Untangling Lepidocyrtus (Collembola, Entomobryidae): New molecular data shed light on the relationships of the European groups

Abstract

European Lepidocyrtus species are usually grouped into five species-groups within two subgenera, Lepidocyrtus s.s. and Lanocyrtus, defined by the distribution of scale covering and dorsal head and body macrochaetotaxy. The discovery of several Lepidocyrtus populations with morphological characters intermediate between two species-groups suggested the need for the present study to test whether molecular data provide support to these groups. The morphology and gene sequences of 110 specimens belonging to 19 species of European Lepidocyrtus have been studied. Our molecular results are congruent with the distribution of the European species into the five groups established in the literature on the basis of morphological characters, but also indicated that subgenus Lanocyrtus is paraphyletic while subgenus Lepidocyrtus s.s. is monophyletic. A new species, Lepidocyrtus intermedius, is described, and a redefinition of European species-groups is proposed based on chaetotaxy.

Full text

Untangling Lepidocyrtus (Collembola, Entomobryidae):
new molecular data shed light on the relationships of
the European groups
Eduardo Mateos
A,D
, Paula Escuer
B
, Galina Bu¸smachiu
C
, Marta Riutort
B
and Marta Álvarez-Presas
B
A
Departament de Biologia Evolutiva, Ecologia i Ciències Ambientals, Facultat de Biologia,
Universitat de Barcelona, Avinguda Diagonal, 643, 08028 Barcelona, Spain.
B
Departament de Genètica, Microbiologia i Estadistica, Facultat de Biologia, Universitat de Barcelona, Spain.
C
Institute of Zoology, Academy of Sciences of Moldova, 2028 Chi¸sinau, Republic of Moldova.
D
Corresponding author. Email: emateos@ub.edu
Abstract. European Lepidocyrtus species are usually grouped into five species-groups within two subgenera, Lepidocyrtus
s.s. and Lanocyrtus,defined by the distribution of scale covering and dorsal head and body macrochaetotaxy. The discovery of
several Lepidocyrtus populations with morphological characters intermediate between two species-groups suggested the
need for the present study to test whether molecular data provide support to these groups. The morphology and gene
sequences of 110 specimens belonging to 19 species of European Lepidocyrtus have been studied. Our molecular results are
congruent with the distribution of the European species into the five groups established in the literature on the basis of
morphological characters, but also indicated that subgenus Lanocyrtus is paraphyletic while subgenus Lepidocyrtus s.s. is
monophyletic. A new species, Lepidocyrtus intermedius, is described, and a redefinition of European species-groups is
proposed based on chaetotaxy.
Additional keywords: chaetotaxy, COXII,EF-1a, cryptic species, DNA, evolution, phylogeny, taxonomy.
Received 22 June 2017, accepted 5 October 2017, published online 8 May 2018
Introduction
Lepidocyrtus Bourlet, 1839 is one of the genera with the highest
number of species within the Collembola; globally, Bellinger
et al. (1996–2017) list 225 species. Up to 10 subgenera have
been proposed by different authors (see Wang et al. 2003), but
because the main character used to define them (presence or
absence and morphology of the dental tubercle) does not seem to
be valid for all lineages, the taxonomic status of Lepidocyrtus
subgenera remains problematic (see Mateos and Greenslade
2015 for a discussion). Among the Entomobryidae, the genus
Lepidocyrtus is characterised by having the body covered with
rounded and finely denticulated scales, antennae with four
segments, 8 + 8 eyes, dentes without spines and with scales in
the anterior region, and bidentate mucro. The dorsal chaetotaxy
of head, thorax and abdomen represent the most informative and
most currently used character set in the species descriptions,
as a result of work pioneered by Szeptycki (1979). Currently,
the European fauna of Lepidocyrtus is represented by a total of
33 species belonging to the subgenera Lepidocyrtus s.s. Bourlet,
1839 (20 species) and Lanocyrtus Yoshii & Suhardjono,
1989 (13 species), which include a large number of species
worldwide (170 and 28 species, respectively sensu Bellinger
et al. 1996–2017). These two subgenera differ from the other
eight subgenera in the lack of dental tubercle in the furca (see
Wang et al. 2003). They differ because Lepidocyrtus s.s.
has scales on the antennae, legs and posterior region of the
manubrium, whereas Lanocyrtus does not. Winkler and Traser
(2012)described the species L. tomosvaryi from Hungary with
chaetotaxic characters of European subgenera, but also with a
small rounded dental tubercle as in other non-European
subgenera of Lepidocyrtus. The validity of the presence and
morphology of the dental tubercle to differentiate between
subgenera has been previously discussed by many authors
(see Winkler and Traser 2012), and its presence in a European
species adds confusion to this topic.
The taxonomic characters normally used at a specific level in
the genus Lepidocyrtus have been discussed by several authors
(Mari Mutt 1986; Snider 1967; Soto-Adames 2000; Mateos
2008a) and, independently of the subgenera, five groups have
been distinguished for European species: lusitanicus-group
(sensu Mateos 2008b), lignorum-group (sensu Mateos 2011),
lanuginosus-group (sensu Mateos 2012), curvicollis-group
(sensu Mateos and Petersen 2012) and pallidus–serbicus-group
(sensu Gisin 1965; Winkler and Traser 2012). Winkler (2017),
Journal compilation CSIRO 2018 www.publish.csiro.au/journals/is
CSIRO PUBLISHING
Invertebrate Systematics, 2018, 32, 639–651
https://doi.org/10.1071/IS17056

