Research Article |
Corresponding author: Robert Puschendorf ( robert.puschendorf@plymouth.ac.uk ) Academic editor: Angelica Crottini
© 2019 James Cryer, Felicity Wynne, Stephen J. Price, Robert Puschendorf.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Cryer J, Wynne F, Price SJ, Puschendorf R (2019) Cryptic diversity in Lithobates warszewitschii (Amphibia, Anura, Ranidae). ZooKeys 838: 49-69. https://doi.org/10.3897/zookeys.838.29635
|
Lithobates warszewitschii is a species of ranid frog distributed from southern Honduras to Panama. This species suffered severe population declines at higher elevations (above 500 m a.s.l.) from the 1980s to early 1990s, but there is more recent evidence of recovery in parts of its range. Here we advocate for the status of Lithobates warszewitschii as a candidate cryptic species complex based on sequence data from mitochondrial genes CO1 and 16S. Using concatenated phylogenies, nucleotide diversity (K2P-π), net between group mean distance (NBGMD) (πnet) and species delimitation methods, we further elucidate cryptic diversity within this species. All phylogenies display polyphyletic lineages within Costa Rica and Panama. At both loci, observed genetic polymorphism (K2P-π) is also high within and between geographic populations, surpassing proposed species threshold values for amphibians. Additionally, patterns of phylogeographic structure are complicated for this species, and do not appear to be explained by geographic barriers or isolation by distance. These preliminary findings suggest L. warszewitschii is a wide-ranging species complex. Therefore, we propose further research within its wider range, and recommend integrative taxonomic assessment is merited to assess species status.
Área de Conservación Guanacaste (ACG), barcoding, biodiversity, CO1, phylogenetics, phylogeography, 16S
Cryptic species are poorly defined and highly heterogeneous. Identification of potential singular, nominal species may be masked when morphological traits are shared within and between sister taxa (
Given that the majority of species remain undescribed, endeavours to explain and catalogue biodiversity are inevitable to both understanding and preventing extinctions (
Whereas some species are presumed to be widely distributed, those within a cryptic complex may have smaller ranges or different ecological requirements (
High levels of genetic diversity in Costa Rican and Panamanian frog populations are well recognized (
Lithobates warszewitschii occurs from Honduras to Panama and has been recorded at elevations up to 1740 meters above sea level (m a.s.l.). They are fairly common, diurnal and generally abundant frogs in forests near streams where they breed (
In this study we expand the research on cryptic diversity within L. warszewitschii, based on published sequence data from two localities in Panama (
Lithobates warszewitschii were sampled from five field sites within the Área de Conservación Guanacaste (ACG), Costa Rica: Pitilla, San Gerardo, Maritza, Cacao, and Caribe (Figure
A total of 34 samples were collected from ACG and obtained from GenBank, but only 29 had both CO1 and 16S available and therefore used in this analysis. All data for L. warszewitschii samples collected in Panamanian sites El Copé and Brewster were obtained from other studies (
Sites | Collection dates | No. tissue samples | Habitat | Longitude / | Elevation(m) | Reference |
---|---|---|---|---|---|---|
Pitilla | August, 2016 | 1 | Rainforest | 10.989, -85.426 | 650–750 | Field data – this study |
June, 2017 | 1 | |||||
San Gerardo | August, 2017 | 2 | Rainforest/pastureland | 10.881, -85.389 | 470–640 | Field data – this study |
Maritza | June, 2015 | 7 | Dry/wetforest | 10.956, -85.495 | 570–610 | Field data – this study |
August, 2015 | 7 | |||||
November, 2016 | 6 | |||||
July, 2017 | 3 | |||||
August, 2017 | 5 | |||||
Cacao | November, 2016 | 4 | Rain/cloud forest | 10.923, -85.468 | 980–1130 | Field data – this study |
August, 2017 | 3 | |||||
Caribe | June, 2015 | 4 | Rainforest | 10.902, -85.275 | 370 | Field data – this study |
El Copé | July, 2010 | NA | Rainforest | 8.667, -80.592 | 700–750 | (KRL0823) |
Brewster | June, 2015 | NA | Rainforest | 9.265, -79.508 | 130–810 | (CH6868) |
In order to extract DNA from tissue samples a standard ammonium acetate protocol was used (
Extracted DNA from a subset of samples was sent to the Canadian Centre of DNA barcoding for PCR amplification and sequencing. These samples used CO1 primers (C_VF1LFt1 + C_VF1LRt1) in PCR reactions (
Concatenated gene alignments were used in the phylogenetic analyses. GENEIOUS v11.0.5 (
