© 2010 The Japan Mendel Society Cytologia 75(4): 467–475, 2010
Chromosomal Features of the Endemic Pill Millipedes (Arthrosphaera: Sphaerotheriidae, Diplopoda) from Western Ghats, India
Bombrana Seetharama Kadamannaya1, Kanale Sreenivasa Sreepada2* and Kandikere Ramaiah Sridhar3
1 Department of Zoology, FMKM Kariappa Mangalore University College, Madikeri 571 201, Karnataka, India 2 Department of Applied Zoology, Mangalore University, Mangalagangothri, Mangalore 574 199, Karnataka, India 3 Department of Biosciences, Mangalore University, Mangalagangothri, Mangalore 574 199, Karnataka, India
Received August 12, 2010; accepted December 4, 2010
Summary Chromosome features and karyotypes of male meiosis of 2 species of pill millipedes belonging to the genus Arthrosphaera (Arthrosphaera dalyi and Arthrosphaera sp.) endemic to the Western Ghats revealed interesting facts. The Arthrosphaera dalyi has diploid chromosome number (2n) 30 with relative lengths ranging from 39–195. It showed 3–4 distinct subtelocentric chromo- some pairs with discernible short arms and the rest are acrocentrics and/or telocentrics. In Arthrosphaera sp. the diploid chromosome number (2n) is 26 and all chromosomes are telocentrics with relative lengths ranging from 36–178. The first pair of chromosome is the largest of all the chromosomes in both the species and is homomorphic in Arthrosphaera sp. and hetromorphic in A. dalyi, wherein one of the chromosomes is the largest and subtelocentric with a clear short arm and the other homologue is a telocentric chromosome. The sex determination mechanism is XX/XY type. A synopsis of the diplopod chromosomes is presented, compared and the possible evolutionary significance have been discussed.
Key words Arthrosphaera, Pill millipedes, Diplopoda, Sphaerotheriida, Chromosomes, Western Ghats.
Millipedes as macroinvertebrates have considerable ecological importance in litter breakdown and nutrient cycle (Wolters and Ekschmitt 1997, Ashwini and Sridhar 2005, Kadamannaya and Sridhar 2009). In spite of their significant contribution in decomposition, biological research on the class Diplopoda lacks taxonomic attention. Linnaeus listed 7 specimen in the genus Julus in 1758. Since then, approximately 12,000 species of millipedes have been described. Hoffman et al. (2002) estimated that there are about 80,000 species exists in Diplopoda. Nearly 68% of the genera are monotypic or consisting of only 2 species. Approximately 715 generic names are currently considered synonyms. This clearly shows that a vast numbers of millipede species are known and there is a lack of taxonomic revision. Most millipede collections are known from the North America and in Europe, which are relatively well documented taxonomically. Australia, South Africa and Japan also have fairly good collections of millipedes. General geographical areas, especially Africa, Central and South America and Asia, are neglected with regard to millipede study (Sierwald and Bond 2007). Millipedes are often microendemic with very small distributional ranges. There are reports indicating the occurrence of different species of millipede within a range of 20 km (Enghoff 1983,
* Corresponding author, e-mail: [email protected] 468 B. S. Kadamannaya et al. Cytologia 75(4)
Hamer and Slotow 2002). The taxonomy and morphology of Diplopoda in general and the order Sphaerotheriida (pill millipedes) in particular, have drawn less attention (Sierewald and Bond 2007). A monograph of the pill millipedes inhabiting in India, Sri Lanka and Burma has been published by Pocock (1899). According to Pocock (1882), pill millipede presence in peninsular India consists of about 27 species of Arthrosphaera. Furthermore, key to the Indian genera of Sphaerotheriida has been revised by Attems (1936). Beginning from this classical work, systematics of Sphaerotheriida have focused mainly on the morphological details, which is the only parameter used in identification. As there are ambiguities in the taxonomy of members of Sphaerotheriida using morphological