Zoosyst. Evol. 99 (1) 2023, 253-260 | DO! 10.3897/zse.99.100461 > PENSUFT. yee BERLIN A new short brown unpatterned moray eel (Anguilliformes, Muraenidae) from the southeast coast of India, Bay of Bengal Paramasivam Kodeeswaran!*, Ganesan Kantharajan', Anil Mohapatra®, T. T. Ajith Kumar’, Uttam Kumar Sarkar! 1 ICAR-National Bureau of Fish Genetic Resources, Lucknow, Uttar Pradesh — 226 002, India 2 Faculty of Fisheries Science, Kerala University of Fisheries and Ocean Studies, Kochi, Kerala — 682 506, India 3 Estuarine Biology Regional Centre, Zoological Survey of India, Gopalpur-on-Sea, Ganjam, Odisha — 761 002, India https://zoobank. org/A86FEFES-7DFD-458E-9E34-616C17E4D06E Corresponding author: T. T. Ajith Kumar (ttajith8 7@gmail.com) Academic editor: Nalani Schnell # Received 12 January 2023 # Accepted 14 March 2023 @ Published 23 March 2023 Abstract Gymnothorax tamilnaduensis sp. nov., anew species of short brown unpatterned moray, 1s described, based on four specimens rang- ing from 272-487 mm total length collected from the trawl bycatch landings at Mudasalodai fish landing centre, off Cuddalore coast, Tamil Nadu, southeast coast of India. The new species is distinguished by the following combination of characters: origin of dorsal fin at middle of rictus and gill opening, anus just before mid-body, series of lines of small dark spots present on head and a single line of black spot-on mid-line of body, jaw pores with white rim, anal-fin margin whitish, 3 pre-dorsal vertebrae, 56—59 pre-anal vertebrae and 139-150 total vertebrae. The new species differs from its known Indian water congeners by having series of lines of small dark spots present on the head and a single line of black spots on the mid-line of the body (vs. absent in all the three congeners in India), serrated teeth (vs. smooth), jaw pores with white rim (vs. black to brown in others) and higher vertebral count (139-150 vs. 134-138 in others). Our morphological and molecular analyses show that the new species forms a distinct clade from its congeners and these data support the status as a new species. Key Words Elopomorpha, molecular analyses, Tamil Nadu, unpatterned moray Introduction The family Muraenidae Rafinesque, 1810 encompasses 223 valid species under two subfamilies, Muraeninae (187 species) and Uropterygiinae (36 species), distrib- uted throughout the tropical and subtropical seas of the world (Fricke et al. 2022). The Indo-Pacific brown unpat- terned moray eels were reviewed by Bohlke (1997, 2000) and she placed 16 species under two categories, viz. elon- gate brown unpatterned moray and short brown unpat- terned moray with eight species each. Later, six species were added to the elongate unpatterned group and three to the short unpatterned group (Mohapatra et al. 2019). Amongst these, eight brown unpatterned morays viz. Strophidon sathete (Hamilton, 1822), S. dorsalis (Seale, 1917), Gymnothorax prolatus Sasaki & Amaoka, 1991 (Mohapatra et al. 2015), G. mishrai Ray, Mohapatra & Smith, 2015, G. indicus Mohapatra, Ray, Smith & Mish- ra, 2016, G. visakhaensis Mohapatra, Smith, Mohanty, Mishra & Tudu, 2017, G. odishi Mohapatra, Mohanty, Smith, Mishra & Roy, 2018 and G. andamanensis Moha- patra, Kiruba-Sankar, Praveenraj & Mohanty, 2019 were described or documented from the Indian waters (Moha- patra et al. 2019). During our recent sampling along the southeast coast of India, four moray eels were collected and subsequent examination suggests that the collected specimens are notably different from their congeners by having a series of dark spots on the head and mid-line of Copyright Kodeeswaran, P. et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 254 the body (vs. absent in all the Indian water species). The present paper describes the collected specimens as a new short brown unpatterned moray eel from the Tamil Nadu