Investment in testes, sperm-duct glands and lipid reserves differs ... · Mazzoldi, 2002). In both...

Journal of Fish Biology (2010) 76, 1609–1625

doi:10.1111/j.1095-8649.2010.02587.x, available online at www.interscience.wiley.com

Investment in testes, sperm-duct glands and lipid reservesdiffers between male morphs but not between earlyand late breeding season in Pomatoschistus minutus

C. Kvarnemo*†‡, O. Svensson*† and W. Manson*§

*Department of Zoology, Stockholm University, SE-106 91 Stockholm, Sweden and†Department of Zoology, University of Gothenburg, Box 463, SE-405 30 Gothenburg, Sweden

(Received 13 April 2009, Accepted 20 January 2010)

This study of the sand goby Pomatoschistus minutus, a nest-holding fish with paternal care, focusedon gonadal investment among males of different sizes collected early and late in the breeding season.All males caught at the nest had breeding colour, whereas trawl-caught fish consisted of males bothwith and without colour. The absence or presence of breeding colour was a good predictor of testesinvestment. Compared to males with breeding colour, males without colour were smaller in bodysize but had extraordinarily large testes. In absolute terms, testes mass of males without breedingcolour was on average 3·4 times greater than those of males with breeding colour. Since smallcolourless males are known to reproduce as sneaker males, this heavy investment in testes probablyreflects that they are forced to spawn under sperm competition. Contrary to testes size, sperm-ductglands were largest among males with breeding colour. These glands produce mucins used formaking sperm-containing mucous trails that males place in the nest before and during spawning.Since both sneakers and nest-holders potentially could benefit from having large glands, this resultis intriguing. Yet, high mucus production may be more important for nest-holders, because it alsoprotects developing embryos from infections. There was no significant effect of season on bodysize, testes or sperm-duct glands size, but colourless males tended to be less common late in theseason. Possibly this may indicate that individual small colourless males develop into their morecolourful counterparts within the breeding season. © 2010 The Authors

Journal compilation © 2010 The Fisheries Society of the British Isles

Key words: accessory glands; Gobiidae; gonado-somatic index; parasitic spawning; seminal vesicles.

INTRODUCTION

Intra-sexual selection before mating is generated by the differential ability of indi-viduals of one sex to compete with each other for mating opportunities and may leadto evolution of traits such as weaponry and large body size (Andersson, 1994). Aftermating, intra-sexual selection can continue through sperm competition (Birkhead &Møller, 1998). Sperm competition is defined as ‘competition between the sperm oftwo or more different males over the fertilization of a given set of ova’ (Parker,

‡Author to whom correspondence should be addressed. Tel.: +46 31 7863479; fax: +46 31 416729;email: [email protected]§Present address: Department of Environment and Conservation, Locked Bag 104, Bentley Delivery Centre,WA 6983, Australia

1609© 2010 The AuthorsJournal compilation © 2010 The Fisheries Society of the British Isles

1610 C . K VA R N E M O E T A L .

1984, 1998). Sperm competition has generated a range of male adaptations (Birk-head & Møller, 1998), for example, toxic seminal fluid in fruit flies (Chapmanet al., 1995), and sclerotized intromittent organs in bean weevils (Crudgington &Siva-Jothy, 2000). Another trait affected by sperm competition is testes size. The-ory predicts that males that face a high risk of mating under sperm competitionshould invest more heavily in sperm numbers and therefore develop larger testesthan males that are rarely exposed to sperm competition (Parker, 1990; Parker et al.,1997). Consistent with this theory, parasitically spawning males, such as sneakers orsatellites that always mate under sperm competition, have been found to have largertestes relative to their body size than dominant males, which in many cases canavoid mating under sperm competition (Stockley & Purvis, 1993; Taborsky, 1998;Simmons et al., 1999, 2000).

In general, the degree of sperm competition is high among fishes, especially inexternally fertilizing, group spawning fishes with no parental care (Stockley et al.,1997; Petersen & Warner, 1998). Sperm competition, however, can also be very highin species where a dominant territorial male mates with a female, since other malescommonly interfere with the spawning pair and fertilize some of the eggs (Taborsky,1998, 2001; DeWoody & Avise, 2001; Avise et al., 2002).

In many taxa, individuals that use alternative routes to achieve reproductive suc-cess show different phenotypes. When the alternative reproductive phenotype isgenetically determined with Mendelian inheritance, it is called a strategy. Thereare few known cases of such genetic polymorphism (Gross, 1996; Shuster & Wade,2003). Alternative reproductive tactics, however, appear to be much more common,both in fishes and in other taxa (Gross, 1996; Tomkins & Hazel, 2007). Alternativereproductive tactics are often condition dependent, sometimes expressed as a thresh-old response to the status of the individual (e.g. age, condition or body size) or anenvironmental cue (e.g. population density, temperature, daylight or food abundance)(Roff, 1996; Tomkins & Hazel, 2007). With a genetic basis to the threshold switchpoint, this point can be under selection and evolve to differ between populations(Tomkins & Hazel, 2007). In addition, although equal fitness for the alternative tac-tics may occur, it is probably not very common (Tomkins & Brown, 2004). Hence,males may simply act as sneakers or satellites as the ‘best of a bad job’ (Gross,1991, 1996; Taborsky, 1994).

