Historical demography and species delimitation in the palm rocket frog in the
1
Colombian Andes.
2 3
Gabrielle Genty13, Carlos Guarnizo1, Astrid Muñoz-Ortiz2, Andrew J. Crawford1. 4
1 Departamento de Ciencias Biológicas, Universidad de los Andes, Bogotá D.C, Colombia. 5
2 Departamento de Ciencias Básicas, Universidad de La Salle, Bogotá, Colombia. 6
Abstract 9
A key factor that promotes biological diversification in terrestrial vertebrates is geographical 10
isolation, which is frequently associated with geographical barriers caused by topographic and 11
climatic heterogeneity. The palm rocket frog (Anura: Aromobatidae: Rheobates) is distributed 12
across heterogeneous landscapes of the Central and Eastern Andean cordilleras of Colombia and 13
thus offers an ideal system to investigate the relative contributions of peaks, valleys, and 14
environmental variables in promoting population genetic divergence. A previous phylogenetic 15
study of this species complex by Muñoz-Ortiz et al. (2015), based on DNA sequences of one 16
nuclear and two mitochondrial genes, revealed five strongly differentiated lineages largely 17
corresponding to geography. In this study, we use a population genetic approach to re-evaluate 18
population delimitation and infer the historical demography of the Rheobates genus based on a 19
new dataset of four rapidly-evolving nuclear markers and two mitochondrial genes. Summary 20
statistics of population parameters revealed a strong genetic differentiation among five inferred 21
genetic populations using mitochondrial data (Fst > 0.8), while nuclear loci displayed 22
idiosyncratic levels of genetic structure. Alternative proposals for delimitation of Rheobates 23
species were evaluated using a Bayesian approach to generate posterior probabilities of species 24
assignments based on the interspecies coalescent. This analysis suggested that the same five 25
main lineages identified previously by Muñoz-Ortiz et al. (2015) are supported as candidate 26
species, i.e., Santa Maria, Central Cordillera, plus three species along the western flank of the 27
Eastern Cordillera (North/Central and South). Fitting an historical demographic model of 28
isolation-with-migration suggests very low levels of migration between the five groups. We 29
discuss the implications of our findings in terms of how the Northern Andes topography shaped 30
the population dynamics and diversification of this deeply differentiated endemic frog genus. 31
Keywords 32
Historical demography; migration rates; Rheobates palmatus; species delimitation; vicariance. 33
Introduction 34
The tropical Andes are an important global biodiversity hotspot, home to the highest 35
density of species per area in the world (Myers et al. 2000). In contrast to the high alpha-36
diversity in Amazonia, the Andes exhibit high beta-diversity, which is attributed to Andean 37
orogenesis (Jaramillo et al. 2006) and the creation of topographic complexity. The history of the 38
enormous changes in South America has been evident with major geological events that have 39
modified continents and oceans, climate changes and associated glaciations (Graham 2009), 40
which impacted the continent, creating complex scenarios for species diversification. The Andes 41
Cordillera has had a complex history in a relatively short time, uplifting from south to north at a 42
fast rate during the last 20 million years (Hartley 2003). This fast and asynchronous uplift have 43
promoted vicariance in several Andean species (Luebert et al. 2011). 44
The important role of the Andes shaping the demographic history of populations have 45
been confirmed by several analyses (Gutiérrez-Pinto et al. 2012). As for this it’s been proposed 46
that high Andean species richness of frogs and other organisms, is explained by rapid speciation 47
associated with the Andean uplift and historical climatic change (Guarnizo et al. 2009; Rosser et 48
al. 2012). These two processes can abruptly interrupt the landscape with low-elevations gaps and 49
high elevation ridgelines that restrict dispersal between isolated montane populations and can 50
lead to population differentiation. 51
The Palm Rocket Frog (Anura: Aromobatidae: Rheobates) is a useful system to elucidate 52
