Description of a new Pangasius (Valenciennes, 1840) species, from the Cauvery River extends distribution range of the genus up to South Western Ghats in peninsular India

A new species of the genus Pangasius, is described based on 17 specimens collected from the Cauvery River, India. It can be distinguished from its sister species from South and Southeast Asia, by its widely placed, small and rounded vomerine and palatine tooth plates, longer maxillary and mandibular barbels, greater vertebrae count 50 (vs. 44–48), and smaller caudal peduncle depth (6.5–8.2% SL vs. 9.89–13.09% SL). The tooth plates of the new species closely resembles that of Pangasius macronema but can be clearly distinguished from the latter by having lesser gill rakers (16–19 vs. 36–45); a smaller eye (2.4–4.4% SL vs. 5.2–9.6% SL); and larger adipose-fin base (1.5–2.9% SL vs. 0.1–1.2% SL). The mitochondrial cytochrome c oxidase (COI) gene sequence of the new species shows the genetic divergence of 3.5% and 5.1% from P. pangasius and P. silasi respectively, the two sister species found in South Asia and India. The species delimitation approaches, Poisson Tree Processes (PTP) and assemble species by automatic partitioning (ASAP) clearly resolved that the P. icaria is distinct from its sister species. Phylogenetic position of the species with its sister species was evaluated using maximum likelihood and Bayesian analysis. The discovery of this previously unknown species of genus Pangasius from the Cauvery River of peninsular India indicates important biogeographical insight that this genus migrated till the southern division of Western Ghats.


INTRODUCTION
The past few decades have witnessed the discovery of several new catfish species from southeast and south Asia (Pouyaud, Teugels & Legendre, 1999;Roberts, 1999;Pouyaud & Teugels, 2000;Pouyaud, Gustiano & Teugels, 2002;Gustiano, Teugels & Pouyaud, 2003) including some from the peninsular India (Radhakrishnan, Sureshkumar & Ng, 2010;Babu, 2012;Ng, 2013;Lal et al., 2017;Dwivedi et al., 2017). Pangasiid catfishes (Pangasius Valenciennes, 1840) are popular for their commercial value in both aquaculture and wild capture fisheries (Roberts & Vidthayanon, 1991) and are considered to be a delicacy. A total of 22 valid species are distributed in the river basins of southeast and south Asia Fricke, Eschmeyer & Fong, 2021). These have also been introduced in many countries for aquaculture. Among the valid species, only two species have been known from the Indian rivers, the most common is Pangasius pangasius (Hamilton, 1822), distributed in the rivers Ganges, Brahmaputra and Godavari, and the other, Pangasius silasi  is described from the Nagarjuna Sagar Dam, in the Krishna River.
Previously, Hora (1951) had mentioned about the presence of Pangasius in the Stanley Reservoir (also known as Mettur Dam), which is one of the largest fishing reservoirs in South India. However, inadequate data led to consideration of the species as Pangasius pangasius only, though this raised hitherto unresolved query for the biogeographers of the time (Silas, 1952b). They assumed the absence of divergence in Pangasius genus, which probably migrated into peninsular India from Himalayan rivers. This is in contrast to some genera which are common between rivers of the Himalaya and the Indian peninsula, however, are represented by different species such as Silonia (family Schilbidae).
The explorations were conducted in the Cauvery River basin, Tamil Nadu, India and specimens of Pangasius were collected. In the present study, we used mitochondrial gene cytochrome c oxidase (COI) to support the morphological analysis. Morphometrics and molecular marker dataset analysis were used for deciphering phylogenetic relationships and species delimitation. Results explicitly indicated a distinct clade of the specimens from the Cauvery River, which is well differentiated from its congeners and described here as Pangasius icaria, a new species.

MATERIALS & METHODS
Specimens described in present study, were collected from the commercial catches from landings along the locations, Mettur Dam, (Tamil Nadu). and upstream of Shivanasamudra falls, Chamarajanagar (Karnataka), on the Cauvery River basin in India. The details of samples and collection localities are presented in Table S1. The right-side pectoral fin was excised and preserved in 95% ethanol for molecular studies form individual specimen. Specimens were tagged with unique code, preserved in formalin (10%) and transported to the laboratory for examination and permanent preservation.
Data for meristic and morphometric measurements were generated following Ng & Dodson (1999). Measurements were taken using a digital caliper (Mitutoyo Digimatic Caliper, Japan) to the nearest 0.1 mm. The body and head subunits were presented as percent (%) of standard length (SL). A total of 31 morphological characters were recorded for comparative assessment with other pangasius species (Roberts & Vidthayanon, 1991;Dwivedi et al., 2017). Individual digital X-ray radiographs were used for vertebrae count following Ng & Kottelat (2013). Morphometric measurements in proportion of standard length (SL) and head length (HL) of the holotype, followed by paratype were documented. The holotype and paratype accessions, examined in the study, were deposited in the notified repository (http://nbaindia.org/uploaded/pdf/notification/1%20designated%20repositories. pdf), National Repository and Fish Museum at ICAR-NBFGR, Lucknow, India.