Condition-dependent alternative reproductive tactics have evolved in many fishspecies, in which the reproductive success depends on aggression and competitionbetween males (Gross, 1996). Usually, small males that are less likely to successfullydefend a nest and attract a female act as sneakers or satellites (Magnhagen, 1992;Taborsky, 1994). Since fishes continue to grow throughout life, the same individualmay go from employing one tactic to another as it grows larger (Taborsky, 1998;Magnhagen, 1999). Among longer-lived fishes, this can result in different age classesshowing different tactics (de Fraipont et al., 1993; Mazzoldi et al., 2000). The tactic,however, can also be facultatively determined from case to case, as found in the Gilatopminnow Poeciliopsis occidentalis (Baird & Girard), in which small males thatact as sneakers in the presence of large and aggressively territorial males becometerritorial themselves when the resident male is removed (Constantz, 1975).

In several species of gobies, e.g. grass goby Zosterisessor ophiocephalus (Pallas),black goby Gobius niger L., lagoon goby Knipowitschia panizzae (Verga) and sandgoby Pomatoschistus minutus (Pallas), nest-holding males attach a sperm-containing

© 2010 The AuthorsJournal compilation © 2010 The Fisheries Society of the British Isles, Journal of Fish Biology 2010, 76, 1609–1625

P O M AT O S C H I S T U S M I N U T U S M O R P H S D I F F E R I N G O NA D S A N D C O N D I T I O N 1611

mucus to the part of their nest where eggs later will be attached (Marconato et al.,1996; Ota et al., 1996; Scaggiante et al., 1999; Rasotto & Mazzoldi, 2002; Svensson& Kvarnemo, 2005). Sperm slowly disperse from the mucus. Motile spermatozoahave been observed after 40–80 min, and eggs can be fertilized for several hours(Marconato et al., 1996; Ota et al., 1996; Scaggiante et al., 1999). Although themicropyle (i.e. the pore in the membrane around the egg through which a spermcan enter) of gobiid eggs face the substratum (Miller, 1984), eggs do not dependon sperm trails for fertilization, but they can be fertilized by sperm released intothe water before, during and after egg deposition (Ota et al., 1996). Small Z. ophio-cephalus and G. niger males have larger testes relative to their body size than largemales (Scaggiante et al., 1999; Rasotto & Mazzoldi, 2002). The opposite is true forthe sperm-duct glands (to use the vocabulary recommended by Miller, 1984: alsoreferred to as seminal vesicles, e.g. Fishelson, 1991). The sperm-duct glands are aspecialized accessory organ near the testes. In Z. ophiocephalus and G. niger, thesperm-duct glands of large males were found to produce large amounts of mucus, theglands of medium-sized males to contain a mixture of sperm and mucus, whereasthe major function of small males’ sperm-duct glands was sperm storage with aminimal production of mucus (Scaggiante et al., 1999; Rasotto & Mazzoldi, 2002).Thus, this morphological pattern seems to reflect adaptations to different behaviouralmating tactics.

Pomatoschistus minutus is a small marine fish that breeds in shallow sandy areasalong the coasts of Europe (Miller, 1986). With a life span of 1–2 years (Healey,1971), it is thought to have only one reproductive season (late April or early May tomid-June or late June), during which individuals breed repeatedly. The males buildnests under a stone or mussel shell by excavating underneath and covering it withsand. A spawning female attaches her clutch in a single layer to the ceiling of the nestand then leaves the subsequent care to the male (Forsgren, 1999). Nests normallycontain eggs of several females (range two to six), and fertilization by parasiticallyspawning males is very common (Jones et al., 2001a, b). During the breeding season,most sexually mature males develop a distinct mating colouration with blue andblack marks on their fins. Some smaller males, however, remain very pale, yet showa sexually active behaviour as sneaker males (Svensson & Kvarnemo, 2007).

In the common goby Pomatoschistus microps (Krøyer) and G. niger, male repro-ductive behaviour depends both on their absolute and relative size and has beensuggested to follow an ontogenetic gradient (Magnhagen, 1992, 1999; Rasotto &Mazzoldi, 2002). In both species, males of medium size may act both as sneakersand as nest builders, while the smallest males do not try to build nests even whenalone and the largest males do not try to sneak fertilizations (Magnhagen, 1992;Mazzoldi & Rasotto, 2002). Furthermore, in G. niger, sneaker males change boththeir behaviour and relative allocation in testes and sperm-duct glands if kept with-out competition from other males (Immler et al., 2004). If P. minutus males wouldfollow also an ontogenetic gradient, it would be expected that they would sneakwhen they are small and become nest-holders when they grow larger. If the thresh-old switch point between the two morphs is unaffected by time of the season, thenfewer sneakers would be predicted late in the breeding season.

