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ZOOTAXA
ISSN 1175-5326 (print edition)
ISSN 1175-5334 (online edition)
Accepted by T. Tiunova: 6 Mar. 2023; published: 21 Mar. 2023 371
Zootaxa 5256 (4): 371–382
https://www.mapress.com/zt/
Copyright © 2023 Magnolia Press Article
https://doi.org/10.11646/zootaxa.5256.4.5
http://zoobank.org/urn:lsid:zoobank.org:pub:DDAF05C4-5D74-4799-85F6-E06082B8BFE2
Who is Andesiops peruvianus (Ulmer, 1920) (Ephemeroptera: Baetidae)?
New insight from the type basin using morphological and molecular analyses
RENZO MERA1*, RAQUEL SICCHA-RAMIREZ1,2,5, JORGE L. RAMIREZ1,2,6,
DANIELA NÚÑEZ-RODRÍGUEZ1,2,7, RICARDO BRITZKE1,2,8, KAREN VELÁSQUEZ-RODRÍGUEZ3,
RINA RAMÍREZ1,2,9 & ANA A. HUAMANTINCO1,10
1Facultad de Ciencias Biológicas, Universidad Nacional Mayor de San Marcos, Lima, Perú.
2Museo de Historia Natural, Universidad Nacional Mayor de San Marcos, Lima, Perú.
3Facultad de Ciencias de la Vida y de la Salud, Universidad Científica del Sur, Lima, Perú.
4
�
kvelasquezr@cientifica.edu.pe; https://orcid.org/0000-0001-9540-796X
5
�
zsicchar@unmsm.edu.pe; https://orcid.org/0000-0002-5597-3948
6
�
jramirezma@unmsm.edu.pe; https://orcid.org/0000-0001-8138-9203
7
�
dnunezrodriguez@gmail.com; https://orcid.org/0000-0002-3638-1752
8
�
rbritzke@unmsm.edu.pe; https://orcid.org/0000-0002-1042-2544
9
�
rramirezm@unmsm.edu.pe; https://orcid.org/0000-0003-1924-5844
10
�
ahuamantincoa1@unmsm.edu.pe; https://orcid.org/0000-0001-6558-1326
*Corresponding author.
�
renzo.mera@unmsm.edu.pe; https://orcid.org/0000-0002-5147-1406
Abstract
Andesiops peruvianus (Ulmer, 1920) is a small minnow mayfly with a distribution throughout the Andes Mountains,
mainly in lotic environments. In recent years, the taxonomic status of the species has been shifting, even finding out
molecular and morphological evidence to consider it as a cryptic species. In this work, we collected seven specimens of A.
peruvianus from the Lake Titicaca in Peru, the type locality and perform molecular and morphological analyses to test their
relationship with specimens from other regions. The species delimitation analysis revealed the existence of five MOTUs
for specimens identified as A. peruvianus along the Andes Mountains, while the Lake Titicaca specimens formed a single
MOTU with high interspecific distance. In this MOTU were found specimens with different types of abdominal gills, from
translucent to markedly tracheated supporting the hypothesis of phenotypic plasticity at the abdominal gill level. Also, an
extension in the range of tarsal claw denticles, from 10–12 to 10–14, were observed, higher than what were found in other
regions. Our results support A. peruvianus as a species complex and reveal A. peruvianus from Titicaca as a single MOTU.
Future morphological revisions of topotype specimens as well as from other Andean regions are required to strengthen the
diagnosis of informative characteristics at both the larval and adult stage to elucidate the real status of the species.
Key words: Andes Mountains, molecular approach, MOTU, species complex, species delimitation
Introduction
Andesiops peruvianus (Ulmer, 1920) is a small minnow mayfly of Baetidae (Ephemeroptera) distributed along the
Andes from Venezuela to both Chile and Argentina (Domínguez et al. 2009) and usually found in rapid and well-
oxygenated waters in high-altitude regions, mainly in rivers or streams. Its presence along the inter-Andean valleys
in lentic environment has also been documented, although not predominant (Lugo-Ortiz & McCafferty 1999). It is
a common part of the aquatic macroinvertebrate richness found in biomonitoring assessments and established as a
species highly sensible to pollution (Figueroa et al. 2003; Ríos-Touma et al. 2014).
