INTERNATIONAL JOURNAL OF AGRICULTURE & BIOLOGY ISSN Print: 1560–8530; ISSN Online: 1814–9596 10–607/SRZ/2011/13–4–553–558 http://www.fspublishers.org
Full Length Article
External Morphological Comparison, Taxonomic Revision and Molecular Differentiation of the Four Economically Important Species of Earthworm in Thailand
WIRIYA LOONGYAI1, PHUWADOL BANGRAK† AND SOMCHAI CHANTSAVANG Department of Animal Science, Faculty of Agriculture, Kasetsart University, Bangkok 10900 Thailand †School of Science, Walailak University, Thasala, Nakhon Si Thammarat 80160 Thailand 1Corresponding author’s e-mail: [email protected]; [email protected]
ABSTRACT
Four economically important species of earthworm were cultured and the external and internal characters of adult clitellate earthworms were studied. Partial sequences for ribosomal 16S rDNA and subunit one for mitochondrial cytochrome c oxidase (COI) of four earthworm species were obtained. The result of sequence analysis combined with taxonomic characters could distinguish the different species of earthworm. Morphology and nucleotide sequence of two genes for the red worm (Pheretima peguana) were distinct from Eudrilus eugeniae but were similar to the blue worm (Perionyx excavatus) and Lao worm (P. excavates) and therefore, it was classified as a new species, Perionyx sp. 1. Moreover, Eudrilus eugeniae was evidently defined as the same genus and species. Interestingly, the blue worm and Lao worm were morphologically similar to Perionyx sp. However, the molecular data of 16S rDNA could not differentiate in taxa of those two species. COI nucleotide sequence analyses showed the presence of divergent lineages between two species, suggesting the blue worm and Lao worm could be described as Perionyx sp. 2 and Perionyx sp. 3, respectively. © 2011 Friends Science Publishers
Key Word: Cytochrome c oxidase subunit I; Eudrilus eugeniae; Perionyx sp.; Ribosomal 16S rDNA
INTRODUCTION the red worm has led to situations in which the same or different species receives names. However, the blue worm Over 10,000 species of earthworms exist around the (P. excavatus) and Lao worm (P. excavatus?) are also world and only 31 described species of earthworms inhabit similar in body size and coloration. Interestingly, the Lao Thailand (Gates, 1939). The culture of worms on a large worm has a smaller tail than the blue worm, although they scale is in high demand for the production of both protein have been identified as same species. Such problems have and biofertilizer. In every region of the world, many species led to misidentification of cultured earthworms species. The of earthworm are cultured namely Eisenia fetida, Lumbricus taxonomic technique was used to identify morphological terrestris, Perionyx excavatus and Eudrilus eugeniae in all characters of earthworm species. The problem of taxonomic part of the world. In Thailand, these four economically identification is morphological characters, both external and important species of earthworm are of great importance in internal, often show high variability between individuals the vermicomposting of a wide variety of organic wastes (Pop et al., 2003; Reynolds, 2004; Chang et al., 2007; and also a potential source of protein for animal Iglesias Briones et al., 2009). However, previous reports consumption. E. eugeniae is widely distributed in warmer have shown the evidence that in earthworm systematics, parts of the world and cultured as the "African reproductive organs including the clitella, male pores, Nightcrawler" (AF). Introduced species are commonly female pores, testes, ovaries, spermathecae, and prostate found over a large area of tropical Asia, namely, the blue glands are believed to be less affected by environmental worm or Indian worm (Perionyx excavates), red worm influences through time and are evolutionarily more (Pheretima peguana) and earthworm from Lao (P. conservative (Gates, 1972; Blakemore, 2002). excavates) as previously described by Ayamuang (2000). In recent times, morphological characters still are used However, the AF earthworm (E. eugeniae) and red worm to characterize earthworm species. Stephenson (1930) (P. peguana) are almost similar in body size and coloration. reported that the genital system is much more conservative The lack of agreement in their ranking as diagnostic and resistant to evolutionary change than the somatic characters for taxonomic and genetic purposes, especially system. Thus, molecular techniques are used to improve the
To cite this paper: Loongyai, W., P. Bangrak and S. Chantsavang, 2011. External morphological comparison, taxonomic revision and molecular differentiation of the four economically important species of earthworm in Thailand. Int. J. Agric. Biol., 13: 553–558