In a study of egg-guarding P. minutus, small males were found to have a higherlipid content in relation to their lean-body mass (body mass minus lipid mass) thanlarge males at the end of the parental care period, despite the fact that they had


1612 C . K VA R N E M O E T A L .

fanned more than the large males did and showed similar rates of filial cannibalism(M. Lissaker & C. Kvarnemo, unpubl. obs.). This intriguing result brought forwardthe hypothesis that small P. minutus males might make up for a high parental expen-diture by building up extra energy reserves early in the breeding season, therebymasking costs that arise later on (Tuomi et al., 1983). Thus, based on this hypoth-esis, an interaction would be predicted between male size and season among themales in breeding colouration (i.e. potential nest builders), such that small maleswould have a higher relative lipid content than large males early but not late in thebreeding season.

The aims of the present study were: (1) to determine whether sneaker morphmales (i.e. small P. minutus males that act as sneakers and lack breeding coloura-tion; Svensson & Kvarnemo, 2007) are sexually mature; (2) to investigate whethernest-holding males invest differently into gonads than do non-nest-holders with andwithout breeding colouration; (3) whether the frequency of sneaker morph males; (4)the relative investment into testes and seminal-duct glands; and (5) the switch-pointsize differs over the breeding season. In addition (6) if lipid content of nest-holdingmales changes over the breeding season; (7) if it differs from non-nest-holders and(8) if there is an interaction between male size and breeding season on lipid contentof males in breeding colouration were investigated.

MATERIALS AND METHODS

This study was carried out near the Tjarno Marine Biological Laboratory on the west coastof Sweden (58◦ 52′ N; 11◦ 10′ E). Using hand-nets, nest-holding P. minutus males werecollected together with their nests while snorkelling in shallow water. This was done duringa period relatively early in the breeding season (15–21 May) and was repeated again laterin the breeding season (9–14 June). At the end of each period, the area was hand-trawled toalso collect males that did not hold nest sites. Snorkelling and hand-trawling were done bythe same people and with approximately similar effort in the two sampling periods. The studyarea was only a small fraction of a much larger area with continuous sand bottom populatedby P. minutus. Thirty-one males were collected at nests and 30 by trawl in May, and 19 malesat nests and 27 by trawl in June (total length, LT, mean ± s.e.: 55·5 ± 0·6 mm, n = 107,range 41–72 mm). In addition, the sex ratio was close to even, with 51% females caught bytrawl during the first and 47% during the second half of the breeding season.

All 50 males that were caught at nests were in breeding colouration, whereas the 58 trawl-caught males (which most probably included both nest-holders and nest-less males) werefound both with and without breeding colouration. Hence, based on how each male wascaught and whether he showed breeding colour, the data were divided into three categories,which will be referred to as male breeding status or simply male status: (1) trawled maleswithout breeding colour, (2) trawled males with breeding colour and (3) nest-caught maleswith breeding colour. One of the males caught by trawl in June showed breeding colour thatwas intermediate to groups 1 and 2. This individual was of similar size as the largest malesthat lacked breeding colour, and his testes and sperm-duct glands were intermediate in size tomales with and without breeding colour. As this male could not be categorized as belongingto group 1 or 2 based on absence or presence of breeding colour, he was excluded from allfurther analyses.

All males were euthanized, stored in a −20◦ C freezer and later dissected under a standardlight microscope (×60–120 magnification). Before removing the intestines from the rest ofthe body, and separating the testes from the sperm-duct glands, it was also noted whetherthe males were in breeding colouration. The advantage of assessing breeding colouration ondead fish is that the colour is independent of social dominance, stress or background colour,and that the melanin-based aspect of their breeding colouration is shown at its maximum,



since all melanin chromatophores are then relaxed. Males that showed no or extremely faintblack colours were classified as having no breeding colouration. Such individuals could stillbe identified as males based on a faint iridescent blue colour on their anal fins. At dissection,the testes were inspected visually for sexual maturity, based on size, texture and colour of thetestes. For comparison, a separate set of 10 males (range 41–71 mm LT) caught mid-May toearly June were used to check whether the testes contained motile sperm. These males weredissected immediately after decapitation. The testes were placed on a microscope slide, a dropof natural sea water was added and the testes were squashed gently under a cover slip, beforemotility of the sperm was examined using a light microscope with ×400 magnification.

