Biochemical Systematics and Ecology 51 (2013) 8–15

Contents lists available at ScienceDirect

Biochemical Systematics and Ecology

journal homepage: www.elsevier .com/locate/biochemsyseco

Low heterogeneity in populations of the terrestrialearthworm, Metaphire peguana (Rosa, 1890), in Thailand, asrevealed by analysis of mitochondrial DNA COI sequencesand nuclear allozymes

Pongpun Prasankok a, Ueangfa Bantaowong b, Samuel W. James c,Somsak Panha b,*

a School of Biology, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, ThailandbAnimal Systematics Research Unit, Department of Biology, Faculty of Science, Chulalongkorn University, Bangkok 10330, ThailandcDepartment of Biology, University of Iowa, 143 Biology Building, Iowa City, IA 52242-1324, USA

a r t i c l e i n f o

Article history:Received 21 March 2013Accepted 7 July 2013Available online 3 August 2013

Keywords:Metaphire peguanamtDNAAllozymeLow heterogeneity

* Corresponding author. Tel./fax: þ66 2 2185273.E-mail address: somsak.pan@chula.ac.th (S. Panh

0305-1978/$ – see front matter � 2013 Elsevier Ltdhttp://dx.doi.org/10.1016/j.bse.2013.07.001

a b s t r a c t

The genetic variation of a widely distributed terrestrial earthworm species, Metaphirepeguana (Rosa, 1890), was examined in Thailand using allozyme andmitochondrial (mt)DNAcytochrome oxidase subunit 1 (COI) sequence analyses. A total of 274 individuals werecollected from 13 localities in Thailand and scored for 12 enzyme systems (18 presumptiveallozyme loci) using horizontal starch gel electrophoresis. Fourteen of these presumed lociwere found to be polymorphic. In addition, a 660 bp fragment of the COI mtDNA gene wasamplified and sequenced from one representative individual for each of the 13 geographicalregions. The expectedheterozygosity (Hexp)was relatively low, ranging from0.059–0.147withan overall mean of 0.092� 0.02 and therewas no significant isolation by distance pattern forthe 13 populations (localities) across the entire study area (P-value > 0.05). Genetic di-vergences among the samples were low, with a low genetic distance (mean ¼ 0.048) anduncorrectedp-distance (mean¼0.02) indicating frequent geneflowamong thesepopulationsin Thailand. The monophyly of M. peguana across these regions was supported by Neighborjoining, Maximum likelihood and Bayesian inference based phylogenetic analysis of themtDNA COI sequences with no geographic location dependent pattern. This may be due toanthropogenic movement ofM. peguana in soil or with plant scions throughout this region.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

It is known that terrestrial earthworms can be found in forest communities, floodplains, upland meadows, pastures, andagricultural lands. They play a major role in the biogeochemical cycles of terrestrial ecosystems due to their influence onmicrobial activity, carbon and nitrogen cycles and alteration of soil. Indeed, worms can adapt to effectively colonize newhabitats differing in soil climate conditions and resource supplies. Consequently, they are expected to exhibit the charac-teristics of invasive species, and this has been shown to be the case for several European earthworm species invading intoNorth America (Hendrix and Bohlen, 2002).

Over the past few decades, earthworms have received increasing attention because they represent the majority of soilbiological invasions. Metaphire peguana is one of the Asian species that have attained wider than natural distributions as a

a).

. All rights reserved.

P. Prasankok et al. / Biochemical Systematics and Ecology 51 (2013) 8–15 9

regional invasive species due to human activity, and these are frequently encountered in Thailand (James, 2008). M. peguanawas originally described from Burma, but has a large recorded range throughout Thailand and nearby countries (Gates, 1972;Shen and Yeo, 2005; Bantaowong et al., 2011). Theworm is found in dipterocarp forests, deciduous forests, and anthropogenicareas, even in the waste water saturated soil from households. These trends, including their successful expansion of theirranges and colonization of new habitats, along with their geographic disjunctive populations, make M. peguana an attractivesubject for evaluating their population and biogeographic structure since theymay exhibit a complex genetic structure acrossdifferent regions. In addition, analysis of the macro-scale distributions of M. peguana in Thailand may provide importantinsights into and identify the potential of such invasive species spreading across Thailand. Moreover, there has been no directexamination of the genetic variation within this earthworm species.

