DOI: 10.5061/dryad.dbrv15f05
title: Introgression and species delimitation in the longear sunfish Lepomis megalotis (Teleostei: Percomorpha: Centrarchidae)

description: Introgression and hybridization are major impediments to genomic-based
 species delimitation because many implementations of the multispecies
 coalescent framework assume no gene flow among species. The sunfish genus
 Lepomis, one of the world’s most popular groups of freshwater sport fish,
 has a complicated taxonomic history. The results of ddRAD phylogenomic
 analyses do not provide support for the current taxonomy that recognizes
 two species, L. megalotis and L. peltastes, in the L. megalotis complex.
 Instead, evidence from phylogenomics and phenotype warrants recognizing
 six relatively ancient evolutionary lineages in the complex. The
 introgressed and hybridizing populations in the L. megalotis complex are
 localized and appear to be the result of secondary contact or rare
 hybridization events between non-sister species. Segregating admixed
 populations from our multispecies coalescent analyses identifies six
 species with moderate to high genealogical divergence, whereas including
 admixed populations drives all but one lineage below the species threshold
 of genealogical divergence. Segregation of admixed individuals also helps
 reveal phenotypic distinctiveness among the six species in morphological
 traits used by ichthyologists to discover and delimit species over the
 last two centuries. Our protocols allow for the identification and
 accommodation of hybridization and introgression in species delimitation.
 Genomic-based species delimitation validated with multiple lines of
 evidence provides a path towards the discovery of new biodiversity and
 resolving long-standing taxonomic problems. 
description: <strong>BPP_Lmeg_pure.ctl</strong> ‘Control’ input for the BPP analysis for the dataset that no specimens of genomic admixture are included.   <strong>BPP_Lmeg_pure.Imap.txt</strong> ‘Imapfile’ input for the BPP analysis for the dataset that no specimens of genomic admixture are included.  <strong>BPP_Lmeg_pure_loci.prn</strong> ‘seqfile’ input for the BPP analysis for the dataset that no specimens of genomic admixture are included.  <strong>BPP_Lmeg_pure_Theta-Tau.csv</strong> Mean and 95% CI of the theta and tau parameter estimates resulting from the BPP analysis for the dataset that no specimens of genomic admixture are included.  <strong>BPP_Lmeg_admix.ctl</strong> ‘Control’ input for the BPP analysis for the dataset that includes specimens of genomic admixture.   <strong>BPP_Lmeg_admix.Imap.txt</strong> ‘Imapfile’ input for the BPP analysis for the dataset that includes specimens of genomic admixture.   <strong>BPP_Lmeg_admix_loci.prn</strong> ‘seqfile’ input for the BPP analysis for the dataset that includes specimens of genomic admixture.   <strong>BPP_Lmeg_admix_Theta-Tau.csv</strong> Mean and 95% CI of the theta and tau parameter estimates resulting from the BPP analysis for the dataset that includes specimens of genomic admixture.   <strong>MEGOZKMO_MSFS.obs</strong> Multisite frequency spectrum input for the Fastsimcoal2 analysis for the dataset containing specimens of <i>Lepomis megalotis</i>, <i>L.</i> sp. Ozark, and genomic admixture between the two species. <strong>MEGPELMP_MSFS.obs</strong> Multisite frequency spectrum input for the Fastsimcoal2 analysis for the dataset containing specimens of <i>Lepomis megalotis</i>, <i>L. peltastes</i>, and genomic admixture between the two species. <strong>MEGSOLMS_MSFS.obs</strong> Multisite frequency spectrum input for the Fastsimcoal2 analysis for the dataset containing specimens of <i>Lepomis megalotis</i>, <i>L. solis</i>, and genomic admixture between the two species. <strong>MEGOZKMO.ctl</strong> ‘Control’ input for the G-PHoCS analysis for the dataset containing specimens of <i>Lepomis megalotis</i>, <i>L.</i> sp. Ozark, and genomic admixture between the two species. <strong>MEGOZKMO.gphocs</strong> ‘seq-file’ input for the G-PHoCS analysis for the dataset containing specimens of <i>Lepomis megalotis</i>, <i>L.