how the Andes have promoted biological diversification. This frog has a peculiar geographic 53
distribution restricted to mid-elevation habitats between 350 and 2200 m above the sea level, yet 54
also has a wide latitudinal distribution and also occurs in two cordilleras, from the cloud forests 55
on the eastern flank of the Central Cordillera to both flanks of the Eastern Cordillera (Rivero & 56
Serna 1988). At the moment this genus comprises two species, Rheobates palmatus that is found 57
in the eastern cordillera and the Rheobates pseudopalmatus that can be found in the central 58
cordillera, although nothing is really known about this second species, even the taxonomic 59
research is needed to determine the validity of this species according to the AmphibiaWeb. 60
In a previous study of Muñoz-Ortiz et al. (2015), hypothesized that valleys have isolated 61
R.palmatus more often than peaks, and also, that the genus Rheobates might be comprised of 62
more than the two currently accepted species (R. palmatus and R. pseudopalmatus), in other 63
words, that the genus is composed by multiple cryptic species. 64
For this study, we expanded on the study by Muñoz-Ortiz et al. (2015) adding more genetic data 65
for a total of four nuclear genes and two mitochondrial genes. These additional data were 66
evaluated from a population genetic and historical demographic perspective with a renewed 67
emphasis on species delimitation. We tested the prediction that given the potential barriers 68
shown in the previous phylogenetic study (Muñoz-Ortiz et al. 2015), there are five possible new 69
species promoted by geographic isolation, and therefore that there is no gene flow between these 70
five groups. 71
Methods 72
73
Molecular genetic analyses 74
Genomic DNA was extracted from 24 tissue samples of Rheobates, obtained through 75
field collection and museum donations (see Table S1 in Appendix S1 of the supporting 76
information) using a DNeasy Blood & Tissue kit (Qiagen, Valencia, CA, USA) following the 77
manufacturer’s protocol. Two mitochondrial genes, cytochrome oxidase I (COI) and 16S 78
ribosomal RNA (16S) were amplified by polymerase chain reaction (PCR) for 24 samples each 79
for the previous study of Muñoz-Ortiz et al (2015). Additionally four nuclear genes were PCR 80
amplified, including the pro-opiomelanocortin (POMC) as published in Muñoz-Ortiz et al. 81
(2015). And three new nuclear genes, the loci SF232, SF328 and SF412 for 23, 23 and 24 82
samples, respectively (given some difficulties we weren’t able to amplified the sequences for all 83
the samples on some genes) found by Tezuka et al. (2012) and not included in the previous study 84
of Muñoz-Ortiz et al (2015). These last ones were chosen for their potentially rapid rates of 85
evolution and the fact that they can be used in any species. All sequences generated for this study 86
will be deposited in GenBank (See Table S2 in Appendix S1 for PCR primers and cycling 87
conditions). Sequences were cleaned manually and alignments were made using MAFFT version 88
7 (Katoh & Standley 2013) and edited by eye with Mesquite version 3.04 (Maddison & 89
Maddison 2001). 90
Summary statistics were calculated for each gene (Table 1) using DnaSP version 5 91
(Rozas et al. 2016), including Tajima’s D (Tajima 1989), nucleotide diversity, Pi (Nei 1987), 92
Theta Watterson per site (Watterson 1975), Fu and Li’s D* (Fu & Li 1993) and the Ramos-93
Onsins and Rozas’s R2 (Ramos-Onsins & Rozas 2002). The Dxy parameter which is the 94
average number of nucleotide substitutions per site between populations (Nei 1987) was 95
confirmed using MEGA version 6.06, Kimura 2 parameters (Table 2) (Tamura et al. 2007). By 96
using this different statistics tests we were able to confirm our results given the fact that each 97
statistic test has their own assumptions. 98
99
Phylogenetic methods 100
The best fitting model of nucleotide substitution was selected for each gene alignment 101
with jModelTest version 2.1 (Darriba et al. 2012) using the corrected Akaike information 102
criterion. The recommended models for each gene were the following, for COI the suggested 103
model was TPM2uf + G; for 16S, TIM3 + G; for POMC, TIM1; for SF232, HKY + I; for SF328, 104