In this study, to evaluate the genetic variability and population structure of M. peguana we used 12 enzyme (allozyme)systems, yielding 18 presumed allozyme loci, to analyze 274 individual samples from 13 distinct geographic locations(populations) in Thailand. To investigate the phylogenetic relationships among these populations ofM. peguana, we examinedone representative of each of the 13 populations ofM. peguana using sequence analysis of a 660 bp coding region fragment ofthe mitochondrial (mt)DNA cytochrome oxidase subunit 1 (COI) gene.

2. Material and methods

2.1. Sample collection

A total of 274 individuals of M. peguana were collected in 2009–2010 from thirteen localities throughout Thailand (Fig. 1and Table 1). After collecting, soil particles were removed from the earthworms by soaking in water and then they wereimmediately immersed and maintained in liquid nitrogen and later stored at �20 �C. The specimens were identified under abinocular stereomicroscope based on Kosavititkul (2005), Gates (1972), Somniyam and Suwanwaree (2009) and Bantaowonget al. (2011). The specimens were used for allozyme electrophoresis and mtDNA COI sequence analyses.

2.2. Allozyme analysis

Each individual was cut through the clitellar region and two segments above the clitellum. Tissue samples of eachearthworm were homogenized and the resulting crude protein extracts from each homogenate were then subjected to

Fig. 1. Map of Thailand, showing sampling localities of M. peguana (1 ¼ Chiang Mai, 2 ¼ Nan, 3 ¼ Tak, 4 ¼ Phitsanulok, 5 ¼ Nakhon Sawan, 6 ¼ Ayutthaya,7 ¼ Bangkok, 8 ¼ Sa Kaeo, 9 ¼ Chanthaburi, 10 ¼ Phetchaburi, 11 ¼ Prachuab Khiri Khan, 12 ¼ Chumphon and 13 ¼ Surat Thani) and M. bahli (14 ¼ Lam Pao Dam,Kalasin). Gray and dark shaded areas indicate land higher than 100 and 400 m asl., respectively.

Table 1Locality, geographical coordinates and sample sizes (N) of Metaphire peguana used in this study. The locality numbers correspond to those in Fig. 1.

Locality Latitude, longitude N

Northern region 1. Chiang Mai 98� 550 45.30 0 E, 19� 230 41.10 0 N 202. Nan 100� 470 240 0 E, 19� 170 10.50 0 N 253. Tak 98� 580 54.80 0 E, 17� 140 22.10 0 N 214. Phitsanulok 100� 310 55.20 0 E, 16� 500 17.80 0 N 16

Central region 5. Nakhon Sawan 100� 70 29.90 0 E, 15� 430 6.60 0 N 306. Ayutthaya 100� 280 11.80 0 E, 14� 270 16.30 0 N 247. Bangkok 100� 310 49.80 0 E, 13� 440 18.90 0 N 20

Eastern region 8. Sa Kaeo 101� 560 52.20 0 E, 13� 470 13.90 0 N 329. Chanthaburi 102� 70 8.70 0 E, 12� 300 26.70 0 N 16

Southern region 10. Phetchaburi 99� 550 2.30 0 E, 13� 10 270 0 N 2811. Phachuab Khiri Khan 99� 360 54.30 0 E, 11� 370 30.50 0 N 2212. Chumphon 99� 20 27.90 0 E, 9� 560 51.40 0 N 913. Surat Thani 98� 310 47.60 0 E, 8� 540 37.80 0 N 11

Outgroup (Metaphire bahli) 14. Lam Pao Dam, Kalasin 103� 340 90 0 E, 16� 430 39.70 0 N 20

P. Prasankok et al. / Biochemical Systematics and Ecology 51 (2013) 8–1510

horizontal starch gel electrophoresis for the indicated allozymes and buffers (Table 2), otherwise according to the principlesand procedures detailed byMurphy et al. (1996) with slightmodifications. Voucher specimenswere deposited in theMuseumof Zoology, Chulalongkorn University. Enzyme nomenclature and E.C. numbers follow those proposed by the InternationalUnion of Biochemistry.

