ZooKeys 1095: 165—I 77 (2022) A peer-reviewed open-access journal
doi: 10.3897/zookeys.1095.74308 RESEARCH ARTICLE #ZooKey S

https:/ / ZOO keys. pensoft.net Launched to accelerate biodiversity research

A new species of Pseudophanerotoma
(Hymenoptera, Braconidae) from Nayarit, Mexico

Armando Falcén-Brindis'’, Jorge L. Leén-Cortés', Rubén E Mancilla-Brindis?,
Mario Orlando Estrada-Virgen?, Octavio J. Cambero-Campos?

| Departamento de Conservacién de La Biodiversidad, El Colegio de La Frontera Sur, Maria Carretera Pa-
namericana y Periférico Sur, CP 29290, San Cristébal de Las Casas, Chiapas, Mexico 2 Department of
Entomology, University of Kentucky Research and Education Center, 348 University Drive, Princeton, KY
42445, USA 3 Posgrado en Ciencias Biolégico Agropecuarias, Unidad Académica de Agricultura, Universidad
Autonoma de Nayarit, Carretera Tepic-Compostela Km 9, CP 63155, Xalisco, Nayarit, Mexico

Corresponding authors: Jorge L. Leén-Cortés (jleon@ecosur.mx),

Armando Falcén-Brindis (armandofalcon123@gmail.com)

Academic editor: Andreas Kohler | Received 11 September 2021 | Accepted 28 March 2022 | Published 14 April 2022
http://z00bank.org/5277C150-DFFF-4CB4-9B7A-CCB8006DDCAC

Citation: Falcon-Brindis A, Leén-Cortés JL, Mancilla-Brindis RE Estrada-Virgen MO, Cambero-Campos OJ (2022)
A new species of Pseudophanerotoma (Hymenoptera, Braconidae) from Nayarit, Mexico. ZooKeys 1095: 165-177.
https://doi.org/10.3897/zookeys. 1095.74308

Abstract

Parasitoid wasps are known to be among the most abundant and species-rich on Earth and thus considered
an ecologically important group of arthropods. Braconid wasps play a key role in regulating the popula-
tions of Lepidoptera, Coleoptera, and Diptera. However, the biology and taxonomy of numerous parasi-
toid species remain poorly known. In Mexico, only 17 species of the subfamily Cheloninae have been de-
scribed. A new species of Pseudophanerotoma Zettel, 1990 (Hymenoptera, Braconidae), P huichol sp. nov.,
is described from Nayarit, Mexico. The tortricid moth Cryptaspasma perseana Gilligan & Brown, 2011 is

reported as the host of this parasitoid wasp. Detailed taxonomic and barcoding information are provided.

Keywords
Cheloninae, COI barcode, integrative taxonomy, Mexican biodiversity, Neotropical region, parasitoid

wasp, Tortricidae

Copyright Armando Falcon-Brindis 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.

166 Armando Falcén-Brindis et al. / ZooKeys 1095: 165-177 (2022)

Introduction

The family Braconidae comprises 21,221 species worldwide (Yu et al. 2016), and
the description of new species is exponentially increasing in the Neotropical region
(Fernandez-Triana and Boudreault 2018; Sharkey et al. 2021). Braconid wasps are
primarily specialized parasitoids of Lepidoptera, Coleoptera, and Diptera, thus play-
ing an important role in regulating their populations. Usually, braconid wasp does not
oviposit in the host egg (Wharton et al. 1997). However, members of the subfamily
Cheloninae oviposit in the eggs of Lepidoptera and emerge from later larval instars or
the pupa (Quicke 2015).

Cheloninae are mostly solitary endoparasitoid wasps of several Microlepidoptera,
i.e., Pyraloidea and Tortricoidea (Shaw 1997). Within this subfamily, the genus Pseu-
dophanerotoma Zettel, 1990 is highly specialized in parasitizing tortricid moths (Costa
Lima 1956; Hoddle and Hoddle 2008; Penteado-Dias et al. 2008). Members of this
genus are restricted to the Americas, occurring from southern Texas to Peru and Brazil
(Zettel 1990; Kittel 2018; Sharkey et al. 2021).

