P Campesino (Sepúlveda-Seguroetal 2022)

Organisms Diversity & Evolution
https://doi.org/10.1007/s13127-022-00549-9
ORIGINAL ARTICLE
Phylogeographic structure suggests environmental gradient

speciation in a montane frog from the northern Andes of Colombia
Ana M. Sepúlveda‑Seguro1 · Carlos M. Marín1 · Adolfo Amézquita2 · Yuly A. García1 · Juan M. Daza1
Received: 26 January 2021 / Accepted: 24 February 2022

© Gesellschaft für Biologische Systematik 2022
Abstract
Allopatric speciation has been posed as the main mechanism affecting amphibian diversification. In contrast, the role of
alternative mechanisms such as ecological speciation has received less attention, and empirical evidence of this mechanism in
shaping species richness gradients remains elusive in the tropics. Highly heterogeneous landscapes can favor both allopatric
and ecological speciation either by producing physical barriers to dispersal or by inducing selective pressures on lineages
distributed along environmental gradients (e.g., climatic gradients). Based on explicit predictions under the ecological
speciation hypothesis, we tested whether the phylogeographic, acoustic, morphological, and climatic features differ across the
range of Pristimantis viejas, a frog species distributed along a wide altitudinal gradient in the northern Andes of Colombia.
In agreement with our predictions, we found two highly differentiated lineages within P. viejas, showing striking differences
in molecular diversity, acoustic, and climatic distribution. Importantly, we found that variation of such characteristics is
highly congruent with variation in elevational ranges of these two lineages (P. viejas distributed in the lowlands and a cryptic
sister lineage distributed in the highlands), and our evidence suggest that the divergence of these lineages was influenced
by temperature-related variables. These results support a temperature-driven speciation process which may influence the
amphibian diversity patterns in tropical mountains. Last, we describe and name the new cryptic lineage distributed in the
highlands. We highlight that an integrative framework in phylogeographic studies should not only focuses on the identification
of hidden lineages and their associated processes but also, when multiple lines of evidence are available, it should conduct
to their formal description as species are the units for multiple biodiversity disciplines.
Keywords Cryptic diversity · Ecological speciation · Morphology · Species description · Taxonomy · Terrarana
Introduction highly heterogeneous landscapes where different types of

geomorphological features (e.g., rivers, mountain systems,
Allopatric speciation, the divergence of lineages favored by depressions) can reduce gene flow resulting in independent
physical barriers, has become a primary hypothesis explain- evolutionary lineages (Smith et al., 2014; Boumans et al.,
ing the diversification of amphibians (Guayasamin et al., 2017; Guarnizo & Canatella, 2014). In contrast, the role
2020; Hua & Wiens, 2010; Kozak & Wiens, 2006, 2007; of alternative mechanisms such as ecological speciation
Valero et al., 2019). This is a reasonable and, in most cases, (i.e., the speciation process resulting from ecologically-
an intuitive interpretation of the observed species richness based divergent selection, sensu Rundle & Nosil, 2005)
patterns, especially for taxa distributed in regions with has received less attention (Caro et al., 2013; Coyne & Orr,
2004; Schluter, 2009), and empirical evidence of this mecha-
nism in promoting lineage diversification remains elusive in
Ana M. Sepúlveda-Seguro and Carlos M. Marín contributed
equally. the tropics (Beheregaray et al., 2015).
The landscape geomorphological complexity is expected
* Juan M. Daza to lead to environmental gradients (Boumans et al., 2017;
[email protected] Caro et al., 2013). This is the case of mountain systems in
1
Grupo Herpetológico de Antioquia, Biology Institute,
the tropics where lineages distributed along altitudinal gra-
Universidad de Antioquia, 050010 Medellín, Colombia dients experience different climatic conditions including
2
Laboratory of Biodiversity and Cloud Forests Conservancy,
temperature, precipitation, oxygen availability, and atmos-
Bioconservancy, Jardín, Colombia pheric pressure (Cadena et al., 2011; Caro et al., 2013). If
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A. M. Sepúlveda‑Seguro et al.
differences in climatic conditions among populations per- and if so, this structure is related to physical or climatic
sists in an evolutionary period, local adaptations to these gradients.
conditions are likely to arise, and gene flow is expected to
decrease as individuals of one population cannot tolerate
environmental conditions where the other population occurs Materials and methods
(Rundle & Nosil, 2005; Valero et al., 2019). In the long run,
this asymmetry in the occupied climatic space among dis- Taxon sampling
junct populations can lead to speciation events (Caro et al.,
2013; Valero et al., 2019). Hence, in tropical mountains, it To test the ecological speciation hypothesis, we assembled
is expected that ecological speciation contributes to lineage four different datasets. First, we assembled a morphological
differentiation at the regional scale. dataset including 69 specimens of Pristimantis viejas.
The Andes mountain range is one of the most diverse Second, we obtained genetic data from 54 specimens.
regions of the world harboring an exceptional number of Third, we evaluated bioacoustics data from eight specimens.
species and endemism of plants and terrestrial vertebrates Last, we assembled a large geographic dataset of P. viejas
(Jenkins et al., 2013; Kreft & Jetz, 2007). For instance, including 341 museum specimens. The morphological,
10.8% of the world amphibian diversity and 27.6% of South genetic, and acoustic datasets are all subsets of this largest
American diversity is concentrated in the northern Andes geographic dataset.
(Armesto & Señaris, 2017). As amphibians have limited
dispersal abilities and are physiologically constrained Molecular laboratory procedures
to occupy narrow environmental conditions (Bonetti &
Wiens, 2014; Lawler et al., 2010; Smith & Green, 2005), Total genomic DNA was extracted from ethanol pre-
climatic gradients resulting from mountain orogeny are served tissues using the GeneJET Genomic DNA Purifi-
expected to be linked to amphibian diversification. Despite cation kit (Thermo Fisher Scientific, Inc.). We amplified
the expectation of ecological speciation affecting amphibian and sequenced two mitochondrial and one nuclear region
species richness patterns on the Andes, only a handful of for 54 specimens corresponding to Pristimantis viejas. In
studies have attributed the observed divergence between addition to the new sequences generated for P. viejas, we
lineages to variations along ecological gradients (Guarnizo sequenced ten specimens corresponding to P. factiosus, a
& Cannatella, 2014; Guarnizo et al., 2009; Guayasamin morphologically similar species that share distribution with
et al., 2017; Marin et al., 2018). Therefore, it is still unclear the highland populations of P. viejas. We included for the
in which proportion ecological speciation relative to others first time, P. factiosus in a phylogenetic analysis. We also
mechanisms such as allopatric speciation contributes to the included for the first time sequences from the type locality
amphibian species richness patterns in this region. for these two nominal species. The three regions include
The predictions under ecological speciation in altitudinal partial sequences of the mitochondrial 16S ribosomal gene
gradients include as follows: (1) populations distributed in (primers: 16SCL–16SDH; Santos et al., 2003), the mito-
different parts of the gradient should belong to differenti- chondrial protein-coding gene cytochrome oxidase subunit
ated lineages, (2) the most recently diverged lineage should I coi (primers: dgLCO and dgHCO; Folmer et al., 1994),
have accumulated less genetic variation as it has inhabited and the nuclear protein-coding gene tyrosinase precursor tyr
the new environment during less time, and (3) differences (primers: Tyr1C–Tyr1G; Bossuyt & Milinkovitch, 2001).
in the use of some ecological resources throughout the Amplification protocols followed Santos et al. (2003) and
gradient (e.g., climate) should emerge among lineages, as Pinto-Sánchez et al. (2012). PCR products were purified and
well as differences in relevant traits related to reproductive sequenced at the Macrogen facilities in Korea (Macrogen,
isolation (e.g., acoustic attributes). Here, we tested these Inc.). Chromatograms were checked and manually edited
predictions by studying the genetic, climatic, acoustic and using Geneious version 9.1.8 (http://www.geneious.com,
morphological variation across most of the known distri- Kearse et al., 2012). GenBank accession numbers for novel
bution range of the rain frog Pristimantis viejas (Lynch & sequences are provided in Table 1.
Rueda-Almonacid, 1999). This species inhabits both well
conserved and secondary forested areas, and it is distributed Phylogenetic analysis
from the eastern slope of the northern Cordillera Central to
the western slope of the Cordillera Oriental in Colombia, We assembled a genetic matrix including 264 terminals rep-
spanning a wide altitudinal distribution ranging from 100 to resenting 13 terminals as outgroups, 185 nominal species
2100 m a.s.l. This makes the species an ideal model to test within Pristimantis, and 56 terminals as either intraspe-
whether this wide distribution belongs to a panmictic species cific variation or undetermined species (Online Resource
or there is phylogeographic structure across its distribution, 1). Character sampling included five genomic regions:
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Phylogeographic structure suggests environmental gradient speciation in a montane frog from…
Table 1 GenBank accession Voucher Epithet Lineage Latitude Longitude 16S COI TYR
numbers of novel sequences
generated during this study AJC0922 adnus 7.682000 −78.202200 ON009871 ON008317
AJC0924 adnus 7.682000 −78.202200 ON009872 ON008318
ARU784 factiosus 5.490120 −75.057140 ON009873 ON008319
MHUAA08075 factiosus 6.304440 −75.144380 ON009874 ON008320 ON000888
MHUAA08080 factiosus 6.302240 −75.140690 ON009875 ON008321
MHUAA08093 factiosus 6.304440 −75.144910 ON008322
MHUAA08100 factiosus 6.305020 −75.144100 ON009876
MHUAA08808 factiosus 6.305310 −75.146580 ON009877 ON008323 ON000889
MHUAA05555 viejas Highlands 6.978301 −75.111101 ON009882 ON008328 ON000890
MHUAA05562 viejas Highlands 6.986900 −75.137500 ON009883 ON008329
MHUAA11733 viejas Highlands 6.991300 −75.119600 ON008350
MHUAA06597 viejas Lowlands 6.185463 −74.995779 ON008361 ON000895
MHUAA07119 viejas Lowlands 6.206710 −74.855000 ON009911 ON008362 ON000896
MHUAA10472 viejas Lowlands 6.139910 −75.066830 ON009915
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Table 1 (continued) Voucher Epithet Lineage Latitude Longitude 16S COI TYR

