Mitophylogeny of Pangasiid Catfishes and its Taxonomic Implications for Pangasiidae and the Suborder Siluroidei

© 2023 Academia Sinica, Taiwan Open Access Mitophylogeny of Pangasiid Catfishes and its Taxonomic Implications for Pangasiidae and the Suborder Siluroidei Thuy Yen Duong1, Linh Thi Khanh Pham2,3, Xuyen Thi Kim Le2,3, Ngoc Tran Thi Nguyen1, Siti Azizah Mohd Nor4, and Thanh Hoa Le2,3,* 1College of Aquaculture and Fisheries, Can Tho University, 3/2 street, Can Tho City, Vietnam. E-mail: [email protected] (Duong) 2Immunology Department, Institute of Biotechnology (IBT), Vietnam Academy of Science and Technology (VAST). 18. Hoang Quoc Viet Rd., Cau Giay, Hanoi, Vietnam. *Correspondence: E-mail: [email protected] (Le) E-mail: [email protected] (Pham); [email protected] (Kim Le) 3Graduate University of Science and Technology (GUST), Vietnam Academy of Science and Technology (VAST), 18. Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam 4Institute of Marine Biotechnology, Universiti Malaysia Terengganu, 21030 Kuala Nerus, Terengganu, Malaysia. E-mail: [email protected] (Nor) Received 2 May 2022 / Accepted 24 July 2023 / Published 22 September 2023 Communicated by Ka Hou Chu Pangasiidae (catfish order: Siluriformes) comprises 30 valid catfish species in four genera: Pangasius, Pangasianodon, Helicophagus, and Pseudolais. Their systematics are frequently revised due to the addition of newly described species. Although Pangasiidae is known to be a monophyletic family, the generic and phylogenetic relationships among the taxa are poorly resolved. This study characterized three newly obtained complete mitogenomes of Mekong River catfishes from Vietnam (Pangasius mekongensis, Pangasius krempfi, and Pangasianodon hypophthalmus), as well as the interand intrafamilial relationships of the Pangasiidae and catfish families in Siluroidei. The genomic features of their mitogenomes were similar to those of previously reported pangasiids, including all regulatory elements, extended terminal associated sequences (ETAS), and conserved sequence blocks (CSBs) (CSB-1, CSB-2, CSB-3, and CSBs, A to F) in the control region. A comprehensive phylogeny constructed from datasets of multiple 13 PCG sequences from 117 complete mitogenomes of 32 recognized siluriform families established Pangasiidae as monophyletic and a sister group of Austroglanididae. The [Pangasiidae + Austroglanididae] + (Ictaluridae + Cranoglanididae) + Ariidae] clade is a sister to the “Big Africa” major clade of Siluriformes. Furthermore, both phylogenies constructed from the single barcodes (83 partial cox1 and 80 partial cytB, respectively) clearly indicate genus relationships within Pangasiidae. Pangasianodon was monophyletic and a sister to the (Pangasius + Helicophagus + Pseudolais) group. Within the genus Pangasius, P. mekongensis was placed as a sister taxon to P. pangasius. Pangasius sanitwongsei was found to be related to and grouped with Pangasianodon, but in single-gene phylogenies, it was assigned to the Pangasius + Helicophagus + Pseudolais group. The datasets in this study are useful for studying pangasiid systematics, taxonomy and evolution. Key words: Pangasius, Pangasianodon, Pangasiidae, Mitogenomes, Mitophylogenetic analysis Citation: Duong TY, Pham LTK, Le XTK, Nguyen NTT, Nor SAM, Le TH. 2022. Mitophylogeny of pangasiid catfishes and its taxonomic implications for Pangasiidae and the suborder Siluroidei. Zool Stud 62:48. doi:10.6620/ZS.2023.62-48. BACKGROUND Siluroidei (Teleostei: Siluriformes), one of the three suborders of Siluriformes (Siluroidei, Loricarioidei, and Diplomystoidei), comprises catfishes throughout the world that have not been exhaustively phylogenetically classified (Diogo 2004; Betancur et al. 2017; Fricke et al. 2023; Schedel et al. 2022). Improvements in phylogeny Zoological Studies 62:48 (2023) doi:10.6620/ZS.2023.62-48 1 © 2023 Academia Sinica, Taiwan testing using molecular markers and the increasing availability of mitogenome data have indicated the Pangaean origin of the siluriform catfishes (Hardman 2005; Sullivan et al. 2006; Nakatani et al. 2011; Kappas et al. 2016; Moreira et al. 2017; Schedel et al. 2022). Interfamilial relationships within Siluroidei are divided into two major clades, “Big Africa” and “Big Asia”, first reported by Sullivan et al. (2006) based on the analysis of the rag1 and rag2 nuclear gene sequences. These big groups have been clarified by mitophylogeny (Kappas et al. 2016) and, more recently, by the additional mitogenomic data of mochokids (Mochokidae) and austroglanidids (Austroglanididae) (Schedel et al. 2022). Furthermore, when phylomitogenomic data from a number of loricarioid species were analyzed, the suborder position of Siluroidei, Diplomystoidei, and Loricarioidei in the Siluriformes was recognized (Moreira et al. 2017). However, in Siluroidei, the intraand interrelationships of the pangasiid species and the monophyly of the family Pangasiidae remain uncertain. Schedel et al. (2022) recovered the (Astroglanididae + Pangasiidae) interfamilial group as a sister to a clade encompassing Ictaluridae and Cranoglanididae. The tentative “Big Africa” membership status of Pangasiidae requires additional support. Morphology