Isbrueckerichthys

Sequences of the COI gene were obtained from the Isbrueckerichthys specimens and deposited in GenBank (PP391830–PP391860). All sequences were of high quality and presented no evidence of insertions, deletions, or stop codons. The obtained sequences were aligned with the sequences of I. saxicola from the GenBank database, and a matrix of 36 sequences with 612-bp was generated. The nucleotide diversity (π) was 0.01369, and haplotype diversity (Hd) was 0.806. A total of ten haplotypes were observed: Isbrueckerichthys alipionis had four haplotypes; I. calvus and I. saxicola shared a unique haplotype; individuals of I. duseni and I. cf. duseni had one exclusive haplotype to each species; and in I. epakmos three haplotypes were observed (Fig. 2A). The intraspecific distance for each group ranged from 0 to 0.23%, and the interspecific distance ranged from 0 to 3.63% (Tab. 2). The BAPs results indicated an optimal partition of four clusters (k = 4; marginal log-likelihood = -240.4536). Individuals of I. calvus and I. saxicola were assigned as a single group, I. duseni, and I. cf. duseni were separated into a second cluster, and I. epakmos and I. alipionis represent independent groups (Fig. 2B). The AMOVA analysis, considering the clusters delimited by BAPs, shows a variance value of 83.98% among groups (Tab. 3), with an FST value of 0.96692 (p <0.05). The pairwise FST values between each population ranged from 0.00 to 1.00 (Tab. 4). The Bayesian phylogenetic tree revealed five supported clades: I. epakmos, I. alipionis, Isbrueckerichthys cf. duseni, I. duseni, and I. calvus + I. saxicola (Fig. 2C). Species delimitation methods returned different results (Fig. 2C). The ABGD method resulted in seven partitions ranging from two to nine species, with partition 4 (prior maximal distance P = 0.004642) indicating six species, including the outgroup. ABGD recognized I. epakmos, I. alipionis, I. duseni, and Isbrueckerichthys cf. duseni as single groups and grouped I. calvus and I. saxicola. The best partition identified by the ASAP method (ASAP-score: 3.50; p-value: 1.74e- 02; W: 3.13e- 05; Threshold dist.: 0.004094) recognized the same six species as the ABGD method. The ML solution of bPTP delimited eight species, including the outgroup. bPTP separated I. alipionis into three species and grouped I. calvus and I. saxicola.