Measurements of testes mass, sperm-duct gland mass and somatic body mass (with liverand gut, as well as testes and sperm-duct glands removed) were taken as dry masses to thenearest 0·001 mg on a Cahn microbalance (Cahn Instruments Inc., Ceritos, CA, U.S.A.), afterhaving been dried for at least 24 h at 70◦ C in a desiccation oven. The bodies were then putindividually in petroleum ether (150 ml per fish in airtight glass jars) for lipid extraction. Thefish were removed after 8 h from the petroleum ether, left in a fume cupboard for 2 h anddried in the desiccation oven for another 12 h. Finally, the body mass was re-measured andsubtracted from the initial dry body mass, to find out the mass of the removed lipid content.

One at a time, testes mass, sperm-duct gland mass and lipid mass were analysed usingANCOVA, with body size as covariate and male status (see above) or month (May or June) asfactor (fixed). This was done to avoid problems associated with indeces and residuals (Tomkins& Simmons, 2002). Nevertheless, a figure based on the gonado-somatic index (IG, derivedfrom: IG = 100 MT M−1, where MT = testes mass and M = total body mass) is included toallow comparison with other studies. Lean-body mass (dry mass of the somatic body afterlipid extraction) was used to estimate body size. This was done to avoid autocorrelations thatcan arise when body size is measured as total-body mass (Tomkins & Simmons, 2002; Stoltzet al., 2005). Lipid mass analysed with lean-body mass as a covariate provides an estimateof body condition. All data of body mass, testes mass, sperm-duct gland mass and lipid masswere log10 transformed before analysis. In the ANCOVA, any non-significant interaction(P > 0·10) between covariate and factor was deleted from the analysis. For analyses of malestatus, which involved three group comparisons, Fisher’s protected least significant difference(PLSD) test was used as a post hoc test. A generalized linear model with a logit link functioncoded for colour (no colour = 0; colour = 1) was used to test whether the body size (LT) atwhich males switch morphs differed between early and late in the breeding season. Based ona logistic regression, mean switch-point size was then calculated from the size at which 50%of the males had switched and s.d. from the size at which 50% had switched minus the sizeat which 16% of the fish had switched.

RESULTS

The testes of all 10 P. minutus males that were dissected directly after decapitationcontained large numbers of highly motile sperm. The sperm-duct glands of all butone male were also found to contain some sperm that were activated by sea water.Without having made a quantitative measurement, the sperm in the sperm-duct glandsof the largest and the smallest males appeared to be less numerous than those ofmost medium-sized males. Nine of these males had breeding colouration and onehad not. The male without breeding colouration had very small sperm-duct glandsbut enormous testes similar to the colourless males described below.

Across male sizes, the general shape of the testes was 6–11 mm long and 1–3 mmwide. The shape of the sperm-duct glands was similar to that of P. microps asdepicted by Miller (1984) but differed from those described for P. minutus byFishelson (1991), in that no evidence of finger-like extensions along the marginwas found, but the edge of the wing-like gland was always smooth. The sperm-ductglands were rigid, with large globular cells and a transparent whitish colour, while


1614 C . K VA R N E M O E T A L .

Table I. Mean ± s.e. of untransformed values of lean-body mass (dry mass after lipidextraction), testes mass and sperm-duct gland mass, measured as dry mass of Pomatoschistus

minutus males collected in May and June

Trawl-caught maleswithout breeding colour

Trawl-caught maleswith breeding colour

Males caught on nestwith breeding colour

Lean-bodymass (mg)

114·31 ± 8·62 219·54 ± 9·76 241·56 ± 12·54

Testes mass (mg) 3·83 ± 0·56 1·05 ± 0·06 1·18 ± 0·06Sperm-duct gland

mass (mg)0·41 ± 0·09 1·61 ± 0·11 1·56 ± 0·10

Lipid mass (mg) 1·86 ± 0·18 3·18 ± 0·16 4·09 ± 0·26Sample size (n) 10 47 50

the testes were soft and varied from white to lemon yellow in colour. Most of thesetestes had a swollen and somewhat cross-streaked appearance.

Similar to the 10 males that were dissected fresh, all 108 males that had beenfrozen before dissection had rather swollen and wide testes that were cross-streaked.This was particularly true for the small males without breeding colouration thathad the largest testes (Table I). Thus, all the small P. minutus males that lacked abreeding colouration appeared fully capable of reproduction. It is also known fromanother study that small males with testes that match the appearance found here wereable to fertilize eggs (Svensson & Kvarnemo, 2007). Based on this visual inspectionof the testes, all 108 males of all sizes were judged to be sexually mature, both inthe samples collected early and late in the breeding season.

