Systematic revision of Gatesona (Crassiclitellata, Lumbricidae), an endemic earthworm genus from the Massif Central (France).

The Massif Central in France could potentially harbor numerous ancient endemic lineages owing to its long history of continuous geological stability. Several endemic earthworm species inhabit the area, with Allolobophora (Gatesona) chaetophora, Helodrilus (Acystodrilus) and Avelona ligra showing hints of a common evolutionary origin. However, the phylogenetic relationships and taxonomic status of the species remain to be studied through integrative molecular and morphological methods. To this end, eight species including most of the known species and subspecies of All. (Gatesona), Helodrilus (Acystodrilus) musicus, and Avelona ligra were sequenced for a set of five molecular markers. The species were grouped on the basis of the molecular findings in a phylogenetic framework. All. (Gatesona) was included within the same clade as Helodrilus (Acystodrilus) and Avelona, separated from Allolobophora sensu stricto, supporting its status as a good genus. Branch lengths and average pairwise genetic distances suggested the subspecies of All. (Gatesona) chaetophora examined should be considered species-level taxa. Thus, a generic diagnosis for Gatesona stat. nov. is provided, along with redescriptions of Gatesona chaetophora comb. nov., Gatesona rutena comb. nov. stat. nov., Gatesona lablacherensis comb. nov. stat. nov. and Gatesona serninensis comb. nov. stat. nov. The study findings highlight the need for further sampling of earthworm diversity in the Massif Central (and Southern France), in addition to an increased focus on the Eastern European species of Helodrilus.

Introduction

The Massif Central in France is an ancient massif which originated in the Variscan orogeny (Late Paleozoic) and has been an emerged continental realm through the Mesozoic and Cenozoic to the present [1]. Owing to this stability over geological time, the region is expected to harbour a large number of ancient endemic lineages (as observed for mite harvestmen in the Iberian Peninsula [2]. Earthworms, whose phylogeography and biogeography are known to reflect paleogeographic events [3], are a good example of this prediction. The Massif Central is inhabited by several endemic earthworm species from narrowly distributed genera: Hemigastrodrilus monicae Bouché 1970 (a basal lineage of Hormogastridae [4]), Ethnodrilus aveli Bouché 1972, Ethnodrilus gatesi Bouché 1972, Zophoscolex graffi Bouché 1972, Zophoscolex micellus Bouché 1972, Allolobophora (Gatesona) chaetophora Bouché 1972, Helodrilus (Acystodrilus) cortezi Qiu & Bouché 1998, Helodrilus (Acystodrilus) musicus Qiu & Bouché 1998, Helodrilus (Acystodrilus) segalensis Qiu & Bouché 1998 and Avelona ligra Bouché 1969.

Among the above-mentioned species, All. (Ga.) chaetophora, Helodrilus (Acystodrilus) and Avelona ligra may share a common evolutionary origin according to different sources of evidence. All. (Ga.) chaetophora and He. (Ac.) cortezi were grouped together in a relatively basal clade in the molecular phylogenetics analysis reported by Domínguez et al. [5]. This position is congruent with their morphological characteristics, as excepting by their nephridial bladder structure (sigmoid vs absent) both taxa are very similar. Av. ligra is also endemic to the (northeastern) Massif Central and morphologically similar to both taxa. The only molecular phylogenetics study including Av. ligra [6] found that it is closely related to All. (Ga.) chaetophora; however, the sparse taxon sampling of Lumbricidae does not provide robust support for such a clade. In order to confirm a clade comprising these species, additional representatives of these taxa with a common molecular marker set must be examined.

In addition to the phylogenetic uncertainty, there remain some taxonomic questions regarding All. (Ga.) chaetophora. Bouché (1972) described several subspecies within All. (Ga.) chaetophora with very restricted ranges and significant morphological differences from the type species. Bouché acknowledged that such morphological heterogeneity should be delimited in further studies (which he was unable to perform). Surprisingly, this type of study was performed in Qiu and Bouché [7] for other species complexes such as Scherotheca -resulting in the elevation of several subspecies to species status- but not for All. (Ga.) chaetophora. However, two closely related species, All. (Ga.) sausseyi Qiu & Bouché 1998 and All. (Ga.) transmatica Qiu & Bouché 1998, were described and added to the subgenus Gatesona [8]. The position of Allolobophora chlorotica (type species of Allolobophora) within a well-supported, independent clade from All. (Ga.) chaetophora [5] suggests that Gatesona could be erected as a proper genus. Molecular phylogenetic analysis of additional species (and subspecies) would contribute to a robust assessment of this hypothesis.

In this study, a systematic revision of Allolobophora (Gatesona) is proposed based on a molecular phylogenetic analysis including several species and subspecies. In addition, the phylogenetic link between Allolobophora (Gatesona), Helodrilus (Acystodrilus) and Avelona ligra was tested by the inclusion of the later in the same phylogenetic data set.

Materials and methods

Specimens, sampling and morphological description

The specimens described in this study were collected in a sampling survey carried out in Southeastern France and Catalonia between October and November 2019, with additional sampling in Spring 2021. The list of species and the locations where they were collected are shown in Table 1.

Table 1

Species sampled and sequenced in this study, with their sampling location and coordinates.

Taxon	Location	Coordinates
Allolobophora (Gatesona) chaetophora chaetophora *	L’Hospitalet du Larzac	43.956, 3.218433
Allolobophora (Gatesona) chaetophora lablacherensis Bouché 1972	Lablachère, France	44.433, 4.22643
Allolobophora (Gatesona) chaetophora serninensis Bouché 1972	Saint-Sernin-sur-Rance	43.87487, 2.60635
Allolobophora (Gatesona) chaetophora rutena Bouché 1972	Rôdez, France	44.415, 2.03943
Allolobophora (Gatesona) sausseyi	Argelès-sur-Mer, France	42.52361, 3.04193
Allolobophora (Gatesona) transmatica	Agullana, Spain	42.39626, 2.84851
Helodrilus (Acystodrilus) musicus	Saint-Germain-de-Calberte, France	44.21667, 3.79999
Avelona ligra	Jargeau, France	Not available
Allolobophora bartolii Bouché 1970	Jouques, France	43.641, 5.63043

*Individuals from this species were newly sampled for morphological study but previously existing sequences were used for the phylogenetic analyses.

