The best is yet to come: six new species within a large-bodied earthworm genus (Scherotheca, Lumbricidae) in a densely sampled region (south-western France)

Abstract

Since the 18^th century, several taxonomists have contributed to knowledge of the French earthworm fauna, one of the best-known earthworm communities in Europe, with 164 species currently described. Surprisingly, new species keep being added constantly, even large ones, which are usually the first to be described. The recent discovery of six undescribed morphospecies of Scherotheca during a sampling survey in southern France suggests that other large Lumbricidae remain to be discovered. We provide here an integrative description of these six species, Scherotheca albina Marchán & Decaëns sp. nov., Scherotheca betharramensis Marchán & Decaëns sp. nov., Scherotheca microthomasi Marchán & Decaëns sp. nov., Scherotheca shaihulud Marchán & Decaëns sp. nov., Scherotheca etcheberryensis Marchán & Decaëns sp. nov. and Scherotheca trezencensis Marchán & Decaëns sp. nov., formally name them and place them in a molecular phylogenetic framework together with their Scherotheca relatives from south-western France. This increases the diversity of Scherotheca to 51 species, including Scherotheca haymozi comb. nov. as a small-sized representative of the genus. The validity of the elevation of several subspecies to specific status was corroborated by molecular phylogenetic evidence, and previously unknown cryptic speciation within Scherotheca was detected. Two geographically coherent clades (roughly matching Nouvelle-Aquitaine and Occitanie) correspond to two existing subgenera. The unusually high taxonomic and morphological diversity of Scherotheca within the Pyrénées-Atlantiques may be related to biotic interactions, climatic factors and historical causes. These features highlight the importance of the conservation of these unique earthworm species.

INTRODUCTION

There is a long tradition of research on earthworm diversity and taxonomy in Europe, which began in the 18^th century. By 1830, two of the earliest French earthworm taxonomists, Marie Jules César Savigny and Antoine Louis Dugès, had already described most of the conspicuous and well-known Lumbricidae Rafinesque-Schmaltz (1815) species, including Allolobophora chlorotica (Savigny, 1826), Aporrectodea caliginosa (Savigny, 1826), Aporrectodea trapezoides (Dugès, 1828), Aporrectodea rosea (Savigny, 1826) and Octodrilus complanatus (Dugès, 1828). Since then, several taxonomists have made further contributions to the knowledge of the French earthworm fauna, with Marcel Bouché making an exceptionally large contribution between 1967 and 1998 with the description of 69 new species. As a consequence, France has one of the best-known earthworm faunas in Europe, with 164 species described to date. Remarkably, new French species keep being described at a steady pace, which is a testament to the extraordinarily high diversity of the native earthworms (families Lumbricidae and Hormogastridae Michaelsen, 1900) in the country and can probably be attributed to the importance of the Franco-Iberian realm for the evolutionary history of these soil animals (Novo et al., 2015; Marchán et al., 2021).

When considering the variables that predict the probability of a species being described, body size has been found to have variable explanatory power across different taxa, with most robust evidence coming from insects, snakes and turtles (Gaston, 1991; Reed & Boback, 2002). In these cases, the most recently described species were of smaller body size. However, in earthworms, there appears to be an inverse correlation between description date and body size, possibly due to the fact that large, deep burrowing earthworms are more difficult to sample, are not usually found in gardens, crops or other easily accessible habitats and tend to have more restricted ranges. Preliminary analysis of the body size and description date of earthworm species from France has shown that although this correlation is not significant, there is a trend towards larger species being discovered more recently (D.F. Marchán, pers. obs.).

This pattern may be strongly influenced by the genus ScherothecaBouché, 1972, as most of its 44 species (54 including subspecies) have been described in the past 50 years (Bouché, 1967, 1972, 1979; Qiu & Bouché, 1998). Most species of Scherotheca (besides some medium-sized representatives mainly found in Corsica, Marchán et al., 2023) are large or giant anecic earthworms which inhabit deep vertical galleries and feed on leaf litter. The discovery of six undescribed morphospecies of Scherotheca during a sampling survey in south-western and southern France, with two found in previously studied localities (Bouché, 1972), suggests that other large Lumbricidae (such as Scherotheca) remain to be discovered in accordance with the aforementioned trend.

An integrative description of these six species is provided here, and the species are placed in a molecular phylogenetics framework together with their Scherotheca relatives from south-western France and Corsica. Two species of unknown affinities and found in sympatry with Scherotheca (Allolobophora haymozi Zicsi, 1977 and Aporrectodea haymoziformis Zicsi & Csuzdi, 1999) were included to test for a possible relationship with them.

MATERIAL AND METHODS

Specimens, sampling and morphological description

Specimens included in this study were collected between March and December 2021 in Nouvelle-Aquitaine and Occitanie (France). The specific localities are listed in Table 1.

Earthworms were collected by digging and hand-sorting the soil, then rinsing with water and fixing in 70% ethanol in order to obtain less drastically retracted specimens. Once immobilized, the specimens were immediately transferred to 100% ethanol for further molecular analyses. Species classification and morphological diagnoses were conducted using the same set of external and internal morphological characters reported by Qiu & Bouché (1998).

The following main external morphological characters were considered: mean length, mean number of segments, mean weight, pigmentation, type of prostomium, position of papillae, position of first dorsal pore, position of spermathecal pores, position of clitellum and position of tubercula pubertatis. The following main internal anatomical characters were considered: position of lateral 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.

Specimens that could not be assigned to previously described species were instead grouped into morphospecies. We considered a morphospecies as a group of specimens sharing the same morpho-anatomical characters, and differing from other groups in more than one of the morpho-anatomical characters.

