Genetic and potential non-genetic benefits increase offspring fitness of polyandrous females in non-resource based mating system

RESEARCH ARTICLE Open Access Gene ic and po en ial non-gene ic bene i s inc ease o sp ing i ness o polyand ous emales in non- esou ce based ma ing sys em Jukka Kekäläinen 1,2* , Gei Rudol sen 3 , Ma i Janhunen 4 , La s Figenschou 3 , Nina Peuhku i 5 , Niina Tampe 6 , Raine Ko e 6,7 Abs ac Backg ound: The adap i e signi icance o emale polyand y is cu en ly unde conside able deba e. In non- esou ce based ma ing sys ems, indi ec , i.e. gene ic bene i s ha e been p oposed o be esponsible o he i ness gain om polyand y. We s udied he bene i s o polyand y in he A c ic cha (Sal elinus alpinus) using an expe imen al design in which he ma e ial in es men s by he si es and ma e nal en i onmen al e ec s we e con olled. Resul s: Emb yonic mo ali y showed a s ong pa e nal gene ic componen , and i was lowe in polyand ously e ilized o sp ing (spe m compe i ion o wo males) han in monand ous e iliza ions. We also ound ha high spe m eloci y was associa ed wi h low o sp ing mo ali y, bu no wi h he size o he o sp ing o hei yolk olume. Al hough no male e ec was ound on he size o he o sp ing yolk ese es, yolk olume was highe in o sp ing om polyand ous ma ings han o sp ing o he ei he o he wo males when ma ed monand ously. Conclusions: In suppo o he “good spe m hypo hesis, we ound ha spe m eloci y was posi i ely associa ed wi h o sp ing i ness. In addi ion, ou esul s sugges ha polyand ous emales gain gene ic ad an age (highe o sp ing su i al) om his beha io , bu ha some bene i s o polyand y (la ge yolk olume) may no be explained solely by he addi i e gene ic e ec s. This sugges s ha spe m compe i ion en i onmen may in ensi y he selec ion on gene ically supe io spe m which in u n may p oduce o sp ing ha ha e supe io yolk ese es. Howe e , as high spe m eloci y was no associa ed wi h la ge yolk olume, i is possible ha also some o he non-gene ic e ec s may con ibu e o o sp ing i ness. The po en ial ole o polyand ous ma ing in inb eeding a oidance is discussed. Backg ound Ma ing wi h se e al males (polyand y) [1-9] can incu a ious cos s o he emales [10]. Mos ly o his eason, he adap i e signi icance o his beha iou has been unde conside able deba e. When emales gain no di ec ma e ial bene i s om ma ing, indi ec (i.e. gene ic) ad an ages a e belie ed o explain he e olu ion o poly- and y [8,11-15] (see also [16]). By ma ing wi h many males, emales may inc ease he p obabili y o hei eggs becoming e ilized by he spe m o gene ically supe io o compa ible male [3,17]. Thus, ce ain genes o gene combina ions should esul in polyand ous emales p o- ducing o sp ing o highe i ness han monand ous emales. “Good spe m”hypo hesis p edic s ha spe m ha a e mo e success ul in compe i ion o e ilising he eggs a ealsomo ee ec i einp oducing iableo sp ing.In o he wo ds, a male’s spe m compe i ion abili y should co ela e wi h he i ness o his o sp ing [18-21]. P e- ious s udies ha ha e demons a ed gene ic bene i s o polyand y ha e o en been con ounded by di e en ial ma e nal in es men s ( e iewed by [8]). Acco dingly, he empi ical e idence suppo ing he “good spe m”hypo h- esis is sca ce [22-24]. Hosken e al.[24] ound ha males ha we e mo e success ul in spe m compe i ion had o sp ing ha de eloped as e han o sp ing o * Co espondence: [email p o ec ed]i 1 Depa men o Biological and En i onmen al Science, Uni e si y o Jy äskylä, P.O. Box 35 (YAC-315.2), FI-40014 Uni e si y o Jy äskylä, Jy äskylä, Finland Kekäläinen e al.BMC E olu iona y Biology 2010, 10:20 h p://www.biomedcen al.com/1471-2148/10/20 © 2010 Kekäläinen e al; licensee BioMed Cen al L d. This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License (h p://c ea i ecommons.o g/licenses/by/2.0), which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed. males wi h lowe quali y spe m. Howe e , no associa