using dorsal cephalic macrochaetotaxy, proposed to separate
this last group into three additional groups, namely,
L. pallidus-group, L. serbicus-group and L. arrabonicus-
group. In the present work, we only analysed one species of
each of the three new groups proposed by Winkler (2017) and,
due to this lack of replication within groups, we considered
the L. pallidus–serbicus-group as originally described by
Gisin (1965) and Winkler and Traser (2012). The species
L. fimetarius Gisin, 1964 is not included in any of the groups
mentioned because of its unique body macrochetotaxy (see
Hüther 1971,1986; Szeptycki 1979; Wang et al.2003). The
discovery of several Lepidocyrtus populations in Catalonia
(Spain) with intermediate morphological characters between
the lignorum and curvicollis groups, initiated the present
study. The main objectives are to analyse the existence
of molecular support for species assignment to the
abovementioned groups, as well as to determine which are
the most suitable diagnostic morphological characters for the
identification of these groups. For this purpose, 19 species
of Lepidocyrtus belonging to the five currently recognised
European groups were studied using both molecular data and
morphological descriptions.
Materials and methods
Collection of specimens
In total, 110 specimens from 19 Lepidocyrtus species collected
from several European localities were studied using
morphological and molecular methods (Tables 1and S1).
Specimens were collected from the soil litter layer and from
underneath logs and stones using a micro-entomological
aspirator. Each sample consisted of aspiration from an area of
several square meters. Specimens captured were immediately
transferred to a 2-cc plastic vial, fixed in absolute alcohol
and labelled with a sample code. The studied species belong
to the currently recognised five European species-groups of
Lepidocyrtus, namely, the lusitanicus,lanuginosus,pallidus–
serbicus,lignorum and curvicollis groups. Unfortunately, it
was impossible to obtain DNA for the species Lepidocyrtus
fimetarius (as fresh specimens or those preserved in absolute
Table 1. Locality data of Lepidocyrtus species studied
Sample code Genus Species Municipality Province Country Position (WGS 84) Collecting date
LP325 Lepidocyrtus arrabonicus Morasti Morasti Romania N4513028.200 E2430006.800 01.iv.2013
LP239 Lepidocyrtus barbulus Kakopetros Creta Greece N3524029.200 E2345019.100 07.iv.2009
LP190 Lepidocyrtus bicoloris Cabrils Barcelona Spain N4132047.800 E221055.100 28.xi.2007
LP115 Lepidocyrtus bicoloris Montsant Tarragona Spain N4114013.200 E053003.500 17.ii.2007
BSa Lepidocyrtus bilobatus Sotoserrano Salamanca Spain N4024039.600 W603012.600 02.viii.2007
LP362 Lepidocyrtus cf. pallidus Tjöme Vestfold Norway N5909007.600 E1025055.600 06.iv.2014
LP379 Lepidocyrtus cf. pallidus Oslo Oslo Norway N5955005.200 E1046013.800 15.v.2014
LP277 Lepidocyrtus cyaneus Torla Huesca Spain N4241002.400 W006042.500 31.v.2009
LP223 Lepidocyrtus flexicollis Alcalá de los Gazules Cádiz Spain N3631019.900 W539000.400 28.ii.2009
LP102 Lepidocyrtus intermedius Cerdanyola del Vallès Barcelona Spain N4128026.800 E208050.300 21.i.2007
LP105 Lepidocyrtus intermedius Aiguafreda Barcelona Spain N4146013.800 E216019.900 10.ii.2007
LP114 Lepidocyrtus intermedius Aiguafreda Barcelona Spain N4147059.600 E218002.500 10.ii.2007
LP141 Lepidocyrtus intermedius Serinya Girona Spain N4210039.400 E244012.100 25.ix.2006
LP229 Lepidocyrtus juliae Georgioupoli Creta Greece N3521037.800 E2415006.500 07.iv.2009
LP141 Lepidocyrtus lanuginosus Serinya Girona Spain N4210039.400 E244012.100 25.ix.2006
LP115 Lepidocyrtus lanuginosus Montsant Tarragona Spain N4114013.200 E053003.500 17.ii.2007
LP130 Lepidocyrtus lanuginosus Vallgorguina Barcelona Spain N4139029.900 E231011.600 18.iv.2007
LP105 Lepidocyrtus lignorum Aiguafreda Barcelona Spain N4146013.800 E216019.900 10.ii.2007
LP069 Lepidocyrtus lignorum Caldes de Boi Lleida Spain N4233012.200 E049055.900 20.vii.2006
LP088 Lepidocyrtus lignorum Canyamars Barcelona Spain N4135053.900 E227018.400 10.x.2006
LP095 Lepidocyrtus lignorum Vall d’en Bas Girona Spain N4207000.800 E224003.200 04.xi.2006
LP109 Lepidocyrtus lignorum Aiguafreda Barcelona Spain N4147021.100 E218040.700 10.ii.2007
LP118 Lepidocyrtus lignorum Barcelona Barcelona Spain N4126056.000 E208026.500 18.ii.2007
LP266 Lepidocyrtus lignorum Drios Paros Greece N3700002.200 E2511049.200 10.iv.2009
LTa Lepidocyrtus lusitanicus Poblet Tarragona Spain N4121033.800 E104049.400 03.i.2007
LP129 Lepidocyrtus montseniensis Vallgorguina Barcelona Spain N4139029.900 E231011.600 18.iv.2007
LP328 Lepidocyrtus paradoxus Chisinau Chisinau Moldova N4702007.400 E2847053.200 20.iii.2013
Se Gi Lepidocyrtus selvaticus Tossa de Mar Girona Spain N4143008.400 E254010.100 21.ii.2008
LP234 Lepidocyrtus serbicus Kalamafka Creta Greece N3504049.100 E2539002.900 06.iv.2009
LP383 Lepidocyrtus sp. Sibenick Miljacka Croatia N4400004.000 E1601005.900 29.iv.2015
LP106 Lepidocyrtus tellecheae Aiguafreda Barcelona Spain N4146013.800 E216019.900 10.ii.2007
LP142 Lepidocyrtus tellecheae Aiguafreda Barcelona Spain N4146013.800 E216019.900 10.ii.2007
LP065 Lepidocyrtus violaceus Vall d’Aran Lleida Spain N4246040.400 E050003.100 19.vii.2006
LP440 Cyphoderus gr. bidenticulati Cellers Lleida Spain N4203041.400 E054004.700 01.xii.2015
Orches Orchesella sp.  
640 Invertebrate Systematics E. Mateos et al.

