DOI: 10.5061/dryad.47d7wm3h8
title: Supplementary materials for: Phylogenomics, lineage diversification rates, and the evolution of diadromy in clupeiformes (anchovies, herrings, sardines, and relatives)

description: Migration independently evolved numerous times in animals, with a myriad
 of ecological and evolutionary implications. In fishes, perhaps the most
 extreme form of migration is diadromy, the migration between marine and
 freshwater environments. Key and longstanding questions are: how many
 times has diadromy evolved in fishes, how frequently do diadromous clades
 give rise to non-diadromous species, and does diadromy influence lineage
 diversification rates? Many diadromous fishes have large geographic ranges
 with constituent populations that use isolated freshwater habitats. This
 may limit gene flow among some populations, increasing the likelihood of
 speciation in diadromous lineages relative to non-diadromous lineages.
 Alternatively, diadromy may reduce lineage diversification rates if
 migration is associated with enhanced dispersal capacity that facilitates
 gene flow within and between populations. Clupeiformes (herrings,
 sardines, shads and anchovies) is a model clade for testing hypotheses
 about the evolution of diadromy because it includes an exceptionally high
 proportion of diadromous species and several independent evolutionary
 origins of diadromy. However, relationships among major clupeiform
 lineages remain unresolved and existing phylogenies sparsely sampled
 diadromous species, limiting the resolution of phylogenetically-informed
 statistical analyses. We assembled a phylogenomic dataset and used
 multi-species coalescent and concatenation-based approaches to generate
 the most comprehensive, highly-resolved clupeiform phylogeny to date,
 clarifying associations among several major clades and identifying
 recalcitrant relationships needing further examination. We determined that
 variation in rates of sequence evolution (heterotachy) and
 base-composition (non-stationarity) had little impact on our results.
 Using this phylogeny, we characterized evolutionary patterns of diadromy
 and tested for differences in lineage diversification rates between
 diadromous, marine, and freshwater lineages. We identified thirteen
 transitions to diadromy, all during the Cenozoic Era (10 origins of
 anadromy, two origins of catadromy, and one origin of amphidromy), and
 seven losses of diadromy. Two diadromous lineages rapidly generated
 non-diadromous species, demonstrating that diadromy is not an evolutionary
 dead-end. We discovered considerably faster transition rates out of
 diadromy than to diadromy. The largest lineage diversification rate
 increase in Clupeiformes was associated with a transition to diadromy, but
 we uncovered little statistical support for categorically faster lineage
 diversification rates in diadromous versus non-diadromous fishes. We
 propose that diadromy may increase the potential for accelerated lineage
 diversification, particularly in species that migrate long distances.
 However, this potential may only be realized in certain biogeographic
 contexts, such as when diadromy allows access to ecosystems in which there
 is limited competition from incumbent species.
description: # Supplementary materials for: Phylogenomics, lineage diversification
 rates, and the evolution of diadromy in clupeiformes (anchovies, herrings,
 sardines, and relatives)
 [https://doi.org/10.5061/dryad.47d7wm3h8](https://doi.org/10.5061/dryad.47d7wm3h8) Supplementary materials associated with manuscript. ### Description of the data and file structure In supplementary alignments and tree files, taxon names were not changed to reflect updated taxonomy to maintain continuity throughout the bioinformatics pipeline. However, taxon names in these files can easily be associated with final species assignments because the tips names in the supplementary tree files contain the tissue numbers associated with each individual. The tissue numbers are provided in Supplementary Table S1 along with the final species assignment. **Files associated with the Full Exon-Capture Dataset:**\  \ **File name:** ASTRAL_03Dec2020_IQTREEGeneTreesSpeciesTree.tre\ **Description:** This file contains a species tree we generated using ASTRAL and input gene trees (02Dec20_IQTREE_Output.treefile) generated via analysis of nucleotide alignments (02Dec2020Alignments.zip) of loci in the Full Exon-Capture Dataset. This tree was not time-calibrated.