Barcoding Eophila crodabepis sp. nov. (Annelida, Oligochaeta, Lumbricidae), a Large Stripy Earthworm from Alpine Foothills of Northeastern Italy Similar to Eophila tellinii (Rosa, 1888)

A new Italian earthworm morphologically close to the similarly large and anecic Eophila tellinii (Rosa, 1888) is described. Distribution of Eophila crodabepis sp. nov. extends over 750 km2 from East to West on the Asiago Plateau and Vittorio Veneto Hills, from North to South on mounts Belluno Prealps (Praderadego and Cesen), Asiago, Grappa and onto the Montello foothills. This range abuts that of Eophila tellinii in northern Friuli Venezia Giulia region. Known localities of both E. tellinii and E.crodabepis sp. nov. are mapped. mtDNA barcoding definitively separates the new western species from classical Eophila tellinii (Rosa, 1888).


Introduction
Study of megadrile earthworms is easily justified due to their key ecological role as drivers of soil formation in association with microorganisms (especially bacteria and fungi) [1,2,3,4].
In 1888 Daniele Rosa described Allolobophora tellinii (now Eophila tellinii) the largest Italian earthworm (up to 800 mm according to Paoletti [5,6]), characterized by a livery of puce and purple bands in the middle of each segment. It was included in the "Classical" taxonomic systems of Michaelsen [7] and Stephenson [8] (under genus Helodrilus Hoffmeister, 1845). Its ecological category is anecic or deep-burrowing (cf. [2,5,9,10,11] with vertical burrows going meters deep although feeding is mostly on decaying litter on the soil surface, especially at night or during rain. E. tellinii is often located at the base or under large rocks or in the roots of trees with a marked preference for hazel (Corylus avellana, L.) and decidous forest [12]. Such microhabitats ensure a greater protection from predators and sudden changes of temperature and humidity [3].
Despite their ecological importance, knowledge of earthworm taxonomy and ecology is remarkably limited in Italy as elsewhere and the specific roles of earthworms in soil formation in rural environments-especially in vineyards but in forests as well-is largely underestimated. The large and charismatically coloured E. tellinii exemplifies this: in the current study "E. tellinii" is found to actually comprise two taxa separable on morphology as well as on genetics and distribution pattern.

Morphology
Earthworms were collected at different stations both by spade-fork digging and expulsion using 0.2-0.5% formaldehyde [16] or mustard powder (25 g/l) [17]. Specimens were preserved in 80% ethanol then stored at +4°C and most are kept in the Biology Department of the University of Padua, Via Ugo Bassi 58b, 35121 Padova (Italy) although some were transferred to other institutions as noted under species' description. Three earthworms (Crevada 6, Clauzetto 2, Ragogna 2) are deposited in the Department of Zoology of the University of Granada (Spain) and six (Grappa Mount 2, Ragogna 3, Ragogna 1, Val Posan 2, HNHM 6899, HNHM 12678) in the Hungarian Natural History Museum, Budapest. The specimen called "Campo Solagna 17" was subjected DNA-barcoding and not kept. Two specimens (Ragogna 1, Grappa Mount 2) were subjected to anatomic dissection to observe the internal features. A specimen (HNHM 6899) was bisected and the middle part sectioned to observe musculature. Three specimens (Crevada 6, Clauzetto 2 and Ragogna 2) were sent to Professor Javier Alba-Tercedor of the University of Granada for micro-tomographic scanning with a Bruker-Skyscan 1172. This technique examined features of specimens without dissection, in particular the intestinal typhlosole shape and extent.
LOMBRI software [18] was used for identification confirmed by scientific literature (listed in synonymy) using family and species systematics of Blakemore [19,20].

Ethics Statement
The earthworm samples were collected in public areas in the provinces of Udine, Treviso and Vicenza on forested areas with no special requirements needed for collection permits. No endangered or protected species were involved.

