Complex Male Mate Choice in Marine Snails Littorina
Sara Hintz Saltin
Licentiate thesis Department of Marine Ecology
University of Gothenburg
Till Mamma, Pappa och Hanna
Abstract
The ability to recognise potential mates and choose the best possible mating-partner is of fundamental importance for most animal species. This thesis presents studies of male mate choice within the genus Littorina. Males of this genus are sometimes observed to initiate mating with other males or with females of other species. How such suboptimal mating patterns can evolve is the theme of this thesis. In one study we investigated pre-copulatory- and copulation behaviour in L. fabalis and between this species and its sister-species L.
obtusata. We found that males preferred to mount and mate with large and more fecund females rather than small females. Males also preferred to track the largest females mucus trails even though these were trails from another species (L. obtusata) although cross-matings were interrupted before completion.
In a second study we found that males of three species (L. littorea, L. fabalis and L. obtusata) preferentially followed female trails. This suggests that females add a “gender cue” in the mucus. In the forth species, L. saxatilis, males followed male and female trails at random.
Along with experimental evidence for high mating costs and abilities for male L. saxatilis to detect females of a related species, this suggests a sexual conflict over mating frequency. To reduce number of matings females avoid advertising their sex by disguise their mucus. The reason for the different species strategies is that L. saxatilis lives in much denser populations than the other species and therefore are the least likely to be sperm-limited. Instead, females probably get more than enough matings and disguise their trails in order to reduce the number of the costly matings, thus letting the males search blindly for mates.
Key words: Mate choice, size preference, fecundity, Littorina fabalis, Littorina obtusata, Littorina saxatilis, intraspecific mating, interspecific mating, reproductive barrier, trail- following, mating behaviour, sexual conflict
Populärvetenskaplig sammanfattning
Förmågan att känna igen och välja en lämplig partner är viktig för djur eftersom det påverkar dess möjligheter att fortplanta sig. Detta är därmed en viktig del av evolutionen av arter. Den här avhandlingen är en studie av partnerval inom familjen Littorina, som är en grupp marina snäckor (strandsnäckor). Hanar i den här familjen kan ibland inleda parning med andra hanar eller honor av en annan art. Hur sådana märkliga beteenden kan uppstå är temat för den här avhandlingen som består av två studier.
I den första studien undersökte vi parningsbarriärer mellan systerarterna L. fabalis och L.
obtusata genom att studera deras parningsbeteende. Vi fann att hanar föredrog att bestiga och para sig med stora och mer fertila honor hellre än små honor. Hanarna följde också gärna slemspåren från stora honor trots att de kom från honor av en annan art, L. obtusata. Därför verkar det som att hanarna inte kan avläsa, från spåret, vilken art som har lagt spåret. Däremot så var parningarna mellan arterna färre och kortare vilket tyder på att det finns någon
artigenkännings mekanism som verkar vid närkontakt.
I den andra studien så presenterar vi data som tyder på att det finns en sexuell konflikt mellan könen hos arten L. saxatilis. Vi fann att hanarna hos övriga tre svenska arter (L. littorea, L.
fabalis och L. obtusata) kunde skilja på hon- och hanspår och att de hellre följde honspår. Det tyder på att det finns någon signal i honornas slemspår som hjälper hanarna att identifiera deras spår och därmed lättare hittar en hona att para sig med (genom att följa slemspåret). Hos den fjärde arten, L. saxatilis, följde hanarna däremot alla spår slumpmässigt, vilket kan
förklaras med att honorna av denna art inte avger någon ”honsignal” i slemspåren och då kan inte hanarna skilja på hon- och hanspår. Anledningen till de olika strategierna är att L.
saxatilis lever i mycket tätare populationer och risken att få för lite parningar är minimal.
Tvärt om så får honorna antagligen mer än nog med parningar och genom att låta bli att skylta med att de är honor så kan de minska kostnaden genom att reducera antalet övertaliga
parningarna som en följd av att hanarna söker i blindo, bland både han- och honspår, efter en partner.
Littorina saxatilis. Foto: Patrik Nilsson
This thesis is based on the following papers:
Paper I:
Saltin S. H, Blom E. L, Johannesson K (2010) Pre-mating behaviours in males of a marine snail; the largest female may not be the best. Manuscript.
Paper II:
Johannesson K, Saltin S. H, Duranovic I, Havenhand J. N, Jonsson P. R (2010) Indiscriminate Males: Mating Behaviour of a Marine Snail Compromised by a Sexual Conflict? PLoS ONE 5(8): e12005. doi:10.1371/journal.pone.0012005
Table of contents
Introduction ...2
Mate choice ...2
The study system of Littorina ...3
Reproductive barriers between closely related species...3
Sexual conflicts ...5
Conclusions ...6
References ...8
Acknowledgements...10
……….
Paper I Paper II
Introduction
Evolution of reproductive strategies and species recognition are central to all sexual reproducing species. In most species mate choice is part of these processes.
This thesis will focus on male mate choice in the genus of Littorina and the aim is to try to explain how the sometimes seemingly suboptimal male mating behaviours can evolve. As will be presented here male mate choice can be complicated by size-preference, which can result in maladaptive interspecific copulations between sister-species. Females that disguise their gender due to a sexual conflict can also hamper male mate search, resulting in males following and initiating copulation with other males.
Mate choice within the genus of Littorina is as far as we know today exclusively male mate choice. Males choose both which female to track, which to mount and which to mate with, while the female stays more or less passive during the process. A complete lack of female choice is very unexpected and this observation will be investigated further in future studies.