waters, Bay of Bengal. Materials and methods Four specimens (ranging from 272-487 mm total length) were collected from the trawl bycatch landings at Mu- dasalodai fish landing centre (11°29’N, 79°46’E), off Cuddalore coast, Bay of Bengal, southeast coast of India (Fig. 1). The specimens were collected at a depth of about 25-30 metres. Meristic counts, morphometric measure- ments and terminology follow Bohlke (1989). Johnson et al. (2012) pointed out that the premaxilla is absent in all Anguilliformes, except Protanguilla. Meanwhile, Bohlke (1989) considered the premaxilla fused to the ethmovomer. Hence, the dentition terminology follows Bohlke (1989) with replacement of premaxillary teeth with ethmovomerine teeth. Measurements were made us- ing a digimatic caliper to the nearest 0.1 mm. Teeth and cephalic pores were examined using Nikon SMZ1270 stereomicroscopes. Vertebral counts were obtained from the digital radiograph and these follow Bohlke (1989). The holotype and one paratype specimen were deposited in the museum of the ICAR — National Bureau of Fish Genetic Resources (NBFGR/MURGTAM), Lucknow, India and two paratype specimens were deposited in the 25°0'0"N 50 15°0'0"N @ - Sampling site @ - Major coastal cities 0 -400 -800 Figure 1. Map showing the collection site of the new species. zse.pensoft.net 100 Kodeeswaran, P. et al.: A new moray from Indian waters Estuarine Biology Regional Centre (EBRC/ZSI/F 14687), Zoological Survey of India, Gopalpur-on-Sea, Odisha, India. The study area map has been prepared using the GEBCO Bathymetry data (https://www.gebco.net/) on the ArcMap 10.8.1 platform. Molecular analyses The genomic DNA was isolated using the salting-out method protocol (Sambrook and Russel 2001). A frag- ment of 16s rRNA (~ 550 bp) was amplified using a uni- versal primer (Palumbi 1996) and the PCR thermal cycle was completed with the following conditions viz. initial denaturation (95 °C for 5 min), denaturation (94 °C for 30 sec), annealing (52 °C for 30 sec), extension (72 °C for 45 sec) for 35 cycles and final extension at 72 °C for 10 min. The obtained PCR results were visualised on agarose gel electrophoresis (2%) containing ethidium bromide, using Gel Doc XR+ (Bio-Rad, India) and sent for sequencing with outsources. The obtained new sequence and other sequences of the genus Gymnothorax retrieved from the public domain, GenBank, were aligned using clustalW Multiple alignments algorithm (Thompson et al. 2003) aiding BioEdit version 5.0.9 (Hall 1999) and the new sequence was deposited in GenBank, accession number: 0Q418115. The Maximum Likelihood tree was recon- structed using a best-fit model of HK Y+G+I (Hasegawa et al. 1985) with 1000 bootstrap replicates and the ge- 80°0'0"E CUDDALORE Mudasalodai \ NAGAPATTINAM@ 10°0'0"N Kilometers —<< GEBCO Bathymetry (m) -1200 -1600 -2000 Zoosyst. Evol. 99 (1) 2023, 253-260 netic divergence analysed aiding the Kimura 2 parameter (Kimura 1980) in MEGA X software (Kumar et al. 2018). The sequence of Conger cinereus (AB910542) was used as an outgroup for the phylogenetic analysis. Abbreviations Total length (TL), head length (HL), supraorbital pores (SO), infraorbital pores (IO), mandibular pores (M), dorsal-fin or- igin (DFO), posterior nostril (PN), anterior nostril (AN), gill opening (GO), branchial pores (B), ethomovomerine (EV). Institute acronyms ICAR-National Bureau of Fish Genetic Resources (ICAR-NBFGR), Zoological Survey of India (ZSI), Ma- rine Aquarium and Regional Centre (MARC), Estuarine Biology Regional Centre (EBRC). Comparative materials Gymnothorax mishrai. Holotype — MARC/ZSI F4210; Gymnothorax odishi: Holotype — ZSI F 12592/2, Para- types. —ZSI F 12593/2, EBRC/ZSI/F 9482, F9483, F9484, F9485, F9486, F9487, F9488, F9489, F9490. Gymnotho- rax andamanensis: Holotype — EBRC/ZSI F11227. 