Received: 11 January 2020

| Accepted: 15 January 2020

DOI: 10.1002/ece3.6075

ORIGINAL RESEARCH

Different effects of mating group size as male and as female onsex allocation in a simultaneous hermaphrodite
Masami M. Tamechika1| Kohei Matsuno1,2 | Satoshi Wada1 | Yoichi Yusa3

1
Graduate School of Fisheries Sciences,
Hokkaido University, Hakodate, Japan

Abstract2

Sex allocation theory predicts that the optimal sexual resource allocation of simulta-

Arctic Research Center, Hokkaido
University, Sapporo, Japan
3

Faculty of Science, Nara Women's
University, Nara, Japan
Correspondence
Masami M. Tamechika, Graduate Schoolof Fisheries Science, Hokkaido University,
3-1-1 Minato-cho, Hakodate, Hokkaido 0418611, Japan.
Email: tamechi555@gmail.com
Funding information
Research Institute of Marine Invertebrates
Foundation; Japan Society for the Promotionof Science, Grant/Award Number:
18K14506 and 19H03284

neous hermaphrodites is affected by mating group size (MGS). Although the original
concept assumes that the MGS does not differ between male and female functions,
the MGS in the male function (MGSm; i.e., the number of sperm recipients the focal
individual can deliver its sperm to plus one) and that in the female function (MGSf;
the number of sperm donors plus one) do not always coincide and may differently
affect the optimal sex allocation. Moreover, reproductive costs can be split into “variable” (e.g., sperm and eggs) and “fixed” (e.g., genitalia) costs, but these have been
seldom distinguished in empirical studies. We examined the effects of MGSm and
MGSf on the fixed and variable reproductive investments in the sessilian barnacle
Balanus rostratus. The results showed that MGSm had a positive effect on sex allocation, whereas MGSf had a nearly significant negative effect. Moreover, the “fixed”
cost varied with body size and both aspects of MGS. We argue that the two aspectsof MGS should be distinguished for organisms with unilateral mating.
KEYWORDS

barnacle, mating group size, sex allocation, simultaneous hermaphrodite, sperm competition

1 | I NTRO D U C TI O N

considered an important factor that affects the optimal sex allocation.
Charnov (1980, 1982) predicted theoretically that female-biased in-

Sex allocation in simultaneous hermaphrodites is defined as thevestments are favored in small mating groups in order to alleviate local

proportion of resources allocated to the male relative to the female

sperm competition (i.e., competition between related sperm; Schärer,functions. It is an important life-history trait affecting the fitness of2009). Since then, empirical studies on various simultaneous hermaph-

an individual and hence is subject to selection pressure under given

rodites (e.g., Annelida, Crustacea, Platyhelminthes) have generally sup-environmental conditions (Charnov, 1982). Since the first formaliza-

ported the prediction (Janicke et al., 2013; Schärer, 2009). However,tion by Charnov, theoretical predictions and empirical tests of the

Yamaguchi, Yusa, Sawada, and Takahashi (2013) pointed out that the

optimal sex allocation under these conditions have been a touch-

MGS concept has two aspects, namely (a) the number of individuals

stone in modern evolutionary biology (Janicke et al., 2013; Leonard,

to which a focal individual can donate sperm (+1 = MGS in the male

2019; Schärer, 2009; West, 2009).

function; MGSm) and (b) the number of individuals from which the indi-

Irrespective of its considerable success, sex allocation theory needs

vidual can receive sperm (+1 = MGS in the female function; MGSf). The

refinement (Schärer, 2009). First, mating group size (MGS) has beenoriginal MGS concept assumes that MGS does not differ between maleThis is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,provided the original work is properly cited.
© 2020 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd
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and female functions as all individuals in the same group mate with all

species (Darwin, 1854; Yusa, 2019; Yusa, Takemura, Sawada, &available individuals. Although this distinction may not be important

