Research Article |
Corresponding author: Somsak Panha ( somsak.pan@chula.ac.th ) Academic editor: Samuel James
© 2019 Teerapong Seesamut, Parin Jirapatrasilp, Ratmanee Chanabun, Yuichi Oba, Somsak Panha.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Seesamut T, Jirapatrasilp P, Chanabun R, Oba Y, Panha S (2019) Size variation and geographical distribution of the luminous earthworm Pontodrilus litoralis (Grube, 1855) (Clitellata, Megascolecidae) in Southeast Asia and Japan. ZooKeys 862: 23-43. https://doi.org/10.3897/zookeys.862.35727
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The luminous earthworm Pontodrilus litoralis (Grube, 1855) occurs in a very wide range of subtropical and tropical coastal areas. Morphometrics on size variation (number of segments, body length and diameter) and genetic analysis using the mitochondrial cytochrome c oxidase subunit 1 (COI) gene sequence were conducted on 14 populations of P. litoralis from Southeast Asia and Japan. Statistical inference on morphometric data revealed significantly different size variations in the body length and diameter among these 14 populations of P. litoralis. However, discordance between the morphometric and mitochondrial COI gene-based phylogenetic analyses was evident, where the size variations in P. litoralis showed a different pattern from the COI genetic differences. The update on the current distribution of P. litoralis is reported and revealed different aspects of the littoral habitat characteristics between Southeast Asia and Japan.
COI, habitat, morphometrics, phylogeny
Earthworms are considered as both ecosystem engineers (
Pontodrilus litoralis has a wide distribution in the tropical and subtropical coastal habitats of the Atlantic, Indian, and Pacific oceans. In Thailand, the first record of the littoral earthworm P. litoralis was from Khanom District, Nakhon Si Thammarat Province (
The study of body size can be helpful in identifying earthworm species, as morphometric characters have been represented as one of the keys for confirming their systematic positions (
Many distribution records have reported size variation within P. litoralis (
From August 2011 to September 2018, samples of P. litoralis were collected throughout the coastal areas of both the east and west sides of the Thai-Malay Peninsula (Thailand and Malaysia) and Japan (Honshu, Kyushu, and Ryukyu islands). Moreover, samples from Myanmar, Vietnam, Malaysia, Singapore, and Indonesia were collected (Figs
Coordinates of each locality were recorded using a GPS receiver, and salinity values were recorded using an ATAGO refractometer. For species identification, the specimens were carefully identified using the taxonomic literature of
Fourteen populations of P. litoralis were selected based on being from different geographic regions (Table
Sampling localities, GPS coordinates and number of specimens of P. litoralis used in the morphometric analysis.
Locality | Latitude, Longitude | Number of adult samples | |
Thailand (TA) | 1. Petchaburi (TA1) | 12°49'36.2"N, 99°59'40.3"E | 16 |
2. Trat (TA2) | 12°05'52.4"N, 102°21'27.9"E | 20 | |
3. Chonburi (TA3) | 12°50'25.1"N, 100°54'18.3"E | 15 | |
4. Songkhla (TA4) | 7°43'30.3"N, 100°22'55.4"E | 18 | |
Malaysia (MA) | 5. Pulau Pinang (MA1) | 5°28'06.7"N, 100°16'41.0"E | 16 |
6. Pahang (MA2) | 3°48'25.0"N, 103°20'29.4"E | 18 | |
Myanmar (MY) | 7. Dawei (MY1) | 14°07'43.5"N, 98°05'50.1"E | 10 |
Indonesia (IN) | 8. Banten (IN1) | 6°00'51.3"S, 106°40'38.4"E | 13 |
Vietnam (VT) | 9. Bến Tre (VT1) | 9°48'11.0"N, 106°37'42.2"E | 15 |
10. Huế (VT2) | 16°13'38.9"N, 108°04'58.4"E | 16 | |
11. Nghệ An (VT3) | 18°46'06.1"N, 105°45'31.0"E | 16 | |
Japan (JP) | 12. Aichi (JP1) | 34°48'00.2"N, 136°51'30.3"E | 18 |
13. Hiroshima (JP2) | 34°17'45.0"N, 132°19'08.0"E | 9 | |
14. Okinawa (JP3) | 26°28'20.0"N, 127°49'54.1"E | 12 | |
Total | 212 |
Three specimens were chosen from each of the same 14 populations as in the morphometric analysis resulting in the total of 42 samples used for the molecular analysis (Table
Details of P. litoralis samples using DNA sequencing, and accession numbers of the COI sequences.