ion be ween o sp ing mo ali y and he compe i i e abili y o spe m was de ec ed. To he bes o ou knowledge, only one s udy, wi h he Aus alian ma supial (An echi- nus s ua ii) has demons a ed ha males gaining high pa e ni y unde spe m compe i ion (i.e. good spe m compe i o s) also si e o sp ing wi h lowe mo ali y a e [25]. Al hough he gene ic emale bene i s o polyand y ha e been demons a ed in nume ous s udies, some ecen s udies ha e also shown ha he bene i s o poly- and y may no always be ansmi ed h ough con en- ional addi i e gene ic pa hways [15,26,27] (see also [28,29]). In addi ion, bene i s o polyand y may depend on ce ain emale cha ac e is ics (e.g. condi ion), which sugges ha gene ic bene i s a e no necessa ily equal o all indi iduals [15]. In many o ipa ous ish wi h no pa en al ca e, s a a- ion du ing he c i ical ansi ion pe iod om endogen- ous eeding o independen o aging is among he mos impo an causes o mo ali y in he cou se o he on o- geny (e.g. [30-33]). Thus, he de elopmen o newly ha ched la ae is highly dependen on yolk ese es [32], la ge ese es gi ing mo e ime o ini ia e ex e nal eeding be o e exhaus ion [34]. In gene al, he amoun o yolk may be c i ical o he su i al o he o sp ing [35]. The size o he egg and yolk has o en been consid- e ed solely as a emale dependen ac o and he possi- ble male e ec s ha e o en been neglec ed [36]. Howe e , he male may con ibu e o egg size a e e i- liza ion by a ec ing he wa e up ake o he eggs [36] (see also [37,38]). In addi ion, males can also a ec o hesizeo heo sp ing(o yolk) indi ec ly, h ough me abolic a e o he emb yo [38] (see also [39]). Hence, males could indi ec ly con ibu e o p ogeny quali y and su i al, e en in species ha p o ide no pa en al ca e. TheA c iccha (Sal elinus alpinus) has a lek-like b eeding sys em, whe e bo h sexes ma e mul iply. Du - ing egg elease one la ge male is usually in close con ac wi h he emale [40] and gene ally e ilizes he g ea es p opo ion o he eggs [41]. Howe e , se e al smalle males a e usually also p esen in he icini y o he emale and hese sneaking males e ilize a p opo ion o he eggs leading o in ense spe m compe i ion be ween males[42].Thus, wo ypeso polyand ousma ing occu : Fi s , emales ma e wi h di e en males in di e - en spawning ac s, and second, se e al males compe e o e ilizing eggs wi hin single ac . As he “good spe m”hypo hesis p edic s, spe m elo- ci y is he mos impo an ac o p edic ing he pa e ni y o hemaleA c iccha ,andalsoino he salmonids [43,44] (bu see [45]). On he o he hand, o a ian luid a ec s he eloci y and longe i y o cha spe m [46], indica ing ha c yp ic emale choice may be an impo - an mechanism a ec ing he e iliza ion success o males [47,48]. Females may c yp ically selec o males ha ha e op imal deg ee o gene ic simila i y (o dissim- ila i y) wi h he emale [8,49,50]. This indica es ha polyand ous ma ing and spe m compe i ion could be adap a ions o a oid inb eeding. Thus, bo h male spe m cha ac e is ics and c yp ic emale choice o good (o compa ible genes) may be impo an mechanisms p e- dic ing o sp ing i ness in A c ic cha (bu see [51,52] o da a on MHC). We s udied he bene i s o polyand y by using he A c ic cha as a model species. Two main ques ions we e: 1) Wha kind o bene i s do emales gain om polyand ous ma ing unde spe m compe i ion ea - men ? 2) Is spe m quali y associa ed wi h highe o - sp ing quali y as he “good spe m”hypo hesis p edic s? To es hese hypo heses we used a ma e nal hal -sib b eeding design, whe e con ounding ma e nal in es - men s and si e en i onmen al e ec s we e con olled. Resul s The i ness bene i s o spe m compe i ion O sp ing mo ali y did no di e among he h ee male ea men s (ANOVA, p= 0.17, Figu e 1a, Table 1), bu he e ec o emale ( o al mo ali y pe cen ages 15.1, 48.3 and 87.6%) and he in e ac ion be ween male ea - men (spli plo s; small, la ge o spe m compe i ion) and male combina ion (whole plo s) we e s a