Yamaguchi, 2013). In barnacles, MGS (not distinguished into MGSm

in reciprocally mating hermaphrodites (but see Pongratz & Michiels,

and MGSf) has different effects on sex allocation depending on

2003 for an exception), these two aspects of MGS do not necessarily

the species. In the balanomorphan barnacle Catomerus polymerus,

coincide in hermaphrodites with unilateral mating (Figure 1). This dis-sex allocation is less female-biased in large mating groups than in

tinction is especially important in simultaneous hermaphrodites where

small groups (Raimondi & Martin, 1991) as predicted by Charnov's

individuals first mature as male and then become hermaphroditic

model. But MGS does not affect sex allocation in other species(protandric simultaneous hermaphrodites; Baeza, 2007).

(Hoch & Levinton, 2012; Kelly & Sanford, 2010). Although the dif-

The second issue that needs refinement in the concept of sex al-

ferent results may reflect species difference, the degree of pro-location is the distinction between “fixed” and “variable” costs (Heath,

miscuity and the different criteria used to distinguish small and1977; Schärer, 2009). The fixed costs include producing and maintain-

large groups may also be relevant.

ing reproductive organs such as genitalia that are not consumed in each

The cost of having a penis may be high in barnacles because it isreproductive event, and the variable costs correspond to the resources

several times longer than the body length in many species (Darwin,allocated to produce the gametes (Heath, 1977; Schärer, 2009). These

1854; Dreyer et al., 2018; Neufeld & Palmer, 2008), and it is discarded

two costs will likely have different responses to environmental factors

after each breeding season at least in such species as Semibalanus

such as the two aspects of MGS; for instance, the variable cost is af-balanoides (previously Balanus balanoides; Crisp & Patel, 1958, 1960;

fected by the MGSs but not the fixed cost. Moreover, the “fixed” cost,

Klepal & Barnes, 1974, but not in other species; Barnes, 1992; Hoch,such as the penis, varies according to the physical environments such

Schneck, & Neufeld, 2016). The penis shows phenotypic plasticity inas wave exposures in animals such as barnacles (Neufeld & Palmer,

morphology in relation to local environmental factors, such as wave

2008). However, these are seldom distinguished in empirical studies

strength and density (Hoch, 2008, 2009, 2010; Neufeld & Palmer,(Lorenzi, Sella, Schleicherova, & Ramella, 2005; Schärer, 2009).

2008). Thus, evaluating the fixed cost as male is important in barna-

Sessilian (balanomorphan) barnacles (Cirripedia: Thoracica)cles. However, few studies have examined such fixed and variable

are sedentary animals that deliver sperm to neighboring individ-

costs in sex allocation (Hoch & Levinton, 2012). In this study, weuals using a long penis (Anderson, 1993; Barnes & Barnes, 1977;

examined the effects of MGSm and MGSf on the fixed and variable

Murata, Imafuku, & Abe, 2001). The size of the penis is an espe-

costs of sex allocation in a simultaneous hermaphrodite, the sessilian

cially important characteristic since it determines the area hencebarnacle Balanus rostratus.

the number of individuals that an individual's sperm are delivered,hence MGSm (Neufeld & Palmer, 2008). Moreover, barnacles are
the original model organisms considered in Charnov (1980, 1982)
and have been used for testing sex allocation theory. They show
various degrees of sex allocation, to the extent that even pure