characteristics in describing the species, it would be of great value if it is considered in the light of chromosome biology and cytotaxonomy. Cytogenetics of Diplopoda is poorly studied and understood due to the technical difficulties in obtaining mitotic chromosomes. Fontanetti et al. (2002) have given a comprehensive review of cytogenetic studies in Diplopoda. Studies are confined to meiosis and spermatogonial metaphase, which limits the use of modern cytogenetical techniques. Only 0.1% of the known Diplopoda species have been cytologically investigated (Fontanetti et al. 2002). A catalogue of the chromosome number and sex determination systems of 16 Brazilian species has been given by Fontanetti (1990–2000). Vitturi et al. (1997) observed heterochromatin content in somatic chromosomes of 2 unrelated species of Diplopoda belonging to the families Schizopetalidae and Julidae with diploid chromosome number 12 and 22 respectively. Karyotypic studies of 4 xystodesmid millipedes from Japan have been performed by Tanabe (1992) and showed the chromosome number ranges between 12 and 16. In the family Polydesmidae, 2 species with 2n 8 and 12 were reported by Bessier (1948) and Achar (1984b). Chromosome number, karyotype studies and behavior of chromosomes during meiotic prophase in several Brazilian species of diplopods were studied by Fontanetti (1990– 2000). The members of the family Rhinocricidae have a diploid chromosome number ranging between 20 and 28. The members of Pseudonannolene belonging to the family Pseudonannolenidae showed chromosome numbers ranging from 12–20, while in the members of the family Spirostreptidae that number ranged between 10 and 26 (Achar and Chowdaiah 1979, Fontanetti 1998). A new species of Urostreptus has been described by Peirozzi and Fontanetti (2006) with a diploid number 24. Thus, the diploid chromosomal number in Diplopoda generally varies from 8 to 30 (Fontanetti et al. 2002). White (1979) discussed the present status of Myriapod cytogenetics and indicated the necessity of cytogenetical polymorphism in this group. Fontanetti (1996c) described the karyotype of a Brazillian Diplopod, Sandalodesmus gasparae and found that the diploid number 12. It is believed that the sex chromosomes of diplopods are more conserved than the sex chromosomes of Chilopoda and the general karyotypic evolution in this group may be of conserved nature (White 1979). Achar and Chowdaiah are the leading researchers in the field of cytogenetics of the Indian Diplopoda and have investigated 6 diplopod families (Chowdaiah 1966–1969, Chowdaiah and Kanaka 1969–1979, Achar and Chowdaiah 1979, 1980, Achar 1983–1987, Natarajan 1959) and 2 species of Diplopoda of the families Pachybolidae and Harpagophoridae showed a diploid chromosome number of 12 and 24 respectively. The diploid numbers ranged between 12 and 30 among members of these families with XX/XY system of sex determination. The cytogenetics of the infraclass Pentazonia is limited to a few studies on Glomerida and Sphaerotheriida. Studies on chromosome biology of Glomerida is confined to only 3 species out of about 669 species, while only 15 species of the Sphaerotheriida have been studied from India (Bessiere 1948, Achar 1987, Warchalowska-Silwa et al. 2004). A consolidated list of chromosome numbers in diplopods species studied so far is given in Table 1. Use of differential cytogentical techniques on diplopod chromosome is rare due to the technical difficulties in obtaining mitotic chromosomes. Achar (1980, 1983a) and Achar and 2010 Chromosomes of Western Ghats Pill Millipedes 469
Table 1. Checklist of chromosome numbers of pill millipedes
Taxon 2n Reference
