3.1 GB-RFLP marker analysis for lake of origin
assignment
As a first step, we chose RFLP markers that are specific for lakes
(i.e., allowing to distinguish one population [ingroup] from all
other populations [outgroup]) (Fig. 1 and Fig. 2). Although not all
of them are (yet) described as separate species, genetic differentiation
is very high among most of the lake populations — mainly because the
crater lakes are completely isolated (and therefore not permitting gene
flow) and because their founder populations were small (30–850
individuals), resulting in strong genetic drift (and thereby more
alternatively fixed alleles) (Kautt et al., 2020). Most variants have
likely been recruited from the standing genetic variation present in the
source populations that was introduced into the crater lakes with the
colonizers, as there was too little time for a substantial amount ofde novo mutations to occur — all crater lakes have been
colonized less than 5,000 years ago (Kautt et al., 2020; Kautt,
Machado-Schiaffino, & Meyer, 2016; Kautt et al., 2018). The two great
lakes Managua and Nicaragua are not isolated from each other, but
intermittently connected by a river (Rio Tipitapa) resulting in limited
ongoing gene flow (based on demographic models from (Kautt et al., 2020)
for A. citrinellus approximately 1 in 12000 individuals per
generation).
To assess the performance of markers, we calculated their predictive
power (see detailed description in Materials and Methods section) based
on bootstrapping using allele frequencies from a previous genomic study
that was based on 453 re-sequenced genomes (Kautt et al., 2020) as well
as GB-RFLP assays of additional samples in this study (that were not
part of the resequenced genomes from the (Kautt et al., 2020) study, but
morphologically assigned to lakes and species). In line with the
discussed potential effect of the described demographic parameters
(Kautt et al., 2020), RFLP markers were most powerful for crater lakes.
For ten out of the twelve RFLP markers for Crater Lakes Apoyeque (Fig.
S3E), Apoyo (Fig. S3G, H), As. Managua (Fig. S3I, J), As. León (Fig.
S3K, L), Masaya (Fig. S3M, N), Tiscapa (Fig. S3O, P), and Xiloá (Fig.
S3Q) more than 90% of the samples were correctly assigned to the lake
(Table 1). The second Apoyeque marker (87%, Fig. S3F) and second Xiloá
marker (69%, Fig. S3R) performed less well. For the great lakes Managua
(both 62%) and Nicaragua (87% and 69%) the percentage of correctly
assigned samples was — as expected — lower (Fig. S3A–D), as many
variants are shared between the two great lakes or can be found at least
in one of the crater lakes (as they have been colonized from the great
lakes).