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

Authors:
RESEARCH ARTICLE
Barcoding Eophila crodabepis sp. nov.
(Annelida, Oligochaeta, Lumbricidae), a Large
Stripy Earthworm from Alpine Foothills of
Northeastern Italy Similar to Eophila tellinii
(Rosa, 1888)
Maurizio G. Paoletti
1
*, Robert J. Blakemore
2
, Csaba Csuzdi
3
, Luca Dorigo
4
, Angelo
Leandro Dreon
1
, Federico Gavinelli
1
, Francesca Lazzarini
1
, Nicola Manno
1
, Enzo Moretto
5
,
David Porco
6
, Enrico Ruzzier
7
, Vladimiro Toniello
8
, Andrea Squartini
9
,
Giuseppe Concheri
9
, Marina Zanardo
9
, Javier Alba-Tercedor
10
1Universitàdegli Studi di Padova, Dipartimento di Biologia, Padova, Italia, 2Hanyang University, College of
Natural Science, Seoul, Korea, 3Eszterházy Károly College, Department of Zoology, Eger, Hungary,
4Museo Friulano di Storia Naturale, Udine, Italia, 5ESAPOLIS, Padova, Italia, 6Université de Rouen,
Laboratoire ECODIV, Mont Saint Aignan Cedex, France, 7Natural History Museum, Department of Life
Science, London, United Kingdom, 8Federazione Speleologica Veneta-G.S. C.A.I., Laboratorio di
Biospeleologia di Villa Papadopoli, Vittorio Veneto, Italia, 9Universitàdegli Studi di Padova, Dipartimento di
Agronomia Animali Alimenti Risorse Naturali e Ambiente, DAFNAE, Padova, Italia, 10 Universidad de
Granada, Departamento de Zoología, Facultad de Ciencias, Granada, España
*paoletti@bio.unipd.it
Abstract
A new Italian earthworm morphologically close to the similarly large and anecic Eophila telli-
nii (Rosa, 1888) is described. Distribution of Eophila crodabepis sp. nov. extends over 750
km
2
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 Mon-
tello 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 Ital-
ian 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 Classicaltaxonomic
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
PLOS ONE | DOI:10.1371/journal.pone.0151799 March 28, 2016 1/15
a11111
OPEN ACCESS
Citation: Paoletti MG, Blakemore RJ, Csuzdi C,
Dorigo L, Dreon AL, Gavinelli F, et al. (2016)
Barcoding Eophila crodabepis sp. nov. (Annelida,
Oligochaeta, Lumbricidae), a Large Stripy Earthworm
from Alpine Foothills of Northeastern Italy Similar to
Eophila tellinii (Rosa, 1888). PLoS ONE 11(3):
e0151799. doi:10.1371/journal.pone.0151799
Editor: Peter Prentis, Queensland University of
Technology, AUSTRALIA
Received: December 22, 2015
Accepted: February 17, 2016
Published: March 28, 2016
Copyright: © 2016 Paoletti et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Data Availability Statement: Relevant data are
within the paper and its Supporting Information files.
All sequences codes are available from GenBank
(KT352925-KT352978) and on BOLD in the dataset
[DS-NEO1] through the following DOI: dx.doi.org/10.
5883/DS-NEO1.
Funding: The authors received no specific funding
for this work.
Competing Interests: The authors have declared
that no competing interests exist.
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 micro-
habitats ensure a greater protection from predators and sudden changes of temperature and
humidity [3].
E.tellinii localities are characterized by mull calcareous grey soils, sometimes stony and
with rock outcrops; the species has an altitudinal range between 1001,200 m. This species col-
onize the Southern Prealpine slopes and hills covered by deciduous woodlands [5,13,14,15].
Eophila predators include the badger Meles meles (Linnaeus, 1758), carabid beetles Abax paral-
lelepipedus (Piller & Mitterpacher, 1783) and Carabus (Procerus)gigas (Creutzer, 1799), Eupo-
lybothrus grossipes (C.L. Koch, 1847), Xerobdella sp. (von Frauenfeld, 1868) that share the same
range [5].
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 environmentsespecially in vineyards but in forests as wellis largely underesti-
mated. The large and charismatically coloured E.tellinii exemplifies this: in the current study
E.telliniiis found to actually comprise two taxa separable on morphology as well as on genet-
ics and distribution pattern.