Lake Titicaca is located in the Peruvian-Bolivian highlands at approximately 3800 m.a.s.l. (Ministerio del
Ambiente [MINAM] 2014). Its diversity is characterized by the presence of different groups and has been reported
encompassing groups considered endemic (Lüssen et al. 2003; Albrecht et al. 2009; Kroll et al. 2012; Jurado-
Rivera et al. 2020; Jaume et al. 2021; Zapelloni et al. 2021) and a significant number of insect families (Dejoux
1991). Among this insect diversity, a scarcely reported species for the lake is Andesiops peruvianus. This species
MERA ET AL.
372 · Zootaxa 5256 (4) © 2023 Magnolia Press
was firstly described by Ulmer (1920) as Baetis peruvianus using only adult specimens from Peruvian-Bolivian
highlands including Lake Titicaca in Guaqui, Bolivia. The first description of the larval stage was made by Needham
& Murphy (1924), though the first detailed description was presented by Berner (1980) using topotypes specimens
from the Peruvian-Bolivian highlands. Later, Lugo-Ortiz & McCafferty (1999) redescribed the species based on the
combination of certain morphological features as non-tracheated abdominal gills or thin tracheated ones and two rows
of denticles in the tarsal claw, although the taxonomy at the genus level has required recent revisions (Nieto 2004a).
DNA Barcoding is meant to deploy a standard molecular marker for the molecular identification of metazoans. To
meet this goal, the cytochrome oxidase subunit I (COI) is being successfully used in a wide variety of groups (Hebert et
al. 2003; Baird & Sweeney 2011; Raupach et al. 2020). However, some species do not show full resolution displaying
overlaps between intraspecific and interspecific distances, as in species of Caenis in North America (Webb et al.
2012) or the genus Baetis in the Canary Islands (Gattolliat et al. 2018) which were found as a species complex. This
uncertainty also appears in A. peruvianus, in which despite the apparent taxonomic resolution within the group, evidence
of inconsistencies has emerged in recent years by morphological details overlooked in the past, and which have been
validated with molecular data (COI and 16S rRNA genes) (Ossa-López et al. 2018). These authors found at least four
molecular operational taxonomic units (MOTUs) correlated with differences in the shape and size of the abdominal
gills, just as in the number of denticles on the tarsal claws supporting the hypothesis of a possible species complex
in A. peruvianus (Ossa-López et al. 2018). However, samples from different regions of The Andes and near the Lake
Titicaca, the type basin locality, were not included in that study. Furthermore, some traits about the genetic structure of A.
peruvianus populations have been described, with panmictic population within basins, strong isolation among mountains,
and reasonable gene flow between nearly headwaters except in areas with deep isolating canyons (Finn et al. 2016).
Thus, our study aims to evaluate the specimens of A. peruvianus, collected from Lake Titicaca and test their
phylogenetic relationship with specimens from other regions. We used an integrative approach, including species
delimitation methods (GMYC, PTP, pPTP) by using COI gene and morphological analysis of abdominal gills,
mouthparts and tarsal claws. Furthermore, this study represents the first molecular data closely related to the
holotype described in 1920 (Ulmer 1920).
Materials and methods
Study area
The study area included two sampling stations, one located in the village Cayñajoni at 3830 m.a.s.l. (15°28′56′′
S, 69°24′16.06′′ W) (Fig. 1a) and the other in Huarisani at 3837 m.a.s.l. (15°16′59′′ S, 69°45′00′′ W) (Fig. 1b), both
in Puno Department, Peru, on the shores of Lake Titicaca. The sampling stations were constituted by rocky substrate
with a depth of 0.5 m to 1 m. The habitat was characterized by lateral rocky mounds covered with mosses and water
with few suspended particles. Sampling was carried out in November 2020, in Cayñajoni, and in August 2021, in
Huarisani both in the dry season.
