Phylogenetic position of the glassfrog "Cochranella" megista (Anura: Centrolenidae) and first records for Ecuador

Abstract

"Cochranella" megista is an Endangered and rarely encountered species of glass frog that, until now, had been only registered in the Colombian Andes. Here we report this species for the first time in Ecuador expanding its known distribution ca. 530 km south of its original range. Additionally, we include C. megista in a molecular phylogeny for the first time and unambiguously place the species in the genus Nymphargus, resulting in a new combination. Habitat in both countries is fragmented and is threatened by mining concessions and agriculture.

"Cochranella" me ista ma perereca de vidro amea ada e raramente encontrada e at o momento só havia sido registrada nos Andes colombianos. Relatamos aqui a presença dessa esp cie pe a primeira ve no ador e pandindo s a distrib i o con ecida para cerca de 530 km ao sul de sua área de distribuição original. Adicionalmente, incluímos C. megista em uma fi o enia mo ec ar pe a primeira ve e a ocamos ine ivocamente a esp cie no nero Nymphargus, resultando em uma nova combinação. abitat em ambos os pa ses fra mentado e está amea ado pelas concessões mineiras e pela agricultura. Palavras-chave: anf bios conserva o esp cie amea ada Nymphargus, perereca-de-vidro-andina-grande, Reserva do Rio Manduriacu.

Full text

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Phyllomedusa - 20(1), June 2021
PROOFS
Received 09 February 2021
Accepted 24 May 2021
Distributed June 2021
Phylogenetic position of the glassfrog “Cochranella”
megista (Anura: Centrolenidae) and rst records for
Ecuador
Scott J. Trageser,1 Ross J. Maynard,1 Jaime Culebras,2 Sebastian Kohn,3,4 Amanda Quezada,5,6
and Juan M. Guayasamin7
1 The Biodiversity Group, Tucson, AZ, USA. E-mail: scott@biodiversitygroup.org.
2 Photo Wildlife Tours, Quito, Ecuador.
3 Fundación Cóndor Andino, Quito, Ecuador.
4 Fundación EcoMinga, Quito, Ecuador.
5 Tropical Herping, Quito, Ecuador.
6 Universidad del Azuay, Museo de Zoología. 24 de Mayo 7-77, Cuenca, Azuay, Ecuador.
7 Universidad San Francisco de Quito - USFQ, Colegio de Ciencias Biológicas y Ambientales COCIBA, Instituto BIOSFERA-
USFQ, Laboratorio de Biología Evolutiva, Campus Cumbayá. Casilla Postal 17-1200-841, Quito 170901, Ecuador.
Abstract
Phylogenetic position of “Cochranella” megista (Anura: Centrolenidae) and rst
records for Ecuador. “Cochranella” megista is an Endangered and rarely encountered
species of glass frog that, until now, had been only registered in the Colombian Andes.
ere e report tis species for te first time in cador epandin its non distribtion
ca. 530 km south of its original range. Additionally, we include C. megista in a molecular
poen for te first time and nambios pace te species in te ens Nymphargus,
resulting in a new combination. Habitat in both countries is fragmented and is threatened
by mining concessions and agriculture.
Keywords: Amphibians, conservation, Greater Andean Glassfrog, Nymphargus, Río
Manduriacu Reserve, threatened species.
Resumo
Posição logenética de “Cochranella” megista (Anura: Centrolenidae) e primeiros registos
para o Equador. “Cochranella” meista  ma pererecadevidro ameaada e raramente encontrada
e at o momento só havia sido registrada nos Andes colombianos. Relatamos aqui a presença
dessa espcie pea primeira ve no ador epandindo sa distribio conecida para cerca de
530 km ao sul de sua área de distribuição original. Adicionalmente, incluímos C. megista em uma
fioenia moecar pea primeira ve e aocamos ineivocamente a espcie no nero Nymphargus,
resultando em uma nova combinação.  abitat em ambos os pases  framentado e está ameaado
pelas concessões mineiras e pela agricultura.
Palavras-chave: anfbios conservao espcie ameaada Nymphargus, perereca-de-vidro-andina-
grande, Reserva do Rio Manduriacu.
Phyllomedusa 20(1):27–35, 2021
© 2021 Universidade de São Paulo - ESALQ
ISSN 1519-1397 (print) / ISSN 2316-9079 (online)
doi: http://dx.doi.org/10.11606/issn.2316-9079.v20i1p27-35

