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Italian Journal of Zoology

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Guide to the microscope analysis of Italian mammals hairs: Insectivora, Rodentia and Lagomorpha Anna M. De Marinis & Paolo Agnelli To cite this article: Anna M. De Marinis & Paolo Agnelli (1993) Guide to the microscope analysis of Italian mammals hairs: Insectivora, Rodentia and Lagomorpha, Italian Journal of Zoology, 60:2, 225-232, DOI: 10.1080/11250009309355815 To link to this article: https://doi.org/10.1080/11250009309355815

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Boll. Zool. 60: 225-232 (1993)

Guide to the microscope analysis of Italian mammals hairs: Insectivora, Rodentia and Lagomorpha ANNA M. DE MARINIS PAOLO AGNELLI Museo di Storia Naturale dell'Università di Firenze, Sezione di Zoologia «La Specola», via Romana 17, 1-50125 Firenze (Italy)

INTRODUCTION

The microscope analysis of hair can be a valuable aid both in ecological research on the relationship between predators and their prey, and in environment quality research on the relationship between small mammal fauna and habitat variability. Much research has been carried out in Europe on the microscopic analysis of hair (Day, 1966; Dziurdzik, 1973, 1978; Vogel & Köpchen, 1978; Keller, 1978, 1980, 1981, 1983; Faliu et al., 1980; Debrot et al, 1982; Teerink, 1991) but not on such mammal species as Erinaceus concolor, Suncus etruscus, Marmota marmota, Dryomys nitedula and Hystrix cristata. Some of these studies require special techniques and equipment such as a scanning electron microscope and microtome which, in turn, require skilled labour, thus slowing down the analyses. The present paper proposes an identification key for Italian insectivores, rodents and lagomorphs. It also illustrates a new method of cross-sectioning hair, thus adding a parameter which can be essential in correctly identifying taxon in routine investigations. As a whole the techniques used in this study can be easily, quickly and economically applied without compromising the results of the research.

MATERIALS AND METHODS

ABSTRACT A key is proposed for identifying the hairs of Italian Insectívora, Rodentia and Lagomorpha to the genera and species level using microscope analysis. The criteria used are: general morphology, scale cuticular pattern, cortex structure, medulla type, and shape in cross-section. A new method of cross-sectioning hair is illustrated. KEY WORDS: Hair identification - Insectívora - Rodentia Lagomorpha. ACKNOWLEDGEMENTS The authors wish to thank Dr. R. Brizzi and Dr. C. Calloni (Department of Animal Biology and Genetics) and Nautilus ltd. of Florence for their expert technical assistance. The authors are also grateful to Dr. M. Poggesi (Florence Natural History Museum, Zoological Section), Prof. B. Lanza and Prof. M. Vannini (Department of Animal Biology and Genetics) for their careful critical review of the manuscript. Thanks are also due to L. Lapini (Friuli Natural History Museum) who kindly supplied specimens of Erinaceus concolor and Dryomys nitedula, and to Sarah Whitman (Florence Natural History Museum, Zoological Section) who corrected the English text.

(Received IS June 1992 - Accepted 15 September 1992)

Hair samples were taken from dry skins or specimens in alcohol in the mammal collection of the Florence Natural History Museum, Zoological Section, except for those of Erinaceus concolor and Dryomys nitedula which came from the Friuli Natural History Museum. The species examined are listed in Appendix. The hair sample was drawn from the ventral region in Erinaceus europaeus, E. concolor and Hystrix cristata, from the latero-dorsal region in the remaining species. The spines of the two Italian species of Erinaceidae were also examined. Land mammals are covered with two distinct types of hair: long, pigmented guard hairs (overhairs) and short, less pigmented and more numerous fine hairs (underhairs). In both guard and fine hairs may be distinguished a proximal and a distal region. The proximal region is usually constricted in the fine hairs but not in the guard hairs, and includes the hair root. Only the distal region has the discriminant characteristics useful to identification. It generally contains a thickened portion called shield, more pronounced in the guard hairs, which was used to separate the taxonomic categories. Both guard and fine hairs, excluding Chiroptera (Tupinier, 1973), consist of three layers of keratin: cutícula (outermost layer), cortex and medulla (innermost layer). The features used in this identification key are the following: external morphology, cuticular scale pattern, structure of cortex, types of medulla, and shape in cross-section. External morphology The following characteristics were recorded using a hand lens: hair profile (straight, zig-zag, or curved) presence or absence of shield; presence or absence of constrictions; greater or lesser hair stiffness. Hair length was measured in millimeters using a calibrated ocular micrometer.

