Identification of Sri Lankan Muroid Rodents using Hair Anatomy

We report here characteristic features of hair anatomy of all fifteen muroid rodent species occurring in Sri Lanka. We examined cuticular scale patterns, cross-sections, medullae patterns, hair profile and made measurements (length and maximum width) of dorsal guard hairs. We also developed a dichotomous key for identification of rodent species based on hair anatomy, supported with photomicrographs of cuticular scales, medullae and illustrations of cross-sections. All species except spiny rats (Mus mayori and M. fernandoni) can reliably be distinguished from each other using hair anatomy, in most cases using a combination of characters. Spiny rats can only be distinguished as a group. Cuticular scale patterns and cross-sections show more heterogeneity among species, compared to other characters. Medullary cell shape, their arrangement, maximum number of cells in the widest region of the hair, hair dimensions and profiles are also useful in species identification.


INTRODUCTION
Epidermal hair is a unique feature of mammals.Anatomy of hairs is known to change according to the habitats in which mammals live, and hence is species-specific and important in taxonomy (Kondo, 2000;Sarkar et al., 2011;Anwar et al., 2012).Identification of species through hair anatomy is useful in many disciplines, such as paleontology, zooarchaeology, anthropology, ecology and forensic science (Marinis and Asprea, 2006).
There are three types of hairs located in different regions of the mammalian body; vibrissae, bristle hairs and coat hairs; of these latter is mostly used in taxonomic studies (Teerink, 1991).Coat hairs are of two types: guard hairs (GH) and under hairs (UH), with the former being of greater taxonomic importance (Marinis and Agnelli, 1993;Teerink, 1991).Of the three guard hair types GH0, GH1 and GH2, the latter two show more heterogeneity among species and are important in taxonomic studies (Teerink, 1991;Menike et al., 2012).A hair consists of three structural units: outermost cuticle, mid-cortex and innermost medulla (Homan and Genoways, 1978;Hausman, 1920).Of these, cuticular scales and medullae patterns are studied in detail for species identification along with crosssections of hairs.In addition, hair profile and dimensions (length and maximum diameter of hairs) are used (Teerink, 1991;Amerasinghe, 1983).
In Sri Lanka, fifteen species of murine rodents (rats and mice) belonging to eight genera, namely, Bandicota, Mus, Golunda, Srilankamys, Rattus, Madromys, Millardia and Vandeleuria and a single species of gerbil, Tatera tatera are recorded.Of these, one genus and three species are endemic to the island (Table 1).All species of muroid rodents, except Mus booduga, were included in a study on hair anatomy of Sri Lankan mammals (Amerasinghe, 1983).However, since the study dealt with 108 mammal species and subspecies in the country, illustrations were limited to examples and only generic level identification was attempted in the keys, hence in most instances not distinguishing among species.
Here, we characterize dorsal guard hairs of all 16 species of muroid rodents in Sri Lanka providing an improved tool for discriminating among the dorsal guard hairs of these species based on cuticular scale patterns, shapes of cross-sections, medullae patterns, hair profile and dimensions.We also considered a single pair of subspecies, Gollunda ellioti ellioti and G. e. nuwara, because the latter subspecies has spinous hairs while the former has soft hairs (Phillips, 1980).Since cuticular scale patterns, shapes of cross-sections and the form of medullae are not uniform from the tip to the base of guard hairs (Amerasinghe, 1983;Teerink, 1991;Menike et al., 2012), we also report charactervariation along the entire length of the hair.