One of the main limitations of studying parasitoid wasps is the large number of
species and high variability in life histories (Shaw 1995; Hanson and Gauld 2006).
The taxonomy of Braconidae has been poorly documented, especially in the tropics
(LaSalle and Gauld 1991). Since the classical description of species started undergoing
the so-called taxonomy crisis almost two decades ago (Godfray 2002), the integrative
taxonomy framework (Dayrat 2005) has brought complementary approaches to tackle
challenging groups. As in most parasitoid wasps, the taxonomy of Cheloninae has
been changing and the use of molecular tools allowed advances for the systematics of
the group. For instance, Pseudophanerotoma and Furcidentia Zettel, 1990 are now two
separate genera based on molecular and morphological characters (Zettel 1990; Kit-
tel et al. 2016). Kittel (2018) essentially cleared up the taxonomy of both genera and
added new species, but a recent work also provided additional species names (Sharkey
et al. 2021).

In Mexico, Pseudophanerotoma members correspond to a poorly documented tax-
on with a few records from the States of Oaxaca, Quintana Roo, Tamaulipas, Veracruz,
and Yucatan (Sanchez-Garcia and Lépez-Martinez 2000; Coronado-Blanco 2011).
However, none of these specimens have been determined to species level, limiting our
knowledge upon such specialized genus but most importantly, reducing our ability to
identifying priority biodiversity areas in the face of human disturbance (Ceballos et
al. 2015). In this work, we describe a new species, Pseudophanerotoma huichol sp. nov.,
which attacks the tortricid moth Cryptaspasma perseana Gilligan & Brown, 2011 oc-
curring in Nayarit, Mexico (Mancilla-Brindis et al. 2019). We provide detailed taxo-
nomic diagnoses for both sexes and support our findings with DNA barcoding. In ad-
dition, we present an updated checklist of Pseudophanerotoma species, their potential
phylogenetic relationship, and discuss elements of their biology and distribution.

A new species of Pseudophanerotoma from Mexico 167

Methods

As part of a survey to evaluate the prevalence of Cryptaspasma perseana among avocado
orchards in Nayarit, Mexico, several fruits and seeds were collected from December
2019 to October 2020 (Mancilla-Brindis et al. 2021). The rearing process was con-
ducted at the Universidad Auténoma de Nayarit. The emerged wasps were preserved
in 96% ethanol and shipped to the Collection of Entomology (ECO-SC-E) of El
Colegio de la Frontera Sur (ECOSUR), San Cristébal de las Casas, Chiapas, Mexico,
for taxonomic identification.

DNA extraction was conducted at the Barcoding Laboratory of Life of
ECOSUR, Chetumal, Quintana Roo, Mexico, using standard protocols (Hebert
et al. 2003; Ivanova et al. 2006). Extracted DNA was amplified for a 650-bp
region near the 5' terminus of the cytochrome c oxidase subunit I (COI) gene
using the primers ZplankF1 (TCT ASW AAT CAT AAR GAT ATT GG) and
ZplankR1 (TTC AGG RTG RCC RAA RAA TCA) (Posser et al. 2013). Amplified
PCR product was then shipped to Eurofins Scientific, Louisville, KY, USA, for
purification and sequencing using Sanger technology. Sequence was cleaned
and aligned using BioEdit (Hall 1999). We performed a phylogenetic analysis
using the neighbor-joining method (Saitou and Nei 1987), based on a bootstrap
consensus tree inferred from 10,000 replicates (Felsenstein 1985) to evaluate
the relationship of 10 Pseudophanerotoma species. In addition, a genetic distance
matrix between pairs of sequences was calculated through maximum composite
likelihood to estimate the evolutionary divergence between species (Tamura et
al. 2004). Pseudophanerotoma alvarengai Zettel, 1990, P longicornia Zettel, 1990,
P. maculosa Zettel, 1990, P peruana Zettel, 1990, and P zeteki (Cushman) 1922
where not included since there is no barcoding information for them. The analysis
involved 10 nucleotide sequences, eliminating positions with missing data.
Bootstrap analysis, genetic distance matrix, and phylogenetic tree were conducted
in MEGA11 (Tamura et al. 2021).