2 | M ATE R I A L S A N D M E TH O DS
2.1 | Sample collection and rearing

males (i.e., sex allocation = 1) and females (0) are known in several
Balanus rostratus has an annual reproductive cycle (Kado, Suzuki,
Suzuki, Nanba, & Ogawa, 2009; Korn, 1985). The ovary develops
most extensively from December to April (Kado et al., 2009). The
development of the testis starts from February and reaches maximum before the onset of the mating season (Kado et al., 2009; Korn,
1985), which is from October to November in Northern Japan (Kadoet al., 2009). Individuals keep long penises even in nonreproductive
seasons (1.89–3.60 cm, mean = 2.53 cm, N = 36 measured in June
2018; personal observation).
Individuals of B. rostratus were obtained from a wave-protectedarea managed by the Kawauchi Fisheries Cooperative Association(Mutsu Town, Aomori Prefecture, Japan; 41°11′50″N; 140°59′21″E)
in May 2018 (N = 173 barnacles). These barnacles had settled naturally on the shells of the scallop Patinopecten yessoensis that were suspended in the sea at a depth of about 20 m in situ using hanging culture
F I G U R E 1 Hypothetical barnacle population consisting oflarge individuals (i.e., hermaphrodite phase) and small ones (i.e.,
protandric male phase). The area to which the penis of focal
individual (gray color) can reach is shown as a meshed circle. The
focal individual has different MGSs, MGSm = 4 and MGSf = 5,
including itself

for almost 4 years. Thereafter, these shells were suspended in the sea
at a depth of approximately 8 m using buoys and ropes in Hakodate
City, Hokkaido Prefecture, Japan (41°56′17″N; 140°56′34″E). Shells
were spaced at 20 cm intervals to avoid barnacles on different shells
from interacting. Forty-two shells, each with 2–7 barnacles (Figure A1),
were reared until late September 2018 (N = 173 barnacles).

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2.2 | Measurements

TAMECHIKA et al.

sex allocation was treated as a response variable (N = 164). The
MGSm and MGSf can be different as penis length varied among

The barnacles were frozen at the end of the rearing period, which

individuals, from 1.67 to 4.46 cm (mean ± SD = 2.77 ± 0.53). The

was just before the onset of the mating season (Kado et al., 2009);

effects of MGSs on all response variables were not artefacts due totherefore, the reproductive organs had completely developed but

multiple collinearity (generally considered to be present if Varianceindividuals had not used their gametes yet. Although they may con-Inflation Factor [VIF] > 10: Chatterjee & Hadi, 2012) between

tinue to produce eggs and sperm during the mating season, it is the

MGSm and MGSf (VIF < 2.45 in all the analyses; R package car; Fox

most suitable season to quantify sex allocation in this species.

& Weisberg, 2019). The correlation coefficient r was 0.75, which was

To calculate MGS, we measured the distance between individ-

significant (p < .001; Pearson's product-moment correlation) but

uals (minimum distance between the two opercula) on the same

was not very high as compared with the traditional assumption ofbivalve shell. Then we dissected out the operculum (scutum andr = 1). Additionally, the signs of estimates for MGSm and MGSf did

tergum), ovary, testis, seminal vesicles, and penis of each specimen

not change between simple and multiple regressions for all responseusing a nipper and tweezers. The penis was photographed with avariables. All these pieces of evidence are in disfavor of the presence

digital camera (Stylus TG-4, Olympus Corporation), and its lengthof multiple collinearity.

and width were measured to the nearest 10 µm using ImageJ (ver-We included the shell ID (N = 41) as a random factor to incorpo-

sion 1.51j8, National Institutes of Health). Then, the operculum andrate differences in the microenvironment (e.g., number of individuals

ovary were each placed on an aluminum pan (35 mm in diameter,

on one shell). Statistically significance was set at p = .05. Interaction

21 mm high) that had been preheated at 480°C and preweighed withterms were not incorporated in the models as all interactions werean electric microbalance (Mettler Toledo MT5) to a precision of 1 µg.

nonsignificant. All analyses were performed using R software ver-The testis, seminal vesicles, and penis were each placed on a smaller

sion 3.6.1 (R Core Team, 2019).

aluminum pan (16 mm in diameter, 15 mm high). Then, the treatedorgans were dried in an oven at 60°C for 12 hr and were weighed todetermine dry mass.

