Work on the Lake Victoria cichlids Pundamilia nyererei (red dorsum males, deeper water), Pundamilia pundamilia (blue males, shallower water) and related species pairs has provided insights into processes of speciation. Here, we investigate female mating behaviour of five Pundamilia species and four of their F1-hybrids through mate choice trials and paternity testing. We discuss the results in the context of the geography of speciation and coexistence. Complete assortative mating was observed among all sympatric species. Parapatric species with similar depth habitat distributions interbred whereas other parapatric and allopatric species showed complete assortative mating. F1-hybrids mated exclusively with species accepted by females of the parental species. Although consistent with reinforcement in sympatry, a closer look at our results suggests otherwise and it is more likely that pre-existing female preferences influence which taxa can co-exist in sympatry. Regardless of the mechanism, mating preferences may influence species distribution in potentially hybridizing taxa, such as in the adaptive radiations of cichlid fish. We suggest that this at least partly explains why some species fail to establish breeding populations in locations where they are occasionally recorded. Our result support the notion that mating preferences of potentially cross-breeding species ought to be included in coexistence theory.

Experimental setup

A 240 x 80 x 40 cm aquarium was divided by grids into ten male compartments with a flowerpot as a standardized spawning site in each. This ‘partial partition’ design allowed females to move freely whereas the larger males were confined to their compartments (Supporting Information Figure S1). We used standard daylight full light spectrum aquarium T5 fluorescent tubes to enable nuptial colours to be visible for all females. The combination of ten males, two from each species, was changed every second month the first year when most clutches were spawned and thereafter every 3-5 months when we collected the remaining clutches (total number of males used: 9 P. azurea, 10 P. igneopinnis, 8 P. nyererei, 8 P. pundamilia and 8 P. sp. ‘red head’, Electronic supplementary material Table S1-S2). All experimental fish were marked with PIT tags and a clip from the dorsal fin provided a DNA sample. Females were introduced when large enough to be PIT-tagged (size differences depended on age). For the first three months only wild-type fish were present; F1 hybrid females and more wild-type fish were added over the following year. Females were stripped of embryos/juveniles once a month and released back into the experimental tank for a second and third clutch and thereafter removed. Females with eggs were placed in a separate aquarium until the eggs hatched. Larvae/juveniles were euthanized using MS-222 (tricaine methanesulfonate) and stored in 95% ethanol prior to paternity analyses. Electronic supplementary material Table S1 shows when each female spawned i.e. were present in the aquarium. The size of males were similar among species and initially 99-109 mm TL although they grew during the experiment. Female sizes when spawning were 65-97 mm TL and we confirmed for each female that it could pass the grids of the dividers.

Paternity analyses

Where possible, four embryos from each of two (sometimes three) clutches per female were genotyped at two to four microsatellite loci, Ppun5, Ppun7, Pun17 and Ppun21. (Taylor et al., 2003) using the methods for DNA extraction and PCR reactions as in Svensson et al. (2017). The amplified DNA samples were genotyped on a Beckman Coulter CEQ 8000 capillary sequencer. Genotypes were received from the CEQ 8000 Series Genetic Analyses System 8.0.52. Paternities were determined manually by inspection of the allele size estimates, and males that possessed two alleles at a microsatellite locus that were both not present in the offspring were excluded as possible fathers. In all analysed offspring, paternity could be assigned with 100 % certainty to one species only (Electronic supplementary material table S1-S3). If a clutch was confirmed to be fathered by more than one male; each sire was considered to represent one mate choice decision by the female.