Morphological determination of Pseudophanerotoma followed Sharkey et al.
(2021). Taxonomic features provided in this study followed van Achterberg (1988)
and Wharton et al. (1997). Images were taken with a Leica MC 170 HD camera,
adapted to a Leica M205C stereoscopic microscope. Morphometric data and image
aligning was performed with the Leica Application Suite software. Holotype and para-
type specimens are deposited in ECO-SC-E.

We used the following abbreviations to describe key ocellar measurements
(Kittel 2018):

LOL lateral ocellar line (distance between lateral and anterior ocelli);
OOL ocular ocellar line (distance between lateral ocellus and compound eye);
POL posterior ocellar line (distance between lateral ocelli).

168 Armando Falcén-Brindis et al. / ZooKeys 1095: 165-177 (2022)

Results

Pseudophanerotoma huichol Falcon-Brindis, sp. nov.

http://zoobank.org/ DB6 DDDDA-E2C6-4DD6-9BCB-4388701 14206

Diagnosis. Pseudophanerotoma huichol sp. nov. can be distinguished from the other
species by the unicolorous head, meso, and metasoma, except for the dark brownish
integument on the apical half of hind femora and a small spot on the mesopleuron
(sometimes absent in males); antenna with 52 antennomeres in females and 46 anten-

nomeres in males, occipital carina complete.
Description (female). Body length 5.5 mm; ratio of length of fore wing to body
0.8; ratio of metasoma to mesosoma 1.2 (Fig. 1A—C).

Figure |. Pseudophanerotoma huichol sp. nov., female A head frontal B lateral habitus C dorsal view of

meso and metasoma. Scale bars: 1.0 mm.

Head. Antenna with 52 antennomeres; ratio of width of face to its height in fron-
tal view 1.4; ratio of width of clypeus to its height 1.8; ratio of length of third antenno-
mere to width 3.7; ratio of length of fourth antennomere to width 3.0; ratio of length
of penultimate antennomere to width 1.3; malar space to base of mandible 0.3 mm.
Clypeus convex and sparsely punctate with two teeth; face straight in lateral view,
punctate; frons and vertex densely punctate; ratio of LOL:POL:OOL 0.1:0.1:0.1:0.4.

Mesosoma. Mesoscutum shiny and densely punctate, mid mesoscutal area coarsely
sculptured; notauli present; mesopleuron punctures shallow and less dense; scutellar
sulcus present, with coarse pits; mesoscutellum convex and punctate; propodeum

A new species of Pseudophanerotoma from Mexico 169

areolate; propodeal tubercles absent; ratio of mesosoma height to its length 0.6; ratio of
hind tibia to hind tarsus 2.1; ratio of length to width of hind coxa 2.0; ratio of length
to width of hind femur 5.2; ratio of length to width of hind tibia 7.5; ratio of length
to width of hind tarsus 6.2; ratio of length of fore wing to body 0.8; fore wing length
4,2 mm; 1RS present; M curved; RS+M spectral (Fig. 2).

Figure 2. Pseudophanerotoma huichol sp. nov., fore wing. Scale bar: 1.0 mm.

Metasoma. Oval in dorsal view; sculpture striate throughout; ratio of metasomal
width to length 0.6; ratio of length of the three metasomal tergites 0.7:0.7:0.8.

Color. Scape, head, meso and metasoma ferruginous; antennomeres and legs pale
orange to yellowish; dark brown integument on tegula, a small spot on the top of
mesopleuron (below the tegula) and apical half of hind femora. Wings hyaline with
egreenish-purplish reflections, wing venation brown to dark brown; parastigma and
pterostigma dark brown.

Type material. Holotype and paratypes specimens are pinned and deposited
in ECO-SC-E. Holotype: Mexico: 2, Lo de Lamedo, Tepic, Nayarit 21.54222,
—104.92833; 880 m elev., 10 Oct. 2020; 21.53388, —104.93722; 860 m elev., 04
Sep. 2020; R.E Mancilla-Brindis leg. Holotype voucher code 69271. Paratypes:
6 2: 69272, 69273, 69274, 69275, 69276, 69277; 5 G: 69278, 69279, 69280,
69281, 69282.