DNA sequencing and phylogenetic analysis

For each locality we selected up to five adult individuals per morphospecies for DNA barcoding. Small ventral integument tissue samples were assembled in 96-well plates and shipped to the Centre for Biodiversity Genomics at the University of Guelph (Ontario, Canada,) for processing. After extraction of total genomic DNA by a CTAB-based approach, the standard DNA barcode for animals (Hebert et al., 2003)—a 658-bp fragment of mitochondrial gene cytochrome c oxidase subunit 1 (COI)—was amplified using the primers C_LepFolF and C_LepFolR (Hernández-Triana et al., 2014). Sequencing reactions were carried out with the same primer pair, and the products were subjected to a PureSeq-MP (Aline Biosciences, Woburn, USA) clean-up before Sanger sequencing in an ABI 3730XL DNA sequencer. Consensus sequences from automatically assembled contigs (subsequently reviewed and manually edited when needed) were uploaded to BOLD (www.boldsystems.org) along with trace files, specimen data and images. The DNA sequences, together with metadata are publicly available in the dataset DS-EWNSPNWF in BOLD (dx.doi.org/10.5883/DS-EWNSPNWF).

The molecular analysis was completed for additional markers. Total genomic DNA was extracted from ventral integument samples, of approximately 5 × 5 mm in size, with a DNeasy Blood & Tissue Kit (Qiagen) from two representative specimens of each putative species. Regions of the nuclear 28S rRNA and mitochondrial 16S rRNA plus tRNAs (Ala-Leu-Ser) and NADH dehydrogenase (ND1) were amplified by polymerase chain reaction (PCR), with the primers and conditions as described in Pérez-Losada et al. (2009, 2015). PCR products were purified and sequenced by the Centro de Apoio Científico-Tecnolóxico á Investigación (C.A.C.T.I.) Genomics service (University of Vigo). The DNA sequences are available in GenBank, under accession numbers OP746034–OP746055, OP747606–OP747655 and OP751157–OP751206.

Previously existing sequences of 17 species of Scherotheca (Domínguez et al., 2015; Marchán et al., 2020, 2023) were included in the dataset. Sequences from representatives of the most closely related Lumbricidae genera (Aporrectodea sensu stricto Orley, 1885, Bimastos Moore, 1893, Eisenoides Gates, 1969, Eisenia Malm, 1877 and Lumbricus Linnaeus, 1758) were downloaded from GenBank and used as reference data (Supporting Information, Table S1). COI sequences are available in the dataset DS-EWNSPNWF in BOLD (dx.doi.org/10.5883/DS-EWNSPNWF).

Sequences were aligned using MAFFT v.7 (Katoh et al., 2013) with default settings and concatenated using BioEdit (Hall, 1999), resulting in a matrix of 3136 bp. The best fitting evolutionary model for each partition was selected using jModelTest v.2.1.3 (Darriba et al., 2012) by applying the Akaike information criterion (AIC; Akaike, 1973) and the Bayesian information criterion (BIC; Schwarz, 1978). GTR+I+G was selected as the best-fitting evolutionary model for COI, 28S and ND1 and HKY+I+G was selected for 16S and tRNAs.

Bayesian inference of the phylogeny was estimated using MrBayes v.3.1.2 (Ronquist & Huelsenbeck, 2003) as implemented in the CIPRES Science Gateway v.3.3. Parameters were set to 50 million generations and sampled every 5000^th generation (10 000 trees). Two independent runs were performed, each with four chains, and 20% of the trees were discarded as burn-in. The remaining trees were combined and summarized on a 50% majority-rule consensus tree. Maximum likelihood phylogenetic inference was performed using RAxML-NG (Kozlov et al., 2019) in the CIPRES Science Gateway platform, from ten random starting trees and 1000 rapid bootstrap replicates.

Average uncorrected pairwise distances within and between morphospecies were calculated using MEGA 11.

Taxonomic acts

New species names were deposited in the databases Taxref (Gargominy et al., 2021) and ZooBank (https://zoobank.org/).

RESULTS

Morphological study of the sampled specimens resulted in the identification of ten morphospecies belonging to the genus Scherotheca—with six of them not corresponding to the diagnosis of any known species in the genus—and Al. haymozi and Ap. haymoziformis (Table 1) .

The morphological characters of the six undescribed morphospecies and the most similar already known Scherotheca are shown in Table 2.

Phylogenetic relationships

The Bayesian phylogenetic tree (Fig. 1) recovered Scherotheca as monophyletic, but Al. haymozi and Ap. haymoziformis were nested deeply within this clade.

Scherotheca albomaculata (Bouché, 1972) was recovered as the earliest branching taxon, followed by two well supported clades comprising Corsican species [with the exception of Scherotheca portcrosana Marchán et al., 2020 and Scherotheca targionii (Baldasseroni, 1906)] and French mainland species, respectively.

The mainland clade displayed a polytomy consisting of Scherotheca aquitania (Bouché 1972) (topotype samples), a clade of species from the centre of the range of Scherotheca (Occitanie: Hérault, Tarn) and a clade of south-western species: this branching pattern was not strongly supported (low to moderate posterior probability values). The maximum likelihood tree recovered a different topology, with Sc. aquitania as the earliest branching taxon within a weakly supported Occitan clade, and a sister south-western clade with strong bootstrap support.

The nine Scherotheca species that were incorporated in a phylogenetic tree for the first time (Sc. Aquitania (Bouché, 1972), Scherotheca dinoscolex (Bouché, 1972), Scherotheca orbiensis (Bouché, 1972), Scherotheca michaelseniQiu & Bouché, 1998, Scherotheca minor (Bouché, 1972), Scherotheca nivicola (Bouché, 1972), Scherotheca occidentalis (Michaelsen, 1922), Scherotheca chicharia (Bouché, 1972) and Scherotheca occitanicaSzederjesi, Pavlicek & Csuzdi, 2021), as well as the six undescribed morphospecies, were supported as independent species by the topology of the phylogenetic tree. One representative identified morphologically as Sc. aquitania appeared as a separate branch rendering Sc. aquitania as polyphyletic: this indicates the existence of cryptic species.

The studied representatives of Al. haymozi and Ap. haymoziformis were separated by short branches, more suggestive of intraspecific divergence than of interspecific divergence.