Materials and Methods
Morphology
Earthworms were collected at different stations both by spade-fork digging and expulsion
using 0.20.5% 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 Uni-
versity of Padua, Via Ugo Bassi 58b, 35121 Padova (Italy) although some were transferred to
other institutions as noted under speciesdescription. 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 Uni-
versity of Granada for micro-tomographic scanning with a Bruker-Skyscan 1172. This tech-
nique 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
Eophila crodabepis sp. nov. from Northeastern Italy
PLOS ONE | DOI:10.1371/journal.pone.0151799 March 28, 2016 2/15
(Cognetti 1905) were also sequenced (Fig 1). Uncertainty of latter taxon name and position
detailed in Blakemore [20].
Sequencing. Specimens were sequenced for mtDNA-barcoding region (658bp of the mito-
chondrial cytochrome oxidase subunit I 5end [21]).
DNA was extracted from one mm
3
of muscle taken from the tailof each specimen and pre-
served 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]. Ampli-
fication used M13 tailed primers (C_LepFolF/C_LepFolR) and followed standard CCDB pro-
tocol 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 assem-
bled 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 Interna-
tional 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.
Results
Taxonomy
Eophila tellinii (Rosa, 1888).
(S1 and S2 Figs)
Allolobophora tellinii Rosa, 1888: 1. Type locality northeast of Italy, Ragogna hills in the
province of Udine (Friuli Venezia Giulia). Syntypes in O1579 ITALIA, Friuli, Ragogna (UD)
Torino (Turin) Regional Museum of Natural Science (not examined) [30].
Allolobophora (Eophila)tellinii:[31]: 10; [32]: 93.
Helodrilus (Helodrilus) tellinii [7]: 500.
Eophila tellinii:[8,14,33,34,35,36]: 73, figs 1and 2(misdated Rosa, 1894); [37,38]: 481
(misdated Rosa, 1886); [11].
Eophila crodabepis sp. nov. from Northeastern Italy
PLOS ONE | DOI:10.1371/journal.pone.0151799 March 28, 2016 3/15
Fig 1. Cluster of the sequences from E.tellinii and E.crodabepis sp. nov sampled in the studied area with Perelia gestroi shown for comparison.
doi:10.1371/journal.pone.0151799.g001
Eophila crodabepis sp. nov. from Northeastern Italy
PLOS ONE | DOI:10.1371/journal.pone.0151799 March 28, 2016 4/15
Rosa [30] said: Questo lombrico,di grandissime dimensioni,me è noto per un solo esemplare
raccolto del signor Achille Tellini laureando in scienze naturali a Ragogna nel Friuli (alt.220 m)
sul finire dello scorso aprile, i.e., he had a single, large specimen collected in April from
Ragogna nel Friuli (= Ragogna) at 220 m of altitude.
Newly collected specimens are from several locations in Friuli Venezia Giulia (S1 Table).
Fig 2 shows geographical distribution of the samples integrated with their barcode clusters.
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).
Fixed adult lengths 170360 mm (syntype 300 mm). First dorsal pore 5/6. Segments 250
341 in adult specimens (syntype 264) and from 193275 in the immature specimens (including
specimens which have probably been victims of predation). Color dark puce with purple bands
in the middle of each segment; sometimes bands are less visible on the terminal portion of the
body or pigment is lacking; intersegments are always pale. Adult specimens weighed 11.428.2
g. Maximum width at clitellum 1014 mm. Clitellar height 911 mm. Length of living
Fig 2. Barcoding Cluster of E.crodabepis sp. nov. (in yellow) in relationship with E.tellinii (in red) corresponding to their geographical
distributions.
doi:10.1371/journal.pone.0151799.g002
Eophila crodabepis sp. nov. from Northeastern Italy
PLOS ONE | DOI:10.1371/journal.pone.0151799 March 28, 2016 5/15
specimens can be 600800 mm. Prostomium epilobous. Setae closely paired. Clitellum
26,27,2840,41,42,44 involving fourteen to eighteen or nineteen segments. Tubercula puberta-
tis 30,31,3237,38,39,40 (syntype 3237). Spermathecal pores paired in 9/10 & 10/11 in cd.
