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Onychogomphus cazuma sp. nov. from Spain: Molecular and morphological evidence supports the discovery of a new European dragonfly species (Odonata: Gomphidae)

Authors:
  • Museo Nacional de Ciencias Naturales MNCN-CSCI Real Jardín Botánico RJB-CSIC
  • Consejería de Agricultura
  • Sociedad Odonatologica de la Comunitat Valenciana

Abstract and Figures

Onychogomphus cazuma Barona, Cardo & Díaz sp. nov. is described from the mountainous inland area of Valencia in central-eastern Spain. The new species presents a combination of morphological characters that distinguishes it from all other species of the genus and can be readily identified by the morphology of the male appendages and the female vulvar scale, and by the shape of the median lobe of the prementum and the labial palps of the exuvia. Molecular analysis of two genetic markers, one nuclear and one mitochondrial (PRMT and COII), supports the full species rank for this new taxon, which is sister to the northwestern African endemic O. boudoti. Despite its small known distribution and the vulnerability of its habitat, available data are still insufficient to place this new species into an IUCN Red List of Threatened Species category.
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Onychogomphus cazuma sp. nov. from Spain 125
Odonatologica 49(1/2) 2020: 125-154
1st June 2020
Odonatologica 49(1/2) 2020: 125-154 – DOI:10.5281/zenodo.3823337
Onychogomphus cazuma sp. nov. from Spain:
Molecular and morphological evidence supports
the discovery of a new European dragony species
(Odonata: Gomphidae)
E. Karen López-Estrada1,2,5, Javier Barona Fernández3, Nuria Cardo-
Maeso4, Santiago Teruel Montejano3 & Cecilia Díaz-Martínez4,5
1 Museo Nacional de Ciencias Naturales, MNCN-CSIC, José Gutiérrez Abascal, 2,
28006 Madrid, Spain
2 Real Jardín Botánico RJB-CSIC, Plaza de Murillo, 2. 28014 Madrid, Spain
3 Parotets, Sociedad Odonatológica de la Comunitat Valenciana,
C/ Padre Vicente Cabanes, 5, 2, 12, 46900 Torrent, Valencia, Spain
4 Sociedad Entomológica de Castilla-La Mancha, C/ Londres, 7,
45003 Toledo, Spain
5 Corresponding authors: <lokaren21@gmail.com>; <cdiaz.cuenca@gmail.com>
Received 31st March 2020; revised and accepted 27th April 2020
Abstract. Onychogomphus cazuma Barona, Cardo & Díaz sp. nov. is described from the
mountainous inland area of Valencia in central-eastern Spain. e new species presents a
combination of morphological characters that distinguishes it from all other species of the
genus and can be readily identied by the morphology of the male appendages and the fe-
male vulvar scale, and by the shape of the median lobe of the prementum and the labial palps
of the exuvia. Molecular analysis of two genetic markers, one nuclear and one mitochondrial
(PRMT and COII), supports the full species rank for this new taxon, which is sister to the
north-western African endemic O.boudoti. Despite its small known distribution and the
vulnerability of its habitat, available data are still insucient to place this new species into an
IUCN Red List of reatened Species category.
Further key words. Anisoptera, Iberia, Valencia, taxonomy, phylogeny
Introducon
Gomphidae, distributed all over the world except in Antarctica, is the third
largest family of Odonata with 1 013 species described (S  P
2020). is species richness is heterogeneously distributed over the globe
(D K 2012); in the Western Palaearctic region, only 22
gomphid species are known, while in similar latitudes in North America,

126
Odonatologica 49(1/2) 2020: 125-154
gomphid diversity is considerably higher (D L 2006;
K et al. 2008; B  K 2015).
At present, 40 species are classied in the genus Onychogomphus Selys, 1854
(S  P 2020), distributed over the Palaearctic, Afrotropical and
Oriental regions (D  K 2012; F et al. 2014b). It is
almost certain that this genus is polyphyletic and probably all tropical species
should be moved to other genera (D  K 2012).
ree species of Onychogomphus have been reported so far from the Ibe-
rian Peninsula but none are endemic to this area. eir life-cycle, behav-
iour, larval and adult morphology, geographic distribution and ecological
dynamics are relatively well known (F-R et al. 1999; R-
 2003; C V 2009; P 2018; B  K
2015; V-V et al. 2018).
Only one phylogenetic hypothesis including Western Palaearctic Onycho-
gomphus species has been developed (F et al. 2014b). In that study,
a new lineage from Morocco was described based on molecular (COII and
PRMT genetic markers) and morphological evidence: Onychogomphus bou-
doti Ferreira, 2014. e relationships of that new taxon were not fully re-
solved in the estimated phylogenies. However, it is not expected that O.bou-
doti is closely related to the Afrotropical fauna, since morphologically it is
similar to the Western Palaearctic species.
In 2017, while carrying out a study on threatened odonates in the Escalona
river basin, in the province of Valencia in Spain (B 2017), individuals
that diered morphologically from all other Western Palaearctic Onycho-
gomphus were observed. In 2019, additional eldwork in eastern areas of the
Iberian Peninsula yielded males, females, larval stages and exuviae of these
particular populations. e hypothesis that these individuals represented an
undescribed taxon is tested in this study using morphological and molecu-
lar data, resulting in the description of a new species.
Material & methods
Data collection
Initial eld trips were carried out in 2017, visiting 15 watercourses in the Es-
calona river basin (B 2017), including rivers Cazuma, Fraile, Grande,
and Barranc de la Manyana (Fig. 1).
Onychogomphus cazuma sp. nov. from Spain 127
Odonatologica 49(1/2) 2020: 125-154
Figure 1           Onycho-
gomphus-
Onychogomphus cazuma sp. nov.

128
Odonatologica 49(1/2) 2020: 125-154
We reviewed specimens in ve entomological collections (collection
acronyms are given in parentheses): Consejo Superior de Investiga-
ciones Cientícas, Museo Nacional de Ciencias Naturales, Madrid, Spain
(MNCN); Institut Cavanilles de Biodiversitat i Biologia Evolutiva, Uni-
versitat de València, Spain (ICBIBE); Colección de Entomología del Área
de Biología Animal del Departamento de Zoología y Antropología Física,
Universidad de Murcia, Spain (UMCZ); Colección de entomología, Cen-
tro Iberoamericano de la Biodiversidad, Universidad de Alicante, Spain
(CEUA); Museu de Ciencias Naturales La Salle, Paterna, Valencia, Spain
(CLS-MHLP).
In addition, we checked the following citizen science websites for Onycho-
gomphus pictures from eastern Spain: https://www.biodiversidadvirtual.
org/; http://www.bdb.gva.es/es; https://observation.org/. Finally, we con-
tacted local photographers and amateur entomologists and reviewed our
own photographic archives, gathering additional pictures of Onycho-
gomphus individuals from the Comunidad Valenciana (Fig. 1).
As a result of the preliminary outcomes of the entomological collection
and photographic les surveys, in 2019 we extended eldwork to two addi-
tional sites (La Tosquilla, Rambla del Ral, Fig. 1) where specimens with the
features under study were expected.
Measurements [mm] were taken using a digital calliper. Laboratory pho-
tographs were taken with a Leica M165C stereoscope coupled with a Leica
DFC450 camera and processed with the program Leica Application Suite X.
e map was created in QGIS v.3.0.1, using Universal Transverse Mercator
(UTM) coordinate system.
DNA extraction and sequencing
Genomic DNA was extracted from a tissue sample of the thoracic muscle
of the hind leg using the ‘DNA Easy extraction Kit’ (Qiagen®) following the
manufacturer’s protocol. Partial sequences of the nuclear arginine methyl-
transferase (PRMT) and mitochondrial cytochrome oxidase subunit II
(COII) genes were amplied by polymerase chain reaction (PCR) using the
following set of primers: ARG_F2/ARG_R3 (F et al. 2014a) and
COII-F-SF/COII-R-SF (F et al. 2014b) respectively. ese mark-
ers were selected based on their proven eciency in previous phylogenetic
Onychogomphus cazuma sp. nov. from Spain 129
Odonatologica 49(1/2) 2020: 125-154
and phylogeographic studies of Odonata (B et al. 2008; F et al.
2014a, 2014b; C et al. 2015). PCRs were carried out in 25 L of nal re-
action volumes containing: 17.5 L of H2O, 2.5 L of a reaction buer with
MgCl2, 1 L of dNTP, 0.1 L of MgCl2, 0.5 L of each primer, 0.2 L of Taq
polymerase (Nzytech©) and 2 L of specimen DNA; reaction thermal cy-
cling conditions were set as same as F et al. (2014b). PCR products
were sent to Macrogen© Spain to be sequenced in both directions by Sanger
sequencing. Chromatograms were assembled and edited using Geneious®
v11.0.5.
Phylogenetic analyses
Phylogenetic reconstruction was performed by assembling separate data sets
for each gene (COII and PRMT) including sequences of Onycho gomphus
and related genera available in GenBank. Both matrices were aligned sepa-
rately using the MAFFT algorithm through their online server (K et
al. 2017).
Bayesian analyses were performed with MrBayes version 3.2.6 (R
et al. 2012). Selection of the best model of substitution for each gene was
calculated by setting the command lset nst to mixed. MrBayes analysis con-
sisted of two simultaneous runs of 100 million generations each, sampling
trees every 10 000 generations. Mixing and convergence among runs were
evaluated by checking the average standard deviation of split frequencies
and the EES values and Potential Scale Reduction Factor for each parameter.
A majority consensus tree was reconstructed aer discarding the rst 20 000
sampled trees as burn-in. COII data set included 35 terminals and 732 pb
while PRMT data set contained 21 terminals and 511 pb. Taxa included in
this analysis, locality, GenBank accession and voucher numbers are pro-
vided in Table 1 and Appendix 1.
All analyses were run in the public resource CIPRES Science Gateway ver-
sion 3.3 (M et al. 2010).
Abbreviations
FW – Fore wing; HW – hind wing; Pt – Pterostigma; Ax – Antenodal cross-veins;
Px– Postnodal cross-veins; S1–S10 – abdominal segments one to ten; m a.s.l. – me-
tres above sea level.