Uncorrected average pairwise distances for the COI sequences between the studied species and morphospecies of Scherotheca (Table 3) ranged between 10.1% and 19.4% (average 15.4%), with a single exception: the distance between the new morphospecies Scherotheca microthomasi sp. nov. and Sc. microthomasi sp. nov. was significantly smaller (7%). Available intraspecific distances were generally low, being much higher for Scherotheca gigas (Duges, 1828), the new morphospecies Sc. microthomasi and Scherotheca albina sp. nov. (4.2%, 4.1% and 4.6%, respectively). The average interspecific distance in the Occitan clade was notably higher than in the south-western clade (15.2% vs. 14%).

SYSTEMATIC RESULTS

Results of the molecular phylogenetics analyses and morphological characters support the six newly found morphospecies as independent, well delimited species. Thus, a taxonomic description is provided below for all of the species.

Phylum Annelida Lamarck, 1802

 

Class Clitellata Michaelsen, 1919

 

Subclass Oligochaeta Grube, 1850

 

Order Crassiclitellata Jamieson, 1988

 

Tribe Lumbricini Qiu & Bouché, 1998

 

Family Lumbricidae Rafinesque-Schmaltz, 1815

 

Genus ScherothecaBouché, 1972

 

Scherotheca albina Marchán & Decaëns sp. nov.

 

(Figs 2a, 3g, h, 4)

Zoobank registration:

urn:lsid:zoobank.org:act:BDD2FFC5-C311-4CA5-B6C8-227910470E04.

Type material:

Holotype. France • adult; Occitanie, Hérault, le Soulié; latitude/longitude: 43.531/2.658; elevation: 853 m asl; 06-May-2021; T. Decaëns, D. Fernández Marchán leg.; BOLD Sample ID: DFM-0572; deposited in the Musée national d’Histoire naturelle, Paris.

Paratypes:

France • five adult specimens, one subadult specimen; same data as holotype (four specimens); BOLD Sample ID: DFM-0573, DFM-0574, DFM-0575, DFM-0576; Occitanie, Hérault, Saint-Etienne-d’Albagnan; latitude/longitude: 43,543/2,86; elevation: 250 m asl; 14-Apr-2021; T. Decaëns, D. Fernández Marchán leg. (two specimens); BOLD Sample ID: DFM-0025, DFM-0026; specimens DFM-0573, DFM-0575, DFM-0576 deposited in Eco&Sols collection, Montpellier; DFM-0025, DFM-0026, DFM-0574 deposited at Musée national d’Histoire naturelle, Paris.

Etymology:

The species name is derived from the lack of cutaneous pigmentation which characterize it by opposition to the usually pigmented continental species of Scherotheca. Treated as adjectival.

Diagnosis:

Scherotheca albina can be distinguished from Sc. rhodana (Bouché, 1972) (the most similar species) by the position of the clitellum (XXVII–XLI vs. (1/2 XXVI) 1/2 XXVII–1/2 XLII (1/2 XLIII)) and tubercula pubertatis (XXXI–1/n XLI vs. 1/n XXXII–XLI), by its smaller body size and by the absence of cutaneous pigmentation (Table 2).

COI uncorrected average pairwise distances and topology of multilocus molecular phylogenetic trees supports the status of Sc. albina as independent from other morphologically related species.

Description

External morphology:

Body pigmentation absent or faint brown in the anterior part of the body (Fig. 2a). Beige with whitish clitellum in fixed specimens (Fig. 3g, h). Average length 15.15 cm (13.16–17.2 cm, N = 2 adults); average diameter 0.64/0.73/0.59 cm (preclitellar/clitellar/postclitellar); body subcylindrical in cross-section, ventrally flattened; average number of segments 249 (244–253, N = 2 adults). Average weight (fixed specimens), 4.88 g (4.37–5.39 g, N = 2 adults). Prostomium epilobous, closed. Longitudinal furrows in segments I and II. Nephridial pores ‘en solfège’ (irregularly distributed). Spermathecal pores at intersegmental furrows 9/10–12/13(13/14). Male pores in segment XV, surrounded by a moderately developed porophore. Female pores in segment XIV. Clitellum saddle-shaped in segments XXVII–XLI. Tubercula pubertatis in segments XXXI–1/n XLI. Setae closely paired.

Internal anatomy:

Septa 5/6–10/11 thickened and muscular. Lateral hearts in segments VI–XI. Calciferous glands in segments X–XIV, with diverticula in segment X. Crop in segments XV–XVI, gizzard in segments XVII–XIX. Typhlosole pinnate. Male sexual system holandric, testes and funnels (not enclosed in testes sacs, but with sperm present) located ventrally in segments X and XI. Four pairs of reniform seminal vesicles in segments IX, X, XI and XII, the first two pairs being smaller. Ovaries and female funnels in segment XIII, ovarian receptacles (ovisacs) in segment XIV. Four or five pairs of small globular spermathecae in segments IX, X, XI, XII (XIII) (intersegments 9/10–12/13(13/14)), simple. Nephridial bladders U-shaped, reclinate in segment XXX.

Distribution and ecology:

Scherotheca albina was found in the vicinities of the Regional Park of Haut-Languedoc, Hérault, Occitanie (Fig. 4), in managed grasslands surrounded by forests.

Scherotheca betharramensis Marchán & Decaëns sp. nov.

 

(Figs 2b, 3e, f, 4)

Zoobank registration:

urn:lsid:zoobank.org:act:6E9B7096-C98F-405B-A5D4-54D2782C8A8B.

Type material:

Holotype. France • adult; Occitanie, Hautes-Pyrénées, Saint-Pé-de-Bigorre; latitude/longitude: 43.10197/-0.18483; elevation: 344 m asl; 11-Dec-2021; T. Decaëns, D. Fernández Marchán leg.; BOLD Sample ID: DFM-1089; deposited at Musée national d’Histoire naturelle, Paris.

Etymology:

The species name is derived from the Grottes de Bétharram near the entry where the type locality is situated.