alcohol were not available), whose chaetotaxy does not match
with any European group.
For the molecular analyses, we selected as outgroups
Orchesella sp. (i.e. a basal genus within the family
Entomobryidae) and Cyphoderus bidenticulati (sensu Delamare-
Deboutteville 1948) because it shows a close relationship with
Lepidocyrtinae in molecular studies (Zhang et al.2015).
The head of each specimen was removed and preserved in 70%
alcohol for morphological identification, while the rest of the
body was prepared primarily for DNA extraction.
DNA sequencing, alignment and phylogenetic tree
reconstruction
We sequenced 2–6 individuals from each sample to perform the
phylogenetic analysis. DNA extractions were obtained with
Speedtools tissue DNA extraction Kit (Biotools, Madrid,
Spain), following the manufacturer’s protocol. Bodies were
degraded with proteinase K and the exoskeleton was preserved
in 70% ethanol for morphological studies. An ~800-bp fragment
of the mitochondrial gene cytochrome coxidase subunit II
(COXII) and fragments of the first and second exon (~500 bp),
including an intron of the nuclear EF1–1agene, were amplified
by PCR and sequenced with specific primers tRNA-K-LcuJ and
tRNA-L-LcuN for COXII, and EFLcuJ and EFLcuN for EF-1a
(Cicconardi et al.2010), applying annealing temperatures of
48C and 55CinCOXII and EF-1aamplifications, respectively.
Finally, PCR products were purified using a vacuum manifold
(Millipore, SA) and sequenced at Macrogen in Europe
(Amsterdam, The Netherlands).
SEQMAN v. 6.00 (DNASTAR) software was used to
revise the chromatograms and obtain the definitive sequences.
Sequences for each gene of the outgroup genus Orchesella were
obtained from GenBank. Gene sequences from species of the
lusitanicus-group (L. selvaticus,L. lusitanicus and L. bilobatus)
were kindly provided by Jesús Lozano (pers. comm.).
Three datasets were used in the phylogenetic inference
(COXII,EF-1aand the concatenation of both genes). The
COXII gene and the coding region (first and second exons) of
EF-1awere aligned with ClustalW (Larkin and Blackshields
2007) in BioEdit 7.2.5. (Hall 1999), following the amino acid
guide. EF-1a’s intron was aligned with MAFFT, and Gblocks
0.91B (Castresana 2000) was used to delete regions ambiguously
aligned or with many gaps. Individual alignments were merged
in a concatenated dataset and missing data were denoted as ‘N’.
Ap-distances matrix was constructed with the mitochondrial
COXII information using the software MEGA v. 7.0.21 (Kumar
et al.2016).
For the phylogenetic analyses, jModelTest 2.1.4 (Darriba et al.
2012) software was used to choose the evolutionary model that
best fitted the substitution probability following the Akaike
information criteria (Akaike 1973). Maximum likelihood (ML)
trees were inferred with RaxML 7.0.4 (Stamatakis 2006) applying
10 000 bootstrap replicates (Felsenstein 1985) and Bayesian
inference (BI) trees were obtained using MrBAYES v. 3.2.2
(Huelsenbeck and Ronquist 2005). Three million generations
for two independent runs were performed to calculate the
consensus tree. The 25% of trees in the sample were removed
as burn-in to avoid including trees sampled before likelihood
values had reached a plateau. Partitions were used by gene and the
parameters for the model were unlinked for each gene.
We used FigTree (Rambaut 2009) to visualise the trees and
the iTOL web server (Letunic and Bork 2007) to prepare and
generate all tree figures.
Morphological analysis
For morphological studies, the body skin and the head of
sequenced specimens were mounted on slides. Additional
specimens were mounted on slides for species L. cf. pallidus
and L. intermedius, sp. nov. The specimens were cleared using
Nesbitt fluid and then slide-mounted in Hoyer medium. The slides
were studied under a phase contrast microscope. The species
identification was done consulting the published original
descriptions and type material when available. Each species
was assigned to a Lepidocyrtus group (Table 2) using the
diagnostic characters of the European species-groups proposed
by Mateos (2008b,2011,2012), Mateos and Petersen (2012),
and Winkler and Traser (2012). The species Lepidocyrtus sp. is
still under study (it could be a new species), but its morphological
characters are well established and perfectly comply with the
definition of the L. lignorum-group. We found contradictory
published information regarding the head chaetotaxy of
L. pallidus Reuter, 1890 (see ‘Systematics’) and for this
reason we have done a detailed study of this chaetotaxy. We
also studied several populations that could be assigned to
the lignorum-group or curvicollis-group depending on the
chaetotaxic characters used.
Nomenclature used in chaetotaxy
The notation of Gisin (1963,1964a,1964b,1967) was followed
for the dorsal body macrochaetotaxy and for labial chaetotaxy.
The dorsal macrochaetotaxic formula follows the notation
system introduced by Gisin (1967), which for head, thorax
(second and third segments) and abdomen (first to fourth
segments) specifies the number of macrochaetae present in
a dorsal position. For the dorsal cephalic chaetotaxy we added
the notation [R
1
s] to designate the presence of ciliated
mesochaetae between macrochaetae R
0
and R
1
(see Figs 2
and 9). For dorsal cephalic chaetotaxy we also used the
‘AMS’nomenclature system originally proposed by Szeptycki
(1973) for macrochaetae in Korean Homidia Börner, developed
in more detail by Mari Mutt (1979) for Dicranocentrus Schött,
and later applied (with modifications) to Entomobrya Rondani
(Jordana and Baquero 2005), Seira Lubbock (Soto-Adames
2008) and Pseudosinella Schäffer (Soto-Adames 2010). For
the dorsal chaetotaxy of the thorax and abdomen, the notation
established by Szeptycki (1972) for S-chaetae (revised by Zhang
and Deharveng 2014) and Szeptycki (1979) for other chaetae
was used.
Abbreviations
The following abbreviations have been used in morphological
descriptions: ant, antennal segment; abd, abdominal segment; cx,
coxae; th, thorax segment; tr, trochanter; I–VI, segments; bcm,
blunt broad ciliated macrochaeta; tcm, acuminate thin ciliated
macrochaeta.
Untangling European Lepidocyrtus Invertebrate Systematics 641

