BARCODES OF SCOLOPENDROMORPHA CENTIPEDES OF TAIWAN
Chao, Jui-Lung (1) and Chang, Hsueh-Wen (2)
National Sun Yat-Sen University, Kaohsiung, Taiwan, R. O. C
70, Lianhai Rd., Gushan Dist., Kaohsiung City 804, Taiwan, R.O.C.
The traditional systematics of Scolopendromorpha centipedes is based on morphological characters.
However, it is not easy to identify some species with morphological characters. In addition, factors
such as the sexual dimorphism and morphological changes of the post larval development in some
species, variations in the regenerations of legs and antennae, all adding difficulty and leading to
errors in the identification and classification of Scolopendromorpha species. In the present study,
we determined 45 DNA barcodes for 14 species scolopendromorphs and 2 of geophilomorpha from
Taiwan, and analyzed their phylogenetic relationship with 23 partial COI sequences of Chilopoda
centipedes from GenBank. The result indicates that the interspecies sequence identities and the
intraspecies sequence identities vary with species. Both high minimum intraspecies identity and
maximum interspecies identity are present in Genus Scolopocryptops. The phylogenetic trees
divided Scolopendromorpha into three families, supported that a close relationship exists between
Scolopocryptopidae and Scolopendridae, and confirmed that Scolopendra multidens should be a
valid species, not a subspecies of S. subspinipes. Although the phylogenetic trees didn’t distinguish
among the genera Rhysida, Otostigmus and Ethmostigmus, the phylogenetic trees and the low
maximum interspecies identifies showed that the DNA barcoding method is a good tool for
identifying some species of Scolopendromorpha. The result indicates that the minimum intraspecies
identity of Rhysida immarginata are very high (0.993), and two of five sequences from Lieyu Islet
which is near China mainland, the other three from Taiwan Island. However, R. immarginata was
never recorded in China mainland.
Keywords: Chilopoda; Myriapoda; molecular data; phylogeny; systemics.
Introduction
The class Chilopoda consists of Scutigeromorpha, Lithobiomorpha, Craterostigmomorpha,
Scolopendromorpha and Geophilomorpha. The order Scolopendromorpha is characterized as with
21 or 23 pairs of legs, and each side of cephalic plate with or without four ocelli. Fourteen species
of 5 genera recognized from Taiwan. The traditional systematics of the Scolopendromorpha
centipedes is based on external morphological characters (Lewis et al. 2005). Their body sizes vary
from 10 to 200 mm, and the body color are bright green, red, blue, dark brown, orange or yellow.
The traditional systematics of the Scolopendromorpha centipedes is based on morphological
characters, for example, number of legs, number of antennomeres, cephalic plate, clypeus, labrum,
mandibles, first maxilla, second maxilla, forcipules, tooth plate, spiracles, tergites, sternites,
coxopleuron, ultimate legs and genito-anal segment (terminal segment), etc. (Lewis et al. 2005).
1
However, it is not easy to identify some species with these morphological characters. In addition,
factors such as the sexual dimorphism and morphological changes of the post larval development in
some species, variations in the regenerations of legs and antennae, adding difficulty and leady to
errors in the identification and classification of Scolopendromorpha species (Lewis 2003).
Furthermore, some holotypes were lost or destroyed, the original descriptions were short and
indefinite, hence many published names are synonymous or homonymous in Taiwan (Chao and
Chang 2003, 2008). Because weighted different characters, different classifications of
Scolopendromorpha were published, e.g. Attems’ system (1930, Fig. 1), Schileyko’s system (1997,
Fig. 2), Shelley’s system (2002, Fig. 3) and Chao & Chang’s system (2006, Fig. 4). Recently,
Giribet and Edgecombe (2002, 2004, 2006) analyzed the phylogeny of centipedes base on
morphology and genetic data (18S rRNA, 28S rRNA, 16S rRNA, COI, EF1a, EF2 and POLII).