Diagnosis:

Scherotheca betharramensis can be distinguished from all of the other species of Scherotheca by the position of the clitellum in segments (1/n XXVIII)XXIX–XLIX and tubercula pubertatis in segments 1/n XXX–XLIII (Table 2).

COI uncorrected average pairwise distances and topology of multilocus molecular phylogenetic trees support the status of Sc. betharramensis as independent from other morphologically related species.

Description

External morphology:

Body pigmentation dark with anteroposterior gradient and neatly interrupted at level of setae cd, clitellum ash grey (Fig. 2b). Dark brown-greyish in fixed specimens, with beige clitellum and ventral part of the body (Fig. 3e, f). Length of the holotype, 37.5 cm (only known specimen); average diameter 1.1/1.25/0.9 cm (preclitellar/clitellar/postclitellar); body subcylindrical in cross-section, ventrally flattened; number of segments 334. Weight (fixed specimen), 20.5 g. Prostomium epilobous, closed. Longitudinal furrows in segments I and II. Nephridial pores ‘en solfège’ (irregularly distributed). Spermathecal pores at intersegmental furrows 12/13–19/20. Male pores in segment XV, surrounded by a well-developed porophore. Female pores in segment XIV. Clitellum saddle-shaped in segments (1/n XXVIII) XXIX–XLIX. Tubercula pubertatis in 1/n XXX–XLIII. Setae large and closely paired.

Internal anatomy:

Septa 5/6–10/11 thickened and muscular. Lateral hearts in segments VI–XI. Calciferous glands in segments X–XIV, with diverticula in segment X. Crop in segments XV–XVI, gizzard in segments XVII–XIX. Typhlosole pinnate. Male sexual system holandric, testes and funnels (not enclosed in testes sacs, but with sperm present) located ventrally in segments X and XI. Four pairs of reniform seminal vesicles in segments IX, X, XI and XII, the first two pairs being smaller. Ovaries and female funnels in segment XIII, ovarian receptacles (ovisacs) in segment XIV. Eight pairs of globular spermathecae in segments XII, XIII, XIV, XV, XVI, XVII, XVIII, XIX (intersegments 12/13–19/20), simple. Nephridial bladders U-shaped, reclinate in segment XXX.

Distribution and ecology:

Scherotheca betharramensis was found in a single location, in a deciduous forest near Saint-Pé-de-Bigorre (Fig. 4).

Scherotheca microthomasi Marchán & Decaëns sp. nov.

 

(Figs 2d, 3k, 4)

Zoobank registration:

urn:lsid:zoobank.org:act:448F5BBB-D330-47C0-A58B-F8D020E64D8B.

Type material:

Holotype. France • adult; Nouvelle Aquitaine, Pyrénées-Atlantiques, Ordiarp, Kalbegi; latitude/longitude: 43.2/-0.942; elevation: 192 m asl; 11-May-2021; W. Perrin leg.; BOLD Sample ID: DFM-0571; deposited at Musée national d’Histoire naturelle, Paris.

Paratypes:

France • one adult specimen; Nouvelle Aquitaine, Pyrénées-Atlantiques, Ahusky; latitude/longitude: 43.10647/-1.01477; elevation: 1075 m asl; 22-May-2021; W. Perrin leg. BOLD Sample ID: AHUSKY; deposited at Eco&Sols collection, Montpellier.

Etymology:

The species name refers to its superficial resemblance with Scherotheca thomasiBouché, 1972 from which it can be rapidly distinguished by its remarkably smaller body size.

Diagnosis:

Scherotheca microthomasi can be distinguished from Sc. thomasi (positions of the clitellum and tubercula pubertatis are similar) by the number and position of spermathecae [(12/13)13/14–18/19 vs. 13/14, 14/15, 15/16] and vastly smaller body size, and from Scherotheca etcheberryensis (in which position of the clitellum is similar) by the position of the tubercula pubertatis in segments [(XLIII)XLV–XLIX,LI vs. XXXIII–XLV], number and position of spermathecae [(12/13)13/14–18/19 vs. 13/14–19/20] and significantly smaller body size at maturity (Table 2).

COI uncorrected average pairwise distances and topology of multilocus molecular phylogenetic trees supports the status of Sc. microthomasi as independent from other morphologically related species.

Description

External morphology:

Body pigmentation dark with anteroposterior and dorsoventral gradients (Fig. 2d). Brown in fixed specimens (Fig. 3k). Average length 16.4 cm (14.0–18.8 cm, N = 2 adults); average diameter 0.98/1.08/0.9 cm (preclitellar/clitellar/postclitellar); body subcylindrical in cross-section, ventrally flattened with dorsoventrally flattened tail; average number of segments 265 (225–304, N = 2 adults). Average weight (fixed specimens), 8.95 g (6.0–11.9 g, N = 2 adults). Prostomium epilobous, closed. Longitudinal furrows in segments I and II. Nephridial pores ‘en solfège’ (irregularly distributed). Spermathecal pores at intersegmental furrows (12/13)13/14–18/19. Male pores in segment XV, porophore inconspicuous. Female pores in segment XIV. Clitellum saddle-shaped in segments (XXVIII)XXIX–L, LII. Tubercula pubertatis in segments (XLIII)XLV–XLIX, LI). Setae small and closely paired.

Internal anatomy:

Septa 5/6–10/11 thickened and muscular. Lateral hearts in segments VI–XI. Calciferous glands in segments X–XIV, with diverticula in segment X. Crop in segments XV–XVI, gizzard in segments XVII–XIX. Typhlosole pinnate. Male sexual system holandric, testes and funnels (not enclosed in testes sacs, but with sperm present) located ventrally in segments X and XI. Two pairs of reniform seminal vesicles in segments XI and XII. Ovaries and female funnels in segment XIII, ovarian receptacles (ovisacs) in segment XIV. Six or seven pairs of small globular spermathecae in segments (XII)XIII, XIV, XV, XVI, XVII, XVIII [intersegments (12/13)13/14–18/19], simple. Nephridial bladders U-shaped, reclinate in segment XXX.

