Mitochondrial DNA and morphological variation in the sentinel earthworm species Lumbricus rubellus

European Journal of Soil Biology 64 (2014) 23e29

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European Journal of Soil Biology journal homepage: http://www.elsevier.com/locate/ejsobi

Original article

Mitochondrial DNA and morphological variation in the sentinel earthworm species Lumbricus rubellus Robert K. Donnelly a, b, *, Georgina L. Harper a, A. John Morgan b, Gabriela A. Pinto-Juma b, Michael W. Bruford b a b

Faculty of Health, Sport and Science, University of Glamorgan, Llantwit Road, Trefforest, Mid Glamorgan CF37 1DL, UK Cardiff School of Biosciences, Cardiff University, PO Box 915, Cardiff CF10 3TL, UK

a r t i c l e i n f o

a b s t r a c t

Article history: Received 6 May 2014 Received in revised form 24 July 2014 Accepted 25 July 2014 Available online 28 July 2014

The common epigeic earthworm Lumbricus rubellus has been found to comprise two genetically distinct lineages and this genetic heterogeneity could compromise its currently widespread use as a sentinel in soil ecotoxicology. An extensive analysis of lineage diversity was conducted on UK populations of L. rubellus, comprising 137 individuals collected from England and Wales. Sequencing of the mitochondrial COI region revealed the widespread occurrence of the two described lineages throughout Britain, which were often found to co-exist at the same site. Morphological characters were investigated to differentiate the two lineages. A rapid genetic test (mitochondrial PCR amplification and restriction digestion) was applied to determine the lineage of each specimen. A blind trial revealed a characteristic glandular tumescence to be effective in differentiating the lineages, particularly in identifying ‘lineage B’ worms. COI sequence analysis was also conducted upon three other Lumbricus species, which failed to uncover any genetic lineages comparable to those found within L. rubellus. © 2014 Elsevier Masson SAS. All rights reserved.

Keywords: Lumbricus rubellus Earthworm Cryptic lineages mtDNA Cytochrome oxidase I Morphology

1. Introduction The increasing evidence that components of soil fauna are sensitive to a variety of anthropogenic activities [30,41] promotes the need for a more accurate understanding of species diversity and its relationship to ecosystem services [37]. Overlooking cryptic species leads to underestimation of ecosystem complexity, and could potentially confound biomonitoring and conservation initiatives [5]. Therefore a reliance on the use of only morphological criteria to identify species, especially in taxa such as earthworms where juvenile life-stages present particular difficulties and others where good keys and taxonomic expertise is limiting [12,38], has resulted in a change towards the widespread application of molecular genetic approaches [16]. The application of molecular techniques to taxonomy has revealed that many common invertebrate morphotypes may actually comprise highly divergent cryptic lineages [13,17,21,22,35,42]. The phenomenon appears to be especially prevalent in sexually reproducing lumbricid earthworms [25,27,28,32,33].

* Corresponding author. Present address: Cardiff School of Biosciences, Cardiff University, PO Box 915, Cardiff CF10 3TL, UK. E-mail address: [email protected] (R.K. Donnelly). http://dx.doi.org/10.1016/j.ejsobi.2014.07.002 1164-5563/© 2014 Elsevier Masson SAS. All rights reserved.

Phylogenetic analyses of European lumbricid earthworms using mitochondrial barcoding primers have revealed evidence of deeply-divergent lineages in several species: Allolobophora chlorotica, Aporrectodea longa, Aporrectodea rosea, Aporrectodea trapezoides, Lumbricus rubellus and Lumbricus terrestris [1,15,18,25,27,28,32,33]. Two distinct genetic lineages have been uncovered within UK populations of L. rubellus, with as many as four other identifiable lineages in mainland Europe (P. Sechi, pers. comm.), each displaying sequence divergences comparable to distinct species. Moreover, the two UK lineages have been confirmed as being present in ecotoxicity studies employing combinations of mitochondrial and nuclear markers [2,14,24]. These high levels of genetic divergence within L. rubellus could have major implications for the role of this epigeic species as a biomonitoring sentinel, especially in highly discriminatory ‘omics’ studies [4,10]. For example [11], emphasised that the substantial genetic heterogeneity that often exists in field populations should be fully considered in the design and interpretation of field-based ecotoxicological assessments. Studies demonstrating genotype-related differential sensitivities and resilience to toxicant exposures highlight the need to account for this variation [3,34,39,44]. From the above examples, the practical and interpretive problems associated with the inclusion of undetected cryptic speciation in molecular-genetic monitoring regimes are clear. For this reason