They (2006) suggested the addition of genetic data dose not produce a more stable hypothesis for
deep centipede relationships. In this study, we just analyzed COI fragment (678 bp) for 68
centipedes (38 species)(Table 1), which including 3 specimens of Scutigeromorpha, 2 of
Craterostigmomorpha, 3 of Lithobiomorpha, 52 of Scolopendromorpha (43 from Taiwan), 8 of
Geophilomorpha (2 from Taiwan) for the phylogeny of Scolopendromorpha.
Materials and methods
Molecular data
Forty-five DNA barcodes of Taiwanese Chilopoda were obtained from freshly collected specimens
or from museum specimens (NMNS). DNA from preserved tissues was extracted using the Wizard®
Genomic DNA purification kit (Promega.com). New DNA barcode fragments, partial cytochrome c
oxidase subunit I, were obtained using primer pair:
LCO1490 (5’-GGTCAACAAATCATAAAGATATTGG-3’)
HCO2198 (5’-TAAACTTCAGGGTGACCAAAAAATCA-3’),
which amplifies a 678 bp fragment. In addition to, we obtained twenty-three COI fragments from
GenBank, which including 3 sequences of Scutigeromorpha (Scutigera, Thereuopoda and
Thereuonema), 2 of Craterostigmomorpha (2 Craterostigmus), 3 of Lithobiomorpha (Anopsobius,
Lithobius and Bothropolys), 6 of Geophilomorpha (Bothriogaster, Himantanum, Mecistocephalus,
Geophilus, Strigamia and Pachymerium) and 9 of Scolopendromorpha (Cormocephalus, Alipes,
Scolopendra, Rhysida, Ethmostigmus, Theatops, Cryptops and 2 Scolopocryptops) (Table 1).
Analytical methods
The DNA barcode data sets were aligned using the multiple alignment program, BioEdit version
7.0.9.0 (Hall 1999) and ClustalX version 2 (Larkin et al. 2007). The molecular data were analyzed
for the phylogeny using the parsimony method, maximum likelihood coding method and
Neighbor-Joining method with the computer program PAUP*, version 4.0b10 for 32-bit Microsoft
Windows, beta-test version as the optimality criterion. Nodal support was measured via 1000
replicates of bootstrap PAUP* for parsimony and Neighbor-Joining trees.
Results
2
Maximum Likelihood Coding tree (Fig. 5) and Neighbor-Joining tree (Fig. 6), support
Scolopendridae+Scolopocryptopidae is a monophyletic group, and Cryptops has a much distant
relationship with it. Therefore, Scolopendromorpha is not monophyletic, and Cryptopidae may be a
separate taxa from the rest of Scolopendromorpha. In addition, the phylogenetic trees and the
sequence identity (Fig. 7) clearly support that S. multidens (Fig.8) should be a valid species, not a
subspecies of S. subspinipes. The phylogenetic trees and the low maximum interspecies identity
showed that the DNA barcoding method is a good tool for identifying some species of
Scolopendromorpha. The result indicates that the minimum intraspecies identity and the maximum
interspecies identity vary with the species (Fig. 6). Some species such as S. multidens, Rhysida
immarginata etc. has high intraspecies identifies minimum (more than 0.99). However, in present
study, none maximum interspecies identify is more than 0.9.
Discussion
In 1903, Kraepelin (1903) suggested both Scolopendra multidens Newport, 1844 and Scolopendra
mutilans L. Koch, 1878 were two subspecies of Scolopendra subspinipes Leach, 1815. We (Chao
and Chang 2003) considered S. multidens as a valid species, not a subspecies. However, these three
phylogenetic trees (Fig. 3, 4, 5) and the sequence identity (Fig. 6) clearly support that S. multidens
should be a valid species, but S. mutilans is a synonym of S. subspinipes. Although the phylogenetic
trees didn’t distinguish among the genera Rhysida, Otostigmus and Ethmostigmus, the maximum
parsimony bootstrap trees and the low maximum interspecies identity showed that the DNA
barcoding method is one of good tools for identifying some species of Scolopendromorpha. The
minimum intraspecies identity of Cryptops japonicus is less than its maximum interspecies identity.