2.3 | Analyses

3 | R E S U LT S
The dry weight of the operculum ranged from 244.98 to 1,807.32 mg,
and the testis and seminal vesicles ranged from 4.63 to 227.84 mg.
The penis weight was much smaller, but varied greatly, from 0.33 to

We measured MGSm and MGSf for each individual as the number

2.63 mg and the length ranged from 1.67 to 4.46 cm. The ovary alsoof individuals within the area reachable by its penis plus one andvaried greatly from 62.91 to 1,949.72 mg; however, all the individuals

the number of individuals whose penises can reach the focal indi-

in this study had developed the ovary to some extent. Both MGSm

vidual plus one, respectively. The distance reachable by the penis

and MGSf ranged from 2 to 7 individuals.

was considered to be 1.82 times the length of the penis of the speci-

The results of LMMs showed that both the weight of the tes-men based on the elongation rate of the congener B. glandula in a

tis and seminal vesicles (i.e., variable cost as male) and that of thewave-protected shore (Neufeld & Palmer, 2008). This species has

penis (i.e., fixed cost as male) increased with body size and MGSm.

a close phylogenetic relationship to B. rostratus as they belong the

Additionally, these male investments decreased with MGSf (Table 1).

same Balanus balanus group (Pitombo, 2004).

On the other hand, the ovary was only positively affected by body

Individuals with no potential mating partner (i.e., MGS = 1) orsize and unaffected by either MGS (Table 1). Sex allocation increased

those with some organs lost were excluded from the analyses.

with increasing MGSm and decreased almost significantly with in-

Following Hoch and Levinton (2012), the ovary was considered as

creasing MGSf (Table 1; Figure 2). The inclusion of MGSf improveda female variable cost. The testis and seminal vesicles were consid-

the model on sex allocation significantly as compared with theered as a male variable cost, and the penis as a male fixed cost (Hochmodel without MGSf (Likelihood chi-square = 3.869, p = .049). This

& Levinton, 2012). The operculum weight was used as the index ofis also true for model on the weight of the testis and seminal vesi-

body weight (Kado et al., 2009) because they were positively cor-cles (Likelihood chi-square = 7.546, p = .006) and that of the penis

related (r = .75, p < .001, N = 162; Pearson's product-moment cor-

(Likelihood chi-square = 10.514, p = .001).

relation). Sex allocation was defined as the total male investment(testis and seminal vesicles) divided by the male and female investments (testis, seminal vesicles, and ovary). Note that the sex alloca-

4 | D I S CU S S I O Ntion in terms of weight as we evaluated is a relative value because we
do not know the actual energetic cost invested in each sex function.

This study shows that sex allocation responded differently to MGSm

To investigate the effects of MGSm, MGSf, and that of body size,and MGSf in the barnacle B. rostratus. Although this needs to be

we used linear mixed-effects models (LMMs, R package lmerTest;

interpreted with considerable care as sex allocation can be driven

Kuznetsova, Brockhoff, & Christensen, 2017) in which each repro-

by only the male function, sex allocation was positively affected

ductive investment (testis + seminal vesicles, penis, and ovary) andby MGSm, which supports the prediction of sex allocation theory

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TAMECHIKA et al.

TA B L E 1 LMM results on effectsof MGSm, MGSf, and body size on
reproductive investment (testis + seminal
vesicles, penis, and ovary) and sex
allocation in Balanus rostratus

Reproductive organ

Parameters

Estimate

SE

Testis and seminal
vesicleIntercept

17.98013.251.178

MGSm

10.799

3.629

.003

MGSf

–10.501

3.784

.006

Penis

Ovary

Body weight (mg)