Sampling
Nymphs were collected by using a D-Net (mesh opening of 500 µm) and small auxiliary nets in a stretch of
10 m, with a distance of five meters towards the lake. The D-net was slightly dragged over the substrate, rocks and
submerged vegetation. Specimens were preserved in 96° alcohol inside 50 ml and 2 ml tubes and labeled with the
location data. Subsequently, they were headed to the Laboratory of Aquatic Invertebrates of Universidad Nacional
Mayor de San Marcos (UNMSM) to be examined.
Morphological analysis
Taxonomic identification of nymphs to the species level followed dichotomous keys found in Domínguez et
al. (2009) and in order to categorize the specimens in larval stages, the classification in Beltrán et al. (2011) was
followed. Before dissecting four specimens for morphological analysis (LSMFI00111, LSMFI00507, LSMFI00508
and LSMFI00509) (Table 1), a stereoscopic microscope with a Nikon (12Mpx) and Leica (5Mpx) digital camera
were used to obtain photos of the specimens. Mouthparts and legs were digested in KOH (10%) for 15 hours.
Permanent slides of mouthparts, legs, abdominal gills and paraproct were prepared using Euparal as mounting
media. A total of four specimens were morphologically analyzed. Three specimens included in this study were only
part of the molecular analysis: LSMFI00169, LSMFI00170 and LSMFI00171 (Table 1).
WHO IS ANDESIOPS PERUVIANUS ?Zootaxa 5256 (4) © 2023 Magnolia Press · 373
TABLE 1. Accession codes, reference and collection data of the specimens used in the molecular analysis.
Species GenBank
accesion
BOLD Systems
accesion
Reference Basin Country MOTU
Andesiops
peruvianus
-LSMFI00111 This study Lake Titicaca
(type locality)
Peru MOTU 2
A. peruvianus -LSMFI00169 This study Lake Titicaca
(type locality)
Peru MOTU 2
A. peruvianus - LSMFI00170 This study Lake Titicaca
(type locality)
Peru MOTU 2
A. peruvianus - LSMFI00171 This study Lake Titicaca
(type locality)
Peru MOTU 2
A. peruvianus - LSMFI00507 This study Lake Titicaca
(type locality)
Peru MOTU 2
A. peruvianus - LSMFI00508 This study Lake Titicaca
(type locality)
Peru MOTU 2
A. peruvianus - LSMFI00509 This study Lake Titicaca
(type locality)
Peru MOTU 2
A. peruvianus KT625456 - Ossa-López et al.,
2018
Chinchiná River Colombia MOTU 3
A. peruvianus KT625457 - Ossa-López et al.,
2018
Chinchiná River Colombia MOTU 1
A. peruvianus KT625458 - Ossa-López et al.,
2018
Chinchiná River Colombia MOTU 3
A. peruvianus KT625459 - Ossa-López et al.,
2018
Chinchiná River Colombia MOTU 7
A. peruvianus KT625460 - Ossa-López et al.,
2018
Chinchiná River Colombia MOTU 7
Andesiops torrens GU175985 - Sabando et al.,
2011
Maule River Chile MOTU 4
A. torrens GU175986 - Sabando et al.,
2011
Maule River Chile MOTU 4
A. torrens GU175987 - Sabando et al.,
2011
Maule River Chile MOTU 4
A. torrens GU175988 - Sabando et al.,
2011
Maule River Chile MOTU 4
A. torrens GU175989 - Sabando et al.,
2011
Maule River Chile MOTU 4
A. peruvianus KU710321 - Finn et al., 2016 Napo River Ecuador MOTU 5
A. peruvianus KU710322 - Finn et al., 2016 Napo River Ecuador MOTU 5
A. peruvianus KU710323 - Finn et al., 2016 Napo River Ecuador MOTU 5
A. peruvianus KU710324 - Finn et al., 2016 Napo River Ecuador MOTU 5
A. peruvianus KU710325 - Finn et al., 2016 Napo River Ecuador MOTU 5
Andesiops sp. - EVTEC1047-13 Polato et al., 2018 Napo River Ecuador MOTU 9
Andesiops sp. - EVTEC1048-13 Polato et al., 2018 Napo River Ecuador MOTU 8
Andesiops sp. - EVTEC1049-13 Polato et al., 2018 Napo River Ecuador MOTU 8
Andesiops sp. - EVTEC1050-13 Polato et al., 2018 Napo River Ecuador MOTU 10
Andesiops sp. - EVTEC1051-13 Polato et al., 2018 Napo River Ecuador MOTU 6
MERA ET AL.