Earthworms were obtained by soil digging and hand-sorting, rinsed with water and fixed in 70% in order to obtain relaxed (as opposed to excessively retracted) specimens. They were subsequently transferred to 100% ethanol to enable further molecular analyses. Species classification and morphological diagnoses were conducted using the same set of external and internal morphological characters used by Qiu & Bouché [8,9]. The following main external morphological characters were considered: mean length, mean number of segments, mean weight, pigmentation, type of prostomium, setal arrangement, position of papillae, position of first dorsal pore, nephridial pore arrangement, position and development of male pores, position and development of female pores, position of spermathecal pores, position of clitellum and position of tubercula pubertatis.

The main internal anatomical characters considered were as follows: position of oesophageal hearts, position and morphology of calciferous glands, position of crop, position of gizzard, type of typhlosole, shape of nephridial bladders, number and position of seminal vesicles, and number and position of spermathecae.

Holotypes of Allolobophora (Gatesona) chaetophora and its different subspecies (see Table 1), temporally available at the personal collection of Marcel Bouché, were studied and compared with the newly sampled material.

DNA isolation, sequencing and molecular analyses

Total genomic DNA was extracted from ventral integument samples of approximately 5 x 5 mm by using the DNeasy Blood & Tissue Kit (Qiagen). For 8 species, regions of the nuclear 28S rRNA and mitochondrial 16S rRNA, tRNAs Ala, Leu and Ser, NADH dehydrogenase (ND1) and COI were amplified by polymerase chain reaction (PCR), with the primers and conditions described in Pérez-Losada et al. [10,11]. PCR products were purified and sequenced by the C.A.C.T.I Genomics service (University of Vigo).

DNA sequences obtained in this study are available in Genbank, under accession numbers MZ153163-MZ153169, MZ156573-MZ156588 and MZ235293-MZ235314.

Sequences reported by Domínguez et al. [5,12], Pérez-Losada et al. [10,11,13], Paoletti et al. [14] and de Sosa et al. [15], including representatives of most of the Lumbricidae genera and two members of the closest families (Hormogastridae and Criodrilidae), were downloaded from Genbank and used as a reference data set. The species included are listed in Supporting Information S1 File.

Sequences were aligned with MAFFT v.7 [16] with default settings and concatenated with BioEdit [17], resulting in a matrix of 3,201 bp. The best fitting evolutionary model for each partition was selected with jModelTest v. 2.1.3 [18] by applying the Akaike information criterion (AIC [19]), and Bayesian information criterion (BIC [20]). GTR + I + G was selected as the best-fitting evolutionary model for COI, 28S and ND1, HKY+I+G was selected for 16S and HKY+G was selected for tRNAs.

Maximum Likelihood analysis was performed with RaxML-NG [21] as implemented in the CIPRES Science Gateway V. 3.3 [22], with 10 random starting trees and estimating the support for the resulting topologies with 1,000 rapid bootstrap replicates. Bayesian Inference of the phylogeny was estimated with MRBAYES v.3.1.2 [23] as implemented in the CIPRES Science Gateway V. 3.3. Parameters were set to 50 million generations and sampled every 5,000th generation (10,000 trees). Two independent runs each with four chains were performed and 20% of the trees were discarded as burn-in. The remaining trees were combined and summarized on a 50% majority-rule consensus tree. Clade support values over 70% and 90% (for Bootstrap and Posterior probability respectively) were considered high (see [15,24]).

Uncorrected average pairwise distances between the studied species for the molecular markers COI and 16S were calculated in MEGA X [25] in order to support their status as separate species and to study genetic distances within and between genera.

Results and discussion

Molecular phylogenetic analyses

Bayesian inference (Fig 1) and maximum likelihood inference phylogenetic trees recovered consistent topologies. Avelona ligra and representatives of Helodrilus (Acystodrilus) and Allolobophora (Gatesona) species formed a well-supported clade, isolated from species assigned to Allolobophora and Helodrilus. Allolobophora (Gatesona) and Helodrilus (Acystodrilus) appeared intermixed in a well-supported genus-level clade, hereafter referred to as Gatesona. Within Gatesona, representatives of the different subspecies of All. (Ga.) chaetophora were separated by long branches. In addition, Ga. sausseyi and Ga. transmatica were nested within a clade including All. (Ga.) chaetophora and its different subspecies.

Fig 1

Detail of the phylogenetic tree obtained by Bayesian phylogenetic analysis of the concatenated sequence of molecular markers COI–16S-tRNAs–ND1–28S (see full tree in S2 File).

Allolobophora (Gatesona) representatives are shaded in blue and Helodrilus (Acystodrilus) representatives are shaded in green. Representatives of the main Lumbricidae genera were collapsed and are shown in boxes. Posterior probability support values are shown besides the corresponding nodes.

COI and 16s uncorrected average pairwise genetic (UAPG) distances between the species of the genera Avelona, Gatesona (comprising All. (Gatesona) and He (Acystodrilus)) and Helodrilus (Helodrilus) included in this study are shown in Table 2. UAPG distances between representatives of Avelona, Gatesona and Helodrilus (Helodrilus) were high, ranging from 15.2% to 21% for COI and from 11.0% to 17.5% for 16s. UAPG distances within the genus-level clades were lower, with values corresponding to Gatesona (8.1%-17.3% COI, 4.1%-13.2% 16s) being generally lower than distances within Helodrilus (Helodrilus) (17% COI, 11.9% 16s). UAPG distances between the different species and subspecies of Gatesona were high (see above) except for All. (Ga.) sausseyi- All. (Ga.) transmatica (8.1% COI, 4.1% 16s).

