TRICHOTAXONOMY OF INDIAN SPECIES OF GENUS RATUFA GRAY ( MAMMALIA : RODENTIA : SCIURIDAE )

Oriental giant squirrels (Genus Ratufa) belong to subfamily Ratufinae and are found in parts of South and South-east Asia. There are four species of oriental giant squirrels : Ratufa affinis (Raffles) (Pale Giant Squirrel), Ratufa bicolor (Sparrman) (Malaya Giant Squirrel), Ratufa indica (Erxleben) (Indian Giant Squirrel) and Ratufa macroura (Pennant) (Grizzled Giant Squirrel). Ratufa affinis (Raffles) (Pale Giant Squirrel) is found in Brunei, Indonesia, Malaysia, Singapore and Thailand (Krapp 1998).

The Malayan Giant Squirrel, Ratufa bicolor (Sparrman) is at home on the Indian subcontinent, north of the Ganges in Nepal, Sikkim, Bhutan and Assam; farther to the east it lives in Burma, Malaya and upto Southern China and on Java.It is deep dark brown, almost black on the back and a light beige on the underside.They are very shy and they live exclusively in forest in the highest trees.They are very agile and jump in great leap from tree to tree, over a distance of almost 22 ft (Krapp 1998).Ratufa bicolor (Sparrman) is a Schedule II species under IWPA 1972, and has category Lc IUCN (Kumar and Khanna 2004).

Medulla
To study the type of medulla, the cleaned hair was mounted in DePeX (Gurr) for whole mount.When mounting, the hair tuft, it is necessary to ensure that the individual hair is well separated.For temporary mounting Paraffin oil is a most convenient medium (Appleyard, 1960).
Longitudinal sections were also prepared for clear picture of medulla type.
Cross section: For the present study, hair cross sections were obtained by simple hand sectioning after mounting the hair in paraffin wax, the method followed as given in reference guide Bahuguna et al 2010.For longitudinal section of medulla, the blocks of hair were prepared in paraffin wax and hand sectioning was done longitudinally.The technique is useful for clear picture of medulla type (Bahuguna 2008) as the presence of pigments generally hides the structure.
Scale casts : Procedures for studying scale pattern usually involve the use of special media to obtain a cast or impression of the actual hair surface.For getting the cast, the cleaned hair was kept with the help of the fine forceps on thin film of the gelatin (3%) medium on slide for some time till the medium was air-dried.After drying of gelatin the hair was removed gently.For very long hair they can be cut into sections to have complete picture of scales at tip, mid and basal region of hair.Another medium polyvinyl acetate (PVA in 50% distilled water) can also be used for this purpose (Appleyard,960).

Rec. zool. Surv. India
For cuticle studies different parts of hair i.e. distal (tip), mid and proximal part (base) of hair were examined.
Photomicrographs were taken for cross section, medulla type and cuticular studies at xl00 to x200 (total magnification) under compound light microscope, Olympus CX41.

Scanning electron microscope study
This was performed for studying details of cuticular pattern.After cleaning the hair, small mid section of hair were kept on adhesive on stub.The stubs were coated with thin film (15-20 A) of gold and kept in the chamber to view details of scale pattern.The electron micrographs thus obtained from Zeiss EV040 were used to find out scale index, scales types and scale margins.
Hair measurements: Hair measurements were noted for calculating mean of thickness of medulla and total thickness of hair and their ratios for medullary index.