the mitochondrial regions 12S, 16S, coi, and the nuclear Lineage assignment
coding regions rag1 and tyr. Each genomic region was
aligned using the G-INS-i method in MAFFT v7 under We examined 341 specimens under the name Pristimantis
default parameters (Katoh & Standley, 2013). We estab- viejas housed at the Museo de Herpetología Universidad de
lished 11 partitions (12S, 16S, and each codon position for Antioquia (MHUA) (see the “Taxon sampling” section and
the coding regions) and inferred the best partition scheme Online Resource 2, Examined specimens). Because of the
and evolution model for each partition using ModelFinder strong evidence suggesting that populations of this species
under the BIC criterion (Kalyaanamoorthy et al., 2017). form two distinctive lineages, we assigned museum speci-
We inferred a phylogenetic tree using maximum likelihood mens to each of these lineages. To do this, we assigned all
in the program IQTREE (Nguyen et al., 2015). Nodal sup- specimens from a particular site where we previously obtained
port was estimated from 10,000 pseudo replicates using the genetic samples to the lineage to which these samples clus-
ultrafast bootstrap approach (Hoang et al., 2018). Phyloge- tered in our phylogenetic analysis. We acknowledge this speci-
netic results showed that samples of P. viejas are structured men classification procedure might lead to the recognition of
in two well-differentiated lineages (see results for more artificial groups thus biasing our results. To overcome this
detailed explanation). Therefore, we designed the following issue, we replicated all analyses with an alternative dataset
analyses to test the ecological speciation hypothesis using including only sequenced specimens. The environmental,
this lineage distinction. morphological, and spatial analyses conducted on both data-
sets led to similar results; thus, to improve data visualization,
we show the results based on the largest dataset (results based
Genetic analyses on the reduced dataset are shown in the Online Resource 2).
Within Pristimantis viejas, we constructed haplotype net- Environmental analysis