We have to obtain more molecular data of Cryptops for answering this difference.
Attems (1930) dividing the order Scolopendromorpha into 2 families: Scolopendridae (ocelli
present) and Cryptopidae (ocelli absent), and family Scolopendridae consists of subfamily
Scolopendrinae (including Scolpendra, Cormocephalus, Trachycormocephalus, Arthrorabdus,
campilostigmus, Rhoda, Scolopendropsis, Asanada, Pseudocryptops) and subfamily Otostigminae
(including Otostigmus, Rhysida, Alipes, Ethmostigmus, Digitipes, Allurops, Arrhabdotus). The
family Cryptopidae consists of three subfamilies: Scolopocryptopinae (Scolopocryptops, Newpartia,
Kethops, Kartops, Dinocryptops), Cryptopinae (Cryptops, Paracryptops, Anethops, Mimops) and
Theatopsinae (Theatops, Plutonium) (Fig. 7).
Based on the number of body segments and pairs of spiracles, Schileyko (1997) suggested new
families Plutoniidae (Plutonium) and Scolopocryptopidae which separated from Cryptopidae, and
grouped Cryptopinae, Theatopsinae, Scolopendrinae, Otostigminae, Sterropristinae (separated from
Otostigminae by 7th segment with spiracles, included Rhysida, Ethmostigmus, Allurops,
Arrhabdotus) into family Scolopendridae (Fig. 8).
Shelley (2002) suggested three families: Scolopendridae, Scolopocryptopidae and Cryptopidae.
The Scolopendrinae (Scolopendra) and Otostigminae (Rhysida, Otostigmus) were belonged to
Scolopendridae, and Scolopocryptopinae (Scolopocryptops, Dinocryptops), Kethopinae (Kethops,
Thalkethops) and Newportiinae were belonged to Scolopocryptopidae, however, Cryptopinae
3
(Cryptops) and Plutoniuminae (Theatops and Plutonium) were belong Cryptopidae by ocelli absent
and 21 pairs of legs (Fig.9). Edgecombe and Girbet (2004, 2006) analyzed the phylogeny of
Chilopoda centipedes using the maximum parsimony method for their morphology and molecular
data (18S rRNA, 28S rRNA, 16S rRNA and COI, but this data includes just 6 COI sequences from
Scolopendromorpha), their summary cladogram show Scolopendromorpha is monophyletic, and
Scolopocryptopinae as sister to Cryptopinae + Plutoniuminae (Fig 10).
In this study, both Maximum Likelihood Coding tree and Neighbor-Joining tree show
Scolopendridae+Scolopocryptopidae is a monophyletic group, and a further relationship between
Cryptops and Scolopendridae+Scolopocryptopidae, and therefore Scolopendromorpha is not a
monophyletic group. The phylogenetic trees disagree with the cladogram of Edgecombe and Girbet,
and support the traditional classification of Scolopendromorpha by has to restructure. Furthermore,
the phylogenetic trees support that genera Rhysida, Otostigmus, Ethmostigmus and Alipes were
grouped into Otostigminae, without respect to the spiracles of 7th body segment absent or present.
The phylogenetic trees grouped Theatops (21 pairs of legs) and Scolopocryptops (23 pairs of legs)
into Scolopocryptopidae. In conclusion, we suggest a new classification that family Cryptopidae is
only with genus Cryptops, family Scolopocryptopidae includes Scolopocryptopinae
(Scolopocryptops) and Theatopsinae (Theatops), and Scolopendridae includes Scolopendrinae
(Scolpendra, Cormocephalus) and Otostigminae (Rhysida, Otostigmus, Ethmostigmus, Alipes) (Fig.
