TRACING THE EVOLUTIONARY HISTORY OF TWO ENDEMIC GROUND FROGS OF TEMPERATE FOREST OF SOUTHERN CHILE, THROUGH MOLECULAR AND CYTOGENETIC APPROACHES
TESIS DE MAGISTER CAMILA ANDREA QUERCIA RATY
VALDIVIA – CHILE
TRACING THE EVOLUTIONARY HISTORY OF TWO ENDEMIC GROUND FROGS OF TEMPERATE FOREST OF SOUTHERN CHILE, TROUGHT MOLECULAR AND CYTOGENETIC APPROACHES
Tesis presentada a la Facultad de Ciencias de la Universidad Austral de Chile en cumplimiento parcial de los requisitos para optar al grado de Magíster en Ciencias mención Genética
por CAMILA A. QUERCIA RATY
Valdivia Chile 2019 Universidad Austral de Chile Facultad de Ciencias INFORME DE APROBACIÓN TESIS DE MAGISTER
La Comisión Evaluadora de Tesis comunica a la Directora de la Escuela de Graduados de la Facultad de Ciencias que la Tesis de Magíster presentada por la candidata
CAMILA ANDREA QUERCIA RATY
ha sido aprobada en el exámen de defensa de Tesis rendido el día ___de _____ de 20__ como requisito para optar al grado de Magíster en Ciencias mención Genética y, para que así conste para todos los efectos firman:
Profesor Patrocinante Dr. José J. Nuñez Instituto de Ciencias Marinas y Limnológicas ______
Profesor Copatrocinante Dr. Elkin Y. Suárez-Villota Instituto de Ciencias Marinas y Limnológicas ______
Comisión Evaluadora Dra. Leyla Cárdenas Instituto de Ciencias Ambientales y Evolutivas ______
Dr. Guillermo D´Elía Instituto de Ciencias Ambientales y Evolutivas ______
DEDICATORIA
A mis padres y todos quienes se convirtieron en mi familia
De aquellos glaciares y hojarascas de quienes tu no podrás saber.
AGRADECIMIENTOS Siento especial gratitud hacia quienes fueron mis mayores guías en este proceso, Dr. José Nuñez y Dr. Elkin Suárez-Villota, cuyos consejos y conocimientos fueron el complemento clave para lograr este trabajo. Más aún, por enseñarme sin recelo parte de sus experiencias y pensamientos. Por la confianza y exigencia en mi puesta, y por haber enriquecido inigualablemente mi camino científico. A todos mis compañeros del Laboratorio de Sistemática UACh, quienes siempre reciben con una sonrisa, dispuestos a contribuir. En especial a la Dr. Leila Díaz por su ayuda en los procedimientos de laboratorio, y al Ingeniero en conservación Nicolás Gonzáles, por su colaboración en la toma de muestras en terreno. Agradezco también la disposición y grata acojida de los investigadores del Laboratorio de Biología e Genética de Peixes (UNESP, Botucatu, Brasil), donde tuve la posibilidad de realizar una pasantía en citogenética. En especial al Dr. Fausto Floresti, Dr. Cristian Araya y Dr. Duilio Zerbinato. A todos aquellas personas que, incluso sin saberlo, me brindaron inspiración y apoyo, mis más sinceros reconocimientos. Y por último, pero no menos importante, infinita gratitud a todos los ejemplares de anuros que fueron utilizados para este trabajo. El financiamiento para este trabajo fue dado por el proyecto FONDECYT 3160328 de EYS-V., beca de magíster CONICYT 22180766 de CAQ, y programa de Magister en Ciencias mención Genética, UACh. CONTENT INDEX