130
Odonatologica 49(1/2) 2020: 125-154
Results
During 2017 and 2019, we collected the following material of the putative
new species: nine adult specimens (6 3), three larvae and ten exuviae.
Additionally, we photographed 19 adult individuals (15 4) and observed
a further 19 adults (17 2) (Table 1). In 2017, adult individuals with dis-
tinct morphology were observed in sympatry with Onychogomphus uncatus
(Charpentier, 1840) and represented from 16.7 % of total Onychogomphus
individuals in the Cazuma river up to 100 % in the Barranc de la Manyana
(B 2017).
Altogether, 184 Onychogomphus specimens were examined in ve en-
tomological collections (104 in MNCN; 67 in ICBIBE; 10 in UMCZ; 3 in
CEUA; 0 in CLS-MHLP). Four dry-preserved specimens apparently related
to the putative new species were found, three in ICBIBE (Table 1) and one in
UMCZ. e former have the diagnostic features of the new species and have
been designated as paratypes. A detailed morphological examination of the
latter specimen, collected in the 1950s in Barinas, Murcia, revealed that it is
a chimaera, as head, thorax and rst segments of the abdomen belong to a
specimen of O. forcipatus that is glued to the last four segments of an abdo-
men with the features of the new species. is specimen was excluded from
the type series because we could not attribute it to a locality (the label can
correspond to either or both specimens).
Type series of adult specimens and exuviae samples have been deposited
in the following institutions: in the MNCN, male holotype and one female
paratype dry-preserved and seven paratypes in alcohol, and in the ICBIBE
three paratypes dry-preserved and two exuviae. In addition, larvae were
reared by the authors.
We gathered pictures of 241 individuals of Onychogomphus, nding eight
adults of the putative new species in 5 UTM 1 × 1 km squares in the Comu-
nidad Valenciana (Table 1). From all data sources combined, we collected
304 records of Onychogomphus individuals from 122 UTM 1 × 1 km squares
(Fig.1). e new species has been found in nine UTM 1 × 1 km squares
(7.4 %) while O. forcipatus and O. uncatus have been found in 60 (49.2 %)
and 79 (64.8 %) squares, respectively.
No additional sites were identied by reviewing 378 pictures of Onycho-
gomphus from eastern Spain on citizen science platforms.
Onychogomphus cazuma sp. nov. from Spain 131
Odonatologica 49(1/2) 2020: 125-154
Table 1. Sites and records for Onychogomphus cazuma -
-


Site UTM Elev. Records Voucher nr GenBank acc.
number
COII PRMT


de la Manyana
30SYJ
0532
100 09-vi-2017: 1
J. Barona

J. Barona & S. Teruel

Bicorp,
Río Cazuma
30SXJ
8531
30SXJ
8431
435 14-vi-2017: 1
J. Barona
14-vii-2017: 4
J. Barona, N. Cardo & C. Díaz

Ent255172
MT
415339
MT
415344
23-vii-2017: 1

26-vii-2017: 1

08-viii-2017: 1
J. Barona
10-viii-201: 1
J. Barona
11-viii-2017: 2
1
J. Barona
12-viii-2017: 1
J. Barona
12-vi-2018: 1

M.J. Tarruella & S. Teruel
08-vi-2019: 1
J. Barona, N. Cardo, C. Díaz &
S. Teruel

Ent255173
MT
415340
MT
415345
16-vi-2019: 1

S. Teruel
22-vi-2019: 4 1
S. Teruel

132
Odonatologica 49(1/2) 2020: 125-154
Site UTM Elev. Records Voucher nr GenBank acc.
number
COII PRMT
29-vi-2019: 1
Y. Maggioto
05-vii-2019: 2
J. Barona

Ent255167
MT
415346
20-vii-2019: 1 coll.,
1
J. Barona

Ent268518

Ent255168
21-vii-2019: 1 1
1
J. Barona

Ent268519

Bicorp,

30SXJ
8830
321 23-vii-2017: 2
J. Barona.
30SXJ
8528
484 24-vii-2017: 1
J. Ordóñez & MJ. Tarruella

Quesa, Río

de Quesa
30SXJ
9128
281 14-vi-2016: 1
J. Pérez

Cortes de Pallás,

30SXJ
7048
434 03-vii-2019: 1

18-vii-2019: 1
1 coll., 1
N. Cardo & C. Díaz

Ent255169

Ent255170

Titaguas,

30SXK
5812
30SXK
5811
561 19-vi-2003: 1


00018882
24-vii-2003: 1


00052471



00050960
02-viii-2008: 1
S. Teruel
07-vii-2019: 10, 1
4 coll., 3 
J. Barona & S. Teruel

Ent255171
Onychogomphus cazuma sp. nov. from Spain 133
Odonatologica 49(1/2) 2020: 125-154
Onychogomphus specimens used for genec comparison
Site UTM Elev. Idencaon Voucher
number
GenBank accession
number
COII PRMT
Spain, Cuenca,

Río Júcar
30SWK6321 850 O. forcipatus 
Ent255162
MT
415341
MT
415348
Spain, Cuenca,

Río Júcar
30SWK6321 850 O. forcipatus 
Ent255163
MT
415342
MT
415349
Spain, Murcia,


 280 O. forcipatus 
Ent255161
MT
415350
Spain, Cuenca,
Uña, Río Júcar
30TWK8652  O. uncatus 
Ent255166
MT
415343
MT
415347
Phylogenetic reconstruction
e topologies of the mitochondrial and the nuclear phylograms were to-
tally congruent with each other (Fig. 2). Phylogenetic relationships for the
taxa analysed coincided with those previously proposed by F et al.
(2014b). Our sequences from specimens found at the Valdeganga and Chí-
camo rivers were recovered within the O. forcipatus clade, while the sample
from the Júcar river grouped with the O. uncatus clade. However, sequences
of specimens from the Cazuma river formed a separate group, sister to the
Moroccan O. boudoti (PP = 1).
Molecular evidence indicates that the specimens from the Cazuma river
represent a well-dierentiated evolutionary unit within the genus Onycho-
gomphus, sister to O. boudoti. is fact, together with the unique morpho-
lo gy of the specimens belonging to this clade, as discussed below, lead us to
describe these populations as representatives of a new species.
Onychogomphus cazuma Barona, Cardo & Díaz sp. nov.

