ZooKeys 1061: | 199 (202 1) A peer-rev iewed open-access journal I

doi: 10.3897/zookeys. 106 1.67458 RESEARCH ARTICLE 7,00Ke y

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

Cotesia cassina sp. nov. from southwestern Colombia:
a new gregarious microgastrine wasp (Hymenoptera,
Braconidae) reared from the pest species Opsiphanes
cassina Felder & Felder (Lepidoptera, Nymphalidae)
feeding on Elaeis oil palm trees (Arecaceae)

Geraldo Salgado-Neto', Consuelo Alexandra Narvaez Vasquez’,
Dillon S. Max?, James B. Whitfield?

| Pés-graduagao em Agronomia, Departamento de Defesa Fitossanitdria, Universidade Federal de Santa Maria,
97105-900, Santa Maria, RS, Brazil 2. Pés-graduagao em Entomologia, Departamento de Entomologia/BIO-
AGRO, Universidade Federal de Vicosa, 36570-900, Vicosa, MG, Brazil 3 Department of Entomology, 320
Morrill Hall, 505 South Goodwin Ave., University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA

Corresponding author: James B. Whitfield (jwhitfie@life.illinois.edu)

Academic editor: J. Fernandez-Triana | Received 16 April 2021 | Accepted 12 July 2021 | Published 28 September 2021
Attp://zoobank. org/8208 BB83-73 1 B-4F97-90F 2-85 15638EC124

Citation: Salgado-Neto G, Vasquez CAN, Max DS, Whitfield JB (2021) Cotesia cassina sp. nov. from southwestern
Colombia: a new gregarious microgastrine wasp (Hymenoptera, Braconidae) reared from the pest species Opsiphanes
cassina Felder & Felder (Lepidoptera, Nymphalidae) feeding on Elaeis oil palm trees (Arecaceae). ZooKeys 1061: 11-
22. https://doi.org/10.3897/zookeys.1061.67458

Abstract

A new species of microgastrine wasp, Cotesia cassina Salgado-Neto, Vasquez & Whitfield, sp. nov., is
described from southwestern Colombia in Tumaco, Narifo. This species is a koinobiont gregarious larval
endoparasitoid, and spins a common mass of cocoons underneath the host caterpillars of Opsiphanes cas-
sina (Felder & Felder) (Lepidoptera, Nymphalidae), feeding on oil palm trees (interspecific hybrid Elaeis
oleifera x E. guineensis) (Arecaceae). While superficially similar, both morphologically and biologically, to
C. invirae Salgado-Neto & Whitfield from southern Brazil, the two species are distinct based on DNA

barcodes, host species, geographical range and morphological characters.

Keywords

Butterfly, DNA barcode, integrative taxonomy, morphology, natural enemy, new species

Copyright Geraldo Salgado-Neto 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.

12 Geraldo Salgado-Neto et al. / ZooKeys 1061: 11—22 (2021)

Introduction

The nymphalid butterfly Opsiphanes cassina Felder & Felder occurs from Mexico to the
Amazon Basin (Brazil, Bolivia, Colombia, Ecuador, French Guiana, Guyana, Peru, Suri-
name and Venezuela) (Lamas 2004). This species is widespread in Colombia but is most
commonly found within the States of Narifio, Cauca and Putumayo (Benavides and Carde-
nas 1970; Fuentes 1973; Pava et al. 1983; Posada 1989; Villegas 1993). Opsiphanes cassina
is considered a pest of oil palm trees (interspecific hybrid Elzeis oleifera x Elaeis guineensis)
(Arecaceae) in south-west Colombia (Genty 1978; Mexz6n and Chinchilla 1996; Posada
and Cardenas 1996; Loria et al. 2000; Gonzalez 2011). In southwestern Colombia, the
occurrence of the subspecies O. cassina numatius Fruhstorfer was recorded by Martinez
(1970). We also recorded the presence of three additional subspecies: O. cassina chiriquensis
Stichel, Opsiphanes cassina periphetes Fruhstorfer, and Opsiphanes cassina caliensis Bristow.

Five species of Braconidae have been recorded as endoparasitoids of species of
Opsiphanes (larval stage): Cotesia biezankoi (Blanchard), Cotesia opsiphanis (Schrottky),
Cotesia alia (Muesebeck) (Mason 1981; Penteado-Dias 1987; De Santis 1989; Salga-
do-Neto 2013), Cotesia invirae Salgado-Neto & Whitfield (Salgado-Neto et al. 2019),
and Rhysipolis sp. (Sauer 1946; Costa Lima 1950, 1962; Silva et al. 1968; Bricefo-
Vergara 1978; De Santis 1980; Mason 1981; Penteado-Dias 1987; Briceno-Vergara
1997; Mexzén 1997; Rodriguez et al. 2006). Here we describe a new species of Cotesia
Cameron, reared from O. cassina feeding on oil palms in southwestern Colombia.

