Two new species of the hillstream loach genus Indoreonectes from the northern Western Ghats of India (Teleostei: Nemacheilidae)

The hill stream loach genus Indoreonectes is endemic to peninsular India south of the Satpura hill ranges and is represented by three species I. evezardi, I. keralensis and I. telanganaensis. Indoreonectes evezardi has been suggested as a species complex based on recent genetic studies; however, due to lack of type material the species delimitation has been difficult. Here we redescribe I. evezardi collected from its type locality and describe two new species from the northern Western Ghats of India. Indoreonectes neeleshi, described from Mula River tributary of Godavari river system, can be diagnosed from all its congeners based on a combination of characters: inner rostral barbel reaching middle of nostril; maxillary barbel reaching midway between eye and posterior border of operculum; dorsal hump behind nape; bars on lateral side of the body wider than inter-bar space; total vertebrae 35 and dorsal fin insertion between 13th and 14th abdominal vertebrae. Indoreonectes rajeevi, described from Hiranyakeshi River of the Krishna river system, differs from all its congeners based on a combination of characters: inner rostral barbel reaching anterior margin of eye; maxillary barbel reaching posterior border of operculum; conspicuous black markings on lower lip, dorsal hump absent; total vertebrae 36 and dorsal fin insertion between 12th and 13th abdominal vertebrae. Further, I. neeleshi differs from its congeners by the raw genetic distance of 6.8–14.4% for the cox1 gene and 5.7–16.2% for the cytb gene, while I. rajeevi differs from its congeners by the raw genetic distance of 10.9–14.0% for the cox1 gene and 11.8–15.8% for the cytb gene.


Introduction
The genus Indoreonectes Rita, Bănărescu and Nalbant of hill stream loaches (Cypriniformes: Nemacheilidae) was initially proposed as a subgenus of Oreonectes Günther, while describing O. (Indoreonectes) keralensis Rita and Nalbant (Rita et al. 1978). Kottelat (1990aKottelat ( , 1990bKottelat ( , 2012 and Prokofiev (2010) considered Indoreonectes as a valid genus endemic to Peninsular India. The genus is diagnosed based on following combination of characters cies complex with several undescribed species. However, lack of type material for I. evezardi (see for detailed discussion, Prasad et al. 2020: 344) has made it difficult to diagnose species in the I. evezardi complex. Although Prasad et al. (2020) recognized this issue and provided photographs of I. evezardi from its type locality, they did not redescribe the species in detail.
In the current study we redescribe Indoreonectes evezardi from its type locality using fresh collections and specimens studied by Prasad et al. (2020). Further, we describe two new species from the I. evezardi complex from the Mula River, a tributary of the Godavari river system and from the Hiranyakeshi River, part of the Krishna river system.

Fish collection
Individuals of the new species of Indoreonectes were collected from two different localities, one in the upper reaches of Godavari river system in Maharashtra at Harishchandragad (19°23.64′N; 73°46.74′E, ca 1180 m a.s.l.)

Morphology, morphometry and osteology
Measurements were taken for each specimen to the nearest 0.1 mm using digital callipers (Mitutoyo, Japan). Morphometric methods follow Keskar et al. (2015). Subunits of the body are presented as a percent of standard length (SL), and subunits of the head as a percent of head length (HL). Fin rays were counted using a stereomicroscope (Magnus, India). Frequency of the count is provided in parentheses after the count. The last two fin rays of the dorsal and anal fins, which articulate with a single pte-rygiophore and often referred to as "last ray split to the base" were counted as one ray. Terminology used for describing the pattern of bars and other markings is defined in Figure 2. Reach of barbels is based on the condition when folded back artificially.