Results
Molecular analyses
We obtained 109 sequences with a total length of 684 bp with no
gaps for COXII of the species mentioned in Table S1. In the case
of the nuclear gene EF-1a, 117 bp of the first exon, 151 bp of the
intron and 390 bp of the second exon were sequenced from 65
specimens (Table S1). Maximum values of p-distance for COXII
(Table S2) ranged between 0.16 (distance between L. bicoloris
and L. lanuginosus-2) and 0.318 (corresponding to L. violaceus
vs L. flexicollis).
We inferred three phylogenetic trees through ML and BI:
one for COXII, one for EF-1aand one for the concatenated
dataset of both genes. The GTR+I+Gmodel was selected as the
most appropriate model for both ML and BI. The COXII dataset
produced more resolved branches at terminal nodes than EF-1a,
which resolved basal nodes better. Support values increase
in the concatenated tree in comparison with the individual
gene trees.
The concatenated phylogeny (Fig. 1) recovered five major
monophyletic clades in both ML and BI trees, coinciding with
the five morphological groups (Table 2). The most basal clade
belonged to the lusitanicus-group, including L. bilobatus Mateos,
2008, L. lusitanicus Gama, 1964 and L. selvaticus Arbea & Ariza,
2007 specimens. In this clade, the position of L. bilobatus was not
as highly supported as the remaining species. The clade of the
lanuginosus-group consisted of the L. bicoloris Mateos, 2012,
L. cyaneus Tullberg, 1871 and L. lanuginosus (Gmelin, 1788)
specimens and was highly supported. The specimens
morphologically identified as L. lanuginosus did not form
a monophyletic entity (specimen LP141–2 appears separated
from the others), indicating the presence of one cryptic
species. In the pallidus–serbicus clade, species L. arrabonicus
Traser, 2000, L. cf. pallidus Reuter, 1890 and L. serbicus Denis,
1933 appeared as sister taxa with good support in the BI tree
(0.97 PP); however, this group was not well supported in the ML
tree (66% bootstrap). The monophyly of the curvicollis-group
including the species L. flexicollis Gisin, 1965, L. montseniensis
Mateos, 1985 and L. paradoxus Uzel, 1890 was highly supported
in both ML and BI, while the lignorum-group had lower support
in the ML tree (71%). Inside this group, we found the species
L. barbulus Mateos, 2011, L. intermedius, sp. nov., L. juliae
Mateos, 2011, L. lignorum (Fabricius, 1793), L. tellecheae
Arbea & Jordana, 1990, L. violaceus Lubbock, 1873 and
L. sp. Specimens identified as L. lignorum split among four
different monophyletic clades in the tree, indicating the
presence of three cryptic species. The curvicollis and lignorum
groups appeared as sister clades with maximum support (PP 1,
bootstrap 100).
The topology of the trees for individual genes was quite similar
to the concatenated tree topology, especially the EF-1adataset.
In the COXII dataset (Fig. S1) the lanuginosus and curvicollis
groups formed well-supported monophyletic clades, and also
the lignorum+curvicollis clade. Specimens of L. bilobatus were
Table 2. Studied Lepidocyrtus species and groups
Head mac, dorsal head macrochaetotaxy; the equivalence between Gisin (1967) and AMS macrochaetae notation is: R
0
=A
0
,R
1
s=A
2
a, R
1
=A
2
,R
2
=A
3
,S=M
2
,
T=S
3
, So = Pa5; body mac, thorax and abdomen macrochaetotaxy; scales: presence of scales on antennae, legs (beyond coxae), and posterior face of the
manubrium; Abd.IV-s: presence or absence of chaeta s on abd.IV; size: maximum body size, without head, in mm; shape: D, body dorsoventrally depressed;
SC, body slightly laterally compressed; C, body laterally compressed; Th.II pro: degree of th.II protrusion over the head: 0, th.II not protruded (Fig. 1A); 1, th.II
protruded (Fig. 1B); 2, th.II highly protruded (Fig. 1C)
Head mac Body mac Scales Abd.IV-s Size Shape Th.II pro
L. lusitanicus-group
bilobatus R
0
[R
1
s] R
1
R
2
S T So 10/0301+3 No No 1.0 D 0
lusitanicus R
0
[R
1
s] R
1
R
2
S T So 10/0301+3 No No 1.0 D 0
selvaticus R
0
[R
1
s] R
1
R
2
S T So 10/0301+3 No No 1.0 D 0
L. lanuginosus-group
bicoloris R
0
[R
1
s] R
1
R
2
S T So 10/0101+2 No No 1.0 D 0
cyaneus R
0
[R
1
s] R
1
R
2
S T So 10/0101+2 No No 1.2 D 0
lanuginosus R
0
[R
1
s] R
1
R
2
S T So 10/0101+2 No No 1.2 D 0
L. pallidus–serbicus-group
arrabonicus R
0
[R
1
s] R
1
R
2
So 00/0101+2 No No 1.4 D 0
cf. pallidus R
0
[R
1
s] R
1
T So 00/0101+2 No No 1.6 D 0
serbicus R
0
[R
1
s] R
1
R
2
S T So 00/0101+2 No No 1.9 D 0
L. lignorum-group
barbulus R
0
[R
1
s] R
1
R
2
So 00/0101+3 Yes No 2.6 SC 1
intermedius, sp. nov. R
0
[R
1
s] R
1
So 00/0101+3 Yes No 1.2 SC 1
juliae R
0
[R
1
s] R
1
R
2
So 00/0101+3 Yes No 1.5 SC 1
lignorum R
0
[R
1
s] R
1
R
2
So 00/0101+3 Yes No 2.0 SC 1
tellecheae R
0
[R
1
s] R
1
R
2
So 00/0101+3 Yes No 2.4 SC 1
sp. R
0
[R
1
s] R
1
R
2
So 00/0101+3 Yes No 1.7 SC 1
violaceus R
0
[R
1
s] R
1
R
2
So 00/0101+3 Yes No 2.1 SC 1
L. curvicollis-group
flexicollis R
0
[R
1
s] R
1
So 00/0101+3 Yes Yes 3.4 C 2
montseniensis R
0
[R
1
s] R
1
So 00/0101+3 Yes Yes 2.5 C 2
paradoxus R
0
[R
1
s] R
1
So 00/0101+3 Yes Yes 3.0 C 2
642 Invertebrate Systematics E. Mateos et al.