Cotesia is easily recognizable morphologically among microgastrine braconids, al-
though the huge variety of species can be difficult to distinguish from each other (Whit-
field et al. 2009), especially those without host data. The wasps have a koinobiont habit
(Kankare and Shaw 2004) and both solitary and gregarious species are known. Cotesia
(Braconidae, Microgastrinae) currently contains roughly 300-400 described species
(Fernandez-Triana et al. 2020), but this number will certainly increase dramatically,
as world estimates range from 1000-2000 species (Mason 1981; Michel-Salzat and
Whitfield 2004; Whitfield et al. 2018; Fernandez-Triana et al. 2020), and a relatively
small number of studies recording Neotropical species of Cotesia and their biology are
available so far (Whitfield 1997; Whitfield et al. 2018), particularly in South America.

As Cotesia species appear to be highly host specialized (Kankare and Shaw 2004),
with many cryptic species and geographically restricted distributions (Fiaboe et al.
2017), the use of an integrative taxonomic approach (combining morphological, mo-
lecular, biological and geographical data) is critical for recognizing and distinguishing
these parasitoid wasps (Smith et al. 2008; Kaiser et al. 2017).

Using such an integrative taxonomic approach, this paper provides a description
of a new species of Cotesia, whose brood was produced from caterpillars of Opsiphanes
cassina (Felder & Felder) (Lepidoptera, Nymphalidae) (Fig. 1) feeding on palm trees
(interspecific hybrid Elaeis oleifera x E. guineensis) (Arecaceae) in Tumaco, Narifo,
south-west Colombia. We compare it with the other described species of Opsiphanes
that have been formally recorded from the Neotropical region, two of which have been
well characterized and two of which are of uncertain identity.

New species of Cotesia from Opsiphanes in Colombia 13

Materials and methods

Between April 2018 and March 2019; we collected 35 larvae of Opsiphanes cassina as
part of a survey carried out on exotic palms in the Palmeiras plantation A.S., 58 km from
San Andrés de Tumaco, Narifio, Colombia (1°47'28.0"N, 78°47'33.9"W, 28 m elev. —
see Fig. 1A, B). The larvae of O. cassina were found on the interspecific hybrid Elaeis
oleifera x E. guineensis (Arecaceae). Upon collecting, larvae were kept in the laboratory
(25 + 1 °C; 70% RH; photoperiod of 14 hours of light) and observed daily until the
emergence of the butterflies or parasitoids, which were then preserved in 70% ethanol.

Photographs of the caterpillar and parasitoid cocoons (Fig. 1C, D) were taken in
the field by CANV. Morphological photographs of the Cotesia (Fig. 2A—F) were taken
by DSM at the University of Illinois, USA using a Leica M205 C stereo microscope
(467 nm resolution) fitted with a 5 megapixel Leica DFC 425 digital microscope cam-
era. Images were stacked using a motor drive on the microscope and Zerene Stacker
software. Morphological terms and measurements of structures are mostly those used
by Salgado-Neto et al. (2019).

To characterize and compare the new species at the molecular level, the mito-
chondrial (DNA barcode) gene cytochrome oxidase I (COI) was analyzed. For the
amplification of a fragment of approximately 460 bp of this gene, we used the fol-
lowing primer pair: COI-F (5’°-GATTTTTTGGKCAYCCMGAAG-3’) and COL-R
(SC CRAATACRGCTCCTATWGATAAWAC-3’) (Gusmao et al. 2010). DNA extrac-
tion of one specimen was performed with the GenElute Mammalian Genomic DNA
Miniprep Kit (Sigma-Aldrich) and followed the manufacturer's protocol. The product
was amplified via Polymerase Chain Reaction (PCR) according to the following sched-
ule: 94 °C for 2 minutes, 40 cycles of 94 °C for 30 seconds, 54 °C for 30 seconds,
72 °C for 40 seconds and 72 °C for 4 minutes. Then the PCR product was purified
using polyethylene glycol precipitation (PEG; Schmitz and Riesner 2006). These sam-
ples were sequenced using the Big Dye 3.1 reagent (Life Technologies) and a 3500 XL

automatic sequencer (Life Technologies).