In this thesis I will present the main results of two studies (Paper I and II) and discuss these results in the context of conceptual models and earlier empirical findings. Starting with a brief introduction of the relevance of studying mate choice and the biological characteristics of the genus of Littorina.
Mate choice
From an evolutionary perspective, mate choice can be the most important decision in an individual’s life. Some animals spend their whole life with one partner (Harris 1973) while most animals (not least invertebrates) meet their partner only briefly during mating. Either way choice of mate is fundamental, in particular if the entire life reproduction is invested in one or a few mating opportunities (Foellmer & Fairbairn 2003). Traditionally researchers have been focusing on female mate choice. The reason is that females invest more in each mating in terms of size of the gametes (Bateman 1948) and sometimes parental care (Trivers 1972). With higher investment follow choosiness since the loss from a bad choice will increase with increasing investment. A good mate choice, on the other hand, can increase the fitness of the offspring through enhanced survival and reproductive success for sometimes several generations.
Besides investments, there can also be costs involved in mating ranging from costs of mate search to increased mortality during mating (Daly 1978). These costs are not exclusive for the female; the male also often suffer from costs of mating and both male and female tend to be more choosy if there is a high cost involved in mating (Ridley 1983).
Discriminate mating (sexual selection) have given rise to many of the most remarkable characters we can see in nature with numerous examples from the avian family, e. g. the tail of the peacock (Marion et al. 1991) and the widowbird (Andersson 1982), or extensive colouration and body-shape in families of teleost fishes (Kodric-Brown 1985; Quinn & Foot 1994). During the evolution of species, sexual selection has been a major selection
mechanism influencing morphology, physiology and behaviour traits in many species (Andersson 1994).
Of course a fundamental and obvious strategy in mate choice must be to mate with
individuals of the opposite sex and of the same species. To achieve this, mate recognition can sometimes be a complex process involving several cues and stimuli (Hankison & Morris 2003). This complexity serves to prevent interspecific matings with the risk of producing inferior hybrids (Coyne & Orr 2004). However, mate choice can sometimes be further
complicated by conflicting interests of preferable traits and species recognition or by sexual conflicts between the sexes, and this is the theme of my thesis.
The study system of Littorina
The 19 species of the genus Littorina are widely spread in the subarctic and temperate regions of the Northern Hemisphere (Reid 1996). They all inhabit the littoral-zone, but some species live mostly submerged on seaweeds or rocks, while other species are confined to rocks above mean-water and up into the splash-zone. Snails in the upper littoral zone can have long intervals of inactivity, sometimes dried up for weeks during calm summer days (Stafford &
Davies 2004). When wetted they move by muscular movements in the foot gliding on secreted mucus. This enables them to actively search for food and to perform mate search.
The secreted mucus remains as a trail after the snail, and other snails may follow in the same trail. From previous studies we know that snails can gain information from mucus trails, e.g.
gender identity (Johannesson et al. 2010), which enables the males to use the mucus trails during mate search.
In Sweden there are four species: Littorina littorea, L. saxatilis, L. fabalis and L. obtusata. All four species have separate sexes and internal fertilisation (Reid 1996). Littorina littorea, which is the species most distantly related to the other three, is the only species in the genus that has pelagic larvae and these are released during spring at the end of the mating period (Reid 1996). Littorina saxatilis is ovoviviparous and is reproductively active year-round, while L. fabalis and L. obtusata have restricted mating seasons from spring to fall, during which they mate and lay egg-masses (Reid 1996). The two latter species are considered sister- species that diverged about 1 Mya (Kemppainen et al. 2009). They are both similar in shape with a flat spire, but adult L. obtusata are mostly larger than L. fabalis and the former also lives several years while the latter lives only one to two years (Reid 1996). The only diagnostic differences between the two species are found in the reproductive organs with males of L. fabalis having an extended tip on the penis compared to L. obtusata (Reid 1996) (Fig. 1).
Both L. saxatilis and L. fabalis are polymorphic species, that is, different ecotypes are adapted to different environments. The ecotypes of L. saxatilis in Sweden comprises of the smaller and more fragile exposed morph (e-morph) living on wave-exposed cliffs and the larger and more robust sheltered morph (s-morph) living in micro-sheltered environments behind and beneath boulders. Littorina. fabalis lives on fucoid algae and inhabit sheltered waters, where the “Small-Sheltered” morph (SS-morph) lives, to moderately exposed shore where the
“Large-Moderate” morph (LM-morph) occur. These morphs are different in morphology, in shell thickness and size, and to a large part these are inherited differences that seem to evolve easily due to strong potentials of local adaptation (L. saxatilis: Janson 1982; Janson & Ward 1984; Johannesson & Tatarenkov 1997, L. fabalis: Reimchen 1982; Tatarenkov &
Johannesson 1998, Kemppainen et al. 2005).
Reproductive barriers between closely related species
Speciation is a key area in biological research and also one of the most debated areas, ever since 1859 when Charles Darwin published “On the Origin of Species by means of natural selection” (Mayer 1942; Maynard Smith 1966; Otte & Endler 1989; Coyne & Orr 2004). To be able to understand speciation we first need to address the question of how to define species. The definitions of species are many, but the most common species concept, The Biological Species Concept states that: “Species are groups of actually or potentially
interbreeding populations, which are reproductively isolated from other such groups” (Mayr
study of reproductive barriers an important part of speciation research. Reproductive barriers can be divided in three major groups (Coyne & Orr 2004):
I. Pre-mating isolating barriers, which are barriers that prevents gene flow before sperm or pollen is transferred. It could be due to behavioural differences in, for example, courtship or due to mechanical isolation where the genitalia are so different in the two species that mating is not completed. It can also be due to spatial or temporal separation of the two species.
