A hybrid genetic linkage map of two ecologically and morphologically divergent Midas cichlid fishes (Amphilophus spp.) obtained by massively parallel DNA sequencing (ddRADSeq)

Abstract

Cichlid fishes are an excellent model system for studying speciation and the formation of adaptive radiations because of their tremendous species richness and astonishing phenotypic diversity. Most research has focused on African rift lake fishes, although Neotropical cichlid species display much variability as well. Almost one dozen species of the Midas cichlid species complex (Amphilophus spp.) have been described so far and have formed repeated adaptive radiations in several Nicaraguan crater lakes. Here we apply double-digest restriction-site associated DNA sequencing to obtain a high-density linkage map of an interspecific cross between the benthic Amphilophus astorquii and the limnetic Amphilophus zaliosus, which are sympatric species endemic to Crater Lake Apoyo, Nicaragua. A total of 755 RAD markers were genotyped in 343 F(2) hybrids. The map resolved 25 linkage groups and spans a total distance of 1427 cM with an average marker spacing distance of 1.95 cM, almost matching the total number of chromosomes (n = 24) in these species. Regions of segregation distortion were identified in five linkage groups. Based on the pedigree of parents to F(2) offspring, we calculated a genome-wide mutation rate of 6.6 × 10(-8) mutations per nucleotide per generation. This genetic map will facilitate the mapping of ecomorphologically relevant adaptive traits in the repeated phenotypes that evolved within the Midas cichlid lineage and, as the first linkage map of a Neotropical cichlid, facilitate comparative genomic analyses between African cichlids, Neotropical cichlids and other teleost fishes.

Keywords: Midas cichlid; RAD markers; double-digest RADSeq; mutation rate; segregation distortion; synteny.

Figure 1

Schematic of the ddRADSeq protocol. Arrows indicate restriction sites for a rare-cutting enzyme in green and a frequent-cutting enzyme in orange. P1 adapters specifically bind to the rare-cutting enzyme (green) and P2 adapters bind to the frequent-cutter (orange) restriction sites. Each individual contains a different barcode of five nucleotide bases specified by the P1 adapter. Sequenced RAD tags start with the barcode (individual one: red barcode, individual two: blue barcode). The bioinformatic software Stacks allows for the detection of single nucleotide polymorphisms between individuals (outlined by red box in sequence alignment).

Figure 2

Linkage groups constructed with 755 RAD markers and ordered by size. Blocks of segregation distortion are indicated by brackets. Markers used for synteny analyses are shown in orange. For further information on marker IDs, position, segregation distortion and markers used in synteny analyses see, Table S3.

Figure 3

Oxford grid showing relationship between Midas cichlid and other teleost fishes. Each linkage group (in the case of medaka each chromosome) is size-normalized to equal one and marker positions were placed relative to their respective linkage group (or chromosome) size. Oxford grids showing marker relationships between (A) tilapia and Midas cichlid, (B) stickleback and Midas cichlid, and (C) medaka and Midas cichlid. Color refers to markers found on a Midas linkage group.