Order Glomerida (Family, Glomeridae) Glomeris annulata 20 Bessiere 1948 G. connexa 16 Warchalowska-Sliva et al. 2004 G. hexasticha 16 Warchalowska-Sliva et al. 2004 Order Sphaerotheriida (Family, Sphaerotheriidae) Arthrosphaera bicolor 30 Chowdaiah and Kanaka 1974 A. craspedota 30 Chowdaiah and Kanaka 1974 A. dalyi 30 Chowdaiah and Kanaka 1974, Achar 1986 A. davisoni 26 Achar 1986 A. distincta 28 Chowdaiah and Kanaka 1974, Achar 1986 A. gracilis 28 Chowdaiah and Kanaka 1974 A. hendersoni 30 Chowdaiah and Kanaka 1974 A. lutescens 26 Achar 1986 A. magna 30 Chowdaiah and Kanaka 1974, Achar 1986 A. nitida 30 Achar 1983b, 1986 A. zebraica 26 Chowdaiah 1966c A. sp. 1 30 Chowdaiah 1966c A. sp. 2 30 Achar 1987 A. sp. (C) 30 Achar 1987 A. sp. (M) 30 Achar 1986
Chowdaiah (1979) have employed air-drying followed by Giemsa differential banding techniques for the first time in Indian diplopods. Based on their studies, Achar (1987) stated that Robertsonian rearrangements and the pericentric inversions have occurred during the evolution of diplopod karyotype. Not much has been added to the cytogenetics of Sphaerotheriida from Indian subcontinent except a few attempts mentioned earlier. The specimens for these studies were obtained from various locations of Southern India: Wynad (Kerala), Alagarkovil hills (Tamil Nadu), Karkala, Thirthahalli and Madikeri (Karnataka) and Khandla hills (Maharashtra). However, no serious attempts have been made to study the chromosome architecture in pill millipedes from different habitats and altitudes of the Western Ghats of Karnataka. Therefore, the present paper describes chromosomal features of 2 endemic pill millipedes in the family Sphaerotheriidae collected from the forest and plantations of Western Ghats, Karnataka, India.
Materials and methods Due to the technical and logistic difficulties only 2 adult males each from Arthrosphaera dalyi and Arthrosphaera sp. were collected during the monsoon and post-monsoon seasons from Ulvi (647 m asl), Varadalli (712m asl) and Kadaba (124 m asl) locations of Western Ghats of Karnataka, India have been subjected to chromosomal analysis. The testes from 4 males were dissected out and fixed in Carnoy’s fluid for 1h, treated with 5N HCl for 20 min and placed directly into the Feulgen solution for 60 min. After thorough mincing, the material was squashed in 45% acetic acid. Different meiotic stages were traced. Due to the difficulty in consistent spreading, air-drying technique was also followed for further analysis. Two to 3 adult males of Arthrosphaera dalyi and Arthrosphaera sp. were used for chromosome preparations following the air-drying technique of Rothfels and Siminovitch (1958). Karyotypes were constructed using 2 well spread spermatogonial metaphase plates and karyometric analysis were carried out based on the method outlined by Levan et al. (1964). Relative length (RL) and centromeric index (CI) were calculated from the karyotypes. Since the sex chromosomes could not be identified the average of all chromosomes, including the largest first pair, is considered to 470 B. S. Kadamannaya et al. Cytologia 75(4)
Fig. 1. Photomicrographs of chromosomes as seen in male meiosis of Arthrosphaera dalyi (2n 30): Spermatogonial metaphase (A), Spermatogonial anaphase (B), Pachytene configurations (C), Diplotene stage (D), Dia-Metaphase-I (E, F), Anaphase-I (two sets) (G), Metaphase-II (polar view) (H), Early anaphase-II (I). calculate the RL: Relative Length (RL) (Length of the chromosome Total length of haploid set) 1000 Centromeric Index (CI) (Length of the short arm Length of the chromosome) 100
Results and discussion Arthrospaera dalyi (2n 30) The spermatogonial metaphase karyotype analysis has revealed that the diploid chromosome number in A. dalyi is 30 (Fig. 1). The pachytene stage is recognized by the pairing of homologues with numerous chromomeres all along their length. Characteristic fuzziness of the chromosomes can be seen in the diplotene stage. At diakinesis, the chromosomes become more condensed and the chiasmata can be seen only at their terminal ends. In metaphase I, the 15 bivalent natures of chromosomes are very clear. However, the largest chromosome pair exhibited pseudo-bivalent nature. At metaphase II, prior to the congregation at the equatorial plate, the chromosomes show characteristic lightly stained gaps flanked on either side by the darkly stained heterochromatic regions.