255 Results Gymnothorax tamilnaduensis sp. nov. https://zoobank. org/9E8E2E62-AE3 1-4538-8272-3BBA 122602F5 Figs 2-5, Tables 1, 2 Proposed common name: Tamil Nadu brown moray Type material. The holotype and paratype specimens are deposited at the National Fish Repository of the ICAR-National Bureau of Fish Genetic Resources (Min- istry of Agriculture and Farmers Welfare, Govt. of In- dia). Holotype. NBEGR/MURGTAM (487 mm TL), col- lected from trawl landings at Mudasalodai fish landing centre, off Cuddalore coast, Bay of Bengal, 11°29’N, 79°46’E, 26 June 2022, coll. by G. Kantharajan and P. Kodeeswaran. Paratypes. Three specimens, NBFGR/ MURGTAM.2 (296 mm TL); EBRC/ZSI/F 14687 (2: 272-451 mm TL), were collected along with the holotype. Diagnosis. A new species of a short brown unpatterned moray eel with the following combination of characters: series of lines of small dark spots present on head and a single line of black spots on mid-line of body, origin of dorsal fin at middle of rictus and gill opening, anus just before mid-body, pre-anal length 45.7-47.4% TL, snout blunt and very short, 6.5—7.7 mm in HL, eye small, teeth serrated, uniserial, ethomovomerine teeth five on each side with one tooth on mid-point, vomerine with eight teeth in a series, jaw pores with white rim, anal-fin margin Figure 2. Gymnothorax tamilnaduensis sp. nov. holotype, NBFGR/MURGTAM, 487.8 mm TL, fresh colouration, collected from Mudasalodai fish landing centre, off Cuddalore, Bay of Bengal. zse.pensoft.net 256 whitish, 3 pre-dorsal vertebrae, 56—59 pre-anal vertebrae, 139-150 total vertebrae. Description (all measurements in mm). A medi- um-sized, moderately elongated eel with tapering body, anus just before mid-body, pre-anal length 45.7-47.4% TL, depth at gill opening 19.8—32.8 in TL, depth at anus 31.1-49.0 in TL. Head moderate, 7.8-8.2 in TL, snout blunt, and very short, 6.5—-7.7 in HL, both jaws almost equal, upper jaw 2.5—3.0 in HL, lower jaw 2.4—2.8 in HL, eye small, 9.1-12.2 in HL, at middle of upper jaw, far from rictus, interorbital space 7.0—12.6 in HL. Dorsal-fin origin before gill opening, at mid-length of rictus and gill opening, pre-dorsal length 9.3-11.0 in TL, anal fin starts just after anus, gill opening a minute diagonal slit, 8.0- 11.3 in HL. Teeth serrated, uniserial; five pointed ethomo- vomerine teeth on each side, one tooth on mid-point, three median ethomovomerine teeth, middle tooth larger than other two, maxillary teeth uniserial, 12-13 on each side, uniserial vomerine with eight teeth; lower jaw teeth unise- rial, 16—17 teeth on each side, three anterior teeth enlarged (Fig. 3). Head pores moderate-sized, three supraorbital Median ethomovomerine Kodeeswaran, P. et al.: A new moray from Indian waters pores, four infraorbital pores, six mandibular pores before rictus, cephalic pores in white rims. Branchial pores two behind the dorsal-fin origin (Fig. 4). Three pre-dorsal ver- tebrae, 56—59 pre-anal vertebrae, 139-150 total vertebrae. Counts and measurements of the holotype in mm: TL 487.8, pre-anal length 223.1, tail length 260.7, trunk length 155, pre-dorsal length 44.4, head length 59.6, depth at anus 15.7, width at anus 12.3, snout length 9.2, eye diameter 5.7, upper jaw length 23.9, interorbital width 8.5, gill opening 6.8. Vertebral formula 3-59-150. Colouration. In fresh condition, body uniform chocolate brown, head with numerous line marks, ventral-most por- tion of lower body pale, dorsal fin brown, dorsal-fin margin black, anal-fin margin whitish, head pores with white rim, series of small dark spots forming several lines on head and a single line of black spot-on mid-line of body (Fig. 2). After preservation, body tanned, series of small dark spot lines remain the same in the head and body (Fig. 5). Distribution. Indian Ocean: off Cuddalore Coast, Bay of Bengal, southeast coast of India. The species were col- lected at a depth of