Material studied
Holotype (MNCN_Ent 268518). España, Valencia, Bicorp, río Cazuma,
39.1108333°N, 0.853055°W (UTM 30SXJ8531), 435 m a.s.l., 20-vii-2019,

134
Odonatologica 49(1/2) 2020: 125-154
leg. J. Barona [white label, printed]; MNCN_Ent 268518 [white label, print-
ed], Holotypus, Onychogomphus cazuma Barona, Cardo & Díaz des. 2020
[red label, printed]. Mature male, dry-preserved in the Entomological Col-
lection of MNCN.
Paratypes (8 3).
5 in ethanol, labelled: »Río Cazuma, Bicorp (V), 30SXJ8531, 435 m, J.Ba-
rona, N. Cardo & C. Díaz, 14-vii-2017«; MNCN_Ent 255172/ »Río Ca-
zuma, Bicorp (V), 30SXJ8531, 435 m, Barona, Cardo, Díaz & Teruel, 08-vi-
-2019«; MNCN_Ent 255173/ »Río Cazuma, Bicorp (V), 30SXJ8531, 435 m,
J. Barona, 05-vii-2019«; MNCN_Ent 255167/ »Rambla del Ral, Cortes de
Pallás (V), 30SXJ7048 – 434 m, Cardo & Díaz, 18-vii-2019«; MNCN_Ent
Figure 2. Onycho-
gomphus        
   -
-
ated species.
Onychogomphus cazuma sp. nov. from Spain 135
Odonatologica 49(1/2) 2020: 125-154
255169/ »La Tosquilla, Titaguas (V), 30SXK5812, 561 m, Barona & Ter-
uel, 07-vii-2019«; MNCN_Ent 255171 [white labels, printed; in MNCN];
1 dry-preserved, labelled: »Río Cazuma, Bicorp (V), 30SXJ8531, 435m,
J. Barona, 21-vii-2019«; MNCN_Ent 268519 [white labels, printed; in
MNCN]; 2 in ethanol, labelled: »Río Cazuma, Bicorp (V), 30SXJ8531,
435 m, J. Barona, 20-vii-2019«; MNCN_Ent 255168/ »Rambla del Ral,
Cortes de Pallás (V), 30SXJ7048 - 434 m, Cardo & Díaz, 18-vii-2019«;
MNCN_Ent 255170 [white labels, printed; in MNCN]; 3 dry-preserved,
labelled: »La Tosquilla, Titaguas (V), 30SXK5812, 600 m, P. González, J.M.
Michelena, 19-vi-2003«; MUVHN_ENV00018882/ »La Tosquilla, Titaguas
(V), 30SXK5812, 600 m, P. González, J.M. Michelena, 24-vii-2003«; MU-
VHN_ENV00052471/ »La Tosquilla, Titaguas (V), 30SXK5812, 600 m,
J.Baixeras, J.M. Michelena, 09-ix-2003«; MUVHN_ENV00050960 [col-
lecting data in white labels, printed; voucher number in red labels, printed;
in ICBIBE].
All paratypes labelled: »Paratypus, Onychogomphus cazuma Barona, Car-
do & Díaz des. 2020« [red labels, printed].
Exuviae (1 1)
Dry-preserved, labelled: »Río Cazuma, Bicorp (V), 30SXJ8531, 435 m,
S. Teruel, 12-vi-2019«; MUVHN_ENV00052558/ »La Tosquilla, Titaguas
(V)/30SXK5812, 600 m, J. Barona & S. Teruel, 07-vii-2019«; MUVHN_
ENV00051056 [collecting data in white labels, printed; voucher number in
red labels, printed; in ICBIBE].
Etymology
e epithet cazuma (noun in apposition and, therefore, invariable) refers to
the type locality, which was also the site where we rst realized that it could
be a new species.
Male (holotype)
Head – Eyes light blue, when alive; frons, anteclypeus, postclypeus and la-
brum yellow, bordered by black; base of mandibles yellow, sclerotized ex-
tremities brown; labium yellow with median lobe bordered by brown; ver-
tex black with a yellow rounded spot in line with the central ocellus and as

136
Odonatologica 49(1/2) 2020: 125-154
wide as it; occiput trapezium-shaped and yellow; 4-segmented antennae
mainly black (agellum and pedicel), except basal segment (scape) yellow
(Fig. 3a).
orax – Prothorax black with yellow marks: anterior lobe black with yel-
low anterior margin; median lobe with four yellow spots (two central and
two lateral); posterior lobe yellow divided into two pieces by a furrow,
which is black at the anterior margin. Synthorax in dorsal view: the yel-
low collar (transverse crest at the front of synthorax dorsum) and the yel-
low area posterior to it, are not interrupted with black, except for a faded
black dot at the junction of the dorsal carina and the collar; dorsal carina
yellow, ante-alar crest black, ante-alar sinus black and yellow. Black mid-
dorsal stripes on both sides of dorsal carina, yellow post-dorsal stripe is
connected with yellow area adjacent to collar and with yellow ante-humeral
stripe. In lateral view, mesepimeron, metepisternum and metepimeron yel-
low and bordered by black stripes. Between the humeral (or mesothoracic
pleural) and metapleural (or metathoracic pleural) sutures, black stripes
frame two characteristic yellow spots: superior hammer-shaped, and infe-
rior bow-shaped with the metastigma in its centre. Yellow patch between
the metapleural suture and the metathoracic coxa (Fig. 3b). Legs: Coxae
and trochanters mainly yellow; black femora with yellow marks, the meta-
thoracic femur has a yellow oval proximal spot at the exterior of the leg.
e joint that articulates femur and tibia is yellow, and the tibiae, tarsi and
pretarsi black (Fig. 3b).
Wings – Hyaline, venation dark brown except for the whitish costa of FW
and HW. Pt black bordered by black veins, above 3 cells in FW and 2 ½–3½
cells in HW; 11 antenodal cross-veins in FW and 8 in HW; 7–8 Px in FW
and 7–8 in HW; anal triangle 3-celled (Fig. 3a).
Abdomen Black with mid-dorsal spots, yellow in S1 and S2, fading to
whitish from S3 to S7, and yellow again in S8–S10, which gives the speci-
men a pale overall appearance. Wider at S1–S2 and from S7 to S10. Two yel-
low auricles in S2. In dorsal view, spot in S1 is yellow and triangular-shaped,
in S2 it is elongated, and in S3–S7 are bilobed, with proximal lobes extend-
Onychogomphus cazuma sp. nov. from Spain 137
Odonatologica 49(1/2) 2020: 125-154
Figure 3. Onychogomphus cazuma
-



138
Odonatologica 49(1/2) 2020: 125-154
ing laterally and ventrally, and distal lobe is lanceolate, except for S7 where it
is bid. In S8 spot is bid and does not extend laterally. In S9, a yellow band
at the distal border extends laterally and ventrally, interrupted in the mid-
dle by a black line. S10 is black at the proximal end and mainly yellow at the
distal end (Fig. 3a). Secondary genitalia are mainly black, with yellow spots
on penis vesicle and anterior face of genital lobe. Anterior lamina rounded,
posterior hamule ends in a prominent and sharp hook; oblong genital lobe
is 1.5 × longer than posterior hamule. Glans of penis ends in two short and
straight agella or cornua (Fig. 3c).
Anal appendages – Superior anal appendages yellow; more than twice the
length of S10. eir angled distal ends do not overlap, and in distal view
they present a jagged margin. Inferior anal appendage (epiproct) yellow,
bid and slightly longer than the superior anal appendages. Its distal end,
curved upwards, is bezel-shaped. In sub-basal position, the epiproct has
two cuticular laminar expansions that in lateral view appear as a ridge as-
cending towards the proximal end, culminating in a peak straight upwards
(Fig. 3d, 4e). ese expansions do not project outwards in dorsal view
(Fig.3e).
Measurements [mm]: Total length (frons to end of anal appendages): 45.2;
Abdomen total length: 32.0; Abdomen (excluding anal appendages): 29.5;
FW: 27.5; HW: 26.8; Pt length: 3.2 in FW and HW.
Variation in male paratypes
On the thorax dorsum, the black dot at the junction of the dorsal carina and
the collar may be more or less marked and even absent. e shape of the
markings on the sides of the thorax is slightly variable (Fig. 4d). Some males
have tiny yellow markings at the proximal end of the tibiae, especially in the
hind pair of legs. CAZ1 has two black symmetrical spots between the mid-
dorsal and ante-humeral black stripes.
Total length varies between 43.1–46.4 mm. Ax FW 10–13, HW 7–10. Px
FW 6–8, HW 8–9. Pt is above 3–4 cells in FW and HW. In HW, anal triangle
has 3 cells in holotype and paratypes, but in one of the photographed indi-
viduals (Fig. 4c) has 4 cells.
Onychogomphus cazuma sp. nov. from Spain 139
Odonatologica 49(1/2) 2020: 125-154
Figure 4. Onychogomphus cazuma    
-

  


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Figure 5.   Onychogomphus cazuma 
   