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[2], advocated that every individual sampled from wild populations should be genotyped prior to eco-toxicogenomic assay. However this will often be impractical due to logistic and financial constraints in routine monitoring, therefore it could be highly advantageous to be able to identify cryptic species in the field The recognition of putative L. rubellus sub-species based on minor differences in external morphological traits [7] highlights that it might be similarly possible to discriminate between individuals of the two UK L. rubellus lineages. The first aim of this study was to apply ‘universal’ COI primers [19] to a relatively large number of L. rubellus from a variety of habitat types across a wide geographic range to evaluate genetic diversity within the two lineages to see whether L. rubellus includes more cryptic diversity than previously recorded in the UK. Mitochondrial analysis was also applied to individuals of three congeners (Lumbricus castaneus, Lumbricus festivus, L. terrestris) in order to establish whether the lineage diversification so commonly encountered in L. rubellus is a feature shared by other members of the genus. This is especially relevant in a European context, since studies have recently indicated the presence of cryptic genetic variation within L. terrestris sampled from Canada, the US and mainland Europe [28]. The second aim of this study was to determine whether variation in the anterior segment number (an evolutionarily conserved trait widely used in earthworm taxonomy [43]), and anteriorlocated glandular tumescences are distinguishing morphological features in L. rubellus lineages. A blind trial was conducted to determine if these traits could be successfully applied to differentiate between the lineages in the field, with a lineage-specific mitochondrial Restriction Fragment-Length Polymorphism (RFLP) test subsequently used to ascertain whether the inferences based on morphology were reliable. 2. Material and methods 2.1. MtDNA analysis Specimens of four Lumbricus species were collected from a field site located at Pontcanna fields, Cardiff (Tables 1 and 2). Further collections were conducted at two sites at Clydach, Swansea, two sites located in Mid-Wales (Wemyss and Ystwyth Source) and two sites in South-West England (Glovers Field and Hallen Hill). The sample sites represented a range of different habitats including forests, pastureland and parkland (Table 2). Approximately 25 mg of earthworm tissue was used for each DNA extraction. DNA was extracted using a QIAGEN DNeasy tissue extraction kit (QIAGEN, UK). A 580 bp sub-unit of the mitochondrial cytochrome oxidase I gene (COI) was then PCR-amplified using the general invertebrate primers LCO1490 (50 -GGTCAACAAATCATAAAGATATTGG-30 ) and HCO2198 (50 -TAAACTTCAGGGTGACCAAAAAATCA-30 ), [19]. PCR reactions were performed using the

Table 2 Location and description of sample sites. Site

Latitude

Longitude

Site description

Glovers field

51:18:42N

02:47:33W

Hallen Hill

51:30:57N

02:39:28W

Ystwyth Source East Cottage

52:21:57N 52:21:35N

03:41:44W 03:44:59W

Wemyss

52:20:58N

03:53:14W

Clydach 1

51:41:47N

03:53:18W

Clydach 2 Dinas Powys 6/Pontcanna

51:41:48N 51:29:37N

03:52:49W 03:12:18W

Rough pastureland near abandoned Zn mine Deciduous woodland near abandoned Pb/Zn smelter Stream bank Derelict building at abandoned Pb/Zn mine Stream bank near abandoned Pb/Zn mine Deciduous woodland and rough grassland close to Ni smelter Playing field Mound of topsoil leaves on mowed parkland

reaction volumes and times suggested by Ref. [25]. All reactions were performed using a GeneAmp PCR System 9700 (Applied Biosystems, UK). The general COI primers were found to successfully amplify only two of the L. rubellus lineage B individuals. A primer pair was therefore developed based upon the COI sequences from these two individuals, which successfully amplified a smaller 356 bp fragment of the COI region of the lineage B individuals (LRB COIF 50 TCTTCTTTCTTGTCATGCCTGT-30 , LRB COIR 50 -TGAAGTATTTAGATTTCGGTCAGTT-30 ). PCR reactions were again performed using the reaction volumes of [25]. All samples were initially denatured for 2.5 min at 94  C with 35 cycles of 94  C for 30s, 45  C for 30s and 72  C for 45s, followed by 72  C for 10 min. Following PCR amplification, PCR products were purified and sequenced in accordance with the procedure detailed by Ref. [14]. Sequencher 3.1.2. (Gene Codes Corporation, USA) and CodonCode Aligner 3.0.1 (CodonCode Corporation, USA) were used to align and edit all sequences and to determine COI haplotypes for each species. The program MEGA 4.0 [45] was used to compute pairwise genetic distances between all haplotypes. Pairwise distances were calculated both as uncorrected p-distances and also using the Tamura-Nei model of substitution (gamma shape distribution parameter ¼ 0.28), selected on the basis of hierarchical likelihood ratio tests performed using the program jModelTest [36]. A maximum-likelihood phylogeny was produced using the program PHYML [20]. This was based upon Tamura-Nei corrected distances between haplotypes with a bootstrap replication of 1000. A Bayesian phylogenetic analysis of haplotypes was also conducted using MrBayes 3.1.2 [40]. The general time reversible model was selected for this analysis with gamma and invariable sites. Bayesian analysis was run using two independent runs consisting of four chains. These analyses were run for 400,000 generations with trees saved every 100 generations. Following the analysis, 1000 trees were removed as burn-in. Consensus trees were viewed using MEGA 4.0 [45].

