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6. Based on the results of their study, Fisher and Hoekstra posit that sperm aggregation could be involved in reproductive isolation between P. maniculatus and P. polionotus. (a) Please classify the type of barrier they are referring to (i.e. premating, postmating-prezygotic, or postzygotic).
Among the extraordinary adaptations driven by sperm competi- tion is the cooperative behaviour of spermatozoa'. By forming cooperative groups, sperm can increase their swimming velocity and thereby gain an advantage in intermale sperm competition Accordingly, selection should favour cooperation of the most closely related sperm to maximize fitness'. Here we show that sperm of deer mice (genus Peromyscus) form motile aggregations, then we use this system to test predictions of sperm cooperation. We find that sperm aggregate more often with conspecific than heterospecific sperm, suggesting that individual sperm can dis- criminate on the basis of genetic relatedness. Next, we provide evidence that the cooperative behaviour of closely related sperm is driven by sperm competition. In a monogamous species lacking sperm competition, Peromyscus polionotus, sperm indiscrimi- nately group with unrelated conspecific sperm. In contrast, in the highly promiscuous deer mouse, Peromyscus maniculatus, sperm are significantly more likely to aggregate with those obtained from the same male than with sperm from an unrelated conspecific donor. Even when we test sperm from sibling males, we continue to see preferential aggregations of related sperm, in P. maniculatus. These results suggest that sperm from promiscuous deer mice discriminate among relatives and thereby cooperate with the most closely related sperm, an adaptation likely to have been driven by sperm competition. In species where females mate promiscuously, sperm competition- in which ejaculates of multiple males compete for fertilization within the female reproductive tractcan drive the evolution of physio- logical, morphological and behavioural adaptations. Although ferti- lization success is largely determined by the relative number of spermatozoa inseminated by competing males, additional sperm traits can also improve fertilization ability. Sperm swimming velocity, for example, is positively correlated with fertilization success in a number of vertebrate species. Morphological adaptations can contribute to improved speed", or more rarely, individual sperm form cooperative aggregates as they move through the female tract'. Spermatozoa of muroid rodents seem uniquely suited for this task; most possess a falciform head with an apical hook" that is thought to facilitate the formation and/or stabilization" of sperm aggregations. In the wood mouse (Apodemus sylvaticus) and Norway rat (Rattus norvegicus), sperm form groups or 'trains' of up to hundreds of cells that exhibit increased swimming velocity in vitro. Here we report cooperation in the sperm of Peromyscus mice and describe a unique adaptive beha- viour: the ability to recognize sperm based on genetic relatedness and preferentially cooperate with the most closely related sperm. Upon initial release from the cauda epididymis, spermatozoa of deer mice, P. maniculatus, are highly motile (29096 progressively motile) single cells, yet within one minute the cells begin forming motile aggregations of 2-40 cells (Supplementary Movie 1), and con- tinue forming groups for approximately one hour in vitro. Aggregates Department of Organismic and Evolutionary Biology, Museum of Comparative Zoology, begin to disperse after approximately 40 min, and by 3 h dispersal is complete. Sperm cells form groups by attaching to one another at the sperm head (Fig. 1a) or head hook to midpiece (Fig. 1b). Aggregates display significantly greater swimming velocity (127.4±3.8 um s (s.c.m.), individuals 10, thotal aggregates 50) than single cells (109.8±3.7 um s (ts.e.m.), ndividuals 10, hotal celle 50; t-3.028, P=0.0039). Thus, in this species characterized by a highly promiscuous mating systems and multiple-paternity litters", sperm groups may gain a fertilization advantage in competitive environments, as they are able to migrate through the female repro- ductive tract at a greater speed. Cooperation, however, may also be a risky strategy for sperm, as a portion of cells in a motile aggregation may undergo a premature acrosome reaction, rendering them unable to fertilize the oocytes'. Although a sperm achieves the greatest fitness advantage with a successful fertilization (direct fitness), it can still improve the probability of transmitting its genes by aiding related Figure 1 Images of Peromyscus sperm in BWW medium. a, b, Phase contrast image (1,000x) of P. maniculatus sperm aggregates attached at sperm heads (a) and head hook to midpiece (b). c, Image (400x) of motile P. polionotus sperm aggregate stained with 400 nM Tubulin Tracker. d, Image (1,000x) of aggregated sperm observed in a mixture containing sperm from one P. maniculatus male and one P. polionotus male (midpiece of P. maniculatus sperm is stained with MitoTracker Red 580 and midpiece of P. polionotus sperm with MitoTracker Green FM). Center for Brain Science, Harvard University, Cambridge, Massachusetts 02138, USA.