374 · Zootaxa 5256 (4) © 2023 Magnolia Press
FIGURE 1. Sampling stations in Cayñajoni and Huarisani, Puno, Peru and across Andes Mountains.
Molecular analysis
DNA was extracted from seven complete specimens, with the Wizard SV Genomic DNA Purification System
Kit (Promega), following the manufacture protocol. The quality and quantity of DNA were measured with NanoDrop
(Desjardins & Conklin 2010). We amplified a fragment of approximately 700 bp of the cytochrome oxidase subunit
I (COI) gene using Zplank primers (Prosser et al. 2013). The final volume of the PCR reaction was 15 µl, with
the following conditions: 1X Taq Buffer (10X), 250 µM dNTPs, 0.4 µM of each primer, 2mM MgCl2, 1 I of Taq
Polymerase, and 2 µl of template DNA. PCR was performed with: an initial denaturation at 95°C for 1 min, 5 cycles
at 94°C for 40 s, 45°C for 40 s, 72°C for 1 min, and then 35 cycles at 94°C for 40 s, 51°C for 40 s, 72°C for 1 min and
a final extension at 72°C for 5 min. Positive amplicons were purified using AmpliCleanTM Cleanup Kit following
the manufacturing instructions and sequenced using an ABI 3730xl System sequencer (Applied Biosystems) in both
directions (forward and reverse) through the Sanger methodology in Macrogen Inc. (Seoul, South Korea).
COI sequences were edited using CodonCode Aligner 9.0.2. (http://www.codoncode.com/aligner). A.
peruvianus sequences (accessions KT625456-KT625460) (Ossa-López et al. 2018), Andesiops torrens Lugo-
Ortiz & McCafferty, 1999 (accessions GU175985-GU175989) (Sabando et al. 2011), A. peruvianus (accessions
KU710321-KU710325) (Finn et al. 2016) from BOLD Systems and Andesiops sp. (accessions EVTEC1047-13 to
EVTEC1051-13) (Polato et al. 2018) from GenBank were included in the analyses (Table 1). The establishment of
molecular operational taxonomic units (MOTUs) was conducted by three molecular approaches: General Mixed
Yule Coalescent (GMYC) (Pons et al. 2006), Poison Tree Processes (PTP) (Zhang et al. 2013) and the Bayesian
version of PTP (bPTP). For this purpose, a fasta file was created with the sequences aligned using the ClustalW
(Thompson et al. 1994) in BioEdit (Hall 1999). Then, an ultrametric Bayesian tree was constructed using BEAST
WHO IS ANDESIOPS PERUVIANUS ?Zootaxa 5256 (4) © 2023 Magnolia Press · 375
2 (Bouckaert et al. 2014). jModelTest v.2.1.10 (Darriba et al. 2012) was employed to select the best evolutionary
model according to the alignment sequences, which turned out to be GTR + G (shape: 0.255). Also, we set a relaxed
clock model (log normal), a Birth Death tree Model, 10 millions of Monte Carlo Markov chain (MCMC) iterations
stored every 1000, and a burn-in of 10%. The MCMC convergence was verified in Tracer v.1.7.1 (Rambaut et
al. 2018). The tree obtained was used as input for the species delimitation analyses (GMYC, PTP and bPTP) in
SPdel (https://github.com/jolobito/SPdel). K2p intraspecific and interspecific genetic distances and consensus of
species delimitation methods were generated in SPdel. All sequences were uploaded in the project DNA Barcoding
of Invertebrates of Titicaca (TITII) on Barcode of Life Data Systems (BOLD Systems) (Ratnasingham & Hebert
2007).