located out of the lusitanicus-group and appeared as sister to the
lanuginosus-group, and the curvicollis-group appeared inside the
lignorum-group. In the EF-1adataset (Fig. S2) the same
monophyletic clades as in the concatenated tree were obtained.
Systematics
Family ENTOMOBRYIDAE Schött
Genus Lepidocyrtus Bourlet
Type species: Lepidocyrtus curvicollis Bourlet, 1839.
Lepidocyrtus cf. pallidus Reuter
(Fig. 2, Tables 1,2, S1)
Material examined
Six specimens from sample LP362 (Vestfold, Norway) and three specimens
from sample LP379 (Oslo, Norway) (see Table 1).
Diagnosis
Body colour blue or light blue. Length 1.6 mm. Mesothorax not
projecting over the head. Antennae, legs and posterior face of
the manubrium without scales. Ant.IV without apical bulb.
Dorsal head macrochaetotaxy R
0
[R
1
s] R
1
T So (Fig. 2).
lignorum-group
lanuginosus-group
lusitanicus-group
curvicollis-group
LP105 1
L. serbicus
L. cyaneus
L. lanuginosus - 2
L. bicoloris
L. lanuginosus - 1
Cyphoderus
L. bilobatus
L. lusitanicus
L. selvaticus
L. flexicollis
Orchesella
L. paradoxus
L. montseniensis
L. tellecheae
L. lignorum - 3
L. intermedius
L. violeaceus
L. lignorum - 2
L. lignorum - 1
L. juliae
L. lignorum - 4
L. barbulus
L. sp.
L. cf. pallidus
L. arrabonicus
LP440 1
LP440 2
Se Gi50
L Ta56
B Sa2
LP277 1
LP277 3
LP277 2
LP141 2
LP115 2
LP190 4
LP115 3
LP130 1
LP190 3
LP190 6
LP130 3
LP130 2
LP234 5
LP362 3
LP325 3
LP234 4
LP234 3
LP223 1
LP223 3
LP223 2
LP129 2
LP328 2
LP129 1
LP129 3
LP065 1
LP065 3
LP065 2
LP328 3
LP328 1
LP118 3
LP383 4
LP383 1
LP383 3
LP239 2
LP266 3
LP229 1
LP069 3
LP105 3
LP239 3
LP239 1
LP266 1
LP069 4
LP118 1
LP102 4
LP105 4
LP105 7
LP105 6
LP105 8
LP102 3
LP141 3
LP114 2
LP109 3
LP109 4
LP095 14
LP106 3
LP106 4
LP106 2
LP105 2
LP142 2
LP088 5
LP088 4
100/1
100/1
100/1
100/1
100/1
100/1
100/1
100/1
100/1
100/1
100/1
100/1
100/1
100/1
100/1
100/1
100/1
100/1
59/0.65 99/1
98/1
82/1
96/1
92/1
66/0.97
71/0.99
pallidus serbicus-group
(A)
(C)
(B)
Animals scale: 1 mm
Tree scale: 0.1
Fig. 1. Tree inferred based on concatenated dataset using Bayesian inference. Numbers at nodes are bootstrap values (maximum likelihood)/posterior
probability (bayesian inference) (in coincidence of topology). Only values higher than 60% bootstrap and 0.65 in posterior probability are shown. Tree
scale indicates number of substitutions per site. Outgroups correspond to the genera Orchesella and Cyphoderus. The three drawings represent the
Lepidocyrtus general body shape of groups (all three in the same scale): (A) mesothorax not protruded (lusitanicus,lanuginosus and pallidus–serbicus
species-groups), (B) mesothorax protruded (lignorum-group), (C) mesothorax highly protruded (curvicollis-group).
Untangling European Lepidocyrtus Invertebrate Systematics 643

Dorsal macrochetotaxy formula of thorax and abdomen
00/0101+2. Without chaeta s on abd.IV. Labial chaetotaxy
M
1
M
2
rEL
1
L
2
(all ciliated chaetae except r chaeta, which is
vestigial). Unguiculus acuminate with finely serrated outer edge.
Inner edge of unguis with a basal pair of teeth 50% along its
length, and two unpaired teeth at 75% and 95%, respectively
(the distal one minute and difficult to see).
Molecular diagnosis. This species includes all populations
that cluster with COXII and EF-1asequences of the individuals
included in this study (Table S1), with significant support in an
adequate molecular delimitation model.
Head chaetotaxy
All specimens studied have the same dorsal cephalic chaetotaxy
(Fig. 2), with ciliated macrochaetae R
0
,R
1
, T and So, and with
ciliated long mesochaetae R
1
s between R
0
and R
1
.
Comparative discussion
The dorsal cephalic macrochaetotaxy of L. pallidus has been
interpreted in several ways according to the authors. The original
description does not include chaetotaxy, since it was not until
the mid-twentieth century when the chaetotaxic characters began
to be used in Lepidocyrtus. Gisin (1965) revised the species
(studying Finnish specimens) and drew a picture of the dorsal
cephalic macrochaetotaxy he interpreted as R
0
R
1
R
2
T So.
Fjellberg (2007) described L. pallidus from Fennoscandia and
Denmark with dorsal head macrochaetotaxy R
0
T (macrochaetae
R
1
and R
2
absent). In the North American specimens of L. pallidus
examined by Christiansen and Bellinger (1980) and Soto-
Adames (2000), the dorsal head macrochaetotaxy is R
0
R
1
T
So (R
2
absent). The specimens examined in the present work
have dorsal cephalic macrochaetotaxy also as R
0
R
1
TSo(R
2
smooth mesochaeta). Before assigning our specimens to species
L. pallidus, the scheme of dorsal head macrochaetotaxy for this
species needs to be clarified, but this is beyond the scope of the
present work, so we named our specimens Lepidocyrtus cf.
pallidus. However, it is clear that our specimens belong to the
Lepidocyrtus pallidus–serbicus-group, and this implies that,
according to the definition of the group by Winkler and Traser
(2012), it should be noted that the cephalic macrochaeta R
2
may
be present or absent.
Lepidocyrtus intermedius Mateos, Escuer & Álvarez-Presas,
sp. nov.
(Figs 3–19; Tables 1,2, S1)
Material examined
Holotype.Spain: Barcelona province, Aiguafreda municipality,
Montseny Natural Park (from LP114, see Table 1): ,on one slide (code
CRBA-51864), position: N 4147059.600 E2
18002.500, 560m a.s.l.,
10.ii.2007, leg. E. Mateos.
Paratypes.Spain: Same locality and same data collection as holotype
(sample LP114): Two specimens in two slides. Barcelona Province,
Cerdanyola del Vallès municipality, Collserola Natural Park: Seven
specimens in seven slides (from sample LP102), N 4128026.800 E2
080
50.300, 85m a.s.l., 21.i.2007, leg. E. Mateos.
Other material examined. Spain: (see Table 1), 47 specimens from
the same locality and same data collection as holotype (sample LP114); 40
specimens from the same locality and data collection as paratypes from
sample LP102; 25 specimens (sample LP105) from Montseny Natural
Park, Aiguafreda municipality, Barcelona province, N 4146013.800 E2