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Variation in female paratypes
In lateral thorax marks, a yellow bow-shaped spot is connected to yellow
mesepimeron (Fig. 5b), and a yellow hammer-shaped spot is isolated in two
female paratypes (Fig. 5b) and connected to bow-shaped spot in the third
one.
Females abdominal colour pattern is similar to that of male, except for
yellow spots on S7–S8: spot in S7 is not deeply divided as in males; spot in
S8 is smaller than in males, variable in shape but never bid (Fig. 5a). In fe-
males, the tibiae of the rst and third pair of legs have yellow lines (Fig. 5b).
e vulvar scale (Fig. 5c) is rounded and divided into two pointed lobes
by a central cle, which is as deep as half the vulvar scale’s length. Postgenae
(surface posterior to the eyes) are smooth, lacking tubercles or other struc-
tures (Fig. 5d).
Total length varies between 44.0 and 47.5 mm. Ax FW 10–13; HW 8–9.
Px FW 7–9; HW 6–11. Pt is above 3–4 ½ cells in FW and above 3 ½–4 cells
in HW.
Exuvia
Coloration is brown with darker wing sheaths, S10 and darker paraprocts in
ventral view (Figs 6a, c).
Head – wider than long, with rounded eyes and sides of occiput rounded
and converging posteriorly. Four-segmented antennae; third segment is the
largest, dorsoventrally attened and concave, densely covered laterally with
long setae; fourth segment tiny and conical (Fig. 6d). Prementum at; pre-
mentum–postmentum joint reaching posterior margin of procoxae. Pre-
mentum subquadrangular, median lobe waved; its apical margin concave
in the middle with a ventral row of 14–16 short and rounded teeth, as well
as dorsal rows of brownish piliform setae. Labial palps curved inwards and
pointed, internal margin with 10-11 teeth. Movable hook acutely pointed
(Fig. 6e).
orax – Prothorax small, narrower than head and paler than synthorax.
Divergent wing sheaths, reaching S5. Stout legs with abundant setae and
spines. Femora distally with a darker marking. Pro- and mesotibiae longer

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Figure 6: Onychogomphus cazuma    

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than femora, metatibiae shorter than femora. A spur is present in pro-and
mesotibiae and absent in metatibiae. Light-coloured tarsi, tarsal formula
2-2-3.
Abdomen – Brown, in ventral view becoming darker distally, in dorsal view
with dark markings on S2–S8, and in lateral view with dark spots on S2–
S8. Mid-dorsal spines absent on S1 and S10, and prominent from S2 to S9
(Fig.6b). Lateral spines present on S7, S8 and S9, which diverge from the
edge of the segment (Fig. 6a). Anal appendages longer than S10. Epiproct
triangular, slightly longer than paraprocts and longer than cerci.
Measurements [mm] total length (including paraprocts) 20.90–22.26; third
antennal segment: length 1.30–1.49 mm, width 0.54–0.60 mm; prementum:
length 3.20–3.49, basal width 1.90–2.10; distal width 3.10–3.40mm; maxi-
mum width 3.10–3.40 mm; prementum movable hook length 1.10; para-
procts length 1.10–1.20; epiproct length 1.43–1.58; cerci length 1.08–1.32
(n = 10).
Diagnosis
By its overall appearance, Onychogomphus cazuma sp. nov. is close to O. un-
catus, O. forcipatus and O. boudoti, sympatric in the Atlanto-Mediterranean
region, but can be easily distinguished by morphological details. In O. cazu-
ma, the male epiproct has cuticle expansions in a sub-basal position, which
in O. uncatus and O. forcipatus are tooth-shaped and project outwards in
dorsal view. In O. boudoti and O. cazuma these expansions are laminar and
do not project outwards, but while in O. boudoti they are smooth, bare-
ly prominent in lateral view, in O. cazuma they appear as a ridge ascend-
ing towards the proximal pole and culminating in a peak straight upwards
(Fig.7).
In females of O. cazuma, postgenae lack post-ocular tubercles as in O.un-
catus, but unlike that species the vulvar scale has two broad lobes (as in
O.forcipatus and O. boudoti) divided by a median cle. e length of this
median cle is almost as long as the vulvar scale in O. forcipatus, half the
length of the vulvar scale in O. cazuma, and two-thirds of the length of the
vulvar scale in O.boudoti (Fig. 7).

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e colour pattern of O. cazuma diers from that of the mentioned conge-
ners by the following combination: (i) black vertex with a rounded yellow
spot, (ii)yellow thoracic collar not interrupted by a thick black line, and
(iii)post-dorsal and ante-humeral yellow stripes are connected.
ese features may show variability, for instance in some individuals of
O. forcipatus and O. cazuma the collar may be narrowly interrupted by a
Figure 7. Onychogomphus
-
   
-

O. for-
cipatus and O. uncatus 2006.
O. cazuma O. boudo O. f. unguiculatus O. uncatus
Vertex
Synthorax
dorsal view
Synthorax
lateral view
Male
appendages
Vulvar
scale
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slight darkening near the mid-dorsal carina. Onychogomphus uncatus most
oen has a totally black vertex, although there are rare individual excep-
tions, while the collar is interrupted by a thick black line. Onychogomphus
Figure 8. Onychogomphus-
O.boudoO. costaeO. forcipatusO. uncatus
e – O. cazuma-
O. cazuma

a b
c d
e

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forcipatus has a yellow spot on the vertex that is not round but elongate, and
the post-dorsal and ante-humeral yellow stripes are not connected. Onycho-
gomphus boudoti has a round yellow spot on the vertex, as in O. cazuma,
but the collar is interrupted and the post-dorsal and ante-humeral yellow
stripes are not connected (Fig. 7).
e diagnostic characters can also be used to separate the nominotypical
O. forcipatus subspecies, O. forcipatus forcipatus (Linnaeus, 1758), recently
recorded in the North of the Iberian Peninsula (M-A 
T-B 2015; P L et al. 2017).
Lastly, O. cazuma exuviae and all larval instars observed so far dier from
all other regional Onychogomphus by the waved shape of the median lobe of
the prementum and the pointed distal end of the labial palps (Fig.8).
Ecology
Individuals of Onychogomphus cazuma sp. nov. were found at six water-
courses in the mountainous inland part of the province of Valencia (cen-
tral-eastern Iberian Peninsula, 39°N, 0.8°W, Fig. 1, Table 1), at elevations of
100–561 m a.s.l., running through Cretaceous and Jurassic limestone (G-
 et al. 1984). e climate is typically Mediterranean with a dry and
hot summer (Csa) according to the Köppen-Geiger classication (A
E  M  I  M  P-
 2011). In local weather stations, mean annual temperature ranges be-
tween 15.4–17.8°C and mean annual rainfall is 418.8–602.6 mm (S
 I G  D A 2020).
Onychogomphus cazuma inhabits springs, streams or upper courses of
small lowland Mediterranean rivers (100–561 m a.s.l.) in well-preserved for-
est environments with barely any agriculture, livestock or habitation. ese
small perennial limestone rivulets are typically 1–5 m wide and 2–60cm
deep, with low ow (mean annual ow <0.1 m³/s, IGME-DGA 2009) but
a torrential regime, with occasional strong oods. Water is very clean and
oligo trophic; calcium bicarbonate is the main mineral present and con-
ductivity exceeds 400µS/cm (IGME 1988; L S P
2019). e high content of calcium salts facilitates both chemical and bio-
logical precipitation of calcareous tufa including calcied plant or mollusc
fragments. e alternation of low slope shallow sections, small ries, falls
Onychogomphus cazuma sp. nov. from Spain 147
Odonatologica 49(1/2) 2020: 125-154
and pools up to 2–3 m deep oers macrohabitat diversity. e substrate is
dominated by bedrock with variable amounts of scattered cobbles, gravels
and sand, as well as limestone silt.
Aquatic vegetation is characterized by various unicellular algae growth in
the riverbed and scarce macrophytes (Chara L., 1753, and Nigella L., 1753).
Riparian vegetation consists of a Mediterranean riparian thicket of Neri-
um oleander L., 1753 (Rubo ulmifolii-Nerietum oleandrii, Bolòs, 1956), in-
terspersed with helophytes like Phragmites australis (Cav.) Trin. ex Steud.,
1841, Cladium mariscus (L.) Pohl, 1882, Schoenus nigricans L., 1753, and
other Poaceae and Cyperaceae. Steep rocky slopes by the river are mainly
occupied by Aleppo pine forests (Pinus halepensis Mill., 1768) and sclero-
phyllous scrub (Quercus coccifera L., 1753, Juniperus phoenicea L., 1753, Pis-
tacia lentiscus L., 1753, Salvia rosmarinus (L.) Schleid., 1852, Chamaerops
humilis L., 1753) (F G 2014).
Sections of the watercourses occupied by O. cazuma are very short, from
150 to 1 800 m. Resident males (1–15 per section) were observed at shal-
low waters (<15 cm) with a low discharge, bordered by dense stands (up to
70 %) of Poaceae and Cyperaceae such as C. mariscus or S. nigricans. While
males could be seen perched on stones and riverine vegetation, females visit
the riverbed only for mating and ovipositing. At the Cazuma river, both
males and females use cleared areas (abandoned elds) far from the river-
bed (30–50 m) as possible maturation areas, where they were seen perching
on vegetation, stones and ground. At La Tosquilla, several males were seen
perched close to each other.
A female was observed ovipositing into a pool (1 m depth) with thick
(30 cm) limestone silt at La Tosquilla (07-vii-2019). Larvae were found in
shallow water (<10 cm depth) and low-discharge sections, with a bottom
of gravel, calcied silt and leaf litter (1–4 cm). Most exuviae were found at
such sites too, clinging to herbaceous vegetation (C. mariscus, S. nigricans),
1–3cm above the water surface. e species was in ight from 08-vi to 09-
-ix, with three emergences recorded between 08- and 16-vi.
e Cazuma river (Fig. 4b) has a diverse community of Odonata, includ-
ing 37 species (B 2017; BDB 2020). In this area O. cazuma is sym-
patric with O. uncatus and O. forcipatus. Onychogomphus uncatus is also
present in La Tosquilla and the Fraile river. Notable is the overlap of O. ca-