works using the median joining method (Bandelt et al.,
1999) as implemented in the program PopART (Leigh, To test whether the recovered lineages were distributed in
2016). Because we found two distinct evolutionary lineages, different parts of the climatic space, we conducted discri-
we inferred haplotype networks for each lineage using the minant analysis coupled to principal component analysis
mitochondrial region coi. We also estimated nucleotide and (DAPC, Jombart et al., 2010). In brief, DAPC aims at pre-
haplotype diversity parameters for these two networks using dicting group membership of a number of individuals by
the R packages haplotypes (Aktas, 2020) and pegas (Para- using a linear combination of intercorrelated descriptors.
dis, 2010). Last, to have an estimate of genetic divergence Principal components analysis (PCA) is often used to graph-
within/among lineages, we included intraspecific sampling ically depict among-groups variation in multiple traits. Yet
of the closest relatives to P. viejas. We calculated uncor- PCA is focused on trait covariation and ignores the group
rected genetic distances among these closely related species identity of individuals, which makes it useful to merely
with 1,000 bootstrap pseudoreplicates using the program describe variation but at risk of overlooking differences
MEGA 7.0 (Kumar et al., 2016). between groups (Jombart, 2008; Jombart & Colins, 2015).
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As output variable, we used lineage identity, and as input and allows to test simultaneously for the effect of geographic
variables, we used the values of the bioclimatic variables and environmental landscapes on the genetic population dif-
associated to each georeferenced locality where individu- ferentiation. We implemented the MMRR method using a
als were recorded. Values from 19 climatic variables were matrix of genetic distances as dependent variable and both
obtained from the WorldClim dataset v.2 (Flick & Hijmans, geographic and environmental distance matrices as predic-
2017) downloaded at 30-arc-second (approximately 1-km) tor variables. We conducted this multiple regression analysis
resolution using the raster R package (Hijmans, 2017). We using the R code provided by Wang (2013), and statistics sig-
first estimated the median number (and 95% confidence nificance was assessed with 10,000 permutations. The genetic
interval) of successful groups assignments obtained by distance matrix was computed on the coi alignment using the
chance. Then, to estimate the minimum number of princi- substitution model K80 implemented in the dist.dna function in
pal components that best summarized climatic covariation the ape R package (Paradis & Schliep, 2019). Although it has
and predicted group membership, we used cross-validation been shown that variation in mtDNA affects the performance of
with 90% of the data as training set, and 300 replicates organisms occurring under different thermal regimes (Camus
at each level of PCA retention. For each added PC, this et al., 2017; Dowling et al., 2008; Lajbner et al., 2018; Mishmar
analysis renders the mean successful assignment and its et al., 2003), we used a coi fragment as a measure of neutral
root mean square error; we thus selected the number of PCs genetic variation among populations. We observed the same
that maximized the former and minimized the latter or a phylogeographic pattern using the other sequenced regions
lower number when the contribution of additional PCs was for these populations (16S, tyr) but with less genetic variation
considered negligible (i.e., below 1% of successful assign- among individuals. To estimate the environmental distance
ments). We then ran the DAPC with the selected number of matrix, we computed the Euclidean distances on seven tem-
PCs and the lowest number of discriminant functions that perature variables (see results of the “Environmental analysis”
eventually led to classification success rates significantly section for details on the selection of these variables) using the
above 90%. These analyses were conducted with the ade- R base function dist (R Core Team, 2020). Last, the geographic
genet R package (Jombart, 2008; Jombart & Colins, 2015). distance matrix (in km) was computed on the georeferenced
Additionally, we tested whether the climatic niche of the records using the distm function in the geosphere R package
two recovered lineages significantly differ when compared (Hijmans, 2019). These matrices were computed with 53 sam-
against the background climate. To do this, we conducted pled haplotypes (all specimens having coi sequence) and their
a niche overlap test based on ellipsoid envelopes follow- associated geographic coordinates and climatic values.
ing the pipeline described by Nuñez-Penichet et al. (2021)
using the ellipsenm R package (Cobos et al., 2020; Online Morphological analysis
Resources 2, Supplementary Methods).
We assembled a morphometric dataset including 17 vari-
Geographic and environment determinants ables, selected following Watters et al. (2016), on 69 speci-
of genetic differentiation mens of Pristimantis viejas (39 for the lowland lineage
and 30 for the highland lineage). Variables abbreviations
Genetic differentiation between lowland and highland lineages are as follow: SVL (snout-vent length), HL (head length),
of P. viejas can result in one out of two patterns: first, isola- HW (head width), ED (eye diameter), UEW (upper eyelid
tion by distance, in which genetic differentiation is explained width), EN (eye-nostril distance), SL (snout length), IND
solely by the geographic distances separating populations, (internarial distance), IOD (interorbital distance), TD (tym-
and second, isolation by environment in which genetic dif- panum diameter), FLL (forearm length), HAL (hand length),
ferentiation is explained by environmental (i.e., climatic) dis- THL (thigh length), TL (tibia length), FL (foot length),
similarities. To support ecological speciation as the underly- Fin4DW (finger IV disk width), and Toe4DW (toe IV disk
ing cause of lineage divergence within P. viejas, a pattern of width). Measurements were taken under the stereoscope
isolation by environment would be recovered indicating that with a digital caliper to the nearest 0.1 mm. To estimate
lineages occurring under different environmental regimes have the degree of morphometric segregation between both the
diverged as they adapted to local climates. Here we addressed two lineages and the sexes, we ran a DAPC (see the “Envi-
the causes of the genetic differentiation within P. viejas by ronmental analysis” section) using morphometric variables
implementing a multiple matrix regression with randomiza- as descriptors and the combination of lineage-sex levels as
tion (MMRR) analysis (Wang, 2013). This method is an exten- a grouping variable. For these analyses we only included
sion of the multiple linear regression for distance matrices adult specimens.
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◂Fig. 1 (Left) Maximum likelihood tree (-lnL = − 116,566.9) depicting Geographic lineage distribution

evolutionary relationships within the Pristimantis ridens species group
sensu Padial et al. (2014) inferred from a partitioned analysis using
4807 sites, five genomic regions, and 254 terminals. The complete tree Our museum specimen classification resulted in the
is provided as Online Resource 1. (Right) Haplotype networks based assignment of 341 specimens to Pristimantis viejas (89
on 675 bp of the coi fragment for the recovered lowland and highland assigned to the lowland lineage and 252 specimens to the
lineages within P. viejas. The number of mutational steps are repre- highland lineage). Distribution of the georeferenced records
sented on the lines connecting haplotypes. One asterisk represents a
specimen collected near the type locality. Two asterisks represent the of all examined specimens shows a striking pattern of
holotype for the highland lineage disjunct altitudinal distribution between lineages (Fig. 2b–c)
with both lineages overlapping in a narrow altitudinal band
near to 1600 m a.s.l. Noticeably, no geographic barrier (e.g.,
Results deep valleys, mountain ranges) separates both lineages, and
instead major differences in geographic distribution between
Phylogenetic relationships lineages occurs along altitudinal axis (Fig. 2b–c).
Sequence alignment resulted in a matrix of 4824 sites: 1061 Environmental analysis

