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Molecular phylogeny of silk-producing insects based on 16S ribosomal RNA

and cytochrome oxidase subunit I genes

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c Indian Academy of Sciences

RESEARCH ARTICLE

Molecular phylogeny of silk-producing insects based on

16S ribosomal RNA and cytochrome oxidase subunit I genes

B. MAHENDRAN, S. K. GHOSH and S. C. KUNDU∗

Department of Biotechnology, Indian Institute of Technology, Kharagpur 721 302, India

Abstract

We have examined the molecular-phylogenetic relationships between nonmulberry and mulberry silkworm species that belong
to the families Saturniidae, Bombycidae and Lasiocampidae using 16S ribosomal RNA (16S rRNA) and cytochrome oxidase
subunit I (coxI) gene sequences. Aligned nucleotide sequences of 16S rRNA and coxI from 14 silk-producing species were
used for construction of phylogenetic trees by maximum likelihood and maximum parsimony methods. The tree topology
on the basis of 16S rRNA supports monophyly for members of Saturniidae and Bombycidae. Weighted parsimony analysis
weighted towards transversions relative to transitions (ts, tv4) for coxI resulted in more robust bootstrap support over un-
weighted parsimony and favours the 16S rRNA tree topology. Combined analysis reflected clear biogeographic pattern, and
agrees with morphological and cytological data.

[Mahendran B., Ghosh S. K. and Kundu S. C. 2006 Molecular phylogeny of silk-producing insects based on 16S ribosomal RNA and
cytochrome oxidase subunit I genes. J. Genet. 85, 31-38]

Introduction on the chorine egg, the arrangement of tubercular setae on

the larvae, and karyotyping data (Jolly et al. 1969, 1970a;
Silk-producing insects of Lepidoptera can be divided into
Sinha et al. 1994). Classification and phylogenetic analysis
two major groups, namely mulberry and nonmulberry silk-
of species on the basis of morphological attributes may be
worms. Mulberry silk is mainly contributed by Bombyx mori
riddled with problems because morphological features may
of the family Bombycidae. It is domesticated, wide in distri-
be variable with environment (Shouche and Patole 2000).
bution, and is a major part of the sericulture industry. Unlike
Molecular features provide an alternative. Different molec-
mulberry silks, nonmulberry silks are more heterogeneous
ular markers are available for characterization of genetic di-
and have apparently originated in the Indo-Australian region
versity of Bombyx mori (Reddy et al. 1999; Nagaraju and
of the Gondwana belt, or may have even wider distribution
Goldsmith 2002; Goldsmith et al. 2005), whereas very little
(Jolly et al. 1974). The nonmulberry silks are tasar, muga,
attention has been focussed on the genetic diversity of non-
eri and fagaria, which are mainly produced by species of
mulberry silkworms. It is essential to develop a DNA marker
the Antheraea and Attacini tribes of the family Saturniidae.
system to study genetic diversity among all varieties of silk-
Most of the species in this family are wild and polyphagous
worms (Prasad et al. 2002; Chatterjee and Tanushree 2004).
in nature, and the silks produced by them are specific to a
Selection of a gene for phylogenetic analysis plays a piv-
particular geographical zone: temperate tasar is produced by
otal role (Whitfield and Cameron 1998). Ribosomal RNA
Antheraea pernyi, A. roylei and A. proylei, tropical tasar by
and cytochrome oxidase genes encoded by the mitochon-
A. mylitta, muga silk by A. assama, eri silk by Philosamia
drial genome are commonly used in molecular-phylogenetic
ricini, fagaria by Attacus atlas, and shashe silk by Gonometa
studies because they have conserved sequences whose evo-
postica. The silk-producing insects have been classified on
lutionary rate is appropriate to resolve phylogenetic relation-
the basis of morphological clues such as follicular imprints
ships between small taxonomic units like genera or fami-
lies (Brower 1994; Canapa et al. 2000; Han 2000; Niehuis
∗ For correspondence. Email: [email protected]. and Wagele 2004). There are several reports on the utility

Keywords. silkworm; Saturniidae; Bombycidae; Lasiocampidae; 16S rRNA; coxI gene.

