Daniel F Marchán

Csaba Csuzdi

Thibaud Decaëns

Timea Szederjesi

Vaclav Pizl

Jorge Domínguez

The disjunct distribution of relict earthworm genera clarifies the early historical biogeography of the Lumbricidae (Crassiclitellata, Annelida)

Keywords: zoobank Systematics, Molecular Phylogenetics, Time-calibrated phylogeny, Ecological Niche Modelling, Earthworms

The historical biogeography of the Lumbricidae, the main Palearctic earthworm family, may be linked to the paleogeography of their putative ancestral range in Iberia-Southeastern France-Corsica-Sardinia. Although molecular phylogenetics has recently been used to analyze most endemic genera in this area, the genus Kritodrilus has not yet been studied. The generotype Kritodrilus calarensis and other closely related species inhabit Southeastern France. However, other species from the Iberian Peninsula (e.g. Kr. osellai) have been assigned to Kritodrilus, the same for the Vindobonic (Hungarian-Austrian-Czech-Slovakian) Dendrobaena mrazeki. In order to unravel the paleobiogeographic, evolutionary and taxonomic significance of Kritodrilus, the aforementioned species (and three potential relatives) were sequenced for seven molecular markers. Molecular phylogenetic inference recovered a clade of French Kritodrilus (including Et. setusmontanus) closely related to the Northwestern Iberian Galiciandrilus and Compostelandrilus, the latter being a sister clade to the Vindobonic species (described as a new genus). Time-calibrated phylogenetic inference estimated that these three clades diverged around 55Ma. Ecological Niche Models for these clades were mainly influenced by the same environmental variables, but showed nonoverlapping highly suitable areas (except for Kritodrilus). Ancestral area reconstruction inferred the origin of the Lumbricidae in Corsica-Sardinia and the Iberian Peninsula, with eventual expansion eastwards towards France, Central Europe and the Carpato-Balkanic area. For Kritodrilus and its relatives, this expansion may have been contemporaneous with the Paleocene-Eocene Thermal Maximum. Later climatic changes may explain the strikingly disjunct ranges. This work highlights the evolutionary and biogeographic significance of these isolated earthworm lineages and the importance of their preservation.

Introduction

Earthworms (Annelida, Megadrili) are an ancient animal group, originating around the Permo-Triassic boundary (about 250 mya) [START_REF] Marchán | Pinpointing cryptic borders: Fine-scale phylogeography and genetic landscape analysis of the Hormogaster elisae complex (Oligochaeta, Hormogastridae)[END_REF], but see [START_REF] Erséus | Phylogenomic analyses reveal a Palaeozoic radiation and support a freshwater origin for clitellate annelids[END_REF] for a slightly younger estimate) and with the major clades predating or splitting during the break-up of Pangea (about 175 mya) [START_REF] Anderson | Phylogenomic analyses of Crassiclitellata support major Northern and Southern Hemisphere clades and a Pangaean origin for earthworms[END_REF]. Due to the apparent correlation between paleogeographic and cladogenetic events in this animal group [START_REF] Fernández | Earthworms, good indicators for palaeogeographical studies? Testing the genetic structure and demographic history in the peregrine earthworm Aporrectodea trapezoides (Dugès, 1828) in southern Europe[END_REF], the origin and diversification of earthworm families is a promising research topic in the fields of zoology, biogeography and geology.

The native earthworm families in the Paleartic region are Criodrilidae Vejdovsky, 1884 Hormogastridae, Michaelsen, 1900 and Lumbricidae Rafinesque-Schmaltz, 1815, with the latter being the most diverse and widely distributed (44 genera and around 670 species: [START_REF] Blakemore | An updated list of valid, invalid and synonymous names of Criodriloidea and Lumbricoidea (Annelida: Oligochaeta: Criodrilidae, Sparganophilidae, Ailoscolecidae, Hormogastridae, Lumbricidae, Lutodrilidae). A Series of Searchable Texts on Earthworm Biodiversity, Ecology and Systematics from Various Regions of the World-Supplemental[END_REF]. Considering the large number of endemic species and genera, these sister taxa are believed to have originated and diversified in Southwestern Europe representing the Franco-Iberian earthworm domain [START_REF] Csuzdi Cs | Earthworms of Hungary (Annelida: Oligochaeta, Lumbricidae)[END_REF]. [START_REF] Novo | Unearthing the historical biogeography of Mediterranean earthworms (Annelida: Hormogastridae)[END_REF] inferred an origin of the family Hormogastridae in Southeastern France using molecular phylogenetic inference and performing ancestral area reconstruction analysis. Thus, it is to be expected that Lumbricidae would share this ancestral area. The presence of several endemic lumbricid genera (Ethnodrilus Bouché 1972, Kritodrilus Bouché 1972, Avelona Qiu & Bouché 1998) and species groups (Zophoscolex (Zophoscolex) [START_REF] Qiu | Révision des taxons supraspécifiques de Lumbricoidea[END_REF], Allolobophora (Gatesona) chaetophora, Helodrilus (Acystodrilus) [START_REF] Qiu | Révision des taxons supraspécifiques de Lumbricoidea[END_REF] supports that hypothesis.