Arthrosphaera sp. (2n 26) The spermatogonial metaphase of Arthrosphaera sp. showed the diploid number of 26 chromosomes (Fig. 2). The large telocentric pair is relatively homomorphic. In zygotene stage, the chromosomes appear as slender threads with several dark stained regions. The dark stained regions 2010 Chromosomes of Western Ghats Pill Millipedes 471
Fig. 2. Photomicrographs of chromosomes as seen in male meiosis of Arthrosphaera sp. (2n 26): Spermatogonial metaphase (A), Spermatogonial anaphase (B), Pachytene configurations (C), Diplotene stage (D), Diakinesis (E), Metaphase-I (F), Sister groups of anaphase-I (G), Metaphase-II (H). can also be seen in pachytene stage. The homologous chromosomes become more thick and condensed. During diakinesis, highly condensed 12 bivalents and 1 pair of pseudo-bivalents, in metaphase I, 13 highly condensed chromosome pairs are seen. Terminalization of chiasmata in the large chromosome pair is not completed even in condensed state as seen in metaphase I.
Karyotype analysis The karyotype analysis from the spermetogonial metaphase plates of 2 species of Arthrosphaera showed diploid chromosome number in 30 and 26. The sex determination mechanism in both the species is XX/XY type. The karyotype analysis of 2 plates of A. dalyi (2n 30) have shown that there are 1 pair of submetacentrics (5th), 3 pairs of subtelocentrics (2nd, 4th and 6th) and 10 pairs (3rd, 7th–15th) of telocentric chromosomes (Fig. 3A). Arthrosphaera sp. (2n 26) has revealed all telocentric chromosomes in decreasing order of size except the first 2 pairs, which are very large (Fig. 3B). The first pair of chromosome is the largest of all the chromosomes in both the species and is homomorphic in Arthrosphaera sp. and hetromorphic in A. dalyi, wherein one of the chromosomes is the largest and subtelocentric with a clear short arm and the other homologue is a telocentric/or acrocnetric chromosome. The sex chromosomes in these species could not be identified. Hence, for the relative length analysis all the chromosomes are 472 B. S. Kadamannaya et al. Cytologia 75(4)
Fig. 3. Karyotypes of Arthrosphaera dalyi (2n 30) with heteromorphic chromosome pair (arrow) (A) and Arthrosphaera sp. (2n 26) (B).
Table 2. Relative length and centromeric index of the Table 3. Relative length of the chromosomes of chromosomes of Arthrosphaera dalyi (2n 30) Arthrosphaera sp.
Chromosome Relative length Centromeric Nature of Chromosome Relative length Nature of pair (RL) index (CI) chromosome pair (RL) chromosome
1 a) 194.74 6.03 15.50 subtelocentric 1 177.88 4.60 telocentric b) 161.03 3.25 – telocentric 2 126.08 6.67 telocentric 2 99.90 5.02 15.15 subtelocentric 3 90.70 3.39 telocentric 3 78.25 6.41 – telocentric 4 84.12 1.41 telocentric 4 76.30 4.68 20.00 subtelocentric 5 83.33 0.29 telocentric 5 67.39 2.40 27.77 submetacentric 6 76.55 8.12 telocentric 6 65.09 4.31 18.23 subtelocentric 7 72.41 3.40 telocentric 7 61.72 0.58 – telocentric 8 60.10 3.88 telocentric 8 56.74 4.90 – telocentric 9 53.65 0.62 telocentric 9 56.26 4.21 – telocentric 10 44.39 5.40 telocentric 10 54.55 2.82 – telocentric 11 43.18 5.93 telocentric 11 50.90 1.80 – telocentric 12 39.13 6.96 telocentric 12 48.40 0.70 – telocentric 13 35.80 2.26 telocentric 13 44.90 1.52 – telocentric 14 43.26 2.80 – telocentric 15 39.40 7.24 – telocentric
taken. The relative lengths of the chromosomes in A. dalyi range from 39.4 to 194.74 (Table 2) and in Arthrosphaera sp. range is between 35.8 and 177.88 (Table 3). As reported by previous researchers, the largest chromosomes can be considered as sex chromosomes. Chowdaiah (1966c) opined that the large size of the sex chromosome is an unique example in Diplopoda. It was proposed that the sex determination mechanism in Diplopoda is in a primitive state, since in most of the species the sex chromosomes are poorly differentiated from the autosomes (Chowdaiah and Kanaka 1974, Achar 1983a). Fontanetti (1991–2000) also opines that in most of the Brazilian diplopods