Ratios of length of hair and thickness of hair were also taken into account to get length index .The measurements were shown under observations as mean ±SD Nomenclature of medulla type was adopted after Wildman (1954)  However, it has been reported by Menon (2003), that color of coat varies from different regions of distribution in case of Ratufa indica (Erxleben) an endemic squirrel.The dorsal region is a mixture of maroon and black with under parts cream and buff.In the northern Western Ghats this squirrel is brownish-maroon in appearance, with an all brown and white tail.In south it is black and dark maroon with a black and brown tail, whereas in the central and Southeastern Indian forms has brown color coat on the back with black hair on forelegs and have black tail with a pale tip.Malayan or Black Giant squirrel, Ratufa bicolor (Sparrman), is deep brown or black on the back and buff beneath.It has large black ears with hairy tufts, a black tail and black marks on its chin.The forelegs are black in front and buff on the back.Grizzled giant squirrel Ratufa macroura (Pennant), this endangered squirrel is comparatively smallest and has brownish gray coat with pale hair tips giving it a grizzled look.Its ventral surface is dirty white and tail has white bands.Ears and head are dark brown or black.Thus the color of hair from all body regions of the species of Ratufa although noted for the record in the present study but not taken into consideration for the key for identification of species.However, in case of Indian species of Ratufa, the banding is not the characteristic feature of the primary guard hair from all regions examined.But, bandwidth was noted to be characteristics of the genus Callosciurus of family Sciuridae and has been utilized in development of the key for identification of the species (Bahuguna 2008) as one of the important physical features.Length indices for all three species for primary guard hair from all body regions were: for Ratufa bicolor (Sparrman) 30.3 ± 0.0 (for dorsal), 24.5 ± 0.0 (for ventral), 16.80 ± 0.0 (for head) and 51.7 ± 0.25 (for tail); for Ratufa indica (Erxleben) they were 39.2 ± 0.1 (for dorsal), 14.6 ± 0.16 (for ventral), 17.81 ± 0.06 (for head), 57.19 ± 0.25(for tail).In case of Ratufa macroura (Pennant) length indices were 46.04 ± 0.5 (for dorsal), 35.45 ± 0.016 (for ventral), 13.76 ± 0.04 (for head) and 39.4 ± 0.07 (for tail).
Length indices were noted to be maximum of Ratufa macroura (Pennant) for dorsal hair, of Ratufa bicolor (Sparrman) for ventral, of Ratufa indica (Erxleben) for head and tail.Length indices were used as an additional physical characteristics of hair (Bahuguna 2008) to know its consistency in identification of the species.In the present study the species of Ratufa examined showed the interspecific variability in length indices as noted in case of Indian species of Callosciurus (Bahuguna 2008).
Cuticular characteristics : In case of Ratufa macroura (Pennant) (figs 45-47, 50-52, 55-57, 60, 61), the pattern was regular wave with rippled margin and near in all dorsal, ventral and head.But in tail region pattern is regular wave with smooth margin and distant.
In case of Ratufa indica (Erxleben), the cuticular pattern was regular wave, with rippled margin and near in case of hair from all body regions (figs 23,24,26,27,29,31,32,34,35,37,39,41,42).The cuticular pattern of Ratufa bicolor (Sparrman), was regular wave, with rippled margin and near in case of primary guard hair of dorsal, ventral but in tail regions it is irregular wave with rippled margin and near at proximal and mid but at distal it was with smooth margin (figs 2-5, 7, 9, 10, 12-15).However hairs from head showed regular wave pattern with scalloped margin and distance between scales were near (figs [18][19][20]22).Cuticular characteristics are known to be species specific in many studies (Chakraborty and De 1995, De et al 1998, Bahuguna and Mukherjee 2000, Pradhan et al 2005, Bahuguna 2007).Scale indices of dorsal primary guard hair of R. indica and R. bicolor were noted to be almost same but in R. macroura it was noted to be 6.24 ± 0.32 in all species examined for dorsal primary guard hair.However they were noted to be different in case of primary guard hair from other body regions.Scale index of the guard hair of ventral region of R. bicolor was noted to be 5.3 ± 0.01 and of R. indica was 6.5 ± 0.01 and that of R. macroura was 6.3 ± 0.0 .Scale index of primary guard hair of head of R. bicolor was 4.17 ± 0.0 and of R indica was 7.3 ± 0.0 and of R. macroura was 4.4 ± 0.0.Scale index of tail of R. bicolor was 2.86 ± 0.01 , R. indica 6.5 ± 0.01 and of R. macroura was 4.25 ± 0.01.