11)
For biogeographic analysis, the result indicates that the minimum intraspecies identity of
Rhysida immarginata are more than 0.993, and two of five sequences from Lieyu Islet which is near
China mainland, the other three from Taiwan Island. However, Lieyu Islet is 220 km further from
Taiwan. Furthermore, we publish two barcodes of Scolopendra morsitans, one is from Kinmen Islet
which next to Lieyu Islet, another one from Magong Islet, the biggest islet of Pescadores. There is
155 km from Magong Islet to Kinmen Islet. However, R. immarginata and S. morsitans were never
recorded in China mainland (Song 2004).
Reference
Attems, G. 1930. Myriapoda 2. Scolopendromoroha. Das Tierreich. Berlin und Leipzig 127-133,
143, 149-154.
Chao, J. L. and Chang, H. W. 2003. The scolopendromorph centipedes (Chilopoda) of Taiwan.
African Invertebrates 44(1): 1-11.
Chao, J. L. and Chang, H. W. 2006. Variation of the poison duct in Chilopoda centipedes from
Taiwan. Norwegian Journal of Entomology 53(2): 139-151.
Chao, J. L. and Chang, H. W. 2008. Neotype designation for two centipedes, Scolopocryptops
curtus (Takakuwa, 1939) and Cryptops nigropictus Takakuwa, 1936, and a review of species of
Scolopendromorpha (Chilopoda) in Taiwan. Collection and Research 21: 1-15.
Edgecombe, G. D. and Giribet, G. 2004. Adding mitochondrial sequence data (16S rRNA and
cytochrome c oxidase subunit I) to the phylogeny of centipedes (Myriapoda: Chilopoda): an
analysis of morphology and four molecular loci. Journal of Zoological Systematics and
4
Evolutionary Research 42: 89-134.
Edgecombe, G. D. and Giribet, G. 2006. Conflict between datasets and phylogeny of centipedes: an
analysis based on seven genes and morphology. Proceedings of the Royal Society B 273:
531-538.
Hall, T. A. 1999. BioEdit: A user-friendly biological sequence alignment editor and analysis
program for Windows 95/98/NT. Nucl. Acids. Symp. Ser. 41:95-98.
Kraepelin, K. 1903. Revision der Scolopendriden Mitt. Natur. Mus. Hamburg 20: 263-264.
Larkin,M.A., Blackshields, G., Brown, N.P., Chenna, R., McGettigan, P.A., McWilliam, H., Valentin,
F., Wallace, I.M., Wilm, A., Lopez, R., Thompson, J.D., Gibson, T.J., Higgins, D.G.. 2007.
Clustal W and Clustal X version 2.0. Bioinformatics, 23:2947-2948.
Lewis, J. G. E. 2003. The problems involved in the characterization of scolopendromorph species
(Chilopoda: Scolopendromorpha). African Invertebrates 44(1): 61-69.
Lewis, J. G. E., Edgecombe, G. D. and Shelley, R. M. 2005. A proposed standardized terminology
for the external taxonomic characters of the scolopendromorpha (Chilopoda). Fragmenta
Faunistica 48(1): 1-8.
Newport, G.. 1844. One new species of Myriapoda. The Annals and Magazine of Natural History 13:
97.
Schileyko, A. A. and I. J. Pavlinov. 1997. A cladistic analysis of the order Scolopendromorpha
(chilopoda). Ento. Scan. Suppl. 51: 33-40.
Shelley, R. M. 2002. A synopsis of the North American centipedes of the order Scolopendromorpha
(Chilopoda). Virginia Museum of Natural History, Martinsville.
Song, Z. S. 2004. “Taxonomic Study on Chinese Centipedes of the Order Scolopendromorpha.”
Diss. Hebei University, China.
5
Table 1. List of taxa, National Sun Yat-Sen University (NSYSU) accession numbers, localities, and
GenBank accession numbers for each COI sequence.
Taxon
Accession number
Locality
Scutigeromorpha
Scutigera coleoptrata gb
GenBank / AJ507061
Italy
Thereuopoda clunifera gb
GenBank / DQ222171
Japan
Thereuonema turkestana gb
GenBank / DQ201427
Uzbekistan
Craterostigmus crabilli gb
GenBank / EU024618
New Zealand
Craterostigmus tasmanianus gb
GenBank / EU024611
Australia
Anopsobius neozelanicus gb
GenBank / AF334313
Unknown
Bothropolys sp gb.