LOONGYAI et al. / Int. J. Agric. Biol., Vol. 13, No. 4, 2011 understanding of disputed taxonomic problems of different at 72°C for 10 min. A fragment of the mitochondrial earthworm groups that are rather scarce and sporadic (Pop et cytochrome c oxidase subunit I sequences (COI) was al., 2004; Heethoff et al., 2004; Dupont, 2009). Recently, amplified using the universal primers HCO2198 (5′- several genes have been chosen for identifying the TAAACTTCAGGGTGACCAAAAAATCA-3′) and earthworm species through DNA barcoding such as LCO1490 (5′-GGTCAACAAATCATAAAGATATTGG- mitochondrial cytochrome-c oxidase I, COI (Pop et al., 3′) (Folmer et al., 1994) following the same procedures as 2003; Chang et al., 2007; Huang et al., 2007; Pop et al., for 16S rDNA fragments. The PCR products were checked 2007; Iglesias Briones et al., 2009; Otomo et al., 2009; on 1.5% agarose gel electrophoresis and cloned into pGEM- Pérez Losada et al., 2009; Richard et al., 2010), 18S (Pop et T Easy (Promega, USA). DNA sequencing was performed al., 2003, 2007), 28S (Pérez Losada et al., 2009) and 16S in both directions by BioDesign Co., Ltd., Thailand. ribosomal DNA (Pop et al., 2003; Pop et al., 2007; Iglesias Sequence analysis: Sequences were aligned using Clustal Briones et al., 2009; Pérez Losada et al., 2009). X, version 1.8 (Thompson et al., 1997), and were improved Mitochondrial DNA (mtDNA) has been widely used manually using BIOEDIT 5.0.9 sequence alignment in molecular taxonomy of animals, because it evolves much software (Hall, 1999). All alignment illustrations were more rapidly than nuclear DNA and it has been used to created with the program GENEDOC, version 2.6.001 identify the differences between closely related species (Nicholas & Nicholas, 1997). The nucleotide divergence of (Brown et al., 1979; Moore, 1995; Mindell et al., 1997; 16S rDNA and COI gene from four species was analyzed Hebert et al., 2003; Otomo et al., 2009). Thus, DNA by PAUP portable version 4.0b10 for Unix (Swofford, barcodes based on a fragment of the COI and 16S rDNA 2002). genes have been demonstrated to work well for species identification. The present study explores the utility of DNA RESULTS sequences from these genes to identify the four species and taxonomic status of poorly known earthworm species. External and internal characters: The adult clitellate earthworms used in this study are illustrated in Fig. 1. MATERIALS AND METHODS External and internal characters were used to identify different species of earthworm as described by Gates Earthworm culture and preservation: The four (1972). Different parameters including length, clitellum economically important species of earthworm, African width, segment number, first dorsal pore position, setae, Nightcrawler, red worm, blue worm and Lao worm were prostomium, position of female and male pore, penail setae, cultured in a plastic box under controlled environmental number and position of spermathecae, position of last heart, conditions. Air-dried powdered chicken manure moistened position of seminal vesicles and position of prostate glands with water and kept for one week for thermal stabilization were used for the identification of earthworm species (Table and microbial initiation of degradation. The culture beds I). The result showed that E. eugeniae was defined as the were prepared by transferring stabilized chicken manure as same genus and species. The morphology of P. peguana food (in the ratio of 1:50 worm to manure) to round plastic was similar to P. macintoshi, but some differences such as boxes. The moisture content of the food was maintained at the number of clitellum, position of first dorsal pore, thick 75 to 80% by sprinkling it with tap water whenever septa at position 6-7 and lack of gizzard. Therefore, the red required. The food was replaced at monthly intervals by worm (P. peguana) was classified as Perionyx sp. 1. The stabilized stock chicken manure, to avoid scarcity of food. species of the blue worm (P. excavatus) and earthworm The adult clitellate earthworms (six individuals for each from Lao (P. excavates?) were morphologically similar to population) were killed in 30% ethanol and preserved in Perionyx tenuis. Many species were compared and it was 95% ethanol at room temperature. All specimens in this found that the position of spermathecae was distinct from P. study were identified following the taxonomic key in Gate tenuis. The two species of the Perionyx species complex, the (1972). blue worm and Lao worm, were morphologically similar DNA extraction, PCR amplification and DNA while body length, clitellum width, number of segments and sequencing: Total genomic DNA was isolated from one to clitellum segments were different. Thus, the blue worm and six individuals per species following the phenol/chloroform Lao worm were described as new species identified as method and washing with ethanol. DNA was diluted to the Perionyx sp. 2 and Perionyx sp. 3, respectively. working concentration with TE-buffer. The 16S rDNA The 16S ribosomal DNA (16S rDNA): The 16S rDNA fragments were amplified using the primers 16sar (5′- sequences were generated from six adult earthworms. The CGCCTGTTTATCAAAAACAT-3′) and 16sbr (5′- samples used in the alignment analyses and the CCGGTYTGAACTCAGATCAYGT-3′) (Palumbi et al., corresponding GenBank accession numbers of the 16S 1991). PCR amplification was carried out in a 50 μL total rDNA sequences (Table II). Nucleotide sites ranging volume, using 1 cycle at 94°C for 1 min, followed by 35 between 482 to 485 bp were sequenced and showed cycles of denaturation for 30 s at 94°C, annealing for 30 s at alignments of nucleotide sequences of E. eugeniae, 54°C, and extension for 50 s at 72°C, with a final extension Perionyx sp. 1, Perionyx sp. 2 and Perionyx sp. 3 (Fig. 2).