226 Cuticular scale pattern The cutícula pattern was determined from a cast of the hair surface. The cast is easily made as follows (Twigg, 1975): after rinsing the hairs in ether for a few minutes to remove the natural oils, lay the hairs in a thin film of transparent nail polish on a microscope slide; when the nail polish has dried, peel away the hairs and observe the scale cast under the microscope (from lOOx to 400x). The complicated nomenclature of the various types of cuticular scale patterns (Chehébar & Martín, 1989; Teerink, 1991) is not used here. Some of these patterns are not easily distinguishable from each other and the chance for confusion is great. In our opinion, except for a few species, such features have little diagnostic value. Cortex After rinsing the hairs in ether for a few minutes, the structure of cortex examined under the microscope was transparent. The cortex allows the identification of shrews guard hairs. Medulla The procedure for examining the medulla types, partially according to Keller (1978), is: after rinsing the hairs in ether for a few minutes, bleach the hairs by dipping them in a solution of hydrogen peroxide (70%) and ammonia (30%); after rinsing the hairs in water to remove such solution, lay the hairs on a slide, mount them in an appropriate reagent and observe the medulla types under the microscope (from lOOx to 400x). The hydrogen peroxide-ammonia solution bleached the hair in all species studied. The degree of bleaching is easily modified by changing the length of time in the solution. The types of medulla observed on the distal part are: - unicellular regular (es. Suncits etruscus, Fig. 9); - unicellular irregular (es. Eliomys quercinus, Fig. 26); - unicellular interrupted (es. Glis glis. Fig. 23); - unicellular vacuolated (es. Erinaceus europaeus, Fig. 2); - multicellular net-shaped (es. Clethrionomys glareolus, Fig. 14); - multicellular column-shaped (es. Lepus europaeus, Fig. 29); - multicellular globe-shaped (es. Hystri.x cristata, Fig. 28). Cross-section The hair cross-section can often be of greater diagnostic value than the cuticular scale pattern and medulla type. The method used here allowed cross-sectioning the shield without wasting time on cross-sections of the proximal part, thus optimizing the data collection and reducing the analysis time in comparison of traditional techniques (Mathiak, 1938; Day, 1966; Dziurdzik, 1973; Brunner & Coman, 1974; Keller, 1980). Hair cross-sectioning is done as follows: cut two strips of Polyurethane (about 60 x 30 x 10 mm) and spread a thin film of vinyl glue over one of the surfaces; lay the hairs on this film with their shields parallel to each other and place the other strip on top; when the glue has hardened, cut the polyurethane strips at the level of the shield with a scalpel blade. The cross-sections examined were classified in the following categories, only partially according to Debrot et al. (1982): 1. Convex profile - round (es. Arvícola terrestris, Fig. 16); - oval (es. Marmota marmota, Fig. 13). 2. Concave profile - one side (es. Microtus arvalis, Fig. 18); - two sides like a dumb-bell (es. Lepus europaeus, Fig. 30); - three sides (es. Apodemus sylvaticus, Fig. 20); - four sides (es. Clethrionomys glareolus, Fig. 15). 3. Angular profile - «H» shaped (es. Sorex araneus, Fig. 4); - square (es. Crocidura leucodon, Fig. 5); - «key hole» shaped (es. Crocidura leucodon, Fig. 6).

A. M. DE MARINIS, P. AGNELLI

Appendix contains 21 cards summarizing the main features identifying insectivores, rodents and lagomorphs.