it is not possible to identify the larger pair of chromosomes as sex chromosomes. The heteromorphism between X and Y chromosomes is also reported by Pierozzi and Fontanetti (2006) in a new species of Urostrepters. Using C-banding technique, the X chromosome was marked by a large C block, whereas the Y chromosome had much smaller block. The C-banding of chromosomes of Pseudonannolene strinatti (a cave millipede) was 2010 Chromosomes of Western Ghats Pill Millipedes 473 performed by Campos and Fontanetti (2004). Out of 8 pairs of chromosomes, 1, 2, 3 and 5 were almost entirely heterochromatic and in 4 and X chromosomes, the short arms were completely heterochromatic. The Y chromosome showed a large heterochromatic band. This clearly reveals that a large amount of genome is in the heterochromatic form and there exists a considerable heteromorphism between X and Y chromosomes. Due to a scarcity of suitable tissue to study chromosomes, the female Diplopoda were not subjected to cytological studies in earlier and in the current investigations. Hence, the identification of sex chromosomes has to be done with some reserve. The meiotic behaviors of the chromosomes in both species of Arthrosphaera studied are similar. At zygotene, well condensed and less condensed states of the chromosomes are visible. At pachytene, the chromosomes are in condensed state and a typical arrangement can be seen. Even the diplotene chromosomes showed highly condensed as well as less condensed regions. In metaphase I, chromosomes are highly condensed and look like a cluster (clumping). This type of clumping was also observed in other species of Arthrosphaera by Achar (1986), Chowdaiah and Kanaka (1974) and in termitophilic species of Diplopoda by Fontanetti (1996c). In addition to the chromatin clumping mentioned above, the phenomenon of heteropycnosis was seen in the present study. The onset of meiotic prophase is marked by the appearance of thin and granular threads traversing the nucleus in all directions. A pycnotic condensation of the chromatin at the end of each thread was evident. The diploid chromosome number 26 has also been reported in A. davisoni, A. lutescens and A. zebraica (Achar 1986, Chowdaiah 1966c). As reported by Achar (1983b), the normal male karyotype of A. davisoni comprises 26 chromosomes including the heteromorphic sex chromosome pairs. Out of 26 chromosomes, 2 pairs are sub-metacentric and the rest are acrocentric. The largest pair is also acrocentric and in this pair one of the chromosomes is slightly larger than the other. However, the largest pair of chromosomes in Arthrosphaera sp. in the present study is homomorphic. Arthrosphaera dalyi showed a diploid chromosome number of 30. A similar chromosome number was reported earlier in 9 species from India (Achar 1986, 1987, Chowdaiah and Kanaka 1974). In the present study, A. dalyi showed the presence of heteromorphic largest pair of chromosomes. This was also reported for A. nitida (2n 30) by Achar (1983b). However, the nature of the chromosomes seems to be different. In A. dalyi, one of the chromosomes is subtelocentric and the other is acrocentric or telocentric, compared to the acrocentric nature of both the chromosomes in A. nitida. It is evident from the earlier cytogenetic studies and the present analysis that the chromosomal alterations involved in the speciation of Indian Sphaerotheriida is poorly understood. White (1979) stated that diplopods are conservative in karyotype evolution. Achar (1987) reports that the Robertsonian changes and pericentric inversions are the main chromosomal rearrangements those have occurred during the evolution of diplopods. Definite conclusions regarding karyotype evolution cannot be drawn owing to the lack of extensive cytogenetic investigations in Diplopoda. Further studies should focus on thorough revision of chromosome behaviour using differential banding techniques of widely collected specimen to clarify the taxonomic uncertainties of Sphaerotheriida.