Table 2

Uncorrected average pairwise genetic (UAPG) distances between species of the genera Avelona, Helodrilus (Acystodrilus), Gatesona and Helodrilus (Helodrilus) included in this study for the molecular markers COI (below the diagonal) and 16S (above the diagonal).

	Av. ligra	He. (Ac.) cortezi	He. (Ac.) musicus	All. (Ga.) chaetophora	All. (Ga.) chaetophora lab.	All. (Ga.) chaetophora ser.	All. (Ga.) chaetophora rutena	All. (Ga.) sausseyi	All. (Ga.) transmatica	He. cernosvitovianus	He. patriarchalis
Av. ligra		14.3	16.9	16.6	13.5	12.4	13.5	14.6	15.7	13.3	13.1
He. (Ac.) cortezi	19.6		7.9	11.2	10.2	11.7	10.6	10.5	10.2	14.0	15.0
He. (Ac.) musicus	19.6	10.6		12.5	12.5	13.1	12.0	9.7	11.6	14.8	16.3
All. (Ga.) chaetophora	15.2	16.0	16.0		13.2	11.3	9.1	11.3	13.2	13.6	14.6
All. (Ga.) chaetophora lablacherensis	*	*	*	*		10.1	9.0	9.7	10.4	11.4	14.2
All. (Ga.) chaetophora serninensis	19.0	16.7	16.2	14.5	*		8.2	6.7	9.0	14.0	17.5
All. (Ga.) chaetophora rutena	17.8	9.1		13.6	*	14.6		7.5	7.9	11.0	13.5
All. (Ga.) sausseyi	18.3	17.3	16.5	14.4	*	14.5	14.1		4.1	14.8	17.1
All. (Ga.) transmatica	18.5	16.7	16.3	13.2	*	12.9	14.1	8.1		14.0	16.3
He. cernosvitovianus	20.5	19.0	18.8	17.5	*	19.5	17.3	19.8	19.1		11.9
He. patriarchalis	19.1	18.6	18.8	17.7	*	20.1	17.2	18.2	18.2	17.0

Red shading indicates higher values and green shading indicates lower values. The COI distance between All. (Ga.) rutena and He. (Ac.) musicus is not displayed as it was unusually low. Asterisks indicate COI sequences were not available for the species.

Morphological analysis

External and internal morphological characters of the species and subspecies of Allolobophora (Gatesona) and related Helodrilus (Acystodrilus) species are shown in Table 3. Specimens were consistent with the holotypes and available descriptions [8,9,26] except for the aspects detailed below.

Table 3

Main morphological characters of the species and subspecies of Allolobophora (Gatesona) and related Helodrilus (Acystodrilus) species under study.

Characters	All. (Ga.) chaetophora	All. (Ga.) chaetophora lablacherensis	All. (Ga.) chaetophora serninensis	All. (Ga.) chaetophora rutena	All. (Ga.) sausseyi	All. (Ga.) transmatica	Helodrilus (Ac.) cortezi*	Helodrilus (Ac.) musicus
Length	68–85 mm	88–100 mm	70–99 mm	98–115 mm	86 mm	66 mm	152–192 mm	127 mm
N. segments	190	184–193	118–147	175–192	183	184	185–191	197
Weight	2.04–3.18 g	1.64–1.72g	1.085–1.675 g	2.16–3.47 g	1.25 g	1.03 g	1.10–1.36 g	1.42 g
Pigmentation	Pigmentary dots	Pigmentary dots	Pigmentary dots	Pigmentary dots	Pigmentary dots	Pigmentary dots	Pigmentary dots	Pigmentary dots
Setal arrangement	10:1.25:9.5:1:12.5	16:1:8.5:1:31	13.3:1:8.3:1:33	15:1:8.5:0.7:27.5	48:1:8:1:12	20:1:10:1:18	11.6:1.6:8.33:1:30	7:1:4:1:22
First dorsal pore	5/6	5/6	5/6	4/5	6/7	(6/7)7/8	4/5	4/5
Spermathecal pores	9/10, 10/11 simple in C	9/10, 10/11 simple in C	9/10, 10/11 simple in C	9/10, 10/11 simple in C	9/10, 10/11 simple in C	9/10, 10/11 simple in C	9/10, 10/11 simple in C	9/10, 10/11 simple in C
Clitellum	26–36	25–32	25–33	26–35	24–33	25–33	1/2 22–33	23–32
Tubercula pubertatis	(29)30-36	27–30, strongly developed	28-32(33)	30–35	28–32	29–32	28–30	27-1/2 31
Oesophageal hearts	5–11	6–11	6–12	6–11	6–11	6–11	6–11	6–11
Calciferous glands	10–12, dilated in 10	10–12, dilated in 10	10–12, diverticles in 10	10–14, dilated in 10	10–14, diverticles in 10	10–12	10–14	½ 10–14, diverticles in 10
Typhlosole	Bifid	Bifid	Bifid	Bifid	Bifid	Bifid	Bifid	Bifid
Nephridial bladders	Sigmoid, proclinate	Sigmoid, proclinate	Sigmoid, proclinate	Sigmoid, proclinate	Sigmoid, proclinate	Sigmoid, proclinate	Absent	Absent
Seminal vesicles	9<10<11,12	10,11,12	9,10<11,12	9,10<11,12	9,10<11,12	9,11,12	9,10<11,12	9,10<11,12
Spermathecae	Simple, globular in 9,10	Simple, globular in 9,10	Simple, globular in 9,10	Simple, globular in 9,10	Simple, globular in 9,10	Simple, globular in 9,10	Simple, globular in 9,10	Simple, globular in 9,10

Species indicated with *: information from Qiu and Bouché [9]. In bold, morphological characters for which differences were observed relative to the original description [26].