CONTENT INDEX I-IV TABLES INDEX V-VI FIGURES INDEX VII-VIII ABBREVIATION INDEX IX RESUMEN X-XIII ABSTRACT XIV-XVII GENERAL INTRODUCTION 1-19 1. RESEARCH CONTEXT 1-17 1.1. DETERMINATION OF BIODIVERSITY ON EARTH 1-3 1.2. SPECIES DELIMITATION 3-7 1.3. PLEISTOCENE HISTORY OF SOUTHERN CHILE 7-8 1.4. FROGS OF THE GENUS Eupsophus 9-10 1.5. CYTOGENETIC WITHIN THE GENUS Eupsophus 12-14 1.6. PHYLOGENETIC RELATIONSHIP OF THE 14-16 vertebralis GROUP 2. HYPOTHESIS AND OBJETIVES 18-19 2.1. HYPOTHESIS 18 2.2. GENERAL OBJECTIVE 18 2.3. SPECIFIC OBJECTIVES 19 2.3.1. Specific objectives: CHAPTER 1 19 2.3.2. Specific objectives: CHAPTER 2 19
I
CHAPTER 1: MOLECULAR PHYLOGENY, SPECIES 20-81 DELIMITATION, AND TIMING OF DIVERGENCE OF THE vertebralis GROUP (Anura: Alsodidae) IN SOUTHERN CHILE ABSTRACT 20-21 1. INTRODUCTION 21-27 2. MATERIALS AND METHODS 27-35 2.1. SAMPLES AND ETHIC STATEMENTS 27-28 2.2. GENETIC DATA 28-29 2.3. PHYLOGENETIC TREE RECONSTRUCTION 29-31 2.4. SPECIES DELIMITATION 31-34 2.5. TIME OF DIVERSIFICATION 34-35 3. RESULTS 35-39 3.1. PHYLOGENETIC PATTERNS INTO THE 35-37 vertebralis GROUP 3.2. SPECIES DELIMITATION ANALYSES 38-39 3.3. DIVERSIFICATION TIME 39 4. DISCUSSION 40-47 4.1. PHYLOGENETIC RELATIONSHIPS AND 40-43 DIVERGENCE TIME IN THE vertebralis GROUP 4.2. SPECIES DELIMITATION ANALYSES OF THE 44-47 vertebralis GROUP 4.3. FIELDWORKS REMARKS 47
II 5. CONCLUSION 48 ACKNOWLEDGMENTS 49 TABLES 50 FIGURES 51-55 SUPPLEMENTARY TABLES 56-62 SUPPLEMENTARY FIGURES 63-64 6. REFERENCES 65-81 CHAPTER 2: COMPARATIVE CYTOGENETICS OF TWO 82-114 GROUND FROGS OF GENUS Eupsophus: E. Emiliopugini AND E. vertebralis (Alsodidae); WITH COMMENTS ON INTRASPECIFIC POLYMORPHISM IN NORs ABSTRACT 82-83 1. INTRODUCTION 83-86 2. MATERIALS AND METHODS 86-90 2.1. SAMPLES COLLECTION AND CYTOLOGICAL 86-87 PREPARATIONS 2.2. CLASSICAL CYTOGENETIC TECHNIQUES 87-88 2.3. MOLECULAR CYTOGENETIC TECHNIQUES 88-90 3. RESULTS 90-91 3.1. CLASSICAL CYTOGENETIC TECHNIQUES 90-91 3.2. CYTOMOLECULAR TECHNIQUES 91 4. DISCUSSION 92-98 4.1. KARYOTYPES DESCRIPTION OF E. vertebralis 92-93 AND E. emiliopugini SPECIES
III 4.2. NUCLEOLAR ORGANIZER REGIONS 93-97 POLYMORPHISM 4.3. HYPOTHESIS ABOUT THE EVOLUTION OF PAIR 97-98 13 5. CONCLUSION 98 ACKNOWLEDGMENTS 99 FIGURES 100-104 SUPPLEMENTARY TABLE 105-106 8. REFERENCES 107-114 GENERAL DISCUSSION AND CONCLUSIONS 115-120 GENERAL REFERENCES 121-139
ANNEX I: Speciation in a biodiversity hotspot: Phylogenetic relationships, species delimitation, and divergence times of Patagonian ground frogs from the Eupsophus roseus group (Alsodidae)
ANNEX II: Mitochondrial genomes of the South American Frogs Eupsophus vertebralis and E. emiliopugni (Neobatrachia: Alsodidae) and their phylogenetic relationships
ANNEX III: Resumen taxonómico de las especies del grupo vertebralis