Males of different breeding status differed in body size (Table I), but there was noeffect of time of the breeding season or any interaction between body size and time(two-factor ANOVA: male status: F2,101 = 13·2, P < 0·001; month: F1,101 = 0·11,P > 0·05; interaction: F2,101 = 0·10, P > 0·05; Fig. 1). Post hoc tests showed thattrawled males lacking colouration were significantly smaller than males with colour,whether caught at nests (P < 0·001) or not (P < 0·001), whereas the two groups ofmales with breeding colouration did not differ in size (P > 0·05). Hence, to controlfor body size, lean-body mass was included as a covariate in the ANOVA below.

Comparing the frequencies (n values given in Fig. 1) of males with and withoutbreeding colouration among the fish caught by trawl in May and June, colourlessmales showed a non-significant trend to be less common later in the breeding season(χ2 = 3·64, d.f. = 1, P > 0·05). Male testes investment did not differ between sam-pling periods (ANCOVA, month: F1,104 = 1·05, P > 0·05; body size: F1,104 = 2·28,P > 0·05; with non-significant interaction removed: F1,103 = 1·68, P > 0·05) or didtheir investment into sperm-duct glands (ANCOVA, month: F1,104 = 1·26, P > 0·05;body size: F1,104 = 148, P < 0·001; with the non-significant interaction removed:F1,103 = 0·51, P > 0·05). Lipid content was also unaffected by sampling period(ANCOVA, month: F1,102 = 0·08, P > 0·05; body size: F1,102 = 222, P < 0·001;with non-significant interaction removed: F1,101 = 0·29, P > 0·05). Thus, on thepopulation level, male condition did not deteriorate between the two sampling peri-ods. Therefore, month was not included in the morphological analyses.



25221·5

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Male status

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* **

28 1931

Fig. 1. Male lean-body size (mean + s.e., log10 dry mass after lipid extraction) of Pomatoschistus minutusmales collected early (May, ) and late (June, ) in the breeding season. Male status refers to whetherthey were caught by hand at the nest or by trawl and whether they showed breeding colour. Sample size(n) is shown at the base of each bar. ** *, a significant difference in male body size (P < 0·001).

Testes mass was influenced by body size and differed depending on male sta-tus [ANCOVA, male status: F2,103 = 70·3, P < 0·001; body size: F1,103 = 17·2,P < 0·001; with non-significant interaction removed: F2,101 = 1·17, P > 0·05;Fig. 2(a) and Table II]. Colourless males had significantly larger testes for theirbody size than males with breeding colour (post hoc P < 0·001 in both compar-isons), but there was no significant difference between the two groups of males withbreeding colour (P > 0·05). Using untransformed values to compare absolute testesmass of males in breeding colour (mean ± s.e., 1·12 ± 0·44 mg, n = 97) and maleswithout (Table I), colourless males were found to have on average 3·4 times largertestes than males in breeding colour. Adjusting for body size using a gonado-somaticindex (IG) gives an even greater difference in testes investment between males withand without breeding colour [Fig. 2(c) and Table II].

Analysing male investment into sperm-duct glands among the three groups ofmales, both male status and body size, had a significant effect [ANCOVA, malestatus: F2,103 = 14·6, P < 0·001; body size: F1,103 = 78·1, P < 0·001; with non-significant interaction removed: F2,101 = 1·82, P > 0·05; Fig. 2(b) and Table II].The significant effect of male status was generated by colourless males having smallersperm-duct glands for their body size than either of the other two groups of males(post hoc P < 0·001 in both comparisons), but there was no difference between thetwo groups of males with breeding colour (P > 0·05).

Comparing male condition, measured as lipid mass with lean-body mass as covari-ate, between males of different status, there was a significant effect (ANCOVA,male status: F2,101 = 4·91, P < 0·01; body size: F1,101 = 163, P < 0·001; with non-significant interaction removed: F2,99 = 0·21, P > 0·05). All three groups of males


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Fig. 2. Investment by Pomatoschistus minutus males into (a) testes (log10 dry mass) and (b) sperm-duct gland(log10 dry mass) in relation to their lean-body size (log dry mass; see Fig. 1), depending on male breedingstatus ( , males caught at nests, with breeding colour; , males caught by trawl, with breeding colour,

, males caught by trawl, no breeding colour). (c) For comparative reasons a gonado-somatic index (IG),is shown. For allometric equations for fitting the curves see Table II.