Specimens of All. (Ga.) rutena differed from the previous description in the number and position of spermathecal pores (2 pairs in 9/10, 10/11 vs 3 pairs in 9/10,10/11 and 11/12). However, the holotype of All. (Ga.) rutena showed 3 pairs of spermathecae on the right side but 2 pairs of spermathecae on the left side. Thus, this character appears to show variability even within the same individual.

Specimens of Helodrilus (Ac.) musicus did not show red pigmentation as stated by [9], but displayed a faint reddish hue instead.

Nephridial pores were defined by [8,9,26] as irregularly distributed; however, newly captured material showed aligned nephridial pores.

Systematic implications

The phylogenetic position of representatives of Gatesona as a well-supported clade unrelated to the generotype of Allolobophora (All. chlorotica) suggests it should be considered its own genus. The inclusion of Helodrilus (Acystodrilus) cortezi and Helodrilus (Acystodrilus) musicus within this clade suggest that they also belong to Gatesona. The latter two species and the other representatives of Gatesona are very similar in most external and internal morphological characters, except for the absence of nephridial vesicles. This character appears in several unrelated lumbricid lineages (Erséus, Csudzi pers. comm.) and is thus homoplasic: the same is observed for the posterior displacement of the male pore, which has stopped being regarded as a key taxonomic character in lumbricid systematics [12,27]. Regression of a complex morphological structure is easier in evolutionary terms than the evolution of such structures [28], so it is likely that loss of nephridial bladders happened repeatedly associated with high moisture environments [29].

Another species placed within Helodrilus (Acystodrilus) by Qiu and Bouché [9] and not included in this work, Helodrilus (Acystodrilus) segalensis, could be expected to belong to the same genus-level clade as Helodrilus (Acystodrilus) cortezi and Ac. musicus, due to their morphological similarity and geographic proximity (100–120 km); however, molecular phylogenetics analyses of this species is necessary to confirm this hypothesis. Furthermore, Helodrilus putricola and tebra have been found to belong to an unrelated clade (Csudzi pers. comm.) and Helodrilus rifensis is considered a synonym of Eiseniella neapolitana (Orley 1885) [30].

The name Helodrilus should be restricted to the clade containing He. oculatus, its generotype. However, He. oculatus was not included in this study, whereas only two Eastern European Helodrilus (Helodrilus) representatives (Helodrilus patriarchalis and Helodrilus cernosvitovianus) among its 18 species (http://taxo.drilobase.org/) were included. Thus, a well-supported systematic revision of Helodrilus should include a more comprehensive sampling of both groups in a molecular phylogenetic framework.

As Avelona ligra was recovered as a sister clade to Gatesona, it could be argued that it should also be included within the same genus. However, the branch lengths separating both taxa in the phylogenetic tree were indicative of separate genera. The 16s UAPG distances (which are less saturated at above-species level than COI distances) between Avelona ligra and representatives of Gatesona were significantly higher than those found within Gatesona, and were within the range observed between well-established earthworm genera (8.6%-20.01%, average 13.4%; Supporting Information S2 File). In addition, Avelona ligra (only known species within Avelona) possesses only two pairs of seminal vesicles in 11, 12 (vs 3 or 4 pairs in (9,10) 11, 12) and displays striking bioluminescence.

The phylogenetic relationships, branch lengths and UAPG distances between the different species and subspecies of Gatesona suggest that they should all be considered different species. The existing morphological differences between these taxa, of similar or higher magnitude than those used by Qiu and Bouché [8] to distinguish All. (Ga.) sausseyi and All. (Ga.) transmatica, support their status as proper species.

All. (Ga.) argentatensis could not be sampled for this study, but following the same criteria it is very likely that it constitutes an independent species from All. (Ga.) chaetophora.

Proposed systematic changes

Phylum Annelida Lamarck, 1802

Class Oligochaeta Grube, 1850/Clitellata Michaelsen, 1919

Order Crassiclitellata Jamieson, 1988

Family Lumbricidae Rafinesque-Schmaltz, 1815

Genus Gatesona Qiu and Bouché 1998 stat. nov.

Allolobophora (Gatesona) Qiu and Bouché 1998

Type species

Allolobophora chaetophora Bouché 1972

Included species

Gatesona chaetophora comb. nov. (Bouché 1972)

Gatesona chaetophora argentatensis comb.nov. (Bouché 1972)

Gatesona rutena comb.nov. stat. nov. (Bouché, 1972)

Gatesona lablacherensis comb.nov. (Bouché, 1972)

Gatesona serninensis comb.nov. stat. nov. (Bouché 1972)

Gatesona sausseyi comb. nov. (Qiu and Bouché 1998)

Gatesona transmatica comb. nov. (Qiu and Bouché 1998).

Gatesona cortezi comb. nov. (Qiu and Bouché 1998).

Gatesona musica comb. nov. (Qiu and Bouché 1998).

Diagnosis

Mid-sized (66 to 190 mm) Lumbricidae, with fewer than 230 segments. Mid-segment pigmentary dots in cephalic (sometimes tail) segments. Numerous chaetophores in the shape of genital papillae between segments 9 and the end of the clitellum. Prostomium epylobous. Closely paired setae. First dorsal pore in 4/5 (3 species), 5/6 (3 species), 6/7 (1 species), 7/8 (1 species). Nephridial pores aligned. Male pores in ½ 15 with developed porophore. Spermathecal pores simple, in 9/10, 10/11 (exceptionally 9/10, 10/11, 11/12). Clitellum starts between segments 23 to 27. Calciferous gland variable, in 10-12(14) usually dilated or with diverticles in 10. Typhlosole bifid. Three to four pairs of seminal vesicles in (9, 10) 11, 12. Nephridial vesicles sigmoid, proclinate, sometimes absent (Ga. cortezi, Ga. musica).