sperm (indirect fitness)". The benefit of aggregating should therefore depend on the genetic relatedness of the cells involved; sperm that are able to recognize relatives, and preferentially associate with them, should gain a selective advantage in a competitive environment. If sperm are able to identify and group with related cells, this should be most pronounced in interspecific pairings, thus we first investigated the ability of sperm to discriminate between conspecific and hetero- specific sperm. In an in vitro assay, we mixed live sperm obtained from a P. maniculatus male and a male from its sister species, the oldfield mouse (P. polionotus, Fig. 1c), each uniquely labelled with a fluorescent probe. Approximately 83% of aggregates included both P. maniculatus and P. polionotus sperm (for example, Fig. 1d), however we found that overall groups were composed of significantly more conspecific sperm than expected at random (₁4 8.68, P<0.0001, n=15; Fig. 2a). Spermatozoa of the two species are morphologically similar, yet not identical, and both are capable of cross-fertilization and hybridiza- tion. These species no longer naturally co-occur, yet for sympatric species the ability to identify and cooperate with related sperm may provide a mechanism for conspecific sperm precedence, whereby con- specific sperm, presumably adapted to the female reproductive tract, cooperate and outcompete heterospecifics". Next we examined intraspecific sperm recognition and took advantage of variation in Peromyscus mating systems to test the pre- diction that sperm competition drives preferential cooperation among closely related sperm. In P. maniculatus, males often copulate with a female in overlapping series; and in semi-natural enclosures, copulations with multiple males can occur less than 1 min apart", providing an opportunity for sperm of different males to interact. When we mixed sperm from two unrelated conspecific P. maniculatus males, each labelled with a unique fluorescent probe, we found that sperm group significantly more often with sperm of the same male than expected at random (t, 11.963, P<0.0001, n-8; Fig. 2b). In contrast to the promiscuous deer mouse, its monogamous sister- species, P. polionotus, experiences little if any sperm competition". Proportion of same colour sperm uoga.60e ue u 0.85 0.80 0.75 0.70 0.65 0.60 0.55 0.50 Control Treatment NS NS Heterospecifics Unrelated ILI Unrelated Related. P. maniculatus P polionotus P maniculatus In a study of 220 wild-caught P. polionotus females, none showed genetic evidence of multiple paternity". Moreover, relative testis size is three times smaller in P. polionotus than in P. maniculatus, consistent with the well-established relationship between relative testis size and sperm competition. In contrast to the behaviour of P. maniculatus sperm, we found that aggregations form indiscriminately in assays involving sperm of two unrelated conspecific P. polionotus males (ty=0.627, P=0.547, n-8; Fig. 2c). Our data, therefore, support the prediction that sperm competition, and thus mating system, drives the evolution of preferential cooperation among related sperm cells. Why then do sperm of P. polionotus aggregate at all if the species is strictly monogamous and lacks sperm competition? Sperm coopera- tion may benefit monogamous males if the increased swimming velocity of aggregated sperm allows them to migrate faster through a potentially hostile female tract' or manoeuvre around obstacles while travelling to the fertilization site". Consistent with these theories, in the wood mouse, A. sylvaticus, >95% of sperm found in the uterine lumen following natural matings were aggregates, not single cells, in over half of the females tested'. Alternatively, it is possible that promiscuity is the ancestral reproductive strategy in Peromyscus and sperm aggregation arose before the divergence of P. maniculatus and P. polionotus, yet the discriminating ability arose after the divergence. Owing to limited dispersal and typically high population densities of P. maniculatus in nature", a female may often mate with multiple males that are closely related to one another. To examine the extent of discriminatory ability of P. maniculatus sperm, we tested the inter- action of sperm from full-sibling littermates. Again we found a greater proportion of sperm from the same male grouped together than was expected at random (1, 3.782, P=0.007, n8; Fig. 2d). Moreover, we