Molecular analysis
Gill tissues were extracted from a paratype of each species preserved in absolute alcohol (Indoreonectes neeleshi -BNHS FWF 1080 and I. rajeevi -BNHS FWF 1089). DNA was extracted using QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) following the protocol given by the manufacturer. Mitochondrial cytochrome oxidase subunit I (cox1) gene was amplified using Polymerase Chain Reaction with primers FishF1 and FishR1 (Ward et al. 2005). Gene amplification, purification and sequencing protocols follow Ali et al. (2013). Sequencing was performed by Apical Scientific SDN, BHD., Malaysia. The raw chromatograms were manually assembled and verified for potential mistakes using free software FinchTV 1.4.0 (Geospiza, Inc.; Seattle, WA, USA; http://www. geospiza.com). The BLAST tool (Altschul et al. 1990) was used to see the similarity to the listed sequences in the database of NCBI (http://www.ncbi.nlm.nih.gov/). Gene sequences are deposited in GenBank with the accession number MW136273 and MW136274. Additional sequences, for cox1 and cytochrome b (cytb) genes from our earlier studies Keskar et al. (2018) and Kumkar et al. (2016) and cytb gene from Prasad et al. (2020), were retrieved from NCBI GenBank database (Appendix I).
Gene sequences were aligned with MUSCLE (Edgar 2004) for individual genes and the aligned sequences were concatenated to get the combined data matrix of cox1 and cytb. The combined matrix had 1747 base pairs. Balitora chipkali (Cypriniformes: Balitoridae) was used as an outgroup. Data were partitioned in two genes and their respective codon positions to create a full partition. The best partitioning scheme and the nucleotide substitution model for the partition scheme were estimated using partition analysis (Chernomor et al. 2016) and Mod-elFinder (Kalyaanamoorthy et al. 2017) implemented in IQ-TREE 1.6.12 (Nguyen et al. 2015). Maximum likelihood (ML) analysis was performed in IQ-TREE 1.6.12 (Nguyen et al. 2015) with the best partition scheme and ultrafast bootstrap support for 1000 iterations (Hoang et al. 2018). The resulting maximum likelihood phylogram was edited in FigTree v1.4.4 (Rambaut 2018). Uncorrected raw (p) distances between pairs of sequences were calculated in MEGA X 10.1 (Kumar et al. 2018).  Diagnosis. Indoreonectes evezardi can be distinguished from all other congeners by having caudal peduncle bar divided as two spots (vs. not divided in I. keralensis, I. telanganaensis, I. neeleshi and I. rajeevi); caudal peduncle deeper than long (vs. longer than deep in I. keralensis, as deep as long in I. telanganaensis and I. neeleshi, slightly longer than deep in I. rajeevi). Further, Indoreonectes evezardi can be distinguished from I. keralensis by having long nasal barbel reaching mid of eye (vs. short nasal barbel barely reaching anterior border of eye); inner rostral barbel reaching middle of nostril (vs. reaching anterior margin of eye); presence of dorsal hump behind nape (vs. absent); dorsal-fin insertion between neural spines of 12 th and 13 th abdominal vertebrae (vs. between 13 th and 14 th ); presence of a dark brown to black spot at base of first dorsal-fin ray and distinct spots on dorsal side of head (vs. absent); spots on cheek below eye absent (vs. present). Indoreonectes evezardi can be distinguished from I. telanganaensis by having inner rostral barbel reaching middle of nostril (vs. reaching further posteriorly to anterior margin of eye); spots on cheek below eye absent (vs. present). Indoreonectes evezardi can be distinguished from I. neeleshi by having lateral bars narrower than inter-bar spaces (vs. wider in I. neeleshi). Indoreonectes evezardi can be distinguished from I. rajeevi by having inner rostral barbel reaching middle of nostril (vs. reaching further posteriorly to anterior margin of eye); maxillary barbel not reaching posterior border of operculum (vs. reaching); absence of conspicuous black marking on lower lip (vs. presence); presence of dorsal hump behind nape (vs. absence); total vertebrae 35 (vs. 36). Figure 3; morphometric data are provided in Table 1.