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Phyllomedusa - 20(1), June 2021
Introduction
The highest diversity of glassfrogs
(Centrolenidae) is concentrated in the northern
Andes, with 83 species (Guayasamin et al.
2020). Still, new records (e.g., Culebras et al.
2020) and descriptions of new species
(Guayasamin et al. 2019a, b, 2020) increase our
knowledge about their diversity and biogeography
nearly every year. Under this context, prolonged
fiedor prorams often rest in te discover
of species that are rare, have low detectability, or
inabit microabitats tat are diffict to access
by herpetologists. Such efforts are particularly
important in Andean forests, where glassfrog
diversity is high and many species are poorly
known, and generally restricted by more narrow
breadths of suitable habitat than lowland species
(Guayasamin et al. 2020). One such species is
“Cochranella” megista, a relatively large
assfro crrent non on from te acific
slopes of the Colombian Andes, from the
Departments of Antioquia, Chocó, Risaralda,
and Valle del Cauca at elevations of 1,400–2,100
m a.s.l. (Rivero 1985, Acosta-Galvis 2000, Rada
and Guayasamin 2008, IUCN SSC Amphibian
Specialist Group 2017). Little is known about
this taxon with only 12 observations reported
since the type specimen and Neotype (MHUA
5851; Rada and Guayasamin 2008) were
collected in 1980 (Rivero 1985). A photo record
of C. megista on iNaturalist (2012) corresponds
to a fied observation made drin te coection
effort that yielded the ICN specimens in the
1990s (M. Rada pers. comm.), from which
Bernal and Lynch (2008) cite 11 museum
specimens (ICN 17242-44, 27763-8, 27718,
28796). Only one additional observation was
recorded after the ICN specimens were collected,
which was a single observation from the type
locality of Parque Nacional Orquideas, Colombia
(IUCN SSC Specialist Group 2017). Moreover,
in the most recent taxonomic review of glassfrog
systematics (Guayasamin et al. 2009),
“Cochranella” megista was considered of
uncertain generic placement within Centrolenidae
because its unusual combination of morphological
traits (i.e., presence of webbing between Fingers
III and IV, absence of humeral spines) and the
lack, at that moment, of complementary
molecular data.
ere e report te first records of C. megista
from Ecuador, representing a substantial range
extension and only the second set of observations
in over 20 years. Additionally, we include, for
te first time D seences of C. megista into
a phylogenetic analysis, thereby improving our
understanding of the evolutionary relationships
of the species. This new record increases the
number of glassfrogs in Ecuador to 61 species
(updated from Guayasamin et al. 2020), and is
the ninth glassfrog species known to occur at the
relatively small Río Manduriacu Reserve
(Maynard et al. 2020).
Materials and Methods
Study Sites
Fieldwork was primarily carried out at the
Río Manduriacu Reserve (RMR), a protected
area managed by Fundación EcoMinga (https://
ecomingafoundation.wordpress.com/about/),
ocated on te acific ndean sopes in estern
Imbabura, Ecuador (see Lynch et al. 2014,
Guayasamin et al. 2019b, Maynard et al. 2020;
Figure 1). Opportunistic surveys were also
carried ot at ptimo araso  
78.7652 W; 1,550 m a.s.l.), a locality 40 km
from RMR, near the town of Mindo, in Pichincha
rovince abitat at  and ptimo araso
consists of primary and mature secondary lower
montane and cloud forest habitat. The habitat
surveyed for C. megista consists of primary
cloud forest and is located along a mountain
crest at elevations 1,800–2,000 m a.s.l.
Sampling time frames at RMR are outlined
in Guayasamin et al. (2019b) and Maynard et al.
(2020); however, sampling concentrated within
cloud forest habitat occurred from 01 to 06
arc      os aria
Loaiza, Rolando Peña, Fabricio Narvaez, and
Trageser et al.