Acknowledgements We are grateful to Mangalore University for granting us permission to carry out this study at the Department of Biosciences, Mangalagangotri. One of us (BSK) is indebted to the University Grants Commission, New Delhi and Mangalore University, Mangalore for granting a fellowship under Faculty Improvement Porgramme. BSK is also thankful to Dr. Puspha Kuttanna, Principal, FMKMK Mangalore University College, Madikeri for genuine support. 474 B. S. Kadamannaya et al. Cytologia 75(4)
References
Achar, K. P. 1980. The use of C-banding technique in the chromosome studies of a millipede species–Carlogonus acifer. Caryologia 33: 185–191. —, 1983a. The use of G-banding technique in the chromosome studies of a millipede species Spirostreptus asthenes. Curr. Sci. 52: 540–543. —, 1983b. Karyological studies in nine species of Indian Diplopoda (Myriapoda). Nucleus 26: 191–197. —, 1984a. Analysis of male meiosis in five species of Indian Diplopoda (Myriapoda). Caryologia 37: 373–386. —, 1984b. Chromosome studies in Indian Diplopoda (Myriapoda). I. A note on the occurrence of polyploidy in Polydesmus gracilis. Curr. Sci. 53: 764–767. —, 1985. Chromosome studies in Indian Diplopoda (Myriapoda) II. A note on the occurrence of translocation in Alacobolus excellens. Curr. Sci. 54: 1057–1060. —, 1986. Analysis of male meiosis in seven species of Indian Diplopoda (Myriapoda). Caryologia 39: 89–101. —, 1987. Chromosomal evolution in Diplopoda (Myriapoda: Arthropoda). Caryologia 40: 145–155. — and Chowdaiah, B. N. 1979. The use of air drying in the study of diplopod chromosomes. In: Camatini, M. (ed.). Myriapod Biology. Academic Press, London. pp. 21–23. — and — 1980. The use of C-banding technique in the chromosome studies of millipede species Carlogonus acifer. Caryologia 33: 185–191. Ashwini, K. M. and Sridhar, K. R. 2005. Leaf litter preference and conversion by a saprophagous tropical pill millipede, Arthrosphaera magna Attems. Pedobiologia (Jen a) 49: 307–316. Attems, C. 1936. Diplopods of India. Memoirs of the Indian Museum 11: 133–167. Bessier, C. 1948. La spermatogenese de quelques Myriapods diplopods. Arch. Zool. Exp. 85: 149–236. Campos, K. A. and Fontanetti, C. S. 2004. Chromosomal characterization of Pseudonannolene strinatii (Spirostreptida, Pseudonannolenidae). Iheringia, Ser. Zool. 94: 53–56. Chowdaiah, B. N. 1966a. Cytological investigation on some Indian Diplopoda (Myriapoda). Nature 210: 847. —, 1966b. Cytological studies of some Indian Diplopoda (Myriapoda). Cytologia 31: 294–301. —, 1966c. Chromosome studies in two species of pill millipedes. Caryologia 19: 135–141. —, 1967. A note on the abnormal sex chromosome behavior in the male of Thyropygous species. Curr. Sci. 36: 105–106. —, 1969. A note on occurrence of chromatid bridges in the male of Arthrosphaera species, a pill-millipede. Curr. Sci. 38: 1969–1970. — and Kanaka, R. 1969. Cytological studies of Indian Diplopoda II. (Myriapoda). Bull. Mus. Nat. D’His. Natur. 41 (Suppl.): 43–47. — and — 1974. Cytological studies in six species of pill-millipedes (Diplopoda, Myriapoda). Caryologia 27: 55–64. — and — 1979. Chromosome cytology of seven species of Indian Diplopoda (Myriapoda). In: Camatini, M. (ed.). Myriapod Biology. Academic Press, London. pp. 9–20. Enghoff, H. 1983. Adaptive radiation of the millipede genus Cylindroiulus on Madeira: Habitat, body size and morphology (Diplopoda, Julida; Julidae). Rev. Ecol. Biol. Sol. 20: 403–415. Fontanetti, C. S. 1990. Meiotic prophase in Diplopoda. Rev. Brasil. Genet. 