K80 + I and for SF412, TrN. We included all sequences and the outgroups when possible, which 105
were an Allobates ranoides and an Allobates femoralis for this test. For genes SF328 and SF412 106
we weren’t able to amplified the sequences for the outgroups so we only used the Rheobates sp. 107
sequences. 108
A BEAUti file was generated specifying the nodes of calibration, and a Bayesian 109
phylogenetic inferences were made with BEAST version 1.8 (Drummond et al. 2012). The 110
nucleotide substitution model for each gene was set as above and a lognormal relaxed clock. The 111
prior for the uncorrelated lognormal relaxed clock mean was with a gamma distribution with 112
shape 0.004, a scale of 1000 and an offset of 0. A coalescence tree prior was selected for 113
modeling individuals of the same population. To constrain the root age we used the results 114
Santos et al. (2009) as a secondary calibration and set prior distribution on the age of most recent 115
common ancestor of dendrobatoid frogs as a normal distribution with mean 43.7 million years 116
and standard deviation of 6.7. Two independent analyses where run each one for 100 million 117
iterations and had a burn-in of 100000 generations. A contrast was made with a partitioned 118
maximum likelihood inference (ML) using RAxML version 8 (Stamatakis 2006) using a 119
GTRGAMMA model of evolution and a bootstrap iteration of 1000. To root the ML trees we 120
used the population of Santa María, Boyacá. 121
The Monte Carlo Markov chain output from BEAST was checked for MCMC 122
convergence and effective sample size for the estimated parameters on Tracer version 1.6 and 123
generated on TreeAnnotator version 1.8 using a majority 50% consensus rule, to be visualized 124
with FigTree version 1.4.2 (Drummond et al. 2012). The same Monte Carlo Markov chain 125
output from BEAST was visualized on DensiTree version 2.0.1 (Bouckaert 2010). 126
127
Species delimitation and migration rate 128
To test the hypothesis of whether their five and not only two species of Rheobates we 129
used the Bayesian species delimitation (BPP) (Rannala & Yang 2013) to statistically test 130
alternative proposals for delimitation of this species. We used three previously conducted prior 131
distributions schemes for ancestral population size θ and root age τ to take into account three 132
types of demographic histories: small population size with shallow divergence, large population 133
size with deep divergence and large population size with shallow divergences among species 134
(Guarnizo et al. 2016). BPP uses the reversible-jump Markov chain Monte Carlo algorithm and 135
it is known that it exhibits mixing problem in some datasets (Rannala & Yang 2013). To avoid 136
known problems with mixing of the rjMCMC we conducted the analyses several times to verify 137
that different starting tree models generated congruent results. Although we only achieved ideal 138
fine-tune adjustment for scheme 1, all runs consisted of 100,000 samples taken every 1 step with 139
a burn-in period of 8000 steps (Guarnizo et al. 2016). 140
Population structure 142
As well as before, we tested the migration rates among lineages (populations) identified 143
previously (Muñoz-Ortiz et al. 2015) to evaluate if their still exists any migration between this 144
lineages or if they are completely isolated one from another as expected. And for this we used 145
IMa2 (Hey 2011) to estimate asymmetric migration rates. IMa2 uses an MCMC sampling 146
strategy to jointly estimate multiple demographic parameters, including the population mutation 147
rate, θ (under ideal population conditions θ = 4Neµ, where Ne is the effective population size and
148
µ the per-generation, per-site mutation rate), asymmetric migration rates (M1=m1/µ and 149
M2=m2/µ, where m is the proportion of individuals in each population that were born in a 150
different population) and population divergence time (t=T/µ, where T is the unscaled divergence 151
time). Runs were conducted with a burn-in of 10,000 generations followed by 100,000 iterations. 152
We used the default mutation rate (µ=1). We tested two prior, first with 10 and afterwards 20 as 153
priors for the parameter of maximum population size (4Nu) and symmetrical migrations (m1→ 154
2= m2→ 1) (Liao et al. 2012). 155