II. Postmating, prezygotic isolating barriers act after sperm or pollen transfer, but before fertilization. It could be due to behavioural differences during copulation that prevents fertilization or to incompatible gametes.
III. Postzygotic isolating barriers are barriers that act after the formation of a zygote, either by zygote mortality, hybrid inviability or sterility, or hybrid breakdown.
In most cases, when two species have split far back in time, there are many different
reproductive barriers and it is difficult to say which barrier was the first and most important for the split. This is the reason why it is good to study speciation using closely related species, or even partially isolated ecotypes, as model organisms. Littorina is therefore an excellent genus to study to learn more about speciation. It is especially interesting to focus on L. fabalis that gives us the unique opportunity to study both reproductive barriers between the sister- species pair (L. fabalis and L. obtusata) as well as reproductive barriers within species, between ecotypes.
In Paper I we studied reproductive barriers between L. fabalis and L. obtusata. In that paper mate choice had a central role since we focused on males’ choice of females and their ability to detect conspecific females both during mate search and mating. We found that there are pre-mating isolating barriers between the species acting during direct contact, although L.
fabalis males seem to be unable to differ between species from mucus trails. On the contrary, males rather followed females of L. obtusata than conspecific females. This could be
explained by males’ preference for large females, since L. obtusata is larger than L. fabalis. In Paper I we also tested and found that L. fabalis males indeed preferred mating with large females of its own species, which concurred with previous studies of other species in the genus showing preference for large females (L. littorea: Saur 1990; Erlandsson &
Johannesson 1994) as well as observations of Spanish L. fabalis mating patterns from the field (Rolán-Alvarez et al. 1995). Even though males could not discriminate between species from mucus trails they could do so in close contact with females. That is, we found that L.
fabalis males initiated mating with L. obtusata females less often compared with conspecific females - and heterospecific copulations were also interrupted after just a few minutes.
Although the mechanism of species recognition remains unknown, it has been observed that males move in a stereotypic way after mounting a female (Saur 1990), which might be a way for males to evaluate their partner and prohibit interspecific copulations. Since we also found that interspecific copulations were interrupted, it is likely that there are species recognition mechanisms acting during copulation. One of the most pronounced morphological difference between the species, and the only diagnostic character, is the dissimilarity in male genitalia, where male L. fabalis have an extended tip that is lacking in male L. obtusata (Fig 1).
Although it is speculative, it is possible that the dissimilarity in genital morphology is involved in species recognition.
Fig. 1. Male genitalia in L. obtusata (to the left) and L. fabalis (to the right). Source: Reid 1996.
In light of the results from Paper I it is intriguing that males are unable to distinguish
between species early in the process; they would obviously benefit from being able to decide the species already from the mucus trail, and in this way avoid following females of the wrong species. One explanation could be that this is not a big problem in nature, e. g. even though their geographic distributions overlap, they could have a microallopatric separation in the shore caused by microscale ecological and behavioural differences. Another possible explanation could be that a species-specific cue has not yet evolved since the colonisation of Swedish waters have happened quite recently (after glaciation, about 10 000 years ago) from allopatric sites where such trait would not be selected for.
Sexual conflicts
Various types of sexual conflicts are found in nature, and a common type is if the number of matings to maximize the individuals´ fitness differs between the sexes. The basis for sexual conflicts originate from the definition of the sexes; males having numerous and small gametes whereas females have few and large gametes. Hence females invest more in each offspring than do males (Bateman 1948). Moreover, males generally have the potential ability to produce many more offspring than females (Parker & Simmons 1996). Males therefore often benefit from repeated matings, while females having received enough sperm to fertilize all her eggs may not benefit from further mating. Sometimes females can obtain higher fitness by receiving additional sperm for example by increasing the genetic diversity among the offspring (Jennions & Petrie 2000). There are also other more specific cases where females receive, for example, a nutrition resource by additional matings (Boggs 1995) or where sexual contact is important for social interactions (Savage-Rumbaugh & Wilkerson 1978). But often the costs of the matings exceed the benefit of the mating at a lower mating frequency for females than for males resulting in a sexual conflict over mating frequency (Fig 2).
Fig. 2. Generally individual fitness for females start to decrease at lower mating frequency compared to males.
In Paper II we argue that the pattern of mating behaviour we observe in four species of the genus Littorina can be explained by an underlying sexual conflict over mating frequency between the sexes in one of the species. In three of the four Swedish species of Littorina (L.
fabalis, L. obtusata and L. littorea) males have the ability to determine if a trail belongs to a male or a female. How this is done is still unknown, but the most likely explanation is that there is a gender specific chemical in the mucus that the males in the three species can detect.