Distribution and ecology:

Scherotheca microthomasi was found in the vicinity of the forest of Ahusky and within it (Fig. 4), both under tree cover and in open grasslands.

Remarks:

Although morphological characters clearly differentiate Sc. microthomasi and Sc. michaelseni, the low COI genetic distance between these species indicates that they are closely related. The less plausible alternative would be to consider Sc. michaelseni as an extremely polymorphic species encompassing both giant and medium-large populations with widely variable positions of the clitellum and tubercula pubertatis. The former explanation appears more consistent with our understanding of earthworm speciation and systematics.

Scherotheca shaihulud Marchán & Decaëns sp. nov.

 

(Figs 2c, 3a, b, 4)

Zoobank registration:

urn:lsid:zoobank.org:act:2DEA2566-1187-40D8-9A7E-0993C4C3C204.

Type material:

Holotype. France • adult; Nouvelle Aquitaine, Pyrénées-Atlantiques, Ispoure, Pic d’Arradoy; latitude/longitude: 43.183/-1.253; elevation: 331 m asl; 15-December-2021; T. Decaëns, D. Fernández Marchán leg.; BOLD Sample ID: DFM-1119; deposited at Musée national d’Histoire naturelle, Paris.

Paratypes:

France • three adult specimens; same data as holotype (two specimens); BOLD Sample ID: DFM-1120, DFM-1121; Nouvelle Aquitaine, Pyrénées-Atlantiques, Khalbegui; latitude/longitude: 43.2/-0.942; elevation: 192 m asl; 09-May-2021; W. Perrin leg. (one specimen); BOLD Sample ID: DFM-0570; specimens DFM-0570, DFM-1120, DFM-1121 deposited in the Eco&Sols collection, Montpellier.

Etymology:

The species name is derived from the fictional giant sandworm Shai Hulud, from the science fiction novel Dune written by Frank Hebert. Noun in apposition.

Diagnosis:

Scherotheca shaihulud can be distinguished from Sc. chicharia by the position of the clitellum (XXXIII–LII, LIII vs. 1/n XXXII–1/n LII) and tubercula pubertatis [XL–XLVI vs. (1/2 XXXVIII)1/3 XXXIX–XLV], and from Sc. occidentalis by the position of the clitellum (XXXIII–LII, LIII vs. XXXV–LI) and tubercula pubertatis (XL–XLVI vs. 1/n XXXIX–XLV), number and type of spermathecae (intraparietal and multiple -5 to 9- vs. supraparietal and multiple -2 to 3-) and larger body size (Table 2).

COI uncorrected average pairwise distances and topology of multilocus molecular phylogenetic trees supports the status of Sc. shaihulud as independent from other morphologically related species.

Description

External morphology:

Body pigmentation dark with anteroposterior and dorsoventral gradients (Fig. 2c), the tail can appear almost white and with intersegmental dark stripes. Grey with beige clitellum and ventral side in fixed specimens (Fig. 3a, b). Length of the holotype, 30 cm (only complete specimen); average diameter 1.48/1.8/1.33 cm (preclitellar/clitellar/postclitellar); body hemicylindrical with a flattened ventral surface and strongly dorsoventrally flattened tail; number of segments 405. Weight (fixed specimen), 41.5 g. Prostomium epilobus, closed. Longitudinal furrows in segments I and II. Nephridial pores ‘en solfège’ (irregularly distributed). Spermathecal pores at intersegmental furrows 13/14 and 14/15. Male pores in segment XV, surrounded by a moderately developed porophore. Female pores in segment XIV. Clitellum saddle-shaped in segments XXXIII–LII, LIII. Tubercula pubertatis in segments XL–XLVI. Setae large and closely paired.

Internal anatomy:

Septa 5/6–10/11 thickened and muscular. Lateral hearts in segments VI–XI. Calciferous glands in segments X–XIV, with diverticula in segment X. Crop in segments XV–XVI, gizzard in segments XVII–XIX. Typhlosole pinnate. Male sexual system holandric, testes and funnels (not enclosed in testes sacs, but with sperm present) located ventrally in segments X and XI. Two pairs of reniform seminal vesicles in segments XI and XII. Ovaries and female funnels in segment XIII, ovarian receptacles (ovisacs) in segment XIV. Very small globular, intraparietal (under body wall musculature) spermathecae, multiple (from five to nine) in intersegments 13/14 and 14/15. Nephridial bladders U-shaped, reclinate in segment XXX.

Distribution and ecology:

Scherotheca shaihulud was found in two localities near Saint-Jean-Pied-de-Port and Ordiarp (separated by 25 km) (Fig. 4), in a steeply sloped deciduous forest and in a grassland near forest patches.

Scherotheca etcheberryensis Marchán & Decaëns sp. nov.

 

(Figs 2e, 3c, d, 4)

Zoobank registration:

urn:lsid:zoobank.org:act:E48C4A75-D55F-44CB-9EB8-5296C1D542F7.

Type material:

Holotype. France • adult; Nouvelle Aquitaine, Pyrénées-Atlantiques, Tardets-Montory; latitude/longitude: 43.112/-0.84; elevation: 284 m asl; 17-Dec-2021; D. Fernández Marchán leg.; BOLD Sample ID: DFM-1155; deposited at Musée national d’Histoire naturelle, Paris.

Paratypes:

France • three adult specimens, three subadult specimens; same data as holotype; BOLD Sample ID: DFM-1156, DFM-1157, DFM-1158, DFM-1159, DFM-1160, DFM-1161; specimens DFM-1156, DFM-1157, DFM-1158, DFM-1159, DFM-1160, DFM-1161 deposited in the Eco&Sols collection, Montpellier.

Etymology:

The species name is derived from the Etcheberry River on the border where the type locality is situated.

Diagnosis:

Scherotheca etcheberryensis can be distinguished from Sc. thomasi (which shows similar position of the clitellum) by the number and position of spermathecae (13/14–19/20 vs. 13/14, 14/15, 15/16) and position of the tubercula pubertatis (XXXIII–XLV vs. XLV–L), and from Sc. microthomasi (which also has a similar position of the clitellum) by the position of the tubercula pubertatis in segments [XXXIII–XLV vs. (XLIII)XLV–XLIX, LI], number and position of spermathecae [13/14–19/20 vs. (12/13)13/14–18/19] and significantly larger body size (Table 2) .

