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DNA Barcoding of Portuguese Lacewings (Neuroptera)
and Snakeflies (Raphidioptera) (Insecta, Neuropterida)
Daniel Oliveira1,2, Cátia Chaves1, Joana Pinto1, Joana Paupério1,
Nuno Fonseca1, Pedro Beja1,3, Sónia Ferreira1
1CIBIO/InBIO – Centro de Investigação em Biodiversidade e Recursos Genéticos da Universidade do Porto,
Vairão, 4485–661 Vairão, Portugal 2Departamento de Biologia, Faculdade de Ciências, Universidade do
Porto, 4169-007 Porto, Portugal 3CIBIO/InBIO, Centro de Investigação em Biodiversidade e Recursos Gené-
ticos, Instituto Superior de Agronomia, Universidade de Lisboa, Lisboa, Portugal
Corresponding author: Sónia Ferreira (hiporame@gmail.com)
Academic editor: S. Winterton|Received 18 February 2021|Accepted 25 May 2021|Published 3 August 2021
http://zoobank.org/D2A38983-2C5A-456A-B5F8-2CE3F7E3121A
Citation: Oliveira D, Chaves C, Pinto J, Paupério J, Fonseca N, Beja P, Ferreira S (2021) DNA Barcoding of
Portuguese Lacewings (Neuroptera) and Snakeies (Raphidioptera) (Insecta, Neuropterida). ZooKeys 1054: 67–84.
https://doi.org/10.3897/zookeys.1054.64608
Abstract
e orders Neuroptera and Raphidioptera include the species of insects known as lacewings and snake-
ies, respectively. In Portugal, these groups account for over 100 species, some of which are very dicult
to identify by morphological analysis. is work is the rst to sample and DNA sequence lacewings and
snakeies of Portugal. A reference collection was built with captured specimens that were identied mor-
phologically. DNA barcode sequences of 658 bp were obtained from 243 specimens of 54 species. e
results showed that most species can be successfully identied through DNA barcoding, with the excep-
tion of seven species of Chrysopidae (Neuroptera). Additionally, the rst published distribution data are
presented for Portugal for the neuropterans Gymnocnemia variegata (Schneider, 1845) and Myrmecaelurus
(Myrmecaelurus) trigrammus (Pallas, 1771).
Keywords
Cytochrome c oxidase subunit I (COI), DNA barcode, mitochondrial DNA, Portugal, taxonomy
ZooKeys 1054: 67–84 (2021)
doi: 10.3897/zookeys.1054.64608
https://zookeys.pensoft.net
Copyright Daniel Oliveira et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC
BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Daniel Oliveira et al. / ZooKeys 1054: 67–84 (2021)
68
Introduction
Neuropterida is a superorder of insects which encompasses the orders Neuroptera,
Raphidioptera and Megaloptera. e present work focuses on DNA barcoding of the
rst two orders in Portugal, while DNA barcoding of Megaloptera in the country was
addressed in Ferreira et al. (2019).
e order Neuroptera includes the holometabolous insects commonly known as
lacewings. With at least 6000 species worldwide, more than 300 of which occur in
Europe, Neuroptera accounts for most of the diversity of the Neuropterida (Aspöck
2002b; Aspöck et al. 2015). For the almost 200 species known in the Iberian Penin-
sula, around half have been recorded in Portugal, spanning 10 families (Aspöck et al.
2001; Letardi and Almeida 2013; Monserrat and Triviño 2013; Oliveira and Ferreira
2020; this work).
e small order Raphidioptera Latreille, 1810, groups about 260 species of in-
sects worldwide (Aspöck 2002a), which are commonly known as snakeies. From the
16 species of Raphidioptera present in the Iberian Peninsula, six species are known
to occur in Portugal (Monserrat and Papenberg 2015; Papenberg 2015). e fam-
ily Raphidiidae is represented by ve species: Atlantoraphidia maculicollis (Stephens,
1836), Harraphidia laueri (Navás, 1915), Hispanoraphidia castellana (Navás, 1915),
Ohmella bolivari (Navás, 1915) and Subilla aliena (Navás, 1915). In contrast, Inocel-
liidae is represented by a single species: Fibla hesperica Navás, 1915 (Monserrat and
Papenberg 2015; Papenberg 2015).