In the fourth species, L. saxatilis, the males seem to be unable to distinguish between conspecific males and females. It is likely that the common ancestor to all four species had this ability to distinguish between male and female mucus trails, but for some reason L.
saxatilis has lost this ability. This is surprising since being able to rule out half of the trails encountered and only follow female trails seems to be a highly adaptive skill for males. The explanation to this seemingly strange pattern lies in the life-history of these species. The three species that have the ability to detect females (L. fabalis, L. obtusata and L. littorea) have higher or much higher egg production and shorter seasons of mating than L. saxatilis and they also live at lower population densities than L. saxatilis. These differences in life-history all contribute to make L. saxatilis the least sperm-limited species among the four. Indeed,
females of L. saxatilis mated in the wild carries offspring sired by around 20 males (Panova et al. 2010). As the cost of mating is high (Paper II) the total cost of mating is higher in L.
saxatilis than in any other of the four species, and to reduce this cost selection has favoured females of L. saxatilis that do not revile the sex in the mucus trail. For male L. saxatilis it is still optimal to mate with as many females as possible, as males have no other possibility to increase its inclusive fitness. For females, it is extremely important to survive at least as long as she is brooding her offspring under her shell. This unbalance gives rise to a sexual conflict within L. saxatilis where males try to mate as much as possible and females try to avoid male harassment by disguising their gender. Further strength for this conclusion was provided when we found that L. saxatilis males could as well differ between mucus-trails of male and female L. fabalis. The females of this species leaved a “female mark” in their trails that male L. saxatilis still recognised and followed, showing that males if they have the chance follow
“female-marked” trails. This shows that the reason for L. saxatilis males following conspecific males and females at random are not that they do not care for searching for females, it is due to females disguising their trails to promote an overall lower mating
frequency. Such sexual conflicts and gender mimicry are not uncommon in nature, especially not in high-density populations (Burley 1981). One example is damselflies where females mimic males in colour and behaviour to reduce number of matings and harassment during mating (Andres et al. 2002). There are many other examples like this among insects and other animals, but Paper II provides us with one of few examples in a marine invertebrate species.
Conclusions
This thesis addressed male mate choice within the genus Littorina and the aim was to try to explain the maladaptive mating-behaviours sometimes observed among males. This adds on to the many examples (summarized by Coyne 2009) showing that nature is not perfect in every sense, which is one of the arguments against the counter theory of evolution – creationism that states that all organisms was created by an intelligent being.
Mate choice can influence reproductive barriers and can therefore be important for speciation.
As shown in Paper I, mate choice is an important part of the reproductive barriers between the sister-species Littorina fabalis and Littorina obtusata. This finding opens for further studies of L. fabalis mate choice focusing on populations within species, namely the different
ecotypes (SS- and LM-morph). This gives us the opportunity to study both complete and ongoing speciation within the same species, even though the existence of reproductive barriers between the morphs is yet to be investigated.
Future studies will also be focused on female mate choice since a complete lack of choice for females is highly unlikely. Even if we have no observations suggesting that females choose among the males prior to mating, cryptic female choice through sperm-selection after copulation is still one possibility.
That males follow and initiate mating with both females and males (as was reported in Paper II) seems to be maladaptive from the males point of view, but this is probably a great
advantage for females since it reduces the mating frequency and thereby also reduces the lifetime costs of mating. Similar sexual conflicts over mating frequency are likely to be found in species where females are unlikely to be sperm limited, for example, species that lives in high population densities with relatively low reproduction rate and long mating season, and for which there are high costs of mating. Although L. saxatilis is the only marine example this far on a sexual conflict over mating frequency, we would expect to find this in additional species with similar demographic characteristics and internal fertilization.
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Acknowledgements
Först vill jag tacka min superba handledare Kerstin Johannesson, som är den bästa läraren, medarbetaren och föredömet. Dessutom är hon en fantastisk fin människa och det är gaska otroligt att ha en handledare som förbihållslöst alltid sätter mig som doktorand högst på sin långa prioriteringslista. Jag skulle kunna fortsätta att skryta en halvsida till om Kerstin men jag vet att hon antagligen bara skulle påpeka att jag ska korta ner den biten eftersom det trots allt bara är rena spekulationer
Jag har ju också andra handledare. Per och Gunilla som är riktiga klippor vad det gäller statistik och experimentdesign. Det känns väldigt skönt att kunna vila på er ibland när det gäller sådana svåra men ack så viktiga bitar! Tack för all hjälp!
Jag har riktigt fina kollegor också; ingen nämnd och ingen glömd brukar det ju heta… Vill ändå passa på att skicka kramar till några speciella vänner; Anna-Lisa och Christin. Sedan finns det också några starka, vackra kvinnor som inspirerar mig; Eva-Marie, Anette, Marina, Ann, Helena, Elisabeth, Anita och Sanna. Och så doktorand-gänget förståss! Robin, Angelica, Daniel, Swantje, Geno, Finn, Micke, Erika, Per och Elin. Tack även till mina studenter som jag handlett; Martin, Sara, Emmelie och speciellt Eva-Lotta som har bidragit till den här avhandlingen!
Sist, men egentligen först; min härliga familj! Thomas och Sebastian som alltid står vid min sida och vars stöd jag inte står utan. Mycket kärlek tack för allt stöd också till storfamiljen;
Mamma och Pappa; Hanna, Marcus och Ester; Mormor och Morfar; Ingegärd, Gunnar, Anna, Emma, Anders och Lisa.
Female choice in snails….?