Table II. Allometric equations for regressions shown in Figs 2 and 3

y variable Equation r2 Male status Figures

Log10 testes mass (mg) y = −1·233 + 0·868x 0·25 Trawl: no colour 2(a)y = −0·621 + 0·268x 0·07 Trawl: coloury = −1·057 + 0·467x 0·20 Nest: colour

Log10 sperm-duct glandmass (mg)

y = −4·041 + 1·740x 0·40 Trawl: no colour 2(b)

y = −2·453 + 1·123x 0·44 Trawl: coloury = −1·876 + 0·862x 0·50 Nest: colour

Gonado-somatic index (IG) y = 3·462–0·004X 0·01 Trawl: no colour 2(c)y = 0·856–0·002X 0·20 Trawl: coloury = 0·675–0·001X 0·17 Nest: colour

Log10 lipid mass (mg) y = −1·705 + 0·955x 0·58 Trawl: no colour 3y = −1·847 + 0·998x 0·734 Trawl: coloury = −1·543 + 0·897x 0·53 Nest: colour

x, log10 lean-body mass (mg); x, lean-body mass (mg; dry mass after lipid extraction).[Correction added after online publication 22 April 2010: In the above table under ‘Gonado-somaticindex (IG)’ three values were corrected from ‘x ’ to ‘X ’].

differed significantly (post hoc P < 0·001) from one another. Males in breedingcolour that were caught at nests had the highest lipid content for their body sizes,trawled males without breeding colour had intermediate levels and trawled maleswith breeding colour showed the lowest lipid content for their body sizes (Fig. 3and Table II). Finally, to test for an interaction between male body size and time ofthe breeding season on lipid content, specifically among males in breeding coloura-tion, the analysis was run on this sub-set of males, but no such effect was found(ANCOVA, interaction: F1,91 = 0·06, P > 0·05). This shows that the hypothesis thatsmall males in breeding colouration have higher lipid content than large males earlybut not late in the breeding season could be rejected.

The body length (LT) at which males switch morph did not differ between earlyand late in the breeding season (likelihood ratio χ2 = 34·7, d.f. = 2, P < 0·001; LT:Wald χ2 = 12, d.f. = 1, P = 0·001; month: Wald χ2 = 2·54, d.f. = 1, P > 0·05).Across both months, mean ± s.d. switch point size was 45·6 ± 3·6 mm.

DISCUSSION

In this study, two distinct male morphs were found: small males without breedingcolouration that have extremely large testes but small sperm-duct glands, and largermales that have breeding colouration and remarkably small testes but large sperm-duct glands.

The finding that small P. minutus males that lack breeding colour have large testesagrees with Miller (1984), who noted that several authors had found small malesof the genus Pomatoschistus to lack nuptial colouration but have large testes andgenital papilla. Miller (1984) also noted that it was unclear whether such males werereproductively active. The present observation that the testes of such a small malewithout breeding colouration contained motile sperm suggests that they are sexuallymature. Even better evidence that these males are sexually mature and reproductively


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Fig. 3. Lipid content (log10 lipid mass) in relation to Pomatoschistus minutus male lean-body size (log10 dry-body mass; see Fig. 1) when comparing males of different breeding status ( , males caught at nests,with breeding colour; , males caught by trawl, with breeding colour; , males caught by trawl, nobreeding colour). Males in breeding colouration caught at nests had the highest lipid content for theirbody size, followed by trawl-caught males without breeding colouration, while trawl-caught males withbreeding colouration had the lowest lipid content. For allometric equations for fitting the curves, seeTable II.

active is found in another study, in which three small males that also lacked breedingcolouration but had very large testes were shown to have fertilized eggs in the nestof another male (Svensson & Kvarnemo, 2007).

Previous work on fishes (Taborsky, 1998; Neff et al., 2003) and other taxa, such asinsects (Simmons et al., 1999, 2000) and mammals (Stockley & Purvis, 1993), showthat males of different sizes often invest very differently into gonads. Consistent withthis, small P. minutus males have large testes and large males have small testes in thisstudy. Absence or presence of breeding colouration, however, proved to be an evenbetter predictor of gonadal investment than body size: males with breeding colourare relatively large in body size and have small testes but large sperm-duct glands.In contrast, males that lack breeding colour are smaller and have small sperm-ductglands but very large testes. The latter is true not only when testes size is consideredin relation to their small body size but also as absolute testes size. In fact, thesemales have on average 3·4 times larger testes than males with breeding colour. Thisrepresents an unusually high investment in testes compared to what has been found inother fishes. Most other studies have only found that small males invest significantlymore than large males only when seen in relation to their body size (Taborsky, 1998;Tomkins & Simmons, 2002; Neat et al., 2003; Neff et al., 2003).

The reason for these small males that lack breeding colour to invest heavily intestes size is obvious, as they are highly unlikely to mate in the absence of spermcompetition. In such cases, theory predicts males will invest in large testes, in orderto produce high numbers of sperm, which is advantageous when mating under a



high risk of sperm competition (Parker, 1990; Parker et al., 1997). In comparison,the smaller testes found in the larger males with breeding colouration might beunderstood since such males often manage to reduce the risk of sperm competitionby defending a territory and building a nest. Even so, nest-holders rarely eliminatethe risk of sperm competition completely. From field-collected P. minutus nests,it is known that every second nest contains some eggs fertilized by another malethan the nest-holder (Jones et al., 2001a). In addition, parasitic spawning is not onlypractised by small, colourless sneaker morph males but by virtually every kind ofmale, regardless of body size, and whether the male has a nest and eggs in it (Singeret al., 2006, Svensson & Kvarnemo, 2007). Therefore, males in breeding colourationwould also be expected to be under some selection for large testes. Thus, the smalltestes found in these males might reflect a trade-off between energetic investmentinto testes v. nest defence and paternal care, a trade-off that sneaker morph malesare not forced to make. In addition, it is possible that nest-holding males investconsiderably less into large testes than sneaker morph males simply because they arelikely to spawn less often. While sneaker morph males might spawn several timesa day, most nest-holders may only spawn with a few females every second week orso in their own nest, plus occasionally in neighbouring nests.