\  \ **Folder name:** 02Dec2020Alignments_V2.zip\ **Description:** This folder contains nucleotide alignments for each locus in the Full Exon-Capture Dataset and Expanded Dataset used as input for IQ-TREE analyses (“FullExonCapture” subdirectory). “Output” subdirectries contain gene trees from IQ-TREE analyses. The “ExpandedDataset” subdirectory contains alignments that include additional NCBI data in Expanded Dataset analyses. We included concatenated and unconcatenated files for mitochondrial loci. We used trees generated from concatenation-based IQ-TREE analyses of mitochondrial loci as input for ASTRAL analyses.\  \ **File name:** 02Dec20_IQTREE_Output.treefile\ **Description:** This file contains gene trees for each locus in the Full Exon-Capture Dataset generated via IQ-TREE and nucleotide alignments (02Dec2020Alignments.zip).\  \ **File name:** 29Mar21_ASTRAL_FullExonCaptureDataset_AA.tre\ **Description:** This file contains a species tree we generated using ASTRAL and input gene trees (26Mar21_IQTREE_OutputExonCaptureOnly_AA.treefile) from analysis of amino acid alignments (AminoAcidAlignments_26March21.zip) of loci in the Full Exon-Capture Dataset. This tree is not time-calibrated.\  \ **File name:** 26Mar21_IQTREE_OutputExonCaptureOnly_AA.treefile\ **Description:** This file contains gene trees for each locus in the Full Exon-Capture Dataset generated via IQ-TREE and amino acid alignments (AminoAcidAlignments_26March21.zip). We used gene trees from analyses of concatenated mitochondrial loci (found in AminoAcidAlignments_26March21.zip) as input for ASTRAL analyses.\  \ **Folder name:** AminoAcidAlignments_26March21_V2.zip\ **Description:** This folder contains amino acid alignments for each locus in the Full Exon-Capture Dataset (“FullExonCapture” subdirectory) and Expanded Dataset (“ExpandedDataset” subdirectory) used as input for IQ-TREE analyses and output gene trees from analyses of concatenated mitochondrial loci. We included concatenated and unconcatenated files for mitochondrial loci. We used trees generated from concatenation-based IQ-TREE analyses of mitochondrial loci as input for ASTRAL analyses.\  \ **File name:** FigTree_ExonCaptureOnly.tre\ **Description:** Species tree (ASTRAL_03Dec2020_IQTREEGeneTreesSpeciesTree.tre) time calibrated with MCMCTree.\  \ **File name:** ExonCaptureOnly_ConcatenationFASTA.fasta\ **Description:** This file contains a concatenated nucleotide alignment for all loci in the Full Exon-Capture Dataset used as input for concatenation-based IQ-TREE analysis.\  \ **File name:** 05Apr21_IQTREE_N_Concatenated.treefile\ **Description:** This file contains a species tree generated via IQ-TREE analysis of a concatenated nucleotide alignment for all loci in the Full Exon-Capture Dataset (ExonCaptureOnly_ConcatenationFASTA.fasta).\  \ **File name:** FullExonCaptureDataset_AA_27March21Concatenation.fasta\ **Description:** This file contains a concatenated amino acid alignment for all loci in the Full Exon-Capture Dataset used as input for concatenation-based IQ-TREE analysis.\  \ **File name:** 27_March_ConcatenatedAA_FullExonCapture_FullAnnotations.treefile\ **Description:** This file contains a species tree generated via IQ-TREE analysis (run with SH-aLRT option) of a concatenated amino acid alignment for all loci in the Full Exon-Capture Dataset (ExonCaptureOnly_ConcatenationFASTA.fasta).\  \ **File name:** CollapsedTrees_FullExonDataset_N.treefile\ **Description:** This file contains gene trees with poorly supported divergences (&lt;33%) collapsed for each locus in the Full Exon-Capture Dataset. We generated gene trees with IQ-TREE and nucleotide alignments.\  \ **File name:** ASTRAL_06Apr2021_N_CollapsedTrees_FullExonDataset.tre\ **Description:** This file contains a species tree generated using ASTRAL and input gene trees with poorly supported divergences (&lt;33%) collapsed (CollapsedTrees_FullExonDataset_N.treefile). Input gene trees were inferred using IQ-TREE and nucleotide alignments for all loci in the Full Exon-Capture Dataset. This tree was not time-calibrated.\  \ **File name:** CollapsedTrees_FullExonDataset_AA.treefile\ **Description:** This file contains gene trees with poorly supported divergences (&lt;33%) collapsed for each locus in the Full Exon-Capture Dataset. We generated gene trees with IQ-TREE and amino acid alignments.\  \ **File name:** ASTRAL_06Apr2021_AA_CollapsedTrees_FullExonDataset.tre\ **Description:** This file contains a species tree generated using ASTRAL and input gene trees with poorly supported divergences (&lt;33%) collapsed (CollapsedTrees_FullExonDataset_AA.treefile). Input gene trees were inferred using IQ-TREE and amino acid alignments for all loci in the Full Exon-Capture Dataset. This tree is not time-calibrated.\  \ **Files associated with the Reduced Locus Dataset:**\  \ **File name:** 02Dec20_IQTREE_OutputReducedExon-CaptureDataset.treefile\ **Description:** This file contains gene trees for each locus in the Reduced Locus Dataset generated via IQ-TREE and nucleotide alignments.