Results 156
The BEAST analyses clearly identified the separation of five lineages within the genus 157
Rheobates. Both concatenated gene tree inference methods shows almost identical topologies. 158
For simplicity, the consensus phylogeny obtained from the concatenated analysis is presented in 159
Figure 1. As it is shown here and as it was expected given the previous results of Muñoz-Ortiz et 160
al. (2015), five highly divergent and well-supported lineages are found. They are divided by their 161
geographic distribution and it is possible to differ the lineages of Santa María separated from the 162
rest of Rheobates, followed by four other evident lineages that are defined as well by their 163
distribution. The clade in the Central Cordillera is divided from the ones on the Eastern 164
Cordillera. And the ones in the Eastern Cordillera are separated in three. Two are separated by 165
the Chicamocha canyon dividing them in two clades, one located in the North and another one 166
located at the South of the Chicamocha. Finally the individuals on the south of the Eastern 167
Cordillera also correspond to a separate clade (Fig 1). 168
Summary statistics of population parameters revealed a strong genetic differentiation 169
among five inferred genetic populations using mitochondrial data (Fst> 0.8), while nuclear loci 170
displayed idiosyncratic levels of genetic structure. Alternative proposals for delimitation of 171
Rheobates species were evaluated using a Bayesian approach to generate posterior probabilities 172
of species assignments based on the interspecies coalescent. By using the concatenated dataset, it 173
revealed five highly supported genetic clusters with a probability of 1. Bayesian species 174
delimitation inferred five well-supported species with the three priors. Also for all different prior 175
distribution used, the BPP method found very high speciation probabilities for all internal nodes, 176
which mean that according to BPP analysis all five groups previously identified have high 177
probability of representing different species. This analysis suggested that the same five main 178
lineages identified on Figure 1 are supported as candidate species i.e., Santa María, Central 179
Cordillera, plus three species along the western flank of the Eastern Cordillera (North/Central 180
and South). 181
Simulations with IMa2 revealed a posterior probability distribution of the demographic 182
parameters. Making clearly the fact that in the present there is no existing significant gene flow 183
as shown in figure 1c between the populations. Even thought we highlight the highest posterior 184
probability with a thick yellow arrow between SwEC and NwEC (see Fig 1 for an explanation of 185
letter codes. So, fitting an historical demographic model of isolation-with-migration suggests a 186
not significant level of migration between the five groups. We discuss the implications of our 187
findings in terms of how the Northern Andes topography shaped the population dynamic and 188
diversification of this deeply differentiated endemic frog genus. 189
Discussion 190
Clearly this separation of lineages was given to the different geographical barriers. The 191
Chicamocha canyon separates two populations. The Magdalena valleys separate also the ones on 192
the Central Cordillera from the rest of the Eastern Cordillera. The mountains surrounding Santa 193
María isolated this population from the rest. And finally we discuss the possibility that the 194
separation between the population on the South of the Chicamocha canyon and the ones at the 195
bottom of the Eastern Cordillera were possibly separated by vicariance, given the same 196
conditions presented by Guarnizo et al. (2009). The high sequence divergent and Fst and Dxy 197
values between southern of the Chicamocha and the ones at the bottom of the Eastern Cordillera 198
suggests that they have been under reproductive isolation. In a previous study (Irwin 2002) the 199
author concluded that the fact that we consistently obtain the two major clades with data from 200
two mitochondrial fragments and in our case, four independent nuclear genes, suggests that both 201
clades are the result of reproductive isolation and not a byproduct of stochastic processes. For 202