sites of 12S, 1906 of 16S, 675 of coi, 639 of rag1, and 543 of
tyr. The best partition scheme included five partitions and the The DAPC successfully assigned 97.4% of individuals to
inferred evolution models were 12S + 16S: TIM2 + F + R7, the appropriate lineage (Fig. 3a) using a subset of 19 biocli-
COIpos2 + RAG1pos1 + RAG1pos2 + TYRpos1 + TYR- matic variables, as summarized by eight principal compo-
pos2: TPM3 + F + R3, COIpos1: TIM3e + G4, COIpos3: nents combined in a single discriminant function (Fig. 3b).
TN + F + G4, and RAG1pos3 + TYRpos3: K2P + R3. The Only two out of 252 individuals were misclassified in the
inferred phylogenetic tree with a log-likelihood value lowland lineage and seven out of 89 in the highland lineage.
of − 116,566.9 recovers well-supported nodes within Pristi- The cross-validation analysis showed that the rate of suc-
mantis (Online Resource 1), and it is in agreement with pre- cessful assignments barely increased to 98.2% when using
vious studies (Padial et al., 2014; Pinto-Sánchez et al., 2012; 13 principal components. The highest contribution (0.25)
Rivera-Correa & Daza, 2016; Rivera-Correa et al., 2017). to the successful assignment by DAPC was represented by
Pristimantis factiosus and P. viejas are nested within the temperature seasonality (BIO4), followed by a group of
Pristimantis ridens species group sensu Padial et al. (2014) seven variables (all around 0.10): mean temperature of dri-
(Fig. 1). est quarter (BIO9), minimum temperature of coldest month
Pristimantis viejas was recovered as sister to a lineage (BIO6), mean temperature of wettest quarter (BIO8), mean
composed by P. cerasinus from Panama and Costa Rica and temperature of coldest quarter (BIO11), annual mean tem-
Pristimantis sp. (voucher AJC 0217) from Panama (Figs. 1 perature (BIO1), maximum temperature of warmest month
and 2a). Pristimantis viejas shows a strong phylogeographic (BIO5), and mean temperature of warmest quarter (BIO10).
structure with two well-supported lineages segregated in Hence, the discriminant function that summarized this cli-
altitude (Fig. 2b–c). Individuals from one lineage were dis- matic space was highly correlated with elevation (Pearson’s
tributed between 100 and 1570 m a.s.l (hereafter referred as r = − 0.927, 95% CI = − 0.898 to − 0.932, Fig. 3c). The
lowland lineage). In contrast, the other lineage is composed results also showed that the highland lineage occupies a
by individuals ranging from 1520 to 2150 m a.s.l (hereafter much narrower temperature space compared to its lowland
referred as highland lineage; Fig. 2b–c). counterpart (Fig. 3b). Additionally, the results of niche over-
lap showed that the niche of the highland lineage is associ-
Genetic analyses ated with both lower seasonality (BIO4) and annual mean
temperature (BIO1) variables (higher seasonality and annual
We found differences between the haplotype networks mean temperature in the lowland lineage; Online Resource
belonging to each lineage within Pristimantis viejas (Fig. 1 2, Fig. S2). The observed overlap value between both niches
right). Both lowland and highland lineages have similar was zero (p = <0.001). This pattern is consistent with the
levels of haplotype diversity (h = 0.9 for lowland lineage, hypothesis of ecological speciation within P. viejas.
h = 0.96 for highland lineage). By contrast the lowland line-
age has significant higher levels of nucleotide diversity in Geographic and environment determinants
comparison to the highland lineage (pi = 0.01 vs pi = 0.005, of genetic differentiation
respectively). Additionally, uncorrected genetic distances
between the two lineages within P. viejas are similar to Results of the MMRR analysis support an isolation by envi-
genetic distances among species in the phylogenetic neigh- ronment pattern in which climatic variables explain most of
borhood (Online Resource 2, Fig. S1). the genetic variation between lowland and highland lineages
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Fig. 2 a Geographic distribution of genetic sampling including the blue dots records from highland lineage. c Geographic distribution of
phylogenetic neighborhood of Pristimantis viejas. Color in symbols P. viejas showing the two recovered lineages. Open circles represent
match lineages depicted in Fig. 1. b Altitudinal distribution across the sequenced specimens. Notice the absence of evident geographic bar-
latitudinal gradient of the two recovered lineages within the nominal riers separating both lineages
species P. viejas. Red dots denote records from lowland lineage and
of P. viejas (Table 2). The MMRR results reject an isolation the sexes, related to the first discriminant function (Online
by distance pattern, thus indicating that geographic distances Resource 2, Fig. S3b), which in turn was contributed by
do not significantly contribute to explain the observed genetic head width (0.24), foot length (0.23), and head length (0.16).
dissimilarities between lineages. Smaller differences occurred between species, particularly
small between males, along the second discriminant function
Morphological analysis (Online Resource 2, Fig. S3b), which was mostly contributed
by thigh length (0.38) and foot length (0.29). All this vari-
The DAPC correctly assigned 76.5% of frogs to the appropri- ation was independent of body size, which contributed less
ate lineage-sex category (Online Resource 2, Fig. S3a) using than 0.005 to both axes.
morphometric traits summarized by four principal compo-
nents, which in turn were combined into two discriminant
functions (Online Resource 2, Fig. S3b). Most misclassi- Discussion
fications occurred among males of both lineages (Online
Resource 2, Fig. S3a), which substantially overlapped in the We provide multiple evidence that is consistent with a spe-
morphometric space (Online Resource 2, Fig. S3b). It also ciation process driven by environment in the rainfrog Pristi-
revealed much higher morphometric differences between mantis viejas. We found two divergent lineages distributed
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Fig. 3 Between lineages differences in the climatic space occupied discriminant function that represent variation in thermal variables of
by two lineages within Pristimantis viejas, as inferred from DAPC. the corresponding localities. c Relationship between the single dis-
a Posterior probability of assignment of encountered individuals to criminant function and elevation of the corresponding localities. To
each lineage (bars) based on the climatic variables, compared to the improve visualization of data, the X axis has been flipped in plots 3b
actual lineage identity. b Distribution of individuals along the single and 3c. See the “Material and methods” section for further details
along an elevational gradient on the northeastern Cordil- a high uncertainty in the temporal paleogeography of this
lera Central in Colombia. These lineages differ in genetics, region in Colombia (Mora et al., 2020). Second, there is a
geographic distribution, environmental space, morphology, high variation in the estimated timing of lineage separa-
and advertisement call (see the “Bioacoustics” section). Our tion. Previous studies in Pristimantis biogeography have
results strongly suggest that this divergence resulted from dated the node between these two lineages around 8 MYA
adaptation to local climatic conditions at the gradients’ (Pinto et al., 2012), 5 MYA (Mendoza et al., 2015) and 4
extremes. A feasible scenario explaining this divergence MYA (Waddell et al., 2018), encompassing the late Miocene
involves ancient lowlands populations initially diverging in through Pliocene.
allopatry (likely as a consequence of the orogenic process), Our results support an ecological speciation process in