The most complete and up-to-date molecular phylogeny of Lumbricidae [START_REF] Domínguez | Underground evolution: New roots for the old tree of lumbricid earthworms[END_REF] includes representatives of most of the genera, helping to untangle the controversial systematics of the family. However, Ethnodrilus, Kritodrilus, Zophoscolex and Avelona were among the few genera not included in the analysis. [START_REF] Jiménez | Sorry atlanticus, you are not my type. Molecular assessment splits Zophoscolex (Lumbricidae, Crassiclitellata) into French and Iberian genera[END_REF] has recently recovered the genus Zophoscolex Qiu & Bouché 1998 as an early-branching, French endemic genus included in a large clade of Franco-Iberian genera (together with Ethnodrilus), adding more evidence regarding the importance of this region in the early evolution of the Lumbricidae.

Avelona has been found to be closely related to Allolobophora (Gatesona) and Helodrilus (Acystodrilus), which together form a clade endemic to the Massif Central (Marchán et al. unpublished).

Thus, Kritodrilus remains a missing piece in the phylogenetic and historical biogeographical puzzle of the Lumbricidae. Kritodrilus calarensis Tetry, 1944 (generotype), K. micrurus Zicsi andCsuzdi, 1999 andK. tetryae Zicsi andCsuzdi, 1999 inhabit Provence-Alpes-Côte d'Azur and Auvergne-Rhône-Alpes (Southeastern France). Ethnodrilus setusmontanus [START_REF] Qiu | Révision des taxons supraspécifiques de Lumbricoidea[END_REF] was described in a location 85 km to the west of the aforementioned range.

Although assigned to Ethnodrilus, there is remarkable disjunction and morphological disparity between this and the other species of the genus. Thus, E. setusmontanus appears closer to Kritodrilus in both morphology and distribution. Some Iberian lumbricids have also been attributed to Kritodrilus (Dendrobaena osellai Zicsi, 1970, Dendrobaena ruffoi Zicsi, 1970, Dendrobaena pseudorrosea Moreno, 1983) by [START_REF] Omodeo | Earthworms of Turkey[END_REF], but this assignment has been criticized by other authors [START_REF] Qiu | Révision des taxons supraspécifiques de Lumbricoidea[END_REF]Bouché 1998, Blakemore 2008). If these lumbricids were actually members of the genus Kritodrilus, they would be expected to be included within the same clade as Kritodrilus calarensis (and the other French species) in a molecular phylogenetics study. Cernosvitov, 1935 andDendrobaena mrazeki Cernosvitov, 1935 are Vindobonic (Hungarian-Austrian-Czech-Slovakian) endemisms with a unique xerophilic-steppe habitat preference and geographically restricted range. Although both species appear to be closely related owing to their morphological characters, their phylogenetic position is unclear, with D. mrazeki eventually placed within Kritodrilus [START_REF] Omodeo | Earthworms of Turkey[END_REF] and A. hrabei possibly being related to Zophoscolex [START_REF] Csuzdi Cs | Earthworms of Hungary (Annelida: Oligochaeta, Lumbricidae)[END_REF]. Even though their current range is far removed from the aforementioned Franco-Iberian genera, such morphology-based links could be explained by these species being a relic of a formerly widely distributed lumbricid lineage encompassing these territories.

Allolobophora hrabei

In this study, we explored the paleobiogeographic, evolutionary and taxonomic significance of Kritodrilus and potentially related taxa Allolobophora hrabei, Kritodrilus mrazeki and Kritodrilus osselai through their inclusion in a molecular phylogenetic context, together with the previously studied Franco-Iberian early-branching genera. Ecological Niche Modelling was used to investigate which environmental variables explain the restricted distribution of these taxa. Time-calibrated phylogenetic inference and ancestral area reconstruction provide a spatiotemporal framework for the diversification of early-branching Lumbricidae, shedding some light on the historical biogeography of this ancient group.

Materials and methods

Specimens and sampling

For the present study, 22 specimens of 6 species of Lumbricidae were studied morphologically, and one specimen of each species was analyzed through molecular phylogenetics (Table 1).

Additionally, 110 specimens of 110 species (108 lumbricids and 2 outgroup taxa) were included as sequences recovered from Genbank (publications of origin shown in Appendix 1).Specimens of the species Kritodrilus calarensis, Kritodrilus micrurus and Ethnodrilus setusmontanus were collected in a sampling survey carried out in Southeastern France between October and November 2019. Specimens of Allolobophora hrabei and Kritodrilus mrazeki were collected in Várbalog, NE Hungary and Podyjí National Park, Czech Republic, respectively. Specimens assigned to Kritodrilus osellai were available in the UCMLT collection of the Universidad Complutense de Madrid. The locations where the samples were found are listed in Table 2.

Earthworms were collected by digging and hand-sorting or by the diluted formaldehyde method [START_REF] Raw | Estimating earthworm populations by using formalin[END_REF], rinsed with water and fixed in 96% ethanol. Sampling and handling of specimens followed ethical considerations and conformed to Directive 2010/63/EU. Species identification was conducted using the set of external and internal morphological characters used by [START_REF] Qiu | Révision des taxons supraspécifiques de Lumbricoidea[END_REF].