e monophyly of the three orders of Neuropterida (Megaloptera as a sister group
of Neuroptera + Raphidioptera) has been solidly established. Nonetheless, taxonomy
of the groups is incompletely resolved and internal relationships are not yet established,
despite recent studies, especially in the case of Neuroptera (Aspöck 2002b; Winterton
et al. 2010; Wang et al. 2017; Engel et al. 2018). Most notably, recent evidence has
been mounting for the integration of Ascalaphidae as a subfamily of Myrmeleontidae
(Winterton et al. 2018; Machado et al. 2019; Vasilikopoulos et al. 2020).
DNA barcoding was proposed in 2003 as a method to rapidly and accurately iden-
tify species (Hebert et al. 2003; Hebert and Gregory 2005). is method relies on the
existence of comprehensive databases of short DNA sequences (the DNA barcodes),
which are attributed to previously identied specimens and used for comparison with
DNA barcode sequences obtained from unidentied specimens or even environmental
samples. For insects, the typical DNA barcode consists of a 658 bp sequence of the
cytochrome c oxidase subunit I (COI) (Folmer et al. 1994), also known as the “Folmer
region”. DNA barcoding has been used in studies involving Neuropterida, namely
in the construction and analysis of DNA barcode databases for the fauna of certain
regions, including Central Europe (Morinière et al. 2014) and Beijing, China (Yi et
al. 2018), in the description of new species (Pantaleoni and Badano 2012; Badano et
al. 2016), and to resolve taxonomic questions (Price et al. 2015). It is important to
accurately identify species, especially the ones with agricultural applications, such as
those in Chrysopidae and Hemerobiidae, as misidentications may compromise bio-
DNA Barcoding of Portuguese Lacewings and Snakeies 69
logical control. Hitherto, DNA barcoding studies of Neuroptera and Raphidioptera
in Portugal were non-existent, despite the considerable number of species known to
occur in the country.
In this work, we present a contribution to the DNA barcode library for the Por-
tuguese species of Neuroptera and Raphidioptera representing about 50% of known
species in the country, alongside new and interesting distributional data. While most
species were found to be identiable through the use of the obtained DNA barcodes,
this was not true for some cases in Chrysopidae. is work was conducted within the
frame of the InBIO Barcoding Initiative, which aims at producing a comprehensive
DNA barcode database for the Portuguese terrestrial invertebrate biodiversity.
Materials and methods
Sampling of specimens
Specimens were collected during eld expeditions throughout continental Portugal,
from 2006 to 2019, and stored in 96% ethanol at the InBIO Barcoding Initiative ref-
erence collection (Vairão, Portugal). Specimens were captured during direct searches of
the environment or lured by light trapping, the latter with UV LEDs or mercury va-
pour lamps. Morphological identication was done based on the most recent literature
on Iberian Neuroptera and Raphidioptera (Monserrat and Acevedo 2012a, b, 2013;
Monserrat 2014a, b, c, 2016a, b; Monserrat et al. 2014; Monserrat and Papenberg
2015), and using an Olympus SZX2-ILLT Stereozoom microscope when necessary.
From each specimen, one tissue sample (a leg) was removed and stored in 96% ethanol
for DNA extraction.
DNA extraction, amplification and sequencing
For each species, we selected six specimens for DNA sequencing based on their loca-
tion of capture, attempting to maximize the geographical coverage of the study. For
species with less than six specimens, all were selected for sequencing.
DNA was extracted from most tissue samples using the EasySpin Genomic DNA
Microplate Tissue Kit. For specimens belonging to species of smaller sizes (such as
those from the Hemerobiidae and Coniopterygidae families), the QIAmp DNA Micro
Kit was used, as it is designed to extract higher concentrations of genetic material from
samples with small amounts of DNA.