2.3. mtDNA analysis

In total, 13 individuals of M. peguana, one from each geographical region, plus one individual of Metaphire bahli collectedfrom a separate locality in Thailand (Fig. 1 and Table 1) were used for the mtDNA COI sequence analyses. The samples ofmuscular tissue from near the anterior endwere homogenized with a microspatula and total DNAwas extracted by a silica gelbased spin column procedure according to the protocol of the DNeasy blood and tissue kit (Qiagen).

The universal primers LCO1409 and LCO2198 (Folmer et al., 1994) were used to amplify a 660-bp portion of the mtDNA COIgene. PCR amplifications were performed in a 50 mL total volume, and thermal cycled using one cycle at 94 �C for 2 min, 36cycles of 94 �C for 30 s, 50 �C for 30 s and 72 �C for 60 s, followed by a final 72 �C for 5min. The PCR products (amplicons) werepurified using a microspin purification kit (Qiagen). Both strands of each reaction were direct sequenced using the aboveprimers (in separate reactions) using the 23 ABI 3730 XLs sequencer (Macrogen). The consensus sequence for each isolate wassubmitted to GenBank (Accession numbers KC404831–KC404843 forM. peguana and KC404844 forM. bahli). TheM. bahli COIsequence was used as the outgroup in all phylogenetic analyses.

2.4. Allozyme analysis

Genotypic frequency at each presumed polymorphic locus was tested for agreement with Hardy-Weinberg expectationsusing an exact probability test. The genetic variability within each population was estimated by calculating the meanobserved heterozygosity (Hobs), the mean expected heterozygosity (Hexp), the mean number of alleles per locus (A), and thepercentage of polymorphic loci (P).

To estimate the heterogeneity among samples, Wright’s (1965) Fst was calculated for each locus. Each Fst value was testedfor significant departure from zero following themethod ofWorkman and Niswander (1970). We also calculated the Fis and Fitfor each locus.

Table 2Enzymes and presumptive allozyme loci screened using the indicated buffer systems used.

Enzyme E.C. number Locus Buffer systema

Aspartate aminotransferase 2.6.1.1 Aat-1,2 TBE8.7Esterase 3.1.1- Est TC8Glucose-6-phosphate isomerase 5.3.1.9 Gpi TC83-Hydroxybutyrate dehydrogenase 1.1.1.30 Hbdh TBE8.7Peptidase (leucyl-glycyl-glycine) 3.4.11.- Pep-lgg-1,2,3,4 LioHMalate dehydrogenase 1.1.1.37 Mdh-1,2 CAPM6Isocitrate dehydrogenase 1.1.1.42 Idh-1,2 CAPM6Mannose-6-phosphate isomerase 5.3.1.8 Mpi CAPM6Malate dehydrogenase (NADPþ) 1.1.1.40 Me CAPM6Phosphoglucomutase 2.7.5.1 Pgm TC8Phosphogluconate dehydrogenase 1.1.1.44 Pgd CAPM6Superoxide dismutase 1.15.1.1 Sod CAPM6

a Buffer systems: CAPM6, Citrate-aminopropylmorpholine, pH 6.0 (Clayton and Tretiak, 1972); LioH, lithium hydroxide-boric acid, pH 8.1 (Ridgway et al.,1970); TC8, tris-citrate, pH 8.0 (Clayton and Tretiak, 1972); TBE8.7, tris-borate-EDTA, pH 8.7 (Boyer et al., 1963).

P. Prasankok et al. / Biochemical Systematics and Ecology 51 (2013) 8–15 11

Nei’s (1978) unbiased genetic distance (D) was calculated for all pairwise comparisons of samples in order to estimate theextent of differentiation among populations. An UPGMA (Sneath and Sokal, 1973) dendrogram was created for M. peguanafrom the Nei’s unbiased genetic distances. Evaluation of the genetic variability and genetic heterogeneity were computed inBIOSYS-1 (Swofford and Selander, 1981).

In order to test for an association between genetic differences and geographic distances, the geographic distances werecorrelated with pairwise values of Fst/(1 � Fst) between all samples. We used the Mantel test (Mantel, 1967) for correlationbetween the two distance matrices based on 10,000 permutations, as implemented in the ISOLDE program in GENEPOP(Raymond and Rousset,1995). GENEPOPwas also used to compute the regression line describing the relationship between Fst/(1 � Fst) and geographic distances.