Description (male). Antenna with 46 antennomeres. Body length 5.2 mm; ratio
of length of fore wing to body 0.8; ratio of length of metasoma to mesosoma 1.2
(Fig. 3A—C).

Head. Antenna with 46 antennomeres; ratio of width of face to its height in fron-
tal view 1.2; ratio of width of clypeus to its height 2.0; ratio of length of third antenno-
mere to width 4.5; ratio of length of fourth antennomere to width 3.6; ratio of length
of penultimate antennomere to width 1.9; malar space to base of mandible 0.3 mm.

170 Armando Falcén-Brindis et al. / ZooKeys 1095: 165-177 (2022)

Figure 3. Pseudophanerotoma huichol sp. nov., male A head frontal B lateral habitus C dorsal view of

meso and metasoma. Scale bars: 1.0 mm.

Clypeus convex and sparsely punctate with two teeth; face straight in lateral view,
punctate; frons and vertex densely punctate; ratio of LOL:POL:OOL 0.1:0.1:0.1:0.3.

Mesosoma. Same sculpture patterns as in female; ratio of mesosoma height to its
length 0.7; ratio of hind tibia to hind tarsus 1.9; ratio of length to width of hind coxa
2.1; ratio of length to width of hind femur 4.4; ratio of length to width of hind tibia
5.3; ratio of length to width of hind tarsus 5.8; ratio of length of fore wing to body 0.8;
fore wing length 4.1 mm.

Metasoma. Oval in dorsal view; sculpture striate throughout; ratio of metasomal
width to length 0.6; ratio of length of the three metasomal tergites 0.6:0.6:0.8.

Color. In general, male is paler than female; scape, head, meso and metasoma light
orange; antennomeres and legs pale yellow to beige; dark brown integument on tegula
and apical half of hind femora. Wings the same color pattern as in female.

Remarks. ‘This species differs from all the other congeners by having a large number
of antennomeres in both sexes: 52 and 46 antennomeres in females and males respectively.

Biology. Parasitoid of Cryptaspasma perseana, a tortricid pest of avocado docu-
mented in Hidalgo, Michoacan, and Nayarit, Mexico (Mancilla-Brindis et al. 2019).

Sequence data. GenBank accession number for this species is COI MZ501206.

Etymology. The species is named in honor to the Huichol culture from
Nayarit, Mexico.

According to the phylogenetic analysis for the barcoded Pseudophanerotoma spe-
cies (Fig. 4), the closest species to P huichol sp. nov. was P alejandromarini Sharkey,
2021 (90%), both being sister species of P paranaensis Costa Lima, 1956 (100%). The
results from the evolutionary distance analysis showed that P alexsmithi and P austini

A new species of Pseudophanerotoma from Mexico 171

are the closest species (0.0051), followed by P huichol sp. nov. — P alejandromarini
(0.0133), P alejandromarini — P. paranaensis (0.0261). In contrast, the pairwise com-
parison between P austini — P allisonbrownae and P alexsmithi — P. allisonbrownae
revealed the largest evolutionary divergence (0.2167) (Table 1).

Table |. Estimates of evolutionary divergence between sequences. The number of base substitutions per

site from between sequences are shown.

u Sy X&
S iv] xy 7 iS} s
® : 8 S s 8 g by .
S 2 N 3 S$ Ss R s S
S S = 5 BS ~~ 8 3 3
> > BS 3 S s S < 5
yg & S sS S 8 “8
xs RS S 8 Kd $ S a =
S 8 Sy a = a es
x a: 8 = x x
a gx
P. albanjimenezi 0.1250
P. alejandromarini 0.1188 0.1277
P. alexsmithi 0.1186 0.1335 0.1120
P. allisonbrownae 0.1843 0.1943 0.2052 0.2167
P. bobrobbinsi 0.1442 0.1534 0.1412 0.1467 0.1663
P. paranaensis 0.1248 0.1402 0.0261 0.1243 ~—:0.2085~—(0.1506
P austini 0.1124 0.1366 0.1088 0.0051 ~—-:0.2167.—S «0.1401 ~— 0.1210
P thapsina 0.0450 0.1371 0.1308 ~=—0.1278 ~— 0.2086 ~—0.1634_ ~— 0.1400 ~— 0.1214
P huichol 0.1220 0.1340 «0.0103. —0.1119 0.2089 0.1477, (0.0314 0.1119 02.141
P. allisonbrownae ACJ220]
88
48 P. bobrobbinsi ADB6487
pl P. albanjimenezi AAV8843
P. alanflemingi ACL3 198
99 . = i
P. thapsina K.J472582
P. alexsmithi ACBI516
100
P. austini KJ472583
P. paranaensis K.J472581
100 P. alejandromarini ACE1984
90