Locality | abbreviation | GenBank accession number |
1. Petchaburi, Thailand(TA1) | TA1 | MK642691 |
TA1_A | MK714106 | |
TA1_B | MK714107 | |
2. Trat, Thailand (TA2) | TA2 | MK642690 |
TA2_A | MK714108 | |
TA2_B | MK714109 | |
3. Chonburi, Thailand (TA3) | TA3 | MK642689 |
TA3_A | MK714110 | |
TA3_B | MK714111 | |
4. Songkhla, Thailand (TA4) | TA4 | MK642688 |
TA4_A | MK714112 | |
TA4_B | MK714113 | |
5. Pulau Pinang, Malaysia (MA1) | MA1 | MK642694 |
MA1_A | MK714100 | |
MA1_B | MK714101 | |
6. Pahang, Malaysia (MA2) | MA2 | MK642693 |
MA2_A | MK714102 | |
MA2_B | MK714103 | |
7. Dawei, Myanmar (MY1) | MY1 | MK642692 |
MY1_A | MK714104 | |
MY1_B | MK714105 | |
8. Banten, Indonesia (IN1) | IN1 | MK642698 |
IN1_A | MK714092 | |
IN1_B | MK714093 | |
9. Bến Tre, Vietnam (VT1) | VT1 | MK642687 |
VT1_A | MK714114 | |
VT1_B | MK714115 | |
10. Huế, Vietnam (VT2) | VT2 | MK642686 |
VT2_A | MK714116 | |
VT2_B | MK714117 | |
11. Nghệ An, Vietnam (VT3) | VT3 | MK642685 |
VT3_A | MK714118 | |
VT3_B | MK714119 | |
12. Aichi, Japan (JP1) | JP1 | MK642697 |
JP1_A | MK714094 | |
JP1_B | MK714095 | |
13. Hiroshima, Japan (JP2) | JP2 | MK642696 |
JP2_A | MK714096 | |
JP2_B | MK714097 | |
14. Okinawa, Japan (JP3) | JP3 | MK642695 |
JP3_A | MK714098 | |
JP3_B | MK714099 |
For sequencing, the PCR products were directly sent to Macrogen Inc. (Japan) without purification. All COI sequences were aligned using the ClustalW algorithm in MEGA7 v. 7.0.18 (
The measurement of 212 individuals of P. litoralis earthworms from all 14 sampling sites revealed a length range between 31.1–125.4 mm (Fig.
The ANOVA analysis revealed a significant difference (p < 0.05) in the mean length and diameter of P. litoralis among the 14 locations (Fig.
Cluster analysis of the 14 populations based on the three morphometric data revealed two clusters, one of which contained 13 populations and was further divided into two subclusters, and the other contained only the JP2 population from Japan (Figure
PCA percentage of the explained variance and weights of morphometric ratios for the 14 populations of P. litoralis.
Variable | PC1 | PC2 | PC3 |
Length | 0.675 | −0.143 | 0.724 |
Diameter | 0.638 | −0.380 | −0.670 |
Segment number | 0.371 | 0.914 | −0.165 |
Eigenvalue | 1.8174 | 0.8882 | 0.2944 |
% total variance | 60.6 | 29.6 | 9.8 |
The COI DNA sequences (658 bp) from 42 individuals, three specimens from each of the 14 populations used in the morphometric analysis, were analyzed. The analysis yielded 158 variable (polymorphic) sites and 139 parsimony informative sites. No insertions, deletions, or stop codons were observed in any of the sequences. The K2P genetic distances among the 14 geographical locations within P. litoralis ranged from 0.3–12.8 % (Table
Between groups mean genetic distances corrected with the Kimura-2 parameter model among the 14 populations of P. litoralis. The bold values represent the genetic distance within group. Sampling site codes are given in Table
IN1 | JP1 | JP2 | JP3 | MA1 | MA2 | MY1 | TA1 | TA2 | TA3 | TA4 | VT1 | VT2 | VT3 | |
IN1 | 0.002 | |||||||||||||
JP1 | 0.102 | 0.001 | ||||||||||||
JP2 | 0.087 | 0.073 | 0.072 | |||||||||||
JP3 | 0.079 | 0.108 | 0.043 | 0.011 | ||||||||||
MA1 | 0.089 | 0.101 | 0.103 | 0.102 | 0.002 | |||||||||
MA2 | 0.064 | 0.128 | 0.100 | 0.083 | 0.112 | 0.001 | ||||||||
MY1 | 0.048 | 0.109 | 0.088 | 0.076 | 0.109 | 0.073 | 0.002 | |||||||
TA1 | 0.067 | 0.128 | 0.099 | 0.082 | 0.111 | 0.003 | 0.074 | 0.005 | ||||||
TA2 | 0.100 | 0.111 | 0.112 | 0.110 | 0.108 | 0.119 | 0.101 | 0.118 | 0.090 | |||||
TA3 | 0.064 | 0.125 | 0.093 | 0.075 | 0.106 | 0.018 | 0.068 | 0.017 | 0.113 | 0.001 | ||||
TA4 | 0.071 | 0.109 | 0.074 | 0.055 | 0.107 | 0.065 | 0.078 | 0.067 | 0.114 | 0.067 | 0.067 | |||
VT1 | 0.067 | 0.127 | 0.098 | 0.081 | 0.109 | 0.005 | 0.074 | 0.004 | 0.116 | 0.013 | 0.066 | 0.000 | ||
VT2 | 0.073 | 0.113 | 0.056 | 0.035 | 0.103 | 0.067 | 0.071 | 0.066 | 0.109 | 0.061 | 0.060 | 0.063 | 0.050 | |
VT3 | 0.067 | 0.119 | 0.088 | 0.072 | 0.102 | 0.030 | 0.060 | 0.030 | 0.105 | 0.024 | 0.066 | 0.026 | 0.052 | 0.000 |
ML phylogenetic tree of P. litoralis based on the mitochondrial COI gene (658 bp) with Pontodrilus longissimus as the outgroup. Only bootstrap values >70% are indicated at each node. Scale bar represents the number of nucleotide substitutions per site. The sample names correspond to those in Table
In Southeast Asia, P. litoralis was found scattered over the coastal areas in Thailand, Myanmar, Vietnam, Malaysia, Singapore, and Indonesia (Figures
Based on field collections within Thailand and some parts of Southeast Asia, P. litoralis was found to occupy several types of habitats (Table
Salinity records (mean ‰ ± SD) and habitat characteristics of the sampling sites of P. litoralis in this study.