2.5. mtDNA COI sequence alignment and phylogenetic analysis

Sequences were edited and aligned with Sequence Navigator version 1.0.1 and were improved manually using MUSCLE asimplemented in MEGA v. 5.05 (Tamura et al., 2011). Amino acid translations were examined to test for accuracy and func-tionality of the sequences. All positions containing gaps and missing data were eliminated. Substitution saturation analyseswere conducted using the Kimura 2-parameter method. The rate variation among sites was modeled with a gamma distri-bution (shape parameter ¼ 0.05), using all COI codon positions and both transitions and transversions were plotted againstthe evolutionary distance of the Kimura 2-parameter. Evolutionary analyses were conducted in MEGA 5.05.

To estimate the genetic diversity of theM. peguana populations based on the COI gene fragment sequence, the nucleotidediversity (p), haplotype diversity (h) and Fst were calculated with DnaSP v 5.10 (Librado and Rozas, 2009). We also estimatedthe evolutionary divergence between sequences amongst populations of M. peguana by calculating the uncorrected p-dis-tances in MEGA 5.05.

The phylogenetic position and degree of evolutionary divergence ofM. peguanawas evaluated by constructing Neighbour-joining (NJ), Maximum likelihood (ML) and Bayesian inference (BI) phylogenetic trees from the COI sequences, and in all casesthe corresponding sequence fromM. bahliwas used as the outgroup to root the tree. NJ analysis was performed by calculatinguncorrected p-distance in MEGA 5.05, and nonparametric bootstrapping (10,000 replicates) to assess the stability of internalbranches.

ML analyses were performed using the heuristic search algorithmwith the GTRmodel of evolution, a g-shape parameter of0.05 and the proportion of invariable position, as selected by JModelTest v1.0 under the Akaike Information Criterion. Cladesupport was assessed using the non-parametric bootstrap procedure with 10,000 bootstrap replicates estimated in PhyML v.3.0 (Guindon and Gascuel, 2003).

BI analyses, using the Markov chain Monte Carlo technique (MCMC), were performed by MrBayes 3.2 (Ronquist et al.,2012) with a random starting tree and run for 4.5 million generations to calculate the Bayesian posterior probabilities(bpp) for each branch.

3. Results

3.1. Population structure

Twelve enzyme systems encompassing 18 assumed allozyme loci (Table 2) were screened for allelic variation (whereelectromorphic variationwas assumed to be allelic variation) inM. peguana. Of these, 14 were found to be polymorphic whilstthe Aat-2, Lgg-1, Idh-1 and Sod loci weremonomorphic (not shown). Genetic variability inM. peguana samples showed highlyvariable A and P values (1.2–1.6 and 23.5–47.1, respectively), whilst the Hobs (0.049–0.119; mean 0.079 � 0.019) and Hexp(0.059–0.147; mean 0.092 � 0.022) values obtained were also relatively low (Table 3). In some contrast, the mtDNA COI

Table 3Localities, mean number of allozyme alleles per locus (A), percentage of polymorphic loci (P), mean observed heterozygosity (Hobs), and mean expectedheterozygosity (Hexp) in (N) samples of M. peguana from each locality. Standard error of Hexp is indicated in parentheses.

Locality A P Hobs Hexp

Northern region 1. Chiang Mai 1.5 47.1 0.119 0.147 (0.051)2. Nan 1.2 23.5 0.059 0.075 (0.040)3. Tak 1.4 41.2 0.073 0.097 (0.039)4. Phitsanulok 1.4 35.3 0.089 0.087 (0.036)

Central region 5. Nakhon Sawan 1.4 35.3 0.055 0.068 (0.029)6. Ayutthaya 1.3 29.4 0.049 0.059 (0.030)7. Bangkok 1.5 47.1 0.097 0.100 (0.036)

Eastern region 8. Sa Kaeo 1.5 35.3 0.088 0.101 (0.040)9. Chanthaburi 1.4 29.4 0.089 0.087 (0.040)

Southern region 10. Phetchaburi 1.6 47.1 0.074 0.117 (0.046)11. Phachuab Khiri Khan 1.5 47.1 0.092 0.085 (0.033)12. Chumphon 1.4 29.4 0.081 0.091 (0.040)13. Surat Thani 1.4 29.4 0.072 0.081 (0.039)

Table 4Summary of F-statistics at all loci of Metaphire peguana from Thailand.