The high investment by males with breeding colour into sperm-duct glands isalso according to expectations, as these glands are important for forming the mucus,which makes it possible for nest-holding males to prepare the inner surface of thenest with sperm-containing mucous trails (Scaggiante et al., 1999). It is reasonableto assume that the presence of sperm trails makes it possible for the nest-holder todevote more time to guarding the nest-opening against intruders while spawning,since he does not need to stay in the closest vicinity to the female at all times,as suggested by Marconato et al. (1996). This should be especially important sincegobiid females attach the eggs one-by-one, into the ceiling of the nest, and thereforea single spawning event often takes several hours. In addition, preparing the nestwith sperm trails may prevent instant dilution of the sperm, which otherwise is aproblem for most external spawners (Petersen, 1991; Levitan, 1998). It may also givethe nest-holder an advantage in numbers and position of sperm, whenever the guard-ing strategy fails and another male manages to enter the nest. In fact, nest-holdingP. minutus males have been shown to increase their mucous trail deposition whensneaker morph males were kept in a vial nearby the nest (Svensson & Kvarnemo,2005), suggesting that sperm trails indeed are an important defence against spermcompetition from parasitically spawning males.

Using microsatellite DNA in a previous P. minutus study, one parasitically spawn-ing male has been documented to fertilize eggs, even though he left the nest 19 minbefore spawning started (Svensson & Kvarnemo, 2007). This shows that parasiticmales also can deposit sperm that stay fertile for long periods of time, possiblyby depositing mucous trails, as suggested for other goby species (Marconato et al.,1996). On the other hand, P. minutus sperm can stay motile in sea water for at least2 h (O. Svensson & H. Elofsson, unpubl. obs.). Thus, it is possible that this malegained his fertilizations by just releasing sperm into the body of water inside thenest. Nevertheless, sneaker morph males have also been observed to turn upsidedown inside the nest, rubbing their belly against the ceiling in a similar way asnest-holders do (Svensson & Kvarnemo, 2007). If sneaker morph males produce


1620 C . K VA R N E M O E T A L .

sperm-containing mucous trails, however, they would be expected to invest sim-ilarly into sperm-duct glands as nest-holding males. The fact that they do not istherefore somewhat intriguing. The explanation might be found in a combinationof (1) strong selection for large testes, when spawning under a high risk of spermcompetition (Parker, 1998), making heavy allocation into sperm-duct glands impos-sible if testes investment is traded-off against sperm-duct gland investment, and (2)long-lived sperm also without mucous trails, making heavy allocation into sperm-duct glands unnecessary. An additional, rather speculative, possibility is that sneakermorph males parasitize on the mucous trails, which the nest-holder has deposited,by adding sperm to existing trails. Finally, an important function of gobiid mucins isto protect the eggs and developing embryos from microbial infections (Giacomelloet al., 2008). The difference in investment into sperm-duct glands between nest-holding males and sneaker morph males may hence reflect difference in paternalcare behaviour rather than different risk of sperm competition.

The data in the present study are insufficient to say whether sneaker morph malescan change tactic. Importantly, however, recently collected data from an experi-mental study do show that individual sneaker morph males can change tactic. Thechange was expressed both behaviourally and morphologically when housed withoutcompeting males (T. Takegaki, O. Svensson & C. Kvarnemo, unpubl. obs.).

It is not yet known what exactly causes males of P. minutus to shift from one tacticto another, but in general, a shift would be likely to correlate with the male’s chanceto breed successfully as a nest-holder. Therefore, potentially important factors includehigh or low levels of social dominance by other males, female attention, nest siteavailability and risk of egg predation. In G. niger, a behavioural and morphologicalshift from sneaker to nest-holder has been found to depend on social context, butnot on male size (Immler et al., 2004). In other cases, a shift from one tactic toanother appears to be size or age dependent, possibly because many of the factorslisted above co-vary with male size. As mentioned in the introduction, this has beenfound in P. microps (Magnhagen, 1992, 1998). Given that the threshold body sizebetween the two male morphs did not differ between May and June, more colourlesssneaker morph males would be expected early than late in the breeding season ifP. minutus males also follow an ontogenetic gradient and change reproductive tacticsas they grow. Consistent with this prediction, sneaker morph males tended to be morecommon early than late in the breeding season. Yet, if P. minutus males continue togrow after maturity, a general increase in size would have been expected betweenMay and June, which was not found. This might indicate that males do not growmuch over the breeding season or at least not between the two times of sampling inthe current study. Alternatively, the same pattern could be generated if the largestmales, which begin breeding early in the season, start to die off halfway throughthe season, and if other males that only reach a large size later in the season thenreplace these males.