accompanied by a subsequent differentiation in the climatic which niche divergence was identified as the main driver of
tolerances spurred by adaptation to local climatic condi- the environmental differentiation between lineages of Pristi-
tions in the ends of the gradients. However, it is too early mantis viejas. Moreover, this mode of speciation is consist-
to correlate the emergence of the northern Cordillera Cen- ent with the observed parapatric distribution range with no
tral with the divergence of these two lineages. First, there is evident geographical barriers separating both lineages. We
acknowledge that other processes might drive a similar pat-
Table 2 Results of the MMRR model testing the effect of geographic tern of geographic distribution. These processes include (1)
and environmental variables on the genetic differences between low- limited dispersal abilities and (2) post-speciation ecological
land and highland lineages within Pristimantis viejas. In bold are divergence. The former process can lead to a parapatric dis-
highlighted the statistically significant results tribution by preventing geographically isolated lineages to
Predictor Coefficient t-statistic p-value overlap thus maintaining them in parapatry (Khimoun et al.,
2013). The latter process can lead to a parapatric distribution
Intercept 0.02 5.46 1.000
via range expansion of allopatric lineages with subsequent
Geographic distances −2.29e −05 −2.21 0.90
displacement of niche-related characters once they reach
Environmental distances 0.010 25.34 <0.001
secondary contact (Blair et al., 2013; Losos & Glor, 2003).
R2 = 0.33, F = 343.6, p = <0.001 Under the limited dispersal abilities scenario, it would be
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expected an isolation by distance process, with interpopula- dispersal), they also favor ecological speciation by inducing
tion divergence being predicted by geography. However, we selective pressures on lineages distributed along environmen-
found that geographic distance does not predict the genetic tal gradients (e.g., climatic gradients; Cadena et al., 2011;
distances within P. viejas, thus, we discard this process Caro et al., 2013). Remarkably, despite allopatric speciation is
as potential explanation. Nevertheless, we cannot discard commonly invoked to explain divergence events across many
the potential role of post-speciation ecological divergence taxa, the effects of geographical barriers on lineage divergence
because of the uncertainty of associate the divergence of have rarely been tested in the Andes (but see Guarnizo et al.,
both lineages within P. viejas with the uprising chronology 2009; Guarnizo & Cannatella, 2014; Muñoz-Ortiz et al., 2015).
of the Cordillera Central. Since allopatric and ecological speciation can be coupled, it is
We found that differences in the occupied climatic space expected that both processes contribute to a similar extent to
between both lineages occurs along temperature rather than promote lineage differentiation. However, the relative contribu-
precipitation variables. This finding suggests that lineage tion of both processes in shaping the species richness gradients
diversification was influenced by temperature, an observation remains currently as an unknown dimension of the Andean
further supported by recent studies showing that temperature- biota diversification.
related variables are the best predictors of amphibian Phylogeographic studies focusing on species-rich taxa
diversification (Rolland & Condamine, 2019; Vasconcelos inhabiting highly complex landscapes often unravel deeply
et al., 2019). Moreover, it has been suggested that in the divergent lineages, thus suggesting that patterns of species
complex Andean landscapes, temperature is associated with richness are largely underestimated across both taxa and
genetic structure of amphibian communities (Guarnizo & regions (Fouquet et al., 2007; Guzmán et al., 2018; Everson
Canatella, 2014). This pattern stems in the trend of amphibians et al., 2020; Jaramillo et al., 2020). Noticeably, the vast major-
to maintain their thermal preferences (i.e., thermal niche ity of authors do not describe and name the newly discovered
conservatism). Along elevational gradients, variation in species. Failing to describe these “new” components of the
temperature is usually larger than variation in precipitation diversity can limit our understanding of lineages diversifica-
(Fernandes, 2016; Guarnizo & Canatella, 2014; Online tion with immediate consequences in our ability to decipher
Resource 2, Fig. S4); therefore, it is expected that temperature- the evolutionary and ecological mechanisms underlying such
driven speciation strongly influences the amphibian diversity diversity patterns (Chenuil et al., 2019; Kozak & Wiens,
patterns in mountain systems (Cadena et al., 2011). 2016). Here, we describe a new species we discovered by
Cryptic diversity, the phenomenon by which either studying the distribution of the genetic diversity of an Andean
sister species or deeply divergent lineages exhibit similar lineage and more importantly, we were able to associate the
morphology, is ubiquitous across the amphibian tree of life distribution of this lineage with climatic factors. We argue that
(Bickford et al., 2007; Meegaskumbura et al., 2002). In an integrative approach in phylogeographic studies should not
the neotropical frog genus Pristimantis, the most speciose only focus on discovering cryptic lineages, but also describing
lineage of terrestrial vertebrates, growing evidence suggests and naming species, especially when supported by multiple
that cryptic diversity is more common than previously lines of evidence. By adopting such approach, we will gain
thought (Elmer & Canatella, 2008; Ortega-Andrade et al., further insights into the evolution of morphological, behavio-
2015; Arteaga et al., 2016; Guayasamin et al., 2017; ral, and ecological traits which ultimately may help understand
Urgiles et al., 2019). The two distinct lineages within P. the evolutionary forces shaping the species richness gradients
viejas provide a good example of cryptic lineages, as both across highly diverse taxa and regions.
entities are largely indistinguishable in external morphology Our phylogenetic, genetic, acoustic, and environmental
(Fig. 5a–h). Lynch and Rueda-Almonacid (1999) reported results consistently support the existence of two divergent
on the presence of P. viejas in the highlands of Guatapé, lineages within the nominal species Pristimantis viejas. We
Antioquia, a region falling inside the known distribution consider the lowland lineage as Pristimantis viejas sensu
range of the highland lineage. To the best of our knowledge, stricto because we included specimens and sequences from
both lineages are only differentiated externally (in life) by the type locality and they were nested in this lineage. Below,
the iris coloration (Fig. 5a, e). In contrast, genetic distance, we describe and name the highland species.
acoustic traits, and geographic distribution strongly support
the recognition of the highland lineage as a new species.
The notion that landscape complexity has shaped species Taxonomic description
richness is pervasive in the literature of Andean taxa diversifi-
cation (Chazot et al., 2018; Esquerré et al., 2019; Guayasamin Pristimantis campesino sp. nov
et al., 2020; Hazzi et al., 2018; Luebert & Weigend, 2014).
Although Andean landscapes can favor lineage divergence Eleutherodactylus viejas (Lynch & Rueda-Almonacid,
via allopatric speciation (i.e., mediated by physical barriers to 1999:311; part).
13
Paratypes 31 specimens, all from Colombia, Department of