Species | Study site | Voucher ID | CO1 Genbank Accession # | 16S Genbank Accession # |
---|---|---|---|---|
L. warszewitschii | Maritza | RP 388 | MH559513 | MH603380 |
Maritza | RP 389 | MH559517 | MH603379 | |
Pitilla | RP 435 | NA | MH603378 | |
San Gerardo | RP 466 | MH559519 | MH603377 | |
San Gerardo | RP 475 | MH559514 | MH603376 | |
Maritza | RP 496 | MH559518 | MH603375 | |
Maritza | RP 500 | MH559515 | MH724925 | |
Cacao | RP 878 | NA | MH724926 | |
Cacao | RP 885 | MH559516 | MH724927 | |
Cacao | RP 887 | NA | MH724928 | |
Caribe | RP Fw142 | MH559500 | MH603393 | |
Caribe | RP Fw144 | MH559501 | MH603392 | |
Caribe | RP Fw147 | MH559502 | NA | |
Maritza | RP Fw455 | MH559503 | MH603391 | |
Maritza | RP Fw457 | MH559504 | MH603390 | |
Pitilla | RP Fw570 | MH559505 | MH603389 | |
Cacao | RP Fw591 | MH559506 | MH603388 | |
Cacao | RP Fw597 | MH559507 | MH603387 | |
Cacao | RP Fw601 | MH559508 | MH603386 | |
Cacao | RP Fw616 | NA | MH603385 | |
Maritza | RP Fw618 | MH559509 | MH603384 | |
Maritza | RP Fw619 | MH559510 | MH603383 | |
Maritza | RP Fw620 | MH559511 | MH603382 | |
Maritza | RP Fw635 | MH559512 | MH603381 | |
Brewster | CH6868 | KR863019 | KR863275 | |
Brewster | AJC1794 | KR863021 | KR863277 | |
Brewster | AJC1798 | KR863026 | KR863282 | |
Brewster | CH6658 | KR863027 | KR863283 | |
Brewster | CH6659 | KR863028 | KR863284 | |
El Copé | KRL 0823 | FJ766749 | FJ84384 | |
El Copé | KRL 1540 | FJ766751 | FJ84552 | |
El Copé | KRL 1508 | KR911913 | KR911916 | |
El Copé | KRL 1496 | KR911914 | KR911917 | |
El Copé | KRL 1567 | KR911915 | KR911918 | |
L. catesbeiana | NA | – | KX686108* | KX686108* |
L. clamitans | NA | – | EF525879 | KY677813 |
L. maculata | NA | – | NA | AY779207 |
L. palmipes | NA | CFBHT12435 | KU494586 | KU495379 |
L. septentrionalis | NA | – | EF525896 | AY779200 |
L. sylvaticus | NA | – | KP222281* | KP222281* |
L. vaillanti | NA | – | KY587190 | AY779214 |
R. maoershanensis | NA | SYNU08030061 | KX1397728 | KX1397722 |
Separate Bayesian consensus trees for the CO1 and 16S alignments were estimated independently using MR BAYES v3.2.6 (Ronquist et al. 2013) to ensure they did not conflict with each other. After establishing that there were no conflicts, columns with gaps were removed from the two individual alignments, which were then concatenated end to end with PhyUtility v.2.7.1 (
Species boundaries were assessed in two ways. The first using the GENEIOUS plugin SPECIES DELIMITATION (
Concatenated phylogenetic trees reconstructed using Bayesian inference and Maximum likelihood (Figure
Phylogenetic reconstruction of Lithobates warszewitschii relationships between Costa Rican and Panamanian populations using concatenated alignments of CO1 and 16S. Node support values (posterior probabilities) and percentages calculated from 1000 bootstrap replicates are annotated at nodes. Samples collected in different localities are represented by different colours: individuals from Área de Conservación Guanacaste (ACG; Cacao, Caribe, Maritza, Pitilla, and San Gerardo) highlighted in red, individuals from Brewster highlighted in purple, and individuals from El Copé highlighted in orange. Sample information can be found in Table
CO1 species delimitation in GENEIOUS yielded three OTUs (Table
OTU | Closest OTU | Monophyletic? | Intra Dist | Inter Dist – Closest | Intra/Inter | PID(Strict) | PID(Liberal) | Av(MRCA-tips) | P(Randomly Distinct) | Rosenberg’sP(AB) |
---|---|---|---|---|---|---|---|---|---|---|
1: ACG | 2: El Copé | yes | 0.01 | 0.109 | 0.08 | 0.97 (0.91,1.0) | 0.99 (0.96,1.0) | 0.0076 | 0.05 | 8.10E-06 |
2: El Copé | 1: ACG | yes | 0.01 | 0.109 | 0.06 | 0.83 (0.69,0.97) | 0.97 (0.86,1.0) | 0.0047 | 0.05 | 8.10E-06 |
3: Brewster & KRL 0823 | 2: El Copé | yes | 0.02 | 0.197 | 0.08 | 0.88 (0.75,1.0) | 0.97 (0.87,1.0) | 0.0211 | 0.05 | 1.10E-07 |
4: L. palmipes | 5: L. vaillanti | yes | 0 | 0.114 | 0 | 0 | 0.96 (0.83,1.0) | 0 | NA | 1 |
5: L. vaillanti | 4: L. palmipes | yes | 0 | 0.114 | 0 | 0 | 0.96 (0.83,1.0) | 0 | NA | 1 |
6: L. catesbeiana | 7: L. clamitans | yes | 0 | 0.057 | 0 | 0 | 0.96 (0.83,1.0) | 0 | NA | 1 |
7: L. clamitans | L. catesbeiana | yes | 0 | 0.057 | 0 | 0 | 0.96 (0.83,1.0) | 0 | NA | 1 |
8: L. septentrionalis | 7: L. clamitans | yes | 0 | 0.092 | 0 | 0 | 0.96 (0.83,1.0) | 0 | NA | 0.33 |
9: L. sylvaticus | 8: L. septentrionalis | yes | 0 | 0.238 | 0 | 0 | 0.96 (0.83,1.0) | 0 | NA | 0.17 |
K2P-π at the CO1 and 16S loci showed a mean value of 7.2% and 3.4%, respectively, within all L. warszewitschii samples (Table
Intraspecific nucleotide diversity (π) within geographic groups of L. warszewitschii.