Table 1 Number of sampled individuals at each site. Collection site

Site L. castaneus L. festivus L. rubellus L. terrestris code

2.2. Blind trial

Pontcanna fields, Cardiff Clydach (site 1), Swansea Clydach (site 2), Swansea Glovers Field, Somerset Hallen Hill, Bristol Ystwth Source, Powys East Cottage, Ceredigion Wemyss, Ceredigion Total

PT 17 CL1 0 CL2 0 GF 0 HH 0 YS 0 EC 0 WM 0 17

One hundred and five L. rubellus individuals were collected from four field sites located across South Wales. The sites were selected by the collectors to include populations of both mitochondrial lineages of L. rubellus based upon the findings of previous studies. All earthworms were placed individually into polythene bags labelled with an identifying code. The code was initially known only to the collector, allowing a blind trial to be conducted by the experimenter.

25 0 11 0 0 0 0 0 36

25 22 22 19 9 10 16 14 137

22 0 9 0 0 0 0 0 31

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Earthworms were then washed with distilled water and depurated for two days. Following depuration each individual was weighed and then separated into anterior and posterior sections that were preserved in ethanol. Posterior sections were preserved in 100% ethanol for DNA extraction whilst anterior sections were preserved in 70% ethanol for morphological analysis. After processing, both the anterior segment number and the location and size of an anterior glandular tumescence (associated with a lack of ventral setae) was recorded for all L. rubellus individuals and the possible lineage of the individual recorded based upon the degree of the glandular swelling (Fig. 1). Sequence analysis of the mtDNA 16s region of lineage A and B L. rubellus yielded two restriction sites present within lineage B but absent from lineage A (A. King, pers. comm.). By applying restriction fragment analysis to PCR amplified fragments, it was possible

25

to efficiently determine the lineage of L. rubellus individuals. A 500e650 bp fragment of the mtDNA 16s RNA coding region was then PCR amplified in a 25 ml reaction using the invertebrate primers 16sar (50 -CGCCTGTTTATCAAAAACAT-30 ) and 16sbr (50 CCGGTCTGAACTCAGATCACGT-30 [31]). PCR reactions were performed using the reaction volumes and times suggested by Ref. [25]. Restriction digestion of the 16s fragments was then performed using the restriction enzymes DraI and HpyI in 10 ml volumes containing 7.8 ml of the PCR product, 1 enzyme buffer (NE Biolabs Ltd., UK), distilled water and 4 units of restriction enzyme (NE Biolabs Ltd., UK). All samples were then incubated at 37  C for 3 h using a GeneAmp PCR System 9700 (Applied Biosystems, UK). Following incubation all fragments were run on a 1.5% agarose gel. Following sequence analysis, alcohol-fixed glandular tumescences of both lineage A and lineage B L. rubellus were imaged with

Fig. 1. Scanning electron microscope images of glandular tumescences of A) Lumbricus rubellus lineage A (segments ieii) B) Lumbricus rubellus lineage B (segment i). Note the absence of ventral setae on these segments.

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a scanning electron microscope. Segments containing the tumescences were excised with a scalpel. The samples were then processed by critical-point drying, mounted onto aluminium stubs and sputter-coated with gold. The specimens were imaged using a Philips XL 20 high vacuum scanning electron microscope.