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Table I: Morphological comparison of Eudrilus eugeniae, Perionyx sp. 1, P. sp. 2 and P. sp. 3 according to the original descriptions
Eudrilus eugeniae Perionyx sp. 1 Perionyx sp. 2 Perionyx sp. 3 Body length (mm) 140-145 122-134 85-120 73-90 Clitellum width (mm) 5.3-5.5 4.0-4.2 1.4-1.7 1.5-1.7 Number of segments 184-188 166-175 149-185 179-181 Clitellum segments 14-18 13-17 13-17 14-16 First dorsal pore position ND 5-6 ND ND Setae Closely paired Lumbricine Lumbricine Lumbricine Prostomium Epilobous Epilobous Epilobous Epilobous Position of female pore 14 14 13 13 Position of male pore 17-18 18 18 18 Penail setae - - 1 pairs 1 pairs Number of spermathecae ND 2 pairs 3 pairs 3 pairs Position of spermathecae ND 7-8, 8-9 6-7, 7-8, 8-9 6-7, 7-8, 8-9 Position of last heart 12 12 13 13 Position of seminal vesicles 11, 12 11, 12 11, 12 11, 12 Position of prostate glands 17-23 18 18 18 ND: no data
Table II: Genes, Species and GenBank accession numbers for sequenced earthworms
Gene Species Nucleotide length (bp) Accession number 16S rDNA Eudrilus eugeniae 482 HM219175 Perionyx sp.1 485 HM219176 Perionyx sp.2 483 HM219177 Perionyx sp.3 483 HM219178 Cytochrome c oxidase subunit I sequences Eudrilus eugeniae 657 HM219171 Perionyx sp.1 657 HM219172 Perionyx sp.2 657 HM219173 Perionyx sp.3 657 HM219174
Table III: Comparison of nucleotide sequence identity of the 16S rRNA and COI of Eudrilus eugeniae, Perionyx sp. 1, Perionyx sp. 2 and Perionyx sp. 3 to those deposited in GenBank
Species Accession number Nucleotide length (bp) Identity (%) 16S rRNA Eudrilus eugeniae Diporochaeta sp. AF406574 463 84 Allolobophora chlorotica AM774393 479 84 Pontodrilus litoralis AF406586 468 84 Perionyx sp. 1 Perionyx excavatus AF406582 455 95 Diporochaeta sp. AF406574 463 87 Spenceriella sp. AF406572 465 87 Perionyx sp. 2 Perionyx excavatus AF406582 455 90 Eisenia fetida DQ257296 455 90 Pontodrilus litoralis AF406586 468 87 Perionyx sp. 3 Perionyx excavatus AF406582 455 90 Eisenia fetida DQ257296 455 90 Pontodrilus litoralis AF406586 468 87 Cytochrome c oxidase subunit I Eudrilus eugeniae Diplocardia komareki EF156634 601 83 Glossoscolecidae sp. GU013961 657 82 Amynthas wulinensis DQ224180 658 82 Perionyx sp. 1 Megascolecidae sp. GU013873 657 83 Metaphire nanaoensis AY960805 1056 83 Metaphire formosae AY960807 1056 83 Perionyx sp. 2 Megascolecidae sp. GU013856 657 83 Metaphire glareosa AY960803 1056 83 Acanthodrilidae sp. GU014211 657 83 Perionyx sp. 3 Megascolecidae sp. GU013856 657 83 Metaphire glareosa AY960803 1056 83 Acanthodrilidae sp. GU014211 657 83
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Table IV: Nucleotide structure of 16S rDNA and Fig. 1: The four economically important species of Cytochrome c oxidase sequences in earthworms earthworm: Eudrilus eugeniae (A), Perionyx sp. 1 (B) Perionyx sp. 2 (C) and Perionyx sp. 3 (D) Species 16S rDNA COI GC% AT% GC/AT GC% AT% GC/AT Eudrilus eugeniae 33.36 66.64 0.50 41.65 58.35 0.71 Perionyx sp. 1 39.18 60.82 0.64 42.60 57.40 0.74 Perionyx sp. 2 37.11 62.89 0.59 40.24 59.76 0.67 Perionyx sp. 3 37.11 62.89 0.59 40.24 59.76 0.67
Table V: The sequence divergence of the 16S rRNA and COI gene between Eudrilus eugeniae, Perionyx sp. 1, Perionyx sp. 2 and Perionyx sp. 3 A B
16S rDNA Perionyx Perionyx Perionyx E. eugeniae sp. 2 sp. 3 sp. 1 Perionyx sp. 3 0.00% Perionyx sp. 1 10.59% 10.59% E. eugeniae 18.62% 18.62% 17.93%
Cytochrome c oxidase subunit I
Perionyx sp. 3 0.16% C D Perionyx sp. 1 18.48% 18.63% E. eugeniae 19.88% 19.72% 19.57% GenBank [accession number AF406582]), respectively. Moreover, the sequence of the COI obtained from the No variation was observed in the sequences between present study, E. eugeniae showed 83% homology to D. Perionyx sp. 2 and Perionyx sp. 3. Interestingly, the komareki (601 bp compared, GenBank [accession number nucleotide of Perionyx sp. 1 differs from Perionyx sp. 2 and EF156634]). The sequence of COI determined for isolates Perionyx sp. 3 at 32 sites in the nucleotide sequence. of Perionyx sp. 1, Perionyx sp. 2 and Perionyx sp. 3 showed Between E. eugeniae and Perionyx