However it has been realized that hair characteristics of primary guard hair from other body regions are equally important and should also be recorded.Since it is difficult to collect hair samples from large number of specimens, (especially for threatened and endangered species), thus it is recommended to have the record of hair characteristics from all body regions for further comparisons from other samples.This is required for dealing with wildlife forensic cases.
Based on characteristics of primary guard hair from all body regions, key was prepared, which is useful in identification of species of Genus Ratufa for various biological studies including Wildlife forensic.Scanning electron micrograph at mid and the same for cuticular scale pattern and cross section types after Brunner and Coman (1974.)OBSERVATIONS Genus Raufa Gray, 1867 Ratufa bicolor (Sparrman), Malayan Giant Squirrel Status IWPA : Schedule II, Part II, CITES: Appendix II; CAMP: VU (Nationally)light brown and dark brown Total thickness (T) : 86.3 ± 1.5 }lm Length index (LIT) : 30.3 ± 0.09 Shape and Nature: Straight and thin B Cuticular Scale Pattern At mid: SP regular wave ,SM rippled, DS near; at proximal, SP regular wave SM rippled , DS near; At distal : SP regular wave, SM rippled, DS near Scale index: 6.0 ± 0Total thickness (T) : 49.8 ± 0.0 }lm Length index: 24.5 ± 0.0 Shape and Nature: Straight and thin B Cuticular Scale Pattern At mid: SP regular wave, SM rippled, DS near; at proximal, SP regular wave SM rippled, DS near; At distal: SP regular wave, SM rippled, DS near Scale index: 5.3 ± 0SP regular wave, SM scalloped margin, DS near; at proximal, SP regular wave SM scalloped, DS near; At distal : SP regular wave, SM scalloped, DS near, Scale index: 4.17 ± 0.0 C Medulla Medullary configuration: Wide Aeriform Lattice Medulla thickness: 89.0 ± 1.0 : }lm Medullary index: 0.51 ± 0Pattern At mid: SP Irregular wave, SM rippled, DS near; at proximal, SP Irregular wave SM slightly rippled, DS near; At distal: SP Irregular wave, SM smooth, DS near Scale index: 2.86 ± 0SP regular wave, SM rippled, DS near; at proximal, SP regular wave SM rippled, DS near; At distal: SP regular wave, SM rippled, DS near Scale index: 6.0 ± 0.45 C Medulla Medullary configuration: Simple medulla Medulla thickness 65.4 ± 0.52 : }lm Medullary index: 0.76 ± 0.0 D Cross section Type of cross section: Oblong Medulla size in cross section: Large Ventral A Physical characteristics Colour : light brown and beige Total thickness (T) : 84.5 ± 0.32 }lm Length index: 14.6 ± 0.16 Shape and Nature: Straight and thin B Cuticular Scale Pattern At mid: SP regular wave, SM slightly rippled, DS near; at proximal, SP regular wave SM slightly rippled, DS near; At distal: SP regular wave, SM rippled, DS near Scale index: 6.5 ± 0.01.C Medulla Medullary configuration : Wide Aeriform Lattice Medulla thickness: 44.5 ± 0.52 }lm Medullary index: 0.52 ± 0.01 D Cross section Type of cross section: Oblong Medulla size in cross section: Medium Head A Physical characteristics Colour: light brown at tips, mid dark brown and black Total thickness (T) : 69.6 ± 0.08 }lm Length index: 17.81 ± 0.06 Shape and Nature: Straight and thin B Cuticular Scale Pattern At mid: SP regular wave, SM rippled, DS near; at proximal, SP regular wave SM rippled, DS near; At distal: SP regular wave, SM rippled, DS near Scale