GenBank / AY691655
Unknown
Lithobius forficatus gb
GenBank / AF309492
Unknown
Mecistocephalus sp. J
NSYSU / J0041409
Kinmen islet (next to China)
Mecistocephalus sp A4
NSYSU / A4091523
South Taiwan
Mecistocephalus guildingii gb
GenBank / AY288747
Unknown
Geophilus electricus gb
GenBank / AY288750
Unknown
Strigamia maritime gb
GenBank / AY288753
Unknown
Pachymerium ferrugineum gb
GenBank / AF370838
Unknown
Bothriogaster signata gb
GenBank / AY288749
Unknown
Himantarium gabrielis gb
GenBank / AY288748
Unknown
Scolopendra multidens F
NSYSU / F0051206
North Taiwan
Scolopendra multidens G
NSYSU / G0051208
North Taiwan
Scolopendra multidens H
NSYSU / H0051212
North Taiwan
Scolopendra subspinipes H1
NSYSU / F1062308
South Taiwan
Scolopendra subspinipes L
NSYSU / L0052601
East Taiwan
Scolopendra subspinipes Z1
NSYSU / Z1081103
South Taiwan
Scolopendra mutilans A1
NSYSU / A1060603
North Taiwan
Scolopendra mutilans B1
NSYSU /B1060605
South Taiwan
Scolopendra mutilans W1
NSYSU / W1080618
East Taiwan
Scolopendra mutilans H1
NSYSU /H1062905
Middle Taiwan
Scolopendra mutilans G1
NSYSU /G1062309
North Taiwan
Scolopendra mutilans M
NSYSU / M0052602
South Taiwan
Scolopendra morsitans E1
NSYSU / E1061617
Kinmen Islet (next to China)
Craterostigmomorpha
Lithobiomorpha
Geophilomorpha
Scolopendromorpha
Scolopendridae
Scolopendrinae
6
Scolopendra morsitans C4
NSYSU / C4090202
Magong Islet (Pescadores)
Scolopendra viridis gb
GenBank / DQ201431
Unknown
Cormocephalus monteithi gb
GenBank / DQ201430
Unknown
Otostigmus scaber D
NSYSU / D0050505
Middle Taiwan
Otostigmus scaber N1
NSYSU / N1070110
South Taiwan
Otostigmus scaber A3
NSYSU / A3081414
Keelung islet (North Taiwan)
Otostigmus scaber M1
NSYSU / M1070109
South Taiwan
Otostigmus aculeatus L1
NSYSU / L1070108
East Taiwan
Otostigmus aculeatus Q1
NSYSU / Q1070116
South Taiwan
Otostigmus aculeatus A
NSYSU / A0050501
Middle Taiwan
Otostigmus astenus K1
NSYSU / K1070107
Lanyu Islet (East Taiwan)
Otostigmus glaber B4
NSYSU / B4092901
Itu Aba Island (South China See)
Rhysida immarginata B
NSYSU / B0050502
Middle Taiwan
Rhysida immarginata O1
NSYSU /O1070112
Middle Taiwan
Rhysida immarginata P1
NSYSU / P1070113
South Taiwan
Rhysida immarginata V1
NSYSU / V1080617
Lieyu Islet (next to China)
Rhysida immarginata Y1
NSYSU / Y1080613
Lieyu Islet (next to China)
Rhysida longipes X1
NSYSU / X1080416
Middle Taiwan
Rhysida longipes C
NSYSU / C0050503
South Taiwan
Rhysida longipes E
NSYSU / E0050506
Middle Taiwan
Rhysida longipes D4
NSYSU / D4090203
Siyu Islet (Pescadores)
Rhysida nuda gb
GenBank / DQ201432
Unknown
Ethmostigmus rubripes gb
GenBank / AF370836
Unknown
Alipes crotalus gb
GenBank / AY288742
Swaziland
Scolopocryptops rubiginosus S1
NSYSU / S1072008
South Taiwan
Scolopocryptops rubiginosus N
NSYSU / N0052610
North Taiwan
Scolopocryptops rubiginosus R1
NSYSU / R1070702
North Taiwan