is ically signi i- can (ANOVA, p< 0.001 in bo h cases). When he sin- gle male ea men s we e combined and es ed agains he spe m compe i ion ea men , he mo ali y o o - sp ing was lowe in spe m compe i ion ials (ANOVA, F 1,9 = 11.36, p= 0.01) and he e ec s o emale as well as male combina ion we e also signi ican (ANOVA, F 2,18 = 498.75, p< 0.001 and F 9,11.5 =3.11,p= 0.04). Again, he e was a s a is ically signi ican in e ac ion be ween male ea men (spe m compe i ion s. single) and male combina ion (ANOVA, F 9,20 =2.92,p=0.02). These esul s indica e o sp ing mo ali y di e ences be ween emales and ha he di e ences be ween small, la ge and spe m compe i ion ials we e no consis en ac oss all 10 male g oups (male combina ions). Thus, al hough spe m compe i ion g oup had lowe mo ali y han in single males on a e age, his was no he case in all indi idual combina ions. The mean body mass and o al leng h o he o sp ing did no di e be ween male ea men s (ANOVA, p= 0.06 o bo h measu es, Figu e 2, Table 1). Female e ec was signi ican o body mass (ANOVA, p< 0.001), bu no o o al leng h. The yolk sac olume a ied signi i- can ly ac oss male ea men s (ANOVA, p= 0.004, Fig- u e 1b). Tukey pos hoc es s e ealed ha he size o he yolk was highe in spe m compe i ion ials han in ei he o he single male ials. Howe e , small and la ge males did no di e om each o he in e ms o he Kekäläinen e al.BMC E olu iona y Biology 2010, 10:20 h p://www.biomedcen al.com/1471-2148/10/20 Page 2 o 9 o sp ing yolk olume (Tukey’s es ). When compa isons we emadebe ween hecombinedsinglemaleand spe m compe i ion g oups, we ound a s a is ically sig- ni ican di e ence in he mean size o he o sp ing (ANOVA, F 1,9 = 10.10 and 14.51, p=0.01andp= 0.004 o body mass and o al leng h, espec i ely), e en hough he mean o sp ing size di e ence be ween spe m compe i ion and single male g oups was negligi- ble (0.068 s. 0.069 g and 17.5 s. 17.7 mm, espec i ely) (Figu e 2). These esul s can mainly be explained by he e y low a iance in wi hin-g oup o sp ing body size. Male and emale e ec s on o sp ing i ness Female iden i y had a signi ican e ec on all o he i - ness ai s we measu ed (ANOVA, F 2,38 = 269.78, p< 0.001 o mo ali y, F 1,19 = 196.07, p< 0.001 o body mass, F 1,19 = 7.35, p= 0.014 o o al leng h and F 1,19 = 129.02, p< 0.001 o yolk ese es). Male e ec s we e signi ican o o sp ing mo ali y (ANOVA, F 19,38 = 6.52, p< 0.001), bu no o he o he h ee esponse a iables (ANOVA, p > 0.15, in all cases). Small and la ge males did no di e om each o he in any o he ou o sp ing i ness ai s (ANOVA, p > 0.40, in all cases). Spe m quali y and o sp ing i ness Spe m eloci y was highe in small males han in la ge males (ANOVA F 1,54 = 26.53, p= 0.04) and he e was also a emale e ec on spe m eloci y (F 2,54 = 28.69, p= 0.03). No male g oup (small and la ge males) × emale in e ac ion was ound o spe m eloci y (F 2,54 = 0.08, p= 0.92), indica ing ha he e ec o he emale was simila o he di e en male g oups. A s a is ically sig- ni ican nega i e co ela ion was ound be ween o - sp ing mo ali y and spe m eloci y (Pea son’s co ela ion o VSL: whole da a wi h h ee emales; = - 0.418, p= 0.001, n = 60, mean alues o emales; =- 0.513, p= 0.021, n = 20) (Figu e 3, Table 2). Spe m eloci y was no associa ed wi h o sp ing body mass, o al leng h o yolk olume (Table 2). Table 1 Pa ly nes ed spli -plo ANOVA s a is ics o ou i ness measu es o he o sp ing. Sou ce d Mo ali y (%) Body mass (g) To al leng h (mm) Yolk olume (mm 3 ) FpFpFpFp ♀2 (1) 414.7 < 0.001 329.3 < 0.001 2.99 0.118 128.3 < 0.001 mc 9 (9) 1.44 0.239 1.63 0.240 1.45 0.295 1.41 0.310 m 2 (2) 1.94 0.173 3.31 0.058 3.28 0.059 7.17 0.004 ♀× mc 18 (9) 1.32 0.228 0.64 0.749 0.45 0.891 0.53 0.836 mc × m 18 (18) 5.77 < 0.001 0.86 0.623 0.47 0.946 1.58 0.161 Mc = male combina ion (1-10), m = male ea men (small male, la ge male o spe m compe i ion, ♀= emale). S a is ically signi ican p- alues a