sp., divergent sequences a homology of 83% with the sequence of Megascolecidae of 16S rDNA were observed (Table V). Sequence sp. (657 bp compared, GenBank [accession number divergence between E. eugeniae and Perionyx sp. 1 was GU013856]). The relative nucleotide structure of analyzed around 18.62%, whereas no sequence divergence between genes is presented in Table IV. Nucleotides base Perionyx sp. 2 and Perionyx sp. 3 was observed. composition was similar for all sequences with a strong AT The cytochrome c oxidase subunit 1 (COI): Nucleotide base (%). The percentage of GC content of 16S rDNA sites of 657 bp COI sequences were determined and ranged from 33.36% to 39.18%, while the GC content of GenBank accession numbers (Table II). The alignment of COI gene was stronger than 16S rDNA, the GC content nucleotide sequences (Fig. 3) was analyzed. The results ranged from 40.24 to 42.60%. showed that the nucleotide sequences of Perionyx sp. 2 and Perionyx sp. 3 differed at one nucleotide sites (A: Perionyx DISCUSSION sp. 2 & T: Perionyx sp. 3). Therefore, it was found that these worms revealed, which confirmed the variations at the COI Recently, four economically important species of region. The result of alignment was also displayed the intra- earthworm were identified in Thailand, namely, the AF, specific variation (49 sites) among Perionyx sp. and the blue worm, Lao worm and red worm. However, data is inter-specific variation in nucleotide sequence between E. lacking for those earthworm species (except E. eugeniae & eugeniae and Perionyx sp. (Fig. 3). The results of nucleotide P. excavatus) concerning morphological and genetic divergence of the COI gene (Table V) indicated 19.57% information that is used to determine the precise species and divergence between E. eugeniae and Perionyx sp. 1. eliminate continued confusion about their taxonomic. As Interestingly, the nucleotide divergence between Perionyx previously reported (The information was published in Thai sp. 2 and Perionyx sp. 3 was 0.16%. journal & Thai website), the AF, blue worm, Lao worm and Sequence analysis: The comparison of nucleotide sequence red worm were identified as E. eugeniae, P. excavatus, P. identity of 16S rDNA and COI sequence in the four taxa excavatus? (Ayamuang, 2000) and P. peguana, was shown in Table III. The sequence of E. eugeniae respectively. In the present study, taxonomic identification exhibited 84% homology with the sequence of D. komareki and molecular techniques were used to identify and improve (463 bp compared, GenBank [accession number the understanding of disputed taxonomic problems of the AF406574]). Similarly, sequence of Perionyx sp. 1, abovementioned four economically important species of Perionyx sp. 2 and Perionyx sp. 3 showed 95%, 90% and earthworm in Thailand. External and internal characters of 90% homology to P. excavatus (455 bp compared, earthworm were used to identify species in this study and
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Fig. 2: The nucleotide sequence alignment of 16S rDNA Fig. 3: The nucleotide sequence alignment of of Eudrilus eugeniae (Eue), Perionyx sp. 1 (Psp1), cytochrome c oxidase subunit I (COI) of Eudrilus Perionyx sp. 2 (Psp2) and Perionyx sp. 3 (Psp3) eugeniae (Eue), Perionyx sp. 1 (Psp1), Perionyx sp. 2 (Psp2) and Perionyx sp. 3 (Psp3)
* 2 0 * 4 0 * 6 0 P s p 2 : T G C C A C A C G C A T A A A - A T G T G T G G T A A C T - C C T G C C C A G T G A C A A T C A G T T C A A C G G C C G : 5 8 P s p 3 : T G C C A C A C G C A T A A A - A T G T G T G G T A A C T - C C T G C C C A G T G A C A A T C A G T T C A A C G G C C G : 5 8 * 2 0 * 4 0 * 6 0 P s p 1 : T G C C A T A T G T A T A C A C A T A T A A G G T A A T T T C C T G C C C A G T G A C G A A - A G T T C A A C G G C C G : 5 9 P s p 2 : A C A C T A T A C T T T A A T T T A G G A G T T T G A G C T G G T A T G A T T G G A G C C G G C A T A A G A T T A C T A : 6 0 P s p 3 : A C A C T A T A C T T T A T T T T A G G A G T T T G A G C T G G T A T G A T T G G A G C C G G C A T A A G A T T A C T A : 6 0 E u e : T G C T T T A T G A A T A A A T A T A T A A A G T A T C T - C C T G C C C A G T G A T A T A - - - T T C A A C G G C C G : 5 6 P s p 1 : A C A C T A T A C T T T A A T A T T G G C G T T T G A G C T G G A A T A G T T G G A G C A G G T A T G A G A C T C C T T : 6 0 E u e : A C A C T G T A C T T T A T T C T A G G T G T G T G A G C T G G T A T A A T C G G T G C T G G T A T A A G A C T T C T T : 6 0