Female pores on segment 14 above setae b. Male pores on segment 15 between setae band c,
with or without small sized tumescences confined to segment 15. Setal papillae in adults on
some segments of 613, 712, 713, 811 or 812. Body shape cylindrical depressed caudally.
Setal ratio on segment 12 in adult specimens: aa:511; ab:1;bc:46; cd: 0.60.9; dd: 26.742;
U(circumference): 3354; mean: aa: 8.9; ab:1;bc:5;cd: 0.8; dd: 33.7; U: 42.7 (S3 Table and
Table 1). Nephridial pores irregularly alternate between setal lines band well above d.
Septa 5/67/8, 12/1314/15 thickened, 8/911/12 strongly strengthened. Excretory system
holoic, first pair of nephridia in 4. Nephridial bladders from 6 proclinate J-shaped, after the cli-
tellum almost U-shaped. Hearts 611 with a pair of extraoesophageal vessels in 12. Calciferous
glands in 1012 with large vertical diverticula in 10. Crop large in 1516 and muscular gizzard
in 1719. Typhlosole large, trifid, begins around segments 2345 and ends variably before
pygidium. Testes and male funnels in 10 & 11 seemingly free. Vesicles four pairs in 912, the
first two pairs quite small and easy to overlook, those in 11 & 12 large. Spermathecae globular
in 10 & 11. Ovaries moderate, pear-shaped in 13, ovaric sac moderate in 14 pendant from sep-
tum 13/14. (S3 Table).
Up to a dozen E.tellinii specimens per square meter were collected from Friuli Venezia Giu-
lia, found either alone or with other species of earthworms (S1 Table). Associates were deep-
burrowing Octodrilus complanatus (Dugés, 1828) and Octodrilus pseudocomplanatus (Omo-
deo, 1962) and other species belonging to different ecological categories (i.e., litter species, top-
soil and/or subsoil species) such as Octodrilus lissaensis (Michaelsen, 1891), Octodriloides
phaenohemiandrus (Zicsi, 1971) and Octolasion lacteum (Örley, 1881).
In the laboratory, E.tellinii can live under water for at least 34 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 plujeor 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 912. 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.
Eophila crodabepis Paoletti, 2016 sp. nov.
Eophila crodabepis Paoletti sp. nov.
urn:lsid:zoobank.org:pub:53662919-7E2D-4DC6-BB89-C60D2FC6C193
Table 1. Setal ratio of Eophila tellinii on 12th segment.
SAMPLE aa ab bc cd dd U
Clauzetto 21 9.5 1 5.5 0.8 42.0 38.0
Ciaurlec Mount 8.0 1 5.0 0.8 38.0 35.0
Ragogna 1 11.0 1 5.3 0.7 26.7 51.7
Ragogna 3 11.0 1 5.4 0.6 29.0 54.0
Ragogna 4 5.0 1 6.0 0.9 36.0 33.0
Travesio 22 10.0 1 4.0 0.8 34.0 42.0
Travesio 23 8.0 1 4.0 0.7 30.0 45.0
MEAN 8.9 1 5.0 0.8 33.7 42.7
doi:10.1371/journal.pone.0151799.t001
Eophila crodabepis sp. nov. from Northeastern Italy
PLOS ONE | DOI:10.1371/journal.pone.0151799 March 28, 2016 6/15
Figs 3and 4;S3 Fig.
Specimens studied were collected in different localities in Veneto region (Table 1)
Holotype: Crevada 3 [BOLD sampleID = IT_MGP_Crevada_3,
Genbank accession = KT352951], in the Verona Natural History Museum.
Paratypes: Crevada 1 (P1), Crevada 2 (P2), Crevada 4 (P3) University of Padua, HNHM
6899 (P4), HNHM 12678 (P5) deposited in the Hungarian Natural History Museum, Budapest.
The name of the new species is taken from an acronym dedicated to Giovanni Canestrini,
Daniele Rosa, Charles Darwin, Antonio Berlese, Pietro Omodeo and Filippo Silvestri, for vari-
ous reasons relating to their scientific contributions to soil biology and earthworm studies in
Italy and further abroad. The new taxon may be cited as Eophila crodabepis Paoletti, 2016 in
Paoletti et al. 2016.