P. huichol sp. nov. MZ501206

Figure 4. Bootstrap consensus tree of Pseudophanerotoma species. Five species are missing due to the lack
of sequence information. The percentage of replicate trees in which the associated taxa clustered together

in the bootstrap test are shown next to the branches. ‘The lower clade has a weak bootstrap support.

ugg Armando Falcén-Brindis et al. / ZooKeys 1095: 165-177 (2022)

Discussion

This is the first work describing a new species of the genus Pseudophanerotoma in Mexico.
Previous reports documented individuals at the generic level (SAnchez-Garcia and Lépez-
Martinez 2000; Coronado-Blanco 2011). It is likely that P thapsina (recorded in the United
States and French Guiana) and P austini (Guatemala) also occur in Mexico (Kittel 2018).
However, further revisions are required to confirm their presence. Moreover, it is certain
that other species of the genus occurring in Neotropical Mexico are deposited in museum
collections (Sanchez-Garcia and Lépez-Martinez 2000; Coronado-Blanco 2011).

According to the most recent key for species of Pseudophanerotoma (Kittel 2018), P
huichol sp. nov., can be separated in couplet four, where the difference with P thapsina
is the dark brownish integument on the apical half of hind femora and the dark spot
present on top of the mesopleuron. Despite the close phylogenetic relationship be-
tween P huichol sp. nov. and P alejandromarini, the latter species lacks dark integu-
ment on the hind femora. In summary, P Auichol sp. nov. has the largest number of
antennomeres known from the species presented by Kittel (2018) and is undoubtedly
different from those recently added by Sharkey et al. (2021).

Members of the subfamily Cheloninae have been used in the biocontrol of ex-
otic pests (Narendran 2001). In Mexico, 704 species of Braconidae have been listed
from 34 subfamilies (Coronado-Blanco et al. 2012), of which, only 17 species of
Cheloninae have been identified (Coronado-Blanco and Zaldivar-Riverén 2014).
However, most of these species belong to the genus Chelonus Panzer, 1806 (Cor-
onado-Blanco et al. 2004), which is well known to control the populations of the
fall armyworm Spodoptera frugiperda (J.E. Smith, 1797) feeding on forage maize
in north and central Mexico (Rios-Velasco et al. 2011; Estrada-Virgen et al. 2013;
Garcia-Gonzalez et al. 2020). Nonetheless, the biology of most chelonines occurring
in Mexico remains unknown.

Likewise, the biology of the 15 known Pseudophanerotoma species has been poorly
documented (Table 2). In most cases (66%), there is no information about host pref-
erences and the same proportion is known from a single sex. The described P huichol
sp. nov. corresponds to the Pseudophanerotoma sp. reported parasitizing C. perseana in
two localities from Nayarit, Mexico (Mancilla-Brindis et al. 2019, 2021). Therefore,
this study has helped to identify an important interaction between P Auichol sp. nov.
and a serious pest of avocado. However, further research is needed to provide details
of, for example, the life history, distribution, and management of P Auichol sp. nov.,
potentially regulating the populations of C. perseana.

Our preliminary phylogenetic analysis indicates that there is strong evidence for
P huichol sp. nov., P. alejandromarini, and P paranaensis being sister species, albeit the
cluster classification showed poor resolution (weak bootstrap support). In addition,
the weak evolutionary divergence between P alexsmithi and P austini (0.0051) reveals
the closest relationship among the genus Pseudophanerotoma. Such clades are naturally
affected by the missing barcoded species, but the explanations could be attributed to

A new species of Pseudophanerotoma from Mexico

Is

Table 2. A checklist of Pseudophanerotoma species according to Zettel (1990), Penteado-Dias et al.
(2008), Kittel (2018), Sharkey et al. (2021), and this work. Only accepted scientific names are included.
Types refer to the known sexes on each species.

