DOI: 10.5061/dryad.2280gb5tf
title: Even more oak species in Mexico? Genetic structure and morphological differentiation support the presence of at least two specific entities within Quercus laeta

description: Differentiation among populations, sometimes despite ongoing gene
 exchange, is a key step in speciation. Therefore, comparison of intra- and
 interspecific differentiation patterns is of great significance to
 understanding speciation. The genus Quercus is an interesting system to
 test speciation models in the presence of gene flow, due to its weak
 interspecific reproductive barriers. The aim of the present study was to
 characterize the degree and pattern of morphological and genetic
 differentiation among different morphotypes in the white oak Quercus
 laeta, some corresponding to the previously described species Q.
 centralis, Q. laeta, Q. prinopsis and Q. transmontana, as well as
 geographically structured variation within Q. transmontana not previously
 described. Our goal was to evaluate if some of these can be considered
 distinct specific entities or are rather part of a continuum of variation.
 Nine microsatellite loci and two intergenic regions of chloroplast DNA
 were analyzed. Morphological differences were evaluated using geometric
 morphometrics. Chloroplast DNA showed low differentiation, suggesting
 introgression or sharing of ancestral haplotypes among the Q. laeta
 morphotypes. Nuclear microsatellites indicated differentiation into two
 distinct main genetic groups, which were congruent with morphological
 differentiation. In conclusion, nuclear markers and morphological
 variation suggest the existence of at least two different entities within
 Q. laeta.
description: A total of 161 adult individuals of the <em>Q</em>.
 <em>laeta </em>complex were sampled from 19 sites (hereafter,
 ‘populations’ refers to samples from these locations as geographically
 defined, unless otherwise specified). At each population, 5-11 randomly
 selected trees were sampled, separated from each other by a minimum of
 30-50 m. Populations almost covered the complete geographical distribution
 of <em>Q</em>. <em>laeta </em>and the five
 morphotypes. At each site, the trees were identified based on the
 above-mentioned diagnostic morphological characters. Collected
 branches were pressed and dried to obtain three herbarium specimens. From
 each tree, we additionally sampled mature leaves with no apparent
 damage from different branches; these leaves were stored in plastic bags
 and placed on ice until final storage at -80 °C in the laboratory for
 genetic analysis. Likewise, between six and eight mature leaves were
 pressed per individual for the corresponding morphometric analyses.
 Vouchers of specimens were deposited in the FCME herbarium.
 To estimate the population genetic diversity and structure, nine
 nuclear microsatellite loci were amplified in three multiplex PCRs and two
 individual reactions. The first multiplex reaction included the QpZAG96
 and QpZAG110 loci; the second the QpZAG36 and QrZAG39 loci and the third
 the quru-GA-IF02, quru-GA-OC11 and quru-GA-OMO5 loci. Finally, the
 quru-GA-OC19 and quru-GA-2F05 loci were amplified separately. Reactions
 were carried out with 3 µL of Taq-Platinum master mix (Qiagen CA, USA),
 0.3 µL of each forward and reverse primer (0.2 µM), 1.4 µL of H2O, and 1
 µL of genomic DNA (10 ng/ µL) in a total volume of 6 µL. The reaction
 began with 3 min denaturation at 94 °C, followed by 40 cycles of 30 s
 denaturation at 94 °C, 45 s annealing at 54 °C for quru-GA-OC19 and the
 first and third multiplex reactions, 48°C for the second multiplex
 reaction and 45 °C for quru-GA-OM05. A final extension step of 10 min at
 72 °C was included. One µL of each PCR product was combined with 9 µL of
 Hi-Di Formamide and 0.3 µL of GeneScan-600 LIZ (Applied Biosystems, CA,
 USA) and run on an ABI-PRISM 3100-Avant sequencer (Applied Biosystems, CA,
 USA) to obtain the size of the microsatellite fragments. Electropherograms
 were analyzed using the GENEMARKER software v.1.91 (Softgenetics LLC,
 State College, PA, USA). The individual genotype assignments of the
 nine nSSR were verified at least three times to corroborate our
 genotyping.
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