RESULTS AND DISCUSSION

Hair profile of Insectívora is distinctly zig-zag with constriction on each curve. The cuticular scales protude on one side of the hair proximally to the constriction and on the opposite side distally to that constriction (Fig. 3), except at the constriction immediately preceding the shield, according to a scheme called crossing-over by Day (1966). Insectivore fur can be recognized examining both guard hairs and fine hairs. The latter far outnumber guard hairs which are therefore difficult to detect. Rodentia and Lagomorpha guard hairs do not present any constrictions and are straight except Glis glis and Muscardinus avellanarais showing slightly curved guard hairs. Rodentia fine hairs (with constrictions but with no crossing-over, except in Sciuridae, Hystricidae and Glis glis which have hairs without constrictions) and Lagomorpha fine hairs (with no constrictions) signal only the presence/absence of these orders. Such hair samples cannot be processed through the key and do not allow identification as to family, genus or species. On the contrary, Rodentia and Lagomorpha guard hairs are the only representatives of their respective families, genera and species and only such hairs are identified in this keys. Knowledge of the geographic range of the species in question tends to facilitate identification. Insectívora The shrew and mole hairs included in this key have two or more constrictions with crossing-over. The shrew guard hairs show a distinct spatulate shape with the distal segment almost twice as long as the adjacent; the fine hairs have a distal segment as long as the adjacent or smaller. Crocidura (Card 3), Sorex and Neomys (Card 2) can be recognized only examining the cortex structure of guard hairs (Vogel & Köpchen, 1978). Suncus etruscus was identified to species level (Card 4) due to its small size (length < 2 mm). The mole guard and fine hairs have a distal segment as long as the adjacent or longer. The medulla type of guard and fine hairs allows identifying the genus Talpa (Card 5). The guard hairs of Erinaceidae, which in Italy is represented by Erinaceus europaeus and Erinaceus concolor (the latter occurring in Friuli Venezia Giulia only), have no constrictions, are stiff and not spatulated. Such features are completely atypical for insectivores. A characteristic medulla type (Fig. 2) with a row of big roundish cells enclosed by a thick cortex wall, facilitated identification to the genus Erinaceus. We attempted to identify the two species by examining the spines but these show the same cuticular scale pattern, medulla type and cross section (Card 1).

227

HAIRS OF INSECTÍVORA, RODENTIA AND LAGOMORPHA

2;

.Ǥ

5

8

Figs 1-10 - 1 - Erinaceus europaeus. Spine cross-section. x83. 2 - Erinaceus europaeus. Hair medulla. x4l6. 3 - Neomysfodiens. Crossing-over of cuticular scales (arrows). x4l6. 4 - Sorex araneus. Cross-section. x4l6. 5-6 - Crocidura leucodon. Cross-section. x4l6. 7 - Sorex araneus. Cortex. xl664. 8 - Crocidura leucodon. Cortex. xl664. 9 - Suncus etruscus. Medulla. x4l6. 10 - Talpa europaea. Medulla. x4l6.

228

A. M. DE MARINIS, P. AGNELLI

13

14

15

17

18

22

20

Figs 11-22 - 11 - Sciurus vulgaris. Cuticular pattern. x4l6. 12 - Marmota marmota. Cuticular pattern. x4l6. 13 - Marmota marmota. Crosssection. x4l6. 14 - Clethrionomys glareolus. Medulla. x4l6. 15 - Clethrionomys glareolus. Cross-section. x4l6. 16 - Arvícola terrestris. Crosssection. x4l6. 17 - Pitymys savii. Cuticular pattern. x4l6. 18 - Microtus arvalis. Cross-section. x4l6. 19 - Mycromys minutus. Cuticular pattern. x4l6. 20 - Apodemus sylvaticus. Cross-section. x4l6. 21 - Rattus rattus. Cuticular pattern. x4l6. 22 - Mus musculus. Cuticular pattern. x4l6.

229

HAIRS OF INSECTÍVORA, RODENTIA AND LAGOMORPHA

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25

26

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r.i /• / / j

~

\ ,

27

29

30

Figs 23-30 - 23 - Glis glis. Medulla. x4l6. 24 - Muscardinus avellanarius. Cuticular pattern. x4l6. 25 - Muscardinus avellanarius. Medulla. x4l6. 26 - Eliomys quercinus. Medulla. x4l6. 27 - Dryomys nitedula. Cuticular pattern. x4l6. 28 - Hystrix cristata. Medulla. x4l6. 29 - Lepus europaeus. Medulla. x4l6. 30 - Lepus europaeus. Cross-section. x4l6.