COI uncorrected average pairwise distances and topology of multilocus molecular phylogenetic trees supports the status of Sc. etcheberryensis as independent from other morphologically related species.

Description

External morphology:

Body pigmentation dark with anteroposterior and dorsoventral gradients (Fig. 2e), the tail can appear almost white and with intersegmental dark stripes. Grey with dark grey clitellum and ventral side in fixed specimens (Fig. 3c, d). Average length 29.6 cm (29.2–30.0 cm, N = 2 adults); average diameter 1.35/1.41/1.25 cm (preclitellar/clitellar/postclitellar); body subcylindrical in cross-section, ventrally flattened with tail moderately flattened dorsoventrally; average number of segments 482 (472–491, N = 2 adults). Average weight (fixed specimens), 32.3 g (32.0–32.6 g, N = 2 adults). Prostomium epilobous, closed, the mouth is unusually large. Longitudinal furrows in segments I and II. Nephridial pores ‘en solfège’ (irregularly distributed). Spermathecal pores at intersegmental furrows 13/14–19/20. Male pores in segment XV, surrounded by an inconspicuous porophore. Female pores in segment XIV. Clitellum saddle-shaped in segments XXIX–LII. Tubercula pubertatis in segments XXXIII–XLV. Setae large and closely paired.

Internal anatomy:

Septa 5/6–10/11 thickened and muscular. Lateral hearts in segments VI–XI. Calciferous glands in segments X–XIV, with diverticula in segment X. Crop in segments XV–XVI, gizzard in segments XVII–XIX. Typhlosole pinnate. Male sexual system holandric, testes and funnels (not enclosed in testes sacs, but with sperm present) located ventrally in segments X and XI. Two pairs of reniform seminal vesicles in segments XI and XII. Ovaries and female funnels in segment XIII, ovarian receptacles (ovisacs) in segment XIV. Seven pairs of globular spermathecae in segments XIII–XIX (intersegments 13/14–19/20). Nephridial bladders U-shaped, reclinate in segment XXX.

Distribution and ecology:

Scherotheca etcheberryensis is known from a deciduous forest in a single locality between Tardets and Montory (Fig. 4).

Scherotheca trezencensis Marchán & Decaëns sp. nov.

 

(Figs 2f, 3i, j)

Zoobank registration:

urn:lsid:zoobank.org:act:02061A8D-8AA1-4968-BC79-E019FE9415B4.

Type material:

Holotype. France • adult; Nouvelle Aquitaine, Charente-Maritime, Saint Loup-La Rochelle, vallée de la Trézence; latitude/longitude: 46.0104/-0.674083; elevation: 4 m asl; 17-May-2021; C. Baron leg.; BOLD Sample ID: DFM-0962; deposited at Musée national d’Histoire naturelle, Paris.

Paratypes:

France • two adult specimens, one juvenile specimen; same data as holotype; BOLD Sample ID: DFM-0963, DFM-0964, DFM-0965; specimens DFM-0963, DFM-0964, DFM-0965 deposited in the Eco&Sols collection, Montpellier.

Etymology:

The species name is derived from the Trézence River, in the valley of which the type locality of the species is located.

Diagnosis:

Scherotheca trezencensis can be distinguished from Scherotheca savignyi (Guerne & Horst, 1893) by the position of the clitellum (XXIX–XLIV, 1/n XLV vs. XXXI–XLIV) and number and position of spermathecae (13/14, 14/15 vs. 13/14–15/16) and from Sc. occitanica by the position of the clitellum (XXIX–XLIV, 1/n XLV vs. 1/2 XXVIII, XXIX–XLVII) (Table 2).

COI uncorrected average pairwise distances and topology of multilocus molecular phylogenetic trees supports the status of Sc. trezencensis as independent from other morphologically related species.

Description

External morphology:

Body pigmentation dark with anteroposterior and dorsoventral gradients (Fig. 2f). Grey with beige clitellum and ventral side in fixed specimens (Fig. 3i, j). Length of the holotype, 19.5 cm (only complete adult specimen); average diameter 1.12/1.24/0.9 cm (preclitellar/clitellar/postclitellar); body subcylindrical in cross-section, tail slightly flattened dorsoventrally; number of segments 246. Weight (fixed specimens), 11.53 g. Prostomium epilobus, closed. Longitudinal furrows in segments I and II. Nephridial pores ‘en solfège’ (irregularly distributed). Spermathecal pores at intersegmental furrows 13/14, 14/15. Male pores in segment XV, surrounded by a well-developed porophore. Female pores in segment XIV. Clitellum saddle-shaped in segments XXIX–XLIV, 1/n XLV. Tubercula pubertatis in segments 1/n XXXIII–XXXIX(1/n XL). Setae closely paired.

Internal anatomy:

Septa 5/6–10/11 thickened and muscular. Lateral hearts in segments VI–XI. Calciferous glands in segments X–XIV, with diverticula in segment X. Crop in segments XV–XVI, gizzard in segments XVII–XIX. Typhlosole pinnate. Male sexual system holandric, testes and funnels (not enclosed in testes sacs, but with sperm present) located ventrally in segments X and XI. Four pairs of reniform seminal vesicles in segments IX to XII, the first two pairs being smaller. Multichambered spermathecae, multiple (three) in segments XIII and XIV (intersegments 13/14, 14/15). Nephridial bladders U-shaped, reclinate in segment 30.

Distribution and ecology:

Scherotheca trezencensis is known from a seasonally inundated (by freshwater) forest in a single locality near La Rochelle (Fig. 4). This constitutes the northernmost population of Scherotheca.

Scherotheca haymozi (Zicsi, 1977) comb. nov.

Allolobophora haymozi Zicsi, 1977.