MATERIALS AND METHODS
Hair samples used in this study were from rodents collected during an island-wide survey of small mammals carried out from 2003-2005, except those of Rattus montanus, R. blanfordi, Bandicota indica, B. bengalensis and G. e. ellioti, which were obtained from the National Museum of Sri Lanka (Table 1).Hair samples of Mus cervicolor and R. norvegicus were from both sources.All specimens considered here were adults.Voucher specimens of collected rodents originally deposited in the collection of the Wildlife Heritage Trust (WHT) are now placed in the Department of Zoology, University of Peradeniya, Sri Lanka.
Samples of dorsal hair from one to four specimens from each species and subspecies were used in this study, depending on the availability.Only GH1 and GH2 were studied as they exhibit considerable variation among species (Teerink, 1991).To observe cuticular scale patterns, cuticular scale imprints were taken on a thin layer of nail polish similar to the method used by Amerasinghe (1983) using a thin solution of Canada balsom in xylene.Cuticular scale patterns of the groove side of the hair were not considered because the nail polish frequently got trapped in the groove and damaged the cast.Cross-sections of hairs were made using thin strips of 'botanical pith' (Amerasinghe, 1983;Teerink, 1991).Nail polish was applied on the pith and the hairs were placed along their longitudinal axis.Another strip of pith was then placed over it, covering the hair.After allowing the nail polish to dry for few minutes, thin transverse sections were cut using a clean, sharp razor blade (Amerasinghe, 1983).The sections were pasted in serial order (from tip to base) on a slide using glue and observed under a stereo-microscope.(Amerasinghe, 1983;Teerink, 1991).Five hairs from each individual were used to observe cuticles, medullae and cross-sections.
Photomicrographs of cuticular scales, crosssections and medullae were taken using a USB camera fixed to a stereo-microscope.Illustrations of cross-sections were made in Adobe Photoshop raster graphic editor.Measurements of hairs were made using Image-J software.Length and maximum width of fifteen hairs of each hair-type (GH1 and GH2) were measured from each species.Nomenclature of hair anatomy follows Teerink (1991) and Amerasinghe (1983).

Hair profile
All the rodents in the study have GH1, GH2 and under hair except spiny rats (M.mayori and M. fernandoni) and M. cervicolor, which lack GH2.Spiny rats along with G. e. ellioti also differ from the other species by bearing spinous GH1.

Cuticular scale patterns
Altogether five cuticular scale patterns: irregular wave, petal, mosaic, diamond and chevron are present in both GH1 and GH2 of species and subspecies studied here (Fig. 1).All species have more than one of the above scale patterns through the length of the hair.Although the cuticular scale patterns belong to one of the above broad categories, the shapes, sizes and arrangement of scales of both GH1 and GH2 are characteristic of each species and subspecies, except for those of two spiny rats.
In all the species considered here, the cuticular scale patterns take an irregular wavy appearance

Cross-sections
Cross-sections of both GH1 and GH2 of all species are of circular or oval shapes towards the tip and base of the hairs except the tip of GH1 in spiny rats, which has a characteristic triconcave shape and tip of GH1 in G. e. ellioti, which has triangular and square shaped cross-sections.Between the tip and base of both GH1 and GH2, shapes of cross-sections vary among species (Table 2).
Altogether there are five shapes present in the mid region: concavo-convex, plano-concave, oblong, biconcave and dumbbell.Concavo-convex shape is common to both GH1 and GH2 of all the species except for GH1 and GH2 of B. indica and GH2 of M. booduga.Plano-concave shape is also found in many species; both GH1 and GH2 of R. montanus, GH1 of B. indica, R. rattus, spiny rats, M. booduga and T. tatera and in GH2 of G. e. ellioti.Oblong shape is seen in both GH1 and GH2 of B. indica, B. bengalensis, R. montanus, M. blanfordi, T. tatera and GH2 of M. booduga.Biconcave shape is restricted to few species: GH2 of B. indica, R. rattus and R. norvegicus, M. booduga and GH1 of M. musculus.
Dumbbell shape is only seen in GH1 of G. ellioti subspecies.

Medullae patterns
Medullae of all species are unicellular towards the tip and the base of both GH1 and GH2 and multicellular in the middle (Table 3).GH1 of spiny rats, G. e. nuwara and G. e. ellioti have characteristic transverse lattice medullae while others have circular to oval shaped cells giving a honeycomb appearance to the medullae.Some Table 3. Medullae patterns useful in identification of species of Sri Lankan muroid rodents.Each photograph was taken under medium power of a light microscope (10X10 power) and is 1.07 mm in length.

Length and maximum diameter
The dimensions of the hairs can be used in combination with other characters to distinguish species (Table 4).Species can be broadly categorized into groups based on lengths or maximum widths of GH1 and GH2; for example, both GH1 and GH2 shorter than 10 mm (M.booduga, M. musculus); GH1 shorter than 10 mm

mm
Ta ble 4. Length and maximum width of muroid rodents in Sri Lanka (range, mean and standard deviation).