3.1. Molecular analysis A 356 bp sub-unit of the mitochondrial COI gene was successfully sequenced for 221 individuals. These sequences were found to align unambiguously and displayed no evidence of insertion/deletions. Twenty-two COI haplotypes were found within L. rubellus (Table 3, Genbank accession nos. JN419211eJN419232). The haplotypes were found to group into two distinct clades corresponding to the previously described lineage A and B of L. rubellus [25]. Nine haplotypes were found for ‘lineage A’ (rubellus 1e9) and 13 haplotypes were found for ‘lineage B’ (rubellus 10e22). Haplotypes of both lineages were uncovered at five of the sampled sites (Table 3). No lineage A haplotypes were uncovered at

Table 3 Haplotype frequency at different sites. EC

GF

HH

PT

WM

YST

9 8

CL2

3

1

6 1

6

2

5

1 1 1

15 3 1 1 1 2 2

L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L. L.

rubellus 1a rubellus 2a rubellus 3a rubellus 4a rubellus 5a rubellus 6a rubellus 7a rubellus 8a rubellus 9a rubellus 10b rubellus 11b rubellus 12b rubellus 13b rubellus 14b rubellus 15b rubellus 16b rubellus 17b rubellus 18b rubellus 19b rubellus 20b rubellus 21b rubellus 22b castaneus 1 castaneus 2 castaneus 3 castaneus 4 castaneus 5 castaneus 6 castaneus 7 castaneus 8 festivus 1 festivus 2 festivus 3 festivus 4 festivus 5 festivus 6 festivus 7 terrestris 1 terrestris 2 terrestris 3 terrestris 4 terrestris 5 terrestris 6 terrestris 7

a

Lumbricus rubellus lineage A. L. rubellus lineage B.

b

1 1. 2. 3. 4. 5.

3. Results

CL1

Table 4 Mean pairwise differences between Lumbricus species and lineages (Tamura-Nei distance above diagonal, non-corrected p distance below diagonal), with mean intraspecific values along the diagonal (non-corrected p distance/Tamura-Nei distance).

8 12 2

1 1

3

7 6

7 1 2 1 1 1 1 1 2

1

4 1 2 1 3 3

2 3 1

4 4 2 1 3 1 1 1 15 1 2 3 2 1 1 18 1 2 1

L. L. L. L. L.

rubellus (lineage a) 0.02/0.03 rubellus (lineage b) 0.14 castaneus 0.21 festivus 0.17 terrestris 0.39

2

3

4

5

0.30 0.01/0.01 0.50 0.38 0.30

0.56 0.20 0.05/0.06 0.60 0.49

0.37 0.18 0.22 0.02/0.03 0.46

0.17 0.15 0.20 0.19 0.05/0.06

the site of Clydach 2. Similarly, no lineage B haplotypes were uncovered at the sites of Clydach 1 and Hallen Hill. No clear relationship could be discerned between either the habitat type or contamination status of a site and its constituent haplotypes (Tables 2 and 3). For the other three species, eight COI haplotypes were found for L. castaneus (Genbank accession nos. JN419196eJN419203), seven haplotypes were found for L. festivus (Genbank accession nos. JN419204eJN419210), and seven haplotypes were found for L. terrestris (Genbank accession nos. JN419233eJN419239). The mean uncorrected p-distance of 0.14 between L. rubellus lineages A and B was found to be slightly higher than that observed by Refs. [25]; and is almost comparable to the p-distances observed between L. rubellus and other Lumbricus species (Table 4). Genetic distances between haplotypes of L. rubellus lineage B were far lower than between the haplotypes of lineage A. Both the ML and Bayesian phylogenies displayed strong support for the monophyly of all of the Lumbricus species analysed (Figs. 2 and 3). The phylogenies also both showed strong support for the monophyly of both lineage A L. rubellus and lineage B L. rubellus. The bootstrap and posterior probabilities of the two phylogenies also supported the existence of haplogroups within L. rubellus lineage A, L. castaneus, L. festivus and L. terrestris. Both the ML and Bayesian phylogenies supported the existence of two major haplogroups within L. terrestris although the divergence between these groupings was not as deep as reported by Ref. [28]. 3.2. Blind trial The blind trial experiment resulted in the successful discrimination of 80% of the individuals (significantly different from random: chi2 test, *P ¼ 0.01). Eighty-nine percent of lineage B individuals were correctly identified although only 71% of lineage A individuals were successfully identified. The location of the glandular tumescence was found between segments 8 and 12 in lineage A and segments 9 and 12 in lineage B. The tumescence was only found on a single segment in all lineage B individuals, whereas lineage A individuals displayed the swelling over two segments. It was also noted that the glandular tumescence appeared more prominent in lineage B individuals where it could often be observed without magnification. All individuals were found to display an anterior segment count of 26, corresponding to the segment count described by Ref. [43]. 4. Discussion The discovery that mitochondrial lineages of L. rubellus often coexist within contaminated sites indicates a potential problem for ecotoxicological studies when using this species as a sentinel. This is especially true given that recent nuclear DNA analyses have indicated that the two lineages appear to be reproductively isolated when coexisting within sites [14]. In order for eco-toxicological