0.059

0.010

<.001

Intercept

0.275

0.104

.009

MGSm

0.105

0.028

<.001

MGSf

–0.097

0.029

.001

Body weight (mg)0.00070.00008<.001

Intercept

0.761

104.840

.994

MGSm

40.75230.598.185

MGSf–39.898

31.512

.207

0.843

0.081

<.001

Intercept

0.10840.0152

<.001

MGSm

0.00890.0040

.0267

MGSf

–0.00830.0042.0501

Body weight (mg)–0.000020

0.000012.0930

Body weight (mg)
Sex allocation (male/
total reproductive
investment)p

On the other hand, when the effects of MGSm and MGSf were
evaluated separately, sex allocation was affected negatively by
MGSf almost significantly. Moreover, the models with both MGSm
and MGSf were better than those with MGSm (and body size) alone.
Combined with the fact that female investment was not affectedby both MGS, the decreased sex allocation was unexpectedly owing
to the decrease in the investment to male function with increasing MGSf (Table 1). Unlike MGSm which is linked to the number ofavailable eggs, MGSf is related to the amount of sperm received.
Therefore, MGSf may be a better predictor of the intensity of spermcompetition than MGSm. Severer sperm competition, with thenumber of available eggs being constant, lowers male fitness curveand hence reducing male allocation in such cases appears to be an
F I G U R E 2 Effects of MGSm (filled circles) and MGSf (opencircles) on sex allocation (male/total reproductive investment;
MGSm; p = .0267, regression: y = 0.0089x + 0.0632, MGSf;
p = .0501, regression: y = –0.0083x + 0.1148; LMM). Regression
lines were drawn based on the mean value of body size(=1,014.18 mg operculum weight) and the values of most frequent
MGS (=3, both as male and as female). Bars and large circles show
standard deviation and mean value, respectively

adaptive response. Although not known in barnacles, MGSf may also
be an important factor affecting sex allocation if individuals digest
received sperm for nutrition (Yamaguchi, Sawada, Nakashima, &Takahashi, 2012).
The mechanisms by which individuals sense their own MGSm
and MGSf are unknown, but the barnacles may use water-borne
chemicals to collect information on the number of neighboring individuals and degrees of their ovary and testis developments. In fact,(Charnov, 1980, 1982, 1987). MGSm is related to the number of eggs

some chemicals are used to attract neighbors (Klepal, 1990) such as

the focal individuals can fertilize and hence directly affects sex allo-

ascorbic acid in Balanus sp. (Collier, Ray, & Wilson, 1956). Barnacles

cation via change in the shape of male fitness curve. Most likely, theeven use their penises to search for functional females (Anderson,increased sex allocation, and male investment as well, was an adap-

1993; Klepal, 1990; Murata et al., 2001). Although the penis is dis-

tive response to the increased number of available eggs (Charnov,

carded after the mating season in some congeners (Crisp & Patel,

1980, 1982, 1987) with increasing MGSm. Similar increases of

1958, 1960; Klepal & Barnes, 1974), B. rostratus keep long penises

male allocation have been reported in many animals (e.g., Annelida;even in nonmating seasons (personal observation). Thus, they are

Schärer & Ladurner, 2003, Janicke et al., 2013, Chrodata; Hart etlikely to collect information on mating partners and adjust sex allo-

al., 2010, Platyhelminthes; Trouvé, Jourdane, Renaud, Durand, &cation even before the onset of mating season.

Morand, 1999, Tan, Govedich, & Burd, 2004) including a barnacle(Raimondi & Martin, 1991).

The different responses of male and female outputs suggest alack of trade-off between male and female functions. This lack may

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TAMECHIKA et al.

have been caused by the absence of synchrony in the development

highly variable. We suggest that MGSm is relevant to the numberof female and male gonads (Korn, 1985). The period of yolk forma-of female-acting hermaphrodites and their eggs available to thetion of B. rostratus overlaps with the period when primary produc-

focal individual as male, whereas MGSf is linked to the number oftion in this area is high (i.e., winter–spring; Osaka, 1985). In contrast,

male-acting neighbors and the total amount of their sperm it re-

the testis is fully developed in autumn, just before the onset of theceives. Hence, MGSf is likely to reflect the intensity of sperm com-mating season (Kado et al., 2009). Therefore, larger amounts ofpetition the focal individual experiences better than MGSm. Such

resources can be used for female investment than for male invest-

distinction may also be important in other hermaphrodites with uni-

ment. Hines (1978) also indicated that egg and sperm production inlateral mating. Accurate evaluation of the two aspects of MGS, andsome balanomorphan barnacles shows different responses to food

fixed and variable costs, will be important in future sex allocation

availability. Furthermore, two other factors may be related to thestudy.

