Phylogenetic relationships within Pangasiidae

The complete mtDNA sequence datasets (concatenated 13 PCGs) recovered Pangasius and Pangasianodon as sister subclades and the Pangasiidae as a monophyletic clade with a 100% bootstrap value (Fig. 2). The ML tree clarified the monophyly of Pangasianodon and Pangasius with 88% bootstrap support. The ML tree clarified the monophyly of Pangasianodon, with 88% bootstrap support. Pangasianodon gigas is identified as a sister taxon to a group of P. sanitwongsei and Pn. hypophthalmus (5 mitogenomes), and Pangasius bocourti (as named in GenBank under no. MN842723) is positioned in the Pn. hypophthalmus group with 100% bootstrap support. Within the Pangasius, P. mekongensis was recovered as a sister taxon to the Indian Pangasius pangasius Hamilton-Buchanan, 1822 (Hossain et al. 2009) with a very high bootstrap (100%), while P. krempfi was placed in between P. larnaudii and the two abovementioned Pangasius species (P. mekongensis and P. pangasius) with a moderate bootstrap value (68%). The most concerning feature is that P. sanitwongsei was positioned as a sister taxon to a subgroup of all the Pn. hypophthalmus sequences (bootstrap 98%), and was in between this subgroup and Pn. gigas (Fig. 2). Several samples may have been misidentified and, therefore, phylogenetically misplaced. In fact, the sequence named “ Pangasianodon_hypophthalmus _(BaijinCo-Foshan)-China-MZ286355” is Pangasius larnaudii, and the “ Pangasius bocourti (QingyuanGD) -China-MN842723” sample is Pangasianodon hypophthalmus. These pangasiid sequences were grouped into their corresponding phylogenetic clades (Fig. 2).

Furthermore, using single-gene datasets, we investigated the close phylogenetic relationships between Pangasius and Pangasianodon. Multiple cox 1 and cyt B barcode sequences are available in GenBank and in previous publications; therefore, we downloaded all cox 1 (551 bp) and cyt B (634 bp) sequences and extracted cox 1 and cyt B, respectively, from the complete mitogenomes (listed in Table S3). The cox 1 and cyt B topologies also revealed that the Pangasiidae were a sister group to the Austroglanididae. All three families (Pangasiidae, Cranoglanididae, and Austroglanididae) together with the Ictaluridae formed a large group that was always a sister group to the Ariidae, as discovered in the mitophylogeny constructed based on the complete mitogenome data in our current study and as in the previously reported analysis (Schedel et al. 2022).

The cox 1 phylogenetic tree (Fig. 3) indicated that P. mekongensis (4 sequences) is a sister taxon to P. pangasius (8 sequences) with relatively high bootstrap support (88%). In the cyt B tree (Fig. 4), this species is sistered with P. pangasius with 100% nodal support. Pangasius krempfi, in the cox 1 tree, was shown to be close to the Helicophagus species (Helicophagus leptorhynchus and Helicophagus waandersii) with a low bootstrap of 44%, and was placed as a sister taxon in the cyt B tree in a non-stable phylogenetic relationship with a 63% bootstrap, to P. macronema and P. polyuranodon. The partial cox 1 datasets (four sequences, consisting of two from South Africa, one from China, and one from Cambodia) and cyt B datasets (two sequences from Thailand and one from China) of the correctly identified P. sanitwongsei species placed this taxon into the Pangasius clade, although with low support (bootstrap 36% and 29%, respectively) (Figs. 3 and 4). It should be noted that several P. sanitwongsei sequences, including Psan-(PB2)-(China)-JN020073 and Psan-(PB1)-CN-JN020086, as well as a P. bocourti sequence, Pboc-(QingyuanGD)-China-MN842723, were placed in the Pn. hypophthalmus cluster, which could be attributed to missampling or misidentification (Figs. 3 and 4).

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In both the cox 1 and cyt B phylogenies, the Pangasianodon was clearly resolved as a sister to the Pangasius with medium nodal support (bootstrap: 86% for cox 1 and 52% for cyt B). While the monophyletic Pangasianodon was resolved with its two members (Pn. gigas was the sister taxon to Pn. hypophthalmus in both the cox 1 and the cyt B phylogenetic topologies), the Pangasius cluster included not only Pangasius but Helicophagus and Pseudolais species as well. Though they had low bootstrap values (28% for cox 1 and 29% for cyt B phylogeny), Helicophagus and Pseudolais species were grouped as sister taxa to some of the Pangasius species.