Brood parasitism is known in birds, fish and insects. This phenomenon is associated with complex adaptations by the brood parasite to use the host, as well as counter adaptations by the brood host to eliminate the deleterious effect caused by the parasites, which can lead to coevolutionary arms races between brood parasites and hosts (Davies et al., 1989). Thus, brood parasite and host systems are intriguing subjects for evolutionary studies. Bitterling fish are obligatory brood parasites of mussels. Specifically, they deposit their eggs in the gill chambers of mussels using a long ovipositor. The ovipositor must be long enough to transfer eggs to the gill cavity through the siphon of a mussel, and thus, should be subject to selection via oviposition success. In addition, egg shape can have a vital role in securing the lodging of eggs in the gill cavity until hatching because mussels respond to bitterling oviposition by rapidly contracting their valves and expelling the eggs. Eggs that are expelled before completing development are quickly eaten or die (Kitamura, 2005). Notably, there is marked variation in egg shape among bitterling species (Nakamura, 1969), which may be adaptive in ensuring eggs are able to remain wedged within the host gill. Mussel gill structure varies among species, and it is hypothesized that bitterling egg shape is under selection to match the locally available mussels (Liu et al., 2006; Reichard et al., 2007). Some species of bitterling are host specialists, while others are generalists that use a range of host species (Smith et al., 2004; Kitamura, 2007; Reichard et al., 2007).
In this study, we focus on the tabira bitterling, Acheilognathus tabira, which exhibits varied ovipositor length (OL) and egg shape among local populations (Arai et al., 2007). This species occurs as disconnected populations on the islands of Kyushu and Honshu of the Japanese archipelago. Tabira bitterling use several mussel species in the Unionidae and Margaritiferidae families (order Unionida) (Hirai, 1964; Kondo et al., 1984; Fukuhara et al., 1998; Kitamura, 2007; Oshiumi & Kitamura, 2009; Kitamura & Morosawa, 2010). Most tabira populations use mussels of either the Unioninae or Anodontinae subfamilies of the family Unionidae, and the differences in host taxa likely influence variations in OL and egg shape. The primary importance of the length of the ovipositor is to enable it to reach the appropriate part of the gill cavity for oviposition through the exhalant siphon of the mussel (Kitamura, 2006a,b). Therefore, OL is likely to be selected for in response to the internal structure of particular mussel species. Shell size is generally larger for Anodontinae species (average shell size, 70–90 mm; maximum, ∼200 mm) than for those of Unioninae (average shell size, 50–70 mm; maximum, ∼100 mm; Kondo, 2008; Masuda & Uchiyama, 2010; Kitamura, 2011). In other bitterling species in Japan, populations using anodontine mussels possess longer ovipositors than those using unionine mussels (J. Kitamura, unpublished data; Kitamura, 2006c, 2007; Kitamura et al., 2009a,b; Oshiumi & Kitamura, 2009; Kitamura & Nishio, 2010). Therefore, a longer ovipositor may be selected for in bitterling populations using Anodontinae. In tabira bitterling, deposited eggs are found mainly in the mussel’s suprabranchial cavity, which is connected to the exhalant siphon (Kitamura & Morosawa, 2010). Because the suprabranchial cavity is also larger in Anodontinae than in Unioninae, a more elongated egg shape with a larger surface area may have been selected for in tabira populations using Anodontinae to enable firm attachment of the egg to the cavity wall, thus reducing the probability of being expelled. The larger egg surface area would enhance the contact with cavity wall and the effect of adhesive chemicals secreted from the egg (Nakamura, 1969; Aldridge, 1999). Japanese bitterling species that use small Unionidae usually produce round eggs, whereas those using large Anodontinae exhibit elongated or uniquely-shaped eggs (Nakamura, 1969; Kondo et al., 1984; Kitamura, 2005, 2006c, 2007; Kitamura et al., 2009a,b; Oshiumi & Kitamura, 2009; Kitamura & Morosawa, 2010; Kitamura & Nishio, 2010). The elongated egg shape may bear some cost in terms of its ability to be deposited into small shells as well as a reduction in the total number of eggs deposited per oviposition trial. Thus, an elongated egg shape may only be advantageous when bitterlings use large-sized mussels. Some eggs may eventually settle in the interlamellar space of the gills. The structures of the interlamellar space and septum differ between Anodontinae and Unioninae (Liu et al., 2006) and among species of Unioninae (Kondo, 2008), although it is difficult to predict how these structures affect egg shape evolution. Here, we tested whether the evolutionary changes in these reproductive features coincide with host changes, as predicted above, using a robust phylogenetic tree of tabira bitterling and comparative phylogenetic analysis.