Antioquia. From municipality of Anorí, vereda El Nevado
(6.9783, -75.1111; 1,650 m a.s.l): MHUA-A 5555 (adult
female) collected on 2007 by Laura Bravo; from vereda
San Benigno (6.9869, -75.1375; 1,600 m a.s.l.): MHUA-A
5562 (adult female) collected on 2007 by Laura Bravo; from
Vereda El Retiro, Porce III hydroelectric project protected
area–EPM (6.9913, -75.1196; 1,689 m a.s.l.): MHUA-A
11731, 11733, 11734, 11914, 12019, 12021, 12027 (adult
males), MHUA-A 11737, 11913, 12023, 12024 (adult
females) collected on 2019 by AMS, CMM, and YAG.
From municipality of Guatape, vereda El Tronco (6.3028,
-75.1434; 1,875 m a.s.l.): MHUA-A 8059, 8088 (subadult
females), MHUA-A 8099 (adult male) collected on 2013
by JMD, MHUA-A 8810 (juvenile) collected on 2015 by
Fig. 4 Holotype of Pristimantis campesino sp. nov. adult male MHUA- JMD, MHUA-A 11164 (adult male) collected on 2018 by
A 12022 in life. SVL 17.40 mm. Photo by Carlos M. Marín Adriana Restrepo; from vereda Los Naranjos: MHUA-A
11385 (adult male) collected on 2018 by Diego Alzate-
Estrada. From municipality of Amalfi, vereda Guayabito
Holotype MHUA-A 12022, adult male (Fig. 4) from Colom- (6.8216, -75.0747; 1,906 m a.s.l.): MHUA-A 9428 (sub-
bia, Department of Antioquia, municipality of Anorí, vereda adult female) collected on 2015 by JMD. From municipal-
El Retiro (6.9912, -75.1196; 1,669 m a.s.l., collected on Sep- ity of Granada, vereda Tafetanes (6.1267, -75.1088; 1,823 m
tember 30, 2019 by AMS, CMM and YAG. a.s.l.): MHUA-A 10473 (adult male) collected on 2016 by
Fig. 5 Variation in the coloration of the iris (photographs) and con- 9744, g MHUA-A 11468, and h MHUA-A 9759; and P. factiosus,
cealed surfaces of thighs (illustrations) of Pristimantis campesino i MHUA-A 11736, j MHUA-A 11735, k MHUA-A 11732, and l
sp. nov., a MHUA-A 11913, b MHUA-A 11734, c MHUA-A 8059, MHUA-A 11736. Photos by Carlos M. Marin. Illustration by Ana M.
and d MHUA-A 8089; P. viejas, e MHUA-A 11261, f MHUA-A Sepúlveda-Seguro
13
CMM; from vereda Los Medios (6.1325, -75.0966; 1,723 m 14.4–19.0 mm (mean ± SD = 16.6 ± 1.42; n = 15) and
a.s.l.): MHUA-A 10477, 10508 (juveniles), MHUA-A females 20.2–30.3 mm (mean ± SD = 25.5 ± 3.2; n = 13).
10489, 10514 (adult males) collected on 2016 by CMM and
MHUA-A 10814 (adult male) collected on 2016 by JMD. Comparisons with other species Pristimantis campesino sp.
nov. shares with P. viejas the presence of lateral fringes,
Etymology The specific name “campesino” (farmer in Span- vocal slits, texture skin of venter areolate, finger I shorter
ish) is a noun used in apposition, in reference to people living than finger II and the color pattern of the concealed surfaces
in the rural areas. The name honors the people who live and of thighs and shanks (having orange and/or red dots usually
work in the Colombian rural areas for their resilience and in a black background, Fig. 5b-d, f–h) but differs from P.
perseverance in the struggle towards achieving a peaceful viejas by having a iris solid reddish-terracotta with thin dark
and equitable country. brown and black reticulations (Fig. 5a) versus iris golden at
the upper and lower portions with coarse black reticulations
Diagnosis We assign the new species to the Pristimantis in P. viejas (Fig. 5e); in having shorter calls (0.027 ± 0.004 s
ridens species group based on our morphological and phy- in P. campesino sp. nov. and 0.053 ± 0.004 s in P. viejas;
logenetic results (Fig. 1). Morphological description follows Online Resource 2, Fig. S5); a wider frequency band (rang-
the terminology by Lynch and Duellman (1997). Description ing from 2.328 to 3.503 kHz in P. campesino sp. nov., and
of the coloration patterns was based on field observations 2.670 to 3.282 kHz in P. viejas); and disjunct distribution
and photos of live specimens. Specimens were determined as in both altitudinal range (from 1500 to 2150 m a.s.l in P.
adults by examination of secondary sexual characters (pres- campesino sp. nov. and 100 to nearly 1600 m a.s.l in P.
ence of vocal slits and vocal sacs) and sex was determined viejas; Fig. 2b–c), and occupied climatic space (Fig. 3b).
by the presence of ovaries or testes. Pristimantis campesino Additionally, the distinctiveness of P. campesino sp. nov.
sp. nov. is characterized by the following combination of and P. viejas is further corroborated by uncorrected pairwise
characters: (1) skin texture of the dorsum weakly tubercu- distances of 6.3% in 16S and 9.6% in coi (Fig. S1).
late; venter areolate; dorsolateral folds presents; discoidal As Pristimantis campesino sp. nov. belongs to the Pristim-
fold absent; (2) tympanic membrane and tympanic annulus antis ridens species group sensu Padial et al. (2014), we com-
visible, supratympanic fold usually prominent; diameter pared the new taxon with the species within this clade. Com-
of tympanum 35–40% of eye diameter; (3) snout short and parisons were conducted by a detailed revision of the original
rounded in dorsal and lateral views; (4) one to three coni- descriptions and, when possible, by direct examination of
cal tubercles on the upper eyelid; without cranial crests; (5) museum specimens. For a summary of diagnostic charac-
vomerine odontophores arched and barely separated, that ters of the species belonging the Pristimantis ridens group
extend laterally from margins of the choanae; (6) in males see Online Resource 2, Table S1. Comparing the remaining
vocal slits and small subgular vocal sac present; nuptial pads species of the Pristimantis ridens group, Pristimantis camp-
absent; (7) finger I shorter than II, with large rounded digi- esino sp. nov. differs from P. crenunguis (Lynch, 1976) and
tal discs and defined pads; (8) fingers with narrow lateral P. jorgevelosai (Lynch, 1994), by lacking cranial crest; from
fringes; (9) small ulnar tubercles present; (10) a short line of P. actites (Lynch, 1979), P. adnus (Crawford et al., 2010),
low tubercles on tarsus; inner tarsal fold absent; calcar tuber- P. almendariz (Brito & Pozo-Zamora, 2013), P. bicolor
cles present (11) inner metatarsal tubercle oval and promi- (Rueda-Almonacid & Lynch, 1983), P. factiosus (Lynch &
nent, four to five times bigger than external circular metatar- Rueda-Almonacid, 1998), P. ocellatus (Lynch & Burrowes,
sal tubercle; smalls supernumerary plantar tubercles present 1990), P. rosadoi (Flores, 1988), P. sanguineus (Lynch,
(12) toes with narrow lateral fringes; third toe shorter than 1998), and P. tanyrhynchus (Lehr, 2007) by lacking inner
fifth; a small webbing between toes IV and V reaching the tarsal folds (P. factiosus is also readily distinguishable
middle-distal portion of the basal subarticular tubercles; from P. campesino sp. nov. by lacking dark-brown vertical
(13) dorsum ranging from dark brown to bright yellow in bands on lips; Fig. 5i); from P. cerasinus (Cope, 1875), P.
males and uniform brown to almost black in females (some colomai (Lynch & Duellman, 1997), P. educatoris (Ryan
individuals present longitudinal stripes or a cream middor- et al., 2010), P. labiosus (Lynch et al., 1994), P. lanthanites
sal stripe); dark brown vertical bands on lips; oblique dark (Lynch, 1975), P. museosus (Ibáñez et al., 1994), P. orpa-
brown bars on the dorsal surface of the hind limbs; males cobates (Lynch et al., 1994), and P. tenebrionis (Lynch &
with bright translucent yellow gular and ventral colora- Miyata, 1980) by having lateral fringes; from P. cremnobates
tion that extends to the middle area of the forelimbs, with (Lynch & Duellman, 1980), P. cruentus (Peters, 1873), P.
dark brown thighs; females with creamy orange belly, mot- erythropleura (Boulenger, 1896), P. ixalus (Lynch, 2003),
tling, with thighs and forelimbs browning towards the distal P. laticlavius (Lynch & Burrowes, 1990), P. latidiscus
region; eye with reddish-terracotta iris with thin dark brown (Boulenger, 1898), P. penelopus (Lynch & Rueda-Almonacid,
reticulations and light blue sclera; (14) adult males small 1999), P. rubicundus (Jiménez de la Espada, 1875), and P.
13
thectopternus (Lynch, 1975) by having vocal slits; from P. Coloration of holotype in life Dorsum dark brown and ochre
epacrus (Lynch & Suárez-Mayorga, 2000) and P. w-nigrum with small and darker spots and a dark ochre patch covering
(Boettger, 1892) by lacking nuptial pads; and from P. ridens the eyelids and the front of the snout. Translucent yellow
