DOI: 10.5061/dryad.k6djh9wbs
title: Limited hybridization and introgression despite stocking among endemic Interior Highlands black basses (Centrarchidae: Micropterus)

description: Aim Smallmouth Bass (Micropterus dolomieu; SMB) are globally popular among
 anglers and have been widely introduced (i.e., stocked) for population
 management and sportfishing. Importantly, stocking was prevalent before
 cryptic diversity within the SMB complex was known, which now includes
 three newly elevated species: Neosho Bass (M. velox; NB), Little River
 Bass (M. sp. cf. dolomieu Little River; LRB), and Ouachita Bass (M. sp.
 cf. dolomieu Ouachita River; OB). We sought to quantify population
 structure and hybridization and introgression in these three recently
 described species. Location Species-level diversity, particularly in the
 basin-restricted LRB and OB in the Ouachita Mountains within the Central
 Interior Highlands (CIH), North America, suggests the presence of distinct
 genetic variation that could be eroded by
 introgression.   Methods We estimated interspecific
 introgression and intraspecific population differentiation in the
 Smallmouth Bass species complex (SMB-C) using 472 specimens comprising
 SMB, NB, LRB, and OB, including the naturally sympatric Spotted Bass (M.
 punctulatus; SPB). Genomic samples were genotyped on a SNP panel of 192
 loci designed to detect allele-sharing on multiple hierarchical
 levels.  Results We found low range-wide hybridization between
 species in the SMB-C and SPB (mostly SMB-C backcrosses), and interspecific
 heterozygosity varied, indicating differential introgression. Range-wide
 hybridization between species in the CIH and SMB was similar overall (but
 mostly F2 and CIH backcrosses) and was observed in streams with known SMB
 stocking in connected reservoirs. Interspecific heterozygosity in SMB
 hybrids was also generally lower, indicating later-generation backcrosses.
 We found strong population structure in the Ouachita Mountains (LRB and
 OB).  Main Conclusions Despite isolated incidences of natural
 (SPB) and human-mediated (SMB) introgression, genomic identity appears
 intact in endemic LRB and OB, suggesting potential ecological or
 behavioral isolating mechanisms preventing cross-species reproduction. Our
 findings reveal that genetic variation remains in cryptic,
 basin-restricted species in the Ouachita Mountains ecoregion that may be
 managed for long-term conservation.
description: Individual fish were captured by hook-and-line angling or boat
 electrofishing. Small clips (~12–15mg) of fin tissue were excised from the
 upper caudal fin of the fish and placed in 95% ethanol buffer solution,
 and fish were immediately released. Genomic DNA was extracted from fin
 clips using DNeasy Blood and Tissue Extraction Kits (QIAGEN), and samples
 were processed at the Center for Aquaculture Technology (CAT; San Diego,
 CA, USA).
description: We recommend the raw .vcf file be processed using standard
 software on a high-efficieny computing cluster. 
description: # Reproduce analyses for the Central Interior Highlands Hybridization
 project \[Gunn et al. 2023] Follow the steps listed below in the Analyses
 section to reproduce analyses for this study. Each step below gives a
 summary of the analysis and directs you to a general code file (e.g.,
 snolh_structure_analysis.Rmd below in Analysis 1) which then works through
 the analysis step-by-step. This general file will usually point you to
 other Rmd code, bash shell scripts, or python scripts. # Project: Central
 Interior Highlands Hybridization: Spotted, Smallmouth, Neosho, Ouachita,
 and Little River Bass (SNOLH) Investigating hybridization and population
 structure among and within the black basses (genus Micropterus) in the
 Central Interior Highlands (CIH) ecoregion, including natural and
 anthropogenic hybridization between Spotted Bass (SPB; Micropterus
 punctulatus) and species within the Smallmouth Bass species complex
 (SMBC): Smallmouth Bass (SMB; M. dolomieu), the newly elevated Neosho Bass
 (NB; M. velox), and two other potentially distinct species in the Ouachita
 River Basin, the Ouachita Bass (OB; <i>M. cf. dolomieu </i>
 Ouachita River), and the Little River Bass (LRB; <i>M. cf. dolomieu
 </i> Little River). ## Abbreviations used in Analysis
 Here we give a brief glossary of abbreviations and acronyms used in
 analyses: CIH: Central Interior Highlands SMBC: Smallmouth Bass species
 complex SPB: Spotted Bass SMB: Smallmouth Bass NB: Neosho Bass OB:
 Ouachita Bass LRB: Little River Bass ## General information on repository
 structure This is a publicly visible GitHub repository storing code (and a
 small amount of data, although we have done our best to avoid uploading
 large amounts of data due to the limited storage) for Gunn et al. (2023).