about 25—30 metres. Ethomovomerine pL fr L A ° y V : Vv Vomerine 0 i Maxillary V A ° a v Z. V we Vv 150 vertebrae) and the short brown unpatterned morays (< 150 vertebrae). The species described herein comes under the short brown unpatterned moray. From Indian waters, only three species of short brown unpatterned morays have been described to date (Mohapatra et al. 2019). All three short brown unpatterned morays reported from India have smooth teeth, and the new species clearly differs from all three Species in having serrated teeth. The new species differs from all the Indian water short brown morays by having jaw pores with white rim (vs. black in G. andamanensis, brown in G. mishrai and dark in G. odishi); having more total vertebrae (139-150 vs. 135—136 in G. andamanensis, zse.pensoft.net 258 Kodeeswaran, P. et al.: A new moray from Indian waters Figure 5. Gymnothorax tamilnaduensis sp. nov. holotype, NBFGR/MURGTAM, 487.8 mm TL preserved colouration. 134 in G. mishrai and 133-138 in G. odishi),; smaller snout (6.5—7.4 in HL vs. 4.4 in G. andamanensis, 6.5 in G. mishrai, 4.3—5.4 in G. odishi); having a distinct series of lines of small dark spots present on head and a single line of black spot on the mid-line of the body (vs. absent in all the other species). The new species shares serrate teeth with Gymnotho- rax atolli (Pietschmann, 1935), G. australicola Lavenberg 1992, G. panamensis (Steindachner, 1876) and G. pindae Smith 1962, but the new species differs from the species mentioned above by having more total vertebrae (139- 150 vs. 127-133 in G. atolli, 122-137 in G. panamensis and 110-124 in G. pindae), fewer predorsal vertebrae (3 vs. 4-6 in G. atolli, 5—6 in G. australicola, 8-12 in G. panamensis and 5-7 in G. pindae) and uniserial max- illary teeth (vs. biserial in G. atolli, G. australicola and G. panamensis). Further, the new species differs from the other unpat- terned morays, such as Gymnothorax herrei Beebe & Tee- Van, 1933, G. kontodontos Bohlke, 2000, G. microstictus Bohlke, 2000 and G. pseudoherrei Bohlke, 2000 by hav- ing serrate teeth (vs. smooth in all the remaining species); more total vertebrae (139-150 vs. 108-122 in G. herrei, 126-135 in G. kontodontos, 113-121 in G. microstictus, zse.pensoft.net 110-118 in G. pseudoherrei);, more pre-anal vertebrae (56— 59 vs. 45—50 in G. herrei, 40-43 in G. kontodontos, 40-44 in G. microstictus, 44—S0 in G. pseudoherrei) (Table 2). Molecular analysis. Based on the 16s rRNA gene sequences, the new species exhibits 8.0% genetic di- vergence with G. reticularis sequences in the Kimura 2 parameter. Further, the new species exhibits 10.1% ge- netic divergence with G. sagmacephalus, followed by G. albimarginatus by 11.2%-12.1%, G. castaneus (11.9%) and G. pictus (12.6%). The Maximum Likelihood tree (Fig. 6) shows the new species forms a strongly distinct clade from the congeners with a high bootstrap value sup- porting the status as a new species. Discussion Moray eels are distinguished from other Anguilliformes fishes by having peculiar characters viz. absence of pec- toral fins; smaller gill opening like a pore; lack of later- al-line pores (some have at the anterior end); absence of pores behind the eye and supratemporal canal; well-de- veloped pharyngeal jaws (Smith 2012). Most of the mo- rays are cryptic and found at shallow waters, coral reef Zoosyst. Evol. 99 (1) 2023, 253-260 97 100 259 1009 AB695701 Gymnothorax berndti °F 148695700 Gymnothorax berndti b= JX242972 Gymnothorax margaritophorus AB695696 Gymnothorax chilospilus JX242980 Gymnothorax zonipectis AB695711 Gymnothorax richardsonii JX242979 Gymnothorax vicinus » AF455770 Gymnothorax moringa AB695698 Gymnothorax rueppellii 100 ¢4B695650 Gymnothorax pictus 21 P7 AB695649 Gymnothorax pictus 42 AF455769 Gymnothorax castaneus AB695653 Gymnothorax thyrsoideus JX242975 Gymnothorax