Description. General morphology is shown in
Body sub-cylindrical, elongate; head and anterior part of body almost cylindrical; body laterally compressed posteriorly; predorsal outline convex, gradually rising up to dorsal-fin origin, a distinct hump behind nape; postdorsal outline straight up to base of caudal fin; ventral profile almost straight. Caudal peduncle deeper than long. Lateral line present (14) or absent (1), when present then incomplete, short, ending above middle of adpressed pectoral fin. Scales minute.
Head small, slightly longer than a quarter of SL. Snout round, its length more than one-third of head length. Eye dorsolaterally positioned, closer to tip of snout than to posterior margin of opercle, its diameter 10-16% HL. Mouth semi-circular, with thick fleshy lips, lower lip interrupted medially by a deep groove (Fig. 4A). Barbels four pairs. Two pairs of rostral barbels, inner rostral barbel extending to middle of nostril, outer rostral barbel reaching anterior margin of eye; Maxillary barbel longest, originating at vertical from nostril, reaching midway between eye and posterior border of operculum. Nasal barbel well developed, reaching middle of eye.
Total vertebrae 35 ( Fig. 5A) with 17 (2) abdominal and 18 (2) caudal vertebrae. Dorsal-fin insertion in both cleared and stained specimens is between neural spines of 12 th and 13 th abdominal vertebrae. Fifth ceratobranchial ( Fig. 6A) with single row of 15 to 16 small curved teeth with pointed tips; anterior teeth longer than posterior ones.

Colouration.
In life (Fig. 3A), background colour pale yellow, slightly darker on dorsal profile than lateral profile; with brownish-black irregular bars on lateral and dorsal side of body, lateral and dorsal bars separated from each other; lateral bars narrower than inter-bar spaces; lateral complete bars 13 (3), 14 (6), 15 (4) or 16 (2); lateral incomplete bars 4 (6), 5 (6), 6 (2) or 7 (1). Head dorsally covered with dark brown spots; cheek spots below eye absent. Caudal peduncle bar split into two conspicuous spots. Dorsal fin anterior spot dark brown to black in colour; dorsal-fin membrane hyaline with three rows of black spots on rays. Pectoral, pelvic and anal fins hy-aline, lacking spots; caudal-fin with membrane hyaline, and three to four rows of dark-brown spots on rays. Ventral surface without any markings. In preservative , colouration similar to that in life, but less conspicuous.
Habitat and distribution. Indoreonectes evezardi was collected from fast-flowing clear streams with substrate consisting of rock, pebbles and sand. Co-occurring fish species include Paracanthocobitis mooreh, Schistura denisoni, Rasbora dandia and Devario malabaricus. Indoreonectes evezardi sensu stricto is currently known only from its type locality in Pune (see Keskar et al. 2018) from Mutha River a tributary of the east flowing Krishna river system, Maharashtra, India (Fig. 1).
Remarks. The types of I. evezardi are not traceable and are suspected to be lost (see for details, Prasad et al. 2020). Specimens we examined, which include the specimens studied by Prasad et al. (2020) from the type locali- Length of anal-fin base 6.9 (0.6) 5.9-7.9 6.9 6.4 (0.6) 5. ty in Pune, closely resemble the specimens in the original description of the species as pointed out by Prasad et al. (2020). However, it should be mentioned that in the original description Day (1872) mentions that the lateral line is absent in I. evezardi. However, this is a relatively rare character state and out of total 15 individuals we examined, the lateral line is absent in only one specimen. In all other specimens the lateral line is present, but incomplete and does not extend beyond the middle of the adpressed pectoral fin.     Figure 7; morphometric data are provided in Table 1. Body sub-cylindrical, elongate; head and anterior part of body almost cylindrical; body laterally compressed posteriorly; predorsal outline convex, gradually rising up to dorsal-fin origin, a distinct hump behind nape; postdorsal outline straight up to base of caudal fin; ventral profile almost straight. Caudal peduncle as deep as long. Lateral line present, incomplete, short, ending above middle of adpressed pectoral fin. Scales minute.