Paper I
Pre-mating behaviours in males of a marine snail; the largest female may not be the best
Sara Hintz Saltin*, Eva-Lotta Blom and Kerstin Johannesson
Department of Marine Ecology – Tjärnö, University of Gothenburg, SE-452 96 Strömstad, Sweden
*Corresponding author. Email: sara.hintz-saltin@marecol.gu.se
Abstract
Mate choice is central to fitness of organisms and hence a key trait in the evolution of most species. As egg production is more costly than sperm production, females are usually the most choosy sex. Nevertheless, male mate choice is expected in species where males have extensive costs of mating. In snails mate search is time- and energy consuming for the males and great benefits can be obtained from being choosy early in this process, for example when choosing a mucus trail to follow. We study male mate choice in the marine snail Littorina fabalis using video recordings and live observations of pre-copulation and copulation behaviours. Upon encounter, males preferred to mate large and more fecund females over small females. When offered a choice of different sized females they most frequently tracked mucus trails of the largest females although these were of the larger sister-species Littorina obtusata. However, copulations between the two species were rare and considerably shorter than intraspecific copulations suggesting species-recognition mechanisms that prevent interspecific copulations. Hence our study shows evidence of a trade-off for males that tend to choose female traits from large females over those of small females. Although fecundity increases by female size, there is also an increased risk to spend time and energy on tracing a female of another species.
Key words: Sexual selection, mate choice, size preference, fecundity, Littorina fabalis, Littorina obtusata, intraspecific mating, interspecific mating, reproductive barrier, trail- following, copulation time, mounting time
Introduction
Sexual selection and mate choice are of central importance in the evolution of mate
recognition systems as well as in the evolution of species (Ratcliffe & Grant 1983; Wiernasz
& Kingsolver 1992; Andersson 1994; Owens et al. 1999; van Doorn et al. 2009). The traditional view of mate choice is that females choose which male to mate with and males mate at every possible occasion. However, indiscriminate mating of the males should occur only if the benefit of indiscriminate mating is greater than the costs of mating and both male and female tend to be choosier about their mates in proportion to how much time and energy they invest in mating (Ridley 1983). In many different taxa males prefer to mate a large female rather than a small one. This is explained by the generally higher fecundity of large females (Parker 1970; Ridley 1983; Crespi 1989). Earlier studies have shown that in the marine snail Littorina there is sexual selection favouring large female size (L. littorea, Saur 1990; Erlandsson & Johannesson 1994; L. fabalis, Rolán-Alvarez et al. 1995b) and this is likely a consequence of a positive correlation between fecundity and female size (L. littorea, Hughes & Answer 1982; L. saxatilis, Janson 1985).
Littorina fabalis and Littorina obtusata are sister-species with a large overlap in their geographic distributions, as well as a partial overlap in the microgeographic distribution on the shore; L. obtusata typically prefers either upper parts or more wave-protected parts of the shore while L. fabalis dwells in the low intertidal and in both wave-protected and more wave- exposed areas. Truly sympatric distributions are found in wave-protected parts of shore and in mid-intertidal levels (Reid 1996). The two species have also overlapping breeding seasons, which stretches from spring to autumn. It can be difficult to tell the two species apart since their colour can be similar and they have overlapping size ranges and shell morphologies, although L. obtusata are generally larger than L. fabalis and the latter has a more flat apex.
The species are best discriminated from the morphology of the genital characters (Reid 1996), and in addition there are several diagnostic allozyme loci (Zaslavskaya et al. 1992;
Tatarenkov 1995; Rolán-Alvarez et al. 1995a). According to genetic data the species do not currently hybridise, although they share most of the common mitochondrial haplotypes, suggesting either that hybridization has taken place after their first separation, or that the two species still share ancestral mtDNA haplotypes (incomplete lineage sorting) (Kemppainen et al. 2009). As hybrids between the sister-species never have been encountered, it is likely that there are one or several isolating mechanisms between the two species that prevent
hybridisation. To depict reproductive barriers between sister-species is fundamental to understand how reproductive isolation can evolve between closely related species.
Littorina fabalis is a polymorphic species and two genetically differentiated morphs or ecotypes, the LM-morph and the SS-morph are found in e.g. UK, France, Norway and Sweden (Reimchen 1982; Tatarenkov & Johannesson 1998; Kemppainen et al. 2005). In Sweden, LM-morph snails are larger and live in moderately wave-exposed habitats and the
smaller SS-morph snails live in sheltered habitats, often in sympatry with L. obtusata. In the zones of species overlap interspecific matings are occasionally observed (pers. obs.).
Snails move by muscular movements in the foot and they glide on mucus that enables this way of locomotion, leaving a trail of mucus behind. It has been known for decades that snails often choose to follow other snails’ trails. The reasons for this behaviour have been addressed in experimental studies over the years and many explanations have been offered. One
explanation could be that this behaviour has evolved as an adaptation to save energy since production of mucus can be quite costly and snails can sometimes invest as much as 23-31 % of consumed energy for mucus production (Edwards & Davies 2002). Moreover, Davies and Blackwell (2007) found that snails that follow trails can save considerable amount of their own mucus production since they only have to produce 27% of the usual amount of mucus when they move in a previously laid trail. Besides being an important part of locomotion, following in another snails mucus trail has also been suggested to provide the snails with a chance to graze microalgae (Davies & Beckwith 1999) and/or bacteria (Peduzzi & Herndl 1991) attached to the sticky mucus. Trail-following have also been proposed to be important for mate finding since males use mucus trails to track females (Erlandsson & Kostylev 1995).
For example, males of three species of Littorina (L. littorea, L. fabalis and L. obtusata) follow females significantly longer then they follow males (Johannesson et al. 2010), suggesting that trail-following is linked with mate search. Most likely, trail-following have many advantages in snails, but in this study we study male Littorina fabalis tracking of female mucus trails as part of their mate-searching behaviour.