nudivomer AB695722 Gymnothorax eurostus AB695728 Gymnothorax buroensis a AB695716 Gymnothorax meleagris 1008 AB695717 Gymnothorax meleagris 100 AB695727 Gymnothorax pseudothyrsoideus AB695726 Gymnothorax pseudothyrsoideus 100 JKX426291 Gymnothorax reticularis 26 73 59 100 AB808696 Gymnothorax minor 0Q418115 Gymnothorax tamilnaduensis sp. nov. AB808695 Gymnothorax sagmacephalus AB808691 Gymnothorax albimarginatus AB808690 Gymnothorax albimarginatus es [3 ()| ()54 Conger cinereus (OQutgroup) rr 0.10 Figure 6. The Maximum Likelihood tree of the genus Gymnothorax plotted with a model of HK Y+G+I with 1000 bootstrap repli- cates, based on 16s rRNA sequences (500 bp). Each node represents branch support values. Table 2. Comparative characters from short brown unpatterned Gymnothorax (data obtained from Bohlke (2000) and specimens examined from India). 2 2 os o “ 1) (7) $ S = 8 o ¢ s w = 2 ® . a gj S So So So °C; Branchial pores 2 2 1 i 1 2 2 2 2 2 2 2 Pre-dorsal vert. 3 3 4-6 5-6 7-11 7-9 4-5 9 4 8-12 5-7 5-9 Pre-anal vert. 56-59 57 51-54 52-56 45-50 40-43 40-44 59 55-58 50-55 42-46 44-50 Total vert. 139-150 135-136 127-133 141-146 108-122 126-135 113-121 134 133-138 122-137 110-124 110-118 Jaw pores White rim Black rim Whiterim Whiterim Palerim Palerim Brownrim Brownrim Darkrim Whiterim Brownrim Brownrim Median EV teeth 0-3 3 2 2 2-3 2 2 3 3 2 2 z Inner EV teeth 0 3 8-14 7-12 8-15 9-13 0-7 0 0 9-12 0-10 8-21 MX teeth Uniserial —_Biserial Biserial Biserial Biserial 2-3 series 1-2 series Uniserial Uniserial _Biserial Uniserial Biserial DFO/GO Before Before Before Before Behind Behind Before Before Before Behind Before Before VM teeth Uniserial Uniserial Uniserial © Uniserial © Uniserial 2-3 series Uniserial Uniserial Uniserial Irregular Staggered Staggered Teeth edge Serrate Smooth Serrate Serrate Smooth Smooth Smooth Smooth Smooth _ Serrate Serrate Smooth and rocky environments, like holes and crevices for hid- ing and appearing outside the bolt-hole for feeding and many species were inhabiting off-shore waters and even up to 500 m depth (Smith 2012). In Indian waters, hith- erto 28 species of Gymnothorax have been documented (Mohapatra et al. 2020). Amongst these, eight species be- long to the brown unpatterned moray group (three short unpatterned and five elongate brown unpatterned) and the remaining species are colour-patterned with spotted, barred, mottled or irregular markings. The species de- zse.pensoft.net 260 scribed herein represents the fourth species in the short brown unpatterned moray group and increases the total amount of species of Gymnothorax to 29. The present de- scription is the first new species of the genus Gymnotho- rax from the south-eastern coast of India, Bay of Bengal, whereas its congeners were described from the following locations viz. G. andamanensis was described from two specimens collected from Port Blair, South Andaman; G. mishrai was described, based on a unique holotype specimen collected from the West Bengal, northern Bay of Bengal and G. odishi was described from 11 speci- mens collected along the Odisha, north-eastern coast of India. The new species differs from its known congeners by having serrate teeth (vs. smooth in all three species), by having jaw pores with white rims and more vertebrae counts. The DNA data of most of the brown unpattern mo- rays from India are not available, so comparing the DNA data of the new species with all Indian brown unpattern moray is not possible. Most of the brown unpatterned mo- rays from India are described from less than 50 m depth, though few species were described from bycatch fishes and the depth is not certain. This species is the second brown unpatterned moray from the southern region of In- dia, along with the earlier reported S. sathete. Mostly, the species of this group were landed as bycatch or trash and do not possess