Cave adaptation has evolved repeatedly across the Tree of Life, famously leading to pigmentation and eye degeneration and loss, yet its macroevolutionary implications remain poorly understood. We use the North American amblyopsid fishes, a family spanning a wide degree of cave adaptation, to examine the impact of cave specialization on the modes and tempo of evolution. We reconstruct evolutionary relationships using ultraconserved element loci, estimate the ancestral histories of eye-state, and examine the impact of cave adaptation on body shape evolution. Our phylogenomic analyses provide a well-supported hypothesis for amblyopsid evolutionary relationships. The obligate blind cavefishes form a clade and the cave-facultative eyed spring cavefishes are nested within the obligate cavefishes. Using ancestral state reconstruction, we find support for at least two independent subterranean colonization events within the Amblyopsidae. Eyed and blind fishes have different body shapes, but not different rates of body shape evolution. North American amblyopsids highlight the complex nature of cave-adaptive evolution and the necessity to include multiple lines of evidence to uncover the underlying processes involved in the loss of complex traits.

Ultraconserved elements dataset

We performed target-capture of ultraconserved element loci using the Acanthomorph MYbaits Probe Kit (2,600 probes for 1,341 UCE Loci). Cleaning and processing was performed through the PHYLUCE pipeline and ABySS was used for assembly. The concatenated dataset was made with a 75% complete datamatrix.

2020_Concatenated_alignments_amblyfull75_nexus.nex

This dataset is a concatenated dataset for ultraconserved element (UCE) loci from the Amblyopsidae fish family. After DNA extraction and sonication shearing, a Kapa HyperPrep Library Preparation Kit was used to create libraries. Then target capture of the UCE loci was performed with the MYbaits Acanthomorph probe set and target capture kit targeting 1,314 loci. Sequencing was performed on an Illumina HiSeq300-PE150 platform at the Oklahoma Medical Research Foundation with ~30x sequencing coverage. Assembly was performed using ABySS. Cleaning and processing was completed through the Phyluce pipeline. This dataset was created with a 75% complete datamatrix and contains 897 UCE loci.

Evolution19-0496_S1.docx

Supplementary Materials 1. Specimen tables for both morphology and molecular samples.

Evolution19-0496_S2.docx

Supplementary Materials 2. Supplementary figures in a single document with figure legends.

Evolution19-0496_S3.docx

Supplementary Materials 3. Explanation of landmark positions for landmark-based geometric morphometrics.

S2_FigS1.pdf

Lateral GM landmark scheme for the amblyopsids based on Burress et al. (2017). LM descriptions can be found in Supplementary Material S3. Black filled circles = static landmarks, gray filled circles = sliding landmarks, unfilled circles = unbend landmarks.

S2_FigS2.pdf

Amblyopsid RAxML phylogenomic hypothesis with all individuals labeled. Species codes are: As = Amblyopsis spelaea, Asay = Aphredoderus sayanus, Ccor = Chologaster cornuta, Fa = Forbesichthys agassizii, Fp = F. papilliferus, Po = Percopsis omiscomaycus, Sp = Speoplatyrhinus poulsoni, Te = Typhlichthys eigenmanni, Tr = Troglichthys rosae, Ts = Ty. subterraneus. Locality codes are included for blind cavefishes and eyed spring cavefishes.

S2_FigS3.pdf

Amblyopsid ASTRAL-II coalescent species tree with all individuals labeled. Species codes are: As = Amblyopsis spelaea, Asay = Aphredoderus sayanus, Ccor = Chologaster cornuta, Fa = Forbesichthys agassizii, Fp = F. papilliferus, Po = Percopsis omiscomaycus, Sp = Speoplatyrhinus poulsoni, Te = Typhlichthys eigenmanni, Tr = Troglichthys rosae, Ts = Ty. subterraneus. Locality codes are included for blind cavefishes and eyed spring cavefishes.

S2_FigS4.pdf

Amblyopsid SVDQuartets consensus coalescent species tree with all individuals labeled. Species codes are: As = Amblyopsis spelaea, Asay = Aphredoderus sayanus, Ccor = Chologaster cornuta, Fa = Forbesichthys agassizii, Fp = F. papilliferus, Po = Percopsis omiscomaycus, Sp = Speoplatyrhinus poulsoni, Te = Typhlichthys eigenmanni, Tr = Troglichthys rosae, Ts = Ty. subterraneus. Locality codes are included for blind cavefishes and eyed spring cavefishes.

S2_FigS5.pdf

Summary of 1000 mapped character states using stochastic character mapping of eye-state. Nodes referenced in-text are numbered with roman numerals (I and II).

S2_FigS6.pdf

Biplots of PC scores per individual for the full GM dataset (N = 146) colored by species. PC1 is located on the X axis and PC2 is located on the Y axis. Warp grids indicate the shape of the minimum and maximum specimen along that axis.

S2_FigS7.pdf

GM Phylomorphospace of the amblyopsid data subset (N = 18) with species colored and ellipses surrounding the eye-states (blue = blind, orange = eyed).