Copulating time is another factor that have been used to find out if a male prefers a certain female (Hollander et al. 2005), and the rationale behind this is that much shorter copulations are observed when males mate improper mates such as another male or a juvenile or another species (Saur 1990; Erlandsson 1998). It is also possible that the time a male spend in copula position is positively correlated to the percent of eggs fertilized by that male, as is the case in another invertebrate taxon (Parker 1970).
In this study we experimentally test L. fabalis male mate choice and we ask the question if in the first place there is mate choice, and secondly, if this choice is likely to improve male fitness. More specifically we were interested in if male L. fabalis would choose large (and more fecund) females over small females, and if so, how they then would avoid the problem of tracking (large) females of the partially sympatric sister-species (L. obtusata).
Materials and methods Trail-following experiment
To test whether male Littorina fabalis would choose between females of different size
an experiment in which male choice was assessed both as trail-following and as mounting time. Snails of L. fabalis (SS-morph, the ecotype that lives in sympatry with L. obtusata) and L. obtusata were sampled at two sites along the shores of two small islands (Långholmen and Lökholmen; west of Tjärnö Marine Biological Laboratory). Sampled females of L. fabalis were measured and sorted in two size intervals (large; 9-14 mm and small; 7-9 mm) while all females of L. obtusata was of size 13-15 mm which is normal for this species and partly overlapping in size with the largest females of L. fabalis.
In each of 18 replicate experimental runs, six males and six females were allowed to move freely in a square shaped arena (390*390 mm) and their movements were recorded with digital video over a period of 15 minutes. The six males were all the same species (L. fabalis) and these were of random sizes in the interval 6-12 mm. In each run the males were given equal chance to choose between females representing both different sizes and species as the setup of females were: two large L. fabalis, two small L. fabalis and two L. obtusata. All snails within a run were from the same locality to maintain familiarity among the snails.
The surface of the arena was wetted with seawater and rinsed carefully between each run.
Male activity (trail-following and mounting) were recorded and analysed from the video using computerised motion analysis (CellTrak for Windows, Motion Analysis Corp.). Male movements were defined as trail-following when following a female trail for more than 3 snail diameters. All other paths (including those along the edges of the arena) were excluded from the analyses. It was not possible to detect copulations with any certainty from the films due to inadequate resolution. Instead we used “mounting-time” as a proxy for the male’s time-investment in the female. Mounting time can include copulations although not all mountings lead to copulation. Mounting time was defined as the time the male stayed on a female shell after crawling up on her shell. Hence from each run we received the total distance male trackers followed female trails from the three different groups of females, as well as the total mounting time for each group of females. We pooled the results from all six males of the same run. The reason for doing this was that each trail following could not be considered as a replicate since the action of one snail could potentially affect the other snails in the same arena and hence the level of replication in our experiments was a run. We compared the result between two female groups at a time using a two-tailed binomial test.
Including three groups of females in the experiment even though we only compared two groups at a time was done to provide the males with a free choice of different females, similar to what males would encounter in nature. The null-hypothesis prior to the experiment was that males would not have any preference for any group of female (large L. fabalis, small L. fabalis or L. obtusata). Since we had prior observations that L. fabalis males readily
aggregated with L. obtusata females we wanted to keep the options for any preference open in this experiment by using a two-tailed test.
Copulation time observations
Size preference
As in nature the first experiment involved both female size and species as factors influencing male mate choice. As a complement, we performed additional experiments in which we treated one factor at a time. Thus in one experiment we tested whether male Littorina fabalis showed any size preference of conspecific females. In this experiment we measured
“copulating time”, which is a more direct estimate of mating effort than “mounting time”
used in the previous experiment. To be able to estimate copulation time we conducted direct observations of snail matings instead of using film recordings.
Prior to the experiments snails of L. fabalis were sampled, sized and sorted as described earlier. Twenty runs were performed, each including ten male and ten female L. fabalis (five large and five small, same size classes as before). All snails within a run were from the same locality. Prior to the runs the males were individually marked with nail polish so that it would be possible to follow each male. During a run, females and males were able to move freely in a circular arena (Ø 23 cm) filled with seawater. During the runs the sexual activity between males and females and the size of the female in action was noted as well as the time the pairs spent in copula position (Saur 1990). The runs lasted for one hour, but copulating pairs active when the hour had passed were followed until the male terminated the copulation. If a snail crawled out of the container it was picked up and put back in the centre of the arena. Between each run the arena was cleaned to eliminate contamination by pheromones and mucus trails.
From each run we received a total copulating time for males copulating with large females and a total copulating time for males copulating with small females. Hence the level of replication in this experiment was a run and we used a two-tailed binomial test to address the hypothesis that males would prefer to mate a female of a specific size (large or small) for longer time than the other group.
Species preference:
The second of the complementary experiments was designed to test whether L. fabalis males would prefer to mate females of their own species rather than females of L. obtusata. We also studied male L. obtusata mate choice.
In this experiment we again measured copulation time from live observations of mating pairs.
In each of six aquaria (50x40x40 cm) containing seaweed (Fucus vesiculosus and F.
serratus), fifteen snails of each sex and species (L. obtusata and L. fabalis) were placed. The aquaria were kept outdoors and supplied with running seawater. The reason for this design, compared to the design in previous experiment, was to provide a more natural environment for the interactions of the two species. Snails from different sites were kept in different aquaria to maintain familiarity among snail groups. The snails in this experiment were of
inspected one at a time in a random order and were as gentle as possible searched through not to disturb any mating couples. As soon as a couple was found mating, inter- or intraspecific, copulation time was measured.