e indica ed wi h bold ace. Deg ees o eedoms (d ) o o sp ing body mass, o al leng h and yolk olume a e gi en in pa en heses. Male combina ion comp ises 10 small males, 10 la ge males and a spe m compe i ion g oup (10 small + 10 la ge males simul aneously). See me hods o de ails. Figu e 1 Uni a ia e s a is ic om boo s apped samples (1000 eplica es) o o sp ing mo ali y (mean % ± 95 CI) and yolk olume (mean mm 3 ± 95 CI) ac oss all emales o di e en spe m ea men s. SC = spe m compe i ion. Kekäläinen e al.BMC E olu iona y Biology 2010, 10:20 h p://www.biomedcen al.com/1471-2148/10/20 Page 3 o 9 Discussion In he p esen s udy, eggs om he polyand ous ma ings had lowe mo ali y a es and also la ge yolk ese es han he eggs om he monand ous e iliza ions. I has been demons a ed ha yolk mass is posi i ely ela ed o s a a ion ime and hus o ea ly pe o mance o o - sp ing [53,54]. Thus, ou esul s sugges ha spe m compe i ion inc eases o sp ing i ness, no only ia mo ali y, bu ha o sp ing may also ha e be e abili y o esis un a ou able eeding condi ions du ing pos - ha ching pe iod which a e no uncommon in A c ic condi ions. Al hough ou esul s clea ly show ha poly- and y is bene icial on a e age, hey also indica e ha his is no necessa ily he case in all emale-male combi- na ions (see e.g. [15]). The “good spe m”hypo hesis o polyand y is belie ed o equi e he p esence o addi i e gene ic a iance o male e ec s on o sp ing i ness [23]. Indeed, we ound a signi ican male e ec on o sp ing mo ali y, which indica es ha su i al bene i s o polyand y migh be explained by supe io gene ic quali y (supe io spe m) o ce ain male geno ypes. Howe e , male iden i y did no a ec o sp ing yolk olume, al hough he esul s sug- ges ed ha yolk olume was highe in o sp ing om polyand ous ma ings han o sp ing o ei he o he wo males when ma ed monand ously. This sugges s ha gene ic di e ences be ween males may no be su icien o explain yolk olume di e ences. Al hough no suppo ed by he p esen esul s, some o he s udies ha e shown ha males can indi ec ly con- ibu e o o sp ing o yolk size [36,37,39,55]. In addi- ion, he me abolic a e o A c ic cha eggs can a y signi ican ly be ween amilies and his me abolic Figu e 2 Uni a ia e s a is ic om boo s apped samples (1000 eplica es) o o sp ing body mass (mean g ± 95 CI) and o al leng h (mean mm ± 95 CI) ac oss all emales o di e en spe m ea men s. SC = spe m compe i ion. Table 2 Pea son co ela ion coe icien s o he ela ionship be ween spe m eloci y and o sp ing i ness ai s. T ai VSL VAP VCL p p p mo ali y - 0.418 0.001 - 0.422 0.001 - 0.416 0.001 body mass - 0.244 0.128 - 0.268 0.095 - 0.292 0.067 o al leng h - 0.096 0.555 - 0.120 0.461 - 0.122 0.454 yolk olume - 0.099 0.544 - 0.130 0.422 - 0.153 0.344 Figu e 3 The ela ionship be ween spe m eloci y o males (VSL, μms -1 ) and o sp ing mo ali y. The eg ession line is gi en by he equa ion: y = - 0.004x + 1.107. Kekäläinen e al.BMC E olu iona y Biology 2010, 10:20 h p://www.biomedcen al.com/1471-2148/10/20 Page 4 o 9 a ia ion can ha e a si e componen [38]. Fu he mo e, ele a ed es os e one concen a ion inside he eggs may inc ease he yolk abso p ion e iciency (i.e. dec eases he consump ion a e o he yolk), bu does no a ec he ha ching size o he o sp ing [39]. I he o al s e oid concen a ion o he seminal luid is highe when wo o mo e males a e eleasing spe m simul aneously (spe m compe i ion), i may lead o inc eased s e oid concen a- ions inside he eggs. This may inc ease he yolk u iliza- ion e iciency [39], enabling highe g ow h a e wi h he same amoun o yolk and hus bigge pos -ha ching yolk olume. Al e na i ely, i he nu ien s ansmi ed h ough seminal luid o he eggs educe he yolk u ili- za ion a e du ing he incuba ing pe iod, a highe p o- po ion o he yolk may be ese ed o pos -ha ching de elopmen . We used he same olume o spe ma ozoa bo h in sin- gle-male and spe m-compe i ion ials, which sugges ha ma e ial bene i s canno