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Even more oak species in Mexico? Genetic structure and morphological differentiation support the presence of at least two specific entities within Quercus laeta

Citation

Morales-Saldaña, Saddan et al. (2021), Even more oak species in Mexico? Genetic structure and morphological differentiation support the presence of at least two specific entities within Quercus laeta, Dryad, Dataset, https://doi.org/10.5061/dryad.2280gb5tf

Abstract

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.

Methods

A total of 161 adult individuals of the Q. laeta 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 Q. laeta 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.

Usage Notes

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Funding

Consejo Nacional de Ciencia y Tecnología, Award: 483720 / 624511

PAPIIT-DGAPA-UNAM, Award: 210020