29
Phyllomedusa - 20(1), June 2021
PROOFS
Figure 1. Geographic distribution of Nymphargus megistus in Colombia and Ecuador: A, type locality (red dot),
published in the original description by Rivero (1985), Parque Nacional Natural “Las Orquídeas”, Antioquia
Department; B, on border of the Chocó and Valle del Cauca Departments; C, Municipality of Pueblo Rico,
Risaralda Department; D, Municipality of Carmen de Atrato, Chocó Department; E, Municipality of Sipí,
Chocó Department; F, new record, Río Manduriacu Reserve, Imbabura Province, (yellow dot); G, new
record, near Mindo, Pichincha Province, (yellow dot). Map constructed using Google Earth Pro (7.3.3.7699).
two assistants) and 02 to 09 December 2019
   os aria oaia oando
Peña, and one assistant). Visual encounter
surveys in RMR were conducted along transects
of various lengths within primary cloud forest,
and aon five narro streams ie  m ide
between 19:00 and 02:00 h. General area
searches were conducted when vegetation was
too dense for transects of the stream. Data
collection included the following: relative
humidity, ambient temperature, date, time of
observation, geographic coordinates, sex, age
class, behavior (if any), snout–vent length, perch
height, and perch diameter (when applicable).
Climate data were collected using a Kestrel 3500
Weather Meter, geographic coordinates with a
Garmin GPSmap 62s handheld unit (WGS84
datum), and SVL with dial calipers.
Collected specimens were euthanized using
benocaine and ere fied and preserved in 
EtOH. Muscle and liver samples were preserved
in 96% EtOH. Specimens were deposited at the
Museo de Zoología of the Universidad San
Francisco de Quito (ZSFQ) under permits No.
018-2017-IC-FAU-DNB/MAE and No. MAE-
DNB-CM-2018-0105 by the Ministerio del
mbiente de cador e identification of
preserved specimens as Cochranella” megista
was based on the following diagnostic
traits: large body size (SVL in adults > 30 mm),
green dorsum with black spots, lack of humeral
spines, and moderate webbing between Fingers
Phylogenetic position of the glassfrog ocranea meista

30
Phyllomedusa - 20(1), June 2021
III and IV (Rada and Guayasamin 2008); no
oter assfro from acific sopes of te ndes
of Ecuador and Colombia has the aforementioned
combination of traits. The study was carried out
in accordance with the guidelines for use of live
ampibians and repties in fied and ab researc
(Beaupre et al. 2004), compiled by the American
Society of Ichthyologists and Herpetologists
(ASIH), the Herpetologists’ League (HL), and
the Society for the Study of Amphibians and
Reptiles (SSAR).
Terminology
For taxonomic nomenclature in glassfrogs,
we follow the proposal by Guayasamin et al.
(2009); note that quotation marks around a genus
name indicate that the placement of a species in
that genus is uncertain. For general terminology
and descriptions of morphological characters we
follow the proposals by Lynch and Duellman
(1973), Cisneros-Heredia and McDiarmid
(2007), and Guayasamin et al. (2009, 2020).
Webbing formulae follow the method of Savage
and eer  as modified b aasamin et
al. (2006).
Molecular Sequences and Phylogenetic Analysis
Genetic sequences for a fragment of the
mitochondrial gene 16S of “Cochranella”
megista were generated at the Laboratorio de
Biología Evolutiva, Universidad San Francisco
de Quito, following Guayasamin et al. (2008).
The phylogeny was inferred with the Maximum
likelihood (ML) criterion using the taxon and
gene sampling described in Guayasamin et al.
(2019a), which includes all 12 genera recognized
within the centrolenid family (Guayasamin et al.
2009). ML trees were estimated using GARLI
0.951 (Genetic Algorithm for Rapid Likelihood
Inference; Zwickl 2006). GARLI uses a genetic
aoritm tat finds te tree topoo branc
lengths, and model parameters that maximize
lnL simultaneously (Zwickl 2006). Default
values were used for other GARLI settings, as
per recommendations of the developer (Zwickl
2006). Bootstrap support was assessed via 1,000
pseudoreplicates under the same settings used in
tree search. Genetic distances (uncorrected p)
between the new species and its closest relatives
were calculated using PAUP v.4.0a (Swofford
2002).
Results
Nymphargus megistus (Rivero, 1985)
Figures 1, 2
Centrolenella megista Rivero, 1985
Cochranella megista Ruiz-Carranza
and Lynch, 1991
“Cochranella” megista Guayasamin et al., 2009
Nymphargus megistus, new combination
Common name.—We propose the common
name of reater ndean assfro for
Nymphargus megistus, following the criteria
defined b ooma and aasamin  e
Greek word megistos means arest or
reatest n panis te common name is rana
de cristal andina grande”.
Identication.—Nymphargus megistus can be
distinguished from all other species of
Nymphargus by exhibiting the following
combination of features: large size (SVL in
adult males = 30.8–37.4 mm SVL; in adult
females = 36.9–40.0 mm), green dorsum with
black spots, moderate webbing between Fingers
III and IV of the hand, absence of humeral spines
in adult males, and lacking iridophores on the
gastrointestinal peritoneum (Rada and
Guayasamin 2008). Nymphargus megistus is an
atypical species of the genus Nymphargus, where
all species have a reduced webbing between
Finger III and IV. In contrast, the hand webbing
in N. megistus is more extensive: III 2—(1 1/2–
1 3/4) IV. No additional variation or sexual
dimorphism has been observed.
Evolutionary relationships and generic
placement.—Our phylogenetic results (Figure 3)
clearly indicate that the species is part of the
genus Nymphargus. Note that, as explained in
Trageser et al.

