<em>Nothofagus</em> hybridization and population structure reveals high fluctuating asymmetry in putative hybrids and surprisingly little differentiation between species

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. Methods We collected leaf samples from Nothofagus antarctica and N. pumillo and putative hybrids in the forests near Estancia Pingo Salvaje, Chile (-51.5359, -72.5669) in December 2023: 10 N. pumilio, 11 N. antarctica, 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 N. pumilio, 29 N. antarctica, 29 N. betuloides, and 6 N. dombeyi trees. In the Northern region, we sampled 1 N. pumilio, 2 N. antarctica, 16 N. dombeyi, and 9 N. nitida. (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)