Key for hair identification
The key is based on cuticular scale patterns, crosssections, medullae patterns, hair profile and dimensions of GH1 and GH2 hairs described above.
Characters shared by all species such as irregular wave cuticular scale pattern and circular to oval cross-sections towards the tip of the shield and the base of the shaft of both GH1 and GH2 (except GH1 of spiny rats and G. e. ellioti) are excluded when developing the key.In between the tip and base, cross-sections take concavo-convex, plano-concave, biconcave, dumbbell and oblong shapes in the species studied.Presence or absence of these shapes in crosssections is useful in identification of each species.

Species
Medullae pattern of the guard hairs can also be used for identification of species in combination with other characters.There are two major types of medullae pattern; transverse lattice and honeycomb lattice.Honeycomb lattice is random in some species, which can be used as an identifying character.Number of medullary cell rows in the widest part of the hair is another useful feature for species identification (Amerasinge, 1983;Teerink, 1991), though there are instances where the number of maximum rows reported differ between studies, for example 4 (Teerink, 1991) and 3-4 (Amerasinge, 1983) for M. musculus.
In addition, lengths and maximum widths of guard hairs, and hair profiles can be used to distinguish species.The presence or absence of different types of hairs, abundance of each type of hair, and presence of spinous hairs are some of the hair profile features that can aid in species differentiation.For example, spiny rats and M. cervicolor lack GH2; GH2 is less abundant compared to GH1 in M. musculus and M. booduga, while others have similar numbers of each; spiny rats and G. e. ellioti have spinous GH1.
We considered a single pair of subspecies; G. e. ellioti and G. e. nuwera, since the former has spinous hairs and the latter has soft hair (Phillips, 1980).The study revealed that the two subspecies could be clearly differentiated from each other using their microscopic hair anatomy.They differ from each other by characteristic cuticular scale patterns (Fig. 1), cross-sections (Table 2) and hair dimensions (Table 4).This is the first instance a difference in hair anatomy is reported at subspecies level.
Hair anatomy reported here can be used for species-identification in ecological surveys such as density, population or habitat surveys using hair tubes and for investigating food habits of carnivores using hair samples from scats (Amerasinghe and Ekanayake, 1990;Dickman, 1986;Toth, 2002;Finnegan et al., 2007;Bertolino et al., 2009).It can also be used to aid in identification of museum specimens.
towards the tip of the shield and the base of the shaft of both GH1 and GH2.Hairs of all species have broad or elongated petal scales between the irregular wave pattern of tip and base of hairs except M. blanfordi, M. musculus, T. tatera and GH1 of M. booduga.Both GH1 and GH2 of M. blanfordi and T. tatera have characteristic chevron wave pattern while GH1 of M. musculus and M. booduga have mosaic scales and GH2 of M. musculus has both mosaic and chevron scales.Chevron pattern is also seen in GH2 of M. meltada, G. e. nuwara and GH1 of S. ohiensis.Both GH1 and GH2 of R. rattus, R. norvegicus and R. montanus have diamond scales.

Figure 1 .
Figure 1.Distinguishing cuticular scale patterns of species of Sri Lankan muroid rodents.Each photograph was taken under medium power under a light microscope (10X10 power) and is 1.07 mm in length.

Figure
Figure 1.continued have random honeycomb appearance to the medullae (GH1 and GH2 of B. indica, B. bengalensis, GH1 of R. rattus, R. norvegicus, R. montanus, S. ohiensis, M. booduga and GH2 of G. e. ellioti).Medullary cells in the mid region of GH1 of B. indica are concentrated on the two sides with no cells in the middle, which is unique to the species.

Table 1 .
List of the species and subspecies used in the study with their collection sites and the voucher specimen reference numbers.Underlined numbers are of specimens obtained from National Museum of Sri Lanka; WHT -Wildlife Heritage Trust.
GH1 are useful to distinguish spiny rats and G. e. ellioti from other species.Spiny rats have triconcave cross-sections and G. e. ellioti has triangular and square shaped cross-sections while other species have circular to oval cross-sections.