It was only ongoing matings that were measured, if a couple started mating while the aquaria was inspected this couple was ignored. Only one aquarium at a time was inspected and as soon as all couples that were observed had finished mating the next aquarium was inspected in the same way. Thus we were not able to record the total copulation time for a mating, but the error is randomly distributed over all the observed pairs. This means that “copulation time” in this experiment is a proxy of the real copulation time, it could, nevertheless, be used as a relative measurement for comparison between interspecific and intraspecific copulations in this experiment.
To test if there was a difference in copulation time within and between species a one-way analysis of variance (ANOVA) was done In this experiment all copulations were recorded, hence we also recorded the copulations performed by males of L. obtusata although they never attempted to mate with heterospecific females. The groups compared was therefore;
male L. obtusata copulating with female L. obtusata, male L. fabalis copulating with female L. fabalis and male L. fabalis copulating with female L. obtusata. A posteriori test (Tukey´s test) was used to evaluate the differences between the three groups. The level of replication was aquaria and to avoid confounding results due to repeated measurements we used data from only one group per aquaria by randomly selecting 2 aquaria per group, in the statistical analysis. The hypothesis was that male L. fabalis would prefer to mate with females of its own species rather than with females of L. obtusata.
Results
Male L. fabalis preferences during trail-following and mounting
In the experiment where males of Littorina fabalis were accompanied by both small and large females of their own species and females of the larger Littorina obtusata there was no
significant difference between distances that L. fabalis males followed large and small conspecific females (binomial test, P = 0.5; Fig. 1). Indeed the data tentatively suggest that the males followed females of different sizes at random, although sample size of this test was smaller than in the other tests and the results should be considered as preliminary. When the males encountered a female, however, they mounted large females more frequently and/or stayed with them longer, resulting in significantly longer total mounting time in each run, compared with small females (binomial test, P = 0.001; Fig. 2).
Unexpectedly, we also found that L. fabalis males not only followed females of the sister- species L. obtusata, but they followed them significantly longer distances than they followed both small and large females of their own species (binomial test, P = 0.003; Fig. 3 and P = 0.001; Fig. 4). Mounting time was however not significantly different between interspecific
pairs and intraspecific pairs (in binomial tests of mounting time: P = 0.09 for males mounting small L. fabalis females compared to L. obtusata females, and P = 0.4 for males mounting large L. fabalis females compared to L. obtusata females, Figs. not shown).
Male L. fabalis copulation time with females of different sizes and species
In the experiment in which males of L. fabalis were mixed with females of L. fabalis of two different size-groups, copulating time was significantly longer for males mating large compared to small females (binomial test, P = 0.002; Fig. 5).
In the experiment where males and females of both species were mixed in equal numbers interspecific matings occurred (exclusively L. fabalis males that mated L. obtusata females) but was much fewer than intraspecific matings (16 % of all matings were between species).
The average copulation time for an interspecific copulation was also shorter; 5 minutes compared with 20-23 minutes for intraspecific couples. Consequently, total copulation time between species was significantly smaller than that within species (1-factor ANOVA, F2, 5 = 16.52, P = 0.02). This result is illustrated in figure 6 where we can see that the total
copulating time for both intraspecific couples is significantly higher than for mixed couples (Tukey´s test, P = 0.03).
Figure 1. Distances that male trackers followed small L. fabalis (y-axis) and large L. fabalis (x-axis) females.
Each point represents the total distance of trail-following of 6 males of one replicate run (15 min). The diagonal indicate the expectation of equal tracking distances of both groups of females. Two-tailed P-values of binomial tests are indicated.
0 50 100 150 200 250
0 50 100 150 200 250
Tracking of small L. fabalis (mm)
Tracking of large L. fabalis (mm)
Figure 2. Time (sec) that males spend mounting small L. fabalis (x-axis) and large L. fabalis (y-axis) females.
Each point represents the total mounting time of 6 males of one replicate run (15 min). The diagonal indicate the expectation of equal mounting time of both groups of females. Two-tailed P-values of binomial tests are indicated.
Figure 3. Distances that male trackers followed small L. fabalis (x-axis) and L. obtusata (y-axis) females. Each point represents the total distance of trail-following of 6 males of one replicate run (15 min). The diagonal indicate the expectation of equal tracking distances of both groups of females. Two-tailed P-values of binomial tests are indicated.
0 200 400 600 800 1000 1200
0 200 400 600 800 1000 1200
Mounting of large L. fabalis (sec)
Mounting of small L. fabalis (sec)
P = 0.001
0 100 200 300 400 500 600 700
0 100 200 300 400 500 600 700
Tracking of L. obtusata (mm)
Tracking of small L. fabalis (mm)
P = 0.003
Figure 4. Distances that male trackers followed large L. fabalis (x-axis) and L. obtusata (y-axis) females. Each point represents the total distance of trail-following of 6 males of one replicate run (15 min). The diagonal indicate the expectation of equal tracking distances of both groups of females. Two-tailed P-values of binomial tests are indicated.
Figure 5. Time (min) that males spend copulating with small L. fabalis (x-axis) and large L. fabalis (y-axis) females. Each point represents the total copulating time of 10 males of one replicate run (60 min). The diagonal indicate the expectation of equal copulating time of both groups of females. Two-tailed P-values of binomial
0 100 200 300 400 500 600 700
0 100 200 300 400 500 600 700
Tracking of L. obtusata (mm)
Tracking of large L. fabalis (mm)
P = 0.001
0 10 20 30 40 50 60 70 80
0 10 20 30 40 50 60 70 80
Copulating time with large L. fabalis (min)
Copulating time with small L. fabalis (min) P = 0.002
Figure 6. Total time for intra- and interspecific mating, with standard error bars. Post-hoc test (Tukey´s test) show significant differences (P = 0.03) between both intraspecific mating means compared with interspecific mating mean.