16019.900, 439 m a.s.l., 10.ii.2007, leg. E. Mateos; five specimens (sample
LP141) from riparian forest of Ser river, Serinyà municipality, Girona
province, N 4210039.400 E2
44012.100, 120 m a.s.l., 25.ix.2006, leg.
E. Mateos.
Holotype and one paratype (slide code CRBA-51865) saved in the
collection of the Centre de Recursos de Biodiversitat Animal, Faculty of
Biology, University of Barcelona, Spain (http://www.crba.ub.edu); other
specimens kept in the E. Mateos collection at the University of Barcelona
(Spain).
R0
R1s
R1
T
So
s
t
q
p
2
0.1 mm
S
R2
Fig. 2. Lepidocyrtus cf. pallidus. Dorsal head chaetotaxy (left side).
3
1 mm
Fig. 3. Lepidocyrtus intermedius, sp. nov. Habitus.
644 Invertebrate Systematics E. Mateos et al.

Diagnosis
Body colour grey-white. Length 0.9–1.2 mm. Mesothorax
slightly projecting over the head. Ant.I-II, legs and posterior
face of the manubrium with scales. Ant.IV without apical bulb.
Dorsal head macrochaetotaxy such as R
0
[R
1
s] R
1
So. Dorsal
macrochaetotaxic formula of thorax and abdomen 00/0101+3.
Without chaeta s on abd.IV. Labial chaetotaxy M
1
M
2
R* E L
1
L
2
,
chaetae R half in length of other chaetae (marked with *).
Unguiculus acuminate, with smooth outer margin. Inner edge
of unguis with basal pair of teeth 56% along its length, and two
unpaired teeth at 74% and 88%, respectively.
Molecular diagnosis. This species includes all populations
that cluster with COXII and EF-1asequences of the individuals
included in this study (Table S1), with significant support in an
adequate molecular delimitation model.
Description
Adult body length (without head and furca) 0.9–1.2 mm.
Mesothorax slightly projecting over the head. Body with grey-
white background colour; blue pigment only present on ant.II–IV
and cx.I–III; densely black pigmented ocular areas (Fig. 3).
Antennae with scales on the dorsal face of ant.I–II.
Ratio antenna : cephalic diagonal = 1. Ratio ant.I : II : III : IV is
1 : 1.8 : 1.9 : 3. Basis of ant.I dorsally and ventrally with three
microchaetae arranged in triangle. Ant.III organ composed of
two curved subcylindrical sensory rods partially covered by an
integumentary fold, and with two more small subcylindrical
sensory rods (one on each side) and one pointed microchaeta
(Fig. 4). Without apical ant.IV bulb.
Labrum (Fig. 5) with ciliated prelabral chaetae and smooth
labral chaetae in typical number 4/5,5,4; chaetae of apical row
branched (with two or three apical branches) and thicker than
those in other rows; inverted U-shaped labral apical intrusion;
four labral papillae with 3–4 projections each (Fig. 6). Lateral
process of outer labial papilla (sensu Fjellberg 1999) curved, tip
not reaching the apex of the papilla. Maxillary palps with two
lobal smooth chaetae and three sublobal smooth chaetae.
Labium (Fig. 7) with anterior row formed by five smooth
chaetae (a1–a5); posterior row formed by ciliated chaetae with
formula M
1
M
2
R* E L
1
L
2
, chaetae R half in length of other
chaetae (marked with *); ventral cephalic groove with 4+4
ciliated chaetae.
Dorsal macrochaetae formula such as R
0
[R
1
s] R
1
So/00/0101
+3 (Fig. 8). Following AMS notation system (see Soto-Adames
2010), the dorsal head macrochaetae are: R
0
=A
0
,R
1
s=A
2
a,
R
1
=A
2
and So = Pa
5
(Fig. 9). Maximum number of macrochaetae
A between ocular areas 10+10. Interocular chaetotaxy with
a1
a5
a2
a3
a4
L2
L1
E
R*
M1
M2
7
0.025 mm
4
0.01 mm
0.025 mm
6
5
0.025 mm
Figs 4–7. Lepidocyrtus intermedius, sp. nov. 4, Ant.II organ. 5, Labrum chaetotaxy. 6, Apical labral papillae.
7, Labium and ventral cephalic groove.
Untangling European Lepidocyrtus Invertebrate Systematics 645