Amplication of the DNA was performed using three dierent primer pairs, that
amplify three overlapping fragments of the same 658 bp region of the COI mitochon-
drial gene. Initially, we used two primer pairs, LCO1490 (Folmer et al. 1994) + Ill_C_R
(Shokralla et al. 2015) and Ill_B_F (Shokralla et al. 2015) + HCO2198 (Folmer et al.
1994) (henceforth referred to as LC and BH, respectively) to amplify two overlapping
fragments of 325 bp and 418 bp, respectively. Following publication of the third primer
Daniel Oliveira et al. / ZooKeys 1054: 67–84 (2021)
70
pair, BF2 + BR2 (422 bp fragment), by Elbrecht and Leese (2017), this started to be
used instead of Ill_B_F + HCO2198 due to higher amplication eciency.
PCRs were performed in 10 µl reactions, containing 5 µl of Multiplex PCR Master
Mix (Qiagen, Hilde, Germany, 0.3 (BF2-BR2) – 0.4 mM of each primer, and 1–2µl
of DNA, with the remaining volume in water. For DNA amplication, an initial de-
naturation at 95 °C for 15 min was performed followed by 5 cycles at 95 °C for 30
sec, 47 °C for 45 sec, 72 °C for 45 sec (only for LC and BH); then 40 cycles at 95 °C
for 30 sec, 51 °C for 45 sec (48 °C for 60 sec for BF2 + BR2), 72 °C for 45 sec; and a
nal elongation step at 60 °C for 10 min. DNA amplication was performed in T100
ermal Cycler (Bio-Rad, California, USA).
All PCR products were analysed by agarose gel electrophoresis and samples select-
ed for sequencing were then organised for assignment of sequencing ‘indexes’. One of
two types of index were used for each run. For Illumina indexes, samples were pooled
into one plate, as described in Shokralla et al. (2015). When using custom indexes
(designed based on (Meyer and Kircher (2010)) no pooling was required. e latter
allow for a maximum of 1920 unique index combinations. A second PCR was then
performed where the ‘indexes’ and Illumina sequencing adapters were attached to the
DNA extract. e index PCR was performed in a volume of 10 µl, including 5 µL
of Phusion High-Fidelity PCR Kit (New England Biolabs) or KAPA HiFi PCR Kit
(KAPA Biosystems, USA), 0.5 µL of each ‘index’ and 2 µL of diluted PCR product
(usually 1:4). is PCR reaction is only of 10 cycles and performed at an annealing
temperature of 55 °C. e amplicons were puried using AMPure XP beads (New
England Biolabs) before quantication using NanoDrop 1000 (ermo Scientic).
is step allows for a normalization of concentrations between samples before the nal
quantication step with a qPCR using the KAPA Library Quantication Kit Illumina
Platforms (KAPA Biosystems, USA) (Paupério et al. 2018).
Sequencing was performed at the CIBIO facilities on an Illumina MiSeq benchtop
system, using a V2 MiSeq sequencing kit (2× 250 bp).
Bioinformatic processing and data analysis
Sequences were ltered and processed with OBITools (Boyer et al. 2014) and the frag-
ments were assembled into their consensus 658 bp-long sequences using Geneious
9.1.8 (https://www.geneious.com). e obtained DNA sequences were then compared
against the BOLD database (Ratnasingham and Hebert 2007) using the built-in iden-
tication engine, based on the BLAST algorithm. Sequences were submitted to the
BOLD database and the Barcode Index Numbers (BIN) for every sequence were re-
trieved and analysed (Suppl. material 1: Table S1).