DNA isolation and sequencing

After morphological identification, one representative specimen of each species was chosen for further analysis. Total genomic DNA was extracted using the DNeasy Blood & Tissue Kit (Qiagen) from ventral integument samples of approximately 5 x 5 mm. Regions of the genes for the nuclear 28S rRNA (28S) and mitochondrial 16S rRNA (16S), tRNAs Ala, Leu and Ser (tRNAs), 12S rRNA (12S), and regions of the mitochondrial genes NADH dehydrogenase subunit 1 (ND1), cytochrome c oxidase subunit 1 (COI) and cytochrome c oxidase subunit 2 (COII) were amplified for 6 species using the polymerase chain reaction (PCR); primers and length of the obtained amplicons are shown in Table 2. PCR condition were as follows: 94°C -3´, (94°C -30´´, X°C -45´´, 72°C -1´) 40 (<35 for ND1) cycles (see Table 2), 72°C -5´, 4°C -∞. 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, and the accession numbers are shown in Table 3.

Sequences reported by [START_REF] Domínguez | Underground evolution: New roots for the old tree of lumbricid earthworms[END_REF], 2018[START_REF] Pérez-Losada | Phylogenetic assessment of the earthworm Aporrectodea caliginosa species complex (Oligochaeta, Lumbricidae) based on mitochondrial and nuclear DNA sequences[END_REF][START_REF] Pérez-Losada | An earthworm riddle: systematics and phylogeography of the Spanish lumbricid Postandrilus[END_REF], 2015), Paoletti et al. (2016[START_REF] De Sosa | Placing Eophila tellinii (Oligochaeta, Lumbricidae) in a molecular phylogenetic context advances the century-old controversy around the problematic genus[END_REF]), Bozorgui et al. (2019), [START_REF] Jiménez | Sorry atlanticus, you are not my type. Molecular assessment splits Zophoscolex (Lumbricidae, Crassiclitellata) into French and Iberian genera[END_REF] and Marchán et al. (2020, in press) 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 dataset. The list of species and reference to the publication on which they were provided are shown in Appendix 1. Table 3.

Phylogenetic analyses and time-calibrated phylogenetic inference

Sequences were aligned with MAFFT v.7 [START_REF] Katoh | MAFFT multiple sequence alignment software version 7: Improvements in performance and usability[END_REF] with default settings and concatenated with BioEdit [START_REF] Hall | BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT[END_REF], resulting in a matrix of 4,183 bp (Suppl. Information Alignment S1). The best fitting evolutionary model for each partition was selected with jModelTest v. 2.1.3 [START_REF] Darriba | jModelTest 2: more models, new heuristics and parallel computing[END_REF] by applying the Akaike information criterion (AIC; [START_REF] Akaike | Information theory and an extension of the maximum likelihood principle[END_REF] and the Bayesian information criterion (BIC; [START_REF] Schwarz | Estimating the dimension of a model[END_REF]. tRNAs were analyzed as a separate partition from 16S. GTR + I + G was selected as best-fitting evolutionary model for COI, 28S and ND1, GTR + G was selected for 12S, and HKY+I+G was selected for 16S, tRNAs and COII.

Bayesian Inference of the phylogeny was estimated with MrBayes v.3.2.6 [START_REF] Ronquist | MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space[END_REF] as implemented in CIPRES Science Gateway V. 3.3 [START_REF] Miller | Creating the CIPRES Science Gateway for inference of large phylogenetic trees[END_REF]. The analysis was performed with default parameters, and each of the two independent runs was set to 50 million generations sampling every 5,000th generation (10,000 trees). Twenty-percent of the trees were discarded as burn-in, with remaining trees combined and summarized on a 50% majority-rule consensus tree. Maximum Likelihood phylogenetic inference was performed using RAxML-NG [START_REF] Kozlov | RAxML-NG: a fast, scalable and user-friendly tool for maximum likelihood phylogenetic inference[END_REF] in the CIPRES Science Gateway V. 3.3 platform, from 10 random starting trees and 1,000 rapid bootstrap replicates.

To generate a suitable starting tree for the time-calibrated phylogenetic inference, the Maximum Likelihood tree was converted into an ultrametric tree by non-parametric rate smoothing (NPRS) using the chronopl function in the R package ape v5.2. The maximum and minimum ages of the clades were the same as those used in the downstream BEAST analysis.

The final ultrametric tree was generated with BEAST v. 1.10 (Suchard et al., 2018) using the NPRS tree as the starting tree. Each partition was trimmed with GBlocks [START_REF] Castresana | Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis[END_REF] under the less stringent parameters, with the best-fitting evolutionary model (shown above) as the evolutionary model for each.

The following calibration points were implemented as uniform priors:

-87-170 mya for the Criodrilidae + Lumbricidae + Hormogastridae clade -82-147 mya for the Lumbricidae+Hormogastridae clade These age intervals correspond to the 95% HPD (highest posterior density) intervals obtained by [START_REF] Marchán | Pinpointing cryptic borders: Fine-scale phylogeography and genetic landscape analysis of the Hormogaster elisae complex (Oligochaeta, Hormogastridae)[END_REF].

A Yule diversification model and an uncorrelated lognormal relaxed clock were specified. Three parallel runs were performed, each including 50 million generations, sampling every 5,000th generation. Tree and log files were combined in Logcombiner v. 1.10 (Suchard et al., 2018) by resampling at a lower frequency (15,000) and the results were visualized in Tracer v. 1.7.1 [START_REF] Rambaut | Posterior summarisation in Bayesian phylogenetics using Tracer 1.7[END_REF]. The final tree was generated by TreeAnnotator v.1.10 (Suchard et al., 2018) with a burn-in of 2000 generations.