Journal of Genetics, Vol. 85, No. 1, April 2006 31

 B. Mahendran et al.

of mitochondrial genes for inferring relationships among 100 µg/ml proteinase K). The digested samples were ex-
closely related species and species groups in butterflies tracted twice with an equal volume of Tris-HCl, saturated
(Davies and Bermingham 2002; Blum et al. 2003). The phy- phenol (pH 8.0), and then centrifuged at 5000 g for 15 min
logenetic relationships among members of Saturniidae and to remove protein contaminants and debris. The supernatant
Bombycidae have earlier been studied on the basis of nu- was transferred into a fresh tube and treated with RNAase at
clear genes (elongation factor 1α, DOPA decarboxylase and 37◦ C for 30 min, followed by chloroform extraction and cen-
arylphorin) and mitochondrial DNA (16S rRNA and RFLP trifugation at 5000 g for 15 min. Finally, the aqueous phase
of mtDNA) (Shimada et al. 1995; Friedlander et al. 1998; from each tube was transferred separately to clean centrifuge
Hwang et al. 1999a,b). The previous analysis was limited to tubes and mixed with 0.1 volume of 3 M sodium acetate, pH
Antheraea pernyi, A. yamamai and Attacus atlas of Saturni- 5.2. Genomic DNA was precipitated with two volumes of
idae, and Bombyx mori and B. mandarina of Bombycidae. cold ethanol, spooled, washed twice with 70% ethanol, dried,
Here we present a phylogenetic analysis of 14 silkworm and suspended in 10 mM Tris-Cl (pH 8.0).
species based on sequences of 16S ribosomal RNA (rRNA)
and cytochrome oxidase subunit I (coxI) genes. Amplification and sequencing of 16S rRNA and coxI genes

Materials and methods A 383-bp region of 16S rRNA gene and a 597-bp re-
gion of coxI gene were amplified by polymerase chain re-
Species collection
action (PCR). The primers used for amplification of
Fresh live cocoons were obtained from different locations in the 383-bp fragment of 16S rRNA gene were for-
India and other countries. The cocoons were stored frozen at ward 5′ -GTGCAAAGGTAGCATAATCA-3′ and reverse 5′ -
−80◦ C. The geographical distribution and host plants of 10 TGTCCTGATCCAACATCGAG-3′ . The primers for am-
collected silk-producing insects are given in table 1. plification of the 597-bp fragment of coxI were for-
ward 5′ -TGATCAAATTTATAATAC-3′ and reverse 5′ -
Genomic DNA isolation
GTAAAATTAAAATATAAAC-3′ (Hwang et al. 1999a,b).
Total genomic DNA was isolated from either fresh or frozen PCR was carried out in 50 µl using 1 U of Taq poly-
pupae by the standard laboratory protocol (Datta et al. 2001). merase (Roche) in a Perkin Elmer 2400 thermal cycler.
Prior to DNA extraction, the digestive tract was taken out The PCR reaction conditions for 16S rRNA were 94◦ C
from the pupal body by dissection. About one gram of for 30 sec (denaturation), 55◦ C for 1 min (annealing) and
fat body tissue was ground in liquid nitrogen and incu- 72◦ C for 1 min for 35 cycles. The PCR conditions for
bated overnight at 50◦ C in 10 ml digestion buffer (0.01 M coxI were identical to those for 16S rRNA except that the
NaCl, 0.1 M Tris-HCl, 0.25 M EDTA and 0.5% SDS with annealing temperature was 50◦ C. The PCR products were

Table 1. Collected silk-producing species.

Species (Host plants) Place of collection Distribution Chromosome number (2n)

Antheraea mylitta (Terminalia West Bengal, India North and Central India 62
arjuna, T. tomentosa, Shorea
robusta)
Antheraea frithi (Lithocarpus Imphal, India Eastern India 62
dealbata, Quercus dealbata)
Antheraea assama (Machilus Assam, India Eastern India 30
bombycina)
Antheraea proylei (Quercus Imphal, India Himalayan belt of India 98
serrata, Q. incana)
Antheraea roylei (Quercus Imphal, India Himalayan belt of India 60, 62, 64, 68
spp.)
Antheraea polyphemus (Quer- Edward Island, Canada North America 60
cus alba, Q. nigra, Q. rubra)
Philosamia ricini (Ricinus com- Assam, India India 28
munis, Manihot utilissima)
Attacus atlas (Ailantus al- Yogyakarta, Indonesia Southeast Indonesia –
tissima, Ligustrum, Syringa)
Gonometa postica (Colophos- Kalahari desert Southeast Namibia –
permum mopane)
Hyalophora cecropia (Acer Edward Island, Canada North America 62
saccharinum, Prunus spp,
Quercus spp.)
(Modified from Jolly et al., 1974).