Results
Morphological analysis
The four specimens morphologically analyzed were classified as Andesiops peruvianus and all of them
categorized as Class 7 (with a head capsule width between 0.92–1.05 mm) (Table 2), the last instar prior to the
hatching of the adult. One of the larvae (LSMFI00509) presented well-developed wingpads, although not yet dark.
The larval mouthparts such as labrum, labium, mandibles, maxilla and the paraprocts did not present differences
with the original larva description by Needham & Murphy (1924), the detailed description of topotypes by Berner
(1980) and the redescription of the species by Lugo-Ortiz & McCafferty (1999). The abdominal gills and the number
of tarsal claws denticles displayed contrasting characteristics with the detailed description of topotypes (Berner
1980) and redescription (Lugo-Ortiz & McCafferty 1999). Abdominal gills are described as colorless and without
obvious tracheae but the gill margin as strongly chitinized by Berner (1980) and as untracheated or poor tracheated,
translucent, with margin usually tinged with brown and held dorsolaterally by Lugo-Ortiz & McCafferty (1999).
In this study, three out of four specimens (LSMFI00111, LSMFI00507 y LSMFI00508) showed poor tracheated
abdominal gills concurring with the redescription, while LSMFI00509 had markedly tracheated abdominal gills.
All specimens had abdominal gills with brown-tinged margins (Fig. 2, Table 2). On the other hand, the tarsal claws
are described bearing 10–12 stout denticles by Berner (1980) and by Lugo-Ortiz & McCafferty (1999) bearing two
rows of denticles with 9–11 denticles each, the first row well developed and the second row reduced in size and both
slightly increasing apically. In Lake Titicaca specimens, the number of denticles in the well-developed first row was
in the range of 10–14, while in the reduced second row it was 8–12, clearly lower than the first in size and number
of denticles and in both row the size increased apically (Fig. 2, Table 2).
TABLE 2. Number of tarsal claws denticles and type of gill in each specimen. *N.V: Not visible.
Specimen
Code
Tarsus claw’s
position on
thorax
First row
(well developed)
Second row
(reduced in size)
Gills Heah capsule
width (mm)
Larval
Stage
LSMFI00111 Leg I 14 N.V Poorly tracheated 0.966 Class 7
Leg II N.V N.V
Leg III 13 9
LSMFI00507 Leg I 14 10 Translucent 0.954 Class 7
Leg II 13 N.V
Leg III 14 N.V
LSMFI00508 Leg I 13 10 Poorly tracheated 0.966 Class 7
Leg II 12 8
Leg III 14 N.V
LSMFI00509 Leg I 12 10 Markedly tracheated 0.983 Class 7
Leg II 10 12
Leg III 12 10
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376 · Zootaxa 5256 (4) © 2023 Magnolia Press
FIGURE 2. Types of abdominal gills on Andesiops peruvianus from Lake Titicaca; A, LSMFI00507. B, LSMFI00111. C,
LSMFI00508. D, LSMFI00509 and tarsal claw of first leg with 14 denticles; E and F, LSMFI00111.
Molecular analysis
There were obtained seven COI sequences (658 base pairs) from the specimens identified as A. peruvianus. The
final alignment consisted of 27 sequences of 658 base pairs with 250 variable sites and 221 informative parsimony
sites. The PTP analysis formed 10 MOTUs, bPTP established the same 10 MOTUs and the GMYC analysis nine
MOTUs. Consensus of the three species delimitation methods resulted in the establishment of 10 MOTUs (Fig. 3,
Table 3). The sequences obtained in this study (Lake Titicaca, Peru) were clustered in a single MOTU not shared
with any other sequence (MOTU 2). The sequences of A. peruvianus from Ecuador made up a single MOTU (MOTU
5). A. peruvianus from Colombia were grouped into three different MOTUs (MOTU 1, MOTU 3 and MOTU 7).