148
Odonatologica 49(1/2) 2020: 125-154
zuma and Oxygastra curtisii (Dale, 1834) at all studied sites except Rambla
del Ral (Fig. 4f) (BDB 2020).
e possible ecological segregation of O. cazuma and O. uncatus was ob-
served in the Cazuma river, where O. cazuma was seen at sections with
slower ow and denser riparian vegetation, and in La Tosquilla, where
O.cazuma was present at a spring while O. uncatus inhabited the Turia river.
A female photographed in 2008 at La Tosquilla was trapped in an orb-
weaver spider (Larinioides Caporiacco, 1934) web. A teneral specimen was
collected on 08-vi-2019 in Río Cazuma while it was devoured by a male of
Gomphus graslinii Rambur, 1842.
Discussion
e discovery of a new species of dragony in Europe is a surprise. e
European fauna of Odonata is well-known, with just a few additions of
new species in the last ve decades, viz. Somatochlora borisi Marinov, 2001,
Cordulegaster helladica (Lohmann, 1993), C. heros eischinger, 1979, and
C.trinacriae Waterston, 1976. Furthermore, with Ischnura graellsii Rambur,
1842, the last new species described from Spain dates from the middle of
the 19th century.
e morphology of the male appendages and female vulvar scale sug-
gests a close relationship between Onychogomphus cazuma sp. nov. and
the Moroccan O. boudoti. Molecular phylogenetic reconstruction concurs
with this morphological evidence, indicating a sister relationship between
O. cazuma and O. boudoti. e discovery of O. cazuma claries the phylo-
genetic position of the Maghrebian O. boudoti, demonstrating the shared
evolutionary history of the south-western European and the North African
odonate species in the Western Palaearctic. Both O. boudoti and O. cazuma
are restricted to small geographic areas (cf. F et al. 2014b;  
M 2018).
Onychogomphus cazuma lives under similar conditions, including places
where emergence was observed, to those described for O. boudoti (F-
 et al. 2014b). We observed that in sympatry with other congeners, O.ca-
zuma seemed to prefer river sections with slower ow and more vegetated
banks, with helophyte and grass cover; but more extensive ecological re-
search is needed to conrm these preferences.
Onychogomphus cazuma sp. nov. from Spain 149
Odonatologica 49(1/2) 2020: 125-154
So far, O. cazuma has been only located within ‘lowland Mediterranean riv-
ers’ (see T et al. 2009 for the typology). is riverine habitat is found
along the Mediterranean Spanish coast from Girona to Cádiz, in the Ebro
and Guadalquivir basins and on the island of Mallorca. It is possible that
populations of O. cazuma could be present at more sites within this wider
area.
e close relationship between O. cazuma and O. boudoti allows us to
speculate that the geographic distribution of their presumed common an-
cestor could have dwelled in similar habitats of both the Iberian Peninsula
and the North of Morocco.
Conservation of the Iberian endemic Onychogomphus cazuma
Onychogomphus cazuma sp. nov. was found in relatively well-preserved
areas, although these are not exempt from human disturbance. e main
threats are water abstraction for domestic use and/or irrigation and recrea-
tional activities (like swimming or canyoning). Given the small size and low
ows of these watercourses, any increase of use can make a substantial dif-
ference in the availability of habitat for this species.
Furthermore, O. cazuma, as the only dragony endemic to Iberia (cf.
K et al. 2010), may need special protection measures. Following
the IUCN Red List Criteria (IUCN S  P S-
 2019), we have determined that the known extent of occurrence
(EOO) of the species is 1273 km² and the known area of occupancy (AOO)
is 32 km². Although the species was recorded at six sites, we consider only
ve locations (sensu IUCN). In these sites, the water level and seasonality
are heavily dependent on the groundwater table, some of them sharing the
same local aquifer – for instance the Cazuma river-Manyana and Fraile-
Grande rivers (IGME 1988; IGME-DGA 2009). Moreover, in recent decades
the length of the Cazuma river that is remaining dry all year (except aer
heavy rain events) has substantially increased, suggesting a decline in ex-
tent and quality of this habitat. However, we assume that the species could
be more widely distributed than currently known. erefore we suggest for
the moment that O. cazuma should be considered as »Data Decient« by
the criteria of the IUCN Red List of reatened Species. Further research is
essential to establish the species’ status and identify its conservation needs.

150
Odonatologica 49(1/2) 2020: 125-154
Acknowledgements
We are indebted to the following curators of Entomological collections:
Sergio Montagud (MUVHN), Emilio de la Fuente (La Salle), Cinta Quirce
(CIBIO), Juan José Presa (U. Murcia) and Mercedes París (MNCN); and
Arabia Sánchez (MNCN), José V. Pérez Santa-Rita and Joaquín Baixeras
(ICBIBE). We specially thank Mario García París for facilitating labora-
tory access at the MNCN, and also for comments and suggestions that
signicantly improved this study. K.-D.B. Dijkstra provided useful insights
on the species description process; Miguel Conesa helped with descrip-
tion and measurements of the exuviae; Alberto Sendra and Fernando
Alonso made useful suggestions on the manuscript; Begoña Cardo kind-
ly improved the pictures. Viola Clausnitzer helped with the IUCN Red
List assessment. Feli Fernández and Ben Crouch reviewed the language
of the manuscript. We are grateful to Florian Weihrauch for his kind as-
sistance and Jean-Pierre Boudot, Sónia Ferreira and Christophe Brochard
as reviewers of the manuscript for their helpful comments; Christophe
also kindly provided comparative photographs of the prementum of other
Onycho gomphus spp. We also thank all the photographers and amateur
entomologists who reviewed their photo archives and sent us pictures of
Onychogomphus: Ana María García, Francisco Cervera, Yanina Maggiotto,
Pablo Ruiz, Iván Moya, José Greño, Roque Belenguer and the following
members of Parotets S.O.C.V.: María José Tarruella, Jesús Ordóñez, Natxo
Sendra, María Jesús Sanchís, Juan Antonio Tornero, Ricardo Menor, Teo-
doro Martínez, Nicolau Mouline, Juan Antonio Tovar. Jesús Evangelio do-
nated a O. forcipatus specimen for comparison. We specially thank Jorge
Pérez and Ezequiel Prieto for eld company and helping in search for in-
formation, and Toni Alcocer for providing the beautiful pictures that led
to the discovery of a new site outside the Escalona river basin. Javier Ba-
rona’s eldwork in 2017 was supported by the Generalitat Valenciana (Dis-
tribución, abundancia y estado de conservación de odonatos amenazados
en la cuenca del río Escalona y su entorno; Canal de Navarrés, Macizo
del Caroche – Expte. CNME 17/0301/16). Subdirecció General de Medi
Natural (Generalitat Valenciana) and Dirección General de Medio Natural
(Región de Murcia) kindly granted capture permits.
Onychogomphus cazuma sp. nov. from Spain 151
Odonatologica 49(1/2) 2020: 125-154
 -

    
aire y precipitación (1971–2000). Minis-
  
-


estado de conservación de odonatos ame-
nazados en la cuenca del río Escalona y su
entorno (Canal de Navarrés, macizo del Ca-
-



BDB 2020. Banco de Datos de Biodiversidad
  
   

Eds) 2015-
      -




    
    Cladiscs 24: 477-
514
 Desarrollo larvario
de Onychogomphus costae Sélys, 1885 en
       
  Ophiogomphus ce-
cilia    -
dae). Bolen de la Sociedad Entomológica
Aragonesa 44: 327-332
       . 
    
ArthropodSystemat-
ics & Phylogeny 73: 281-301
-
    -
    -
OrganismsDiversity&Evo-
luon 12: 209-227

   -

   -
-
       

    -
     
Internaonal Journal of Odonatology 17:
135-147
    
      
       
    
Morocco: Onychogomphusboudosp. nov.
  Zootaxa 3856 (3):
349-365

-
-
   

    
 Onycho-
gomphus uncatus  
 -
     -

 Archiv für Hydrobio-
logie 144: 215-228
      
     Mapa geológico de
  
References

152
Odonatologica 49(1/2) 2020: 125-154
  -

Minero de España
     
-
 
y Minero de España. Madrid
    -
     -
    


   -
    
    
 
   -
       
España
    -
      

     
     
 

     
      -
        
  European Red List of dra-
 -

 
       
2008.  -
   Hydrobiologia 595:
351-363

    

BriengsinBioinformacs 20:
1160-1166
  -
-

  -
-
     -
ciana
   
. Primera cita de Onychogom-
phus forcipatus forcipatus (Linnaeus, 1758)
    
Bolende laSociedadentomológi-
ca aragonesa 57: 365-366
      
2010. 