13: 697–703. —, 1991. Karyotype of some Brazilian diplopods. Rev. Bras. Genet. 14: 645–651. —, 1996a. Description of new species and karyotype of cavernicolous millipedes Pseudonannolene strinatii Mauries, 974 (Diplopoda, Pseudonannolenida, Pseudonannolenidae). Rev. Bras. Zool. 13: 419–426. —, 1996b. The use of cytogenetics to certify a Diplopoda species (Pseudonannolenida, Pseudonannolenidae). Rev. Bras. Biol. 56: 775–781. —, 1996c. Karyotype of a termitophilic species of Diplopoda (Polydesmida, Chelodesmidae). Rev. Bras. Genet. 19: 593–595. —, 1998. Chromosome numbers of some Brazilian species of diplopods (Diplopoda,Arthropoda). Cytologia 63: 149–154. —, 2000. Description and chromosome number of a species of Pseudonannolene selvestri (Arthropoda, Diplopoda, Pseudonannolenidae). Rev. Bras. Zool. 17: 187–191. —, Campos, K. A., Prado, R. A. and Souza T. D. S. 2002. Cytogenetic studies in Diplopoda. Cytologia 67: 253–260. Hamer, M. L. and Slotow, R. H. 2002. Conservation applications of existing data for South African millipedes (Diplopoda). Afr. Entomol. 10: 29–42. Hoffman, R. L., Golovatch, S. I., Adis, J. and de Morais, J. W. 2002. Diplopoda. In: Adis, J. (ed.). Arachnida and Myriapoda. Pensoft, Sofia-Moscow. pp. 501–533. Kadamannaya, B. S. and Sridhar, K. R. 2009. Leaf litter ingestion and assimilation by two endemic pill millipedes (Arthrosphaera) of the Western Ghats, India. Biology and Fertility of Soils 45: 761–768. Levan, A., Fredga, K. and Sandburg, A. A. 1964. Nomenclature for centromeric position on chromosomes. Heriditas 52: 201–220. Natarajan, R. 1959. Cytological studies in Diplopoda (Myriapoda). J. Zool. Soc. India 11: 91–101. Pierozzi, P. H. B and Fontanetti, C. 2006. A new species of Urostreptus (Diplopoda, Spirostreptidae): Description and 2010 Chromosomes of Western Ghats Pill Millipedes 475
chromosome number. Iheringia, Ser. Zool. 96: 209–212. Pocock, R. I. 1882. Report upon two collections of Myriyapoda sent from Ceylon by Mr. E. E. Green and from various parts of southern India by Mr. Edger Thurston of the govt. central museum, Madras. Bombay Nat. Hist. Soc. 17: 131–175. —, 1899. A monograph of the pill-millipedes (Zephroniidae) inhabiting India, Cylon and Burma. Bombay Nat. Hist. Soc. 12: 269–285. Rothfels, K. H. and Siminovitch, L. 1958. An air drying technique for flattening chromosomes in mammalian cells grown in vitro. Stain Technol 33: 73–77. Sierwald, P. and Bond, J. E. 2007. Current status of the Myriapod Class Diplopoda (millipedes): Taxonomic diversity and phylogeny. Annu. Rev. Entomol. 52: 401–420. Tanabe, T. 1992. Karyotypes of four xystodesmid millipeds from Japan. Acta Arachnologica 41: 87–90. Vitturi, R., Colomba, M. S., Caputo, V., Sparacio, I. and Barbieri, R. 1997. High heterochromatin content in somatic chromosomes of two unrelated species of Diplopoda (Myriapoda). Chrom. Res. 5: 407–412. Warchalowska-Sliwa, E., Maryanska-Nadachowska, A. and Kania, G. 2004. Cytogenetic studies of Glomeris (Diplopoda; Glomeridae). Folia Biologica (Krakow) 52: 1–2. White, M. J. D. 1979. The present status of myriapod cytogenetics. In: Camatini, M. (ed.). Myriapoda Biology. Academic Press, London. pp. 3–8. Wolters, V. and Ekschmitt, K. 1997. Gastropods, isopods, diplopods and chilopods: Neglected groups of the decomposer food web. In: Beneckiser, G. (ed.). Fauna in Soil Ecosystems. Marcel Dekker, New York. pp. 265–306.