Ancestral area reconstruction

Ancestral area reconstruction was performed in RASP 4 [START_REF] Yu | RASP 4: Ancestral State Reconstruction Tool for Multiple Genes and Characters[END_REF], and the most suitable model was chosen using BioGeoBEARS (DEC+j) and allowing a maximum of 3 areas for each node. The areas considered were coded as follows: A-Iberian Peninsula, B-France, C-Tyrrhenian (Corsica, Sardinia and Italian Penninsula), D-Central Europe (including Alpine Italy), E-Carphato-Balkanic, F-Easten Eurasia, G-North America. Species ranges were mainly obtained from DriloBase Taxo (http://taxo.drilobase.org/); for cosmopolitan/peregrine species, the original range was included when available in the literature, if not they were removed from the analysis. Species ranges are shown in Table 2 andAppendix 1.

Ecological Niche Modelling

Presence data (Table S1) were aggregated according to the clades recovered in the phylogenetic analyses (see Results) as follows: Northwestern Iberian, Mediterranean and Vindobonic.

The following large-scale variables, used in [START_REF] Marchán | Macroecological inferences on soil fauna through comparative niche modeling: the case of Hormogastridae (Annelida, Oligochaeta)[END_REF] Human disturbance (ANTHRO) was incorporated through the 'Human footprint' data setrepresenting the human influence on land surface [START_REF] Sanderson | The human footprint and the last of the wild[END_REF].

Ecological niche models were obtained using the R package 'SSDM' [START_REF] Schmitt | ssdm: An r package to predict distribution of species richness and composition based on stacked species distribution models[END_REF].

Ensemble species distribution models (ESDMs) were built by combining the algorithms ('MAXENT', 'GLM', 'CTA', 'MARS', 'SVM', 'GAM', 'GBM', 'ANN') producing kappa values greater than 0.5, with 5 repetitions for each algorithm.

Results

Phylogenetic analysis and time-calibrated phylogenetic inference

Bayesian phylogenetic inference (Figure 1 Thus, Kritodrilus in the most inclusive sense appears to be polyphyletic, with some representatives placed within loosely-related clades and a well-delimited and strongly supported clade including the generotype (K. calarensis) as well as a species originally assigned to a different genus (Et. setusmontanus).

Figure 1

The time calibrated tree (Figure 2) enabled comparison of the estimated ages of the different genus-level clades within the Lumbricidae. Although the crown age of Kritodrilus sensu stricto appeared relatively young (8Ma, 4.8-12.3 95% Highest Posterior Density interval) and similar to the crown age of Postandrilus (13.7Ma, 9.2-19.4), its divergence from its sister clade was remarkably early (58.6Ma,). Crown ages of the Vindobonic clade (A. hrabei + Kr. mrazeki) and the two Northwestern Iberian genera Galiciandrilus and Compostelandrilus were comparatively ancient (41Ma,38.9Ma,7Ma,, with the splits between these genera occurring relatively close in geological time (53.6Ma,).

The most recent common ancestor of all Lumbricidae (A, see

Ancestral area reconstruction

The Iberian Peninsula, France and Corsica-Sardinia were inferred as the most likely ancestral area of Hormogastridae and Lumbricidae (Fig. 2). The ancestral area of Lumbricidae (node A) was located at Corsica-Sardinia, with subsequent dispersal and vicariance: the Iberian Peninsula

Ecological Niche Modelling

The three ecological niche models obtained displayed high predictive power, with high AUC and kappa values (0.89-0.96 and 0.7-0.92 respectively), high sensitivity and specificity (0.90-1 and 0.88-0.92 respectively) and low omission rates (0.00-0.10) (Table S2).

The geographical representation of the predicted suitability values is shown in Fig. 3. The predicted highly suitable areas was significantly broader than the known range of the studied taxa, but the difference was particularly remarkable for the Mediterranean clade. There was a large amount of overlap between the highly suitable areas of the Mediterranean and Northwestern Iberian clades, while the highly suitable area predicted for the Vindobonic clade was clearly isolated from the other two areas.

The relative contributions of the predictor variables to each model are shown in Table S2.

Discussion

Systematic implications

The inclusion of the type species of Kritodrilus (K. calarensis) within a well-delimited, strongly supported clade together with K. micrurus and Ethnodrilus setusmontanus suggest that the genus should be restricted to these species, excluding K. mrazeki and K. osellai. The morphological similarity and geographical distribution suggest that K. tetryae should probably be included in Kritodrilus proper, while Iberian species assigned to Kritodrilus (K. ruffoi, K. pseudorrosea) would be expected to form a clade with K. osellai. The latter should be confirmed by molecular phylogenetic analysis, as K. pseudorrosea was also assigned to Iberoscolex (Qiu and Bouché 1998X). In any case, K. osellai and its closest relatives should be described as a new genus, as they do not belong to Dendrobaena (the genus they were originally assigned to): the generotype Dendrobaena octaedra Savigny 1826 belongs to a distant clade, as shown by the phylogenetic trees. However, reassignment is not advisable until the aforementioned species (and others like Iberoscolex microepigeus [START_REF] Qiu | Révision des taxons supraspécifiques de Lumbricoidea[END_REF]

Species included:

Kritodrilus calarensis Tetry, 1944

Kritodrilus micrurus Zicsi and Csuzdi, 1999 Kritodrilus setusmontanus (Qiu and Bouché,1998)

Differential diagnosis:

Vindoboscolex shares with Kritodrilus sensu stricto the shape of the nephridial bladders (bilobous) and the presence of calciferous diverticula in segment 11, but it is clearly separated from Kritodrilus sensu stricto by the presence of porphyrin pigment (Kritodrilus species are unpigmented) and pinnate musculature (vs fasciculate musculature).

