Since the time of Darwin, biologists have sought to understand the evolution and origins of phenotypic variation. To understand the genetic and molecular sources of morphological differences, we capitalize on the cichlid fish system. Cichlids of the East African Rift Lakes have undergone an extensive adaptive radiation, including variation in body shape, head shape, and pigmentation. These morphological differences are often intimately linked to the ecology and behavior of these animals. Here, we investigate the genetic basis of these phenotypes using quantitative trait loci (QTL) mapping using four genera of Lake Malawi cichlids and two F₂ hybrid populations. The first hybrid cross is between Aulonocara koningsi, which lives in the open sandy region and feeds insects from the open sand, and Metriaclima mbenjii, an omnivore rock-dwelling fish. The second cross is between Labidochromis caeruleus, a suction-feeding insectivore that swims continuously searching for prey, and Labeotropheus trewavasae, which feeds by biting or scraping attached algae from the rocks in its benthic habitat. Such work can provide insights into the molecular basis of phenotypic adaptation, the genetic architecture of morphology, and the evolution of cichlid fishes.

For the first hybrid cross, a single Metriaclima female crossed to two Aulonocara males; the inclusion of the second grandsire was inadvertent and resulted from an unexpected fertilization event in these species with external fertilization. This single F₁ family was subsequently incrossed to produce a hybrid F₂ population of 491 fishes. For the second cross, a single Labidochromis caeruleus female was crossed with a single Labeotropheus trewavasae male to create one F₁ family, which was subsequently incrossed to produce a hybrid F₂ population of 447 fishes. Hybrid fish and 10 each of the pure parental species were euthanized and 2D imaged at five months, with each image including a color standard and scale. Sex of each animal was determined based on gonad dissection and omitted if there was any ambiguity. Linear measures were collected from these images using the program ImageJ as pixels, converted to cm using measures of a scale in each picture using Excel, and processed to residual after regression to standard length (snout to caudal peduncle) in R. Landmark data was collected from 2D images using TPSdig, converted to XY coordinates using TPSutil, and geometric morphometrics including size correction was conducted using the geomorph package in R.

Genotype data came from DNA extracted from caudal fin tissue, that was sequenced as RADseq libraries, and processed using the R program Stacks. The A allele is designated as coming from the Metriaclima granddam and the B allele from the Aulonocara grandsire. In the second cross, the A allele is designated as coming from the Labidochromis granddam and the B allele from the Labeotropheus grandsire. QTL mapping of residual phenotypes using this genotype data was conducted using MQM in R/qtl.

File names with "MmAk" indicates the Metriaclima x Aulonocara cross, while "LcLt" refers to Labidochromis x Labeotropheus.

.CSV files can be opened in Microsoft Excel, OpenOffice, or a data analysis environment such as R (https://www.r-project.org/). .TPS files can be opened and analyzed using software available at https://sbmorphometrics.org/, specifically TPSutil, TpsDig, and TPSrelw for file management (e.g. sample removal), landmark digitization, and geometric morphometric analysis, respectively. .TPS files can be converted in TPSutil to be analyzed in R. Genotype files are formatted for upload and analysis in R/qtl. File names with "MmAk" indicates the Metriaclima x Aulonocara cross, while "LcLt" refers to Labidochromis x Labeotropheus.