data have led to a division of the family Pangasiidae Bleeker, 1858, comprising 30 valid species, into four recognized genera: Pangasius Valenciennes, 1840; Pangasianodon Chevey, 1931; Helicophagus Bleeker, 1858; and Pseudolais Vaillant, 1902 (https:// researcharchive.calacademy.org/research/ichthyology/ catalog/SpeciesByFamily.asp) (Fricke et al. 2023). The genus Pangasius possesses 23 species, the highest number of species in Pangasiidae, while the genus Pangasianodon includes only two species, the genus Helicophagus contains three species, and Pseudolais encompasses two species (Froese and Pauly 2021; Fricke et al. 2023). Catfishes of the family Pangasiidae are widely distributed in river systems throughout South Asia (Pakistan and India), Southeast Asia (Myanmar, Malaysia, Thailand, Cambodia, Laos, Vietnam, and Indonesia), the southern part of China, and recently in a river in South Africa (Mäkinen et al. 2013; Wei et al. 2020; Fricke et al. 2023). In general, pangasiids are primarily restricted to freshwater, but a few species, including Pangasius mekongensis Gustiano, Teugels & Pouyaud, 2003 and Pangasius krempfi Fang & Chaux, 1949, spend part of their life cycle in brackish water in the Lower Mekong Basin (Rainboth 1996; Poulsen et al. 2004; Hogan et al. 2007). Some large-sized pangasiid catfish species, particularly migratory species inhabiting the Mekong River system, have been considered vulnerable or critically endangered in the wild. Among these threatened species are Pangasianodon hypophthalmus Sauvage, 1878; Pangasius mekongensis; and Pangasius krempfi (Vidthayanon 2012; Vidthayanon and Hogan 2011; Baird 2011). In addition to their ecological importance, Pangasius and Pangasianodon catfish have become economically important aquaculture species in Vietnam, Thailand, Indonesia, and India (Phan et al. 2009). Pangasianodon hypophthalmus (Pn. hypophthalmus) is the most widely cultured pangasiid species, accounting for the eighth-highest freshwater finfish production in the world (FAO 2022). In fish taxonomy, this species was formerly listed in the genus Pangasius (Roberts and Vidthayanon 1991; Pouyaud and Teugels 2000; Zhao et al. 2014). However, it was later revised to Pangasianodon based on the unique character of 8 to 9 pelvic fin rays, compared to 6 pelvic fin rays in other genera of the same family (Rainboth 1996; Gustiano 2009; Kottelat 2013). Pangasius krempfi and Pangasius mekongensis are important for capture fisheries in the Lower Mekong River basin (Poulsen et al. 2004; Hogan et al. 2007). Both species exhibit anadromous behavior (Hogan et al. 2007; Vu et al. 2022) and similar morphological characteristics, particularly at early stages (Karinthanyakit and Jondeung 2012; Tran and Duong 2019; NAGAO 2021). These characteristics make these two species suitable subjects for studying the congeneric relationships of the genus Pangasius and their phylogenetic relationships with Pangasianodon species in the Siluroidei suborder. There are still some difficulties surrounding the taxonomic classification and phylogenetic relationships of several species in the genera Pangasius and Pangasianodon. Furthermore, the systematics of Pangasiidae are frequently revised due to the addition of newly described species (Gustiano et al. 2003 2021; Mohindra et al. 2015; Dwivedi et al. 2017; Fricke et al. 2023). Mitochondrial markers have been tested to infer the mitophylogenetic interfamilial relationship in the suborder Siluroidei, which involves the placement of Pangasiidae. Many previous studies have investigated the phylogenetic relationships of the familial and multifamilial clades of Siluriformes and reported the intraand intercontinental diversification within catfishes. These analyses comprised partial or complete mitochondrial DNA (mtDNA) sequences, which were used to construct the phylogeny of Siluriformes (Sullivan et al. 2006; Hardman 2005; Karinthanyakit and Jondeung 2012; Miya and Nishida 2015; Kappas et al. 2016; Moreira et al. 2017; Kim et al. 2018; Quyen et al. 2018; Zhang et al. 2021; Schedel et al. 2022). The partial or complete single-gene sequence and phylogenetic analyses included cytochrome b (cytB, approximately 1,150 bp) (Hardman 2005; Karinthanyakit and Jondeung 2012), cytochrome page 2 of 17Zoological Studies 62:48 (2023) © 2023 Academia Sinica, Taiwan oxidase subunit 1 (cox1, 551 bp or 1,551 bp) (Quyen et al. 2018; Zhang et al. 2021), and a growing number of works using the complete mitogenomes (Jondeung et al. 2007; Miya and Nishida 2015; Kappas et al. 2016; Moreira et al. 2017; Kim et al. 2018; Schedel et al. 2022). Although partial mtDNA sequences effectively resolve relationships among catfish taxa (Karinthanyakit and Jondeung 2012; Hardman 2005; Kartavtsev et al. 2007; Nakatani et al. 2011), full-length mtDNA sequences provide a higher level of resolution. In some studies, the complete mitogenomes were used to clarify the intergeneric, interfamilial, and intersuborder phylogenetic relationships related to Siluroidei, Diplomystoidei, and Loricarioidei in Siluriformes (Miya and Nishida 2015; Kappas et al. 2016; Villela et al. 2017; Kim et al. 2018; Schedel et al. 2022). Representatives of pangasiids, however, were