found that the average proportion of aggregated cells from the same male does not differ significantly when we mixed sperm of two siblings versus two unrelated conspecifics (t=1.447, P=0.191; Fig. 2, horizontal line) or two heterospecifics (t = 0.412, P=0.693; Fig. 2, horizontal line), suggesting that P. maniculatus sperm discriminate equally against sperm of a brother and a hetero- specific. Such highly selective aggregations are similar to cooperative phenotypes seen in social amoebas (Dictyostelium discoideum) and budding yeast (Saccharomyces cerevisiae)". In these microbes, a single gene encodes for a homo- philic adhesion protein, suggesting that sperm aggregation may also operate under a simple genetic mechanism. In competitive environments, the male (diploid genome) benefits if any one of his sperm fertilizes the egg; thus selection should favour adaptations that help his sperm reach the egg, such as sperm aggrega- tions. The addition of any motile sperm, related or not, to an aggregate should increase the speed at which his sperm reach the egg; however, as all but one sperm fail to fertilize each oocyte, the chance that his . sperm will fertilize the egg decreases as the number of unrelated cells. join the group. From the sperm's perspective (the haploid genome), there is also a benefit to joining a group of related or unrelated sperm to improve its swimming speed. However if that sperm is unable to
in an aggr Proportion of sam 0.85- 0.60- 0.55- 0.50- Unrelated Heterospecifics Unrelated Related P. maniculatus P. polionotus P. maniculatus Figure 2 Preferential sperm aggregations. Proportion of cells in a sperm aggregate labelled with a single probe (data show means.e.m.). Black bars indicate treatments in which sperm of one male is labelled green and sperm of another male is labelled red; white bars indicate controls in which sperm from a single male is labelled with both red and green probes. Pairwise comparisons between treatment and control groups are by paired two-tailed t-test with Bonferroni correction, asterisks indicate P<0.01. Labels below bars show treatments (left to right): heterospecific mixtures containing live sperm from one P. maniculatus male and one P. polionotus male (n=15); conspecific mixtures of sperm from two unrelated males of the promiscuous P. maniculatus species (n=8); conspecific mixture of sperm from two unrelated males from the monogamous P. polionotus (n-8); and conspecific mixture of sperm from two full-sibling P. maniculatus males (n=8). Horizontal lines above bars indicate comparisons between aggregations of sperm by unpaired two-tailed t-tests with Bonferroni correction NS, non-significant. in competitive environments, the male (diploid genome) benefits if any one of his sperm fertilizes the egg; thus selection should favour adaptations that help his sperm reach the egg, such as sperm aggrega- tions. The addition of any motile sperm, related or not, to an aggregate should increase the speed at which his sperm reach the egg; however, as all but one sperm fail to fertilize each oocyte, the chance that his sperm will fertilize the egg decreases as the number of unrelated cells join the group. From the sperm's perspective (the haploid genome), there is also a benefit to joining a group of related or unrelated sperm to improve its swimming speed. However if that sperm is unable to fertilize the egg, it can still increase the probability of transmitting its genes by aiding related sperm (inclusive fitness). Thus selection on both the diploid and haploid genomes should favour recognition and cooperation among related cells if fitness benefits (direct and indirect fitness) outweigh costs (for example, sperm incapacitation due to a premature acrosome reaction). Although it is unclear whether the genotype of the diploid male or haploid sperm determines the observed aggregation phenotype, our results suggest that relatedness matters for cooperative behaviour in P. maniculatus sperm. In this system, sperm discriminate against those from a sibling where the probability of sharing a gene is 25%, and preferentially aggregate with sperm from the same male where the probability is 50%. By contrast, in the monogamous P. polionotus, sperm group indiscriminately with unrelated conspecifics. Our results, therefore, support the long- standing prediction that sperm competition drives the evolution of ©2010 Macmillan Publishers Limited. All rights reserved