economic value, as well as commercial importance. Few patterned morays secure minimal posi- tion in the aquarium trade, but distinct data on the aquar- ium market are meagre. Hence comprehensive studies on the genus Gymnothorax are warranted to find the precise diversity and distribution of this group in Indian waters. Acknowledgements The authors are grateful to the Director of ICAR—Na- tional Bureau of Fish Genetic Resources (NBFGR), Luc- know, for providing the necessary facilities and support. A.M. thanks Dr. Dhriti Banerjee, Director of the Zoolog- ical Survey of India for providing support to carry out the work. References Bohlke EB (1989) Methods and terminology. In: Bohlke EB (Ed.) Fish- es of the Western North Atlantic. Memoir of the Sears Foundation for Marine Research 1(9): 1-7. Bohlke EB (1997) Notes on the identity of elongate unpatterned In- do-Pacific morays, with description of a new species (Muraenidae, Subfamily Muraeninae). Proceedings. Academy of Natural Sciences of Philadelphia 147: 89-109. zse.pensoft.net Kodeeswaran, P. et al.: A new moray from Indian waters Bohlke EB (2000) Notes on the identity of small, brown, unpatterned Indo-Pacific moray eels, with descriptions of three new species (An- guilliformes: Muraenidae). Pacific Science 54(4): 395-416. Fricke R, Eschmeyer WN, Fong JD (2022) Species by Family/Sub- family. Eschmeyer’s Catalog of Fishes. http://researcharchive. calacademy.org/research/ichthyology/catalog/SpeciesByFamily.asp [accessed 24 December 2022] Hall TA (1999) BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 41: 95-98. Hasegawa M, Kishino H, Yano TA (1985) Dating of the human-ape splitting by a molecular clock of mitochondrial DNA. Journal of Molecular Evolution 22(2): 160-174. https://doi.org/10.1007/ BF02101694 Johnson GD, Ida H, Sakaue J, Sado T, Asahida T, Miya M (2012) A ‘living fossil’eel (Anguilliformes: Protanguillidae, fam. nov.) from an undersea cave in Palau. Proceedings of the Royal Society B: Biological Sciences 279(1730): 934-943. https://doi.org/10.1098/ rspb.2011.1289 Kimura M (1980) A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide se- quences. Journal of Molecular Evolution 16(2): 111-120. https:// doi.org/10.1007/BF01731581 Kumar S, Stecher G, Li M, Knyaz C, Tamura K (2018) MEGA X: Mo- lecular Evolutionary Genetics Analysis across computing platforms. Molecular Biology and Evolution 35(6): 1547-1549. https://doi. org/10.1093/molbev/msy096 Mohapatra A, Ray D, Smith DG (2015) First occurrence of the mo- ray eel Gymnothorax prolatus Sasaki & Amaoka, 1991 (Teleostei: Anguilliformes: Muraenidae) from the Northern Indian Ocean. Marine Biodiversity Records 8(e106): 1-3. https://doi.org/10.1017/ $1755267215000834 Mohapatra A, Kiruba-Sankar R, Praveenraj J, Mohanty SR (2019) A new short brown unpatterned moray eel Gymnothorax anda- manensis (Muraenidae: Muraeninae) from Andaman waters, In- dia. Zootaxa 4661(1): 189-196. https://doi.org/10.11646/zoot- axa.4661.1.11 Mohapatra A, Mishra SS, Bineesh KK, Rajendra S, Ray D, Mohanty SR, Roy S (2020) Pisces. In: Chandra K, Raghunathan C, Mondal T (Eds) Faunal Diversity of Biogeographic Zones: Coasts of India. Zoological Survey of India, Kolkata, 655-722. Palumbi SR (1996) Nucleic acids I: the polymerase chain reaction. In: Hillis DM, Moritz C, Mable BK (Eds) Molecular Systematics. Si- nauer & Associates Inc., Massachusetts, 205-247. Sambrook J, Russel DW (2001) Molecular cloning: a laboratory manual (Vol. 1). Cold Spring Harbor, NY, 112 pp. Smith DG (2012) A checklist of the moray eels of the world (Teleostei: Anguilliformes: Muraenidae). Zootaxa 3474(1): 1-64. https://dot. org/10.11646/zootaxa.3474.1.1 Thompson JD, Gibson TJ, Higgins DG (2003) Multiple sequence align- ment using ClustalW and ClustalX. Current Protocols in Bioinfor- matics 00(1): 2-3. https://doi.org/10.1002/0471250953.bi0203s00