Description. General morphology is shown in
Head small, less than a quarter of SL. Snout round, its length more than one-third of head length. Eye dorso-laterally positioned, closer to tip of snout than to posterior margin of operculum, its diameter about 15% HL. Mouth semi-circular, with thick fleshy lips, lower lip interrupted medially by a deep groove (Fig. 4B). Barbels four pairs. Two pairs of rostral barbels, inner rostral barbel extending to middle of nostril, outer rostral barbel reaching anterior margin of eye; Maxillary barbel longest, originating at vertical from nostril, reaching midway between eye and posterior border of operculum. Nasal barbel well developed, reaching middle of eye.
Total vertebrae 35 (Fig. 5B) with 17 (2) abdominal and 18 (2) caudal vertebrae, but vertebral column malformed in both cleared and stained specimens. Dorsal-fin insertion between 13 th and 14 th abdominal vertebrae. Fifth ceratobranchial ( Fig. 6B) with single row of 12 to 17 small curved teeth with pointed tips; anterior teeth longer than posterior.
Colouration. In life (Fig. 7A), background colour yellow ochre slightly darker on dorsal profile of anterior side; with grey irregular bars on lateral and dorsal side of body, lateral and dorsal bars separated from each other; lateral bars wider than inter-bar spaces; lateral complete bars 12 (4), 13 (2), 14 (3) or 16 (1); lateral incomplete bars 4 (2), 5 (5) or 6 (3). Head dorsally studded with dark brown spots; cheek spots below eye absent. Caudal peduncle bar continuous but sometimes faint in the middle. Dorsal fin anterior spot dark brown to black in colour; dorsal fin membrane hyaline with three rows of black spots on rays. Pectoral, ventral and anal fins hyaline, lacking spots; caudal fin with membrane hyaline, and three to four rows of dark-brown spots on rays. Ventral surface without any markings. In preservative (Fig. 7B-D), colouration similar to that in life, but faded.
Etymology. The species name honours Neelesh Dahanukar researcher from Indian Institute of Science Education and Research (IISER), Pune, India, for his remarkable contributions to the understanding of the systematics and evolution of Indian freshwater fishes. Habitat and Distribution. Indoreonectes neeleshi was found in a fast-flowing clear stream with a rocky substrate. Co-occurring fish species include Parapsilorhynchus sp. and the exotic Gambusia affinis. Currently, In-doreonectes neeleshi is known only from its type locality in the Mula tributary of East flowing Godavari River at Harishchandragad, Maharashtra, India (Fig. 1).

Indoreonectes rajeevi sp. nov.
Common name: Rajeev's hill stream loach 8 h t t p : / / z o o b a n k . o rg / 2 9 9 3 0 F 6 A -A 8 7 B -4 C 3 5 -9 A D A -9172A1739BFA Indoreonectes rajeevi is further distinguished from I. keralensis by having long nasal barbel reaching middle of eye (vs. short nasal barbel barely reaching anterior margin of eye); dorsal-fin origin vertical from pelvic-fin base (vs. posterior to vertical at pelvic-fin base); dorsal-fin insertion between neural spines of 12 th and 13 th abdominal vertebrae (vs. between 13 th and 14 th ); presence of a dark brown to black spot on base of first dorsal-fin ray and distinct spots on dorsal side of head (vs. absence); spots on cheek below eye absent (vs. present); total vertebrae 36 (vs. 35).
Indoreonectes rajeevi is distinguished from I. evezardi by having inner rostral barbel reaching anterior margin of eye (vs. reaching middle of nostril); absence of dorsal hump behind nape (vs. presence); dorsal fin origin at vertical from pelvic-fin base (vs. posterior to vertical at pelvic-fin base); caudal peduncle slightly longer than deep (vs. deeper than long); caudal peduncle bar not divided into two spots (vs. divided); total vertebrae 36 (vs. 35).
Indoreonectes rajeevi distinguished from I. telanganaensis by lacking spots on cheek below eye (vs. spots present); absence of dorsal hump behind nape (vs. presence); caudal peduncle slightly longer than deep (vs. as long as deep).
Indoreonectes rajeevi distinguished from I. neeleshi by having inner rostral barbel reaching anterior margin of eye (vs. reaching middle of nostril); absence of dorsal hump behind nape (vs. presence); dorsal-fin insertion between neural spines of 12 th and 13 th abdominal vertebrae (vs. between 13 th and 14 th ); caudal peduncle slightly longer than deep (vs. as long as deep); lateral bars narrower than inter-bar spaces (vs. wider); total vertebrae 36 (vs. 35). Figure 8; morphometric data are provided in Table 1.