Locality | Collection time | Salinity (‰) | Habitat |
Thailand | January 2015 – March 2018 | 19.29 ± 12.14 | Salty mud margins of estuaries, brackish lakes, damp mud under stones, mangrove swamps, under the root of the tree near the shore, under the trash or leaf litter on the sandy beach, sanitary sewer emptying to the sandy beach |
Myanmar | April 2016 | 18 ± 12.82 | Estuaries, under the trash on the sandy beach |
Malaysia | January 2016 | 15.94 ± 9.85 | Estuaries, damp mud under stones and the beach, under the trash or leaf litter on the sandy beach |
Vietnam | May 2018 – July 2018 | 19.38 ± 10.57 | Estuaries, under the trash or leaf litter on the sandy beach |
Indonesia | August 2017 | 12 | Sanitary sewer emptying to the sandy beach |
Singapore | December 2017 | 22 | Under the root of the tree near the shore |
Japan | August 2011 – September 2018 | 17.5 ± 9.85 | Sand beach facing to the ocean (sand mixed with seaweed debris), estuaries |
This study is the first attempt to integrate morphometric variations and molecular marker analyses together in the cosmopolitan littoral earthworm P. litoralis. The specimens investigated in this study were within the variation range previously reported by
According to the results of the one-way ANOVA, there was a significant difference in the body length and diameter among specimens from the different geographical sites. In addition, the PCA results supported that length and diameter had a higher influence than the number of segments in the 14 studied populations of P. litoralis. However, the phylogenetic tree did not show any congruent pattern with the size variation of the specimens analyzed in this study. For instance, in both the PCA and cluster analysis the longest (JP2) and the shortest samples (VT2) formed separate groups with statistical differences in their size, whereas a low genetic distance between the two samples from each respective population was detected, revealing that the size variation of P. litoralis was independent of the genetic (COI gene) differences.
Differences in the body length, diameter, and number of segments have also reported in other earthworms. The terrestrial earthworm Metaphire peguana (Rosa, 1890) from Penang and neighboring states of Malaysia revealed significant differences in their morphometric variations that were not matched by their genetic difference but rather were affected by the type of habitat (
This study is a comprehensive report on the occurrence, distribution and habitat characteristics of the luminous littoral earthworm, P. litoralis, in the coastal areas of Thailand, Japan (Honshu, Kyushu, and Ryukyu islands), and some parts of Southeast Asia (Myanmar, Vietnam, Malaysia, Singapore, and Indonesia) based on field collections. This survey supported the assumption that P. litoralis is widely distributed in subtropical and tropical coastal ecosystems (
In general, the distribution of earthworms is mostly affected by environmental factors, such as the temperature, organic matter content, and soil moisture (
In Japan, beach-cast seaweeds have been reported as important habitats and food for a diverse community of marine and terrestrial organisms, such as amphipods, isopods, and copepods (
In conclusion, although morphometric examinations of size variation could make reliable distinctions among different populations of P. litoralis, this distinction was not congruent with the phylogenetic relationship based on COI gene sequence analysis, reflecting that the size variation of P. litoralis did not correlate with their genetic (COI) differences. Thus, we propose that the food resource is the key factor underlying size variation in P. litoralis. Future analyses on the type of habitats, sand texture, and components of the food resources are necessary. Moreover, studies on salinity tolerance are needed to confirm the habitat preference of this littoral earthworm species.
This work was mainly supported by Center of Excellence on Biodiversity (BDC-PG2-161001), WCU-58-016-FW and Overseas Research Experience Scholarship for Graduate Student of Graduate School Chulalongkorn University. We would like to thank the Human Resource Development in Science Project (Science Achievement Scholarship of Thailand, SAST) for support and encouragement. We are indebted to Yano Daichi, Ikuhiko Kin and all members of the Animal Systematics Research Unit, Chulalongkorn University for assisting in the fieldwork. We are grateful to anonymous reviewers of this paper for their invaluable comments and suggestions.