31
Phyllomedusa - 20(1), June 2021
PROOFS
the methods section, our taxon sampling included
taxa from all the currently recognized genera in
Centrolenidae. Thus, we formally place
Centrolenella megista Rivero 1985 in the genus
Nymphargus sensu Guayasamin et al. (2009).
Nymphargus megistus and N. grandisonae are
recovered as sister taxa; the genetic distance
between these two species is 2.0%.
New records.—ECUADOR: one adult male,
SVL: 30.8 mm; Ecuador, Imbabura Province, Río
Manduriacu Reserve; 00°20.217 N, 78°51.445 W,
1,857 m a.s.l.; 21:14 h, 01 March 2019; Ross
Maynard, Jaime Culebras, and Scott Trageser;
primary cloud forest, perched on broad-leaf stalk
2 cm diameter, 1 m high, 15.6°C, 95% relative
humidity, light rain occurred earlier in the day;
  fied nmber 
ECUADOR: one adult female, SVL: 38.9 mm;
Ecuador, Imbabura Province, Río Manduriacu
Reserve; 00°20.206 N, 78°51.452 W, 1,850 m
a.s.l.; 20:19 h, 07 December 2019; Ross
Maynard, Scott Trageser, and Leslie Rochefort;
primary cloud forest, perched on leaf, 60 cm
high, 14.8°C, 100% relative humidity, 5 mph
ind fond drin eav rain   fied
number SCA 1600). ECUADOR: one adult
Phylogenetic position of the glassfrog ocranea meista
Figure 2. Nymphargus megistus. (A) Adult %, ZSFQ-4071, SVL = 30.8 mm, dorsal view. (B) Adult %, ZSFQ-4071,
SVL = 30.8 mm, ventral view. (C) Adult &, ZSFQ-3924, dorsal view, SVL = 38.9 mm. (D) Adult &, ZSFQ-
3924, ventral view, SVL = 38.9 mm.
A
C
B
D

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Phyllomedusa - 20(1), June 2021
Figure 3. Phylogenetic placement of Nymphargus megistus inferred under Maximum Likelihood using the
mitochondrial gene 16S. Genbank accession numbers for N. megistus are as follow: MZ314501 (ZSFQ
4071) and MZ314502 (ZSFQ 3924).
Trageser et al.