Remarks

Gatesona chaetophora argentatensis (Bouché 1972) remains a subspecies of Gatesona chaetophora owing to failure to include representatives in the integrative systematic revision. However, it is to be expected that Ga. chaetophora argentatensis could represent an independent species.

The name Gatesona was chosen over Acystodrilus for the genus encompassing species of both former subgenera for three main reasons: first, currently there are more species of the former Allolobophora (Gatesona) than of Helodrilus (Acystodrilus); second, the name Acystodrilus (meaning “without vesicle”) does not represent the more common character state within the redefined genus Gatesona. The third reason is purely nomenclatural: the name Gatesona has priority over Acystodrilus, as far as [31] proposed Gatesona in page 201, whereas Acystodrilus was proposed in page 209.

Gatesona chaetophora comb. nov stat. nov.

Allolobophora chaetophora chaetophora Bouché 1972

Redescription

Diagnosis

Specimens of Gatesona chaetophora can be distinguished from other known species of Gatesona by the position of the clitellum in segments 26–36 and tubercula pubertatis in segments (29)30-36 (Table 3).

Etymology

The name chaetophora refers to the multiple chaetophores (glandular structures surrounding genital chaetae) this species possess.

Material examined

Holotype. FRANCE • Adult; Occitanie, Aveyron, L’Hospitalet du Larzac; 11/03/1968; leg. Marcel Bouché; Locality 1: 43.956 3.218433 Prairie; deposited in the collection of the Muséum National d’Histoire Naturelle de Paris (MNHN) (voucher: 87/550/0711).

Additional material. FRANCE • 2 specimens, adults; Occitanie, Aveyron, L’Hospitalet du Larzac; 11/02/2021; leg. Daniel F. Marchán; Locality 1: 43.956, 3.218433 Prairie; deposited in the CEFE earthworm collection (voucher: DFM-0676, DFM-0677).

Morphological description

External morphology. Body pigmentation faint brown. Beige to faint brown with pigmentary dots scattered along the mid-segment line in the cephalic region in fixed specimens (Fig 3A and 3E).

Fig 3

External and internal morphology of fixed specimens of Gatesona chaetophora (a, e, i) Gatesona rutena (b, f, j) Gatesona lablacherensis (c, g, k) and Gatesona serninensis (d, h, l). The insets show a detailed view of the pigmentary dots characteristic of Gatesona species (e, f, g, h), and their nephridial bladders (i, j, k, l). Abbreviations: cl. = clitellum, m. p. = male pore, t. p. = tubercula pubertatis.

Mean length 76.5 mm (68–85 mm, n = 2 adults); body cylindrical in cross-section; mean number of segments 190 (n = 2 adults; 190 segments in the holotype). Mean weight (fixed specimens): 2.60 g (2.035–3.175 g, n = 2 adults). Prostomium epilobous, closed. Longitudinal furrows in segments 1 and 2. First dorsal pore at intersegmental furrow 5/6. Nephridial pores aligned in B close to b. Spermathecal pores at intersegmental furrows 9/10 and 10/11 in c. Male pores in segment 15, surrounded by a well-developed porophore. Female pores on the posterior part of segment 14, inconspicuous. Clitellum saddle-shaped in segments 26–36. Tubercula pubertatis in segments (29)30-36. Chaetae small and closely paired, with interchaetal ratio aa: 10, ab: 1.25, bc: 9.5, cd: 1, dd: 12.5 at segment 40. Chaetophores/genital papillae in segments 11, (14, 16, 17, 18) 20, 25–28, (29), 32–36 with those in 11, 20, 25–28 and 35,36 especially well developed and constant.

Internal anatomy. Septa 5/6-9/10 strongly thickened. Hearts in segments 5–11, oesophageal. Calciferous glands in segments 10–12, dilated in segment 10. Crop in segments 15–16, gizzard in segments 17–18. Typhlosole bifid. Male sexual system holandric, testes and funnels (not enclosed in testes sacs, but with sperm present) located ventrally in segments 10 and 11. Four pairs of reniform seminal vesicles in segments 9, 10, 11 and 12, with the latter two pairs being larger. Ovaries and female funnels in segment 13, ovarian receptacles (ovisacs) in segment 14. Two pairs of globular spermathecae in segments 9 and 10. Nephridial bladders sigmoid, proclinate in anterior and posterior segments (Fig 3I).

Distribution and ecology:Gatesona chaetophora is known from the region of Larzac in the southern Massif Central, France. This species was found at an altitude of 710 meters in a prairie on silty soil, with close to neutral pH (7.4), 2.83% organic matter and 0.308% nitrogen content [26].

Gatesona rutena comb. nov stat. nov.

Allolobophora chaetophora rutena Bouché 1972

Specimens of Gatesona rutena can be distinguished from other known species of Gatesona by the position of the first dorsal pore in 4/5, the position of the clitellum in segments 26–35 and tubercula pubertatis in segments 30–35, calciferous glands in 10–14 and a higher mean body weight (Table 3).

Gatesona rutena was named after the Gallic tribes (the Ruten) that inhabited French lands before Roman invasion (see Bouché [26] page 5).

Holotype. FRANCE • Adult; Occitanie, Aveyron, Rodez; 17/04/1968; leg. Marcel Bouché; Locality 1: 44.415 2.039433 Pasture; deposited in the collection MNHN (voucher: 91/619/0712).

Additional material. FRANCE • 7 specimens, 4 adults, 3 juveniles; Occitanie, Aveyron, Rodez; 10/11/2019; leg. Daniel F. Marchán; Locality 1: 44.416565 2.041011 Oak forest; deposited in the CEFE earthworm collection (voucher: 70257).

External morphology. Body pigmentation very faint brown, white-creamy spots can be observed through the body wall (Fig 2A). White-beige with pigmentary dots scattered along the mid-segment line in the cephalic (sometimes tail) region in fixed specimens (Fig 3B and 3F).