Locus Fis Fit Fst

Hbdh �0.063 0.136 0.187a

Pgd 0.082 0.274 0.210a

Lgg-2 �0.034 �0.005 0.028Lgg-3 �0.032 �0.007 0.024Lgg-4 �0.177 �0.030 0.125a

Me �0.029 0.157 0.181a

Est 0.020 0.441 0.429a

Aat-1 0.021 0.139 0.120a

Idh-2 �0.051 �0.016 0.034Mdh-1 �0.075 �0.012 0.059a

Mdh-2 �0.086 0.094 0.166a

Gpi �0.044 �0.005 0.037Mpi 0.084 0.396 0.340a

Pgm 0.200 0.428 0.285a

Mean 0.030 0.285 0.263

a Significant at the level of p < 0.05.

P. Prasankok et al. / Biochemical Systematics and Ecology 51 (2013) 8–1512

sequences of the 13 individuals (one from each geographical population) yielded seven haplotypes with a fairly highhaplotype diversity (h ¼ 0.926). However, the nucleotide diversity was low (p ¼ 0.009).

The F-statistics based analyses of the allozyme data are presented in Table 4. The mean Fis value among the 13M. peguanapopulations assayed was low (0.030), whilst the mean Fst value was higher (0.263) and was significantly higher than zero for10 of the 14 polymorphic loci. Likewise, the mean Fit value at all loci was relatively low (0.285). In comparison with the COIanalysis (not shown), comparison of the central and southern samples showed a high Fst (0.440), whereas, in contrast, thatbetween the southern and northern samples and between the central and northern samples showed very low Fst (�0.075 and0.079, respectively).

Matrices of Nei’s (1978) genetic distances (D) among the 13 M. peguana populations derived from the allozyme locianalysis are presented in Table 5. The D values obtained within theM. peguana populations were relatively small (D ¼ 0.002–0.170, mean 0.048 � 0.040). Likewise, the overall uncorrected p-distance values of the M. peguana samples derived from themtDNA (COI) analysis were low (mean 0.02; range, 0–0.043). In contrast, the allozyme based genetic divergences betweeninterspecific samples were high (12–14%), as shown in Table 5. The UPGMA dendrogram, based upon these allozyme derivedD values, showed no concordance with the geographic area of the samples of M. peguana (Fig. 2A).

In agreement, no significant isolation by distance (IBD) pattern was found for the 13 M. peguana populations analyzed byallozymepolymorphismfromthegraphof Fst/(1� Fst) versusgeographicdistanceof the1480kmstudyarea (P-value>0.05) (Fig. 3).

3.2. Phylogenetic analysis

With respect to the mtDNA COI fragment sequence dataset, the 660 nucleotide sites revealed 548 nucleotides wereconstant, 80 (12.2%) were variable but parsimony uninformative, and 32 (4.86%) were parsimony informative. The graph oftransitions and transversions versus Kimura 2-parameter revealed no significant crossing of transitions and transversions(data not shown), suggesting rejection of substitution saturation.

Table 5Matrix of genetic distances among the 13 samples ofMetaphire peguana, one from each locality in Thailand, plus the related speciesM. bahli as an outgroup.Below diagonal: Nei (1978) unbiased genetic distance. Above diagonal: uncorrected p-distance based on COI gene.