 In the home directory of the repository (SNOLH_Genomics), you will find a
 README.md file (the source script for this information), the R Project
 file (SNOLH_Genomics.Rproj), and three different "analysis"
 directories, each of which corresponds with a specific analysis conducted
 in our study: 1. 01_filtering_analysis 2. 02_structure_analysis 3.
 03_introgress_analysis Within each analysis directory, you will find an R
 markdown script (.Rmd) with the name of the analysis, which contains all
 of the code needed to run the full analysis. Additionally, you will find:
 1. code The code directory will store all source code, shell scripts,
 lists of bash commands, and software packages needed for analysis. Once
 you have downloaded the repository and located the code directory, you
 should create two additional sub-directories within each analysis (on the
 same level as the code directory): 1. data 2. figures The data directory
 will store all processed data and metadata needed for analysis. The
 figures folder will contain any raw figures generated in ggplot for each
 analysis. Ideally, the .Rmd script should have paths set up so that the
 code reads all data and scripts and generates figures seamlessly. ## Using
 the code To reproduce all analyses in Gunn et al. (2023), download this
 data repository and place in a desired home directory. This may be done on
 your local machine, but we recommend downloading to a high-performance
 computing cluster so that all code will run seamlessly in one environment,
 as long as Rstudio is installed and the GUI can be called on the cluster.
 Once all directories are downloaded, create a new sub-directory within the
 home directory (same level as the seven analysis directories, .Rproj,
 README.md, etc.) called `/raw_data`. This is where you will store the raw
 genomic data and associated sample metadata (see
 <i><b>Data</b></i> section below). ## Data Raw
 genotype data and accompanying metadata are available at Zenodo.org:
 [LINK] Download these data into to your `/raw_data` directory within the
 home working directory. You should have 2 new items in the directory: 1.
 snolh_genotype_data.xlsx 2. snolh_metadata.xlsx If you have any questions
 or issues with data and/or code, please don't hesitate to contact me:
 ## Analyses ### Analysis 1: Filtering Analysis In
 this analysis, we clean and filter the full genotype data for 487 black
 bass individuals, which was derived from the diagnostic SNP panel
 developed by Long et al. (2021) and prepare the data for analysis in
 Structure and NewHybrids (See Analysis 2). Specifically, we filter the
 dataset based on three criteria: 1) out poor quality SNP loci (loci that
 failed to genotype in over 20% of samples); 2) poor quality samples
 (samples that failed to genotype in over 20% of loci); and 3) potential
 duplicate samples (samples that are greater than 95% identical across
 loci). #### Follow the Code:
 `01_filtering_analysis/snolh_filtering_analysis.Rmd` ### Analysis 2:
 Population Structure and Hybrid Assignment In this analysis, we assess
 hierarchical population genomic structure among and within Interior
 Highland species using the diagnostic SNP panel published by Long et al.
 (2021). We begin with a holistic analysis of population structure among
 Spotted Bass and all other Interior Highland species (Smallmouth Bass,
 Neosho Bass, Ouachita Bass, and Little River Bass) and diagnose hybrids
 between these species using SNPs diagnostic for Spotted Bass. We then
 exclude detected hybrids and continue with an analysis of population
 structure and hybridization among Smallmouth Bass, Neosho Bass, Ouachita
 Bass and Little River Bass. We again exclude any detected hybrids and move
 on to a final analysis of all Interior Highland species, excluding Spotted
 Bass and Smallmouth Bass. #### Follow the Code:
 `02_structure_analysis/snolh_structure_analysis.Rmd` ### Analysis 3:
 Introgression Analysis In this analysis, we further investigate
 hybridization and introgression within populations that were inferred to
 contain hybrids based on NEWHYBRIDS analysis (Analysis 2). We use the R
 package Introgress to regress interspecific heterozygosity on hybrid index
 for inferred F1, F2, and back-cross individuals at each hierarchical level
 of hybrid analysis conducted in Analysis 2. With this analysis, we
 determine whether hybrids are of very recent origin (first or second
 generation) or if they show a genetic signature of deeper time
 hybridization. We also infer from this analysis the extent to which
 non-native alleles have introgressed into the native distribution of each
 Smallmouth Bass species complex (SMBC) species. #### Follow the Code:
 `03_introgress_analysis/snolh_introgress_analysis.Rmd`