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Phyllomedusa - 20(1), June 2021
PROOFS
male, SVL: 35.9 mm; Ecuador, Pichincha
rovince ptimo araso nearb te ton of
Mindo; 00°1.758 S, 78°45.912 W, 1,550 m
a.s.l.; 22:00 h, 11 February 2020; Amanda
Quezada and Frank Pichardo; primary cloud
forest, perched on leaf 20 cm high, found during
a cloudy night, about 2 meters from creek; TH-
822.
Discussion
The two records we report of Nymphargus
megistus from the Río Manduriacu Reserve and
the one individual from near Mindo represent the
first observations of te species in cador
extending its known distribution ca. 530 km
south from the nearest record in the southern
Chocó Department of Colombia (Figure 2).
Although a substantial range extension, it is not
unexpected; a number of anuran species are
known to have similar distributions along the
western slope of the Cordillera Occidental of
Colombia and northern Ecuador, including other
glassfrog species that have been reported from
the Río Manduriacu Reserve (i.e., Centrolene
peristicta, Espadarana prosoblepon, Nymphargus
balionotus, and N. grandisonae; Maynard et al.
2020). Despite the seemingly low detectability
of N. megistus at the localities in which it has
been observed, the absence of records between
the Ecuadorian localities and those from
Colombia demonstrate the need for additional
survey efforts within this region.
Our observations of Nymphargus megistus at
RMR were made nine months apart (March and
December 2019) in primary cloud forest near the
upper reaches of RMR just below the ridgeline.
Interestingly, both individuals were observed ca.
175 m from the nearest stream, and within 25 m
of one another. Similar observations are recorded
in te fied notes of aria ristina rdia
Robayo from August 1987 reporting three
individuals (ICN 17242–4) found in mature
forest, away from bodies of water, and 0.5–2 m
above the ground. Rada and Guayasamin (2008)
suggest, based off these observations, that during
non-breeding periods the adults migrate towards
the interior of the forest away from streams.
During early December and early March, our
surveys of four streams at the upper reaches of
RMR did not yield observations of N. megistus
or their advertisement calls. Although this
suggests a lack of breeding activity, the
observation of the gravid female in December
2019 indicates that reproduction in this species
likely does occur during this time frame, despite
the fact that we did not observe this. In fact, call
data for this species has yet to be recorded and
described; however, males were recorded in the
notes by either Maria Cristina Ardila or Pedro
M. Ruiz (M. Rada pers. comm.) to have been
vocalizing at an unknown date on streamside
branches in Araceae plants 1.5–1.8 m above the
ground (Rada and Guayasamin 2008). The
incidental observation near Mindo of a male near
a stream in mid-February does correspond to
potential rainy season breeding activity, though
it was not observed calling. Considering the
proximity of our RMR observations away from a
stream, and at different times of year, it is
possible this area serves as an important
migration corridor.
The IUCN currently recognizes N. megistus
as Endangered (IUCN SSC Amphibian Specialist
Group 2017). Although our two records from
RMR extend the range of N. megistus
considerably, it is unknown if the Ecuadorian
populations are disjunct from those in Colombia.
Results from the Colombia Red List Assessment
Workshop (2016) indicate that the majority of
the population resides in small subpopulations
with no genetic exchange. Furthermore, severe
framentation ma aread eist aon specific
corridors at elevations necessary for this species
to disperse, as Andean cloud forests in Colombia
are disproportionately excluded from regional
and national protected area networks (Forero-
Medina and Joppa 2010). Additionally, in light
of increasing mining activity in Ecuador (Roy et
al. 2018) and immediate threats facing RMR
(Guayasamin et al. 2019b, Maynard 2020),
additional surveying of the vast area between the
Phylogenetic position of the glassfrog ocranea meista

34
Phyllomedusa - 20(1), June 2021
Colombian and Ecuadorian occurrences is
necessary to better understand its distribution,
natural history, and conservation status.
Acknowledgments
The authors thank Carolina Reyes-Puig and
Diego F. Cisneros-Heredia (ZSFQ) for assistance
in organizing the logistics and museum accession
of reated fiedor at  e are ratef to
Fundación EcoMinga for their continued
partnership and efforts to protect and manage
 and to os ara oaia oando ea
arceo esano imm vare os ieira
and Leslie Rochefort for their invaluable help
drin portions of te fied or e are aso
grateful to the Villamizar family, Socio Bosque
Program, IUCN Netherlands, The Dutch National
Postcode Lottery, World Land Trust, Fundación
Cóndor Andino and other donors for the
continued funding of the reserve and the purchase
of additional properties to protect. We are
especially grateful to the Santa Rosa de
Manduriacu community for their openness to our
research, providing access to private property,
logistical assistance, and their incredible
hospitality. RJM and ST are especially grateful
for a generous donation provided by Stephanie
Fogel to The Biodiversity Group, which helped
fnd a portion of te fiedor tat ed to data
presented herein. Genetic sequences were
generated at the Laboratorio de Biología
Evolutiva at Universidad San Francisco de
Quito, with the help of Nathalia Valencia and
Daniela Franco. We also thank the Ministerio
de Ambiente y Agua for granting all required
research permits. JMG’s research is supported
by USFQ (Collaboration Grant 11164, 16871;
COCIBA grants: 5467, 5521, 16808; Programa
ndita  espestas a a crisis de
biodiversidad: La descripción de especies
como herramienta de conservación”). We are
also grateful to the reviewers for their time,
effort, and knowledge during the peer review
process.
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Editor: Vanessa K. Verdade
Phylogenetic position of the glassfrog ocranea meista