Scolopocryptops capillipedatus T1
NSYSU / T1072016
East Taiwan
Scolopocryptops capillipedatus E4
NSYSU / E4093006
Middle Taiwan
Scolopocryptops capillipedatus F4
NSYSU / F4093007
North Taiwan
Scolopocryptops curtus J1
NSYSU / J1070106
North Taiwan
Scolopocryptops curtus U1
NSYSU / U1072017
East Taiwan
Scolopocryptops nigridius gb
GenBank / AY288744
Unknown
Scolopocryptops sexspinosus gb
GenBank / AY288745
Unknown
Theatops posticus gb
GenBank / AY288746
Unknown
Cryptops japonicus I
NSYSU / I0041407
South Taiwan
Cryptops japonicus G4
NSYSU / G4093009
East Taiwan
Otostigmidae
Scolopocryptopidae
Cryptopidae
7
Cryptops japonicus H4
NSYSU / H4093010
East Taiwan
Cryptops spinipes gb
GenBank / AY288743
Unknown
Fig. 1. The classification of Scolopendromorpha by Attems (1930), the genera in parentheses are
used in this study.
Fig. 2. The classification of Scolopendromorpha by Schileyko (1997), the genera in parentheses are
used in this study.
8
Fig. 3 The classification of Scolopendromorpha by Shelley (2002), the genera in parentheses are
used in this study.
Fig. 4 The classification of Scolopendromorpha by Chao and Chang (2006), the genera in
parentheses are used in this study.
9
Fig. 5. Cladogram derived from Maximum Likelihood Coding analysis of the barcoding sequence
data using a distance search followed by TBR branch swapping. Cladogram is rooted from 3 taxa of
Scutigeromorpha.
10
Fig. 6. Neighbor-Joining tree corresponding to the barcoding sequence data analyses. At left is
phylogram, it is rooted from 3 taxa of Scutigeromorpha; using a bootstrap search with 1000
replicates of random addition sequences followed by TBR branch swapping, and with 50%
majority-rule consensus tree at right, displays the bootstrap proportions at the nodes.
11
Fig. 7. Maximum Parsimony tree corresponding to the barcoding sequence data analyses. At left is
rectangular cladogram, it is rooted from 3 taxa of Scutigeromorpha; using a bootstrap search with
1000 replicates of random addition sequences followed by TBR branch swapping, and with 50%
majority-rule consensus tree at right, displays the bootstrap proportions at the nodes.
12
1
0.8
Identity
C_jap_H4
C_jap_G4
C_jap_I
Sc_cur_U1
Sc_cur_J1
Sc_rub_S1
Sc_rub_R1
Sc_rub_N
Sc_cap_E4
Sc_cap_F4
Sc_cap_T1
S_mul_G
S_mul_F
S_mul_H
S_sub_L
S_sub_C1
S_sub_F1
S_mut_H1
S_mut_B1
S_mut_W1
S_mut_A1
S_mut_G1
S_mut_M
S_mor_C4
S_mor_E1
R_imm_Y1
R_imm_V1
R_imm_B
R_imm_P1
R_imm_O1
R_lon_D4
R_lon_X1
R_lon_E
R_lon_C
O_sca_M1
O_sca_A3
O_sca_N1
O_sca_D
O_acu_L1
O_acu_Q1
O_acu_A
O_gla_B4
O_ast_K1
B.
A.
0.9
0.7
Specimen
maximum interspecies identity
minimum intraspecies identity
Fig. 8. The minimum intraspecies identity and the maximum interspecies identifies of the barcoding
sequence (678 bp) for 43 specimens of 13 taxa of Scolopendromorpha.
Fig. 9. Scolopendra multidens Newport, 1844 from Taiwan. A. a juvenile. B. an adult.
13