Population Locality

1 2 3 4 5 6 7 8 9 10 11 12 13 14

1. Chiang Mai – 0.035 0.002 0.041 0.008 0.033 0.042 0.008 0.003 0.003 0.003 0.002 0.002 0.1442. Nan 0.012 – 0.033 0.033 0.03 0.005 0.035 0.03 0.035 0.035 0.035 0.033 0.033 0.1453. Tak 0.029 0.036 – 0.039 0.006 0.032 0.041 0.006 0.002 0.002 0.002 0.00 0.00 0.1424. Phitsanulok 0.015 0.023 0.054 – 0.038 0.032 0.002 0.038 0.041 0.041 0.041 0.039 0.039 0.1265. Nakhon Sawan 0.016 0.010 0.073 0.012 – 0.029 0.039 0.00 0.008 0.008 0.008 0.006 0.006 0.1416. Ayuttaya 0.058 0.051 0.144 0.053 0.031 – 0.033 0.029 0.033 0.033 0.033 0.032 0.032 0.1427. Bangkok 0.013 0.017 0.071 0.021 0.009 0.045 – 0.039 0.042 0.042 0.042 0.041 0.041 0.1278. Sa Kaeo 0.023 0.009 0.072 0.023 0.007 0.028 0.016 – 0.008 0.008 0.008 0.006 0.006 0.1419. Chanthaburi 0.036 0.040 0.053 0.081 0.064 0.086 0.067 0.058 – 0.003 0.003 0.002 0.002 0.14410. Phetchaburi 0.038 0.031 0.113 0.028 0.014 0.041 0.017 0.010 0.096 – 0.003 0.002 0.002 0.14411. Prachuab

Khiri Khan0.042 0.031 0.119 0.028 0.014 0.026 0.026 0.009 0.086 0.009 – 0.002 0.002 0.144

12. Chumphon 0.020 0.009 0.071 0.026 0.009 0.035 0.003 0.004 0.070 0.013 0.015 – 0.00 0.14213. Surat Thani 0.029 0.023 0.052 0.058 0.044 0.090 0.021 0.034 0.107 0.045 0.062 0.013 – 0.14214. M. bahli 0.459 0.470 0.460 0.471 0.505 0.520 0.474 0.462 0.434 0.503 0.460 0.466 0.432 –

Fig. 2. (A). UPGMA phenogram for the sampled Thai populations of M. peguana based on Nei’s (1978) genetic distance evaluated from 14 polymorphic allozymeloci. (B). Phylogenetic relationships ofM. peguana inferred frommtDNA COI sequence analysis and rooted withM. bahli. The Bayesian consensus tree was obtainedusing the GTR model. Nodal numbers correspond to bootstrap support from NJ (10,000 replicates): above row and non-parametric bootstrap values from ML(10,000 replicates)/bipartition’s posterior probabilities, respectively: below row. Scale bar represents substitution/site.

P. Prasankok et al. / Biochemical Systematics and Ecology 51 (2013) 8–15 13

ML analysis under the GTR model produced a topology with ln L ¼ �1475.68365 (g-shape parameter with four discreterate categories ¼ 0.78746); proportion of invariable sites ¼ 0.18269; nucleotide frequencies: A ¼ 0.2879, C ¼ 0.2345,G ¼ 0.17606 and T ¼ 0.30154.

NJ, ML and BI generated almost identical topologies (not shown), all revealing themonophyly ofM. peguana and supportedby strong posterior probability (BI) or bootstrap support (NJ, ML) (Fig. 2B).

4. Discussion

The percentage of polymorphic allozyme loci (P) in M. peguana was highly variable among the 274 samples and 13populations (23.5–47.1%). However, overall M. peguana appeared to show a relatively lower P level than other annelids, suchas Eisenia fetida and Eisenia andrei at 68.7% and 87.5%, respectively (McElroy and Diehl, 2001). Likewise, the mean number ofallozyme alleles per locus in M. peguana in this study varied from 1.2 to 1.6, which is lower than the 2.7 to 3.0 reported forLumbricus rubellus (Peles et al., 2003). The levels of overall allozyme genetic variability inM. peguana, as indicated byHexp, was

0

0.5

1

1.5

2

2.5

0 500 1000 1500 2000

Fs

t/(1

-F

st)

Geographic Distance (km)

Fig. 3. Isolation by distance of the 13 M. peguana populations analyzed by 14 polymorphic allozyme loci. Comparisons (85) are pairwise between all populations.