All DNA barcode sequences were aligned in Geneious 9.1.8. with the CLUSTALW
(ompson et al. 1994) plugin. Nucleotide composition of all sequences, as well as intra
and interspecic p-distances were calculated in MEGAX (Kumar et al. 2018). Neigh-
bour-joining trees were constructed in PAUP* 4.0a167 (Swoord 2003), with 1000
bootstrap replicates, as a simple way of visualizing genetic distance between sequences,
DNA Barcoding of Portuguese Lacewings and Snakeies 71
while detecting possible misidentications and incongruences. First, a tree with all
obtained DNA barcode sequences of Neuroptera and Raphidioptera was constructed.
For this, the outgroup sequences IBIMP001-19 and AGRID020-10 from the BOLD
database (of Sialis fuliginosa Latreille, 1803 and Agriotes proximus Schwarz, 1891, re-
spectively) were used to root the tree. ese outgroups refer, respectively, to a species
of Megaloptera (the third order within the Neuropterida) and a species of Coleop-
tera, the closest order to Neuropterida (Wang et al. 2017). Additionally, a NJ tree was
constructed for Chrysopidae and Hemerobiidae, utilizing the sequences FBNE073-11
and FBNE001-11 (of Osmylus fulvicephalus (Scopoli, 1763) and Sisyra nigra (Retzius,
1783), respectively) as outgroups. e latter set of outgroups was used for family-level
trees as representative of Osmylidae Linnaeus, 1758 and Sisyridae Banks, 1905.
An analysis of the data with the Automatic Barcode Gap Discovery (ABGD) meth-
od (Puillandre et al. 2012) was performed at the dedicated website (https://bioinfo.
mnhn.fr/abi/public/abgd/abgdweb.html), as a test of the existence of a barcoding gap
between species, which is fundamental to species identication using DNA barcodes
(Hebert et al. 2003, 2004).
Results
DNA barcode sequences of 658 bp were obtained for 243 specimens of Neuropterida,
representing 54 of the 104 species known to occur in continental Portugal (Fig. 1;
Suppl. material 1: Table S1). ese species are representative of 9 of 10 families of
Neuroptera, and one of two families of Raphidioptera recorded in the country. ese
sequences represent 21 new species of Neuroptera and one of Raphidioptera for the
BOLD database. Furthermore, of the already available sequences in BOLD only six
originate from continental Portugal (accessed on 19/01/2021).
Neuroptera Linnaeus, 1758
Neuroptera specimens were collected from 67 sampling locations, in 12 districts (Fig.1
and Suppl. material 1: Table S1). From the 51 species, 12 were captured only once and
are therefore represented by a single DNA barcode sequence in the dataset. Two of the
species were hitherto without published records in scientic literature for the country:
Gymnocnemia variegata and Myrmecaelurus trigrammus (Suppl. material 1: Table S1),
despite being widespread in the whole Euro-Mediterranean area and their presence
well known in Spain.
For the DNA barcode sequences of Neuroptera, average nucleotide composition is
39.4% thymine (T), 16.2% cytosine (C), 28.6% adenine (A) and 15.8% guanine (G).
Base frequencies analysis revealed GC-contents of 32% for the DNA barcode frag-
ment. Average genetic p-distances between captured species ranged from 0.46% be-
tween Pseudomallada picteti (McLachlan, 1880) and Pseudomallada avifrons (Brauer,
1851) to 25.91% between Dilar meridionalis Hagen, 1866 and Aleuropteryx iberica
Daniel Oliveira et al. / ZooKeys 1054: 67–84 (2021)
72
Figure 1. Map of continental Portugal with sampling locations A sampling locations of the 8 captured
specimens of Raphidioptera (N = 8) B sampling locations of the 235 captured specimens of Neuroptera
(N = 67).
Monserrat, 1977 (Suppl. material 2: Table S2). Intraspecic distances ranged from 0%
in Palpares hispanus Hagen, 1860 (N = 3), Cunctochrysa baetica (Hölzel, 1972) (N=5)
and Italochrysa italica (Rossi, 1790) (N = 2) to 3.6% in Dilar meridionalis (N = 4)
(Suppl. material 2: Table S2).