The five specimens of Andesiops sp. (no taxonomic resolution to species) sampled in Ecuador formed four different
MOTUs (MOTU 6, MOTU 8, MOTU 9 and MOTU 10) and one MOTU corresponded exclusively to specimens
of A. torrens Lugo-Ortiz & McCafferty, 1999 (MOTU 4) (Fig. 3, Table 3). In the GMYC analysis Andesiops sp.
(MOTU 9) and Andesiops sp. (MOTU 10) merged to form a single MOTU, unlike what was attained in the other
methods (PTP and bPTP) in which they split into two MOTUs (MOTU 9 and MOTU 10) (Fig. 3). Considering only
specimens identified as A. peruvianus, the conformation of five MOTUs was noticed (MOTU 1, MOTU 2, MOTU
3, MOTU 5 and MOTU 7) (Fig. 3). The tree topology shows A. peruvianus as a species complex with MOTUs well-
defined and related to hydrographic basins.
WHO IS ANDESIOPS PERUVIANUS ?Zootaxa 5256 (4) © 2023 Magnolia Press · 377
FIGURE 3. Bayesian tree showing the MOTUs obtained by species delimitation analyses. Values on branches indicate posterior
probabilities > 0.9. The scale bar indicates nucleotide substitutions per site.
Each MOTU had a genetically closest group (Nearest Neighbor) whose distance inter-MOTUs varied from
1.67% to 23.12% (Table 3). The lowest genetic distance was found between MOTU 9 and MOTU 10 (1.67%),
both from Napo River, Ecuador. The highest distance was found between MOTU 1 (Chinchiná River, Caldas) and
MOTU 6 (Napo River, Ecuador) with a value of 23.12%. MOTU 2 (Lake Titicaca, Peru) generated in this study,
was close to MOTU 3 (20.34%), formed by specimens from Chinchiná River, Colombia (Table 3). The intragroup
distances of each MOTU had low values while the intergroup values showed high values. There was no evidence of
an overlap between these two values (Fig. 4).
MERA ET AL.
378 · Zootaxa 5256 (4) © 2023 Magnolia Press
FIGURE 4. DNA Barcoding gap analysis showing intragroup distance (red bars) and intergroup distance (blue bars)
TABLE 3. Distance to the nearest neighbor MOTU. Mean: mean intraspecific distance, Max: maximum intraspecific
distance, NN: Nearest Neighbor, DtoNN: Distance to the Nearest Neighbor.
Intragroup and Intergroup distances
Basin Mean Max NN DtoNN
MOTU 1 A. peruvianus (Chinchiná River) - - MOTU 6 23.12
MOTU 2 A. peruvianus (Lake Titicaca) 0.22 0.496 MOTU 3 20.344
MOTU 3 A. peruvianus (Chinchiná River) 0 0 MOTU 5 18.349
MOTU 4 A. torrens (Maule River) 0.386 0.645 MOTU 10 21.221
MOTU 5 A. peruvianus (Napo River) 0.243 0.304 MOTU 6 15.225
MOTU 6 Andesiops sp. (Napo River) - - MOTU 5 15.225
MOTU 7 A. peruvianus (Chinchiná River) 0.714 0.714 MOTU 8 4.042
MOTU 8 Andesiops sp. (Napo River) 0 0 MOTU 7 4.042
MOTU 9 Andesiops sp. (Napo River) - - MOTU 10 1.673
MOTU 10 Andesiops sp. (Napo River) - - MOTU 9 1.673
Discussion
The previous study of A. peruvianus carried out in the Chinchiná River basin, in Colombia, gave the first lights
of crypticism within the species with evidence at the molecular and morphological level (Ossa-López et al.
2018). Pursuing to clarify A. peruvianus taxonomy, this work found fundamental evidence that contributes to the
understanding of the possible crypticism of this species by analyzing, for the first time, specimens within the type
locality basin (Lake Titicaca) (Ulmer 1920).