explain he di e ences in yolk size. Fu he mo e, he ma e nal hal -sib design allowed us o con ol o di e ences in ma e nal in es - men s [56-58], which he e o e did no con ibu e o he a ia ion in o sp ing i ness obse ed in ou s udy. We also minimized he biasing e ec o o sp ing age di e - ences on yolk ese es by sampling all he ish a he same ime a he beginning o he yolk sac s age, when all he o sp ing had ha ched. In addi ion, ha ching was a he synch onous owing o he wa m wea he wi h mos o he ish ha ching wi hin one week. These ac s oge he wi h he obse a ions ha ea ly ha ching indi i- duals a e o en less de eloped han la ae ha ha ch la e [59,60] sugges s ha he obse ed yolk size di e - ences in he p esen s udy we e no ela ed o he age o he o sp ing. As he highe yolk olume o o sp ing in spe m compe i ion ials was no associa ed wi h he la - ge size o hese ish, we canno comple ely ule ou he possibili y ha some mino di e ences in de elopmen al a e be ween male ea men s may ha e occu ed. How- e e , a ailable da a does no allow us o d aw i m con- clusions on his ques ion a his s age, and hus, his ques ion is an in e es ing a enue o u he esea ch. As he e ec s o po en ial ma e ial bene i s and ma e - nal e ec s a e p ecluded in he p esen s udy, wha migh explain he obse ed la ge yolk olumes in spe m compe i ion ea men s? Theo e ically, he p e- sence o o eign spe m du ing spe m compe i ion should lead o s onge compe i ion be ween spe m cells and also o in ensi ied selec ion o good spe m, wi h only he highes quali y spe m (i espec i e o he male) e ilizing he eggs [61]. The e o e, i seems plausible ha spe m compe i ion may in ensi y he selec ion on high quali y ( as ) spe m also wi hin ejacula es (wi hin male). In heo y, his could lead o highe i ness o o - sp ing in spe m compe i ion compa ed o single ma ings. I is well known ha only a mino p opo ion o spe m is capable o e ilizing he eggs [62,63]. Thus, spe m compe i ion by males may bene i he emale ia wo di e en mechanisms: i s , by c yp ic selec ion o high quali y males and second, by selec ion o highes quali y spe m wi hin each ejacula e. Howe e , as we did no ound any associa ion be ween spe m quali y ( elo- ci y) and o sp ing yolk olume, he e is also a possibili y ha some o he , cu en ly unknown e ec may ha e con ibu ed o ou inding. When he low numbe o ounde s o he s udy popu- la ion is aken in o accoun i seems possible ha he obse ed i ness bene i s could also be pa ly ela ed o gene ic incompa ibili y a oidance o he emales [64]. Thus, polyand ous emales could educe he isk o inb eeding ia c yp ic selec ion o spe m, which would esul in pa e ni y bias owa ds un ela ed o gene ically compa ible males [65,66]. As he gene ic a iabili y is ex emely low in many na u al A c ic cha popula ions [67] inb eeding a oidance can be an impo an mechan- ism explaining he e olu ion o polyand y in he A c ic cha . As i has been demons a ed ha good genes and compa ible genes may bo h be in ol ed in emale choice a he same ime [68-70], hese wo mechanisms a e no mu ually exclusi e mechanisms o sexual selec ion. The “good spe m”hypo hesis p edic s ha he e should be a gene ic co ela ion be ween male e iliza- ion success and o sp ing iabili y [21,71,72]. In suppo o his iew we ound a nega i e associa ion be ween spe m eloci y and o sp ing mo ali y a e. In addi ion, small males had as e spe m han la ge males (bu see [73]), p obably indica ing s a us-dependen shi s in ep oduc i e ac ics [42,74-76] (see also [77]). Spe m eloci y is a good p edic o o male e iliza ion success in he absence o spe m compe i ion [78-81]. Addi ion- ally, in A c ic cha , spe m eloci y is he mos impo - an pa ame e explaining a ia ion in male e iliza ion success wi hin in i o spe m compe i ion ials [44]. The posi i e associa ion be ween spe m eloci y and male spe m compe i ion success is demons a ed also in a a ie y o o he axa, including he domes ic