ciliated chaetae (s, t, p) and 1–3 scales (Fig. 9). Eyes G and H
somewhat smaller than the other. Dorsal chaetotaxy from th.II
to abd.III as in Figs 10–15; th.II without macrochaetae (p3
mesochaeta; Fig. 10). Abd.II m3 and m5 broad ciliated
macrochaetae, trichobothrium m2 with associated chaeta mi
ciliated, trichobothrium a5 with associated chaetae lm and
ll ciliated (Fig. 13); abd.III pm6 and p6 broad ciliated
macrochaetae, m7a and p8p thin ciliated macrochaetae,
trichobothrium m2 with associated chaetae mi, ml and a2
ciliated, trichobothria a5 and m5 with associated chaetae li,
lm, ll, a6, im, em and am6 ciliated (Figs 14,15). Abd.IV
dorsal chaetotaxy as in Fig. 16; trichobothrium T2 with
associated chaetae D1, a and m ciliated, without accessory
chaeta s; trichobothrium T3 with associated chaetae pi and
pe ciliated; dorsal macrochaetae of two distinct morphologies:
B4, B5, B6, C1, D3, E2, E3, E4, F1, F2, F3 broader and with
broad socket (bcm in Fig. 16); T6, T7, D2, De3, E1, E4p, F3p,
Fe4 shorter or longer, but always thinner and with socket of
minor diameter (tcm in Fig. 16); ratio T2–T4/C1p = 6.2 (see
Mateos and Greenslade 2015); ratio C1–B4/B4–B6 = 0.95 (see
Mateos 2008a).
Ventral tube with scales only on anterior side, with 6+6 ciliated
chaetae on anterior side, 17 ciliated chaetae on posterior side, and
with seven ciliated chaetae and four smooth chaetae on each
lateral flap.
Legs with scales. V-shaped trochanteral organ (leg III) with
a maximum of eight smooth, straight chaetae arranged in
triangular shape (Fig. 17). Unguis with basal pair of teeth 56%
along the inner edge, and two inner teeth 74% and 88% from the
base of the inner edge, respectively. Unguiculus lanceolate with
smooth outer margin. Spatulate tibiotarsal tenent hair (Fig. 18).
Furca with scales on anterior and posterior surfaces.
Mucro bidentate; mucronal basal spine without spinelet. Ratio
manubrium : dens : mucro as 15 : 17 : 1. Manubrial plate with two
pseudopores, two inner chaetae and 3–5 outer chaetae (Fig. 19).
Distribution
North-eastern Spain (Table 1).
Comparative discussion
The new species is very close to L. lignorum (sensu Mateos 2011)
and L. juliae. Of these two species, L. intermedius, sp. nov. clearly
differs by the absence of dorsal cephalic macrochaeta R
2
, and
also by the presence of abd.III chaeta d3 and its smaller body
size. From L. juliae, it also differs by the absence of ocular
chaeta q, unguiculus with smooth outer margin and absence of
body pigment.
The current literature describes dorsal head macrochaetotaxy
of the lignorum and curvicollis groups as R
0
[R
1
s] R
1
R
2
So and R
0
[R
1
s] R
1
So, respectively (see Mateos 2011; Mateos and Petersen
2012). In addition, species of curvicollis-group are characterised
by the presence of chaeta s associated with the anterior
trichobothrium of abd.IV. The new species has an intermediate
set of characters, with dorsal head macrochaetotaxy R
0
[R
1
s] R
1
So (as curvicollis-group) and absence of chaeta s on abd.IV
o
o
o
o
o
o
o
o
o
o
o
o
o
o
##
##
##
##
##
##
8
0.5 mm 0.05 mm
9
R0
R1s
R1
So
s
t
p
Figs 8–9. Lepidocyrtus intermedius, sp. nov. 8, Dorsal head and body macrochetotaxy, black dots indicate
macrochaetae, # indicates pseudopori, lines indicate trichobothria. 9, Dorsal head chaetotaxy (left side).
646 Invertebrate Systematics E. Mateos et al.

(as lignorum-group). So, using only morphological characters
is not possible to assign this new species to any of the two
abovementioned groups. Genetic data place this species in
lignorum-group with high support.
Etymology
The species name refers to the intermediate position of the new
species’chaetotaxy with respect to the lignorum and curvicollis
species-groups. ‘Intermedius’in Latin means to be in the middle
of two things. Its lexical components are the prefix‘inter’
(between) and ‘medius’(medium).
Discussion
Thanks to the combination of molecular data and morphological
descriptions, it has been possible to untangle the relationships of
the European members of a very diverse genus of Collembola.
Other studies have already shown that molecular data can help to
distinguish lineages of these animals (Katz et al.2015;Yuet al.
2016) and, again, the application of integrative taxonomy has
proven useful for resolving group classifications. What was more
important in this study, however, was deciding which are more
decisive characters (in this case related to the chaetotaxy) to
diagnose each of the species-groups of the genus Lepidocyrtus
included in this study, thanks to the phylogenies obtained with
molecular data.
The status of the European subgenera of Lepidocyrtus
The only character that allows differentiation between subgenera
Lepidocyrtus s.s. and Lanocyrtus is the presence of scales on
antennae, legs (beyond coxae) and posterior surface of the
manubrium in the former, and their absence in the latter (see
Wang et al.2003). All specimens studied in the lignorum and
curvicollis groups have scales on these regions, and all studied
species of groups lusitanicus and lanuginosus have no scales on
these regions. The three species of the pallidus–serbicus-group
(L. arrabonicus,L. cf. pallidus and L. serbicus) have scales on
coxae, but not on antennae or posterior face of the manubrium.
This distribution of appendicular scales allows assignment of
groups lignorum and curvicollis to subgenus Lepidocyrtus s.s.,
and groups lusitanicus,lanuginosus and pallidus–serbicus to
subgenus Lanocyrtus.
Our molecular results indicated that subgenus Lanocyrtus
is paraphyletic while subgenus Lepidocyrtus s.s. is
monophyletic. This implies that Lanocyrtus is not a natural
group and its taxonomic validity as subgenus is questionable,
while Lepidocyrtus s.s. is a natural group representing the most
recent lineage within European Lepidocyrtus.
Position of Lepidocyrtus intermedius, sp. nov. within
European groups
Genetic data clearly place the new species, L. intermedius, within
lignorum-group, indicating that dorsal head macrochaetotaxy
cannot be used for differentiating between these two groups,
and that the only chaetotaxic character differentiating the
lignorum and curvicollis groups is the absence (in lignorum-
group) or the presence (in curvicollis-group) of chaeta s
associated with anterior trichobothrium in abd.IV. All
currently known species of the lignorum-group have dorsal
cephalic macrochaeta R
2
, and the new species differ from the
other species of this group by the absence of this cephalic
macrochaeta R
2
. This implies that, in the definition of the
lignorum-group by Mateos (2012), should be added that the
dorsal cephalic macrochaeta R
2
can be present or absent.
Characters defining European Lepidocyrtus species-groups
Our molecular results are congruent with the distribution of the
European species in the five groups established in the literature on
the basis of morphological characters. Nevertheless, these results
also indicate that not all characters normally used in the literature
are useful for group differentiation.
Dorsal head macrochaetotaxy is variable within several
species-groups (see Table 2), making this character not
appropriate for group definition. Species in groups lusitanicus,
lanuginosus and curvicollis have no variability in the dorsal
head macrochaetotaxy scheme, while those of groups
pallidus–serbicus and lignorum may possess differences in
several dorsal head macrochaetae. The three species of the
pallidus–serbicus-group have different head macrochaetotaxy,
0.1 mm
ms
al
10
p3
al
11
ms 12
Figs 10–12. Lepidocyrtus intermedius, sp. nov. 10, Th.II chaetotaxy (left
side). 11, Th.III chaetotaxy (left side). 12, Abd.I chaetotaxy (left side).
Untangling European Lepidocyrtus Invertebrate Systematics 647

