214 P. R. Shashank et al.
eppendorf tube and powdered well using liquid
nitrogen. The DNA extraction was done using
Cetyltrim ethyl ammonium bromide (CTAB)
procedure and DNA amplification was done
with procedure described by Doyle and Doyle
( 1990 ). Extracted DNA pellet was dissolved in
500 μl TE and quantified using Nanodrop DNA
quantifier and electrophoresis method (0.8 %
agarose gel). Two μl of isolated DNA were
diluted to 1 ml with TE buffer and the absor-
bance at 260 and 280 nm were recorded against
a buffer blank for assessment of DNA purity. A
260/280 nm ratio for all the samples was calcu-
lated to check the purity. DNA was quantified
using: μg ds DNA/μl = (A260 * 40)/2. Further all
samples were diluted to a final concentration of
10 ng/μl.
DNA Bar coding
The CBOL established the “All-Leps Barcodes
of Life” project because the Lepidoptera is the
second most diverse order of insects. There are
about 180,000 known species, and it is likely that
there are another 300,000 species awaiting de-
scription. The initiative involves campaigns on
three geographic scales: Global (Geometridae,
Saturniidae and Sphingidae), Continental (North
America and Australia), and Regional (Great
Smoky Mountains National Park (USA) and Area
de Conservation Guanacaste) (Bravo et al. 2008 ).
Until now, 632,006 lepidopteran species are with
bar codes; of them 4443 are Crambidae (Interna-
tional Barcode of Life 2012). Hebert et al. ( 2004 )
studied the morphological and DNA bar coding
of Astraptes fulgerator Walch widely distributed
neotropical skipper butterfly (Lepidoptera: Hes-
periidae) in north western Costa Rica with mu-
seum specimens. They showed that A. fulgerator
is a complex of at least ten species in Costa Rica.
Largely sympatric, these taxa have mostly dif-
ferent caterpillar food plants, mostly distinctive
caterpillars, and different ecosystem preferences
but only subtly differing adults with no genitalic
divergence.
Conogethes Bar Code
Bar codes were generated on Conogethes from
specimens collected on castor and cardamom
from six and eight locations, respectively. The
locations were chosen including all the spots in a
cultivated zone of the crops. The DNA bar codes
were appended to an existing dataset from BOLD
for Conogethes species. The entire data set on
Conogethes included 115 DNA signatures cat-
egorized into four clades and these further have
been classified into more than 50 clusters. The
Conogethes on castor and cardamom belonged
to two distinct clades, moths were distinguished
based on the host plants. With castor speci-
men matching 91 % of the standard signature in
BOLD, the signature corresponded to C. punc-
tiferalis. The Conogethes specimen on carda-
mom is a new signature, this DNA signature did
not match with any signatures in BOLD deposit-
ed earlier. So this indicated that the moths reared
on cardamom possibly belong to a new species.
This needs to be further confirmed. The mean
within species divergence for C. punctiferalis
was 2.102 %. However, the pairwise divergence
between C. punctiferalis and the moths reared on
cardamom was > 5 % (Shashank 2012 ) (Fig. 2 ).
The family Crambidae, subfamily Pyrausti-
nae, has 4585 species with bar codes. The genus
Conogethes has 138 bar code sequences and
19 species (Table 2 ). Armstrong ( 2010 ) com-
pared DNA bar coding of different populations
of Conogethes and revealed that Australian and
Asian specimens form separate clades divergent
by ~ 6 %. The bar code data successfully distin-
guished C. punctiferalis and C. pluto, but unex-
pectedly revealed divergence between the Asian
and Australian populations. Morphologically
these were determined to be the same species,
and distinct from other closely related species
found on the east coast of Australia such as C.
haemactalis Walker, C. semifascialis Walker, or
C. tharsalea Walker.
The neighbour joining tree (NJ tree) was
constructed based on all the 33 DNA bar codes
using BOLD analysis tool. Based on NJ tree, two
clades were recognized and those clades which
includes all the Conogethes individuals breeding