still placed in a non-stable phylogenetic relationship within the family Pangasiidae and between the associated siluroid families in Siluroidei (Jondeung et al. 2007; Nakatani et al. 2011; Kappas et al. 2016; Kim et al. 2018; Wei et al. 2020). Concerning genuslevel relationships in Pangasiidae, based on a complete cytochrome b analysis of 13 pangasiids and six schilbids in Thailand, Karinthanyakit and Jondeung (2012) determined that the four genera have Pangasianodon as a basal group and Pseudolais and Helicophagus as sister groups of Pangasius. This conclusion aligned with findings by Quyen et al. (2018), based on partial cox1 and 16S sequences of 30 species belonging to nine families distributed in the Lower Mekong Basin, including 14 species of Pangasiidae. In both studies, Pangasius pangasius was absent from the analysis. However, in a phylogenetic analysis using the partial cytB by Tran et al. (2017), this species showed a sister taxon relationship with P. mekongensis. Another Indian Pangasius silasi was found to be a sister species to P. pangasius (Dwivedi et al. 2017), but no molecular data were available from this newly described species in GenBank for further investigation. Insufficient numbers of complete mtDNA sequences of Pangasiidae have been used for phylogenetic analyses of pangasiids and siluriforms, therefore requiring further work (Jondeung et al. 2007; Miya and Nishida 2015; Kappas et al. 2016). Early molecular phylogenetic studies by Jondeung et al. (2007) and Nakatani et al. (2011) used two complete mtDNA sequences of Pangasianodon gigas (AY762971) and Pangasius larnaudii (AP012018), respectively. In later taxonomic assessments, a few more complete mitogenomes of pangasiids were used. For example, three sequences of Pn. gigas (AY762971), P. larnaudii (AP012018), and Pn. hypophthalmus (NC_021752) were reported in Kappas et al. (2016), and two Pangasius and three Pangasianodon mtDNA sequences were used in Kim et al. (2018). In recent studies, Wei et al. (2020) employed Pn. hypophthalmus and three Pangasius species, including P. sanitwongsei (MN809630), while Villela et al. (2017) and Schedel et al. (2022) included Pangasius pangasius in their phylogenetic analyses. More coverage of complete mitogenomic sequences from each species, as well as more comprehensive sequences and phylogenetic analyses within and between Pangasius and Pangasianodon, is required. This study aimed to reconstruct a mitophylogeny of pangasiids and all representative siluriform species in order to resolve Pangasiidae’s placement within Siluriformes, and clarify interand intrageneric relationships within the monophyletic Pangasiidae among the key families within the suborder Siluroidei. To complete these analyses, we used the available siluriform mitogenomic data and expanded the topology presentation of the siluriform mitophylogeny, combined with our recently sequenced mitosequences from the three Mekong River catfishes (i.e., Pangasius mekongensis, Pangasius krempfi, and Pangasianodon hypophthalmus). Furthermore, we used the available partial cox1 and cytB datasets, plus newly obtained sequence data, to reconstruct phylogenies showing the most comprehensive interspecific and intergeneric relationships of the pangasiid species. MATERIALS AND METHODS Sample collection and species identification Samples were obtained from wild-caught adults from the Mekong River in Vietnam, including Pangasius mekongensis (P. mekongensis) from Vam Nao River in An Giang province (9°53'30.2"N, 105°57'39.3"E); Pangasianodon hypophthalmus (Pn. hypophthalmus) from Hau Giang River in Can Tho City (10.0452°N, 105.7469°E); and Pangasius krempfi (P. krempfi) from Phong Nam in Soc Trang province (10°32'16.79"N, 105°19'22.20"E). After the morphological examination, a piece (50 mg) of muscle was excised from the fish and individually stored at -20°C until use. Total genomic DNA was extracted from each fish sample according to the manufacturer’s instructions using the GeneJET™ Genomic DNA Purification Kit (Thermo Scientific Inc., MA, USA). DNA extract was eluted in 100 μL and stored at -20°C until use. Subsequently, species’ identities were confirmed by molecular phylogenetic analysis using mitochondrial DNA sequences and compared with the sequences available in GenBank, e.g., for P. mekongensis (cytB: page 3 of 17Zoological Studies 62:48 (2023) © 2023 Academia Sinica, Taiwan KY451465; KY451466; KY451467; cox1: KT289880), for Pn. hypophthalmus (cytB: GQ856796/KC846907; cox1: MK216612/MK216603), and for P. krempfi (12S-16S: HM355773/MG076881; cytB: MN087451/ MN087461/ MN087471/ HM236386/ HM236390) (Karinthanyakit and Jondeung 2012; Tran and Duong 2019; Zhao et al. 2014; Kim et al. 2018; Quyen et al. 2018). The annotation and sequence analysis of the pangasiid mitogenomes Long-range PCRs (L-PCRs) were applied using commercial kits (Thermo Fisher Scientific Inc., Waltham, MA, USA) and sequenced using primers listed in table S1. The complete mitogenome was obtained after assembling all the sequences from the sequencing. Protein-coding genes (PCGs) were identified by alignment with available mitogenomes of other Pangasius/Pangasianodon catfish species, with ATG/GTG as start