and the new species, L. intermedius,inlignorum-group, has a
different pattern than the other species in this group (see Table 2).
Body dorsal macrochaetotaxy is highly conservative within
groups, and all species from the same group have the same scheme
(Table 2). Also, the position of each macrochaeta is always
the same in all the species within a group. On th.II, in species
with one dorsal macrochaeta (those from groups lusitanicus
and lanuginosus), p3 is always the macrochaeta. On abd.II, all
European species (except L. fimetarius) have m3 as macrochaeta,
and species from lusitanicus-group have in addition a2 and m3e
as macrochaetae. On abd.IV, all European species (except
L. fimetarius) have C1 as macrochaeta; in addition species from
groups lusitanicus,lignorum and curvicollis have B4, B5 and B6
as macrochaetae, while species from groups lanuginosus and
pallidus–serbicus have only B5 and B6 as macrochaetae
(although some species might have B4 and B5 as macrochaetae
instead of B5 and B6).
Scales on antennae, legs beyond coxae and posterior face
of the manubrium are always present in groups lignorum and
curvicollis and absent in the other groups. However, L. szeptyckii
Rusek, 1985 can be assigned to the pallidus–serbicus-group by
its chaetotaxy, and has scales on the dorsal face of ant.I–II, all
leg segments and the posterior face of the manubrium. This
undermines the value of this character for defining species-
groups and subgenera. Unfortunately, L. szeptyckii could not
be included in the present study, and its relationship with the other
European species remains pending for further analysis. Soto-
Adames (2000), for Neotropical species of Lepidocyrtus,
also found that the distribution of scales in the appendages is
not a good character for defining subgenera. It remains to be
confirmed whether this is the case with European fauna.
Cryptic species among European Lepidocyrtus
As several authors have concluded (Soto-Adames 2002;
Cicconardi et al.2010; Cicconardi et al.2013), the actual
number of Lepidocyrtus species could be much higher than
the species number currently described, possibly due to the
occurrence of cryptic species and species-groups that cannot
be resolved using only morphological characters. Our results
are in concordance with this cryptic diversity within European
Lepidocyrtus, and several cryptic species lay inside the
lanuginosus and lignorum groups. In the three phylogenetic
trees obtained, the same cryptic species are detected (Figs 1,
S1 and S2). Specimens identified as L. lanuginosus split in two
clades, and specimens identified as L. lignorum split in four
clades. A revision of the characters defining these two species is
needed. Moreover, values of genetic p-distance between these
lanuginosus and lignorum clades in comparison with all the
other species are in the same range as the other lineages,
and are congruent with values found in other studies as
Cicconardi et al.(2010).
0.02 mm
a5
m5
pm6
p6 ms
p5
li
lm
ll
im
em
a6
am6
d3
15
13
m2
a5
m3
m5
as
0.1 mm
m2
a5
m5
m7a
pm6
p6
p8p
as
14
mi
lmll
p6
mi
ml
a2
Figs 13–15. Lepidocyrtus intermedius, sp. nov. 13, Abd.II chaetotaxy (left side). 14, Abd.III chaetotaxy (left side). 15, Detail
of Abd.III chaetotaxy (left side).
648 Invertebrate Systematics E. Mateos et al.

Evolution within European Lepidocyrtus
Within the European Lepidocyrtus, the general morphology of
the body differs depending on the species-group. In Table 2, three
body variables for each species studied are summarised (body
length without head, body shape and mesothorax protrusion), and
the schematic illustration in Fig. 1relates to the phylogenetical
and morphological data obtained.
Our phylogenetic results indicate that in European species
the lusitanicus-group occupies a basal position. As successively
derived groups we obtained the lanuginosus-group, the pallidus–
serbicus-group, and finally the lignorum and curvicollis groups
as the most recent. This is indicative of a progressive increase
in body size, a tendency to lateral compression of the body, and
increased mesothoracic protrusion in the speciation process in
European species (Table 2; Fig. 1).
Key to European Lepidocyrtus species-groups
With the new data provided in the present study we conclude
that dorsal body macrochaetotaxy characters are good
candidates for European species-groups diagnosis. As dorsal
head macrochaetotaxy is variable in the pallidus–serbicus and
lignorum groups, it is not suitable to be used for group diagnosis.
We propose the following key for European Lepidocyrtus
species-groups:
1. Lacking dorsal macrochaetae between bothriotricha m2 and a5 on
abd.II ................................................................................L. fimetarius
With one or three dorsal macrochaetae between bothriotricha m2 and a5
on abd.II .............................................................................................2
2. With one dorsal macrochaeta on th.II...................................................... 3
Lacking dorsal macrochaeta on th.II........................................................4
3. Three macrochaetae between bothriotricha m2 and a5 on abd.II ..............
.................................................................................lusitanicus-group
One macrochaeta between bothriotricha m2 and a5 on abd.II ..................
...............................................................................lanuginosus-group
4. Two dorsomedial macrochaetae on abd.IV ........ pallidus–serbicus-group
Three dorsomedial macrochaetae on abd.IV, antennae, legs and posterior
face of manubrium scaled ..................................................................5
5. Lacking chaeta s on abd.IV ..............................................lignorum-group
With chaeta s on abd.IV..................................................curvicollis-group
Fig. 16. Lepidocyrtus intermedius, sp. nov. Abd.IV chaetotaxy.
0.025 mm
18 19
0.025 mm
17
0.025 mm
Figs 17–19. Lepidocyrtusintermedius, sp. nov. 17, Trochanteral organ. 18, Unguis and unguiculum
of third leg. 19, Manubrial plate.
Untangling European Lepidocyrtus Invertebrate Systematics 649

Conflicts of interest
The authors declare no conflicts of interest.
Acknowledgements
We are grateful to Marko Luki
c and Arne Fjellberg for the Lepidocyrtus
specimens they provided. We are indebted to Jesus Lozano for providing
molecular sequences of several specimens. Three anonymous reviewers
provided helpful comments that improved the manuscript. This research
was supported by the Ministerio de Economía y Competitivad, Spain
(project CGL2011–23466).
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Handling editor: Gavin Svenson
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