In many species of fishes, the condition of parental males deteriorates over longerperiods of parental care (Chellappa et al., 1989; Marconato et al., 1993; Takahashi& Yanagisawa, 1999). In the present study, however, there was no evidence ofreduced condition, based on lipid content of males collected early and late in thebreeding season. Naturally, if it had been possible to measure condition of the sameindividual twice (which was not possible with the current method), there mightstill be a decline. Such a drop in condition could have remained undetected in the



estimates, in particular if there are different sets of males breeding early and late.Another reason why no seasonal effect on condition was found might be that maleswere sampled in two periods only, and that no males were sampled right at theonset of the breeding season, in the middle or at the very end. Nevertheless, as thelate-season sample was collected towards the end of a 2 month breeding season,it could have been expected that some effect was found. It is, however, possiblethat the effects of prolonged care on lipid content of the body appear later than, forexample, glycogen levels, or lipid levels of liver and gonads, as was found in thethree-spined stickleback Gasterosteus aculeatus L. (Chellappa et al., 1989). Giventhat filial cannibalism is common among egg-guarding P. minutus males (Lissaker,2006), it is also possible that they replenish their energy storage by eating someof their eggs, as has been shown in other studies on both fishes and insects withpaternal care (Okuda & Yanagisawa, 1996; Lindstrom, 1998; Neff, 2003; Thomas &Manica, 2003; Manica, 2004).

While there was no effect of time on condition, males differed significantly inlipid content depending on whether they were nest-holders or trawl-caught with orwithout breeding colouration. Based on data from other studies (Taborsky, 1998;Takahashi & Yanagisawa, 1999), both egg-guarding nest-holders and sneaker maleswould have been expected to show reduced condition. In the present study, however,the group of fish found to have the least amount of lipids for their body size was thetrawled males with breeding colour. It is difficult to speculate about the reason why,since it is not known whether they were nest-holder. Yet, given that they differed incondition, but not in size, from the males that were caught at nests, it may suggestthat these males were not in good enough condition to hold a nest, or that they hadrecently been holding a nest and thus had temporarily depleted their energy reserves,or possibly that they were investing their energy into growth, to become successfulnest-holders in the future. Since there was no nest-site limitation in the study area,any male could probably claim a nest site, but males of poor quality or conditionmight have had difficulties attracting females to spawn (Forsgren et al., 1996).

Based on the hypothesis presented in the introduction that among nest-holderssmall males might build up extra energy reserves early in the breeding season, smallmales were predicted to have higher relative lipid content than large males, early,but not late, in the breeding season. Such an interaction, however, was not foundbetween male size and season among males in breeding colouration, making this anunlikely explanation for the unexpectedly good condition found among small nest-holders. Furthermore, in other species of fishes, larger individuals (i.e. individualsborn early during the previous reproductive season) have been found to be in bettercondition than smaller ones after the winter (Cargnelli & Gross, 1997). Alternativeexplanations to the result of small males having more lipid for their body size thanlarge males (M. Lissaker & C. Kvarnemo, unpubl. data) may instead include that thesimilar level of filial cannibalism found for these two groups of males would benefitsmall males more than large males (Rohwer, 1978) or that small nest-holders, whichuse a slightly different technique when fanning, might manage to do this at a lowerenergetic cost.

In conclusion, two very distinct phenotypes of male P. minutus were found,namely small males without breeding colouration that had huge testes and smallsperm-duct glands, and a range of larger males with breeding colouration that had


1622 C . K VA R N E M O E T A L .

tiny testes and large sperm-duct glands. Based on this study, it cannot be con-cluded whether these male morphs represent two alternative reproductive strategiesor tactics, but a separate study provides support for the sneaker morph being a tactic.In addition, the trend in the present study with colourless males being less commonlate in the breeding season provides some indication that it may be an age or size-dependent tactic, such that sneaker morph males shift into nest building males withbreeding colouration later in the season.

We are grateful to Tjarno for research facilities, to P. Lindberg for help in the field, whenmost needed, to M. Miettinen for help in the laboratory, to J. Tomkins for discussion, adviceand help with statistics, and to the Crafoord Foundation and the Swedish Research Councilfor funding. This study complies with Swedish law and was done under permit Dnr N60/00from the Swedish Animal Welfare Agency.

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