owl, Gal- lus domes icus [82], A lan ic salmon, Salmo sala [43] and malla ds [83]. Gage e al. [43] ha e also shown ha e iliza ion success o male salmon is no ela ed o spe m numbe o o al leng h and ha he spe m long- e i y is nega i ely co ela ed o compe i ion success (bu see [84-86]). The e o e, al hough he ela i e pa e ni y success o compe ing males is no known, i is likely ha spe m eloci y has impo an e ec s on o sp ing su i al also unde spe m compe i ion. Conclusions Di e en i ness bene i s o polyand y documen ed in hep esen s udywe ep obablycausedbydi e en Kekäläinen e al.BMC E olu iona y Biology 2010, 10:20 h p://www.biomedcen al.com/1471-2148/10/20 Page 5 o 9 mechanisms. We demons a e ha he mo ali y o he emb yos p obably had a gene ic male componen and ha acco ding o “good spe m”hypo hesis, spe m elo- ci y was posi i ely associa ed wi h o sp ing su i al. On he o he hand ou esul s sugges ha he pa e nal e ec s on o sp ing i ness a e no necessa ily unde di ec gene ic con ol. Ins ead, in addi ion o po en ial mino de elopmen al a e di e ences o o sp ing in di - e en male ea men s, yolk size di e ences may esul om in ensi ied selec ion p essu e on high quali y spe m wi hin ejacula es o some o he non-gene ic ac o . Me hods Expe imen al ish The expe imen was ca ied ou a he Tai alkoski Game and Fishe ies Resea ch s a ion o he Finnish Game and Fishe ies Resea ch Ins i u e. The cha des- cended om he Lake Ina injä i (no he n Finland, 69° 70 N, 27°29 E) popula ion and ep esen ed a ou h ha che y gene a ion. The numbe o ounde amilies o he popula ion s a ed in 1982 was only 12, which indi- ca es ha inb eeding is common. In Oc obe 2007, 10 small (mean o al leng h 50.5 cm, ange 46.6-53.6 cm) and 10 la ge (mean o al leng h 60.1 cm, ange 55.7-70.2 cm) males as well as h ee emales (50.8, 55.3 and 57.9 cm) we e andomly selec ed om a common b ood s ock o he expe imen . The males we e s ipped o all a ailable mil and placed in disc e e male-g oup-speci ic anks o 12 days o p ohibi spawning ac i i y du ing he eplenishmen o hei spe m ese es. This p oce- du e ensu ed ha all he males p oduced mil o simila age p io o he expe imen . A i icial insemina ion The ish we e anaes he ized wi h bu e ed icaine me hanesul ona e (MS-222, Sigma®, Sigma Chemical Co., S . Louis, Missou i, U.S.A.) a e which hei o al leng hs and body masses we e measu ed. A e he mea- su emen s, he eggs o each emale we e s ipped as gen ly as possible and di ided in 30 equally la ge ba ches (app ox. 100 eggs/ba ch) o which 10 we e e i- lized wi h he spe m o small males, 10 wi h he spe m o la ge males and 10 wi h he spe m o bo h small and la ge males simul aneously (spe m compe i ion ea - men ). To allow polyand ous e iliza ion, he spe m o he bo h males was mixed oge he p io o insemina- ion. Be o e mil collec ion, he abdomen o males was ca e ully wiped o p e en wa e con amina ion and ac i- a ion o he spe m cells. P io o e iliza ions he spe - ma oc i alues o each male we e de e mined (wi hin one hou a e he mil collec ion) acco ding o [42]. Same olume (i.e. app oxima ely equal numbe ) o spe - ma ozoa was used in e e y case. In consequence, he o al olume o spe ma ozoa/male in spe m compe i ion ials was only hal o he olume used in single male ials. The e iliza ion was done in Pe i dishes, by injec ing he spe m wi h mic opipe es di ec ly on he eggs. Immedia ely a e his, 50 ml o wa e was pou ed on he Pe i dish and each dish was shaken o 5 s o allow he eggs o be e ilized. The same p ocedu e was used also in spe m compe i ion ea men s, wi h he spe m o he wo males being injec ed on he eggs simul aneously. A e s aying 60 s in Pe i dishes, e i- lized eggs we e andomly di ided in wo eplica es so ha he o al numbe o egg ba ches was 180 (3 emales × 30 ba ches × 2 eplica es). All egg ba ches we e incu- ba ed in independen incuba ing con aine s un il ha ch- ing in he nex Ap il (app oxima ely 180 days). Immedia ely a e