Description. General morphology is shown in
Body sub-cylindrical, elongate; head and anterior part of body almost cylindrical; body laterally compressed posteriorly; pre-dorsal outline slightly convex, gradually rising up to dorsal-fin origin, no hump behind nape; post dorsal outline straight up to base of caudal fin; ventral profile almost straight. Caudal peduncle as deep as long or slightly longer. Lateral line present, incomplete, short, ending above middle of adpressed pectoral fin. Scales minute.
Head small, about one fifth of SL. Snout round, its length more than one-third of head length. Eye dorsolaterally positioned, closer to tip of snout than to posterior margin of operculum, its diameter about 14-20% HL. Mouth semi-circular, with thick fleshy lips, lower lip interrupted medially by a deep groove with conspicuous black markings on the either side of the groove (Fig. 4C). Barbels four pairs. Two pairs of rostral barbels, inner rostral barbel extending to anterior margin of eye, outer rostral barbel reaching posterior margin of eye; Maxillary barbel longest, originating at vertical from nostril, reaching posterior border of operculum. Nasal barbel well developed, reaching middle of eye.
Colouration. In life (Fig. 8A), background colour golden yellow; with umber-coloured irregular vertical bars on lateral and dorsal side of body, lateral and dorsal bars usually separated from each other or rarely continuous; lateral bars narrower than inter-bar spaces; lateral complete bars 14 (4), 15 (2), 16 (3) or 17 (3); lateral incomplete bars 5 (4), 6 (3), 7 (2), 8 (2) or 11 (1). Head dorsally studded with dark brown spots; no spots on cheeks below eye. Caudal peduncle bar continuous. Dark brown to black spot on base of first dorsal-fin ray; dorsal fin membrane hyaline with three rows of black spots on rays. Pectoral, ventral and anal fins hyaline, lacking spots; caudal fin membrane hyaline, with three to four rows of dark-brown spots on rays. Ventral surface without any markings. In preservative (Fig. 8B-D), colouration similar to that in life, but faded.
Etymology. The species name honours Rajeev Raghavan from Kerala University of Fisheries and Ocean Studies (KUFOS), Kochi, India, for his remarkable contributions to the understanding of the Systematics and Evolution of Indian freshwater fishes.
Habitat and Distribution. Indoreonectes rajeevi was found in a slow-flowing clear stream with boulders, pebbles and mud as major substratum. Co-occurring fish species includes Parapsilorhynchus sp., Schistura sp., Balitora laticauda, Rasbora dandia and Garra mullya. Currently, Indoreonectes rajeevi is known only from its type locality in the Hiranyakeshi tributary of east flowing Krishna River at Amboli, Maharashtra, India (Fig. 1).

Species comparison and key to the species of Indoreonectes
The type species of the genus I. keralensis can be distinguished from all its congeners based on the following characters: nasal barbels short, barely reaching anterior border of eye (vs. long, reaching middle of eye or beyond); dorsal fin origin posterior to pelvic fin origin (vs. dorsal fin origin opposite to pelvic-fin origin); no spot on base of first dorsal-fin ray (vs. dark brown to black spot on base of first dorsal-fin ray); dorsal surface of head uni- formly coloured with no distinct spots (vs. distinct spots on the dorsal side of head). In addition to these characters, I. keralensis differs from I. evezardi and I. rajeevi in the dorsal fin insertion between 13 th and 14 th abdominal vertebrae (vs. dorsal fin insertion between 12 th and 13 th abdominal vertebrae) and from I. rajeevi in having 35 total vertebrae (vs. 36).
Indoreonectes neeleshi can be distinguished from all its congeners based on characters such as lateral bars wider than inter-bar spaces (vs. narrower than inter bar spaces). In addition, I. neeleshi differs from I. evezardi in caudal peduncle bar not divided into two spots (vs. divided into two spots). Indoreonectes neeleshi further differs from I. evezardi and I. rajeevi in dorsal fin insertion between 13 th and 14 th abdominal vertebrae (vs. between 12 th and 13 th ). Indoreonectes neeleshi also differs from I. telanganaensis and I. keralensis in the absence of spots on cheek below eye (vs. presence).
Indoreonectes rajeevi differs from all its congeners in having very long barbels, in particular nasal barbels long reaching posterior border of eye (vs. reaching between anterior border to mid of eye); maxillary barbels long reaching posterior border of operculum (vs. maxillary barbels short not reaching beyond midway between eye and posterior border of operculum); inner rostral barbel long reaching anterior margin of eye (vs. inner rostral barbel short reaching middle of nostril); conspicuous black marking on lower lip of mouth (vs. no marking on lower lip of mouth). In addition, I. rajeevi differs from I. evezardi, I. neeleshi and I. telanganaensis in the absence of a distinct hump behind nape (vs. present). It also further differs from I. evezardi, I. neeleshi and I. keralensis in having 36 (3) vertebrae (vs. 35 (2), 35 (2), 35 (1) respectively).
Based on the comparison provided above, the species of Indoreonectes can be identified using the following key modified from Prasad et al. (2020):