Fig 3. Eophila crodabepis sp. nov. specimen found upon the litter layer.
doi:10.1371/journal.pone.0151799.g003
Fig 4. Holotype of Eophila crodabepis sp. nov. (Crevada 3 specimen).
doi:10.1371/journal.pone.0151799.g004
Eophila crodabepis sp. nov. from Northeastern Italy
PLOS ONE | DOI:10.1371/journal.pone.0151799 March 28, 2016 7/15
Body cylindrical, caudally depressed. Adult length (fixed): 100240 mm by 10 mm diameter
(holotype 130 mm, P1 120 mm, P2 110 mm, P3 140 mm, P5 240). Adult living specimens
length can be 400600 mm.
Colour: Purplish-brown bands in the middle of each segment with puce intersegments;
bands are less visible or absent caudally in some specimens, or pigment is lacking from poste-
rior half of ventral side or form ventral and lateral sides or entirely lacking ventrally; it is always
lacking along setal lines cd. Adult specimens weigh 2.5812.5 g (holotype 5.07 g, P1 2.64 g, P2
2.58 g, P3 4.927 g). Fixed specimens diameter at the clitellum: from 612 mm (holotype 10
mm, P1 and P2 8 mm, P3 9 mm, P5 11 mm). Fixed specimens height at the clitellum 610 mm
(holotype 9 mm, P1 6 mm, P2 6 mm, P3 8 mm). Total segments 139260 in adult specimens
and 132267 in immatures (including probable predation amputees) (holotype 209, P1 219, P2
220, P3 191, P5 260). First dorsal pore in 5/6. Prostomium epilobous. Setae closely paired. Setal
papillae were not recognized (in holotype, P1, P2, P3) or setal papillae ab on segments 812 or
813 (P5). Clitellum on 24,25,2636,37,38 (2537 holotype and P1, 2437 P2, 2637 P3, 2637
P4, 2638 P5), involving twelve to fourteen segments. Tubercula pubertatis on 29,3036 (holo-
type, P1, P2, P3 3036, P5 ½2936). Spermatheca pores paired in 9/10 & 10/11 in cd. Female
pores on 14 above setae b. Male pores on 15 between setae band c, confined to the segment
and with (P5) or without (holotype, P1, P2, P3) small tumescences. Setal ratio at segment 12, in
adult specimens aa: 6.4; ab:1;bc:4;cd: 0.8; dd: 20.8; U: 29.8 after clitellum aa:ab:bc:cd:dd:
U= 5.5: 1: 2.2: 0.7: 18.8: 25 (S3 Table and Table 2).
Septa 5/67/8, 12/1314/15 thickened, 8/911/12 strongly strengthened. Excretory system
holoic, first pair of nephridia in 4. Nephridial bladders from 6 proclinate J-shaped; after the cli-
tellum almost U-shaped. Hearts 611 with a pair of extraoesophageal vessels in 12. Calciferous
Table 2. Setal ratio of Eophila crodabepis sp. nov. specimens and its mean on 12th segment.
SAMPLE aa ab bc cd dd U
Crevada 1 5.2 1 3.2 0.7 21.0 26.0
Crevada 2 5.0 1 4.2 0.8 20.0 23.0
Crevada 3 5.5 1 2.2 0.7 18.8 25.0
Crevada 4 6.0 1 6.0 0.8 20.0 22.0
Crevada 5 5.2 1 3.5 0.8 20.0 32.0
Fratte 50 6.0 1 3.5 0.8 19.0 28.0
Grappa Mount 2 6.8 1 4.2 0.6 20.0 38.4
Praderadego 24 7.0 1 4.0 0.8 21.0 28.0
Praderadego 25 6.0 1 3.5 0.7 21.0 31.0
Roncavezzai 11 7.0 1 5.0 0.8 27.0 28.0
Roncavezzai 18 4.3 1 3.2 0.8 19.5 23.0
Roncavezzai 2 7.0 1 4.0 0.9 19.0 25.0
Roncavezzai 5 6.0 1 3.7 0.8 25.0 27.0
Val Posan 1 7.0 1 4.0 0.9 23.0 27.0
Val Posan 2 6.8 1 4.7 0.5 18.7 37.9
Val Posan 3 7.0 1 3.0 0.5 18.0 37.0
Val Posan 4 8.0 1 5.0 0.9 23.0 38.0
Val Posan 5 7.0 1 4.0 0.8 19.5 30.0
Ekar 5 8.0 1 4.5 0.7 22.5 40.0
HNHM 12678 8.1 1 4.5 0.6 20.0 40.3
MEAN 6.4 1 4.0 0.7 20.8 30.3
doi:10.1371/journal.pone.0151799.t002
Eophila crodabepis sp. nov. from Northeastern Italy
PLOS ONE | DOI:10.1371/journal.pone.0151799 March 28, 2016 8/15
glands in 1012 with large vertical diverticula in 10. Crop large in 1516 and muscular gizzard
in 1719. Typhlosole large, trifid. Testes and male funnels in 10 & 11 seemingly free. Vesicles
four pairs in 912, the first two pairs are quite small, easy to overlook, those in 11 & 12 large.