</i> sp. Ozark, and genomic admixture between the two species. <strong>MEGPELMP.ctl</strong> ‘Control’ input for the G-PHoCS analysis for the dataset containing specimens of <i>Lepomis megalotis</i>, <i>L. peltastes</i>, and genomic admixture between the two species. <strong>MEGPELMP.gphocs</strong> ‘seq-file’ input for the G-PHoCS analysis for the dataset containing specimens of <i>Lepomis megalotis</i>, <i>L. peltastes</i>, and genomic admixture between the two species. <strong>MEGSOLMS.ctl</strong> ‘Control’ input for the G-PHoCS analysis for the dataset containing specimens of <i>Lepomis megalotis</i>, <i>L. solis</i>, and genomic admixture between the two species. <strong>MEGSOLMS.gphocs</strong> ‘seq-file’ input for the G-PHoCS analysis for the dataset containing specimens of <i>Lepomis megalotis</i>, <i>L. solis</i>, and genomic admixture between the two species. <strong>Kim_et_al_Lepomis_megalotis_complex_Morphology_data.csv</strong> Table with all measured morphological traits of the <em>Lepomis megalotis</em> complex. <strong>SNAPP_Lmeg_divtime.xml</strong> Input for the divergence time analysis using SNAPP. <strong>SNAPP_Lmeg_speciestree.xml</strong> Input for the species-tree estimation using SNAPP. <strong>SNMF_Lmeg_only.ugeno</strong> Dataset for the population structure analysis with a sparse non-negative matrix factorization (snmf) algorithm. <strong>Treemix_Lmeg.snps.hdf5</strong> Sequence data for the TreeMix analysis. <strong>Treemix_Lmeg_imap.txt</strong> ‘imap’ input for the TreeMix analysis. <strong>IQtree_Lepomis_ddRAD.nex</strong> Concatenated nexus alignment for the dataset containing individuals of all <i>Lepomis</i> that were examined in the phylogenetic analyses. <strong>Kim_et_al_Supplementary_MatMeth.docx</strong> Supplementary Materials and Methods <strong>Supplementary Figure S1</strong> Map presenting inland water bodies discussed in the manuscript. Numbers indicate: 1, Cuatrociénegas Basin; 2, Rio Grande; 3, Colorado River; 4, Brazos River; 5, Sabine River; 6, Mississippi River; 7, Red River; 8, Ouachita River; 9, Little River; 10, Kiamichi River; 11, Lake Saint John; 12, Yazoo River; 13, Lake Providence; 14, Arkansas River; 15, White River; 16, Fourche La Fave River; 17, Canadian River; 18, Neosho River; 19, Verdigris River; 20, Hatchie River; 21, Tennessee River; 22, Elk River; 23, Emory River; 24, Clinch River; 25, Ohio River; 26, Kentucky River; 27, Scioto River; 28, Meramec River; 29, Missouri River; 30, Osage River; 31, Illinois River (upper Mississippi River system); 32, Lake George; 33, Iowa Fairport Fish Hatchery; 34, St. Lawrence River; 35, Lake Pontchartrain; 36, Mobile River; 37, Choctawhatchee River; 38, Altamaha River. <b>Supplementary Figure S2</b> Maximum likelihood phylogeny for the <i>Lepomis megalotis</i> complex and its sister group, <i>L. marginatus</i>, inferred from IQ–TREE analysis of concatenated ddRAD loci dataset. Bootstrap supports are shown next nodes. Figure 2 is a simplified version of this figure. <b>Supplementary Figure S3</b> Population structures inferred from snmf analysis for the <i>Lepomis megalotis</i> complex (K=7, 8, 9, 11) are aligned according to IQ–TREE phylogeny. <b>Supplementary Figure S4</b> G-PhoCS analyses results for three populations characterized by genetic admixture with the largest geographic range: a) <i>Lepomis solis</i> x <i>L. megalotis</i> in the middle and upper Tennessee River; b) <i>L. peltastes</i> x <i>L. megalotis</i> in the upper Ohio River; and c) <i>L.</i> sp. Ozark x <i>L. megalotis</i> in the Osage River. The 95% highest posterior density for each parameter (theta, tau, or migration) is shown in parenthesis next to the mean. Arrows indicate direction of gene flow. Width of boxes is proportional to the mean for the theta parameter estimate. <b>Supplementary Figure S5</b> Models tested in Fastsimcoal2 analyses for three populations characterized by genetic admixture with the largest geographic range: a) <i>Lepomis solis</i> x <i>L. megalotis</i> in the middle and upper Tennessee River; b) <i>L. peltastes</i> x <i>L. megalotis</i> in the upper Ohio River; and c) <i>L.