* 8 0 * 1 0 0 * 1 2 0 P s p 2 : C G G T A T C C T A A C C G T G C A A A G G T A G C A T A A T C A T T T G C T C A T T A A T T G T G G G C T A G C A T G : 1 1 8 * 8 0 * 1 0 0 * 1 2 0 P s p 3 : C G G T A T C C T A A C C G T G C A A A G G T A G C A T A A T C A T T T G C T C A T T A A T T G T G G G C T A G C A T G : 1 1 8 P s p 2 : A T T C G A A T T G A A C T A A G A C A G C C G G G G G C A T T C C T T G G C A G C G A T C A A C T A T A T A A T A C A : 1 2 0 P s p 3 : A T T C G A A T T G A A C T A A G A C A G C C G G G G G C A T T C C T T G G C A G C G A T C A A C T A T A T A A T A C A : 1 2 0 P s p 1 : C G G T A T C C T A A C C G T G C A A A G G T A G C A T A A T C A C T T G C C T A T T A A T T G T A G G C T A G A A T G : 1 1 9 P s p 1 : A T T C G A A T C G A A C T A A G A C A A C C T G G A G C A T T T C T A G G A A G T G A T C A A C T A T A T A A C A C A : 1 2 0 E u e : C G G T A T C C T A A C C G T G C G A A G G T A G C A T A A T C A C T T G C C T A T T A A T T G T A G G C T A G A A T G : 1 1 6 E u e : A T T C G A A T T G A A C T A A G A C A G C C G G G T G C T T T T C T A G G A A G A G A C C A A C C C T A T A A C A C T : 1 2 0
* 1 4 0 * 1 6 0 * 1 8 0 * 1 4 0 * 1 6 0 * 1 8 0 P s p 2 : A A A G G A T A A A C G A A A T A A A A T C T G T C T C A T T T A G T A A C C T A A A A A T T A T T C T C C A T A T G A : 1 7 8 P s p 2 : A T T G T A A C C G C A C A C G C A T T C T T A A T A A T T T T C T T C C T A G T A A T A C C A G T A T T C A T T G G T : 1 8 0 P s p 3 : A A A G G A T A A A C G A A A T A A A A T C T G T C T C A T T T A G T A A C C T A A A A A T T A T T C T C C A T A T G A : 1 7 8 P s p 3 : A T T G T A A C C G C A C A C G C A T T C T T A A T A A T T T T C T T C C T A G T A A T A C C A G T A T T C A T T G G T : 1 8 0 P s p 1 : A T C G T G A C A G C C C A C G C A T T C T T A A T A A T T T T C T T C T T A G T T A T G C C A G T A T T T A T C G G C : 1 8 0 P s p 1 : A A C G G A T C A A C G A A A T A A A A C C T G T C T C A G T T A G C A A C C T A A A A A T T A T T C T C C A T A C G A : 1 7 9 E u e : A T C G T T A C A G C T C A T G C T T T T C T A A T A A T C T T T T T C C T T G T A A T A C C A G T T T T T A T T G G C : 1 8 0 E u e : A A T G G A T G A A C G A A A G A T A A G C T G T C T C A G T T A G T A A T C T A A A A A T T A A T A T C T A A T T G A : 1 7 6
* 2 0 0 * 2 2 0 * 2 4 0 * 2 0 0 * 2 2 0 * 2 4 0 P s p 2 : G G G T T C G G A A A C T G A C T G C T C C C A C T A A T A C T A G G T G C T C C C G A C A T A G C A T T C C C A C G A : 2 4 0 P s p 2 : A G A A A T A T G G A T A C A A T C G A A G G A C A A G A A G A C C C T A T A G A G C T T T A - - T T T A A A T C A T A : 2 3 6 P s p 3 : G G G T T C G G A A A C T G A C T G C T C C C A C T A A T A C T A G G T G C T C C C G A C A T A G C A T T C C C A C G A : 2 4 0 P s p 3 : A G A A A T A T G G A T A C A A T C G A A G G A C A A G A A G A C C C T A T A G A G C T T T A - - T T T A A A T C A T A : 2 3 6 P s p 1 : G G A T T C G G A A A T T G G T T A C T C C C G C T C A T A C T A G G A G C A C C G G A C A T A G C A T T C C C A C G T : 2 4 0 E u e : G G A T T T G G A A A T T G A T T A C T T C C A T T A A T A C T G G G A G C G C C C G A T A T G G C A T T C C C C C G A : 2 4 0 P s p 1 : A G A A A T A T G G A T A A A A T C G A A G G A C A A G A A G A C C C T A T A G A G C T T T A - - T T T A A G T C A C A : 2 3 7 E u e : A G A A G A T T A G A T A A A A T C G A A G G A C A A G A A G A C C C T A T A G A G C T T T A A T T T T T A A C T A T A : 2 3 6 * 2 6 0 * 2 8 0 * 3 0 0 P s p 2 : T T A A A T A A C A T A A G A T T C T G A T T A T T A C C G C C A T C T C T A A T T T T A C T T G T T T C C T C A G C A : 3 0 0 * 2 6 0 * 2 8 0 * 3 0 0 P s p 3 : T T A A A T A A C A T A A G A T T C T G A T T A T T A C C G C C A T C T C T A A T T T T A C T T G T T T C C T C A G C A : 3 0 0 P s p 2 : G A T T A T A - T C T A A T G T T A A A A T T C G G T T G G G G C G A C C A A G G G A - A A A T A A A T C A C C C T A T : 2 9 4 P s p 1 : T T A A A T A A T A T G A G A T T C T G A C T G C T A C C T C C G T C A C T C A T T C T A C T A G T C T C T T C A G C T : 3 0 0 P s p 3 : G A T T A T A - T C T A A T G T T A A A A T T C G G T T G G G G C G A C C A A G G G A - A A A T A A A T C A C C C T A T : 2 9 4 E u e : C T A A A T A A C T T A A G A T T T T G A T T A T T A C C T C C T T C A C T A A T T C T C T T A G T T T C G T C A G C T : 3 0 0