P. Prasankok et al. / Biochemical Systematics and Ecology 51 (2013) 8–1514

relatively low among populations, ranging from 0.059 to 0.147 (mean ¼ 0.092 � 0.022) compared to that reported in otherworm, such as for E. fetida (Hexp¼ 0.094–0.262), E. andrei (Hexp ¼ 0.068–0.187) and L. rubellus (Hexp ¼ 0.112–0.160) (McElroyand Diehl, 2001; Peles et al., 2003). Thus, it seems more plausible that the low P and mean Hexp values in M. peguana may beassociated with their rapid expansion from a small number of individuals and the loss of variation following colonization dueto low effective population numbers, as suggested by Porco et al. (2012). In addition, the low nucleotide diversity in M.peguana (mtDNA COI dataset) was associated with high Fst values between the central and southern samples, suggestingpotential genetic drift. If so, the high heterozygosity value in some populations may reflect multiple introductions to theregion providing the genetic variation. Alternatively, moderate genetic variability in M. peguana may be driven by a largepopulation size as a consequence of its abundance, high density, wide geographic range and its ability to effectively colonizenew habitats (Marinissen and den Bosch, 1992; Bantaowong et al., 2011).

The overall allozyme Fis was low at 0.030, which is close to zero, indicating only a small amount of non-random mating.Although earthworms are hermaphrodites, nevertheless, field observations and published information indicate reciprocalmating is the normal case, at least, forM. peguana. We, therefore, consider that inbreeding can be excluded in the present caseand the low (non-zero) Fis can be better explained by fertilized eggs developing in a protective cocoon. Thus, though M.peguana avoids inbreeding within populations, the Wahlund effect creates an overall heterozygosity deficiency(Fit ¼ 0.285 > 0).

On the basis of the allozyme data, the genetic distance among M. peguana samples was found to be low, including low Dand Fst values, and this was supported by the low uncorrected p-distance value based on the sequence variation of themtDNACOI gene fragment. This suggests a high degree of gene flow/migration between populations across the geographic regions ofThailand, and so the apparent geographic barriers (Fig. 1) do not appear to exert any significant restriction on gene flow, assupported by the absence of any IBD and also by the absence of any geographical dependent pattern in the phylogeneticrelationships of this monophyletic species (in Thailand).

This unexpected finding could be explained if earthworms and/or their cocoons were dispersed across regions, either byanimals, including especially humans, or by abiotic means, such as floods. However, the most significant contribution to theirintroduction into widely remote localities is likely to be by human activities, such as soil movement across mountains andother non-flood plain barriers.

Considering the absence of any IBD pattern, the wide scatter in the plots, likely to be due to the random differentiation ofeach population, and the statistically significant higher than zero level for 10 of the 14 polymorphic loci, these imply thatgenetic drift has a much more significant influence than gene flow in the local populations. In fact, M. peguana is a widelydistributed and apparently broad-niched species (Marinissen and den Bosch, 1992).

Although, the mtDNA COI analysis is not fully concordant with the nuclear allozyme analysis, both supported themonophyly of M. peguana and showed very low genetic distances among the 13 geographical locations (populations). Inaddition, average sequence divergences within the M. peguana clade were generally lower than 4%.

Acknowledgments

This work was financially supported by the TRF Senior Scholar of the Thailand Research Fund (2012–2015) RTA5580001 toSP, and by an initial funding for specimens collecting from Rachadapiseksompoj Fund, Chulalongkorn Project, in 2012. Weappreciate the critical reading and invaluable comments on the manuscript by Dr. Robert Butcher of the Publication Coun-seling Unit (PCU), Faculty of Science, Chulalongkorn University.

References

Bantaowong, U., Chanabun, R., Tongkerd, P., Sutcharit, C., James, S.W., Panha, S., 2011. A new species of the terrestrial earthworm of the genus MetaphireSims and Easton, 1972 from Thailand with redescription of some species. Trop. Nat. His. 11, 55–69.

P. Prasankok et al. / Biochemical Systematics and Ecology 51 (2013) 8–15 15

Boyer, S.H., Fainer, D.C., Watson, E.J., 1963. Lactate dehydrogenase variation from human blood: evidence for molecular subunit. Science 141, 642–643.Clayton, J.W., Tretiak, D.N., 1972. Amine-citrate buffers for pH control in starch gel electrophoresis. J. Fish Res. Board Canada 29, 1169–1172.Folmer, O., Black, M., Hoeh, W., Lutz, R., Vrijenhoek, R., 1994. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse

metazoan invertebrates. Mol. Marine Biol. Biotech. 3, 294–299.Gates, G.E., 1972. Burmese earthworms: an introduction to the systematics and biology of megadrile oligochaetes with special reference to Southeast Asia.