Descriptive taxonomy

Cotesia cassina Salgado-Neto, Vasquez & Whitfield, sp. nov.
http://zoobank.org/3C8E3CC9-C4D2-422 1-BE00-6652F17F5BD4

Material examined. Holotype Female, Colombia: Narifo, San Andrés de Tumaco
(1°47'28.0"N, 78°47'33.9"W, 28 m elev.), March 2019, coll. Consuelo Vasquez, ex
larva Opsiphanes cassina Felder & Felder (Lepidoptera, Nymphalidae). Deposited in
the collection of the National University of Colombia (UNC, Dr Fernando Fernandez,
curator). Paratypes 2 males, deposited in UNC, same data as holotype. 1 female, also
same data as holotype, deposited in the Illinois Natural History Survey (INHS). Non-
types. 2 females, in poor condition, also deposited at INHS.

14 Geraldo Salgado-Neto et al. / ZooKeys 1061: 11—22 (2021)

Table |. Diagnostic morphological characters distinguishing Cotesia cassina sp. nov. from the Brazilian
C. invirae Salgado-Neto & Whitfield.

Character C. invirae C. cassina
Color Generally lighter. T3 and all tergites posterior to T3 mostly Generally darker. T3 and all tergites posterior to T3 are more
bright yellow orangish. Mesopleuron with some light yellow/ brown to black rather than orangish. Mesopleuron almost
brown on ventral side entirely black
T2 Sculpture} Mostly smooth. Sculpture is more uniform across width; less More punctate laterally, smooth medially
punctate laterally
T2 Shape Posterior margin/groove straight Posterior margin slightly convex apically, with length greatest
medially

Diagnosis. As discussed above, Cotesia is a huge worldwide genus of hundreds of
species, with many morphologically similar species. While useful world identification
keys are not available, it is currently possible to successfully diagnose species region-
ally, especially combined with molecular and host data. ‘The closest described species,
morphologically, biologically and within the region, is Cotesia invirae from southern
Brazil, which also parasitizes Opsiphanes on palms (different species). The table below
provides a diagnostic comparison between the two species.

Cotesia alia (Muesebeck), also recorded from Opsiphanes, resembles these two spe-
cies but has a relatively longer first metasomal tergite (see illustration in Muesebeck
1958). Like C. cassina, the second tergite has the medial part of the second tergite
longer than the lateral portions, and the tergites tend to both be blackish (tending
to be mostly orangish in C. invirae). The other two named Cotesia species recorded
from Opsiphanes, C. biezankoi (Blanchard) and C. opsiphanis (Schrottky), are both very
poorly characterized in their descriptions and their type locations are unknown (Fer-
nandez- Triana et al. 2020), so they are not compared here. There is a possibility that
C. invirae might prove to be a junior synonym of C. biezankoi, based on shared host
and geographic region, if the holotype of the latter were to resurface and be examined.
Our understanding of the correct nomenclature for the entire complex would benefit
from a full review of the named and putative unnamed species across all of Central and
South America, especially if all the types could eventually be located. In the meantime,
it is possible to characterize the relationships among the species for which we can
clearly establish the identity.

Description. Female. Body length 3.1—3.3 mm; fore wing length 2.9-3.1 mm.
Coloration (Fig. 2A—F). General body coloration black except: scape shading from
light to dark brown, palps pale yellow, tegulae brown, fore legs all yellowish, middle legs
all yellowish, hind legs all yellowish except distal end of femur brown/black dorsally;
distal end of tibia brown, coxae translucent yellowish, laterotergites yellowish ventral-
ly, shading to brown dorsally; sternites and hypopygium translucent yellowish. Head
(Fig. 2A, E). Facial sculpture weakly punctate; vertex sculpture smooth to very weakly
punctate; distance between posterior ocellae nearly identical with distance from outer
ocelli to compound eyes. Mesosoma (Fig. 2A, B, F). Pronotum with both dorsal and
ventral grooves present, ventral groove crenulate. Mesoscutum fully and distinctly but
shallowly punctate; scutoscutellar scrobe slightly sunken groove and formed by 8 pits.

New species of Cotesia from Opsiphanes in Colombia 15

ECUADOR

Figure |. A simplified map of Colombia, showing rough location of Tumaco B close-up of southwestern

Colombia, with location of Tumaco highlighted C caterpillar of Opsiphanes cassina on frond of the palm
Elaeis oleifera x E. guineensis D same as C but with cocoons of emerged Cotesia cassina arranged below
(normally underneath caterpillar).

