indirect defenses in mallotus japonicus . in low light conditions , both the size and the productivity of efns
these results seem to support cnbh , as pearl bodies are rich in protein and represent a significant nitrogen investment ( heil et  al .
our observations that efn production in s.  chapmanii is reduced in low light conditions seem to reflect resource availability and contradict rah .
however , it remains possible that reduced efn production may coincide with an increased investment in nitrogenbased defensive compounds such as alkaloids .
future studies should seek to understand these dynamics , in s.  chapmanii and other species .
the simplest and most elegant explanation for the observed pattern of efn production may come from millncaongo et  al .
( 2014 ) , who observed reduced efn production in shaded leaves of ricinus communis .
changes in efn production were shown to be mediated by cell wall invertase , an enzyme involved in the unloading of sucrose from the phloem into the nectary .
light conditions did not appear to affect cell wall invertase activity , so it was suggested that reduced efn production was a result of lower sucrose content available in the phloem ( millncaongo et  al . 2014 ) .
changes in r / fr light ratio had no effect on efn production in s.  chapmanii .
plants exposed to low r / fr light ratios produced slightly less efn in both damaged and undamaged plants , but the differences were not significant .
these results were surprising as light spectral quality has been observed to affect efn production significantly in both lima beans ( radhika et  al .
it is possible that our shade treatments reduced overall light intensity to such an extent that the effects of light spectral quality were tempered .
( 2010 ) showed that r / fr light ratio affected jainduced efn production even at low light intensities .
our results suggest that s.  chapmanii downregulates indirect defenses in response to shade , but that it does not do so in response to specific spectral signals that indicate competition .
this study contributes to an improved understanding of plant resource allocation , and the dynamics of defensive traits .
spatiotemporal patterns of efn production are often adapted to optimize plant defense ( tilman 1978 ; heil 2015 ) .
these patterns , however , are driven by simple physiological mechanisms that respond to environmental conditions ( heil 2015 ) .
we add to a growing understanding of how changing environmental conditions affect indirect plant defenses , and the interactions that they support .
understanding how efn production responds to changing light conditions is of particular importance in pine rockland habitats , where roughly 27% of plants bear efns ( koptur 1992b ) . over the last century , roughly 98% of pine rockland habitat in south florida ( with the exception of everglades national park ) has been destroyed for agriculture and urban development ( barrios et  al .
in particular , the fires that are necessary to maintain healthy pine rocklands are often suppressed ( possley et  al .
pine rockland habitats are characterized by an open canopy , with high levels of light reaching the speciesrich herb layer . in the absence of fire ,
trees and shrubs quickly become overgrown , and understory plants are shaded . with this experiment
, we hoped to create a clearer understanding of how changing light conditions in the pine rocklands might affect insect 
plant interactions , and the fitness of plants that rely on these interactions for defense .
 abstractextrafloral nectar ( efn ) mediates foodforprotection mutualisms between plants and insects and provides plants with a form of indirect defense against herbivory . 
 understanding sources of variation in efn production is important because such variations affect the number and identity of insect visitors and the effectiveness of plant defense . 
 light represents a potentially crucial tool for regulating resource allocation to defense , as it not only contributes energy but may help plants to anticipate future conditions . 
 low red / farred ( r / fr ) light ratios can act as a signal of the proximity of competing plants . 
 exposure to such light ratios has been shown to promote competitive behaviors that coincide with reduced resource allocation to direct chemical defenses . 
 little is known , however , about how such informational light signals might affect indirect defenses such as efn , and the interactions that they mediate . through controlled glasshouse experiments , we investigated the effects of light intensity , and r / fr light ratios , on efn production in senna mexicana var . 
 chapmanii . 
 plants in lightlimited conditions produced significantly less efn , and leaf damage elicited increased efn production regardless of light conditions . ratios of r / fr light , however , did not appear to affect efn production in either damaged or undamaged plants . understanding the effects of light on indirect defenses 
 is of particular importance for plants in the threatened pine rockland habitats of south florida , where light conditions are changing in predictable ways following extensive fragmentation and subsequent mismanagement . around 27% of species in these habitats 
 produce efn and may rely on insect communities for defense . 
Introduction
Methods
Statistical analysis
Results
Discussion
Conflict of Interest