he in oduc ion in he con aine s, all he eggs we e pho og aphed o de e mine hei ini ial numbe s. Spe m eloci y measu emen s To es ima e a ia ion in spe m eloci y among s udy g oups, we used compu e -assis ed spe m analysis (see [42] o de ails). O a ian luid o he analysis was ob ained om he egg ba ches o he h ee emales (see abo e) wi h a pipe e and s o ed sepa a ely unde he same condi ions as he mil samples. B ie ly, spe m ac i i y was ini ially ideo- eco ded o 40 seconds a e ac i a ion, ha is, om he p ecise momen he subsam- ple o pu e mil was exposed o 4,5 μlo o a ian luid (2:1, OF:wa e ) o he h ee emales on a cooled (ca. 5° C) mic oscope slide (Leja P oduc s BV, Nieuw-Vennep, The Ne he lands). Reco dings we e made using a CCD B/W ideo came a (Sony XC-ST50CE PAL, Tokyo, Japan) a ached o a nega i e phase-con as mic oscope (Olympus CH30, Tokyo, Japan) wi h a 10× magni ica- ion objec i e. Video eco dings we e la e analysed using he HTM-CEROS spe m acke so wa e (CEROS .12, Hamil on Tho ne Resea ch, Be e ly, MA, USA). The a iables measu ed included: a e age pa h eloci y (VAP), s aigh line eloci y (VSL) and cu ilinea elo- ci y (VCL) [87]. The eloci y es ima es we e based on he mean eloci y o all mo ile cells (i.e., hose exceeding he p e-de e mined h eshold alues VAP > 10 μms -1 and a VSL > 20 μms -1 ) eco deda 10,20,30and40s ollowing ac i a ion. Fo s a is ical analyses, he a e age alue o eplica ed measu es wi hin each male was used. As he h ee eloci y pa ame e s we e highly co ela ed (Pea son, > 0.95 in all cases), only VSL was used in s a is ical analyses. Mo ali y and size o he o sp ing The numbe o dead eggs was coun ed and emo ed weekly, om one day a e e iliza ion un il all he ish we e ha ched (Ap il 2008). Emb yonic mo ali y was de ined as a pe cen age o he o sp ing emaining om he ini ial numbe o eggs. A e ha ching a haphaza d sample o 20 (10 × 2) o sp ing om each o he single Kekäläinen e al.BMC E olu iona y Biology 2010, 10:20 h p://www.biomedcen al.com/1471-2148/10/20 Page 6 o 9 male ba ches and 40 (20 × 2) o sp ing om he spe m compe i ion ba ches we e collec ed. Due o high mo al- i y (87.3%) among one emale’s eggs, o sp ing o only wo emales we e sampled o u he (body mass, o al leng h and yolk olume) analyses. The o sp ing o his emale mus be excluded as he e was no enough ish le ha eliable es ima e o hese o sp ing i ness ai s could be de e mined (in nea ly 50% o e iliza ion com- bina ions he numbe o su i ing o sp ing was less han i e). O sp ing o he o he emales we e killed wi h an o e dose o icaine me hanesul ona e (MS- 222), pho og aphed and hei body mass was indi idu- ally measu ed o 0.001 g p ecision. P io o weighing, each ish was ca e ully d ied using hin pape owels o p e en he possibili y ha ex amois u ewoulda ec he eliabili y o ou measu emen s. The o al leng h o he ish o he nea es 0.01 mm and he size o he yolk we e measu ed om he digi al images using Image-P o Plus 3.0 g aphic so wa e (Media Cybe ne ics, Inc., Sil e Sp ing, MD, USA). The olume o he yolk was calcu- la ed acco ding he equa ion o a p ola e sphe oid: V = 0.5236 × leng h × heigh 2 (e.g. [88]). All he ish we e p ese ed in 95% e hanol o la e pa e ni y analyses ( esul s a e p esen ed elsewhe e). The s udy was ca ied ou acco ding o Finnish legisla ion. S a is ical analyses We used a pa ly nes ed spli -plo ANOVA o analyze he di e ences in mo ali y and he o sp ing size a i- ables (body mass, o al leng h and yolk olume) be ween single male and spe m compe i ion ials. Females we e used as blocks in he analysis. Each block was di ided in o 10 whole plo s (10 small, 10 la ge and 10 small + la ge males), whe e each plo include one small male, one la ge male and one spe m compe i ion ea men . Each whole plo was di ided in o h ee spli plo s; la ge male, small male and spe m compe i ion (i.e. h ee male ea men s we e nes ed wi hin 10 male combina ions). Because o high mo ali y in