(Cope, 1866) and P. caryophyllaceus (Barbour, 1928) by belly with dark brown spots in the medial and posterior area;
having skin texture on dorsum weakly tuberculate (smoot in throat region with few small dark brown spots near the lips.
P. ridens and P. caryophyllaceus). Rostral dark-brown edge that lightens towards the area of the
Two species distributed in the Cordillera Central which lips, with dark-brown vertical bands on lips. Supratympanic
can be potentially confused with P. campesino sp. nov. are dark brown membrane in the upper area, which becomes
P. actinolaimus (Lynch & Rueda-Almonacid, 1998) and lighter with the tympanum towards the lower area. Extremi-
P. suetus (Lynch & Rueda-Almonacid, 1998) both species ties dark brown with multiple dark spots with no defined pat-
described from Selva de Florencia, Department of Caldas tern. Concealed surfaces the of thighs and shanks with orange
(the same type locality from P. factiosus). However, P. blotches on a black background. Axillary and groin region
campesino sp. nov. differs from P. actinolaimus by having with dark brown spots on a yellow background. Reddish-
vocal slits and texture skin of the dorsum weakly tuberculate terracotta iris with thin dark brown reticulations and light
(shagreen in P. actinolaimus) and from P. suetus by lacking blue sclera (Fig. 5a).
nuptial pads and skin texture of the dorsum weakly tubercu-
late (finely shagreen in P. suetus).
Coloration of the holotype in ethanol Dorsum pale brown
with light brown blotches and granules dark brown. Flanks
Measurements of the holotype (in millimeters) SVL:
dark brown. Loreal region cream; snout dark brown. Lower
17.4; HL: 6.1; HW: 6.9; ED: 2.6; UEW: 1.9; EN: 2.1; SL:
lip cream with some scattered gray dots. Throat and anterior
2.7; IND: 1.8; IOD: 2.2; TD: 1.0; FLL: 4.2; HAL: 4.9; THL:
portion of chest cream. Belly with light cream granules. Dor-
9.8; TL: 10.6; FL: 8.4; Fin4DW: 0.5 and Toe4DW: 0.5.
sal surface of upper arm cream with dark brown blotches;
anterior surface cream, posterior surface with dark brown
Description of the holotype A male, 17.4 mm SVL. Tex-
blotches; ventrally cream. Dorsal, anterior, and posterior sur-
ture of skin on dorsum and flanks weakly tuberculate; thin
faces of forearm cream lacking distinctive marks. Concealed
and broken dorsolateral folds; venter areolate; discoidal fold
surface of forearm cream. Palmar surfaces brown. Dorsal
absents. Head barely wider than long; rounded snout in dor-
surface of hand with pale brown blotches on fingers. Dorsal
sal and lateral views, relatively short (snout length 15.3% of
surface of thigh light cream with dark brown blotches. Ven-
SVL); protuberant and sideways-facing nostrils; canthus ros-
tral surface of thigh and concealed surfaces of shank and
tralis distinct; loreal region weakly concave; small subgular
foot cream. Plantar surfaces dark brown (Online Resource
vocal sac. Flattened interorbital area, narrower than upper
2, Fig. S6).
eyelid width; one to three conical tubercles on upper eyelid;
cranial crests absent; eye diameter greater than eye-nostril
distance. Supratympanic fold present; tympanic membrane Morphological variation Adult males of Pristimantis camp-
and tympanic annulus distinct and rounded; diameter of tym- esino sp. nov. are smaller than females (Online Resource
panum 40% of eye diameter. 2, Fig. S3b) and exhibit variation in dorsal coloration rang-
Forearm slender, 24.9% of SVL; ulnar tubercles present; ing from dark brown (MHUA-A 08810) to bright yellow
hand length 28.2% of SLV; finger I shorter than II; fingers (MHUA-A 10814). Dorsal coloration in females is variable,
with defined pads and broadly expanded elliptical digital with individuals having a uniform brown (MHUA-A 11737)
discs; narrow lateral fringes on fingers; smalls supernu- to almost black (MHUA-A 12024) dorsal surface and others
merary palmar tubercles; nuptial pads absent. Hind limbs having either a broad dark brown dorsal stripe (MHUA-
relative proportional to body; tibia length 60.7% of SVL; A 08819) or a narrow cream middorsal stripe (MHUA-A
thigh length 56.3% of SVL; tarsal fold absent; tubercles on 08088; Online Resource 2, Fig. S7). Variations in morpho-
tarsus, lows; calcar tubercles. Foot length 79.4% of TL; lat- metric measurements of the type series of P. campesino sp.
eral fringes on toes; basal webbing between toes IV and nov. is shown in Online Resource 2, Table S2.
V; inner metatarsal oval, about 4 × times bigger than exter-
nal metatarsal tubercle; low rounded supernumerary plan- Bioacoustics We recorded and analyzed 94 calls of
tar tubercles; defined pads and broadly expanded elliptical eight individuals: four from the lowland and four from
digital discs on toes; toe III shorter than toe V; tip of toe III the highland lineage. Sound files were obtained using
reaching the middle of penultimate subarticular tubercle of a digital TASCAM DR-40 recorder and the built-
toe IV; tip of toe V reaching the distal border of the distal in microphone. Spontaneously calling males were
subarticular tubercle of toe IV. recorded as near as 1 m of distance from the recorder.
13
We analyzed the sound files using the Raven pro software Distribution of P. campesino sp. nov. in these protected
version 1.5 (Center for Conservation Bioacoustics, 2014) areas can favor its conservation status.
using a Hanning sampling window, window size of 512
points, sampling rate of 44.1 kHz, 16-bit of precision, and
overlap of 50%. Following Köhler et al. (2017), we measured Supplementary Information The online version contains supplemen-
tary material available at https://d oi.o rg/1 0.1 007/s 13127-0 22-0 0549-9.
five temporal and spectral variables: call duration in seconds
and low frequency, high frequency, peak frequency, and Acknowledgements This study was funded by the research Lab Grupo
delta frequency in KHz. Average values were estimated for Herpetológico de Antioquia (GHA) and the Universidad de Antioquia
each individual, our biological, and statistical unit of analy- under “Estrategia de Sostenibilidad 2019–2020” and project CT-2017-
sis. We compared the advertisement call variables which 001714 between the Universidad de Antioquia and Empresas Públicas
de Medellín. We thank the members of the GHA who have collected
showed the highest inter-lineage variation by implementing the new species over the years. We also thank Fernando Vargas-Salinas
the non-parametric Wilcoxon test after check for normality. for providing a tissue sample of Pristimantis factiosus from its type
The advertisement call of Pristimantis campesino sp. nov. locality and Andrew J. Crawford for providing sequences for P. adnus.
consists of a single chirp-like (i.e., pulsed) note lasting about Collecting permits were granted by Resolución 0524/2014 of ANLA-
MADS to the Universidad de Antioquia.
0.027 ± 0.004 s (mean ± SD, n = 53 calls from four individu-
als), with at least four visible harmonics but no appreciable Author contribution JMD conceived the study. AMS, CMM, YG, and
frequency modulation. The calls are uttered in bouts with no JMD collected data. AMS, CMM, YG, AA, and JMD analyzed the
apparent temporal structure. The note oscillogram (Online data. AMS, CMM, YG, AA, and JMD wrote the manuscript.
Resource 2, Fig. S5a) further reveals asymmetric amplitude
modulation, with a slow rise and fast decrease of ampli- Data availability The molecular data generated during the current study
are available in the GenBank repository [https://w
ww.n cbi.n lm.n ih.g ov/
tude, which peaks at about 80–90% of the note duration. The genbank/]. Morphological, climatic and acoustic datasets are available
frequency band of the fundamental harmonic ranges from from the corresponding author on reasonable request.
2.328 ± 0.056 to 3.503 ± 0.246 kHz with frequency peaking
at about 2.649 ± 0.175 kHz. The advertisement call of P. Declarations
campesino sp. nov. is readily distinguished from the call of
P. viejas by the presence of pulses (absent in P. viejas), the Conflict of interest The authors declare no competing interests.
absence of frequency modulation (present in P. viejas), the
shorter note duration (0.027 s in P. campesino sp. nov. and
0.053 s in P. viejas; Wilcoxon test = 0, p < 0.005), and the
lower peak frequency (2.649 kHz in P. campesino sp. nov. References
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Organisms Diversity & Evolution