230

Rodentia Rodents can be divided into two groups: those with unicellular medulla and those with multicellular medulla. Among rodents only Gliridae have unicellular medulla. Glis glis and Eliomys quercinus are easily recognized by their different medulla (Figs 23-26). Muscardinus avellanarius and Dryomys nitedula have identical medulla types (Fig. 25) and their identification is possible by examining the arrangement of the cuticular scales. The cutícula has a chevron pattern in Muscardinus avellanarius (Fig. 24) and an irregular wave pattern in Dryomys nitedula (Fig. 27). Sciuridae and Arvicolidae show a multicellular netshaped medulla. The longest hairs (> 15 mm) are found in Sciuridae, Arvícola terrestris and Rattus sp. The cuticular scale pattern on the proximal part of the shield provides data for the classification of Sciuridae (Figs 11-12). Rats and ground vole are easily recognized measuring hair width. The shortest hairs (< 12 mm) are found in the remaining rodents. Murinae (Apodemus sp.) and Arvicolinae {Microtus sp. and Pitymys sp.) can be recognized to the genus level by examining the crosssection silhouette (Figs 18-20) and cuticular scale pattern (Fig. 17). The identification may proceed to species for the genera represented in Italy by a single species: Clethrionomys glareolus (Card 8) of Arvicolinae and Micromys minutus (Card 12) and Mus musculus (Card 15) of Murinae. The family Hystricidae is represented in Italy only by Hystrix cristata. The unique medullary configuration (multicellular globe-shaped) together with external morphology allow to classify such species (Fig. 28). Spines are not included in the key. Lagotnorpha The medulla type of lagomorph guard hairs is unique. The medullary cells are in longitudinal columns with an extremely characteristic pattern (Fig. 29). Lagomorphs are therefore the easiest order to recognize but the guard hairs of the genus Lepus and Oryctolagus cuniculus cannot be reliably distinguished, due to their close similarity (Day, 1966). Reynolds & Aebischer (1991) and Teerink (1991) proposed cross-section and cuticular scale pattern as discriminant character. These features are not easily distinguishable. Identification below the family level is therefore tentative.

KEY TO THE HAIRS OF THE ORDERS: INSECTÍVORA, RODENTIA AND LAGOMORPHA

la. Hairs with constrictio'ns 2 lb. Hairs without constrictions 3 2a. Hairs with crossing-over of the cuticular scales at each constrictions (Fig. 3) except the one immediately preceding the shield 4

A. M. DE MARINIS, P. AGNELLI

2b. Hairs without crossing-over of the cuticular scales at each constrictions Fine hairs of Rodentia (excluding Sciuridae, Hystricidae and Glis glis). Look for guard hairs (see text). 3a. Hairs bent Fine hairs of Lagomorpha, Hystricidae, Sciuridae and Glis glis. Look for guard hairs (see text). 3b. Hairs not bent 5 4a. Unicellular regular medulla on the shield (Fig. 9). 6 4b. Unicellular irregular medulla on the shield Talpa sp. (Fig. 10) 5a. Unicellular medulla on the shield (Fig. 25) 7 5b. Multicellular medulla on the shield (Fig. 14) 8 6a. Length > 4 mm 9 6b. Length < 2 mm Suncus etruscus 7a. Unicellular not vacuolated medulla on the shield (Fig. 25) 10 7b. Unicellular vacuolated medulla on the shield .. Erinaceus europaeus as well E. concolor (Fig. 2) 8a. Multicellular net-shaped medulla on the shield (Fig. 14) 11 8b. Multicellular column-shaped medulla on the shield Leporidae (Fig. 29) 8c. Multicellular globe-shaped medulla on the shield .. Hystrix cristata (Fig. 28) 9a. Cortex of the shield with longitudinal lines sloping inwards Sorex sp. as well Neomys sp. (Fig. 7) 9b. Cortex of the shield without longitudinal lines (Fig. 8) 12 10a. Unicellular regular medulla on the shield (Fig. 25) 13 10b. Unicellular irregular medulla on the shield Eliomys quercinus (Fig. 26) lia. Length s= 15 mm 14 lib. Length 25 mm - Irregular wave cuticular pattern on the proximal part of the hair (Fig. 12) - Multicellular net-shaped medulla on the shield - Oval or one concave side in shield cross-section (Fig. 13)

Card 8 - Clethrionomys glareolus - Length 8 - 1 0 mm - Multicellular net-shaped medulla on the shield (Fig. 12) - Four concave sides in shield cross-section (Fig. 13)

Card 9 - Arvícola terrestris -Length 15 - 16mm - Shield width 60 - 70 urn - Diamond petal pattern of cuticular scales on the proximal part of the hair - Multicellular net-shaped medulla on the shield - Round or oval cross-section of the shield (Fig. 16)

Note: the nomenclature is according to Corbet & Hill (1991).