-
 
(Louisiana): 1-8


seguimiento de la calidad de los ríos gui-
    -
 
BolendelaSociedadentomológicaarago-
nesa 61: 278-280
   Onychogomphus costae


Odonatologica 47: 1-22
      
     
gomphus uncatus  
Internaonal Journal of Odonatology 7:
65-71
      
       -

J.P. 2012.    
    
across a large model space. SystemacBio
lology 61: 539-542
Onychogomphus cazuma sp. nov. from Spain 153
Odonatologica 49(1/2) 2020: 125-154
 2020. World Odo-
nata List. 
 

    

     
   
     

 

3200. Tipo Ecológico Nº 9. Ríos minerali-
     
 

de interés comunitario en España. Minis-
 
Marino, Madrid
       
gedrag van Onychogomphus boudo in
     Bra-
chytron 19: 93-99

-
cles of Boyeria irene and Onychogomphus
uncatus
  Euro-
peanJournalofEntomology 115: 684-696

154
Odonatologica 49(1/2) 2020: 125-154
Appendix 1. 
Voucher
number
ID Reference GenBank accession code
COII PRMT
 Gomphus graslinii  KM222699 –
 Gomphus pulchellus  KM222700 –
 Gomphus pulchellus  KM222701 –
 Gomphus simillimus  KM222702 –
 Gomphus simillimus  KM222703 –
 Icnogomphusferox  KM222705 –
 Lindenia tetraphylla  KM222704 –
Df269 Onychogomphus assimilis  KM222694 KM222665
 Onychogomphusboudo  KM222674 KM222654
 Onychogomphus costae  KM222695 KM222672
KM222671
 Onychogomphus costae  KM222696 –
 Onychogomphus costae  KM222697 KM222662
 Onychogomphusexuosus  KM222675 –
 Onychogomphus forcipatus  KM222676 KM222656
 Onychogomphus forcipatus  KM222677 KM222656
 Onychogomphus forcipatus  KM222678 KM222661
 Onychogomphus forcipatus  KM222679 KM222660
 Onychogomphus forcipatus  KM222680 KM222661
 Onychogomphus forcipatus  KM222681 KM222660
 Onychogomphus forcipatus  KM222682 KM222670
 Onychogomphus forcipatus  KM222683 KM222660
 Onychogomphus forcipatus  KM222684 KM222656
 Onychogomphus forcipatus  KM222685 KM222656
 Onycogomphus uncatus  KM222686 KM222666
 Onycogomphus uncatus  KM222687 KM222659
 Onycogomphus uncatus  KM222688 KM222659
 Onycogomphus uncatus  KM222689 KM222659
 Onycogomphus uncatus  KM222690 KM222659
 Onycogomphus uncatus  KM222691 KM222659
 Onycogomphus uncatus  KM222692 KM222659
 Onycogomphus uncatus  KM222693 KM222655
... El género Onychogomphus Selys, 1854 contaba en la península ibérica con tres especies de anisópteros de carácter reófilo: Onychogomphus uncatus (Charpentier, 1840), Onychogomphus forcipatus (Linnaeus, 1758) y Onychogomphus costae Selys, 1885, esta última muy escasa a nivel penin-sular e incluida en la Lista Roja de los Invertebrados de España en la categoría "Vulnerable" (Prunier 2018, Verdú et al. 2011. Con el reciente descubrimiento de O. cazuma, se eleva a cuatro el número de especies del género para el territorio peninsular (Boudot & Kalkman 2015, López-Estrada et al. 2020. ...
... El conocimiento de la biología y ecología de O. cazuma se encuentra todavía en un estado muy incipiente. Hasta la fecha solo se había localizado en manantiales, arroyos y cursos superiores de pequeños ríos mediterráneos del interior de la provincia de Valencia, situados en entornos forestales bien conservados de zonas montañosas calizas, entre 100-561 msnm de altitud (López-Estrada et al. 2020). Por este motivo, cualquier dato obtenido a nivel peninsular fuera de su área de distribución principal resulta muy interesante, no solo para conocer mejor su distribución, biología y ecología, sino también para tomar las medidas de conservación oportunas en caso de que fueran necesarias. ...
... A pesar de que el arroyo Tobarra-rambla de Minateda, al igual que el resto de localidades donde se ha detectado O. cazuma, se incluye dentro de los "ríos mineralizados de baja montaña mediterránea" (Toro et al. 2009), el cercano arroyo de la Manga, caracterizado en Toro et al. (2009) dentro de los "ríos mediterráneos muy mineralizados", es uno de sus tributarios. Las aguas del arroyo de Tobarra-rambla de Minateda discurren por un ambiente algo distinto al descrito para las localidades valencianas, especialmente al que rodea al río Cazuma, localidad del holotipo de la especie (López-Estrada et al. 2020). Cruza el saladar de Agramón de norte a sur (ZEC "Saladares de Cordovilla, Agramón y laguna de Alboraj) La mayoría de los ejemplares se observaron entre el camino del Molino del Tabay (que cruza el cauce) y la desembocadura en el río Mundo, posados en ramas de carrizo (Phragmites australis) o en rocas y gravas de la orilla o del cauce (Fig. 1C), comportamiento este último más propio del género (Dijkstra & Lewington 2006). ...
Article
En el presente documento se aportan los primeros datos sobre la biología de Onychogomphus cazuma Barona, Cardo et Díaz, 2020 en la región de Castilla-La Mancha (centro-este de España), confirmando su reproducción en dicho territorio mediante el hallazgo de exuvias y una hembra recién emergida. Hasta la fecha la especie solo se había detectado en la provincia de Valencia, en localidades situadas a más de 100 km. The first data on the biology of Onychogomphus cazuma Barona, Cardo et Díaz, 2020 in Castilla-La Mancha region are provided. Its reproduction in this territory is confirmed by the detection of exuvians and a teneral female. The species had been only detected in the province of Valencia, in locations more than 100 km away.
... This is due to the occurrence of cryptic species and difficulties in larval and exuvial identification, which determine that most of studies rely on adults and are restricted to the their (often short) flying period (e.g., Bried et al. 2012;Solano et al. 2018;Galimberti et al. 2021). Therefore, as in other insect orders (e.g., Lepidoptera, Platania et al. 2020;Coleoptera, Kajtoch et al. 2018;and Hymenoptera;Lecocq et al. 2020), the integration of morphology and DNA-based identification approaches represent a promising and powerful tool for rigorous species identification and surveying of odonates, also in well-investigated areas (e.g., in the Mediterranean, see Ferreira et al. 2014a;López-Estrada et al. 2020). DNA-based approaches strongly rely on the correct taxonomic identification of the organisms of interest, since misidentifications may cause taxonomic inconsistencies in reference databases, and the work done by specialized taxonomists is therefore crucial for the success of such methods (e.g., Salvi et al. 2020). ...
Article
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Molecular-based approaches for species identification and delimitation strongly relies, in terms of universality and efficiency, on the selected markers. Conventionally, when adopting a DNA barcoding approach to discriminate (or identify) metazoans species, the marker choice falls on the 658 base pair region at the 5’ end of the mitochondrial COI gene. However, a growing number of studies suggest to use alternative and more variable genetic regions, even from the same gene, such as the 3’ end of the COI. In this work, we compared the identification performance of the 5’ and 3’ end COI regions on a large sequence dataset of odonate species, an order of arthropods among the most studied in terms of conservation importance for aquatic ecosystems. The genetic datasets comprised a total of 236 specimens, 113 species, 51 genera and 12 families spanning the two odonate suborders Zygoptera and Anisoptera, and were analysed under an integrative multiple approach including descriptive statistics and variability of the sequences, phylogenetic reconstructions, DNA-based species delimitations, genetic distances, identification of diagnostic characters and saturation plots. All analyses were congruent in recovering the COI-3’ region to be slightly more variable than the COI-5’ one, and both regions showed a saturation of transversion at the third codon position. However, phylogenetic reconstructions, genetic distances, and diagnostic characters identification resulted in a similar discrimination power for the two COI regions. Therefore, the COI-3’ region does not add much information to the standard barcode region, which has in turn largely been demonstrated to successfully delineate invertebrate communities through DNA and eDNA metabarcoding, and to have a much more extensive taxonomic coverage in public databases. Overall, the DNA barcoding inventory assembled in this study will provide valuable insights into the systematics and conservation of many odonate species with implications for future DNA and eDNA monitoring-based studies.
... Anisoptera, BOLD, cryptic species, Odonata, species delimitation, Zygoptera López-Estrada, Fernández, Cardo-Maeso, Montejano, & Díaz-Martínez, 2020;Viganò, Janni, & Corso, 2017). Traditionally, odonate identification has relied on morphological data that could be biased by difficulties in larval/exuviae recognition, the occurrence of cryptic species and even high introgression rates (Bried, D'Amico, & Samways, 2012;Bried & Hinchliffe, 2019;Marinov, 2001;Solano et al., 2018). ...
Article
The Odonata are considered among the most endangered freshwater faunal taxa. Their DNA‐based monitoring relies on validated reference datasets that are often lacking or do not cover important biogeographical centres of diversification. This study presents the results of a DNA barcoding campaign on Odonata, based on the standard 658 bp 5’ end region of the mitochondrial COI gene, involving the collection of 812 specimens (409 of which barcoded) from peninsular Italy and its main islands (328 localities), belonging to all the 88 species (31 Zygoptera and 57 Anisoptera) known from the country. Additional BOLD and GenBank data from Holarctic samples expanded the dataset to 1294 DNA barcodes. A multi‐approach species delimitation analysis involving two distance (OT and ABGD) and four tree‐based (PTP, MPTP, GMYC, bGMYC) methods were used to explore these data. Of the 88 investigated morphospecies, 75 (85%) unequivocally corresponded to distinct Molecular Operational Units, whereas the remaining ones were classified as ‘warnings’ (i.e., showing a mismatch between morphospecies assignment and DNA‐based species delimitation). These results are in contrast with other DNA barcoding studies on Odonata showing up to 95% of identification success. The species causing warnings were grouped in three categories depending on if they showed low, high, or mixed genetic divergence patterns. The analysis of haplotype networks revealed unexpected intraspecific complexity at the Italian, Palearctic, and Holarctic scale, possibly indicating the occurrence of cryptic species. Overall, this study provides new insights into the taxonomy of odonates and a valuable basis for future DNA and eDNA‐based monitoring studies.