P s p 1 : G A C T A T A A T C T A G T G T A A T A A T T C G G T T G G G G C G A C C A A G G G A - A T A A A T A T C A C C C T G T : 2 9 6 E u e : A A G C - - - - T A T A G A A T A A A T A T T - G G T T G G G G C G A C C A A G G T A T A A A A A A A T C A T C C T T A : 2 9 1 * 3 2 0 * 3 4 0 * 3 6 0 P s p 2 : G C C G T A G A A A A G G G A G C T G G A A C C G G A T G A A C A G T T T A T C C A C C A C T A G C A A G A A A T A T T : 3 6 0 P s p 3 : G C C G T A G A A A A G G G A G C T G G A A C C G G A T G A A C A G T T T A T C C A C C A C T A G C A A G A A A T A T T : 3 6 0 * 3 2 0 * 3 4 0 * 3 6 0 P s p 1 : G C T G T A G A A A A A G G T G C T G G C A C A G G A T G A A C A G T T T A C C C C C C A C T C G C A A G A A A T A T T : 3 6 0 P s p 2 : A T A A - C A A A G A T T T A T T A A T C T A G T A G C T G A C C C T T A T - - A A G A C C A C A A G A T C A A G C T A : 3 5 1 E u e : G C A G T T G A A A A G G G T G C A G G T A C A G G A T G A A C T G T T T A C C C G C C A C T T G C A A G A A A T C T T : 3 6 0
P s p 3 : A T A A - C A A A G A T T T A T T A A T C T A G T A G C T G A C C C T T A T - - A A G A C C A C A A G A T C A A G C T A : 3 5 1 P s p 1 : A A C A - C A A A G A T C T A T A A A T C C T G T A A T T G A C C C T T A T - - A A G A T C A C A A G A T C A A G C T A : 3 5 3 * 3 8 0 * 4 0 0 * 4 2 0 E u e : A C T A T C A A C G A T A A A T T C A T C T T T A T A A A G A C C C T T T T T A A A G A T C A T A A G T T T A - G C T A : 3 5 0 P s p 2 : G C A C A T G C C G G G C C A T C A G T A G A T C T G G C A A T T T T T T C T T T A C A C T T A G C T G G T G T A T C A : 4 2 0 P s p 3 : G C A C A T G C C G G G C C A T C A G T A G A T C T G G C A A T T T T T T C T T T A C A C T T A G C T G G T G T A T C A : 4 2 0 P s p 1 : G C C C A T G C C G G C C C T T C A G T A G A T T T G G C C A T T T T C T C T C T A C A C C T T G C A G G T G C A T C A : 4 2 0 * 3 8 0 * 4 0 0 * 4 2 0 E u e : G C C C A T G C C G G G C C C T C A G T A G A C C T A G C C A C T T T C T C T C T T C A T C T T G C A G G G G C A T C A : 4 2 0 P s p 2 : C C T T A G G G A T A A C A G G C T A A T C T C A T T C A A G A G T T C A T A T C T A C A A T G G G G T T T G G C A C C : 4 1 1
P s p 3 : C C T T A G G G A T A A C A G G C T A A T C T C A T T C A A G A G T T C A T A T C T A C A A T G G G G T T T G G C A C C : 4 1 1 * 4 4 0 * 4 6 0 * 4 8 0 P s p 1 : C C T T A G G G A T A A C A G G C T A A T C C C A C T C A A G A G T C C A T A T C A A C A G T G G G G T T T G G C A C C : 4 1 3 P s p 2 : T C T A T C T T A G G C G C C A T C A A C T T T A T T A C A A C A G T A A T T A A T A T G C G A T G A T C A G G A C T T : 4 8 0 E u e : C C T T A G G G A T A A C A G G C T A A T C C T A T T A A A G A G T C C T T A T C T A T A A T G G G G A T T G G C A C C : 4 1 0 P s p 3 : T C T A T C T T A G G C G C C A T C A A C T T T A T T A C A A C A G T A A T T A A T A T G C G A T G A T C A G G A C T T : 4 8 0 P s p 1 : T C C A T T T T A G G T G C T A T C A A T T T T A T T A C T A C A G T A A T C A A C A T A C G A T G A T C G G G A C T T : 4 8 0 E u e : T C T A T T T T G G G G G C A A T T A A C T T T A T T A C A A C A G T G A T T A A T A T A C G A T G A T C C G G T C T T : 4 8 0 * 4 4 0 * 4 6 0 * 4 8 0 P s p 2 : T C G A T G T T G G C T T A G G - T A A C T A T A T G G C G C A A A A G C T A T A C T A T G T T G G T T T G T T C A A C : 4 7 0 * 5 0 0 * 5 2 0 * 5 4 0 P s p 3 : T C G A T G T T G G C T T A G G - T A A C T A T A T G G C G C A A A A G C T A T A C T A T G T T G G T T T G T T C A A C : 4 7 0 P s p 2 : C G T T T A G A A C G A A T T C C A C T A T T T G T C T G A G C A G T T G T A A T T A C T G T A G T A T T A C T A C T A : 5 4 0 P s p 1 : T C G A T G T T G G C T T A G G - T A A C T A T G C A G C G C A A A A G C T A C A T T A T G T T G G T T T G T T C A A C : 4 7 2 P s p 3 : C G T T T A G A A C G A A T T C C A C T A T T T G T C T G A G C A G T T G T A A T T A C T G T A G T A T T A C T A C T A : 5 4 0 E u e : T C G A T G T T G G C T T A G G A T A T A A A T A T G G C G C A A A A G T C A A A T T - T A A T G G T T T G T T C A A C : 4 6 9 P s p 1 : C G A T T A G A A C G A A T T C C A C T A T T T G T A T G A G C A G T C G T T A T T A C C G T A G T A T T A T T A C T T : 5 4 0 E u e : C G A T T A G A G C G A A T T C C A C T A T T T G T A T G G G C A G T A G T A A T C A C T G T A G T A T T A C T T C T A : 5 4 0
