Barcodes of scolopendromorpha centipedes of taiwan chao, Jui-Lung




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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). 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

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 (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 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.



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

Craterostigmomorpha







Craterostigmus crabilli gb

GenBank / EU024618

New Zealand

Craterostigmus tasmanianus gb

GenBank / EU024611

Australia

Lithobiomorpha







Anopsobius neozelanicus gb

GenBank / AF334313

Unknown

Bothropolys sp gb.

GenBank / AY691655

Unknown

Lithobius forficatus gb

GenBank / AF309492

Unknown

Geophilomorpha







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

Scolopendromorpha







Scolopendridae







Scolopendrinae







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)

Scolopendra morsitans C4

NSYSU / C4090202

Magong Islet (Pescadores)

Scolopendra viridis gb

GenBank / DQ201431

Unknown

Cormocephalus monteithi gb

GenBank / DQ201430

Unknown

Otostigmidae







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

Scolopocryptopidae







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

Cryptopidae







Cryptops japonicus I

NSYSU / I0041407

South Taiwan

Cryptops japonicus G4

NSYSU / G4093009

East Taiwan

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.



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.



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.



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.



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.



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.

A. B.

Fig. 9. Scolopendra multidens Newport, 1844 from Taiwan. A. a juvenile. B. an adult.








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