32 Journal of Genetics, Vol. 85, No. 1, April 2006

 Phylogeny of silk-producing insects

gel-purified using Qiagen gel extraction kit (Qiagen, Hilden, tree length (L), consistency indices (CI; Kluge and Farris
Germany). The purified DNA fragments were cloned into 1969), and retention indices (RI; Farris 1989) were reported.
a pCR2.1 TOPO TA cloning vector (Invitrogen, USA). The The internal stability of the inferred MP tree was measured
ligated products were used to transform E. coli DH5α. The by bootstrapping using 1000 replications (Felsenstein 1985).
plasmids were isolated, and the inserts were verified accord- Further, the phylogenetic analysis was conducted on a com-
ing to Sambrook and Russell (2001) and sequenced using our bined data set (16S rRNA and coxI). To examine the ef-
automated cycle sequencing facility (ABI Prism 3770). The fect of transition-to-transversion weighting schemes on tree
sequencing was performed using 400 ng of plasmid DNA topology, further analyses were run with transitions down-
as a template and 2.0 pmol of primer (M13 forward or re- weighted relative to transversions by a factor of four to com-
verse). The Sequencher (Gene Codes Corp.) was used for pensate for the observed transition bias. The transition–
sequence assembly and evaluation. The sequences of 16S transversion ratio was calculated using the program MEGA
rRNA and coxI genes were analysed using BLAST (Altschul 1.01 (Kumar et al. 2001).
et al. 1990). The obtained sequences were aligned with The most appropriate substitution model for ML analysis
their orthologues from B. mori (AB070264 for 16S rRNA was determined by using Modeltest version 3.06 (Posada and
and coxI), B. mandarina (AB070263 for 16S rRNA and Crandall 1998) for data on each gene and for the combined
coxI), A. yamamai (AF027952 for 16S rRNA and AF029067 data. Sequence divergence values were also calculated from
for coxI), A. pernyi (AY242996 for 16S rRNA and coxI), the selected model. Rate of heterogeneity was expressed us-
Hyalophora cecropia (AY165720 for coxI) and Spodoptera ing gamma distribution with the shape parameter. In both the
frugiperda (M76713 for 16S rRNA and U72976 for coxI) approaches insertions, deletions and missing data were com-
using ClustalW (Thompson et al. 1994). Sequence statis- pletely excluded and the sequences were analysed in favour
tics were obtained using MEGA 2.1 (Kumar et al. 2001). of the outgroup selection. Spodoptera frugiperda, a Noctu-
The sequences of 16S rRNA and coxI genes have been idae species, was considered as outgroup. One hundred repli-
submitted to GenBank (accession numbers AY598830 (A. cates were performed in ML to find the phylogenetic confi-
mylitta 16S rRNA gene), AY604239 (H. cecropia 16S rRNA dence at each node of the tree. Minimum evolutionary trees
gene), AY604240 (Genometa postica 16S rRNA gene), were constructed for combined data analysis.
AY601275 (A. frithi 16S rRNA gene), AY601276 (A. assama
16S rRNA gene), AY601277 (A. proylei 16S rRNA gene), Results and discussion
AY601278 (A. polyphemus 16S rRNA gene), AY601279 (At-
tacus atlas 16S rRNA gene), AY601280 (Philosomia ricini Molecular genetics and biology of the domesticated silk-
16S rRNA gene), AY960275 (A. roylei 16S rRNA gene), worm Bombyx mori are better understood than those of wild
AY605248 (A. frithi coxI gene), AY605249 (A. assama nonmulberry silkworms. Mitochondrial genes have been
coxI gene), AY605250 (A. proylei coxI gene), AY605251 used to study the relationship between wild and domesticated
(A. polyphemus coxI gene), AY605252 (Attacus atlas coxI species (Hwang et al. 1999a,b; Yukuhiro et al. 2002), but
gene), AY605253 (P. ricini coxI gene), AY605254 (G. pos- those studies considered only a few species. In this study
tica coxI gene), AY605255 (A. mylitta coxI gene), AY960274 emphasis has been given to the phylogenetic relationships
(A. roylei coxI gene)). The saturation analysis and filtering of more economically important wild species on the basis of
of data in relation to the type of substitution and codon po- 16S rRNA and coxI gene sequences. BLAST analysis of am-
sition were performed for all three codon positions for coxI plified and cloned 383-bp region of 16S rRNA and 597-bp re-
(Griffiths 1997). gion of coxI from 10 species of silkworms (table 1) showed
significant homology with A. pernyi and A. yamamai (Sat-
urniidae), and with B. mori and B. mandarina (Bombycidae).
Phylogenetic analysis Both sequence regions showed AT-rich nucleotide composi-
The two genes (16S rRNA and coxI) were initially analysed tion typical of mitochondrial genes. The average percent-
separately, because different genes may experience different age nucleotide composition among silk-producing taxa for
evolutionary pathways. The phylogenetic analyses were per- 16S rRNA is A 39.2, T 40.4, G 6.9, C 13.5, and for coxI
formed with PAUP∗ 4.01 version beta 10 (Swofford 2001) A 30.0, T 39.3, G 13.8, C 16.8. In comparison with coxI,
using maximum parsimony (MP) and maximum likelihood 16S rRNA showed high AT bias and also possessed higher
(ML) approaches. MP trees were produced using heuristic variability. Multiple sequence alignment of 16S rRNA and
searches with 50 repetitions using random stepwise addition coxI sequences from 14 silkworm taxa was performed using
of taxa and gaps were also treated as missing characters. ClustalW (Thompson et al. 1994). No stop codons, inser-
Tree-bisection-reconnection (TBR) branch swapping, Mul- tions or deletions were found in the coxI sequence.
trees option, and accelerated transformation (ACCTRAN)
Phylogenetic analysis
character-state optimizations were in effect. Multiple-state
taxa were interpreted as uncertain and the branches were col- Earlier studies on the basis of morphological clues such as
lapsed if the maximum length was zero. For all analyses, follicular imprints over the chorion of the egg, tubercular