* 5 0 0 P s p 2 : C A A T A A T T T C C T A ------: 4 8 3 * 5 6 0 * 5 8 0 * 6 0 0 P s p 2 : T T A T C A C T C C C A G T T C T A G C A G G T G C A A T C A C T A T A C T T C T A A C A G A C C G A A A C T T A A A T : 6 0 0 P s p 3 : C A A T A A T T T C C T A ------: 4 8 3 P s p 3 : T T A T C A C T C C C A G T T C T A G C A G G T G C A A T C A C T A T A C T T C T A A C A G A C C G A A A C T T A A A T : 6 0 0 P s p 1 : C A A T A A T T T C C T A ------: 4 8 5 P s p 1 : C T T T C C C T G C C A G T A T T A G C G G G A G C T A T T A C C A T A C T A C T A A C A G A T C G A A A C C T T A A C : 6 0 0 E u e : C A A T A A C T T C C T A ------: 4 8 2 E u e : C T A T C A C T A C C A G T C T T A G C G G G G G C A A T C A C A A T A C T T C T C A C A G A T C G T A A T C T C A A T : 6 0 0
* 6 2 0 * 6 4 0 * 6 6 0 P s p 2 : A C T T C A T T C T T T G A T C C T G C T G G T G G T G G C G A C C C T A T T T T A T A C C A A C A C C T A T T C - - - : 6 5 7 P s p 3 : A C T T C A T T C T T T G A T C C T G C T G G T G G T G G C G A C C C T A T T T T A T A C C A A C A C C T A T T C - - - : 6 5 7 the results are shown in Table I. DNA sequences of the P s p 1 : A C C T C A T T T T T C G A C C C T G C T G G A G G A G G G G A C C C A A T T C T A T A T C A A C A C C T A T T T - - - : 6 5 7 E u e : A C T T C A T T C T T C G A C C C A G C T G G G G G T G G A G A C C C A A T T C T T T A T C A A C A T T T A T T C - - - : 6 5 7 mitochondrial COI and 16S rDNA gene were used to evaluate whether the specimens of the four earthworms (P. peguana?) showed homology to Megascolecidae sp. species that differ in external and internal morphology (83% identity) and Perionyx excavatus (95% identity), belong to different genetic lineages. We obtained DNA respectively but was not matched to the sequence of sequences for two fragments of the mitochondrial COI and Pheretima sp. present in the NCBI database. Finally, after 16S rDNA genes for four species. combining the results of taxonomic characteristics, our After comparing larger numbers of specimens, we study concluded that the AF was defined as the same genus found that taxonomic identification can lead to correct and species as previously described, E. eugeniae (Sims & identification within the AF and red worm species. External Gerard, 1985) and P. peguana was described as a new and internal characters of these two earthworms were species, Perionyx sp. 1. determined and identified the AF as E. eugeniae and red On the basis of morphological identification (Table I), worm as a new species, Perionyx sp. 1. In addition, the the blue worm could be distinguished (P. excavatus) from results of sequence analysis of the AF and red worm the Lao worm (P. excavatus?), and therefore, these showed the value of sequence variation was 17.93% and earthworms were identified as two new species, Perionyx 19.57% for 16S rDNA and COI genes, respectively and the sp. 2 and Perionyx sp. 3, respectively. Unfortunately, these relative nucleotide structure of analyzed genes (Table IV) species were indistinguishable by molecular techniques for was different. This abundant evidence indicated that the AF 16S rDNA gene. This indicated that no variation existed in was distinguished as a different genus and species from the the 16S rDNA sequence over these amplified regions. red worm. According to the results from the BLAST search Although, many report have shown that the 16S rDNA gene (Table III), the sequences of COI and 16S genes for the AF could be distinguished among the taxa of same species matched Diporochaeta sp., because the only a few studies group (Pop et al., 2003, 2007; Iglesias Briones et al., 2009; have been published concerning DNA in E. eugeniae and no Pérez Losada et al., 2009). However, the analyzed region of data of COI and 16S rDNA genes have been published. The the mitochondrial 16S rDNA gene (Palumbi et al., 1991) nucleotide sequence of COI and 16S genes for the red worm was not suitable to identify the complex species in this