DNA barcoding
Sampling. Nine specimens of E. tellinii and 25 of E. crodabepis sp. nov. were sequenced. In order to have a comparison point for specific divergence, 20 specimens of Perelia gestroi (Cognetti 1905) were also sequenced (Fig 1). Uncertainty of latter taxon name and position detailed in Blakemore [20].
DNA was extracted from one mm 3 of muscle taken from the 'tail' of each specimen and preserved in 98% ethanol. The extraction took place following the standard Canadian Center for DNA Barcoding (CCDB) automated protocol [22] using 96-well glass fibre plates [23]. Amplification used M13 tailed primers (C_LepFolF/C_LepFolR) and followed standard CCDB protocol for PCR reactions [24] with end products checked on a 2% E-gel 96Agarose (Invitrogen). Unpurified PCR amplicons were sequenced in both directions using M13 tailed primers, their products subsequently purified using Agencourt CleanSEQ protocol and processed using Big-Dye version 3.1 on an ABI 3730 DNA Analyzer (Applied Biosystems). Sequences were assembled and edited with Sequencher 4.5 (GeneCode Corporation, Ann Arbor, MI, USA). Alignments used BIOEDIT version 7.0.5.3 [25]. Sequences are publicly available on GenBank (KT352925-KT352978) and on BOLD in the dataset [DS-NEO1] through the following DOI: dx.doi.org/10.5883/DS-NEO1.
Data analysis. Distance analyses were performed with MEGA6 [26], using a Neighbor-Joining [27] algorithm with the Kimura-2 parameter model [28] to estimate genetic distances. The robustness of nodes was evaluated through bootstrap re-analysis of 1000 pseudoreplicates. Molecular Operational Taxonomic Units (MOTUs) were defined with the software 'mothur' [29].

Nomenclatural Acts
The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature, and hence the new names contained herein are available under that Code from the electronic edition of this article. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix "http://zoobank.org/". The LSID for this publication is: Eophila crodabepis Paoletti sp. nov. urn:lsid:zoobank.org:pub:53662919-7E2D-4DC6-BB89-C60D2FC6C193 The electronic edition of this work was published in a journal with an ISSN, and has been archived and is available from the following digital repositories: PubMed Central.
Newly collected specimens are from several locations in Friuli Venezia Giulia (S1 Table). Other data in the literature originate from specimens deposited in the Museo Civico di Zoologia, Roma (Italy) [14] some of which are now missing (S2 Table).
In the laboratory, E. tellinii can live under water for at least 3-4 weeks, this possibly linked to its particular hemoglobin [39]. Sometimes under very wet field conditions, E. tellinii and E. crodabepis sp. nov. have been found moving on the soil surface, earning them a local name of "vier de la pluje" or worm of the rain in Carnia region of Friuli Venezia Giulia.
Our new specimens comply within acceptable limits of the original description of E. tellinii except in the number of seminal vesicles which was stated as two pairs in segments 11 & 12 by Rosa [30] compared to four pairs found in 9-12. However, in our specimens the first two pairs in 9 & 10 are small and easily overlooked which might be the reason why Rosa missed them.
Analysing the sequences produced for this paper along with those of the other species of Lumbricidae in the previous publication Porco et al. 2013 [40], we were able to recover three MOTUs corresponding to P. gestroi, E. tellinii and the new species E. crodabepis sp. nov. using a 11% threshold value (data not shown). The mean intraspecific divergence found in these three species (P. gestroi 4.79% (ranging from 0% to 8.19%), E. tellinii 1.25% (ranging from 0% to 4.09%), E. crodabepis sp. nov. 4.55% (ranging from 0% to 7.43%), contrasted with a high interspecific mean divergence reaching 18.64% (range 13.86% to 21.97%- Fig 1) confirming the existence of a clear barcode gap for the dataset (Table 3). These ranges of genetic divergence are consistent or exceed those measured among species in Lumbricidae in previous studies [40,41] further confirming the separate specific status of the two taxa concerned.

Ecological observations
• Some specimens have been victims of predation and one of the causes is the leech collected in Val Posan and Roncavezzai: Haemopis sanguisuga were found at the collection places.
• The fact that Octodrilus complanatus and O. pseudocomplanatus specimens were collected together with Eophila tellinii (S1 Table) is interesting as they are both classed as deep-burrowing species.
• The new species is defined on differences both in morpho-genetic characters as well as in its geographic range [42].
• Differences in DNA are used to separate the earthworm species based on their primary types and topotypes as initially advocated in Blakemore et al. [43] allowing full species characterization as in Blakemore [44].

Conclusions
Eophila crodabepis sp. nov. is clearly distinguished from Eophila tellinii both for diagnostic morphological characters (Figs 5 and 7) and for the definitive genetic data (Fig 1) plus their geographical distributions (Figs 2 and 6). Its superficial affinity with Eophila tellinii, especially in macro-morphology (bands brown-purple), led to initial misidentification in the field as Eophila tellinii [5,12,13]. Further ecological assessment is now possible on the objectively differentiated taxa.  Table 4. T-test of the setal ratios of the two groups.