Phylogeographic structure suggests environmental gradient

Received: 26 January 2021 / Accepted: 24 February 2022

Introduction highly heterogeneous landscapes where different types of

Table 1 (continued) Voucher Epithet Lineage Latitude Longitude 16S COI TYR

Within Pristimantis viejas, we constructed haplotype net- Environmental analysis

◂Fig. 1 (Left) Maximum likelihood tree (-lnL = − 116,566.9) depicting Geographic lineage distribution

Sequence alignment resulted in a matrix of 4824 sites: 1061 Environmental analysis

Paratypes 31 specimens, all from Colombia, Department of

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Organisms Diversity & Evolution

Phylogeographic structure suggests environmental gradient

Received: 26 January 2021 / Accepted: 24 February 2022

Introduction highly heterogeneous landscapes where different types of

Table 1 (continued) Voucher Epithet Lineage Latitude Longitude 16S COI TYR​

Within Pristimantis viejas, we constructed haplotype net- Environmental analysis

◂Fig. 1 (Left) Maximum likelihood tree (-lnL = − 116,566.9) depicting Geographic lineage distribution

Sequence alignment resulted in a matrix of 4824 sites: 1061 Environmental analysis

Paratypes 31 specimens, all from Colombia, Department of

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Table 1 (continued) Voucher Epithet Lineage Latitude Longitude 16S COI TYR

◂Fig. 1 (Left) Maximum likelihood tree (-lnL = − 116,566.9) depicting Geographic lineage distribution