lack of a trade-off between the sexes: (a) investments in functions
other than gamete production and (b) the effect of resource bud-

AC K N OW L E D G M E N T S

get. Hermaphrodites often invest their resources to factors otherWe thank Ariana Gaskin, Kota Sawada, and Hiroaki Tamura for pro-than gamete production, such as parental care and mating behavior

viding helpful comments on this research. We are grateful to Atuya(Baeza, 2007; Lorenzi, Schleicherová, & Sella, 2006). For example,

Miyajima (University of Hokkaido) and laboratory members, espe-

there exists a trade-off between male behavior and egg production

cially, Kanoko Ogawa, Hajime Morimura, and Banryu Murakami forin the polychaete worm Ophryotrocha diadema (Picchi & Lorenzi,

support during this experiment. We also appreciate the reviewers for

2019; Santi, Picchi, & Lorenzi, 2018). In addition, a large varia-

these insightful comments and Chiaki I. Yasuda and Sachi Yamaguchition in reproductive resource budget among individuals may maskfor input. Part of this study was supported through a Grant-in-Aid

the underlying trade-off (Schärer, Sandner, & Michiels, 2005; Vanfor Scientific Research 18K14506 (Early-Career Scientists) and

Noordwijk & de Jong, 1986). However, we made an effort to control

19H03284 (B) from the Japan Society for the Promotion of Sciencesuch effects statistically by incorporating body size and attachment(JSPS). This work was funded by a grant from the Research Institutesite (scallop shells) in the model.

of Marine Invertebrates Foundation (RIMI), Japan.

Previous empirical studies on barnacles evaluated sex allocation without considering fixed cost, or both variable and fixed costs

C O N FL I C T O F I N T E R E S T

were incorporated together as resource allocation to male function

The authors declare that they have no conflict of interests.

(Hoch & Levinton, 2012; Kelly & Sanford, 2010; Raimondi & Martin,
1991). This is acceptable as long as the fixed cost (a) does not vary

AU T H O R C O N T R I B U T I O N S

much among individuals and (b) is negligible (Schärer, 2009). HochSW, YY, and MT designed the experiment; KM developed theand Levinton (2012) also used the total male investment in S. bal-method and MT collected and analyzed the data with help fromanoides and B. glandula after ensuring that the inclusion of penis

SW; MT and YY led the writing of the manuscript. All authors

weight did not change the outcome of the results, and there were

contributed critically to the drafts and gave final approval forno differences in the mean weight of the penis among treatments

publication.

or sites. In our study, penis weight accounted for on average 1.24%(=1.00/80.75 × 100) of the total male output. Nevertheless, fixed

DATA AVA I L A B I L I T Y S TAT E M E N T

cost varied greatly with body size and both aspects of group size. As

All data that support this article are accessible in the Dryad reposi-

for the fixed cost, when penis size is not negligible and there is vari-

tory: https​://doi.org/10.5061/dryad.9zw3r​229t.

ation in penial morphology, this investment cannot be considered as“fixed” as it varies with the environment (Schärer, 2009). The cost

ORCID

of building and keeping the penis can be large for barnacles living in

Masami M. Tamechikawave-exposed shores or in dense populations (Hoch, 2008; López,Yoichi Yusa

https://orcid.org/0000-0002-2233-5560

https://orcid.org/0000-0001-5743-3657

Catalán, Barriga, & López, 2014; Neufeld & Palmer, 2008). Moreover,as some barnacles mature at very small sizes (dwarf males; Yusa,

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APPENDIXFIGURE A1
per shell

Histogram of the number of barnacle individuals

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ict016

How to cite this article: Tamechika MM, Matsuno K, Wada S,
Yusa Y. Different effects of mating group size as male and as
female on sex allocation in a simultaneous hermaphrodite. Ecol
Evol. 2020;10:2492–2498. https​://doi.org/10.1002/ece3.6075