Vindoboscolex can also be differentiated from its closest relatives Galiciandrilus and

Compostelandrilus by the presence of porphyrin pigment (Galiciandrilus and Compostelandrilus species are unpigmented).

Remarks.

In the classical system of the Lumbricidae [START_REF] Pop | Zur phylogenie und Systematik der Lumbriciden[END_REF] the setal arrangement was one of the most important characters separating different genera. However, it has recently been shown that several genera include species both with widely and narrowly paired setae, as in Dendrobaena Eisen, 1873 [START_REF] Zicsi | Regenwürmer aus Bulgarien (Oligochaeta, Lumbricidae)[END_REF]) Dendrodriloides Kvavadze, 2000, Bimastos Moore, 1893 [START_REF] Csuzdi | Molecular phylogeny and systematics of native North American lumbricid earthworms (Clitellata: Megadrili)[END_REF] or Castellodrilus [START_REF] Jiménez | Sorry atlanticus, you are not my type. Molecular assessment splits Zophoscolex (Lumbricidae, Crassiclitellata) into French and Iberian genera[END_REF]. The new genus Vindoboscolex follows the same pattern, highlighting the high degree of homoplasy of the the setal position in the family Lumbricidae.

Historical biogeography and evolution

The time-calibrated phylogeny of the Lumbricidae, together with the paleogeographic settings of the corresponding geological times, enables reconstruction of the early evolution of the family. These evolutionary scenarios should be regarded as working hypotheses, as the estimated times of divergence showed wide HPD intervals due to the use of external calibration points in the absence of ingroup fossil record. However, the congruence between some estimated times of divergence ages and vicariant events (separation between Corsican and French Scherotheca at around 27 Ma, diversification of Balearic Postandrilus after 13.6 Ma) supports the general fit of the time-calibrated phylogeny to the historical biogeography of Lumbricidae.

The most recent common ancestor of all studied Lumbricidae would have inhabited Corsica-Sardinia, which was located within a continuous landmass together with France and Northeastern Spain and isolated from surrounding landmasses at the time (Maastrichtian, Upper Cretaceous, Fig. 2). Interestingly, [START_REF] Novo | Unearthing the historical biogeography of Mediterranean earthworms (Annelida: Hormogastridae)[END_REF] estimated that the ancestral area of Hormogastridae was located in Southern France and the Northeastern Iberian Peninsula: this suggests the two sister families could have originated by vicariance within that ancient terrane.

During the late Cretaceous and early Paleocene, paleogeographic changes (uplift of the European Cenozoic Rift System, ECRIS -Ziegler and Dèzes, 2007) led to the connection of the Franco-Ibero-Corso-Sardinian landmass to the adjacent Rhenish and Bohemian massifs and the East European Platform. This would have enabled the eastern expansion of Lumbricidae lineages around this time. This is consistent with the ancestral areas inferred for clade G (Fig. 2) and its internal clades. The biogeographical scenario reconstructed by our analysis does not exactly correspond with that expected from such a West-East colonization route, as the earlierbranching ancestral area (Carpato-Balkanic) is located further than the Central European area.

However, this could be explained by a more crownward phylogenetic position of the Carpato-Balkanic clade (comprising Cernosvitovia, Serbiona, Karpatodinariona and related taxa) or the extinction/failure to sample of Central European taxa.

The same paleogeographic event is also compatible with the inferred historical biogeography of clade E: from an ancestral area located in the Iberian Peninsula, it would have expanded to Southeastern France (Kritodrilus) and to Central Europe (Vindoboscolex), with the Northwestern Iberian Galiciandrilus and Compostelandrilus as remnants of the original range.

However, the 1700 km disjunction between the current ranges of Vindoboscolex and its Iberian relatives is difficult to understand. One possible explanation can be found in the large global climatic changes during the Tertiary. There is a significant overlap between the estimated times of divergence between these clades (including Kritodrilus) and the climatic event known as the Paleocene-Eocene Thermal Maximum (at around 55.5 Ma - [START_REF] Bowen | Two massive, rapid releases of carbon during the onset of the Palaeocene-Eocene thermal maximum[END_REF]. Superimposed on a general trend of long-term global warming, this episode resulted in increased temperatures of up to 8º C. The resulting climatic conditions were very different from those found from the Eocene to the present day. It is possible that, under these conditions, suitable habitat for the ancestors of the disjunct clades was distributed continuously across their ranges, unlike at present (Fig. 3). Under a progressively cooler, drier climate, the ecological niche of these taxa would have diverged and become more restricted, explaining their current isolation.

Intermediate species would probably have become extinct, contributing to the biogeographical gap.