some male- emale combi- na ions missing alues we e also p esen . The e o e, we used he mean alues o he wo eplica es o achie e balanced da a in all analyses (i.e. when missing alues we e p esen mean alues we e eplaced wi h he alue o he o he eplica e). To s udy whe he polyand ous emales ha e a highe i ness han he a e age i ness o single ma ing emales, we combined la ge and small male g oups and compa ed he mean mo ali y, body mass and o al leng h o he o sp ing be ween spe m compe i ion e sus single male ials. The e ec s o indi idual males and emales on o - sp ing i ness (mo ali y, body mass, o al leng h and yolk olume) we e s udied using wo-way ANOVAs wi h h ee (o wo) emales and 20 males as andom ac- o s and he mean alues o he wo eplica es as da a poin s o each emale-male combina ion. To e eal indi idual male e ec s, only single-male ials we e used in hese analyses. Fu he mo e, he di e ences be ween small and la ge male wi h espec o o sp ing mo ali y, body mass, o al leng h, yolk olume and spe m VSL we e es ed using wo-way ANOVA, wi h male g oup (small s. la ge) as a ixed ac o and emale as a an- dom ac o . The ela ionships be ween spe m quali y and o sp ing i ness we e s udied wi h Pea son’s co ela ion analyses. P io o s a is ical analyses, he mo ali y pe cen ages we e ans o med wi h he a csine-squa e- oo ans o - ma ion o achie e no mali y. The ul illmen o he assump ions o all s a is ical es s was de e mined acco ding o [89]. Acknowledgemen s We hank he s a o he Finnish Game and Fishe ies Resea ch Ins i u e (FGFRI), Tai alkoski Game and Fishe ies Resea ch o he help du ing he incuba ing pe iod o he eggs. Jy ki Pusenius is hanked o helping wi h he s a is ical analyses. Ann Hed ick, I a Fols ad, Panu Välimäki, Ch is ophe Eizagui e and one anonymous e e ee p o ided aluable commen s on he ea lie e sion o he manusc ip . This s udy was inancially suppo ed by he Academy o Finland (JK, p ojec 121694; RK, p ojec 127398), he Uni e si y o Oulu (RK), Jenny and An i Wihu i Founda ion (MJ), FGFRI (NP) and he No wegian Resea ch Council (GR,177744/V40; LF,166581/V40). Au ho de ails 1 Depa men o Biological and En i onmen al Science, Uni e si y o Jy äskylä, P.O. Box 35 (YAC-315.2), FI-40014 Uni e si y o Jy äskylä, Jy äskylä, Finland. 2 Ecological Resea ch Ins i u e, Uni e si y o Eas e n Finland, P.O. Box 111, FI-80101 Joensuu, Finland. 3 Depa men o E olu ion and Ecology, Ins i u e o Biology, Uni e si y o T omsø, N- 9037 T omsø, No way. 4 Finnish Game and Fishe ies Resea ch Ins i u e, Joensuu Game and Fishe ies Resea ch, Yliopis onka u 6, FI-80100 Joensuu, Finland. 5 Finnish Game and Fishe ies Resea ch Ins i u e, Viikinkaa i 4, P.O. Box 2, FI-00791 Helsinki, Finland. 6 Depa men o Biology, Uni e si y o Eas e n Finland, P.O. Box 111, FI-80101 Joensuu, Finland. 7 Depa men o Biology, Uni e si y o Oulu, P.O. Box 3000, FI-90014 Uni e si y o Oulu, Oulu, Finland. Au ho s’con ibu ions RK, GR, LF and NP designed he expe imen s. Expe imen s we e ca ied ou wi h he coope a ion o all au ho s. JK, MJ, GR and NT measu ed he i ness pa ame e s. JK and MJ pe o med he s a is ical analyses and JK w o e he pape . 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Camb idge: Camb idge Uni e si y P ess 2002. doi:10.1186/1471-2148-10-20 Ci e his a icle as: Kekäläinen e al.: Gene ic and po en ial non-gene ic bene i s inc ease o sp ing i ness o polyand ous emales in non- esou ce based ma ing sys em. BMC E olu iona y Biology 2010 10:20. Submi you nex manusc ip o BioMed Cen al and ake ull ad an age o : • Con enien online submission • Tho ough pee e iew • No space cons ain s o colo igu e cha ges • Immedia e publica ion on accep ance • Inclusion in PubMed, CAS, Scopus and Google Schola • Resea ch which is eely a ailable o edis ibu ion Submi you manusc ip a www.biomedcen al.com/submi Kekäläinen e al.BMC E olu iona y Biology 2010, 10:20 h p://www.biomedcen al.com/1471-2148/10/20 Page 9 o 9