and TAA/TAG as stop codons. For some genes, T-- or TAincomplete stops were considered. PCGs were translated using the vertebrate mitochondrial genetic code (translation Table 2 in GenBank). The tRNAscan-SE 1.21 program (www. genetics.wustl.edu/eddy/tRNAscan-SE/) (Lowe and Chan 2016) and ARWEN (http://mbio-serv2.mbioekol. lu.se/ARWEN/) were used to identify transfer RNA genes (Laslett and Canback 2008). The mitoribosomal genes (MRG), i.e., 16S (rrnL) and 12S (rrnS) RNA genes, were recognized by the data inference from the previous publication by Jondeung et al. (2007). The circular map was generated by the MitoAnnotator at: http://mitofish.aori.u-tokyo.ac.jp/annotation/input.html (Iwasaki et al. 2013). The stem-loop secondary structure of the OL origin site in each mitogenome was identified based on the prediction from RNAfold with minimum free energy (http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/ RNAfold.cgi). The extended terminal associated sequences (ETAS), central conserved sequence blocks (CSB-F, CSB-E, CSB-D, CSB-C, CSB-B, CSB-A), conserved sequence blocks (CSB-1, CSB-2, CSB-3), and putative promoters of the mtDNA control region were identified by alignment of the conserved domains in teleost fishes (Guo et al. 2003; Li et al. 2012; Fischer et al. 2013; Villela et al. 2017). The MEGA X program (Kumar et al. 2018) was used to determine the mitogenomic characteristics of pangasiid species. These included the nucleotide composition, AT and GC content of the complete mtDNAs and 13 PCGs, and the pairwise nucleotide comparison (%) of each PCG and each MRG among P. mekongensis, Pn. hypophthalmus, P. krempfi, Pn. gigas and P. larnaudii. Genetic distances were inferred by the analysis of 15,566–15,576 coding nucleotide sequences. The AT and GC skewness values (ranging from −1 to +1) were calculated using the following formula: AT skew = (A − T)/(A + T) and GC skew = (G − C)/(G + C) (Perna and Kocher 1995). Mitophylogenetic and synteny analyses We used 117 complete or near-complete mitogenomes from 109 species of 32 families of Siluriformes for ingroup data for phylogenetic analysis, and two species from Gonorynchiformes as an outgroup (information and author reference for each is given in Table S2), which provided all 13 PCGs for nucleotide sequence alignment. The siluriform families were Pangasiidae (n = 13), including one each of the newly obtained mitogenomes from P. mekongensis, Pn. hypophthalmus, and P. krempfi. Other siluriform families were Ailiidae (n = 1), Amblycipitidae (n = 4), Amphiliidae (n = 1), Ariidae (n = 6), Aspredinidae (n = 1), Astroblepidae (n = 1), Auchenipteridae (n = 3), Auchenoglanididae (1), Austroglanididae (n = 3), Bagridae (n = 10), Callichthyidae (n = 2), Cetopsidae (n = 2), Chacidae (n = 1), Clariidae (n = 5), Claroteidae (n = 2), Cranoglanididae (n = 1), Diplomystidae (n = 1), Doradidae (n = 2), Heteropneustidae (n = 1), Horabagridae (n = 2), Ictaluridae (n = 9), Loricariidae (n = 9), Malapteruridae (n = 1), Mochokidae (n = 4), Pimelodidae (n = 5), Plotosidae (n = 2), Ritidae (n = 1), Schilbeidae (n = 2), Siluridae (n = 11), Sisoridae (n = 9), and Trichomycteridae (n = 1). Outgroup species were taken from Gonorynchiformes (Chanos chanos and Gonorynchus greyi) (Saitoh et al. 2016), as used in a previous report (Kim et al. 2018). The taxon collection was targeted at the taxa/families surrounding the Pangasiidae and the representatives of the catfish clade “Big Africa” and “Big Asia” (Sullivan et al. 2006), including the newly described families (Austroglanididae and Mochokidae) by Schedel et al. (2022). The growing catfish mitodataset enabled us to include representative members of all three major lineages, e.g., Diplomystoidei (n = 1), Siluroidei (n = 102), and Loricarioidei (n = 14), for analysis. The nucleotide sequences of the PCGs were concatenated in the order of ND1, ND2, COX1, COX2, ATP8, ATP6, COX3, ND3, ND4L, ND4, ND5, ND6, and cytB (Schedel et al. 2022). The complementary ND6 sequence was automatically converted into a sense sequence when downloaded from GenBank. The final block alignment of about 11,410 bp was used for phylogenetic analysis according to the procedure described in Schedel et al. (2022). We used a software package for phylogenetic services, available at https://ngphylogeny.fr/workflows/ page 4 of 17Zoological Studies 62:48 (2023) © 2023 Academia Sinica, Taiwan (Lemoine et al. 2019) for constructing a maximum likelihood tree. Briefly, the input 119 sequences in FASTA format were uploaded for multiple alignment by MAFFT v7.407 (Katoh and Standley 2013), then curated by BMGE v1.12 (Criscuolo and Gribaldo 2010), and inferenced by PhyML v3.3 with maximum likelihood phylogeny with 1000 bootstrap replicates (Guindon et al. 2010). The output final tree was rendered and displayed in the Newick v1.6 format (Junier and Zdobnov 2010), and this Newick tree (nwk format) was visualized and parameterized with the FigTree v1.4.4 program (Rambaut 2018). To investigate the taxonomic and generic relationships of the pangasiid taxa, we used the partial cox1 and cytB sequences. These sequences were downloaded from GenBank and extracted from the mitogenomes of the newly sequenced pangasiids and