... Anisoptera, BOLD, cryptic species, Odonata, species delimitation, Zygoptera López-Estrada, Fernández, Cardo-Maeso, Montejano, & Díaz-Martínez, 2020;Viganò, Janni, & Corso, 2017). Traditionally, odonate identification has relied on morphological data that could be biased by difficulties in larval/exuviae recognition, the occurrence of cryptic species and even high introgression rates (Bried, D'Amico, & Samways, 2012;Bried & Hinchliffe, 2019;Marinov, 2001;Solano et al., 2018). ...
Preprint
The Odonata are considered among the most endangered freshwater faunal taxa. Their DNA-based monitoring relies on validated reference datasets that are often lacking or do not cover important biogeographical centres of diversification. This study presents the results of a DNA barcoding campaign on Odonata, based on the standard 658 bp 5’ end region of the mitochondrial COI gene, involving the collection of 812 specimens (409 of which barcoded) from peninsular Italy and its main islands (328 localities), belonging to all the 88 species (31 Zygoptera and 57 Anisoptera) known from the country. Additional BOLD and GenBank data from Holarctic samples expanded the dataset to 1294 DNA barcodes. A multi-approach species delimitation analysis involving two distance (OT and ABGD) and four tree-based (PTP, MPTP, GMYC, bGMYC) methods were used to explore these data. Of the 88 investigated morphospecies, 75 (85%) unequivocally corresponded to distinct Molecular Operational Units, whereas the remaining ones were classified as ‘warnings’ (i.e., showing a mismatch between morphospecies assignment and DNA-based species delimitation). These results are in contrast with other DNA barcoding studies on Odonata showing up to 95% of identification success. The species causing warnings were grouped in three categories depending on if they showed low, high, or mixed genetic divergence patterns. The analysis of haplotype networks revealed unexpected intraspecific complexity at the Italian, Palearctic, and Holarctic scale, possibly indicating the occurrence of cryptic species. Overall, this study provides new insights into the taxonomy of odonates and a valuable basis for future DNA and eDNA-based monitoring studies.
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Around 27 species of predominantly riverine Gomphidae occur in the vast region encompassing Europe as far as the Urals, the Maghreb, the Mediterranean Basin, and the Middle East up to the west side of the Indus valley, including Arabia, Iran, and Balu-chistan. They are the remains of a pre-Pleistocene fauna that we estimate at twice the current number. We analyse the relationships, losses, and their causes at the molecular level and, not surprisingly, confirm the widely held opinion that the ice age is overwhelmingly responsible .
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Odonata is considered a "flagship" group of insects and its investigation is of primary importance especially for protected areas where freshwater ecosystems occur. In this study, we focused on Odonate fauna in the "Cansiglio Forest" (Veneto, Italy), a karst area where the only checklist available dates back more than 40 years ago. In order to update the Odonate adult distribution in the area, we selected 21 ponds that were sampled monthly, from May to August, during a 2-years survey. In total, 21 species (belonging to 14 genera and 5 families) have been recorded: we confirmed 15 species from the previous species list and we added to the whole species list 6 new species. Dominant families were represented by Libellulidae (33%) and Aeshnidae (23%), the most common genus was Sympetrum (19%), and the most frequent species was Coenagrion puella (63%). In term of patterns of species richness, highly grazed and pastured ponds exhibited the lower number of species and individuals, as a probable response to the high level of animal disturbance on the vegetation and due to the eutrophication processes. Our results are important also in terms of conservation and management of freshwater sites belonging to Natura 2000 site.
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Background. Dragonflies and damselflies (Odonata) are important components in biomonitoring due to their amphibiotic lifecycle and specific habitat requirements. They are charismatic and popular insects, but can be challenging to identify despite large size and often distinct coloration, especially the immature stages. DNA-based assessment tools rely on validated DNA barcode reference libraries evaluated in a supraregional context to minimize taxonomic incongruence and identification mismatches. Methods. This study reports on findings from the analysis of the most comprehensive DNA barcode dataset for Central European Odonata to date, with 103 out of 145 recorded European species included and publicly deposited in the Barcode of Life Data System (BOLD). The complete dataset includes 697 specimens (548 adults, 108 larvae) from 274 localities in 16 countries with a geographic emphasis on Central Europe. We used BOLD to generate sequence divergence metrics and to examine the taxonomic composition of the DNA barcode clusters within the dataset and in comparison with all data on BOLD. Results. Over 88% of the species included can be readily identified using their DNA barcodes and the reference dataset provided. Considering the complete European dataset, unambiguous identification is hampered in 12 species due to weak mitochondrial differentiation and partial haplotype sharing. However, considering the known species distributions only two groups of five species possibly co-occur, leading to an unambiguous identification of more than 95% of the analysed Odonata via DNA barcoding in real applications. The cases of small interspecific genetic distances and the observed deep intraspecific variation in Cordulia aenea (Linnaeus, 1758) are discussed in detail and the corresponding taxa in the public reference database highlighted. They should be considered in future applications of DNA barcoding and metabarcoding and represent interesting evolutionary biological questions, which call for in depth analyses of the involved taxa throughout their distribution ranges.
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Aquatic macroinvertebrates are a primary component of freshwater ecosystems and one of the most threatened by anthropogenic pressures. Among them, dragonflies are a charismatic group of growing scientific and social interest. However, little is known about the natural history of several species. One paradigmatic example is the declining Orthetrum nitidinerve, a Western Mediterranean endemic anisopteran. We reviewed published and new data on this species, addressing distribution, autecology, and conservation (with a focus on Italy), and provide its first genetic characterization and phylogenetic placement within the genus. In Italy, the species is known from 50 sites so far (only 17 breeding populations) located in Sardinia and Sicily (1841–2019, only 22 from 1990 onward). Records from continental Italy are due to misidentification. The flight period in Italy spans between May and September. Habitat consists of permanent freshwater (mostly helocrene sources, seepages, and small brooks), slow-flowing, shallow, with muddy bottom deposits at elevation from the sea level up to 1000m asl. All the breeding populations are found in open and sunny landscapes, almost invariably in extensive pasturelands. The species has strongly declined in Sicily, whereas several large populations still occur in Sardinia. The major threats identified so far are agriculture and grazing intensification or abandonment and drought/source desiccation determined by water overexploitation and climate change. The first ever provided mitochondrial COI barcode and ITS nuclear sequences allowed a first tentative phylogenetic placement of the species as a sister group of the O. brunneum/O. lineostigma lineage.
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Co-occurrence of species with similar trophic requirements, such as odonates, seems to depend both on them occupying different microhabitats and differing in their life-cycles. The life cycles of the dragonflies Boyeria irene and Onychogomphus uncatus were studied in two consecutive years, mainly by systematic sampling of larvae in seven permanent head courses that constitute the upper basin of the River águeda, western Spain, in the central part of the ranges of these two species. The size ranges of the last five larval stadia of both species were established based on biometric data. The eggs of the egg-overwintering aeshnid hatched in late spring and early summer and for the gomphid hatching peaked in middle-late summer. Both species showed mixed voltinism with "cohort splitting". B. irene had a dominant three-year development (partivoltinism), with some developing in two years (semivoltinism). O. uncatus requires four, sometimes three years to complete development (all partivoltine). B. irene larvae spent the winter before emergence in the last three, maybe four stadia, as a "summer species". O. uncatus mainly behaved as a "spring species", most larvae spending the last winter in the final larval stadium. © Institute of Entomology, Biology Centre, Czech Academy of Sciences, české Budějovice.