this process Guarnizo et al. (2009) suggested a mechanism of differentiation given during the 203
Late Miocene in the Northern Andes, where the elevations stayed relatively stable, but given the 204
temperature at that time the optimal conditions for Rheobates to live in was on the peak 205
elevations. Afterwards the Northern Andes started to rise very quickly and by becoming colder it 206
stimulated a population range expansion, as it did with the D. labialis (Guarnizo et al. 2009). 207
The genetic divergences found among these groups that match their geographic distribution. We 208
were able to confirm the important role of the Eastern Andes uplift, the Magdalena Valley, and 209
the Chicamocha Canyon as promoters of genetic divergence among population. 210
211
We were able to confirm with more data what Muñoz et al. (2015) suggested in a recent 212
study, that the genus Rheobates is composed of at least five highly divergent groups. The BPP 213
analysis supports clearly the presence of the same five groups as previously identified, and 214
suggests that they are possible cryptic species, are valid species. However, further observations 215
on morphology and acoustics should be explored before naming the species. We were able to 216
show that these five groups display very low migration rates among them i.e., very high genetic 217
structure. 218
Our results also support the suggestion that Andean diversity is underestimated. Genetic 219
species discovery is useful for identifying groups needing appraisal and for higher level 220
taxonomic resolution in diverse groups. Finally, we suggest that cryptic species are rampant, and 221
that more integrative analyses that include DNA as well as natural history and ecology need to 222
be conducted. 223
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Table 1. Table containing information related with demographic parameters for all populations 286
for each gene nuclear and mitochondrial. The ones with double asterisk are those that have 287
statistical significance with a p-value< 0.02. 288
16S COI POMC SF232 SF328 SF412
Samples (N) 23 22 22 21 23 24
Number of
sequences (n) 23 22 44 42 46 48
bp 575 658 431 375 404 352
Number of
haplotypes (h) 16 12 12 8 22 6
Polymorphic sites (S) 74 183 20 8 31 8
Tajima's D 1.413 2.70674 0.12915 0.09567 -0.62577 -0.30211 Nucleotide diversity
(Theta-W) per site 0.02991 0.06393 0.01067 0.00497 0.02204 0.00512 Fu y Li D 1.67791** 2.12427** 1.67303** -0.0656 -0.19988 -0.11249
R2 0.1582 0.1999 0.1195 0.1172 0.0879 0.1016 *P<0.05
Figure 1. a) Bayesian gene tree and node support values. Each lineage is named accordingly to its geographic distribution. The 289
colors of the boxes match the colors in the map. b) Colombian Andes map indicating the sampling localities. The line between the 290
lineages of Eastern cordillera Western flank/north and south represent the Chicamocha Canyon. c) Densitree with lineages suffixes 291
specified at the end of the branches. The yellow arrows represent the migration rate between pairs of populations given by IMa2. 292
Depicted inside the arrows is the relative migration rate. The arrows width represents the mean migration rate. 293
Figure 2. a) Heatmap showing Fst/Dxy comparisons among populations. b) Fst/Dxy key. 295
Supporting Information 297
Historical demography and species delimitation in the palm rocket frog in 298
the Colombian Andes. 299
Gabrielle Genty, Carlos Guarnizo, Astrid Muñoz-Ortiz, Andrew J. Crawford.
300
301
Appendix S1 Genetic samples and primers used on the DNA sequence analyses.
302
This appendix contains Table S1 and S2.
303 304
Table S1 Species, field and institutional numbers, locality, geographical coordinates and
305
GenBank accession numbers for ingroups and outgroups samples. Abbreviations for field series:
306
AAV, Álvaro Andrés Velásquez; AJC, Andrew J. Crawford; CG, Carlos E. Guarnizo; LSB,
307
Lucas Santiago Barrientos; MAR, Marco Antonio Rada. Acronyms for museums are: ANDES-A
308
and ANDES-T, Amphibians Collection and Tissues Collection, Museo de Historia Natural
309
ANDES, Universidad de los Andes, Bogotá, Colombia; MHUA, Museo de Herpetología de la
310
Universidad de Antioquia, Colombia. See Fig 1 for an explanation of letter codes.