the associated families (Cranoglanididae, Schilbeidae, Austroglanididae, and Ictaluridae), and two outgroup sequences were used for each phylogeny (information on mtDNA sequences and country of origin is given in Table S3). Due to the constraints in the composition of marker sequences, we took the outgroup sequences from the distant orders, such as Gymnotiformes for the cox1 and Clupeiformes for the cytB analysis. These supplementary datasets contained 83 partial cox1 (551 bp) and 80 partial cytB (634 bp), respectively, for phylogenetic analyses. The phylogenetic tree was reconstructed based on the MAFFT 7.471 alignment by the MEGA X program using the maximum likelihood method with the GTR + I + G model and 1000 bootstrap resamplings (Kumar et al. 2018). RESULTS Mitogenome features of the pangasiid species The complete mitogenome was 16,462 bp in length for P. mekongensis (GenBank: MZ272451), 16,523 bp for Pn. hypophthalmus (MZ272452), and 16,475 bp for P. krempfi (MZ272453). The circular mtDNA comprised 13 PCGs (atp8, atp6, cox1−3, cytB, nad1−6, nad4L), two MRGs (12S or rrnS and 16S or rrnL), and 22 tRNAs, similar in gene order and gene length to those of other fish mitogenomes (Fig. 1A) (Jondeung et al. 2007; Zhao et al. 2014; Satoh et al. 2016). Two MRGs, including 12S (957 bp for P. mekongensis and 958 bp for both Pn. hypophthalmus and P. krempfi), and 16S (1,677 bp for P. mekongensis; 1,674 bp for Pn. hypophthalmus; and 1,681 bp for P. krempfi) were identified. The twenty-two tRNAs in the mtDNAs ranged from 67 to 75 nucleotides in length, and one tRNA specifying serine (tRNASer(GCT)) lacked the DHU-arm (dihydrouridine-arm). All the remaining twenty-one tRNAs had the common ‘cloverleaf’ secondary structures with the complete four arms. Twelve PCGs in P. mekongensis, Pn. hypophthalmus, and P. krempfi used ATG as a start codon, only cox1 used GTG. Nine PCGs used complete TAG or TAA and four (cox2, cox3, nad4, and cytB) used incomplete T-- or TAcodons for gene termination. Short intergenic tracts of 1−5 bp were common, and larger spacers of 14 bp were observed between PCGs. The base composition and skewness values for the mtDNA of seven pangasiid species are listed in table S4. Similar to other Siluroidei and common fishes, for example, in the mtDNA of P. mekongensis, the use of A+T was 55.67% and G+C was 44.03%; the AT-skew was 0.096/mtDNA and 0.030/PCGs, and the GC-skew was -0.293/mtDNA and -0.309/PCGs. A similar pattern for base composition and skewness with a minimal difference was observed in other species in the genera Pangasius and Pangasianodon. Pairwise nucleotide differences (%) for each PCG and MRG among five species, P. mekongensis, P. krempfi, P. larnaudii, Pn. hypophthalmus, and Pn. gigas are shown in table S5. The lowest sequence divergence (2.44%) was found in atp8 between P. mekongensis and P. krempfi, whereas the highest (17.47%) was in nad3 between Pn. gigas and P. krempfi. Genetic distances (p-distances) were estimated from the alignment of nucleotide sequences from 11 mitogenomes from eight pangasiid species (using sequences of 15,566 bp–15,571 bp, excluding tRNAPro and the CR) (Table S6). The lowest level of divergence was between P. mekongensis (Vietnam) and P. pangasius (India) at 4.24%–4.31%, and the highest was between Pangasianodon species and both P. krempfi (at 9.19%– 9.34%) and P. larnaudii (at 9.25%–9.31%). Within each pangasiid species, pairwise genetic distances were small: only 0.07%–0.34% within Pn. hypophthalmus (three mitogenomes available) and 0.29% within P. pangasius (two mitogenomes available). P. sanitwongsei exhibited a lower distance to Pn. hypophthalmus (6.32%–6.45%) while other members of Pangasius did a higher distance (8.90%–9.30%) (Table S6). The origin (OL site) of L-strand replication was a short, non-coding sequence of 30 or 31 nucleotides in the typical tRNAW-A-N-C-Y region (Kartavtsev et al. 2007; Boore 1999). As is the case in all vertebrates, including fish, it has the potential to form a stable stem-loop structure. The conserved stem (hairpin) was rich in G and C and was formed by base-pairing of 9 nucleotides (nt) and ending with an A-rich loop of 9 nt (in P. mekongensis and Pn. hypophthalmus) and 10 nt (in P. krempfi) (Fig. 1B). The major non-coding region, termed the “control page 5 of 17Zoological Studies 62:48 (2023) © 2023 Academia Sinica, Taiwan region” (CR), is 823 bp in length for P. mekongensis, 887 bp for Pn. hypophthalmus, 830 bp for P. krempfi, and 827 bp for P. bocourti. It is 829 bp for P. pangasius, 822 bp for P. larnaudii, 887 bp for P. sanitwongsei, and 897 bp for Pn. gigas (Table S3). The CRs of Pangasius were 44–48 nucleotides shorter than those of Pangasianodon species. By comparative alignment and sequence analysis of the conserved domains in teleost fishes reported in previous studies (Guo et al. 2003; Li et al. 2012; Fischer et al. 2013; Villela et al. 2017; Cui et al. 2020), we successfully identified all regulatory elements in the mtDNAs of the pangasiid taxa. These included ETAS, six central conserved sequence blocks (CSB-F, CSB-E, CSB-D, CSB-C, CSB-B, and CSB-A) and three conserved sequence blocks (CSB-1, CSB2, and CSB-3). The origin site of H-strand