\  \ **File name:** 10May21_ASTRAL_ReducedExon_N.tre\ **Description:** This file contains a species tree we generated using ASTRAL and input gene trees (02Dec20_IQTREE_OutputReducedExon-CaptureDataset.treefile) generated via analysis of nucleotide alignments of loci in the Reduced Locus Dataset. This tree was not time-calibrated.\  \ **Files associated with the Expanded Dataset:**\  \ **File name:** ASTRAL_12Jan2021_IQTREEGeneTrees_SuperMatrixSpeciesTree.tre\ **Description:** This file contains a species tree we generated using ASTRAL and input gene trees (02Dec2020Alignments.zip) generated via analysis of nucleotide alignments of loci in the Expanded Dataset. This tree was not time-calibrated.\  \ **File name:** 29Mar21_ASTRAL_ExpandedDataset_AA.tre\ **Description:** This file contains a species tree we generated using ASTRAL and input gene trees (26Mar21_IQTREE_OutputExpandedDataset_AA.treefile) generated via analysis of amino acid alignments of loci in the Expanded Dataset.\  \ **File name:** ExpandedDataset_AA_27March21Concatenation.fasta\ **Description:** This file contains a concatenated amino acid alignment of all loci in the Expanded Dataset used as input for concatenation-based IQ-TREE analysis.\  \ **Folder name:** AminoAcidAlignments_26March21_V2.zip\ **Description:** This folder contains amino acid alignments for each locus in the Full Exon-Capture Dataset (“FullExonCapture” subdirectory) and Expanded Dataset (“ExpandedDataset” subdirectory) used as input for IQ-TREE analyses and output gene trees from analyses of concatenated mitochondrial loci. We included concatenated and unconcatenated files for mitochondrial loci. We used trees generated from concatenation-based IQ-TREE analyses of mitochondrial loci as input for ASTRAL analyses.\  \ **File name:** 26Mar21_IQTREE_OutputExpandedDataset_AA.treefile\ **Description:** This file contains gene trees for each locus in the Expanded Dataset generated via IQ-TREE and amino acid alignments (AminoAcidAlignments_26March21.zip).\  \ **Folder name:** 02Dec2020Alignments_V2.zip\ **Description:** This folder contains nucleotide alignments for each locus in the Full Exon-Capture Dataset and Expanded Dataset used as input for IQ-TREE analyses (“FullExonCapture” subdirectory). “Output” subdirectries contain gene trees from IQ-TREE analyses. The “ExpandedDataset” subdirectory contains alignments that include additional NCBI data in Expanded Dataset analyses. We included concatenated and unconcatenated files for mitochondrial loci. We used trees generated from concatenation-based IQ-TREE analyses of mitochondrial loci as input for ASTRAL analyses.\  \ **File names:** FigTree_Subset(1-18).tre\ **Description:** Species trees generated via ASTRAL analysis of 65-gene subsets of the Expanded Dataset. We ensured that all species were represented in every subset by including 6 genes with high species sampling in all subsets. We randomly assigned the remaining 59 genes to each subset. Each species tree was time-calibrated using MCMCTree.\  \ **File name:** ExpandedDataset_DistributionUltrametric.nexus\ **Description:** A file containing all species trees generated via ASTRAL analysis of 65-gene subsets of the Expanded Dataset. Each species tree was time-calibrated using MCMCTree. All trees have identical sampling and tip names, allowing the use of this file as input for phylogenetic comparative analyses. **File name:** CommonAncestorHeights.tre **Description:** A file containing an ultrametric maximum clade credibility species tree generated from the 18-Tree Distribution in TreeAnnotator with the “common ancestor heights” option. **Files associated with the 30-Species Dataset:**\  \ **Folder name:** 30SpeciesDatasetAlignments.zip\ **Description:** This folder contains nucleotide alignments for each locus in the 30-Species Dataset used as input for IQ-TREE analyses and output gene trees from analyses of concatenated mitochondrial loci. We concatenated mitochondrial loci for IQ-Tree analyses to constrain mitochrdial loci to a single evolutionary history.\  \ **File name:** GhostAlignments.treefile\ **Description:** This file contains gene trees for each locus in the 30-Species Dataset generated via IQ-TREE Ghost analyses.\  \ **File name:** GhostAlignments_NonGhostAnalysis.treefile\ **Description:** This file contains gene trees for each locus in the 30-Species Dataset generated via standard IQ-TREE analyses.\  \ **File name:** GhostAlignments_ASTRALAnalysis.tre\ **Description:** This file contains the species tree generated via ASTRAL analysis of gene trees from IQ-TREE Ghost analyses and the 30-Species Dataset.