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LOONGYAI et al. / Int. J. Agric. Biol., Vol. 13, No. 4, 2011 study. Keeping in view the sequence divergence, the Hall, T.A., 1999. BioEdit: a user-friendly biological sequence alignment mitochondrial COI gene was used as a tool for resolving editor and analysis program for Windows 95/98/NT. Nucleic Acids Sym. Series, 41: 95–98 differences among the many earthworm species as Hebert, P.D.N., A. Cywinska, S.L. Ball and J.R. De Waard, 2003. previously described (Pop et al., 2003; Chang et al., 2007; Biological identifications through DNA barcodes. Proc. Royal Soc. Huang et al., 2007; Pop et al., 2007; Iglesias Briones et al., London B, 270: 313–321 2009; Otomo et al., 2009; Pérez Losada et al., 2009; Heethoff, M., K. Etzold and S. Scheu, 2004. Mitochondrial COII sequences indicate that the parthenogenetic earthworm Octolasion tyrtaeum Richard et al., 2010). Intra-species sequence polymorphisms (Savigny 1826) constitutes of two lineages differing in body size and of the COI gene between blue worm and Lao worm were genotype. Pedobiologia, 48: 9–13 the most conserved and presented the identical length with Huang, J., Q. Xu, Z.J. Sun, G.L. Tang and Z.Y. Su, 2007. Identifying only 0.16% (one base substitution). These results confirmed earthworms through DNA barcodes. Pedobiologia, 51: 301–309 Iglesias Briones, M.J., P. Morán and D. Posada, 2009. Are the sexual, that the mitochondrial COI gene could be used to somatic and genetic characters enough to solve nomenclatural distinguish complex species. The sequence identity of problems in lumbricid taxonomy? Soil Biol. Biochem., 41: 2257– nucleotide sequences (COI gene) for the blue worm (83%) 2271 compared with the Lao worm (83%) may be related to Mindell, D.P., M.D. Sorenson, C. Huddleston, H.C. Miranda and A. Knight, 1997. Phylogenetic relationships among and within select avian Megascolecidae sp., while the identity of the 16S rDNA orders based on mitochondrial DNA. In: Mindell, D.P. (ed.), Avian gene was related to P. excavatus (90%). Additionally, the Molecular Evolution and Systematics, pp: 214–217. Academic Press, nucleotide sequences of the 16S rDNA gene for the blue New York worm (P. excavatus?) did not match the data of P. Moore, W.S., 1995. Inferring phylogenies from mtDNA variation: mitochondrial gene trees versus nuclear gene trees. Evolution, 49: excavates, which were reported in GenBank (accession 718–726 number AF406582). Nicholas, K.B. and Jr. H.B. Nicholas, 1997. GENEDOC: A Tool for Editing In conclusion, all these evidences proved that the blue and Annotating Multiple Sequence Alignments. http://www.cris.com/ worm and Lao worm are biologically distinct species and ketchup/genedoc.html, accessed January 24, 2010 Otomo, P.V., B.J. Van Vuuren and S.A. Reinecke, 2009. Usefulness of have been classified as the new species as Perionyx sp. 2 DNA barcoding in ecotoxicological investigations: resolving and Perionyx sp. 3, respectively. taxonomic uncertainties using Eisenia Malm 1877 as an example. Acknowledgement: We are most grateful to Dr. Prasuk Bull. Environ. Contam. Toxicol., 82: 261–264 Kosavititkul, Department of Biology, Faculty of Science, Palumbi, S., A. Martin, S. Romano, W.O. McMillan, L. Stice and G. Grabowski, 1991. The Simple Fool's Guide to PCR, Version 2.0. Naresuan University, Phitsanulok for his useful suggestions Honolulu, HL 96822: Department of Zoology and Kewalo Marine on earthworm taxonomy identification. This work was Laboratory, University of Hawaii supported by the Kasetsart University Research and Pérez Losada, M., M. Ricoy, J.C. Marshall and J. Domnguez, 2009. 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