replication (OH), a putative promoter, and the TATA boxes were aligned in the mtDNA CRs of eight species (P. mekongensis, P. krempfi, P. pangasius, P. larnaudii, P. bocourti, P. sanitwongsei, Pn. hypophthalmus (3 mitogenomes), and Pn. gigas) (Fig. S1). The ETAS is a palindromic sequence of 47 nucleotides, containing TAS (TACAT) and cTAS (reverse complementary ATGTA) motifs. The CSB-2, CSB-3, CSB-A, and CSB-B are the most conserved. Other CSBs share 90%–95% sequence identity among the pangasiids. Interestingly, P. sanitwongsei possesses a more similar CR in terms of length (887 bp), sequence composition, and CSB patterns to Pn. hypophthalmus than to Pangasius species (Fig. S1). Pangasianodon hypophthalmus(16,523 bp) Pangasius krempfi(16,475 bp) Pangasius mekongensis (16,462 bp) T T A AA T T TG CA AC C C A T T TAA GC GC GC GC CG GC G C AT A C G A AA A A A A T A C C A CA AT T C A A A Pkre Phyp Pmek trnN(c) trnC(c) loop hairpin (stem) 1 12 loop loop 22 29 23 12 12 22 ’3’5 Pmek: 5’ CTTTCCCCGCCT CGACAATAA AGGCGGGGA 3’ (30 nt) Phyp: 5’ CTTTCCCCGCCT CCAACAAAA AGGCGGGGA 3’ (30 nt) Pkre: 5’ CTTTCCCCGCCT CAATAATTAAAGGCGGGGA 3’ (31 nt) OL A B Fig. 1. A schematic circular map of the mitochondrial genome of three Mekong River pangasiid catfishes catfishes in Vietnam, Pangasius mekongensis, Pangasianodon hypophthalmus, and Pangasius krempfi, and the OL origin site of the light (L) strand’s replication. A, The circular map and gene abbreviations were generated by the MitoAnnotator software in the MitoFish database (http://mitofish.aori.u-tokyo.ac.jp/annotation/ input.html). Protein-coding genes (PCGs) are denoted by two capital letters or full names, and transfer RNA genes (tRNAs) are marked with threeletter amino-acid abbreviations. The heavy (H) strand is indicated by the outer line of the circle and the light (L) strand by the inner line. The D-loop (control region) is located between tRNAPro and tRNAPhe. The pangasiid photos were taken by the authors from the naturally caught fish on site. B, A schematic presentation of the stem-loop secondary structure of the OL origin site in mitogenomes of three pangasiid species based on the RNAfold predicted structure with the lowest free energy (http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi). On the L-strand, between the two flanking tRNAs (trnN (c) and trnC (c)), there is a conserved stem (hairpin) formed by 9-nucleotide (nt) base-pairing and ending with a loop of 9 nt (in Pmek and Phyp) and 10 nt (in Pkre). page 6 of 17Zoological Studies 62:48 (2023) © 2023 Academia Sinica, Taiwan Phylogenetic relationships of Pangasiidae within Siluroidei and Siluriformes The ML tree (Fig. 2) clearly demonstrated the monophyly of Siluroidei in Silurifomes with 80% bootstrap support, distinct from Loricarioidei and Diplomystoidei with 70% and 43% support, respectively. The family Trichomicteridae was not recovered within the Siluroidei. Some siluroid families, such as Ritidae and Ceptopsidae, were shown to be polyphyletically split at the beginning of the descending topology of the tree with a low bootstrap (49%). Rita rita catfish, uniquely reported from Pakistan (GenBank: KF670723), was classified into the family Bagridae (Fricke et al. 2023) but was not recovered in this family in the present study. This species represented the separate Ritidae family, recently used by Schedel et al. (2022), which showed its phylogenetic placement far from Bagridae (Fig. 2). The remaining siluroids were classified into eight multiple family clusters, as follows: i) the Plotosidae and Chacidae with 95% bootstrap; ii) the polyphyletic Pimelodidae, Heteropneustidae, and Clariidae (100% bootstrap); iii) the monophyletic Siluridae (100% bootstrap); iv) the polyphyletic Aspredinidae, Doradidae, and Auchenipteridae with low bootstrap (50%); v) the Bagridae, Horabagridae, and Aillidae (75% bootstrap); vi) the Amblycipitidae and Sisoridae (100% bootstrap); vii) the Claroteidae, Auchenoglanididae, Schilbeidae, Amphillidae, Malapteruridae, and Mochokidae, or also termed as the “Big Africa” major group (59% bootstrap) (Sullivan et al. 2006); and viii) the Pangasiidae, Austroglanididae, Ictaluridae, Cranoglanididae, and Ariidae (72% bootstrap). The “Big Asia” major clade contained the clusters (Amblycipitidae + Sisoridae) and (Bagridae + Horabagridae + Aillidae) (35% bootstrap) (Sullivan et al. 2006), which is positioned as a sister group to the “Big Africa” major clade (Fig. 2). The Pangasiidae and their related group Austroglanididae are monophyletically sistered with the major group (Ictaluridae and Cranoglanididae), and this major group is placed as a sister the Ariidae with a moderate boostrap value (75%), according to the topology depicted in figure 2. 