\  \ **File name:** GhostAlignments_NonGhostASTRALAnalysis.tre\ **Description:** This file contains the species tree generated via ASTRAL analysis of gene trees from standard IQ-TREE analyses and the 30-Species Dataset.\  \ **Additional files associated with analyses:**\  \ **File names:** SamplingFractions_MaxSpecies272_V2.txt -SamplingFractions_MaxSpecies272_V6.txt\ **Description:** Sampling fraction files used for BAMM analyses for each tree in the 18-Tree Distribution. File for trees 1, 3, 4, 6, 7, 8, 10, 11, 12, 13, 14, 15, 16 = V2, file for tree 2 = V3, file for tree 5 = V4, file for tree 9 = V5, and fiel for trees 17 and 18 = V6. **SUPPLEMENTARY FIGURES** **Supplementary figure files:** SupplementaryFigures_Final.zip **Supplementary figure captions:** **Supplementary Figure S1.** Summary of divergence times for select clupeiform clades showing variation in divergence times inferred via analyses of the Full Exon-Capture dataset and 18-Tree Distribution. **Supplementary Figures S2-S19.** Ancestral state reconstruction plots summarizing character state changes inferred by simmap analyses of the two-state diadromy character estimated by stochastic character mapping analyses on each tree from the 18-Tree Distribution and ARD models of discrete character evolution (see Supplementary Table S4). Each tree in the 18-tree distribution was used in 100 iterations of stochastic character mapping. Thus, each visualization summarizes 100 mapped character histories. Figure S18 contains simmapping results for tree 17 from the 18-Tree distribution and includes tip labels and demarcates clupeiform families. **Supplementary Figure S20.** Density maps summarizing character state changes inferred by simmap analyses of the two-state diadromy character estimated by stochaster character mapping analyses using the 18-Tree Distribution and and ARD models of discrete character evolution (see Supplementary Table S4). Each tree in the 18-tree distribution was used in 100 iterations of stochastic character mapping. Thus, each visualization summarizes 100 mapped character histories. **Supplementary Figures S21-S38.** Summary of character state changes inferred by simmap analysis of the three-state diadromy character estimated by stochastic character mapping analyses using the 18-tree distribution and an irreversible model of discrete character evolution, mapped onto tree 17 from the 18-Tree Distribution. Each tree in the 18-tree distribution was used in 100 iterations of stochastic character mapping. Therefore, this visualization using a single tree summarizes a total of 1800 mapped character histories. Figure S18 contains simmapping results for tree 17 from the 18-Tree distribution and includes tip labels and demarcates clupeiform families. **Supplementary Figure S39.** Box plots showing estimated transition times to diadromy inferred using stochastic character mapping, the two-state diadromy character, and the 18-tree distribution. Each tree in the 18-tree distribution was used in 100 iterations of stochastic character mapping, yielding 1800 mapped character histories containing estimates of transition times. Box plots are labeled according to the taxa (either species or genera) involved in each transition to diadromy. Supplementary Figs. S2 and S38 show the phylogenetic positions of each transition and the species involved. **Supplementary Figure S40.** Representative phylorate plots of mean net diversification rates mapped onto the branches of phylogenies estimated using RevBayes (left) and BAMM (right) and the first four trees from the 18-Tree Distribution. Numbers to the right of phylogeny tips demarcate lineages with the highest net diversification rates: (1) *Alosa *+ *Brevoortia *and (2) a largely tropical, marine clade of anchovies primarily distributed in the Gulf of Mexico, Caribbean Sea, and the northern Atlantic coasts of South America. **Supplementary Figure S41.** Violin plots showing variation in tip-specific net diversification rates within individual diadromous lineages, all diadromous taxa, marine taxa, and freshwater taxa estimated with BAMM using the 18-tree distribution. Contents of diadromous lineages: (1) *Ilisha filigera*, (2) *Hilsa kelee*, (3) *Gilchristella aestuaria*, (4) *Ethmalosa fimbriata*, (5) *Coilia nasus*, (6) *Clupeonella caspia* + *Clupeonella cultriventris*, (7) *Tenualosa ilisha* + *Tenualosa macrura* + *Tenualosa reevesii* + *Tenualosa toli*, (8) *Anchoviella lepidentostole*, (9) *Clupanodon thrissa, (10)* Thrissina scratchleyi, (11) *Potamalosa richmondia*, (12) *Lycengraulis grossidens*, and (13) *Alosa* *aestivalis* + *Alosa alabamae* + *Alosa algeriensis* + *Alosa alosa* + *Alosa caspia* + *Alosa immaculata* + *Alosa mediocris* + *Alosa pseudoharengus* + *Alosa sapidissima*.