DNA extraction, sequencing and data clustering
A total of 189 individuals from 33 populations (Fig. 1; Table S1) of the
four species of C. gr. laevigata (plus one sample each ofC. mairei –sister group–, C. distans , C. boryanaand C. borbonica as outgroup) were sampled for
Restriction-site-Associated DNA sequencing (RAD-seq). This technique has
been proven as a useful High Throughput Sequencing (HTS) approach for
investigating recent evolutionary processes in non-model organisms,
including different scenarios of speciation among closely related
species (Otero et al., 2022 and references therein), in which
traditional Sanger-sequenced nuclear and plastid markers have been
unable to solve phylogenetic relationships (Escudero et al., 2013b). A
special effort was conducted for C. laevigata in order to obtain
a wide intraspecific genomic sampling representative of chromosome
number variation within and across populations. Thus, 130 individuals
from 20 populations of C. laevigata , most of which with available
chromosome counts, were included. DNA was extracted using a DNeasy Plant
Mini Kit (Qiagen, Valencia, CA, USA). RAD-seq libraries preparation
using restriction enzyme PstI were followed by sonication and barcoding
performed by Floragenex (Eugene, Portland, OR, USA) following Bairdet al. (2008).
Raw data were demultiplexed based on the code for each sample and
adapters were removed from the reads using ipyrad v0.7.24 (Eaton &
Overcast, 2020) using a reference genome (Carex scoparia ; Planta
et al., 2022). We used the matrix resulting from a clustering threshold
of 90% similarity and a minimum sequencing depth of 6 sequences per
locus for within-sample clustering (c90m6). We compared this matrix to
others with a clustering of 95% and sequencing depth of 6 and 11, but
no significant difference was found. Then we discarded those samples
with higher missing data. Finally, we reanalyzed the data according to
the c90m6 parameters and a minimum number of samples with data at a
given locus of 90 out of 156 (ca. 60%). We discarded those samples with
>95% of missing data for the phylogeny construction, so a
final matrix of 156 accessions was retained (three outgroup taxa, one
sister taxa and 152 Carex gr. laevigata samples). The
phylogenetic relationships of the retained samples were reconstructed
using IQTREE v1.6.11 software (Nguyen et al. , 2015; Hoanget al ., 2018).