Journal of Genetics, Vol. 85, No. 1, April 2006 33

 B. Mahendran et al.

setae of the larvae, eyespots on wings, karyotype, and silk urniidae and Bombycidae. Within Saturniidae two distinct
gland (Jolly et al. 1969, 1970a,b; Gupta 1985) inferred that clades are observed with significant bootstrap. One includes
A. mylitta, A. frithi, A. pernyi, A. roylei, A. proylei, A. assama A. proylei, A. pernyi and A. roylei, and the second includes
and A. yamamai are closely related. A. proylei is a hybrid be- A. mylitta and A. frithi, followed by A. yamamai. More-
tween A. pernyi of China (2n = 98) and A. roylei of the Hi- over, the bootstrap analysis showed low confidence values
malayan belt of India (2n = 60) (Nagaraju and Jolly 1985). between Antheraea and Attacus. This analysis does not sup-
A controversy arose during the 1970s, since one group as- port monophyly for members of Antheraea and Attacini.
signed the hybrid a status of species, calling it A. proylei. The North American large silkmoth Hyalophora cecropia
Some taxonomists considered A. proylei as just a hybrid. De- clusters with Philosamia ricini (the only semidomesticated
spite these facts, A. proylei parents produce fertile A. proylei species included in our study) and the two form a clade with
offspring, and A. proylei is considered as a species in east- Attacus atlas. In this MP tree the relationship of A. assama
ern India. However, Bhagirath et al. (1988) have observed and A. polyphemus with other Antheraea species is not clear.
that the chromosome number of the present-day A. proylei is Gonometa postica (Lasiocampidae) clusters with members
2n = 98. Our study examines the phylogenetic relationships of Bombycidae.
among some of these silk-producing insects on the basis of
16S rRNA and coxI. Phylogenetic analysis on the basis of coxI
Phylogenetic analysis on the basis of 16S rRNA
Unweighted MP analysis resulting in a single most-
Phylogenetic analysis of the 14 silk-producing species was parsimonious tree with low bootstrap value at each node
carried out using PAUP∗ 4.01 (Swofford 2001) software. Be- did not resolve the position of A. yamamai, A. assama and
sides the small differences in the bootstrap values, heuristic A. polyphemus and displayed polytomy (data not shown).
maximum parsimony (MP) and maximum likelihood (ML) It might be due to effect of transition/transversion ratio at
methods yielded similar trees (figure 1). These trees sup- the third codon position in coxI. Therefore, we examined
port monophyly for the major silk-producing families, Sat- the effects of saturation in phylogenetic analysis (figure 2).