 

Aporrectodea haymozi Zicsi, 1977: in Zicsi and Csuzdi, 1999.

 

Aporrectodea haymoziformis Zicsi and Csuzdi, 1999.

Remarks:

The original descriptions of Al. haymozi and Ap. haymoziformis differ slightly in the position of the clitellum (XXXIII–1/2 XL, XL vs. 1/2 XXXIII, XXXIII, XXXIV–XL, XLI), tubercula pubertatis (XXXV, XXXVI–XL vs. 1/2 XXXIV, XXXIV–XL, 1/2 XLI) and spermathecae (XII, XIII, XIV vs. XI, XII, XIII, XIV). However, the results of the molecular phylogenetic analysis and COI genetic distances clearly indicate that these morphological differences correspond to intraspecific variability between different populations, as specimens fitting both descriptions showed a small genetic divergence. These justify considering Ap. haymoziformis as a junior synonym of Al. haymozi. The phylogenetic position of the species further justifies its placement within the genus Scherotheca (Fig. 1).

DISCUSSION

Systematic implications

The addition of six new species and a species previously belonging to a different genus (Sc. haymozi) has increased the diversity of Scherotheca to 51 (including the eight recently described species from Corsica; Marchán et al., 2023). This makes Scherotheca the third most diverse genus of Lumbricidae after Dendrobaena (Eisen, 1873)—111 species—and Allolobophora (Eisen, 1873)—69 species. However, the latter includes several species which have recently been assigned to new or redefined genera, and the revised total would be considerably smaller.

Molecular phylogenetic results and genetic divergence values confirmed the validity of Sc. dinoscolex, (originally Sc. gigas dinoscolex), Sc. orbiensis (originally Sc. gigas orbiensis), Sc. minor (originally Sc. savignyi minor), Sc. nivicola (originally Sc. savignyi nivicola) and Sc. chicharia (originally Sc. occidentalis chicharia) as species rank taxa. This was previously corroborated for Sc. rhodana (originally Sc. gigas rhodana) (Marchán et al., 2020) and lends further support to the systematic revision of Scherotheca performed by Qiu & Bouché (1998). Integrative systematics thus appear as a useful tool to challenge the status of subspecies, a controversial taxonomic category which is losing support in the face of current views of cladistics and molecular species delimitation (but see De Queiroz, 2020).

Cryptic lineages have been found in several lumbricid genera [Aporrectodea (Fernández et al., 2012), Lumbricus (James et al., 2010; Martinsson & Erséus, 2017), Allolobophora (Dupont et al., 2011), Eiseniella (de Sosa et al., 2017)] but were previously unknown for Scherotheca. However, the wide distribution of some species such as Sc. savignyi or Sc. aquitania, which contrasts with the narrow distribution of other congeneric species, makes the existence of cryptic lineages within these species likely. Hence, it was not surprising to find evidence of cryptic speciation in Sc. aquitania, with topotype specimens and individuals from a geographically distant population appearing as phylogenetically unrelated but morphologically indistinguishable lineages. In fact, the morphology of the individual from Spain labelled Scherotheca sp. MPL212 (Domínguez et al., 2015) is somewhat compatible with Sc. aquitania and could constitute a third cryptic lineage. Further sampling across the Franco-Iberian range of this species (and Sc. savignyi) followed by molecular phylogenetic analysis would help to corroborate that their unusually wide range is an artefact caused by several cryptic species being lumped together [as usually occurs with polychaetes (Barroso et al., 2010; Kawauchi & Giribet, 2014)].

The increased number of represented species within the molecular phylogeny of Scherotheca enables a preliminary evaluation of the validity of the subgenus system proposed by Bouché (1972). CorsicadrilusQiu & Bouché, 1998 and RosanusQiu & Bouché, 1998 appear only partially supported by the molecular phylogenetic trees, with Sc. albomaculata separated from the other Corsicadrilus representatives, and Sc. brevisella (Bouché, 1972) and Sc. targionii (belonging to Rosanus) included within the Corsicadrilus clade. On the other hand, both Scherotheca and OpothedrilusQiu & Bouché, 1998 appear to be supported by our results, as representatives from both subgenera formed well delimited clades. Several representatives of Rosanus and most Spanish representatives of Scherotheca remain to be included in molecular phylogenetic analysis, and a comprehensive revision of the taxonomy of the genus, including modification or rejection of subgenera, will only be possible after these species are included in the existing data.

Biogeography and evolution

New findings support south-western France as a hotspot of earthworm diversity, with the Pyrénées-Atlantiques (most south-westerly region) being especially diverse. Eighteen endemic (or near endemic) taxa (belonging to the genera Scherotheca, Prosellodrilus, Coventina, Orodrilus and Lumbricus, see Supporting Information, Table S2) were already reported from the region, with a new total of 23. The restricted ranges and extremely difficult sampling of giant species of Scherotheca probably explain why species new to science are still discovered in this area. It is possible that environmental, biotic or historical factors explain the comparatively high diversity and endemicity of these earthworms.

Sampling surveys performed during this work, as well as previous data, have revealed that it is common for two or more species of Scherotheca to coexist in the same habitat within the Pyrénées-Atlantiques, unlike in the rest of their range, where such a co-occurrence pattern is rare. In these cases, the sympatric species are of markedly different sizes, sometimes ranging across up to three size classes (small, large, giant): the most extreme case is that of Sc. shaihulud and Sc. haymozi which were found to co-occur in one locality, with the former weighing 100 times more than the latter. This suggests that Scherotheca species in this area may have evolved towards non-overlapping body sizes to avoid interspecific competition and to exploit different niches in a complementary way in the same habitats (Lu et al., 2011). Interestingly, several independent body size diversification events can be observed in the molecular phylogenetic trees, with other functional traits such as pigmentation and dorsoventral flattening evolving in a somewhat concerted way. Initial hypotheses of the early-branching mid-sized endogeic species from Corsica representing the ancestral morpho-ecological state of the genus (Marchán et al., in press) become less clear when further mainland species are added, suggesting a more complex evolutionary history than previously thought.