It is worth noting that most cladogenetic events within other lumbricid genera (for example Castellodrilus, Octolasion-Octodrilus, Allolobophora s.s. Aporrectodea s.s, Scherotheca or Eophila) are younger than the estimated ages of divergence between the species of Vindoboscolex, Galiciandrilus and Compostelandrilus. This could signal a replacement of the latter by the former genera in earthworm fauna. In order to support this hypothesis, further information about the interaction between these species (including putative competition) and about the environmental variables influencing their distribution is required.

Finally, the recovery of Pietromodeona and Kritodrilus sensu lato osellai as sister taxa of Postandrilus could solve the biogeographic riddle of the latter being isolated from their relatives in the Balearic Islands [START_REF] Pérez-Losada | An earthworm riddle: systematics and phylogeography of the Spanish lumbricid Postandrilus[END_REF]. Said work identified Galiciandrilus and Postandrilus as sister taxa, thus demonstrating a strong disjunction between them. However, the authors hypothesized the presence of intermediate forms in the Eastern Iberian Peninsula. The addition of further representatives of this clade (Compostelandrilus, -described in 2018-, Vindoboscolex -described here-, Kritodrilus sensu stricto and Kritodrilus sensu lato osellai) apparently filled the gaps in the evolutionary and biogeographic scenario of these earthworms.

The main range of Pietromodeona janueargenti is located in Sardinia, and Kritodrilus sensu lato osellai inhabits Southeastern Spain (Teruel, Valencia): these terranes and the Balearic Islands were contiguous and connected land masses until around 30Ma. Time-calibrated phylogeny estimated an earlier split between these species and Postandrilus, possibly due to unrepresented, potentially closer relatives (e.g. Kritodrilus sensu lato ruffoi). Phylogeographic sampling of Pietromodeona janueargenti, Kritodrilus sensu lato osellai and Kritodrilus sensu lato ruffoi would help to pinpoint the phylogenetic and geographic origin of Postandrilus species and their colonization of the Balearic Islands.

Conclusions

Molecular phylogenetic analysis of the type species (and additional ones) of Kritodrilus allowed to delimit it as a Southeastern France endemic genus. This genus is phylogenetically close to other early-branching Franco-Iberian endemic genera.

Species not included in this work (K. tetryae, K. ruffoi and K. pseudorrosea), and also the generotype of Iberoscolex (Ib. microepigeus) should also be analysed to complete the taxonomic revision of the genus.

The close phylogenetic relationship of Kritodrilus, the Northwestern Iberian Galiciandrilus and Compostelandrilus, and the newly described genus Vindoboscolex (comprising V. hrabei and V. mrazeki, from Czech Republic, Slovakia, Austria and Hungary) is biogeographically puzzling due to the large disjunction between their ranges. The estimated time of divergence of these clades enables proposal of a hypothetical connection between the expansion and retraction of their range and global climatic changes from the Paleocene-Eocene to this day. The phylogenetic uniqueness and restricted range of these relict genera highlights the need for research on their conservation status and putative necessary conservation measures.

Ancestral area reconstructions of representatives of most Lumbricidae genera provided a promising overview of the historical biogeography of the family. On one hand, some genera within the ancestral range of the family (Corsica, Sardinia and the Iberian Peninsula) should be sampled more thoroughly (i.e. Prosellodrilus, Cataladrilus, Zophoscolex, Iberoscolex, Eumenescolex) to better represent their diversity. On the other hand, unsampled species from Central Europe, the ancestral area for a large portion of known Lumbricidae genera should be included in further studies to confirm the biogeographical importance of this area. In addition, some Eastern Europe-Middle East-Central Asia species and genera (such as Perelia, Healyella, Dendrobaena and Eisenia) remain underrepresented in molecular phylogenetic analyses, and examination of these would shed some light on the expansion and evolution of Lumbricidae further from their center of origin. Table 1. Specimen list with species, number of studied specimens (N) and method (M: morphology, DNA: molecular phylogenetics) and Genbank accession numbers. UCM-LT: Universidad Complutense de Madrid Lombrices de Tierra (earthworm collection of the Complutense University of Madrid). HMNH:

Figure legends

Tables

  for the large-bodied, endogeic Hormogastridae) were chosen as predictor variables: -Bioclimatic variables (downloaded from Worldclim -http://www.worldclim.org/ accessed 01/12/2020) Mean Diurnal Range (BIO2, TRANGE) Isothermality (BIO3, ISOTHER) Mean Temperature of the Coldest Quarter (BIO11, TCOLD) Precipitation of the Driest Month (BIO14, PRDRY) -Topographical variable Lithology (LITHO), represented by the PAR-MATDOM2 (Second level code for the dominant parent material of the STU) layer obtained from the European Soil Database Raster Library 1 km _ 1 km (http://eusoils.jrc.ec.europa.eu/ESDB_Archive/ESDB_data_1k_raster_intro/ESDB_1k_raster_d ata_intro.html accessed 01/12/2020). -Biotic variables Vegetation and dominant land use (VEGET) were represented by the CORINE 2006 Land Cover layer (version 12/2009: http://www.eea.europa.eu/data-and-maps/data/corine-land-cover-2006-clc2006-100-m-version-12-2009 accessed 01/12/2020).