(a) (b)

Figure 1. Maximum parsimony (a) and maximum lilkelihood (b) trees for 16S rRNA. Maximum parsi-
mony analysis yielded a single tree with a length of 202, CI = 0.58 and RI = 0.59. The selected model
for maximum likelihood analysis is GTR+G+I (−ln L = 857.55221). Incorporating this model, the base
frequencies were unequal: 0.394 (A), 0.079 (C), 0.061 (G), 0.465 (T). The estimated shape parame-
ter for the gamma distribution was α = 0.326. The tree was rooted using S. frugiperda as outgroup.
Numbers inside the branches are bootstrap values for 1000 and 100 replications.

34 Journal of Genetics, Vol. 85, No. 1, April 2006

 Phylogeny of silk-producing insects

The figure shows that substitutions at the first codon posi- preferential accumulation of further substitutions at the first
tion can be easily fitted to a straight line, indicating that they and second positions and saturation starts at the third codon
are unsaturated (Griffiths 1997; Guryev et al. 2001). On the position. The data were also evaluated excluding the third
other hand, substitutions at the second and third codon posi- position, and also considering the third position alone, but
tions are in mutation pressure. In the case of transversions we could not resolve the deeper nodes and also the question
two clouds in the third-codon-position plot indicate that one of monophyly between Saturniidae and Bombycidae. To un-
group has no transversions and another has several. The derstand the necessity and usefulness of differential weight-
more distantly related species might have lower third-codon- ing we estimated the relative frequencies of transitions and
position divergences than closer relatives. This illustrates the transversions using Kimura two-parameter distance method

Figure 2. Plots illustrating saturation assessments for three data sets of coxI. Pairwise sequence differences within each partition
are plotted against total sequence divergence. The dark and square symbols used in all saturation plots indicate comparison of
substitution rate between silk-producing insects with outgroup.

Table 2. Transition/transversion ratio for coxI for all species studied.

Species 1 2 3 4 5 6 7 8 9 10 11 12 13 14

1. A. mylitta
2. A. frithi 1.02
3. A. assama 0.85 0.64
4. A. pernyi 1.03 1.13 1.12
5. A. roylei 0.98 0.93 1.12 2.03
6. A. yamamai 1.35 1.03 0.99 2.64 2.09
7. A. proylei 1.18 1.31 1.26 1.50 3.74 3.17
8. A. polyphemus 1.27 0.77 0.92 0.75 0.77 0.93 0.88
9. P. ricini 0.98 0.87 0.91 0.53 0.50 0.81 0.62 0.86
10. A. atlas 1.12 0.94 0.97 1.02 0.99 1.28 1.12 0.90 1.08
11. H. cecropia 1.03 1.15 1.22 0.91 0.89 1.15 1.02 0.82 1.94 1.02
12. B. mori 0.55 0.55 0.71 0.60 0.74 0.73 0.63 0.50 0.42 0.67 0.67
13. B. mandarina 0.54 0.54 0.67 0.56 0.70 0.70 0.59 0.51 0.41 0.67 0.68 6.06
14. G. postica 0.51 0.53 0.69 0.59 0.51 0.59 0.62 0.67 0.64 1.05 0.86 0.67 0.66
15. S. frugiperda 0.48 0.46 0.52 0.64 0.53 0.58 0.72 0.62 0.61 0.64 0.86 0.64 0.63 0.56