Fig 2

External morphology of living specimens of a) Gatesona rutena b) Gatesona lablacherensis c) Gatesona serninensis.

Mean length 106.5 mm (98–115 mm, n = 2 adults); body cylindrical in cross-section; mean number of segments 183 (175–192, n = 2 adults; 192 segments in the holotype). Mean weight (fixed specimens): 2.82 g (2.16–3.47 g, n = 2 adults). Prostomium epilobous, closed. Longitudinal furrows in segments 1 and 2. First dorsal pore at intersegmental furrow 4/5. Nephridial pores aligned in B close to b. Spermathecal pores at intersegmental furrows 9/10 and 10/11 (plus 11/12 according to Bouché 1972 page 439) in c. Male pores in segment 15, surrounded by a well-developed porophore. Female pores on the posterior part of segment 14, inconspicuous. Clitellum saddle-shaped in segments 26–35. Tubercula pubertatis in segments 30–35. Chaetae small and closely paired, with interchaetal ratio aa: 15, ab: 1, bc: 8.5, cd: 0.7, dd: 27.5 at segment 40. Chaetophores/genital papillae in segments 12, 16, 17, 27, 24–36.

Internal anatomy. Septum 4/5 thickened, septa 5/6-8/9 strongly thickened. Hearts in segments 6–11, oesophageal. Calciferous glands in segments 10–14, dilated in segment 10. Crop in segments 15–16, gizzard in segments 17–18. Typhlosole bifid. Male sexual system holandric, testes and funnels (not enclosed in testes sacs, but with sperm present) located ventrally in segments 10 and 11. Four pairs of reniform seminal vesicles in segments 9, 10, 11 and 12, with the latter two pairs being larger. Ovaries and female funnels in segment 13, ovarian receptacles (ovisacs) in segment 14. Two pairs of globular spermathecae in segments 9 and 10 (an additional spermathecae can appear in 11 in one or both sides of the body). Nephridial bladders sigmoid, proclinate in anterior and posterior segments (Fig 3J).

Distribution and ecology:Gatesona rutena is known from the region of Rodez, in the western Massif Central, France. This species was found at an altitude of 360 meters in a prairie/oak forest transition on silty soil, with close to neutral pH (7.2), 4.79% organic matter and 0.414% nitrogen content [26].

Gatesona lablacherensis comb. nov stat. nov.

Allolobophora chaetophora lablacherensis Bouché 1972

Specimens of Gatesona lablacherensis can be distinguished from other known species of Gatesona by the position of the clitellum in segments 25–32 and tubercula pubertatis in segments 27–30, as well as by possessing three pairs of seminal vesicles (in segments 10, 11 and 12) rather than four (Table 3).

The name lablacherensis refer to Lablachère, the region of France the species is known to inhabit.

Holotype. FRANCE • Adult; Rhône-Alps, Ardèche, Lablachère; 20/11/1968; leg. Marcel Bouché; Locality 1: 44.433, 4.22643 Oak forest over rocky outcrops; deposited in MNHN (voucher: 90/1056/0716).

Additional material. FRANCE • 12 specimens, adults; Rhône-Alps, Ardèche, Lablachère; 6/11/2019; leg. Daniel F. Marchán; Locality 1: 44.433, 4.22643 Oak forest over rocky outcrops; deposited in the CEFE earthworm collection (voucher: 70256).

External morphology. Body pigmentation absent, very conspicuous white-creamy spots can be observed through the body wall (Fig 2B). White-beige with pigmentary dots scattered along the mid-segment line in the cephalic region in fixed specimens (Fig 3C and 3G).

Mean length 94 mm (88–100 mm, n = 2 adults); body cylindrical in cross-section; mean number of segments 189 (184–193, n = 2 adults; 193 segments in the holotype). Mean weight (fixed specimens): 1.68 g (1.64–1.72 g, n = 2 adults). Prostomium epilobous, closed. Longitudinal furrows in segments 1 and 2. First dorsal pore at intersegmental furrow 5/6. Nephridial pores aligned in B close to b. Spermathecal pores at intersegmental furrows 9/10 and 10/11 in c. Male pores in segment 15, surrounded by a well-developed porophore. Female pores on the posterior part of segment 14, inconspicuous. Clitellum saddle-shaped in segments 25–32. Tubercula pubertatis in segments 27–30 strongly developed as downward projections of the body wall. Chaetae small and closely paired, with interchaetal ratio aa: 16, ab: 1, bc: 8.5, cd: 1, dd: 31 at segment 40. Chaetophores/genital papillae in segments 13, 14, 16, 17, 19, 23–33.

Internal anatomy. Septum 4/5 thickened, septa 5/6-8/9 strongly thickened. Hearts in segments 6–11, oesophageal. Calciferous glands in segments 10–12, dilated in segment 10. Crop in segments 15–16, gizzard in segments 17–18. Typhlosole bifid. Male sexual system holandric, testes and funnels (not enclosed in testes sacs, but with sperm present) located ventrally in segments 10 and 11. Three pairs of reniform seminal vesicles in segments 10, 11 and 12, with the latter two pairs being larger. Ovaries and female funnels in segment 13, ovarian receptacles (ovisacs) in segment 14. Two pairs of globular spermathecae in segments 9 and 10. Nephridial bladders sac-like or J shaped in the first segments, which gradually change to sigmoid, proclinate (Fig 3K).

Distribution and ecology:Gatesona lablacherensis is known from the region of Lablachère, in the eastern Massif Central, France. This species has been found in an oak forest, and thus may prefer natural habitats.

This species was found at an altitude of 180 meters in a prairie/oak forest transition on silty soil over rocks, with close to neutral pH (7.6), 1.9% organic matter and 0.166% nitrogen content [26].

Gatesona serninensis comb. nov stat. nov.