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they frequently coexist in contrasting habitats. This study found no evidence for more than two lineages in the UK, a noteworthy reduction in diversity compared with the L. rubellus complex in mainland Europe (P. Sechi, pers. comm.) The mean divergence between the haplotypes of L. rubellus lineages A and B was relatively high (uncorrected p-distance ¼ 0.14) and was comparable to the level of divergence found between L. rubellus and the three congeneric species featured in the study (uncorrected pdistances ¼ 0.17e0.39). Although none of the other species analysed were found to contain haplogroups comparably divergent to those found within L. rubellus, the average levels of genetic variation were still found to be relatively high compared with general values from COI barcoding studies of earthworms, which show levels of intraspecific variation to be largely below 1% [23]. Several well-supported lineages were found within L. castaneus and L. terrestris, and the L. rubellus lineage A haplotype, rubellus 7, was found to diverge from the other haplotypes by p-distances of between 0.01 and 0.05. Studies involving earthworm identification from mitochondrial DNA extracted from the gut contents of predators [8,9,26] and directly from soil [6] will doubtless increasingly explore this aspect of earthworm diversity. Similarly, the taxonomy of juvenile earthworms will be both facilitated and enriched by molecular approaches [38]. Screening with appropriate lineage-specific morphological traits during field sampling is a feasible way of ensuring that the appropriate number of individuals belonging to a given L. rubellus lineage is collected to satisfy an eco-toxicogenomic experimental

Fig. 2. Maximum Likelihood tree of Lumbricus mtDNA COI haplotypes. Genetic distances calculated using the Tamura-Nei model of substitution. Numbers on tree indicate bootstrap support obtained with 1000 replicates.

studies to yield meaningful observations about the effects of contaminants, it has been proposed that the subject organisms should be individually genotyped so that the assays are performed on a genetically homogeneous sample [2]. The importance of this assertion is underlined by a study of the aquatic oligochaete Tubifex tubifex, which showed that individuals belonging to different lineages diverging at 16S rDNA by up to 13%, display significant differences in their susceptibility to cadmium contamination [44]. Similarly, the marine polychaete Capitella capitata has been demonstrated to comprise several reproductively isolated lineages that exhibit differences in their sensitivity and biochemical responses to hydrocarbons [3,29]. The recent report that the two lineages of L. rubellus display differences in their tolerance to arsenic in the field [24] is, therefore unsurprising. It also underlines the potential value in terms of sampling effort of a lineagescreening method based on subtle morphological traits that is applicable in the field with a high discriminatory reliability for distinguishing the two cryptic L. rubellus lineages extant in the UK. Analysis of genetic variation in the mtDNA DNA barcoding (COI) gene of L. rubellus confirmed the existence of the two cryptic mitochondrial lineages originally uncovered by Refs. [25]; and that

Fig. 3. Majority-rule consensus tree from the Bayesian analysis of Lumbricus mtDNA COI haplotypes. Genetic distances calculated using the GTR þ IþG model of substitution. Numbers on tree indicate posterior probabilities.

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design. It is likely, for example, that many such future studies will focus on lineage B because the draft L. rubellus genome (earthworms.org) is based on an individual belonging to this lineage. Morphological analysis revealed anterior segment number to be a largely well-conserved trait within both L. rubellus lineages, with only a very small proportion of individuals showing any deviation from the numbers described by Ref. [43]. Much more useful for pragmatic lineage-screening is the position and size of the anterior glandular tumescence. This was found to display a wide degree of variation within lineage A, indicating it to be a morphologically plastic trait, but it is a more pronounced and far less variable secondary sexual character in lineage B. It is noteworthy that differences in the size, segment position and extent of the glandular tumescence were used by Ref. [7] to characterise two possible taxonomic sub-species of L. rubellus. These inter-lineage anatomical differences suggest a useful means of assigning L. rubellus to one of the two lineages (B) with almost 90% confidence during field collection. This approach offers a practical solution for extensive field-sampling to provide material for laboratory bioassays. It is envisaged that this assay could be done in the field by the examination of alcohol-fixed worms with either a hand-lens or a simple USB-microscope. Such samples are compatible with DNA-based analytical protocols. It is at yet uncertain whether the distinguishing anatomical features can be examined reliably in living or mildly anaesthetised worms. The advantage of the latter approach is that unwanted worms could be returned to the field.

Acknowledgements This work was supported by the Leverhulme Trust (Grant no. F/ 00407/AI). We would like to thank Andy King and Pete Kille for their help with the project.

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