  ) placed the studied taxa (Kritodrilus calarensis, Kritodrilus micrurus, Ethnodrilus setusmontanus, Allolobophora hrabei, Kritodrilus mrazeki and Kritodrilus osellai) within an early-branching clade sister to another one comprising Castellodrilus, Zophoscolex, Ethnodrilus, Prosellodrilus and Cataladrilus. The former clade split into two moderately supported clades: one of them comprised Pietromodeona januaeargenti, Kr. osellai and Postandrilus representatives, while the other comprised the Northwestern Iberian genera Galiciandrilus and Compostelandrilus, the Vindobonic species A. hrabei and Kr. mrazeki, and the other Kritodrilus species (calarensis and micrurus) together with Et. setusmontanus.

  Figure 2) was estimated to have occurred at 71.3Ma (55.5-87.5), which places it in the Maastrichtian. A clade restricted to the Iberian Peninsula, France and Sardinia (with the exception of the Vindobonic taxa) -B-and a widespread clade with genera distributed across Eurasia and North America -G-were estimated to have diverged at 67Ma (53.5-84), close to the K-T boundary. The most recent common ancestor of the Carpato-Balkanic clade and the majority of the Lumbricidae genera (G) was estimated to have occurred at 64Ma (49.6-79.3). Within clade B, the most recent common ancestor of the Franco-Iberian clade D (Castellodrilus, Zophoscolex, Ethnodrilus, Prosellodrilus and Cataladrilus) and clade C was estimated to have occurred at around 63.3Ma (49.2-78).Kritodrilus sensu stricto and clade F (comprising the Vindobonic species, Galiciandrilus and Compostelandrilus) are estimated to have diverged at), while the most recent common ancestor of the disjunct clades within F was estimated to have occurred at) corresponding to the Late Paleocene-Early Eocene.

Figure 2

 2 Figure 2

  was identified as the ancestral area of the clade comprising all Lumbricidae except Diporodrilus. Another dispersal-vicariance event would have led to the ancestral area of clade G, in the Carpato-Balkanic region. Within clade G, the ancestral area for most of the nodes corresponded to Central Europe, with further dispersal or dispersal-vicariance events explaining ancestral areas in the Iberian Peninsula (Iberoscolex, Satchellius), Corsica (Scherotheca) and North America (Bimastos+Eisenoides). The ancestral area of most nodes within clade B corresponded to the Iberian Peninsula. In node D, a dispersal-vicariance event would have separated Castellodrilus (ancestral range: Iberian Peninsula) and Zophoscolex+Ethnodrilus+Prosellodrilus+Cataladrilus (ancestral range: France). Further dispersal-vicariance would have led to an Iberian ancestral area for Cataladrilus. The ancestral area for clade E (comprising Kritodrilus, Vindoboscolex gen. nov., Galiciandrilus and Compostelandrilus) was probably located in the Iberian Peninsula, with consecutive dispersal-vicariance events leading to the French and Central Europe ancestral areas of Kritodrilus and Vindoboscolex gen. nov. (respectively).

  Isothermality and Lithology were amongst the three most influential variables for the three clades, and Mean Diurnal Range was one of the three most influential variables for the Mediterranean and Northwestern Iberian clades (replaced by Human disturbance for the Vindobonic clade).

Figure 3

 3 Figure3

Family

  , type species of its genus) are analysed.The Vindobonic species (Allolobophora hrabei, Kritodrilus mrazeki) formed an independent, well-supported clade with an estimated time of divergence consistent with other genus-level clades. This, together with their morphological resemblance, justifies the establishment of a new genus to include both species.Phylum AnnelidaLamarck, 1802 Class Oligochaeta Grube, 1850/Clitellata Michaelsen, 1919 Order Megadrili Benham, 1890/Haplotaxida Michaelsen, 1900

  comb. nov.Kritodrilus tetryae?Zicsi and Csuzdi, 1999 Remarks K. tetryae was provisionally included in the genus due to the morphological similarity and geographical proximity with the confirmed representatives. K. osellai was excluded as it belonged to a different clade in molecular phylogenetic analyses. K. ruffoi and K. pseudorrosea were provisionally excluded as their morphological similarity and geographical proximity to K. osellai suggest they are more closely related to the latter than to K. calarensis. All three aforementioned species (osellai, ruffoi, pseudorosea) differ from Kritodrilus by having more or less intensive red pigmentation, and are temporally placed back to their original genus Dendrobaena. However, our results suggest that they represent a different, as yet undescribed genus.GenusVindoboscolex gen. nov. Marchán 2021 LSID urn:lsid:zoobank.org:act:8550AECF-648C-4AA6-9B17-1D241259308A Type species: Eophila hrabei Cernosvitov, 1935 Species included: Vindoboscolex hrabei comb. nov. (Cernosvitov, 1935) Vindoboscolex mrazeki comb. nov. (Cernosvitov, 1935) Diagnosis: Medium to large-sized (80-300 mm) and thin (4-6 mm) Lumbricidae, with up to 615 segments with more or less intensive porphyrin based red pigmentation. Prostomium pro-epilobous. Setae closely paired or separate. First dorsal pore in segments 6/7-7/8. Male pores in segments ½ 15 with well-developed porophore. Spermathecal pores simple, in 9/10 and 10/11. Clitellum between segments 29 and 60. Tubercula pubertatis between segments 37 and 54. Calciferous glands in segments 11-14 with lateral diverticula in 11. Typhlosole large, multilobate. Two pairs of small, globular spermathecae opening in 9/10and 10/11. Two or three pairs of seminal vesicles in segments (9), 11 and 12. Nephridial vesicles after clitellum bilobous. Cross section of longitudinal musculature pinnate.