Discussion
In this study we found that Littorina fabalis males prefer large females since males significantly mount and mate longer with large females compared to small females. This supports earlier observations of Spanish L. fabalis mating patterns from the field (Rolán- Alvarez et al. 1995b). Similar size preference is also known from studies of other species of the genus Littorina (L. littorea, Saur 1990; Erlandsson & Johannesson 1994). A most likely explanation is that large females of Littorina are more fecund than small females and mating a large female thus result in more offspring being sired (L. littorea, Hughes and Answer 1982; L. saxatilis, Janson 1985).
Previous studies have shown that trail-following is an important way of finding a mate (Erlandsson & Kostylev 1995; Johannesson et al. 2010). In our investigations of snail trail- following behaviour we did not find any preference for size when male L. fabalis tracked different sized females of their own species. Part of the explanation could be that the size difference between the two size classes of females was too small to be appreciated by the males from the trail, although size differences were indeed detectable when mates were in physical contact prior and during mating. An alternative explanation to the lack of a choice in tracking small and large females may have been that our experiment was too small, that is, we had to few measurements of intraspecific trail-following. In contrast, L. fabalis males
0 100 200 300 400 500 600 700
IntraspeciBic copulation
for L. obtusata IntraspeciBic copulation
for L. fabalis InterspeciBic copulation for L. fabalis male and L.
obtusata female
Total copula*ng *me (min)
followed the heterospecific Littorina obtustata females significantly longer than their conspecific females. The evolutionary rationale behind this observation seems puzzling, but one explanation could be that males are unable to separate conspecific females from females of the sister-species L. obtustata from their mucus trails, and that the L. obtusata female trails were only interpreted as coming from a large (and therefore preferable) female. Alternatively, males followed these large trails with other purposes than encountering a mate, for example, feeding or moving while saving own mucus production (Peduzzi & Herndl 1991; Davies &
Beckwith 1999; Davies & Blackwell 2007). On the other hand, we would have expected also female L. fabalis to follow trails of L. obtusata for these purposes, but this was not the case.
Preference for large females together with the lack of a species specific cue in the mucus could have the side effect of occasional fallacies of tracking females of the wrong species; L.
obtusata. On the other hand, most of the time this preference for large females would
probably compensate this disadvantage by increasing fitness through favourable matings with large and more fecund conspecific females. In this context it is interesting to consider why no species specific cue in the mucus trail have evolved? If the males were able to identify their own species from mucus trails, the waste of time and energy following and mounting females of another species could have been avoided. There are plenty of examples of the existence of species-specific pheromones in gastropods (Croll 1983) as well as in other taxa (copepods:
Frey et al. 1998, fish: Plenderleith et al. 2005). Indeed, we recently found that males of several Littorina species including L. fabalis can discriminate between male and female trails from a gender-specific cue in mucus (Johannesson et al. 2010), showing that these type of presumably chemical cues have been evolved also in this genus. Notably, however, previous study found that L. saxatilis males followed female rather than male trails of L. fabalis indicating a lack of a strong species-recognition cue also between these species (Johannesson et al. 2010).
The two species Littorina fabalis and L. obtusata have a relatively recent common ancestry about 1 million years ago (Kemppainen et al. 2009). Their current geographic distributions are to a large extent overlapping, although L. obtusata is the only species present along the North American coast (Reid 1996). However, it is unclear if they speciated in sympatry or in allopatry and possibly large parts of their postglacial distribution (including Sweden) is a result of a recent colonization about 10 000 years ago from allopatric sites in which species specific cues would not be selected for. Then it is possible that such cue has not yet evolved even F it would be a favourable adaptation. An alternative explanation could be that the geographic sympatry is nevertheless accompanied by a microallopatric distribution in the shore caused by microscale ecological and behavioural differences.
In this study we also looked at copulation time and found that intraspecific couples copulated significantly longer (average 20-23 minutes) compared with interspecific couples (average 5 minutes) and the latter category was also less frequent. It seems likely that a short copulation
process driven by ciliary movements (Buckland-Nicks pers. commun.). Thus it seems as if any post-zygotic barrier that is likely to be present (as hybrids are not found) is accompanied by a pre-copulatory reproductive barrier that comes into action after the trail-following phase.
It is likely that there are one or several species recognition mechanisms working during close contact, counteracting interspecific copulation. A similar finding of heterospecific attraction due to size preference in swordtail fish (Hankison & Morris 2003) emphasises the importance of multiple cues in species recognition. In that study females rather choose large
heterospecific males when accessing size and chemical cue and only choosing smaller conspecific males provided with multiple cues (chemical and skin-pattern).
This study gives a view of the complex mechanisms contributing to reproductive barriers in early stages of speciation. Future studies of the mating behaviour of L. fabalis should also involve experimental tests of reproductive behaviours involving the two size morphs of L.
fabalis as in light of our current results size seems to be an important component of mate choice in this species as well as in other species of Littorina.
Acknowledgements
We thank Per Jonsson, Mats Lindegarth and Gunilla Toth for help with the statistical analyses. We would also like to thank the Motion Analysis Corp, Santa Rosa, CA for their support with software used for trail-following analysis. This work was performed within the Linnaeus Centre for Marine Evolutionary Biology (http://www.cemeb.science.gu.se/).
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