Regarding the neighbour-joining analysis (Fig. 2), most species were recovered as
monophyletic except for seven species of Chrysopidae, which were separated into two
polyphyletic groups of morphologically identied species. One group encompassing
P. picteti and P. avifrons, another encompassing Chrysoperla carnea (Stephens, 1836),
Chrysoperla lucasina (Lacroix, 1912), Chrysoperla pallida Henry et al., 2002, Chrysop-
erla agilis Henry et al., 2003 and Chrysoperla mediterranea (Hölzel, 1972) (Fig. 3).
e ABGD method yielded partitions generally congruent with morphological
identication. Nonetheless, some exceptions were noted. Regarding the Chrysopidae,
the ABGD analysis yielded 15 partitions (P = 0.0055) (Fig. 3). While congruent with
the NJ analysis (by considering the aforementioned polyphyletic groups of species as
two separate species), it also grouped the DNA barcoding sequences of Pseudomallada
prasinus and Pseudomallada abdominalis (Brauer, 1856), which NJ analysis separates
into three clades (in congruence with three detected morphospecies; see Discussion),
into one single “species”. In the Hemerobiidae family, the ABGD analysis recovered
only eight partitions (P = 0.0492), grouping Wesmaelius subnebulosus (Stephens, 1836)
and Wesmaelius nervosus (Fabricius, 1793) (Fig. 4).
DNA Barcoding of Portuguese Lacewings and Snakeies 73
0.03
ae
a
a
a
e
e
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Berothidae
B
B
B
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Citgidae
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i
t
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t
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Chrysopidae
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C
C
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h
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r
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y
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Myrmeleontidae
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Ntid
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Figure 2. Neighbour-joining tree of all obtained DNA sequences for Portuguese Neuroptera and Raphid-
ioptera. Neighbour-joining tree constructed in PAUP* 4.0a167. Non-highlighted terminal branches rep-
resent the two outgroup sequences.
Similar to the other two methods used, BIN allocation using BOLD Systems
yielded congruent results for most species, with some particular cases of incongruence.
In Ascalaphidae, the sequences belonging to the two species of Libelloides were grouped
under the same BIN. For Chrysopidae, the BIN framework clustered sequences simi-
Daniel Oliveira et al. / ZooKeys 1054: 67–84 (2021)
74
Figure 3. Neighbour-joining tree of Chrysopidae DNA barcode sequences. Neighbour-joining tree con-
structed in PAUP* 4.0a167 and contrasted with the results from the ABGD analysis and BIN attribution.
Bootstrap values under 90% omitted.
DNA Barcoding of Portuguese Lacewings and Snakeies 75
Figure 4. Neighbour-joining tree of Hemerobiidae DNA barcode sequences. Neighbour-joining tree
constructed in PAUP* 4.0a167 and contrasted with the results from the ABGD analysis and BIN attribu-
tion. Bootstrap values under 90% omitted. Subtrees were collapsed for the monophyletic morphologically
identied species. Triangle size for each species is proportional to the intraspecic distance.
larly to ABGD, except for two sequences of Pseudomallada prasinus (INV10273 and
INV07344), which were assigned BINs dierent from each other and the other se-
quences for the species, as well as one sequence from both Pseudomallada genei and
Pseudomallada venosus which were not grouped in the same BIN as the other sequences
of the same species (Fig. 3 and Suppl. material 1: Table S1). e sequences of Hemero-
biidae yielded 10 BINs, one more than the number of morphologically identied spe-
cies, as sequences of Sympherobius pygmaeus are in two BINs (Fig. 4).
Raphidioptera Latreille, 1810
DNA barcode sequences were obtained for eight specimens of Raphidioptera, account-
ing for three of the six species known to occur in Portugal.
Average nucleotide composition of all DNA barcode sequences of Raphidioptera
was calculated as 37.2% thymine (T), 18.1% cytosine (C), 29.6% adenine (A) and
Daniel Oliveira et al. / ZooKeys 1054: 67–84 (2021)
76
15.1% guanine (G). Base frequencies analysis revealed GC-contents of 33% for the
DNA barcode fragment. Genetic distances between species ranged from 12.3% be-
tween A. maculicollis and H. castellana to 15.9% between H. laueri and H. castellana.