Scutellum shield-shaped to subtriangular (anteriorly straight and posteriorly rounded)
and weakly convex, weakly punctate. Mesopeuron smooth and polished throughout.
Propodeum generally finely rugose/punctate with indistinct longitudinal medial ca-
rina. Legs (Fig. 2A—C). Hind coxa mostly smooth with faint sculpture on dorsal face;
inner hind tibial spurs slightly longer than outer. Wings (Fig. 2B, C). Fore wing hyaline
with dark brownish vein pigmentation; stigma more than 2x as long as broad, without
obvious pale spot at proximal end. Metacarp extending 0.60—0.70 to end of 3Rs fold
along wing edge; r approximately same length as 2RS vein and meeting it at a distinct
shallow angle; vannal lobe edge roughly semicircular with distal end slightly flattened;

16 Geraldo Salgado-Neto et al. / ZooKeys 1061: 11-22 (2021)

Figure 2. Cotesia cassina, sp. nov. A lateral habitus B dorsal view of mesosoma and anterior metasomal
tergites C fore wing D lateral view of metasoma with hind leg removed, showing laterotergites, sternites,
hypopygium and ovipositor sheaths E frontal view of head F dorsal view of posterior portions of meso-

soma, especially propodeum.

vannal lobe fringe even and dense. Metasoma (Fig. 2B—D, F). Tergite 1 roughly as
long as broad, evenly widening from anterior margin then rounding over posterior
half, mostly rugulose; tergite 2 very weakly rugulose peripherally, mostly smooth and
slightly raised centrally, roughly twice as broad as long, subrectangular with posterior
margin slightly longer medially than laterally. Hypopygium with angled but blunt tip,
not extending past dorsal end of metasoma; ovipositor with very sparse setae at tip.

New species of Cotesia from Opsiphanes in Colombia ies

Male. Similar to female except with slightly narrower metasoma.

Molecular data. COI barcode deposited in GenBank (MW405620). Using
the identification tools in the Barcode of Life Database (Ratnasingham and Hebert
2007), C. cassina is closest to C. salebrosa (Marshall), a primarily Eurasian species
attacking geometrid larvae, at a similarity level of 97.4%. Interestingly, C. invirae
appears closest (97.02% similarity) to Cotesia Whitfield78 and Cotesia Whitfield20,
two apparently conspecific sets of rearings of an undescribed species from the Lepi-
doptera Inventory of the Guanacaste Conservation Area (ACG) in northwest Costa
Rica (Janzen et al. 2009); these rearings are from another species of Opsiphanes. It
thus appears that there is a complex of at least four closely related species attack-
ing different Opsiphanes species in a variety of geographically dispersed Neotropical
habitats, as suggested by Salgado-Neto et al. (2019). BOLD and NCBI use slightly
different criteria to make cutoffs in sequence comparisons, and to calculate % simi-
larity. They also contain different sets of sequences. We checked the BOLD inves-
tigations of related species by using BLASTn (Altschul et al. 1990) to query the
NCBI nucleotide database (NCBI 1988). The same most closely related species to
Cotesia cassina and C. invirae, respectively, were recovered, with the exception that
for C. cassina, C. melitaearum (Wilkinson) and C. koebelei (Riley), both attacking
other genera of Nymphalidae but in the Holarctic region, joined C. salebrosa as
closest, at roughly 94.6-95.6% similarity for all of them. In neither the BOLD nor
the NCBI search did C. cassina come within 2.5% similarity of any other known
Cotesia species.

Host. Opsiphanes cassina (Felder & Felder) (Lepidoptera, Nymphalidae) (Fig. 1C, D).

Biology/ecology. Cotesia cassina is a gregarious parasitoid wasp that occurs mainly
in the wet season (March-May); however, their host, O. cassina, occurs throughout
the year, mainly in the rainy season (March-July). Cotesia cassina \arvae kill the host
larva before the end of the last instar and form their cocoons in a regular mass of dirty
whitish cocoons, regularly arranged disposed under the host (Fig. 1B). The larvae of
this gregarious species all emerge from the host in a short time through many different
holes in the host cuticle and spin a common woolly cocoon mass within which the
individual cocoons can be distinguished.

Distribution. Known so far from San Andrés de Tumaco, Narifio, Colombia (Ne-
otropical Region).

Etymology. ‘The specific epithet cassina, is a reference to Opsiphanes cassina (Felder
& Felder) (Lepidoptera, Nymphalidae), the host caterpillar name. The word cassina is
the feminine of cassino which in Italian means playhouse.

Acknowledgements

We are grateful to Dr Ricardo Harakava of Instituto Biolégico de Sao Paulo for analy-
ses of the DNA barcode, and to Joshua C. Gibson at the University of Illinois for as-
sistance with the wasp photography.

18 Geraldo Salgado-Neto et al. / ZooKeys 1061: 11-22 (2021)

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