The first description of the larva was made by Needham & Murphy (1924) using specimens from Puente del
Inca, in Mendoza, Argentina. This first description covered mainly external morphological characters of the abdomen
and tail. The first detailed description of the larval stage was carried out by Berner (1980) using topotype specimens
from the Lake Titicaca basin. In Berner (1980), characters are detailed at the level of abdominal gills, tarsal claws
and some mouthparts. In addition, a variant is mentioned within the species, whose abdominal gills are described
as rust colored in the central area and faintly tracheated (Berner 1980). After that, Lugo-Ortiz & McCafferty (1999)
made the most complete redescription of the larval stage, giving thorough details on the mouthparts. However,
the analyzed specimens in Lugo-Ortiz & McCafferty (1999) came from sampling stations throughout the Andes,
covering all its conforming countries from Venezuela to Argentina without including topotype specimens. Lugo-Ortiz
& McCafferty (1999) noted a remarkable plasticity in size and body coloration between the different populations,
but a consistency in mouthpart characters. Conversely, the specimens of A. peruvianus in Ossa-López et al. (2018)
showed a correlation between the conformation of four MOTUs and the morphological differences at the abdominal
gill level and denticles of the tarsal claw. The four specimens analyzed here were categorized as the last instar (Class
7), therefore homogeneous morphological characters were expected. Nevertheless, one specimen (LSMFI00509)
WHO IS ANDESIOPS PERUVIANUS ?Zootaxa 5256 (4) © 2023 Magnolia Press · 379
showed well-developed wingpads evidencing its proximity to hatching unlike the others. Our results show specimens
with different types of abdominal gills, from translucent to markedly tracheated in the same collection site (the
only one with markedly tracheated abdominal gills was LSMFI00509 also with well-developed wingpads), but
molecularly identical forming a single MOTU, supporting the hypothesis given by Berner (1980), who considered
these differences at the gill level as a phenotypic plasticity. The abdominal gill analysis evidenced the presence of
a much more tracheated type than the one described for the species (Berner 1980; Lugo-Ortiz &McCafferty 1999).
The tracheated abdominal gills are very common in species of Baetidae (Vilela et al. 2013; Suttinun et al. 2020),
although the precise definition of its specific function is still unknown (Remy 1925) and it is presumed that it ranges
from a purely mechanical function to directly participating in respiratory processes (Remy 1925; Wingfield 1939).
The decrease in dissolved oxygen has been proposed as one of the two most influential factors in modeling diversity
in the Peruvian-Bolivian highlands (Jacobsen & Marín 2008). In this sense, the occurrence of adaptations as more
tracheated abdominal gills that maximize oxygen uptake in a species such as A. peruvianus successfully linked
to these high-altitude regions is possible (Molina et al. 2008; Ossa-López et al. 2018). Another factor to consider
is that the most tracheated abdominal gills were found in the specimen close to hatching, so markedly tracheated
abdominal gills could be merely related to the growth of the specimen of this population in Lake Titicaca.
For the tarsal claw, our results show that the denticles in the well-developed first row were in a range of 10–14,
similar to the range mentioned in the description of topotypes of 10–12 denticles (Berner 1980), but higher than the
range of 9–11 in the redescription of Lugo-Ortiz & McCafferty (1999) and the range in Ossa-López et al. (2018),
with rows of 7 or 11 denticles. The number of denticles of the tarsal claws of the Lake Titicaca MOTU in some
cases doubled what was found in MOTUs of the Chinchiná River. In Baetidae, tarsal claws have been widely used
as a taxonomic character due to their wide range of variation in size, shape, and number of denticles (Nieto 2010).