Allolobophora chaetophora serninensis Bouché 1972

Specimens of Gatesona serninensis can be distinguished from other known species of Gatesona by the position of the clitellum in segments 25–33 and tubercula pubertatis in segments 28-32(33), as well as by the significantly lower number of segments (Table 3). Gatesona serninensis is similar to Gatesona transmatica in the position of tubercula pubertatis (28-32(33) vs 29–32) and they share the same position of the clitellum, but they are easily differentiated by the position of the first dorsal pore (5/6 vs (6/7) 7/8) and the number and position of seminal vesicles (4 in 9, 10, 11, 12 vs 3 in 9, 11, 12).

The name serninensis refer to Saint-Sernine-sur-Rance, the town in the vicinities of whichr the species are known to inhabit.

Holotype. FRANCE • Adult; Occitanie, Aveyron, Saint-Sernin-sur-Rance; 26/11/1968; leg. Marcel Bouché; Locality 1: 43.875 2.606433 Prairie; deposited in MNHN (voucher 88/1100/0714)

Additional material. FRANCE • 12 specimens, 8 adults, 4 juveniles; Occitanie, Aveyron, Saint-Sernin-sur-Rance; 23/04/2021; leg. Daniel F. Marchán; Locality 1: 43.956, 3.218433 Prairie; deposited in the CEFE earthworm collection (voucher 70258).

External morphology. Body pigmentation absent, conspicuous white-creamy spots can be observed through the body wall. White-beige with pigmentary dots scattered along the mid-segment line in the cephalic (sometimes caudal) region in fixed specimens (Fig 3D and 3H).

Mean length 85 mm (70–99 mm n = 6 adults); body cylindrical in cross-section; mean number of segments 128 (118–147, n = 8 adults; 121 segments in the holotype). Mean weight (fixed specimens): 1.32 g (1.085–1.675 g, n = 4 adults). Prostomium epilobous, closed. Longitudinal furrows in segments 1 and 2. First dorsal pore at intersegmental furrow 5/6. Nephridial pores aligned in cd. Spermathecal pores at intersegmental furrows 9/10 and 10/11 in c. Male pores in segment 15, surrounded by a well-developed porophore. Female pores on the posterior part of segment 14, inconspicuous. Clitellum saddle-shaped in segments 25–33. Tubercula pubertatis in segments 28–32 (33). Chaetae small and closely paired, with interchaetal ratio aa: 13.3, ab: 1, bc: 8.3, cd: 1, dd: 33 at segment 40. Chaetophores/genital papillae in segments 11, 16, 17, 19–33, those in 11, 16, 32 and 33 especially well developed and constant.

Internal anatomy. Septa 5/6-9/10 strongly thickened. Hearts in segments 6–12, oesophageal. Calciferous glands in segments 10–12, with diverticles in segment 10. Crop in segments 15–16, gizzard in segments 17–18. Typhlosole bifid. Male sexual system holandric, testes and funnels (not enclosed in testes sacs, but with sperm present) located ventrally in segments 10 and 11. Four pairs of reniform seminal vesicles in segments 9, 10, 11 and 12, with the latter two pairs being larger. Ovaries and female funnels in segment 13, ovarian receptacles (ovisacs) in segment 14. Two pairs of globular spermathecae in segments 9 and 10. Nephridial bladders J shaped in the first segments, which gradually change to sigmoid, proclinate (Fig 3L).

Distribution and ecology:Gatesona serninensis is known from the region of Saint-Sernin-sur-Rance, in the southern Massif Central, France. This species has been found at an altitude of 350 meters in the transition between an oak-chestnut forest and a prairie on sandy soil over rocks, with acid pH (5.6), 3.78% organic matter and 0.350% nitrogen content [26].

Conclusions

Integrative systematics supported the status of the previously described subgenus Allolobophora (Gatesona) as a genus, including some representatives of Helodrilus (Acystodrilus). Additionally, a clade comprising Gatesona and Avelona was recovered by molecular phylogenetics, an early branching clade with most of its species being endemic to the Massif Central. Morphological and molecular evidence indicated most of the previously described subspecies of Gatesona chaetophora should actually be regarded as proper species. The presence of this diverse assemblage of endemic earthworms, with several taxa remaining to be studied by molecular methods, highlights the interest of this region of Southern France for further sampling effort across their range.

The phylogenetic distinctness of representatives of Helodrilus (Helodrilus) and Helodrilus (Acystodrilus) indicate that a more comprehensive sampling of species (including He. oculatus) is required in order to establish a solid systematic revision of the elusive genus.

Species included in the reference dataset.

(DOCX)

Click here for additional data file.

Phylogenetic tree obtained by Bayesian phylogenetic analysis of the concatenated sequence of molecular markers COI–16S-tRNAs–ND1–28S.

(DOCX)

Click here for additional data file.

Uncorrected average pairwise (UAPG) distances for the marker 16S between representatives of different well-established genera of Lumbricidae.

(DOCX)

Click here for additional data file.

15 Apr 2021

PONE-D-21-07434

Systematic revision of Gatesona (Crassiclitellata, Lumbricidae), an endemic earthworm genus from the Massif Central (France)

PLOS ONE

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PLOS ONE

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Reviewer #1: Yes

Reviewer #2: Yes

**********

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #2: Yes

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Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Authors present a very interesting paper about the identity of several Lumbricidae species and subspecies that are placed in a new genus. These new arrangements are due to the use of an integrative approach (molecular genetics, phylogenetic analysis and morphological revisions). English is ok, as well as molecular and phylogenetic methods and results.

There are, however, some modifications that should be done (mainly in regard to classical nomenclatural arrangements) before the paper could be accepted. The paper, thus, is ACCEPTED WITH MINOR CHANGES.

I directly wrote several comments and suggestions over the main text; following are some of my main points:

1) In the title it is mentioned the word Crassiclitellata; in the classification of the new genus Gatesona, however, this word is not included. Thus, include in the supraorder classification this word with its hierarchical associated level.