- Length 8 - 1 2 mm - Chevron cuticular pattern under the shield (Fig. 24) - Unicellular regular medulla on the shield (Fig. 25) - Round cross-section of the shield

Card 18 - Eliomys quercinus -Length 10- 15 mm - Unicellular irregular medulla on the shield (Fig. 26) - Oval cross-section of the shield

Card 19 - Dryomys nitedula -

Length 10 - 15 mm Irregular wave cuticular pattern under the shield (Fig. 27) Unicellular regular medulla on the shield Round or oval cross-section of the shield

Card 20 - Hystrix cristata - Stiffhairs without shield - Length > 50 mm - Multicellular globe-shaped medulla (Fig. 28)

Card 21 - Oryctolagus cuniculus, Lepus sp. - Length 25 - 35 mm - Multicellular column-shaped medulla on the shield (Fig. 29) - One or two concave sides (like a dumb bell) in shield cross section (Fig. 30)

232

shield Muscardinus avellanarías (Fig. 24) 16b. Irregular wave pattern of cuticular scales under the shield Dryomys nitedula (Fig. 27) 17a. 60-70 urn shield width Arvícola terrestris 17b. 80-100 um shield width Rattus sp. 18a. Cuticular scale margins «M» shaped on the concave side of the shield (Fig. 17) 19 18b. Cuticular scale margins «U» shaped on the concave side of the shield Mus musculus (Fig. 22) 18c. Irregular wave pattern of cuticular scales under the shield Micromys minutus (Fig. 19) 19a. Length 6-7 mm Pitymys sp. 19b. Length 10-12 mm Microtus sp. REFERENCES Brunner H., Coman B. J., 1974 - The identification of mammalian hair. Inkata Press, Melbourne, 176 pp. Chehébar C., Martin S., 1989 - Guía para el reconocimiento microscópico de los pelos de los mamiferos de la Patagonia. Doñana, Acta Vert., 16: 247-291. Corbet G. B., Hill J. E., 1991 - A world list of mammalian species. Oxford University Press, Oxford, 243 pp. Day M. G., 1966 - Identification of hair and feather remains in the gut and faeces of stoats and weasels. J. Zool., Lond., 148: 201-217. Dziurdzik B., 1973 - (Key to the identification of hairs of Mammals from Poland). Acta zool. Cracov., 18: 73-92. Dziurdzik B., 1978 - Histological structure of hair in the Gliridae (Rodentia). Acta zool. Cracov., 23: 1-10.

A. M. DE MARINIS, P. AGNELLI

Debrot S., Fivaz G., Mermod C., Weber J. M.; 1982 - Atlas des poils de mammifères d'Europe. Institut de Zoologie de l'Université de Neuchâtel, 208 pp. Faliu L., Lignereux Y., Barrat J., 1980 - Identification des poils des mammifères pyreneens. Doñana, Acta Vert., 1: 125-212. Keller A., 1978 - Détermination des mammifères de la Suisse par leur pelage. I. Talpidae et Soricidae. Rev. suisse Zool., 85: 758-761. Keller A., 1980 - Détermination des mammifères de la Suisse par leur pelage. III. Lagomorpha, Rodentia (partim). Rev. suisse Zool., 87: 781-796. Keller A., 1981 - Détermination des mammifères de la Suisse par leur pelage. IV. Cricetidae et Muridae. Rev. suisse Zool., 88: 463-473. Keller A., 1983 - Note sur la structure fine des piquants et des poils de jarres chez «Erinaceus europaeus L.» et «Erinaceus algirus Lereboullet» (Insectivora: Erinaceidae). Rev. suisse Zool., 90: 501-508. Mathiak H. A., 1938 - A rapid method of cross-sectioning mammalian hairs. J. Wildl. Manage. 2: 162-164. Reynolds J. C., Aebischer N.J., 1991 - Comparison and quantification of carnivore diet by faecal analysis: a critique, with recommendations, based on a study of the Fox Vulpes vulpes. Mammal Rev., 21: 97-122. Teerink B. J., 1991 - Hair of west European Mammals. Cambridge University Press, Cambridge, 224 pp. Tupinier Y., 1973 - Morphologie des poils de Chiroptères d'Europe occidentale par ètude au microscope èlectronique à balayage. Rev. suisse Zool., 80: 635-653. Twigg G. I., 1975 - Finding Mammals. Their sign and remains. Mammal Rev., 5: 77-78. Vogel P., Köpchen B., 1978 - Besondere Haarstrukturen der Soricidae (Mammalia, Insectivora) und ihre taxonomische Deutung. Zoomorphologie, 89: 47-56.