In Baetodes Needham & Murphy, 1924, the range of denticles varies from 5–6 denticles to 11–12 (Nieto 2004b). In
Camelobaetidius Demoulin, 1966 the number of denticles ranges from 7–11 in C. phaedrus (Traver & Edmunds,
1968) to 30–31 in C. ipaye Nieto, 2003, with almost all species having a single range of denticles distributed in a
spatulate tarsal claw (Nieto 2003), while in Callibaetis Eaton, 1881 the variation lies in the size of the tarsal claw in
relation to the size of the tarsus (Nieto 2008). In Andesiops, A. peruvianus has two rows of denticles with a similar
range in both, in A. ardua Lugo-Ortiz & McCafferty, 1999 the second row has half the denticles of the first row and
A. torrens has only one denticles in the second row (Nieto 2004a). Tarsal claws have always been associated with
adhesion function mainly to the rocky substrate in lotic environments (Caspers 1979). This adaptive requirement
would not be necessary in waters such as Lake Titicaca, where the lentic environment with only a slight tide would
not mean a force capable of modeling an increase in the number of denticles in the tarsal claws as an adhesive
instrument; therefore other adaptive requirements must be influencing these changes in this population of Lake
Titicaca.
A. peruvianus from Lake Titicaca established a single MOTU (MOTU 2) being close to MOTU 3 (20.34%),
whose specimens were collected in the Chinchiná basin in Caldas, Colombia. The result of the molecular analysis
shows the existence of five MOTUs considering only specimens morphologically identified as A. peruvianus,
indicating that the species constitutes a species complex. The possibility of a complex of species within A. peruvianus
has already been evidenced in Ossa-López et al. (2018), where the MOTUs had genetic divergences between 17.4
and 24.5% for the COI gene. The number of MOTUs (and potential undescribed species) within A. peruvianus could
be bigger if we consider the specimens reported as Andesiops sp. or if intermediate localities were included. It is
necessary to consider that A. peruvianus distribution ranges from the north of the Andes Mountains, in Venezuela
to the south, in Argentina (Domínguez et al. 2009). The specimens analyzed here only belong to specific regions
of Colombia, Ecuador, and Peru, leaving many unexplored regions in the Andes, whose specimens could help to
understand the precise status of the species at the morphological and molecular level. The inclusion of specimens
from lacking areas could reduce (if we consider A. peruvianus as a clinal diversity occasioned by distances) or
increase (in a case of species complex isolated by basins) the number of MOTUs, however the high distances found
herein (up to 23%) support the latter case. Also, similar cases of cryptic diversity within the family Baetidae have
been reported, as in Gattolliat et al. (2018), who found out that Baetis (Rhodobaetis) canariensis Müller-Liebenau,
1971 considered until then as a single species, was actually composed of four closely related species, revealing
morphological details not yet described in larval stage. By a detailed morphological analysis, characters which
were not considered in the original description showed up, matching with the establishment of different MOTUs
(Gattolliat et al. 2018).
MERA ET AL.
380 · Zootaxa 5256 (4) © 2023 Magnolia Press
Species delimitation performed by molecular approaches has helped to exhibit crypticism within many insect
species (Ekrem et al. 2010; De León et al. 2020; Raupach et al. 2020). Following this same path, A. peruvianus
could be a case of crypticism supported by molecular evidence found out in Caldas, Colombia (Ossa-López et al.
2018) and now strengthened by our findings made in specimens from the type locality basin. Our results support A.
peruvianus as a species complex and reveal specimens from Titicaca as a different MOTU than the ones described
as A. peruvianus from other basins. Future morphological revisions of topotype specimens as well as from other
Andean regions are required to strengthen the diagnosis of informative characteristics at the larval stage and adult
to elucidate the real status of the species.
Acknowledgement
We thank Fondecyt and the World Bank for the funding of the Project “Environmental DNA Metabarcoding: A
powerful tool for the evaluation and monitoring of contaminated ecosystems in Peru” through the agreement 022-
FONDECYT-BM-INC.INV which made this research possible and the Department Ichthyology of the Natural
History Museum of the Universidad Nacional Mayor de San Marcos for the field equipment used during the
sampling. We appreciate the Peruvian Barcode of Life (PeBol) for the use of the CodonCode software.We also
thank Katherin Mamani, Gerson Quispe y Manuel Silva for the help in the field.
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