Gene amplification and sequencing
Genomic DNA was extracted from the fin (stored in 95% ethanol) following the salting-out method (Sambrook & Russel, 2001). The partial mitochondrial cytochrome c oxidase subunit 1 (COI) gene was amplified using universal primers, forward primer Fish F1 (5 TCAACCAACCACAAAGACATTGGCAC3 ) and reverse primer Fish R1 (5 TAGACTTCTGGGTGGCCAAAGAATCA3 ) (Ward et al., 2005). The amplification was performed in 25 µL reaction which consisted of 1X reaction buffer (10 mM Tris, 50 mM KCl, 0.01% gelatin, pH 9.0), 1.5 mM MgCl 2 , 200 µM of each dNTP, 5 pmol of each (F1 and R1) primer, 3U Taq polymerase (Genei, India) and approximately 50 ng genomic DNA. Thermal cycling was performed with an initial denaturation at 95 • C for 4 min, 35 cycles of 94 • C for 30 s., annealing at 52 • C for 40 s., extension at 72 • C for 45 s and final extension at 72 • C for 10 min. The amplicons were visualized on agarose gel and purified for bidirectional sequencing on ABI automated sequencer (Applied Biosystems, Waltham, MA, USA).

Genetic analysis
The generated sequences were aligned using the ClustalW algorithm in BioEdit version 5.0.9 (Hall, 1999) along with other gene sequences of the genus Pangasius downloaded from NCBI GenBank. The sequences used for analyses are provided in Table S2. The estimates for inter-species genetic divergence was obtained using the Kimura 2P model (Kimura, 1980) implemented in MEGA-X (Kumar et al., 2018). Maximum likelihood phylogenetic analysis was performed (500 bootstraps) using the software MEGA-X (Kumar et al., 2018). The outgroups used for rooting the tree included the COI sequences of Horabagrus brachysoma (Family: Horabagridae) and Clupisoma garua (Family: Schilbeidae) both belonging to order Siluriformes. The best fit model and the partition scheme, for the present dataset was obtained from PartitionFinder2 (Lanfear et al., 2017), which revealed HKY+G to be the optimum for phylogenetic analysis. Data partitioning was done for all three codon positions, of which the first position was optimum. Bayesian analysis was performed using MrBayes v.3.1.2 (Huelsenbeck & Ronquist, 2001) with Markov Chain Markov Chain Monte Carlo sampling with four chains. A total of one million MCMC runs were executed and the trees and parameters sampled at every 1000 generations. The analysis was run until the deviation from split frequencies remained 0.01. The potential scale reduction factor was closer to one. A total of 25% of the samples were discarded as burn-in. The convergence of runs was observed in MCMC trace analysis package v1.7.2 (Drummond & Rambaut, 2007), while the tree topology was visualized in Figtree v1.4.4 (FigTree, 2018).
The species delimitation (PTP) analysis (this study and downloaded sequences) resulted in delimitation of 11 groups (Fig. 8) excluding the outgroup species. P. icaria was delimited clearly from others. The findings of distance-based ASAP were also concordant to PTP. The graphical pattern clearly differentiated the samples of P. icaria, with P. silasi and other P. pangasius in the Krishna River (Ramakrishnaya, 1986) were not confirmed by the later studies Ajith Kumar, Santosh & Lal, 2020). The Mahanadi, Godavari, Krishna and Cauvery rivers are the large independent basins in peninsular India, which flow eastward to drain into Bay of Bengal. The Krishna River has the Mahanadi and Godavari rivers on its northern side and the Cauvery River on its south. The discovery of this new species, P. icaria from the Cauvery River confirms that the genus Pangasius has extended distribution upto the southern division of the Western Ghats and has undergone speciation, like other Himalayan species that have same genus but are represented by alternate species in the peninsular India (Silas, 1952b) for example Silonia silondia (Ganges and Mahanadi) were represented as S. childreni in peninsular rivers. This addresses the earlier paleobiogeographic conflict that the genus Pangasius might be a late migrant and has not undergone the species divergence (Silas, 1952b;Menon, 1980).
In the present study, the P. icaria, is described in collections from two different and distant locations in the Cauvery River. The rivers of peninsular India flows through the Western Ghat region, which is part of the designated mega biodiversity hotspots and is known for endemism. The future programs on fine-scale explorations of these rivers will be significant in delimiting the distribution ranges of the newly discovered pangasius species (P.silasi and P. icaria) for documentation of within species genetic diversity, their conservation strategies and also with the possibility of new undescribed species.

CONCLUSIONS
A new species of the genus Pangasius is described that was collected from the Cauvery River, Tamil Nadu, India. This new discovery of P. icaria clearly highlights the native presence of genus Pangasius in peninsular India and is represented by two recorded divergent species. Future research and explorations are needed to ascertain the distributional range of this endemic species for devising conservation and management of the species and also to evaluate for its aquaculture utilization potential.