Molecular analysis
The best partitioning scheme and nucleotide substitution model for the partition scheme for concatenated sequences was identified as TN+F+I for combined partition of first two codon positions of cox1 and cytb genes (BIC = 10659.524), HKY+F for third codon position of cox1 (BIC = 2158.262) and TN+F+I for third codon position of cytb (BIC = 3651.901). In the maximum likelihood tree all the species formed reciprocally monophyletic groups (Fig. 9). Genetically, I. neeleshi showed a sister group relationship with I. telanganaensis, while I. rajeevi was recovered as the sister group to the clade containing I. evezardi, I. neeleshi and I. telanganaensis. Indoreonectes neeleshi differs from all its congeners, for which genetic data are available, by a raw genetic distance of 6.8% and above for cox1 gene (Table 2) and 5.7% and above for cytb gene (Table 3). In particular, I. neeleshi differs from its sister taxon I. telanganaensis by a raw genetic distance of 6.5-6.7 % in the cytb gene (Table 3). Indoreonectes rajeevi differs from all its congeners, for which genetic data are available, with a raw genetic distance of 10.9% and above for the cox1 gene (Table 2) and 11.8% and above for the cytb gene (Table 3). Keskar et al. (2018: fig. 3) delineated 13 clades of Indoreonectes from northern Western Ghats of India based on multiple molecular methods. Of these, clade 4 containing specimens from Harishchandragad and clade 13 containing specimens from Amboli are described in the current study as I. neeleshi and I. rajeevi, respectively. Both, clades 4 and 13, are distinct from clade 3, which contained topotypic I. evezardi, in all the molecular delimitation methods used by Keskar et al. (2018). However, it is important to note that clades 11, 12 and 13 in Keskar et al. (2018) were not distinct in one of the four genetic species delimitation methods and contained specimens from adjacent areas, namely Gaibi, Mahabaleshwar, Patan, Medha, and Kumbharli (see Keskar et al. 2018 for locality details), suggesting that I. rajeevi could be distributed in areas other than its type locality. However, further taxonomic sampling is necessary to test this hypothesis. Genetically, the new species are quite distinct from their congeners. Although the genetic data for cox1 gene are not available for I. telanganaensis, both I. neeleshi and I. rajeevi are distinct from it in cytb gene sequence. For loaches, the interspecific genetic distances for cytb gene have been observed to range from 2.0% in Balitoridae (Tang et al. 2006) and 3.0% in Cobitidae (Perd-ices et al. 2018) to 5.4% in Nemacheilidae (Bohlen et al. 2020). As a result, the pairwise genetic distance of more than 5.7% between I. neeleshi from all its known congeners and 11.8% between I. rajeevi from all its congeners is high. Interestingly, although both I. rajeevi and I. evezardi are known from Krishna river system and both I. neeleshi and I. telanganaensis are known from Godavari river system, they show large genetic divergence.

Discussion
Consistent with the arguments raised by Dahanukar et al. (2011), Keskar et al. (2018) and Katwate et al. (2020) among others, our study further suggests that the earli-  er notion that northern Western Ghats are species poor is the result of a lack of extensive taxonomic reviews rather than an actual species poverty. Further studies in the northern Western Ghats are likely to reveal much higher diversity among loaches in general and in the genus Indoreonectes in particular.