Trans. Am. Philos. Soc. 62, 1–326.Guindon, S., Gascuel, O., 2003. A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst. Biol. 52, 696–704.Hendrix, P.F., Bohlen, P.J., 2002. Exotic earthworm invasions in North America: ecological and policy implications. Bioscience 52, 801–811.James, S.W., 2008. Earthworm: their diversity and applications to science and technology. Proc. B.R.T. Annual Con. 12, 46–51.Kosavititkul, P., 2005. Species Diversity of Terrestrial Earthworms in Khao Yai National Park. Dissertation. Suranaree University of Technology.Librado, P., Rozas, J., 2009. DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics 25, 1451–1452.Mantel, N., 1967. The detection of disease clustering and a generalized regression approach. Cancer Res. 27, 209–220.Marinissen, J.C.Y., den Bosch, F., 1992. Colonization of new habitat by earthworms. Oecologia 91, 371–376.McElroy, T.C., Diehl, W.J., 2001. Heterosis in two closely related species of earthworm (Eisenia fetida and E. andrei). Heredity 87, 598–608.Murphy, R.W., Sites Jr., J.W., Buth, D.G., Haufler, C.H., 1996. Protein: isozyme electrophoresis. In: Hillis, D.M., Moritz, C., Mable, B.K. (Eds.), Molecular Sys-

tematics. Sinauer Association, Sunderland, Massachusetts, pp. 51–120.Nei, M., 1978. Estimation of average heterozygosity and genetic distance from a small sample number of individuals. Genetics 89, 583–590.Peles, J.D., Towler, W.I., Guttman, S.I., 2003. Population genetic structure of earthworms (Lumbricus rubellus) in soil contaminated by heavy metals. Eco-

toxicology 12, 379–386.Porco, D., Decaëns, T., Deharveng, L., James, S.W., Skar _zy�nski, D., Erséus, C., Butt, K.R., Richard, B., Hebert, P.D.N., 2012. Biological invasions in soil: DNA

barcoding as a monitoring tool in a multiple taxa survey targeting European earthworms and springtails in North America. Biol. Invasions. http://dx.doi.org/10.1007/s10530-012-0338-2.

Raymond, M., Rousset, F., 1995. An exact test for population differentiation. Evolution 49, 1280–1283.Ridgway, G.J., Sherbrune, S.W., Lewis, R.D., 1970. Polymorphisms in the esterase of Atlantic herring. Trans. Am. Fish Soc. 99, 147–151.Ronquist, F., Teslenko, M., van der Mark, P., Ayres, D.L., Darling, A., Höhna, S., Larget, B., Liu, L., Suchard, M.A., Huelsenbeck, J.P., 2012. MrBayes 3.2: efficient

bayesian phylogenetic inference and model choice across a large model space. Syst. Biol. 61, 539–542.Shen, H.P., Yeo, D.C.J., 2005. Terrestrial earthworms (Oligochaeta) from Singapore. Raffles Bull. Zool. 53, 13–33.Sneath, P.H.A., Sokal, R.R., 1973. Numerical Taxonomy. W. H. Freeman, San Francisco, p. 573.Somniyam, P., Suwanwaree, P., 2009. The diversity and distribution of terrestrial earthworms in Sakaerat Environmental Research Station and adjacent arae,

Nakorn Ratchasima, Thailand. World Appl. Sci. J. 6, 221–226.Swofford, D.L., Selander, R.B., 1981. BIOSYS-1: a FORTRAN program for the comprehensive analysis of electrophoretic data in population genetics and

systematics. J. Heredity 72, 281–283.Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., Kumar, S., 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood,

evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28, 2731–2739.Workman, P.L., Niswander, J.D., 1970. Population studies on southwestern Indian tribes. II. Local genetic differentiation in the Papago. Am. J. Hum. Genet. 22,

24–49.Wright, S., 1965. The interpretation of population structure by F-statistics with special regard to systems of mating. Evolution 19, 395–420.