Intraspecic distances ranged from 0.2% in H. castellana to 1.2% in A. maculicollis
(Suppl. material 2: Table S2). e NJ tree constructed with the calculated genetic
distances recovered all species as monophyletic (Fig. 2). Analysis with the BOLD BIN
system yielded three BINs, congruent with the morphological identication. Similarly,
three partitions were recovered from ABGD analysis.
e eight specimens of Raphidioptera were captured in six sampling locations in
Bragança and Leiria (Fig. 1 and Suppl. material 1: Table S1)
Discussion
In this work, DNA barcode sequences and their respective analyses, as well as novel
distributional data are provided based on 235 specimens of 51 species of Neuroptera
and 8 specimens of 3 species of Raphidioptera. is is the rst study focusing on DNA
barcoding for these orders in Portugal.
e main goal of this work was to compile a DNA barcode reference collection for
the Portuguese species of Neuroptera and Raphidioptera. About 50% of the faunal di-
versity of the groups is represented in the collection, and DNA barcode sequences were
added to the BOLD database for species hitherto unrepresented. e analyses con-
ducted suggest that most of the encompassed species can be identied with the COI
gene-based DNA barcodes. is is the case for the Ascalaphidae, Berothidae, Mantispi-
dae, Myrmeleontidae and Nemopteridae families. For the other families, Chrysopidae
and Hemerobiidae, further scrutiny is necessary.
Interestingly, despite the congruence of taxonomy and the obtained DNA bar-
codes for the families Ascalaphidae and Myrmeleontidae, the genetic distances and
phylogenetic tree (Fig. 2) show the latter group as paraphyletic. ese results may
provide further evidence for the integration of current Ascalaphidae species into the
family Myrmeleontidae, a taxonomic change that has seen growing support in recent
years (Winterton et al. 2018; Machado et al. 2019; Vasilikopoulos et al. 2020)
Regarding the Chrysopidae, the results show four groups of species with conicting
results between morphological identication, NJ and ABGD analysis, and BIN attri-
bution. e rst consists of the DNA barcode sequences belonging to P. avifrons and
P. picteti, whose sequences were recovered as a single clade (NJ) and placed by ABGD
analysis into a single group. Despite possessing distinctive morphological characteristics
these are closely-related species with high degree of morphological variation (Aspöck et
al. 2001; Monserrat 2016b; Duelli et al. 2017). e obtained results suggest that P. picteti
and P. avifrons share mitochondrial haplotypes, which may be due to incomplete line-
age sorting or mitochondrial genome capture as a result of introgressive hybridization.
e morphospecies P. venosus and P. genei were recovered as monophyletic and
ABGD considered each of the species as single units, although two dierent BINs were
attributed to each species.
DNA Barcoding of Portuguese Lacewings and Snakeies 77
e Pseudomalla “prasinus” species complex, where P. prasinus and P. abdominalis
are included, is the third group with conicting results between NJ, ABGD and mor-
phological analysis, and has been a subject of interest and contention in Neuropter-
ology for over a century (McLachlan 1886). Recent molecular genetics works have
supported the existence of a species complex (Duelli et al. 2017), showing cryptic
diversity in the group. One of the prasinoid morphotypes is known as “marianus” and
was previously considered as a valid species. e specimens INV10273 and INV07344
were identied as Pseudomallada marianus, by utilizing the key available for the Iberian
Peninsula in Monserrat (2016b). Duelli and Obrist (2019) established the synonymy
of P. marianus with P. prasinus, previously proposed by Hölzel (1973), based on Central
European specimens. In the former, authors state that the morphological characters
previously attributed to the “marianus” morph (i.e., larger size and bundled egg place-
ment) are the ones that dene the “real” P. prasinus. As such, smaller specimens belong-
ing to the “prasinus” species complex can’t yet be identied conclusively to species level
until the prasinoid morphotypes are well-dened and described as a species (Duelli and
Obrist 2019). However, the implications of this work on the Iberian Peninsula’s speci-
mens of the “prasinus” species complex are not clear and require further research. In
the present work, the NJ analysis was congruent with the morphological identication
based on the characteristics described in Monserrat (2016b) since it separately grouped
INV7344 and INV10273, which were identied as the “marianus” morphotype, but
failed to retrieve P. prasinus as monophyletic. In contrast, the ABGD analysis grouped
all specimens of P. prasinus and P. abdominalis. Additionally, the intraspecic distance
between DNA barcode sequences of P. prasinus (2.25%) was higher than expected rela-
tive to the other species in our dataset. Our results, albeit limited, provide additional
support to the existence of cryptic diversity in P. prasinus. Identication through DNA
barcoding may prove problematic until the taxonomy of the group is better resolved,
and will likely benet of the use of other DNA markers.