Population | Mean(π) | Range(π) |
---|---|---|
CO1 | ||
ACG | 0.004 | 0-0.008 |
El Copé | 0.063 | 0.002-0.154 |
Brewster | 0.001 | 0-0.002 |
L. warszewitschii | 0.072 | 0-0.166 |
16S | ||
ACG | 0.003 | 0-0.009 |
El Copé | 0.032 | 0-0.076 |
Brewster | 0.002 | 0-0.006 |
L. warszewitschii | 0.034 | 0-0.079 |
At the CO1 and 16S loci the largest NBGMD (πnet) was 15.4% and 6.9%, respectively, between ACG and Brewster samples (Suppl. material
The concatenated phylogenetic trees consistently outlined three distinct clades within Lithobates warszewitschii supported by high posterior probabilities, bootstrap values, and taxonomic distinctness at the CO1 locus. No field sites within the ACG exhibited any well-defined cladistic structure, indicating it is a larger panmictic population. The individuals from El Copé were polyphyletic, revealing the presence of two OTUs at this site. Geographic groups within L. warszewitschii also exhibited greater genetic distances than many other recognized species pairs within the genus, suggesting cryptic species may be present.
In the analyses of nucleotide diversity and NBGMD, isolation by distance (IBD) (
Isolation by distance may be the main driver of divergence or speciation among conspecific populations (
Lithobates warszewitschii is widely distributed throughout Central America, and the possibility of vicariance may explain mechanisms for genetic divergence. The Talamanca mountain range divides the Pacific and Atlantic versants at ~2000m altitude (
Although vicariance does divide sister species (
Polyphyly can be used as indication of undescribed species in a lineage (
At both CO1 and 16S loci, K2P-π mean (
The type specimen of Lithobates warszewitschii originated from Volcán Chiriqui, western Panama (
Thank you to Roger Blanco, Maria Marta Chavarría, Felipe Chavarría, Alejandro Masis, Daniel Janzen, Winnie Hallwachs, Sigifredo Marín, Luz María Romero, Alejandro Marín and the parataxonomists across ACG for help with fieldwork logistics and your continued commitment to conserving these habitats into perpetuity. Many thanks to Caroline Palmer, Federico Bolaños, Gerardo Chaves, and the reviewers whos comments helped improved this paper. Thank you to Ian Gardiner for all his help and support in the field, and to Benjamin and Sofia Puschendorf for spending endless hours driving around ACG and always being cheerful and helpful when working on this project. All samples were collected under permit No. ACG-PI-022-2017 and R-037-2017-OT-CONAGEBIO.
Supplementary tables and figures
Data type: molecular data
Explanation note:
Table S1. ABGD analysis from CO1 using all species presented in Table S2.
Table S2. Estimates of evolutionary divergence (π), and net evolutionary divergence (πnet) over CO1 sequence pairs between groups.
Table S3. Estimates of evolutionary divergence (π), and net evolutionary divergence (πnet) over 16S sequence pairs between groups.
Figure S1. CO1 phylogenetic tree. Geographic populations ACG (red), Brewster (orange), El Copé (purple) of L. warszewitschii are represented.
Figure S2. 16S phylogenetic tree. Geographic populations ACG (red), Brewster (orange), El Copé (purple) of L. warszewitschii are represented.
Figure S3. Prior intraspecific genetic divergence and number of OTUs using the ABGD algorithm.