Spermathecae globular in 10 & 11. Ovaries moderate, pear-shaped in 13, ovarial sac moderate
in 14, pendant from septum 13/14. Typhlosole trilobed in Crevada specimen (Crevada 6) com-
mencing in 45 and terminating nine segment from pygidium (S3 Table) or begins around seg-
ment 23 and ends in segment 204 in the Grappa Mount 2 specimen. Cross section of
longitudinal musculature pinnate (S3 Fig).
Eophila crodabepis sp. nov. specimens collected in Treviso and Vicenza provinces were
found alone, or in association with other species (S1 Table). It was collected in association with
deep-burrowing species Octodrilus complanatus and O.pseudocomplanatus and with species
belonging to other ecological categories, viz, Aporrectodea sineporis (Omodeo, 1952), Octodri-
lus lissaensis,Eisenia spelaea (Rosa, 1901) [12], Aporrectodea caliginosa and Lumbricus
rubellus.
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.
Comments
The new species differs from E.tellinii in its smaller mean size (100230 vs. 170360 mm),
lower number of segments (139252 vs. 250341) (Fig 5), lesser weight (2.612.5 vs. 11.428.2
g) and different locations of clitellum and tubercula pubertatis. In E.crodabepis sp. nov. the cli-
tellum covers segments 24,25,2636,37,38 and tubercles of puberty are on segments 29,3036
instead of 26,2741 and 3237 as in the syntype of E.tellinii (Rosa, 1888) (Fig 6). In other E.
tellinii specimens, the clitella are located on segments 2740, 2741, 2640, 2742, 2744 and
2644 (S3 Table), which are within range of the position described by Rosa as on segments 27
41. The same applies to the tubercula pubertatis found on segments ½29,29,3036 in E.croda-
bepis sp. nov., instead of 30,31,3237,38,39,40 in E.tellinii (S3 Table). Also, the average weight,
length and number of segments of Eophila crodabepis sp. nov. is lower compared with (E.telli-
nii) specimens collected in Friuli Venezia Giulia (S3 Table). Many other features appear the
Table 3. Intra-interspecific divergence between Eophila crodabepis sp. nov. and Eophila tellinii using Perelia gestroi as outgroup (note threshold
value of 13.86%).
Species Intraspecic Interspecic
E.crodabepis E.tellinii P.gestroi
Eophila crodabepis sp. nov. 4.55
Eophila tellinii 1.25 13.86
Perelia gestroi 4.79 20.10 21.97
doi:10.1371/journal.pone.0151799.t003
Eophila crodabepis sp. nov. from Northeastern Italy
PLOS ONE | DOI:10.1371/journal.pone.0151799 March 28, 2016 9/15
same between the two species, such as the coloration, the shape of the prostomium, the posi-
tion of the first dorsal pore, and the body shape (S3 Table).
NMDS analysis for the setal ratios of the of E.tellinii and E.crodabepis sp. nov. show the
two groups are evidently different (Fig 7). T- test (Tables 1and 2) evaluate the NMDS analysis
and, except for cd, all setal ratios are significantly different for p<0.05 or p<0.001 (Table 4).
From these analyses it is possible to establish feature differences between E.tellinii (which is
present in Friuli Venezia Giulia) and the other population living in different locations in
Veneto. Previously, all the specimens were field identified as E.tellinii based on typical coloura-
tion however, clitella and tubercula pubertatis location as well as the biometry are now proven
to be different.