Species Locality (m.a.s.l) Geographic region Clade Latitude Longitude number Field Institutional number COI 16S POMC SF232 SF328 SF412
Rheobates palmatus
Santa María
(870) eEC SM 4.848 -73.272 AJC 4235 ANDES-A 1486 KJ130682 - KJ130740
4.848 -73.272 AJC 4239 ANDES-A 1487 KJ130683 KJ130718 KJ130742
4.858 -73.264 AJC 4232 ANDES-A 1485 - KJ130714 -
4.858 -73.264 AJC 4233 -
Rheobates pseudopalmatus
Amalfi (1550) CC CC 6.800 -75.086 - MHUA
4732 KJ130692 KJ130726 KJ130746
San Rafael (1250) 6.390 -75.011 LSB 337 Voucher
lost KJ130679 KJ130715 KJ130738
Maceo (575) 6.547 -74.644 - MHUA
4348 KJ130678 KJ130713 KJ130737
Anorí (1530) 6.978 -75.111 - MHUA
5162 KJ130694 KJ130727 KJ130747
6.978 -75.111 - MHUA
5357 KJ130695 KJ130728
Rheobates palmatus
San Vicente
(300) wEC SwEC 7.079 -73.552 AJC 3526
ANDES-A
1481 KJ130670 KJ130706 KJ130733
Virolín (1748) 6.105 -73.199 CG 001 ANDES-T
2350 KJ130681 KJ130716 KJ130739 Puente Nacional
(1623) 5.882 -73.678 AJC 3398
ANDES-A
1476 KJ130665 KJ130701 KJ130729
5.882 -73.678 AJC 3403 ANDES-A
1478 KJ130690 KJ130724
5.882 -73.678 AJC 3404 ANDES-A
1479 KJ130676 KJ130711 Piedecuesta
(940) wEC NwEC 6.783 -73.017 AAV153
ANDES-A
1472 KJ130685 KJ130720 KJ130743
6.783 -73.017 AAV 154 ANDES-A
1473 KJ130688 KJ130722 KJ130744
6.783 -73.017 AJC 3860 ANDES-A
1482 KJ130666 KJ130702 KJ130730
Suratá (1740) 7.367 -72.983 CG 003 ANDES-T
2352 KJ130675 KJ130709 KJ130735
7.367 -72.983 CG 004 ANDES-T
2353 KJ130672 KJ130708 San Francisco
Cáqueza (1515) eEC eEC 4.414 -73.948 CG 007 ANDES-T 2354 KJ130684 KJ130719 KJ871617
4.414 -73.948 CG 013 ANDES-T 2355 KJ130677 KJ130712 KJ130736
Las Brisas (2000) 4.437 -73.919 AJC 2106 ANDES-A 1474 KJ130668 KJ130704 KJ130731
Allobates
ranoides Sabanalarga (320) - Outgroup 4.773 -73.038 AJC 3383 ANDES-A 1073 KJ130661 KJ130697 KJ871619
Allobates
Table S2 The conditions used for the PCR amplification for the mithocondrial markers and 312
POMC were the ones used by Muñoz-Ortiz et al. (2015) appendix S1 table S2. And to 313
amplify the other three nuclear genes it included an initial denaturing step of 2 min at 95 ºC 314
followed by 30 amplification cycles (30 s at 95 ºC, 30 s at 51 ºC, 60 s at 73 ºC) and a final 315
extension of 20 min at 72 ºC. PCR products were cleaned with exonuclease I and SAP 316
enzymes (Werle et al. 1994), and sequenced directly with Sanger technology. 317
318
Gene region Primer Primer sequence (5'-3') Source Mitochondrial
COI (658 bp) dgLCO-1490 GGT CAA CAA ATC ATA AAG AYA TYG G (Meyer & Paulay, 2005)
dhHCO-2198
TAA ACT TCA GGG TGA CCA AAR AAY CA
Chmf4
TYT CWA CWA AYC AYA AAG AYA
TCG G (Che et al., 2012) Chmr4 ACY TCR GGR TGR CCR AAR AAT CA
Mitochondrial
16S (569 bp) Sbr-H Sar-L CCG GTC TGA ACT CAG ATC ACG T CGC CTG TTT ATC AAA AAC AT (Kessing et al., 2004) Nuclear POMC
(472 bp) POMC_DRV_R1
GGR RTT YTT GAA WAG AGT CAT
TAG WGG (Vieites et al., 2007)
POMC_DRV_F1 ATA TGT CAT GAS CCA YTT YCG CTG GAA Nuclear SF232
(375 bp) AGT CAT AAT GGT GCC ACT AAA AG (Tezuka et al., 2012) TGT GGT CCT TGT ATG GGT TG Nuclear SF328
(404 bp) GCC TCA CAA ACA ACC ACA GA CCC AAA AGA AGT TTT GCT GA (Tezuka et al., 2012) Nuclear SF412