Within the Scherotheca species inhabiting the Pyrénées-Atlantiques, an extremely wide range of positions of the clitellum (start: 27–35; end: 41–55, total length: 8–20 segments), tubercula pubertatis (start: 30–45; end: 39–51, total length: 6–14 segments) and number and arrangement of spermathecae (present in two to seven segments, two to 18 per segment) was observed. An unusually large evolutionary plasticity in reproductive organs may be related to the high speciation rate observed within these earthworms. The position of the spermathecae and tubercula pubertatis (which are closely correlated) determine the success of mating between individuals of the same species. Changes in these traits would act as pre-mating barriers, facilitating sympatric speciation. The function of the clitellum during mating is less clear, but it could still play a role in intraspecific recognition (as pre-mating behaviour usually includes exploration of the partner’s clitellum: Nuutinen & Butt, 1997).

It appears likely that the climate in the Pyrénées-Atlantiques and adjacent regions may play an important role in the remarkably high diversity of Scherotheca (14 known species). Mild winters (with average low temperatures above 0 ºC) and summers (daily mean 19.72–22.28 ºC), with high, evenly distributed precipitations across the year (monthly mean 65.52 mm, average 136 days with rainfall per year) appear to match the requirement of high, constant soil moisture and mild temperatures for earthworm activity (Lee, 1985; Edwards & Bohlen, 1996; Edwards & Arancon, 2022). This should be even more favourable for large and giant earthworms such as the Scherotheca which thrive in the area and would need to spend long periods of the year acquiring resources and devoting these to body growth. By comparison, the climate in Hérault (in Mediterranean France), where five species of Scherotheca exist (including Sc. albina), is characterized by warmer summers (daily mean 22.17–24.94 ºC) and less, more irregularly distributed precipitation (monthly mean 36.48 mm, 80.81 days with rainfall per year). The effect of climatic variables on the seasonal activity, reproduction, growth and abundance of different species of Scherotheca is currently unknown. Nevertheless, it is expected that more extreme conditions associated with climate change predictions (greater precipitation seasonality with longer drought periods) could negatively affect these species (Hughes et al., 2018; Singh et al., 2019). Evaluation of the potential impact of such changes on these evolutionarily and ecologically unique species is a priority for soil diversity conservation and maintenance of ecosystem services.

Finally, the importance of the geological history of the region for the diversification of Scherotheca cannot be overlooked. The Pyrenean orogeny, associated with the convergence of the Iberian and European crustal blocks from Late Cretaceous to the Early Miocene (Calvet et al., 2021), has determined the palaeogeography of the region. In the western Pyrenees, collision actually started in the Mid-Eocene (around 45 Mya). Since then, a corridor connecting the area currently inhabited by the Scherotheca endemics in south-western France with the rest of the range of the genus towards the east was uninterrupted. Conversely, this mountain range was relatively isolated from the terranes to the north and south by the intermittently marine Aquitanian and Ebro basins (Calvet et al., 2021). In addition, the presence of permafrost and discontinuous frozen soils across a large portion of France during the Last Glacial Maximum appears to have eliminated most of the endemic fauna, except in glacial refuges such as south-western France (Mathieu & Davies, 2014).

Comprehensive sampling of representatives of Scherotheca in north-eastern Spain, Occitanie and Provence, together with an explicit time-calibrated phylogenetic framework would showcase the expected stepwise colonization of the western Pyrenees from their ancestral range (putatively located between Provence and Corsica; Marchán et al., 2023). Only then will the correlation between geological events and the diversification of Scherotheca be fully understood.

CONCLUSION

A concerted sampling effort in the densely sampled area of south-western France led to the discovery of six species of Scherotheca (including giant ones), which were corroborated and described through integrative systematics. High diversity and morphological diversification of Scherotheca within the small region of Pyrénées-Atlantiques suggest that particular biotic interactions (such as competition avoidance), climate conditions and historical factors may have been in force in the area. The evolutionary and ecological uniqueness of this earthworm fauna highlights the importance of increasing our knowledge about these remarkable soil organisms in order to guarantee their conservation.

[Version of record, published online 11 April 2023; http://zoobank.org/urn:lsid:zoobank.org:pub:41F4B069-C531-4976-A575-02A8DFDE27FF]

ACKNOWLEDGEMENTS

We are grateful to Sylvain Gérard and Raphaël Della Vedova for their help during part of the sampling work, and to William Perrin and Clément Baron for providing specimens of some of the newly described species.

This work was supported by the Xunta de Galicia (Consellería de Cultura, Educación e Ordenación Universitaria, Secretaria Xeral de Universidades under grant ED431B 2019/038), and by subproject Fauna Ibérica XII. Oligochaeta, Lumbricoidea: Lumbricidae, Hormogastridae #PGC2018-095851-B-C66 from the Spanish Ministry of Sciences, Innovation and Universities. D.F.M. was funded by a María Zambrano Postdoctoral Grant from the Spanish Ministry of Sciences, Innovation and Universities and by a Make Our Planet Great Again Postdoctoral grant from Campus France. M.N. was supported by Ramon y Cajal Fellowship (RYC2018-024654-I) from MCIN/AEI/10.13039/501100011033 and by ‘ESF: Investing in your future’.

AUTHOR CONTRIBUTIONS

Franco-Iberian Earthworm Systematics and Evolution Group: Daniel F. Marchán, Marta Novo, Jorge Domínguez, Thibaud Decaëns.

DATA AVAILABILITY

The data underlying this article are available in the dataset DS-EWNSPNWF in BOLD (dx.doi.org/10.5883/DS-EWNSPNWF) and in GenBank with accession codes OP746034–OP746055, OP747606–OP747655 and OP751157–OP751206.

REFERENCES

SUPPORTING INFORMATION

Additional supporting information may be found in the online version of this article on the publisher’s website.

Table S1. GenBank accession numbers for representatives of the genera closely related to Scherotheca.

Table S2. List of endemic lumbricid species from south-western France.