Discussion
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.
Fig 5. Adult specimens total segment counts. Data of the adult specimens which were probably damaged by predation (Roncavezzai 11,Val Posan 2,Val
Posan 5) were not included.
doi:10.1371/journal.pone.0151799.g005
Eophila crodabepis sp. nov. from Northeastern Italy
PLOS ONE | DOI:10.1371/journal.pone.0151799 March 28, 2016 10 / 15
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-bur-
rowing 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 characteri-
zation as in Blakemore [44].
Conclusions
Eophila crodabepis sp. nov. is clearly distinguished from Eophila tellinii both for diagnostic
morphological characters (Figs 5and 7) and for the definitive genetic data (Fig 1) plus their
geographical distributions (Figs 2and 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 dif-
ferentiated taxa.
Fig 6. Clitellum location of adult Eophila tellinii and Eophila crodabepis sp. nov. specimens with geographic distribution from East to West.
doi:10.1371/journal.pone.0151799.g006
Eophila crodabepis sp. nov. from Northeastern Italy
PLOS ONE | DOI:10.1371/journal.pone.0151799 March 28, 2016 11 / 15
Supporting Information
S1 Fig. Eophila tellinii morphological and anatomical details. Livery pattern in Travesio
specimen and peristomial detail (Ragogna 1 specimen). 7.98 μm and 4.35 μm ventral, dorsal
and lateral views of specimen (Ragogna 2); virtual sections of hindmost segments: Schematic
and transversal sections of last ten segments lacking typhlosole, hindmost body segment- (mid-
dle) mesial-middle section (upper right), and sagittal medial section (bottom) from Clauzetto
specimen.
(DOC)
Fig 7. NMDS scatter plot of the Setal ratio in E.tellini and E.crodabepis sp. nov. specimens. Splitting between E.tellinii group (broken line) and E.
crodabepis group (solid line) (Stress = 0.06956; First axis R
2
= 0.8664). The two groups have different setal ratios.
doi:10.1371/journal.pone.0151799.g007
Table 4. T-test of the setal ratios of the two groups.
T test aa ab bc cd dd U
p(value) *n.s. *n.s. *** **
Differences between two groups E.tellinii and E.crodabepis sp. nov. for P(value)<0.05 *. Setal ratio dd
signicant for P(value)<0.001***. cd ratio appears with no signicant differences (ab is used for standard
unit in each groups). P(value)<0.05 *; P(value) <0.01 **; P(value)<0.001***.
doi:10.1371/journal.pone.0151799.t004
Eophila crodabepis sp. nov. from Northeastern Italy
PLOS ONE | DOI:10.1371/journal.pone.0151799 March 28, 2016 12 / 15
S2 Fig. Eophila tellinii ecological details. Wood near Travesio (PN) in which Eophila tellini
was found along with its casts.
(DOC)
S3 Fig. Eophila crodabepis sp. nov. anatomical details. 7.98 μm on ventral, dorsal and lateral
views and 3.08μm from Crevada (Crevada 6). DataViewer´s virtual sections of hindmost seg-
ments: Schematic and transversal sections of last ten segments where no typhlosole occurs;
hindmost body segment- (middle) mesial-middle section (upper right), and sagittal medial sec-
tion (bottom). In the middle right is a transversal sections at level of the penultimate segment;
plus cross section of longitudinal pinnate musculature (HNHM 6899 specimen).
(DOC)
S1 Table. Geographical and ecological information for each specimen analyzed.
(DOC)
S2 Table. Morphological and anatomical features from the literature.
(DOC)
S3 Table. Morphology and anatomy of specimens inspected and from the literature.
(DOC)
Acknowledgments
We want to thank PhD candidates Silvia Fusaro, Valentina Braido and Giulio Nicolussi for
assistance in this research.
Author Contributions
Conceived and designed the experiments: MGP. Performed the experiments: FL FG ER JAT.
Analyzed the data: RJB CC LD VT ALD FG FL. Contributed reagents/materials/analysis tools:
FG FL AS GC MZ NM RJB CC. Wrote the paper: MGP FL FG RJB CC DP. Taxonomy and bar-
coding: RJB CC DP JAT. Field collections: LD VT ALD EM ER. Micro-tomography: JAT. Pho-
tography: EM.
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