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Onychogomphus costae is an Ibero-Maghrebian endemic, which is rare in the Ibe-rian Peninsula. This study updates its distribution in Andalusia, southern Spain, based on a targeted survey carried out in 2015-2017 and the compilation of all available records. The species appears to be more widespread than previously documented, with a core distribution along the river Guadalquivir and its tributaries in the province of Córdoba. The altitudinal distribution of Onychogomphus costae reflects its general preferences for permanent, seasonally flooding, lowland rivers. The period of most observations of adults stretches over two months from mid-May to mid-July. Factors likely to explain why the species has been overlooked in past decades are discussed. These include recording effort, habitat features, adult behaviour, larval ecology and general water quality.
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Se aportan datos sobre la distribución de las especies de odonatos reófilos procedentes del análisis de las larvas incluidas en las muestras de macroinvertebrados bentónicos recolectadas en las campañas de seguimiento de la calidad de los ríos de Gipuzkoa (España) de los últimos años. Son especialmente interesantes las citas de Coenagrion mercuriale (Charpentier, 1840), Onychogomphus forcipatus forcipatus (Linnaeus, 1758), Onychogomphus forcipatus unguiculatus (Vander Linden, 1820) y Oxygastra curtisii (Dale, 1834). Palabras clave: Odonata, Coenagrion mercuriale, Oxygastra curtisii, Onychogomphus forcipatus forcipatus, Onychogomphus for-cipatus unguiculatus, larvas, calidad de los ríos, distribución, Gipuzkoa, España. Contribution of the Guipuzcoan river quality monitoring network to the knowledge of the distribution of Odonata in Gipuzkoa (Spain) Abstract: Data on the distribution of rheophilic Odonata are presented, extracted from the analysis of the larvae included in the samples of benthic macroinvertebrates collected in a series of river quality monitoring campaigns conducted in Gipuzkoa (Spain). Records of special interest are those of Coenagrion mercuriale (Charpentier, 1840), Onychogomphus forcipatus forcipatus (Linnaeus, 1758), Onychogomphus forcipatus unguiculatus (Vander Linden, 1820) and Oxygastra curtisii (Dale, 1834).
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This article describes several features in the MAFFT online service for multiple sequence alignment (MSA). As a result of recent advances in sequencing technologies, huge numbers of biological sequences are available and the need for MSAs with large numbers of sequences is increasing. To extract biologically relevant information from such data, sophistication of algorithms is necessary but not sufficient. Intuitive and interactive tools for experimental biologists to semiautomatically handle large data are becoming important. We are working on development of MAFFT toward these two directions. Here, we explain (i) the Web interface for recently developed options for large data and (ii) interactive usage to refine sequence data sets and MSAs.
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The European Red List is a review of the conservation status of ca. 6,000 European species (dragonflies, butterflies, freshwater fishes, reptiles, amphibians, mammals and selected groups of beetles, molluscs, and vascular plants) according to the IUCN regional Red Listing guidelines. It identifies species that are threatened by extinction at the regional level - so that appropriate conservation action can be taken to improve their status. This Red List publication summarises the results concerning the European dragonflies.
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Understanding the evolutionary history of living organisms is a central problem in biology. Until recently the ability to infer evolutionary relationships was limited by the amount of DNA sequence data available, but new DNA sequencing technologies have largely removed this limitation. As a result, DNA sequence data are readily available or obtainable for a wide spectrum of organisms, thus creating an unprecedented opportunity to explore evolutionary relationships broadly and deeply across the Tree of Life. Unfortunately, the algorithms used to infer evolutionary relationships are NP-hard, so the dramatic increase in available DNA sequence data has created a commensurate increase in the need for access to powerful computational resources. Local laptop or desktop machines are no longer viable for analysis of the larger data sets available today, and progress in the field relies upon access to large, scalable high-performance computing resources. This paper describes development of the CIPRES Science Gateway, a web portal designed to provide researchers with transparent access to the fastest available community codes for inference of phylogenetic relationships, and implementation of these codes on scalable computational resources. Meeting the needs of the community has included developing infrastructure to provide access, working with the community to improve existing community codes, developing infrastructure to insure the portal is scalable to the entire systematics community, and adopting strategies that make the project sustainable by the community. The CIPRES Science Gateway has allowed more than 1800 unique users to run jobs that required 2.5 million Service Units since its release in December 2009. (A Service Unit is a CPU-hour at unit priority).
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Since its introduction in 2001, MrBayes has grown in popularity as a software package for Bayesian phylogenetic inference using Markov chain Monte Carlo (MCMC) methods. With this note, we announce the release of version 3.2, a major upgrade to the latest official release presented in 2003. The new version provides convergence diagnostics and allows multiple analyses to be run in parallel with convergence progress monitored on the fly. The introduction of new proposals and automatic optimization of tuning parameters has improved convergence for many problems. The new version also sports significantly faster likelihood calculations through streaming single-instruction-multiple-data extensions (SSE) and support of the BEAGLE library, allowing likelihood calculations to be delegated to graphics processing units (GPUs) on compatible hardware. Speedup factors range from around 2 with SSE code to more than 50 with BEAGLE for codon problems. Checkpointing across all models allows long runs to be completed even when an analysis is prematurely terminated. New models include relaxed clocks, dating, model averaging across time-reversible substitution models, and support for hard, negative, and partial (backbone) tree constraints. Inference of species trees from gene trees is supported by full incorporation of the Bayesian estimation of species trees (BEST) algorithms. Marginal model likelihoods for Bayes factor tests can be estimated accurately across the entire model space using the stepping stone method. The new version provides more output options than previously, including samples of ancestral states, site rates, site d(N)/d(S) rations, branch rates, and node dates. A wide range of statistics on tree parameters can also be output for visualization in FigTree and compatible software.
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The behaviour of Onychogomphus uncatus, including flight and mating activity, was studied at a fast-flowing irrigation canal. During the day, males perched in sections of the canal with a strong current and a turbulent water surface. During short flights, interactions with other con-specific and hetero-specific males occurred, particularly with Orthetrum coerulescens. Under conditions of high population density, the frequent occurrence and disturbances by this species often resulted in male O. uncatus leaving a particular section of the canal. In the late afternoon and evening, males concentrated on ground perches in the vicinity of the water. The reproductive system of O. uncatus was found to be ‘encounter limited’. The operational sex ratio of imagines at the water was always strongly biased in favour of the males. Individual females were observed at the water during the morning and evening hours. Following pair formation there was a prolonged period of copulation away from the water. Most pair formations were observed in the morning and evening hours. They took place over water, and in the evening hours also away from the water.
Global diversity of dragonflies (Odonata) in freshwater
  • V J Kalkman
  • V Claustnitzer
  • K.-D B Dijkstra
  • A G Orr
  • D R Paulson
  • J Van Tol
Kalkman V.J., Claustnitzer V., Dijkstra K.-D.B., Orr A.G., Paulson D.R. & van Tol J. 2008. Global diversity of dragonflies (Odonata) in freshwater. Hydrobiologia 595: 351-363
Unpublished report, Conselleria de Sanitat Universal i Salut Pública. Generalitat Valenciana Mezquita-Aramburu I. & Torralba-Burrial A. 2015. Primera cita de Onychogom phus forcipatus forcipatus (Linnaeus, 1758) (Odonata: Gomphidae) para la Península Ibérica
Laboratorio de Salud Pública de Valencia 2019. Informe de análisis físico-químico a la salida del manantial del Nacimiento del Río Cazuma (Bicorp, Valencia). Unpublished report, Conselleria de Sanitat Universal i Salut Pública. Generalitat Valenciana Mezquita-Aramburu I. & Torralba-Burrial A. 2015. Primera cita de Onychogom phus forcipatus forcipatus (Linnaeus, 1758) (Odonata: Gomphidae) para la Península Ibérica. Boletín de la Sociedad entomológi ca aragonesa 57: 365-366