DOI: 10.5061/dryad.79cnp5j6m
title: <em>Nothofagus</em> hybridization and population structure reveals high fluctuating asymmetry in putative hybrids and surprisingly little differentiation between species

description: Interspecific hybridization is documented in various tree species, such as
 the Nothofagus (N. pumilio and N. antarctica), located in Chile.
 Hybridization can sometimes lead to developmental instability, resulting
 in increasing leaf fluctuating asymmetry. However, the extent of
 Nothofagus hybridization throughout Chile and its impact on leaf
 fluctuating asymmetry between N. pumilio and N. antarctica hybrids remains
 unexplored. We collected N. pumilio, N. antarctica, and putative hybrid
 leaves for morphological and chloroplast DNA analyses and conducted
 population genetic analysis on N. pumilio, N. antarctica, N. betuloides,
 N. dombeyi, and N. obliqua using microsatellite markers (eight loci). The
 putative hybrids with intermediate phenotypes displayed significantly
 higher fluctuating asymmetry than morphologically unambiguous N. pumilio
 and N. antarctica, suggesting a higher developmental instability. However,
 despite clear phenotypic differences, our DNA analyses of this sympatric
 population did not show the two species or their putative hybrids to be
 genetically differentiated; all three groups shared the same mtDNA and
 almost identical microsatellite composition. Our microsatellite analyses
 of five Nothofagus species found high allelic variation within species and
 subpopulations, but surprisingly low structure between species and
 populations. Further studies are needed to determine whether the N.
 pumilio and N. antarctica genomes are distinct enough to be classified as
 distinct species under the phylogenetic species concept.
description: We collected leaf samples from <em>Nothofagus
 antarctica</em> and <em>N. pumillo</em> and putative
 hybrids in the forests near Estancia Pingo Salvaje, Chile (-51.5359,
 -72.5669) in December 2023: 10 <em>N. pumilio,</em> 11
 <em>N. antarctica</em>, and 23 putative hybrid individuals
 (appendix A) During the following March, October, and
 November, we collected additional samples from 125 trees in 14 populations
 in northern (41-42 °S) and southern regions (51-53 °S; Figure 2A).  In the
 southern region, we sampled 33 <em>N. pumilio</em>, 29
 <em>N. antarctica</em>, 29 <em>N. betuloides</em>,
 and 6 <em>N. dombeyi</em> trees. In the Northern region, we
 sampled 1 <em>N. pumilio</em>, 2 <em>N.
 antarctica,</em> 16 <em>N. dombeyi,</em> and 9
 <em>N. nitida.</em> (appendix B) For the
 samples from Estancia, we collected 3 chloroplast genes (Matk, rbcL, and
 atpB-rbcL intergenic spacer regions) as well as genotype data for 8
 microsatellites. For the remaining samples, we
 collected genotype data for 8 microsatellites (Appendix C)
description: # *Nothofagus* hybridization and population structure reveals high
 fluctuating asymmetry in putative hybrids and surprisingly little
 differentiation between species
 [https://doi.org/10.5061/dryad.79cnp5j6m](https://doi.org/10.5061/dryad.79cnp5j6m) ## Description of the data and file structure Datasets from paper: *Nothofagus* hybridization and population structure reveal high fluctuating asymmetry in putative hybrids and surprisingly little differentiation between species. ### Files and variables #### File: FieldData_2022_appendixA.csv **Description:** Collection of information for individuals used in the morphological analyses.   ##### Variables * Individual:  Individual label * Latitude: GPS coordinates * Longitude:  GPS coordinates * DBH (cm): diameter of trunk at breast height in the field * Height (m): height of the tree in the field * Species: *Nothofagus antarctica, Nothofagus pumilio, or putative hybrid* #### File: FieldData_2023_appendixB.csv **Description:** Collection sites for individuals used in population genetic analyses ##### Variables * ID: individual plant ID * Latitude: GPS coordinate * Longitude: GPS Coordinate * Species: Species identity #### File: Microsatellite_reads_appendixC.csv **Description:**  ##### Variables * ID: individual label * Species: the species of the individual * Collection Group: location information * location: location * 6fam1: 1st allele of microsatellite Npum1 * 6fam1_2: 2nd allele of microsatellite Npum1 * 6fam2: 1st allele of microsatellite Npum9 * 6fam2_2: 2nd allele of microsatellite Npum9 * vic1: 1st allele of microsatellite Npum3 * vic1_2: 2nd allele of microsatellite Npum1 * vic2: 1st allele of microsatellite Npum11 * vic2_2: 2nd allele of microsatellite Npum11 * ned1: 1st allele of microsatellite Npum10 * ned1_2: 2nd allele of microsatellite Npum10 * ned2: 1st allele of microsatellite Npum18 * ned2_2: 2nd allele of microsatellite Npum18 * pet1: 1st allele of microsatellite Npum13 * pet1_2: 2nd allele of microsatellite Npum13 * pet2: 1st allele of microsatellite oak_pum43 * pet2_2: 2nd allele of microsatellite oak_pum43 #### File: intergenic.fasta **Description:** Chloroplast DNA sequences from the intergenic region and rbcl from our *Nothofagus antarctica* and *N. pumilio* samples aligned against existing GenBank sequences. Individual labels refer to labels in Appendix B. #### File: MatK_nothofagus.fasta **Description:** Chloroplast DNA sequences from the MatK gene from our *Nothofagus antarctica* and *N. pumilio* samples aligned against existing GenBank sequences.  Individual labels refer to labels in Appendix B. #### File: rbcl.fasta **Description:** Chloroplast DNA sequences from the rbcl gene from our *Nothofagus antarctica* and *N. pumilio* samples aligned against existing GenBank sequences.  Individual labels refer to labels in Appendix B. ## Access information Other publicly accessible locations of the data: * Genbank accession number PV239901-PV239906 for chloroplast markers for one representative N. pumilio and one N. antarctica Data was derived from the following sources: * For Chloroplast sequencing fasta files we have new collections (Sanger sequencing) and for fasta files Genbank access:  AB015464, AJ581438, U92859, U92860, AY263924, LC677699, L13341, L13342, L13343, L13344, L13345, L13346, L13347, L13348, L13349, L13350, L13351, L13352, L13353, L13354, L13355, L13356, L13357, L13358, L13359, L13360, L13361, L13362, L13363, L13339, AF015686, AF015687, AF015688, AF015689, AF015690, AF015691, AF015692, AF015693, AF015694, AF015695, AF015696, AF015697, AF015698, AF015699, AF015701, AF015703, AF015704, AF015705, AF015706, AF015707, AF015708, AF015709, AF015710. #### Code/Software: Structure_Clean.R: This R script reads STRUCTURE output files, extracts individual ancestry (Q) values, and saves them as a CSV for a chosen number of clusters (K). It then plots these ancestry proportions as stacked bar plots using ggplot2 and exports the figure as a PDF. Nothofagus_Rcode_clean.R: This script performs exploratory plots, ANOVA, and Tukey post-hoc tests to compare multiple leaf traits among species, visualizing results with density plots and boxplots. It also runs species-specific linear regressions to examine relationships between leaf dimensions (length, width, area) and visualizes them with scatter plots and fitted lines. Fst_calculations.R: This code loads the hierfstat package and checks its citation and version. It then calculates allelic richness, allele counts, and basic population genetic statistics (including F-statistics) from genetic data.