Species Occurrence Host data Types Sequence
accession number
P. alanflemingi Sharkey, 2021 Guanacaste, Costa Rica Unknown 2 BOLD:ACL3198
P. albanjimenezi Sharkey, 2021 Alajuela, Costa Rica Episimus ortygia (Tortricidae) feeding S BOLD:AAV8843
on Vismia baccifera (Hypericaceae)
P. alejandromarini Sharkey, 2021 Guanacaste, Costa Rica Unknown 2 BOLD:ACE1984
P. alexsmithi Sharkey, 2021 Alajuela, Costa Rica Cosmorrhyncha albistrigulana 2 BOLD:ACB1516
(Tortricidae) feeding on Dialium
guianense (Fabaceae)
P. allisonbrownae Sharkey, 2021 Guanacaste, Costa Rica Unknown 6 BOLD:ACJ2201
P. alvarengai Zettel, 1990 Bahia, Sao Paulo, Brazil Cydia tonosticha (Tortricidae) ed NA
feeding on Stryphnodendron
adstringens (Fabaceae)
P austini Kittel, 2018 Petén, Guatemala Unknown 3 GenBank
KJ472583
P. bobrobbinsi Sharkey, 2021 Guanacaste, Costa Rica Unknown 2 BOLD:ADB6487
P. huichol Falcén-Brindis, sp. Nayarit, Mexico C. perseana (Tortricidae) feeding od MZ501206
nov. on Persea americana var. Drymifolia
(Lauraceae)
P. longicornia Zettel, 1990 Paraguay; Ecuador Unknown 2d NA
P maculosa Zettel, 1990 Barro Colorado, Panama Unknown 3 NA
P. paranaensis Costa Lima, 1956 Parand, Brazil; Saiil, French © Olethreutes anthracana (Tortricidae) 3 GenBank
Guiana KJ472581
P. peruana Zettel, 1990 Alto-Samboroi, Peru Unknown 3 NA
P. thapsina Walley, 1951 Texas, California, Arizona, Unknown od GenBank
Florida, USA; Mt. Chevoux, KJ472582
French Guiana
P. zeteki (Cushman, 1922) St. Bernardino, Panama Unknown od NA

several factors (e.g., lack of alternative barcoded regions, different gene history, or early
diverging lineages) (Doyle 1992). Thus, our phylogenetic analysis should be inter-
preted with caution, as it only attempted to compare the position of the new species
within the sequenced congeners.

Even though divergent opinions, molecular methods are now an important part
of taxonomy, allowing integrative approaches (Padial et al. 2010). In this regard,
many braconid species have been described using DNA barcoding (Smith et al.
2007, 2008; Sharkey et al. 2021). Sharkey et al. (2021) recently added more than
400 species to the list of Braconidae, including six new species of Pseudophaner-
otoma, with descriptions based on the COI barcoded region. This approach can
certainly be helpful when describing large numbers of species, but morphological
descriptions are handy when molecular tools are not available. In this sense, whereas
the last taxonomic key to Pseudophanerotoma species did not thoroughly include
molecular data (Kittel 2018), the six new species lack of morphological descrip-
tion (Sharkey et al. 2021) and thus further research is required to strengthen their
presumed relationships.

174 Armando Falcén-Brindis et al. / ZooKeys 1095: 165-177 (2022)

Acknowledgements

We thank Dr Manuel Elias Gutierrez for his guidance with the molecular work. Dr
Alma Estrella Garcia Morales for her gentle support with the DNA extraction and
amplification. Collect permissions were granted by the Comité Estatal de Sanidad Veg-
etal del Estado de Nayarit (CESAVENAY). AF-B received a post-doctoral scholarship
(grant number 30223), through a grant from the National Council for Science and
Technology in Mexico (CONACYT) to JLL-C (258792: CB-2015-01).

Funding from the Universidad Auténoma de Nayarit (UAN) supported the pro-
ject: “Cryptaspasma sp. (Lepidoptera, Tortricidae): its distribution and natural ene-
mies in avocado orchards from Nayarit, Mexico” and provided the necessary resources

through the UAN Special Taxes.

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