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_(BaijinCoFoshan)-China-MZ286355” is Pangasius larnaudii, and the “Pangasius bocourti (QingyuanGD)-ChinaMN842723” 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 cox1 and cytB barcode sequences are available in GenBank and in previous publications; therefore, we downloaded all cox1 (551 bp) and cytB (634 bp) sequences and extracted cox1 and cytB, respectively, from the complete mitogenomes (listed in Table S3). The cox1 and cytB 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 cox1 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 cytB tree (Fig. 4), this species is sistered with P. pangasius with 100% nodal support. Pangasius krempfi, in the cox1 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 cytB tree in a non-stable phylogenetic relationship with a 63% bootstrap, to P. macronema and P. polyuranodon. The partial cox1 datasets (four sequences, consisting page 7 of 17Zoological Studies 62:48 (2023) © 2023 Academia Sinica, Taiwan Fig. 2. PhyML-phylogeny of the order Siluriformes, including 32 catfish families (117 sequences) of three suborders, Siluroidei, Loricarioidei, and Diplomystoidei based on the complete concatenated nucleotide sequences of all 13 mitochondrial protein coding genes (about 11,408 bp in length) (Table S2). Two sequences of Gonorynchiformes were used as an outgroup. The alignment was performed by MAFFT (Katoh and Standley 2013), curated by BMGE v1.12 (Criscuolo and Gribaldo 2010), the tree was reconstructed in PhyML 3.3 (Guindon et al. 2010) using a maximum likelihood method and 1000 bootstrap resamplings, and the output Newick tree was extracted and visualized using FigTree v1.4.4 (Rambaut 2018). The nodal bootstrap support values (shown at each node) were interpreted from the concurrently constructed tree using the above MAFFT-BMGE alignment by MEGA X (Kumar et al. 2018). The basal nodes of the three suborders (Diplomystoidei, Loricarioidei, and Siluroidei) as well as the two major “Big Asia” and “Big Africa” groups (background highlighted) are shown by arrows. The Pangasius mekongensis, Pangasianodon hypophthalmus, and Pangasius krempfi sequences in this study are indicated by stars and with the associated families’ background highlighted. The taxa were presented with their full names. The abbreviations of the isolates are given in brackets, including the geographical origin or voucher records of each sequenced specimen (where available), which were retrieved from the previous studies (Saitoh et al. 2003; Nakatani et al. 2011; Kappas et al. 2016; Zhang et al. 2021; Schedel et al. 2022). The country of origin or geographical regions where the sample was reported are given in full name, if available. Accession numbers are given at the end of each sequence label. The scale bar represents the number of substitutions per site. page 8 of 17Zoological Studies 62:48 (2023) © 2023 Academia Sinica, Taiwan Fig. 3. Detailed PhyML-phylogeny based on the analysis of the partial cox1 sequences (551 bp) showing the detailed relationships of the family Pangasiidae and related families (Austroglanididae, Ictaluridae, and Cranoglanididae). In total, 83 sequences, including 81 from Pangasius and Pangasianodon and 2 outgroup sequences from the order Gymnotiformes, were included (Table S3). The alignment was performed by MAFFT (Katoh and Standley 2013), curated by BMGE v1.12 (Criscuolo and Gribaldo 2010), the tree was reconstructed in PhyML 3.3 (Guindon et al. 2010) using a maximum likelihood method and 1000 bootstrap resamplings, and the output Newick tree was extracted and visualized using FigTree v1.4.4 (Rambaut 2018). The basal nodes of the Pangasiidae and two sister groups (Pangasianodon and (Pangasius + Helicophagus + Pseudolais)) are shown by arrows. The Pangasius mekongensis, Pangasianodon hypophthalmus, and Pangasius krempfi sequences in this study are bolded. The taxonmisidentified sequences were added with a question mark at the end. The taxa from Pangasiidae were shortened and those from other related families were presented with their full names. The abbreviations of the isolates are given in brackets, including the geographical origin or voucher records of each sequenced specimen (where available), which were retrieved from the previous studies (Karinthanyakit and Jondeung 2012; Tran and Duong 2019; Schedel et al. 2022). The country of origin or where the sample was reported is given in full or in brackets, if available. Accession numbers are given at the end of each sequence label. The scale bar represents the number of substitutions per site. 0.03 Ppan-(NBFGR-Pp32)-India-EU871047 Ppan-(NBFGR-Pp38)-India-EU871048 Ppan-(UC-PP1)-India-JX997836 Ppan-(NBFGR-Pp27)-India-EU871046 Ppan-(NBFGR-Pp02)-India-EU871045 Ppan-(Odisha)-India-KX950698 Ppan-(Lucknow)-India-KC572135 . Ppan-(11049)-Bangladesh-MK572424 Pmek-(PNST)-Vietnam-MZ272451 Pmek-(Hap2)-Vietnam-KY398032 Pmek-(Hap1)-Vietnam-KY398031 Pmek-(Hap3)-Vietnam-KY398033 Pn-hyp-(BaijinCo-Foshan)-China-M Z286355 (?) Plar-(byMiya-JP)-Thailand-AP012018 Plar-(PL063202)-Cambodia-KY118576 Plar-(PLARN)-Vietnam-MN073459 Plar-(AnGiang)-Vietnam-MG981066 Pboc-(PB061702)-Thailand-KY118574 Pboc-(PBOCO)-Vietnam-MN073456 Pboc-(BW-1791)-Vietnam-EF609425 Pboc-(FRIGL-55PH)-Malaysia-KP036428 Pdja-(FRIGL 53PH)-Malaysia-KP036427 Pboc-(DongThap)-Vietnam-MG981068 Psan-(KW11T126)-South Africa-KC627283 Psan-(WB12-AT136)-South Africa-KC627282 Psan-(LancangR)-China-MN809630 Psan-(StrungTreng)-Cambodia-MG981065 Pcon-(HU19)-Vietnam-KT289886 Pnas-(SLM-PN(PH)-02)-Malaysia-JF781173 Pcon-(HB 03CR)-Malaysia-KP036414 Pcon-(FRIGL 23PH)-Malaysia-KP036413 Pcon-(FNP126)-Thailand-MK448175(CO1) Pnas-(SLM-PN(PH)-03)-Malaysia-JF781174 