Journal of Genetics, Vol. 85, No. 1, April 2006 35

 B. Mahendran et al.

(K2P) and reanalysed the data giving transversions four gies obtained under the optimality criteria of MP (figure 4a)
times more weight than transitions (table 2). The resultant and ML (figure 4b) were almost similar to the weighted MP
weighted tree topology (figure 3, left) differs from the un- tree for coxI. Like in ML analysis of 16S rRNA and coxI,
weighted MP tree and supports monophyly of Saturniidae GTR+G+I was found to be the best model by Modeltest for
and Bombycidae. The relationship of A. yamamai, A. as- combined data also, with likelihood ratio −lnL = 3766.4749
sama and A. polyphemus clade with other species in the same and shape parameter α = 0.8538. In this model the reso-
genera supports monophyletic origin for both Antheraea and lution of nodes is generally high within and between tribes
Attacini tribes. A. assama and A. polyphemus resolved at the of Saturniidae. In both MP and ML analysis monophyletic
root of the Antheraea cluster. For ML analysis the justified relationships were found between Saturniidae and Bombyci-
model GTR+G+I was selected using Modeltest version 3.06 dae members. A similar observation was made on the basis
(Posada and Crandall 1998). In this model, the base frequen- of two nuclear genes (Regier et al. 2002). Within Saturni-
cies were unequal: 0.302 (A), 0.143 (C), 0.145 (G), 0.408 idae Antheraea and Attacus also showed sister group rela-
(T). In comparison with the weighted MP tree this tree could tionship. The different silk-producing species of Antheraea
not resolve the position of A. mylitta, A. frithi, A. assama and fall into distinct clusters according to the type of silk (fig-
A. polyphemus, and displayed polytomy. This analysis re- ure 4). For example the Chinese oak silkmoth A. pernyi
veals that sequence evolution is under different constraints in makes a distinct clade with the Indian oak tasar silkmoth
these species (Dobler and Muller 2000). A. proylei, and also shows close relationship with another
Indian oak tasar silkmoth, A. roylei. Two other tasar-silk-
Combined analysis
producing species, A. mylitta and A. frithi, make a distinct
There is considerable disagreement over whether data sets clade with significant bootstrap value. In this combined anal-
should be combined or considered separately in phylogenetic ysis also, the status of muga-silk-producing species A. as-
analysis (Kluge and Farris 1969). The partition-homogeneity sama and A. polyphemus is not clear. The representative gen-
test did not indicate a significant conflict between the molec- era of Attacini (Philosamia, Attacus and Hyalophora) form
ular data sets (P = 0.93), and thus the 16S rRNA and coxI a monophyletic clade. B. mori and B. mandarina of the
data were combined for total evidence analysis. The topolo- Bombycidae also group together, like in the individual tree

(a) (b)

Figure 3. Weighted parsimony (a) and maximum likelihood (b) analysis for coxI. The weighted tree
topology differs from the unweighted MP tree, and has a length of 1105, with CI = 0.487 and RI =
0.550. The justified model is GTR+G+I (− lnL = 1713.338). The estimated shape parameter for the
gamma distribution was α = 0.968. The tree was rooted using S. frugiperda as outgroup. Numbers
inside the branches are bootstrap values for 1000 and 100 replications.

36 Journal of Genetics, Vol. 85, No. 1, April 2006

 Phylogeny of silk-producing insects

(a) (b)

Figure 4. Phylogenetic relationships among species of Saturniidae, Bombycidae and Lasiocampidae

based on the combined sequence data from 16S rRNA and coxI genes. (a) Combined data set comprises
633 constant characters, and 227 sites are parsimony informative. (b) Maximum likelihood analysis;
the tree was rooted using S. frugiperda as outgroup. Numbers inside the nodes are bootstrap values.

topologies. The combined data also support the close resem- was obtained from the Council of Scientific and Industrial Research
blance of G. postica with members of Bombycidae. (CSIR), New Delhi. B. M. also received a fellowship from CSIR.
The tasar-producing species A. mylitta showed resem- We also thank Dr B.R.P.P. Sinha (Director, Central Tasar Research
blance to the geographically neighbouring species A. frithi. and Training Centre, Ranchi), Mr P. Mukherjee (Directorate of Ser-
From karyotype data, A. assama, with the lowest chromo- iculture, Government of West Bengal, Midnapore (W)), Bill Oehlke
some number, 2n = 30 (Deodikar et al. 1962), has been (Prince Edward Island, Canada), and Mr S. Dey (Orissa), Dr Ibohal
considered as a probable ancestor of all species in the genus and Dr Ibotombi (Manipur) and I. Cummings (Namibia) for pro-
Antheraea. However, A. polyphemus (the only Antheraea viding some of the silkworm samples. We would like to express our
species available in the Palaearctic region) occupies the basal sincere thanks to Professor A.K. Ghosh for providing bioinformat-
position within the genus Antheraea and is very close to ics support and Mr Baranidharan for excellent technical assistance
A. assama, owing to probable occurrence of rapid specia- on bioinformatics. We thank Dr Papoucheva and Andrews Zwick
tion in association with colonization of new continents, and for providing valuable suggestions.
continental drift. A similar phenomenon has been reported
in Chironomus (Diptera) species (Guryev et al. 2001). The References
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Received 16 May 2005

38 Journal of Genetics, Vol. 85, No. 1, April 2006

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