A more complex situation is that of Chrysoperla carnea, C. lucasina, C. pallida,
C. agilis and C. mediterranea, in which all obtained sequences are grouped by NJ,
ABGD and BIN analysis in a single unit. e ve species belong to the so-called
C.carnea species complex (ierry et al. 1998; Henry et al. 2002, 2013). So far, the
most reliable way to identify the species in this group is by their substrate-borne vi-
brational songs, produced by tremulation (Henry and Wells 2015). Even though these
are not used for attraction of mates at long-distances as in many other animals, these
signals are produced for close-range recognition of sexually receptive mates (Henry et
al. 2002, 2003, 2012). e obtained results for the species of the group are congruent
with previous studies (Lourenço et al. 2006; Morinière et al. 2014) and might be a
result of the pre-copulatory reproductive isolation and the recent and rapid speciation
of this group of species (Henry et al. 2013). Considering the obtained data and the
available literature, a COI-based DNA barcode is not a feasible tool for species identi-
cation in this species complex.
e analysis of the sequences obtained from Dilaridae specimens yielded the highest
intra and interspecic genetic distances of all studied species. e intraspecic genetic
diversity in Dilar meridionalis was 3.67% (N = 4), while the genetic distance between the
Daniel Oliveira et al. / ZooKeys 1054: 67–84 (2021)
78
D. meridionalis and D. saldubensis was 17.7%. Since previous works on DNA barcoding
of Neuroptera have poorly (Yi et al. 2018) or not represented (Morinière et al. 2014) the
family at all, further sampling and sequencing would be needed to access the validity of
DNA barcoding based on the COI gene for identication of species in this family.
e two species of Wesmaelius were separated in the NJ analysis as by morphology,
though ABGD failed to recover two distinct groups. Furthermore, both the ID engine
and BIN analysis in BOLD systems clearly separated the species and grouped the se-
quences in BINs with other sequences available in the BOLD database of the same two
species. Considering these results, we suggest that COI DNA barcode sequences may
be used in the identication of these two species.
Another species that presents more than one BIN is Sympherobius pygmaeus. e
genetic diversity observed is congruent with previous work (Morinière et al. 2014) and
further research is needed to verify if it is a case of cryptic diversity.
In our dataset, all species of Raphidioptera showed relatively low intraspecic di-
vergence when compared with the respective interspecic distances. Despite the low
number of DNA barcode sequences available and the absence of three of the six species
in the dataset, the obtained results suggest that a DNA barcoding approach using a
COI gene fragment may be used to discern between species of Portuguese Raphidiop-
tera. is assumption is reinforced by the fact that all six species in the country belong
to six dierent genera and are, as such, predicted to show relatively high interspecic
distances between them.
For the large majority of encompassed species, DNA barcoding appears to be a re-
liable method of identication. While DNA barcoding cannot replace morphological
taxonomy experts entirely, especially in taxa where the taxonomy still needs revision, it
can aid in species identication in cases where morphology cannot be used. For exam-
ple, in diet analyses, where only small body parts (or none at all) can be retrieved, using
DNA barcoding may be the only method suitable for species identication, allowing
the understanding of species interactions and their roles in the ecosystems.