Pcon-(CanTho)-Vietnam-MG981067 Pkre-(PKREM)-Vietnam-MN073460 Pmek-(PMEKO)-Vietnam-MN073458 Pkre-(Hap2)-Vietnam-KY398026 Pkre-(Hap1)-Vietnam-KY398025 Pkre-(BLD2)-Vietnam-KT289877 Pkre-(VNAG)-Vietnam-MZ272453 Pkre-(Hap3)-Vietnam-KY398027 Pkre-(CanTho)-Vietnam-MG981062 Hlep-(CanTho)-Vietnam-MG981071 Hvaa-(FRIGL-24PH)-Malaysia-KP036417 Pmac-(byTruong)-Vietnam-KU747162 Pmac-(Hap2)-Vietnam-KY398030 Pmac-(XS27)-Vietnam-KT289890 Pmac-(XS04)-Vietnam-KT289889 Pmac-(XS71)-Vietnam-KT289892 Pmac-(XS02)-Vietnam-KT289888 Pmac-(XS70)-Vietnam-KT289891 Pmac-(FNP050)-Thailand-MK448111 Pelo-(D80)-Vietnam-KT289879(CO1) Pelo-(StrungTreng)-Cambodia-MG981069 Pelo-(D52)-Vietnam-KT289878 Ps-mic-(BIF3758)-Indonesia-KU692816 Ps-ple-(Pakse)-Laos-MG981072 Ps-mic-(FRIGL 59PH)-Malaysia-KP036422 Ps-mic-(FRIGL 85PH)-Malaysia-KP036423 Pn-hyp-(SAMN)-Indonesia-CM018571 Psan-(PB2)-(China)-JN020073 (?) Pboc-(QingyuanGD)-China-MN842723 (?) Pn-hyp-(VN-RIA2-2014)-Vietnam-CM01 0854 Pn-hyp-(CTIBT)-Vietnam-MZ272452 Pn-hyp-(byZhao)-China-KC846907 Pn-hyp-(CanTho)-VN-MG981070 Pn-gig-(MekongR)-Thailand-AY762971 Pn-gig-(PG063403)-Thailand-KY118586 Austroglanis barnardi-(RB14-A019)-South Africa-Z930069 Austroglanis gilli-(RB14-A041)-South Africa-MZ930072 Austroglanis sclateri-(IRB-513)-South Africa-MZ930070 Pareutropius debauwi-(Uerre)-Congo-AP012017C Clanoglanis bouderius-(Guangxi)-China-AY898626 Ictalurus punctatus-(NEFC-F16-568)-United States-MF621721 Ictalurus punctatus-(Norris)-United States-AF482987 Ictalurus punctatus-(NEFC-F16-262)-United States-MF621722 Ictalurus pricei-(TNHC-21704-10)-Mexico-KJ496298 Ictalurus furcatus-(WabashR)-United States-KM576102 Ameiurus catus-(NEFC-F16-261)-United States-MH324425 Ameiurus-nebulosus-(NEFC-F16-113)-United States-MF621733 Pylodictis olivaris-(NEFC-F16-277)-United States-MF621728 Apteronotus albifrons-(CBM-ZF-10621)-AB054132 Gymnotus carapo-(SAmerica)-AP011979 99 99 87 99 99 88 99 99 37 99 99 99 88 99 36 99 99 68 59 40 99 98 99 86 99 84 99 64 99 38 89 40 99 93 68 89 91 71 Pangasiidae Austroglanididae Cranoglanididae Ictaluridae Pangasius pangasius Pangasius mekongensis Pangasius larnaudii Pangasius bocourti Pangasiusdjambal Pangasius sanitwongsei Pangasius conchophilus Pangasius krempfi Pangasius macronema Helicophagus spp. Pangasius elongatus Pseudolais micronemus Ps. pleurotaenia Pangasianodonhypophthalmus Pangasianodongigas 44 99 Pangasius Helicophagus Pseudolais Pangasianodon 28 page 9 of 17Zoological Studies 62:48 (2023) © 2023 Academia Sinica, Taiwan Liobagrus obesus (Siluriformes, Amblycipididae): Genome description and phylogenetic considerations inferred from the Cyt b and 16S rRNA genes. 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The mitogenome of Pangasius sutchi (Teleostei, Siluriformes: Pangasiidae). Mitochondrial DNA 25(5):342–344. doi:10.3109/19401736.2013.800492. Zhuang X, Qu M, Zhang X, Ding S. 2013. A Comprehensive Description and Evolutionary Analysis of 22 Grouper (Perciformes, Epinephelidae) Mitochondrial Genomes with Emphasis on Two Novel Genome Organizations. PLoS ONE 8(8):e73561. doi:10.1371/journal.pone.0073561. Supplementary materials Fig. S1. Alignment of ten control region sequences from eight species, Pangasius mekongensis (Pmek), Pangasius krempfi (Pkre), Pangasius pangasius (Ppan), Pangasius larnaudii (Plar), Pangasius bocourti (Pboc), Pangasius sanitwongsei (Psan), Pangasianodon hypophthalmus (Phyp, three sequences), and Pangasianodon gigas (Pgig). Types of conserved sequence blocks (CSBs) within a species (CSB-C), within a genus (CSB-B), and within both genera (Pangasius and Pangasianodon) (CSB-A) are presented. Aligned nucleotide sequences and typical CSBs are shown as boxes. For species, see table S2; for descriptions of the blocks, see text. (download) Table S1. Primers for amplification and sequencing of fragments of the mitochondrial genome of Pangasius mekongensis, Pangasianodon hypophthalmus, and Pangasius krempfi of the Mekong River, Vietnam. (download) Table S2. List and information of 117 siluriform sequences (109 species in 32 families) and two outgroup species with complete mitogenomes providing 13 protein-coding sequences used in this study for phylogenetic and sequence analysis of catfishes of the Pangasiidae and families in the order Siluriformes. (download) Table S3. Accession numbers and country report for the reference cox1 and cytB markers from the GenBank database and those from this study used for the pangasiid comparative phylogenetic studies. (download) Table S4. Nucleotide composition and skewness value for the complete mitochondrial genome (mtDNA) and the protein-coding genes (PCGs) of seven species members of the family Pangasiidae. (download) Table S5. Pairwise nucleotide differences (%) among five catfish species of the family Pangasiidae present in the Mekong Basin for individual mitochondrial proteincoding genes and mitochondrial ribosomal genes (12S and 16S). (download) Table S6. Pairwise genetic distances (%) estimated between Pangasius mekongensis, Pangasianodon hypophthalmus, Pangasius krempfi, and the published or GenBank-deposited representative Pangasiidae species based on mitogenome coding nucleotide sequences. (download) page 17 of 17Zoological Studies 62:48 (2023)