2) There are several mistakes in regard of the “types” of the two re-described species. Please attend comments of the text.

3) As authors do not reviewed types of these species, maybe the name (from an already described species) in fact does not correspond to the collected and molecular sequenced material. I strongly suggest contact Dr. Bouché in order to obtain in loan the types (or paratypes) to confirm that the morphology of Gatesona chaetophora rutena and Gatesona chaetophora lablacherensis really correspond to the species you collected. Even more because there is some variation that could be an indication of different morphological species (e.g. the number of spermathecae).

4) In benefit of readers, please explain in the discussion why the genus was named as Gatesona and not Acystodrilus.

Reviewer #2: There are a few small corrections of grammar and content in the MS that I uploaded with this review. The revision of the taxonomy has to be modified to make it clear that G. chaetophora is the type species, and in particular, that G. chaetophora chaetophora is the type species. The subspecies G. c. argentatus could be tentatively excluded. The remaining species are "other species" under a heading.

**********

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Reviewer #1: No

Reviewer #2: No

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Submitted filename: PONE-D-21-07434_reviewer 1.pdf

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Submitted filename: PONE-D-21-07434_reviewerSJ.pdf

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20 Jun 2021

Reviewer #1: Authors present a very interesting paper about the identity of several Lumbricidae species and subspecies that are placed in a new genus. These new arrangements are due to the use of an integrative approach (molecular genetics, phylogenetic analysis and morphological revisions). English is ok, as well as molecular and phylogenetic methods and results.

We want to thank the reviewer for his/her interest on our manuscript and his/her constructive suggestions in order to improve it. All of them have been considered and included.

There are, however, some modifications that should be done (mainly in regard to classical nomenclatural arrangements) before the paper could be accepted. The paper, thus, is ACCEPTED WITH MINOR CHANGES.

I directly wrote several comments and suggestions over the main text; following are some of my main points:

1) In the title it is mentioned the word Crassiclitellata; in the classification of the new genus Gatesona, however, this word is not included. Thus, include in the supraorder classification this word with its hierarchical associated level.

We have included Crassiclitellata in the taxonomy section.

2) There are several mistakes in regard of the “types” of the two re-described species. Please attend comments of the text.

We have corrected the taxonomic errors regarding the types.

3) As authors do not reviewed types of these species, maybe the name (from an already described species) in fact does not correspond to the collected and molecular sequenced material. I strongly suggest contact Dr. Bouché in order to obtain in loan the types (or paratypes) to confirm that the morphology of Gatesona chaetophora rutena and Gatesona chaetophora lablacherensis really correspond to the species you collected. Even more because there is some variation that could be an indication of different morphological species (e.g. the number of spermathecae).

We have contacted Marcel Bouché and revised the holotypes for lablacherensis, rutena but also serninensis and chaetophora. Thanks to that, we can state with certainty that the (morpho-molecularly) studied material is morphologically compatible with the types of the original taxa. The morphological differences initially detected could be actually due to faulty/incomplete descriptions (in the end, it was done through tables and not through an orthodox text); when comparing with the types, the differences were non-existant or comprised within intra-individual variation (Gatesona rutena holotype possessed three spermathecae on one side but only two on the other).

4) In benefit of readers, please explain in the discussion why the genus was named as Gatesona and not Acystodrilus.

We have included a sentence explaining that point in the “Remarks” section of the Systematic changes to the genus Gatesona.

Reviewer #2: There are a few small corrections of grammar and content in the MS that I uploaded with this review. The revision of the taxonomy has to be modified to make it clear that G. chaetophora is the type species, and in particular, that G. chaetophora chaetophora is the type species. The subspecies G. c. argentatus could be tentatively excluded. The remaining species are "other species" under a heading.

We are thankful for the constructive revision and suggestions for improvement of our manuscript. As seen in the document with tracked changes, we have aknowledged and included all the suggested changes.

Most importantly, we contacted Marcel Bouché and revised the holotypes for lablacherensis, rutena but also serninensis and chaetophora. This allowed to check for morphological differences, actually ruling out most of them (holotypes were more consistent with our material tan suggested in the incomplete, informal descriptions provided in Bouché 1972). In addition, we sampled additional material of Gatesona serninensis, which allowed us to provide a formal redescription.

We have decided to mantain argentatensis within Gatesona chaetophora as it clearly belongs to the genus but we lack enough information to robustly elevate it to its own species.

Submitted filename: Response to reviewers.docx

Click here for additional data file.

26 Jul 2021

PONE-D-21-07434R1

Systematic revision of Gatesona (Crassiclitellata, Lumbricidae), an endemic earthworm genus from the Massif Central (France)

PLOS ONE

Dear Dr. Marchán,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by Sep 09 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

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Michael Schubert

Academic Editor

PLOS ONE

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Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewer #1: All comments have been addressed

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**********

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Reviewer #1: Yes

**********

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The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

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Reviewer #1: Yes

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Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The paper is now ready for its publication. I'm, however, sending in the 2nd round reviewed paper very few suggestions and changes (mainly of style) to improve the paper. These suggestions include change of a word, italics, improve of figure 3 legend, a doubt about the identity of a personal communication and (and this is up to the author to include or not) the addition of a nomenclatural comment.

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Reviewer #1: No

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Submitted filename: PONE-D-21-07434_R1_reviewer 1 2nd round.pdf

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26 Jul 2021

We have accepted all the proposed changes

Submitted filename: Response to reviewers.docx

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28 Jul 2021

Systematic revision of Gatesona (Crassiclitellata, Lumbricidae), an endemic earthworm genus from the Massif Central (France)

PONE-D-21-07434R2

Dear Dr. Marchán,

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25 Aug 2021

PONE-D-21-07434R2

Systematic revision of Gatesona (Crassiclitellata, Lumbricidae), an endemic earthworm genus from the Massif Central (France)

Dear Dr. Marchán:

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on behalf of

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PLOS ONE