Currently 73 species of Neuropterida present in Portugal have DNA barcoding
data available, comprising the 54 species encompassed in this work and the 19 already
available in the BOLD database from other countries. Nonetheless, 29 species known
to occur in Portugal remain without DNA barcode available and further eorts are
needed to ll this gap.
Conclusion
is work provides novel data on the DNA barcoding and geographical distribution of
Neuroptera and Raphidioptera species in Portugal. Our results suggest that DNA bar-
coding using COI Folmer region may be used to identify the great majority of species
of Neuroptera and Raphidioptera species recorded in the country. It is not, however,
suitable for identication of several species of the Chrysopidae family. In total, there
were 22 cases where the rst publicly available DNA barcode sequence for a species
DNA Barcoding of Portuguese Lacewings and Snakeies 79
was obtained but further sampling and sequencing eorts are still needed for many.
e completion of DNA barcode databases is an ongoing eort and, in the cases of
Neuroptera and Raphidioptera, still require much work, including in Europe, where
several species are not yet sequenced. e future, however, looks bright as international
initiatives are promoting and aiding in the development of DNA barcode sequences
databases for particular regions worldwide (Letardi 2019).
Acknowledgements
InBIO Barcoding Initiative is funded by the European Union’s Horizon 2020 Research
and Innovation programme under grant agreement No 668981 and by the project
PORBIOTA – Portuguese E-Infrastructure for Information and Research on Biodiver-
sity (POCI-01-0145-FEDER-022127), supported by Operational ematic Program
for Competitiveness and Internationalization (POCI), under the PORTUGAL 2020
Partnership Agreement, through the European Regional Development Fund (FED-
ER). SF was supported by individual research contract (2020.03526.CEECIND)
funded by FCT. e eldwork beneted from EDP Biodiversity Chair, the project
“Promoção dos serviços de ecossistemas no Parque Natural Regional do Vale do Tua:
Controlo de Pragas Agrícolas e Florestais por Morcegos” funded by the Agência de De-
senvolvimento Regional do Vale do Tua, and includes research conducted at the Long
Term Research Site of Baixo Sabor (LTER_EU_PT_002).
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Supplementary material 1
Summary table of all used sequences and specimens, with country of origin
Authors: Daniel Oliveira, Sónia Ferreira
Data type: Occurences and access codes to DNA barcodes
Explanation note: For captured specimens, sex, latitude and longitude coordinates
(WGS 84), date of capture and IBI reference collection code (IBI) are presented.
BOLD accession numbers and BINs (when available) presented for all DNA se-
quences used..
Copyright notice: is dataset is made available under the Open Database License
(http://opendatacommons.org/licenses/odbl/1.0/). e Open Database License
(ODbL) is a license agreement intended to allow users to freely share, modify, and
use this Dataset while maintaining this same freedom for others, provided that the
original source and author(s) are credited.
Link: https://doi.org/10.3897/zookeys.1054.64608.suppl1
Daniel Oliveira et al. / ZooKeys 1054: 67–84 (2021)
84
Supplementary material 2
Genetic distances
Authors: Daniel Oliveira, Sónia Ferreira
Data type: Genetic distances between analysed specimens
Explanation note: Estimates of average genetic divergence (uncorrected p-distances)
for species of Neuropterida. Values under the diagonal refer to interspecic diver-
gence while values in the diagonal and in bold represent intraspecic divergence.
Copyright notice: is dataset is made available under the Open Database License
(http://opendatacommons.org/licenses/odbl/1.0/). e Open Database License
(ODbL) is a license agreement intended to allow users to freely share, modify, and
use this Dataset while maintaining this same freedom for others, provided that the
original source and author(s) are credited.
Link: https://doi.org/10.3897/zookeys.1054.64608.suppl2
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