index: 7.3 ± 0.0 C Medulla Medullary configuration : Wide Aeriform Lattice Medulla thickness: 49.2 ± 0.21 }lm Medullary index: 0.706 ± 0.026 Rec.zool.Surv.India D Cross section Type of cross section: Oblong Medulla size in cross section: Large Tail A Physical characteristics Total thickness (T) : 79.2 ± 0.16 }lm Length index: 57.19 ± 0.25 Shape and Nature: Straight and thin B Cuticular Scale Pattern At mid: SP regular wave, SM rippled, DS near; at proximal, SP regular wave SM rippled, DS near; At distal : SP regular wave, SM rippled, DS near Scale index: 6.5 ± 0.01 C Medulla Medullary configuration: Wide Aerifom Lattice Medulla thickness: 60.7 ± 0.16 }lm Medullary index: 0.76 ± 0.01 D Cross section Type of cross section: Circular, Oblong Medulla size in cross section: Large Ratufa macroura (Pennant, 1769) (Grizzled Indian (Giant) Squirrel) Status: IWPA: Schedule 1, Part 1; RDB : EN; CITES: Appendix II; CAMP: EN (Nationally), DD (Globally) Dorsal A Physical characteristics Colour: Dark brown/black with light brown tips Total thickness (T) : 69.72 ± 0.06 }lm Length index: 46.04 ± 0.05 Shape and Nature: Straight and thin B Cuticular Scale Pattern At mid: SP regular wave, SM rippled, DS near; at proximal, SP regular wave SM rippled, DS near; At distal: SP regular wave, SM rippled, DS near Scale index: 6.24 ± 0.32 C Medulla Medullary configuration: Wide Aeriform Lattice Medulla thickness: 60.3 ± 0.08 }lm Medullary index: 0.86 ± 0.013 D Cross section Type of cross section: Oblong Medulla size in cross section: Large Ventral A Physical characteristics Colour: dark brown/black and light brown Total thickness (T) : 29.9 ± 0.03 }lm Length index: 35.45 ± 0.01 Shape and Nature: Straight and thin B Cuticular Scale Pattern At mid: SP regular wave, SM rippled, DS near; at proximal, SP regular wave SM rippled, DS near; At distal : SP regular wave, SM rippled, DS near Scale index: 6.3 ± 0.0 C Medulla Medullary configuration: Simple medulla Medulla thickness: 20.0 ± 0.0 }lm Medullary index: 0.66 ± 0.0 D Cross section Type of cross section: Oblong Medulla size in cross section: Medium Head A Physical characteristics Colour: Slightly light brown at tips, mid is dark Total thickness (T) : 87.27 ± 1.0 }lm Length index: 13.76 ± 0.047 Shape and Nature: Straight and thin B Cuticular Scale Pattern At mid: SP regular wave, SM rippled, DS near; at proximal, SP regular wave SM rippled, DS near; At distal : SP regular wave, SM rippled, DS near Scale index: 4.4 ± 0.0 C Medulla Medullary configuration: Wide Aeriform Lattice Medulla thickness: 67.2 ± 1.0 }lm Medullary index: 0.77 ± 0.01 D Cross section Type of cross section: Oblong Medulla size in cross section: Large Tail A Physical characteristics Colour: black/dark brown throughout Total thickness (T) : 102.7 ± 0.46 }lm Length index: 39.4 ± 0.07 Shape and Nature: Straight and thin B Cuticular Scale Pattern At mid: SP regular wave, SM smooth, DS distant; at proximal, SP regular wave SM smooth, DS distant; At distal: SP regular wave, SM smooth, DS distant Scale index: 4.25 ± 0.01 C Medulla Medullary configuration: Wide Aeriform Lattice Medulla thickness: 86.3 ± 0.50 }lm Medullary index: 0.84 ± 0.01 D Cross section Type of cross section: Oblong Medulla size in cross section: Large RESULTS AND DISCUSSION Physical characteristics : Coat color is known to vary from juveniles to adults in many mammals.