Figure 1 .

 1 Figure 1. Detail of the Bayesian inference of the phylogenetic relationships between the Kritodrilus species under study, Allolobophora hrabei and their closest relatives (the whole phylogenetic tree displaying the relationships with representatives of Lumbricidae is shown in Suppl. Material 4). The clades highlighted in colour correspond to the groups used for the Ecological Niche Modelling analysis: Mediterranean (red), Vindobonic (blue) and Northwestern Iberian (green).

Figure 2 .

 2 Figure 2. Ancestral area reconstruction and estimated times of divergence of Lumbricidae genus-level clades. The target taxa (Kritodrilus, Vindobonic species and Northwestern Iberian species) are shown in bold. Important nodes referred to in the main text are indicated by the letters A-G. Only the most likely reconstruction for each node is shown for key nodes. Inferred dispersal or vicariance events are shown by blue and green rings respectively. Vertical grey stripes indicate the approximate geological ages corresponding to the paleogeographical settings of the Western Mediterranean shown to the right (modified from Ziegler and Dèzes 2007; Vacherat et al. 2017). PETM: Paleocene-Eocene Thermal Maximum. *Carpato-Balkanic clade: includes representatives of Cernosvitovia, Serbiona and Karpatodinariona. ** Representatives of Dendrobaena were split into two different clades.

Figure 3 .

 3 Figure 3. Ecological Niche Models for the "Mediterranean" clade (comprising Kritodrilus sensu stricto species) and its sister clades, "Northwestern Iberian" (Galiciandrilus and Compostelandrilus species) and "Vindobonic" (hrabei and mrazeki). Only high suitability values (over 0.5) are shown. Dots, squares and triangles depict the presence localities used as input for the models.

Table 1

 1

Table 2

 2

  Hungarian Museum of Natural History. 28S: 28S rRNA; 16S: 16S rRNA; tRNAs: tRNAs Ala, Leu and Ser; 12S: 12S rRNA; ND1: NADH dehydrogenase subunit 1 gene; COI; cytochrome c oxidase subunit 1 gene; COII: cytochrome c oxidase subunit 2 gene.

	Vouchers	Analysis (N) COI	COII	16S	tRNAs	12S	ND1	28S
	Kritodrilus calarensis	M (6)	MZ300979	-	MZ299011 MZ299143 -	MZ299149 MZ299021
	UCM-LT 70051	DNA (1)
	Kritodrilus micrurus	M (9)	MZ300980	-	MZ299012 MZ299144 -	MZ299150 MZ299022
	UCM-LT 70052	DNA (1)
	Ethnodrilus setusmontanus	M (1)	MZ300978	-	MZ299010 MZ299142 -	MZ299148 MZ299020
	UCM-LT 70001	DNA (1)
	Allolobophora hrabei	M (3)	MZ300976	MZ299138 MZ299008 MZ299140 MZ299014 MZ299146 MZ299018
	HMNH-12621, 16316/1, 16478	DNA (1)
	Kritodrilus mrazeki	M (2)	MZ300977	-	MZ299009 MZ299141 MZ299015 MZ299147 MZ299019
	UCM-LT 70053	DNA (1)
	Kritodrilus osellai	M (1)	MZ300981	MZ299139 MZ299013 MZ299145 MZ299016 MZ299151 MZ299023
	UCM-LT 70054	DNA (1)

Table 2 .

 2 Localities of the newly sampled and species ranges used for the Ancestral Area Reconstruction analysis. A: Iberian Peninsula; B: France; D: Central Europe (including Veneto).

	Vouchers	Geographic origin	Range Latitude	Longitude
	Kritodrilus calarensis	Malataverne, Drôme, Rhône-Alps, France	B	44.496	4.73943333
	UCM-LT 70051
	Kritodrilus micrurus	Buisson, Drôme, Rhône-Alps, France	B	44.29884	4.98591
	UCM-LT 70052
	Ethnodrilus setusmontanus	Sète, Occitaine, Herault, France	B	43.396677 3.693158
	UCM-LT 70001
	Allolobophora hrabei	Várbalog, Hungary	D	47.838056 17.055556
	HMNH-12621, 16316/1,
	16478
	Kritodrilus mrazeki	Podyjí National Park, Czech Republic	D	48.877472 15.837667
	UCM-LT 70053
	Kritodrilus osellai	Villalba de los Morales, Teruel, Aragón,	A	40.851678 -1.392155
	UCM-LT 70054	Spain

Table 3 .

 3 Amplified gene fragments (DNA), primer sequences, annealing temperature (AT) and amplicon length in base pairs (AL). List of species included in the reference dataset, species ranges used for the Ancestral Area Reconstruction analysis and reference to the original publication which provided

	28S: 28S rRNA; 16S: 16S

Acknowledgements

This work was supported by a grant from the Systematic Research Fund, 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.

DFM was funded by a Juan de La Cierva-Formación grant (FJCI-2017-32895) from the Spanish Ministry of Sciences, Innovation and Universities.

Data Availability Statement

The data that support the findings of this study are openly available in GenBank at https://www.ncbi.nlm.nih.gov/genbank/, accession numbers MZ300976-MZ300981, MZ299008-MZ299023, MZ299138-MZ299139 and MZ299146-MZ299151.

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