</i> sp. Ozark x <i>L. megalotis</i> in the Osage River. Best-fit model for each population is highlighted with a red box. <strong>Supplementary Table S1</strong> List of described species that were synonymized with <i>Lepomis megalotis</i> prior to the present study. Current status follows the results of the present study. <b>Supplementary Table S2</b> List of specimens used in molecular analyses, GPS coordinates of sampling locations, and voucher and tissue catalog numbers. Specimen names correspond to IQ-TREE phylogeny shown in Supplementary Fig. S2. Museum catalog abbreviations: INHS, Illinois Natural History Survey; MMNS, Mississippi Museum of Natural Science; RNYFH, New York State Fish Hatchery at Randolph; SLU, Southeastern Louisiana University Vertebrate Museum; TCWC, Biodiversity Research and Teaching Collections (formerly Texas Cooperative Wildlife Collection); TNHC, Texas Natural History Collections; UF, University of Florida, Florida Museum of Natural History; UMSNH, Michoacan University of Saint Nicholas of Hidalgo; UT, University of Tennessee Etnier Ichthyological Collection; YPM, Yale Peabody Museum of Natural History; YFTC, Yale Fish Tissue Collection. Species abbreviations: Laqu, <i>Lepomis aquilensis</i>; Loua, <i>Lepomis</i> sp. Ouachita; Lsol, <i>Lepomis solis</i>; Lozk, <i>Lepomis</i> sp. Ozark; Lpel, <i>Lepomis peltastes</i>; Lmeg, <i>Lepomis megalotis</i>. NA, not available. <b>Supplementary Table S3</b> List of specimens used in morphological analyses, number of specimens (N), and GPS coordinates of sampling locations. Species identification was made according to the river basin or system, following the results of our IQ-TREE analysis of the concatenated ddRAD loci data. Some of the specimens identified as hybrids may represent genetically pure species of one of the parental species. Museum catalog abbreviations: ANSP, Academy of Natural Sciences of Philadelphia; AUM, Auburn University Natural History Museum; CMNFI, Canadian Museum of Nature; FMNH, Field Museum of Natural History; INHS, Illinois Natural History Survey; JFBM, Bell Museum of Natural History; KU, University of Kansas Biodiversity Institute; MMNS, Mississippi Museum of Natural Science; MPM, Milwaukee Public Museum; NYSM, New York State Museum; OSUM, Ohio State University, Museum of Biological Diversity; MRNF, Ministry of the Natural Resources and Quebecois Fauna; ROM, Royal Ontario Museum; TNHC, Texas Natural History Collections; UF, University of Florida, Florida Museum of Natural History; UMMZ, University of Michigan Museum of Zoology; USNM, National Museum of Natural History, Smithsonian Institution; UT, University of Tennessee David A. Etnier Ichthyological Collection; UWZM, University of Wisconsin Zoological Museum; YPM, Yale Peabody Museum of Natural History. NA, not available. <b>Supplementary Table S4</b> Models tested in the Fastsimcoal2 analysis for three populations characterized by genetic admixture with the largest geographic range. Parameter estimates of the highest estimated log-likelihood (MaxEstLhood) for each model are shown. Best-fit model for each population is highlighted. Model parameter abbreviations correspond to those shown in Supplementary Fig. S5. NA, not applicable. <b>Supplementary Table S5</b> Fastsimcoal2 best-fit models for three populations characterized by genetic admixture with the largest geographic range and the 95% confidence interval of each parameter from 100 parametric bootstrap replications. Model parameter abbreviations correspond to those shown in Supplementary Fig. S5 and model names correspond to those shown in Supplementary Fig. S5 and Supplementary Table S4. NA, not applicable. <b>Supplementary Table S6</b> Means (bold) and 95% confidence interval of means (in parenthesis) for variable morphometric and meristic traits among species in the <i>Lepomis megalotis</i> complex identified in Tukey honestly-significant difference test (<i>p</i> &lt; 0.001).  <b>Supplementary Table S7</b> Categorical morphological traits and the number of specimens for species in the <i>Lepomis megalotis</i> complex for each category.