IV TABLES INDEX GENERAL INTRODUCTION Table 1 Classification of Eupsophus groups according to 11 their karyotypes and advertisement calls. CHAPTER 1: MOLECULAR PHYLOGENY, SPECIES DELIMITATION, AND TIMING OF DIVERGENCE OF THE vertebralis GROUP (Anura: Alsodidae) IN SOUTHERN CHILE
Table 1 Taxonomic index of congruence (Ctax) among 50 species delimitation analyses. SUPPLEMENTARY TABLES Table 1 Sampling locations of E. vertebralis and E. 56-57 emiliopugini. Table 2 Species delimitation scenarios and results obtained 58 for Tr2 and BPP. Table 3 Bayes factor estimates for Molecular Clock/Tree 59 priors and Species delimitation scenarios. Table 4 Total nucleotide sites, partition schemes and 60 nucleotide substitution models for sequences used in this study. Table 5 Nucleotide substitution rates used for sequences of 61 the vertebralis group in this study. Table 6 Likelihood scores and Akaike’s information 62 criterion (AIC) results for STEM analysis.
V CHAPTER 2: COMPARATIVE CYTOGENETICS OF TWO GROUND FROGS OF GENUS Eupsophus: E. Emiliopugini AND E. vertebralis (Alsodidae); WITH COMMENTS ON INTRASPECIFIC POLYMORPHISM IN NORs. SUPPLEMENTARY TABLE Table 1 List of the specimens used for cytogenetic analyses 105-106 in this study.
VI FIGURES INDEX
GENERAL INTRODUCTION Figure 1 Morphological similarities and distribution range of 17 Eupsophus vertebralis and E. emiliopugini. CHAPTER 1: MOLECULAR PHYLOGENY, SPECIES DELIMITATION, AND TIMING OF DIVERGENCE OF THE vertebralis GROUP (Anura: Alsodidae) IN SOUTHERN CHILE Figure 1 Maps of sampling locations for the vertebralis group. 51 Figure 2 Phylogenetic relationships among the vertebralis group. 52-53 Figure 3 Phylogenetic dated tree by BEAST and species 54-55 delimitation analyses. SUPPLEMENTARY FIGURES Figure 1 Phylogenetic relationships among species of the 63 vertebralis group. CHAPTER 2: COMPARATIVE CYTOGENETICS OF TWO GROUND FROGS OF GENUS Eupsophus: E. emiliopugini AND E. vertebralis (Alsodidae); WITH COMMENTS ON INTRASPECIFIC POLYMORPHISM IN NORs. Figure 1 Map portraying 15 collection localities of the vertebralis 100 group specimens, in Southern Chile. Figure 2 Classical Giemsa and C- banding of the vertebralis group. 101 Figure 3 Results of Ag-NOR staining, and FISH of 28S rDNA probe 102 for the vertebralis group.
VII Figure 4. Telomeric signal obtained by FISH for the vertebralis 103 group. Figure 5 Proposed hypothesis to explain the nucleolar 105 dominance phenomena observed in one specimen from Puyehue locality of E. emiliopugini species.
VIII ABBREVIATIONS INDEX
2n Diploid chromosome number Ag-NOR Silver staining for NORs BC Bibrachial chromosomes BFD Bayes factor delimitation BIC Bayesian information criterion BPP Bayesian phylogenetics and phylogeography program Ctax Congruence index DNA Deoxyribonucleic acid e.g. Exampli gratia FISH Fluorescent in situ hybridization FN Fundamental number GMYC General mixed yule coalescent model HPD Highest posterior density interval IB Bayesian inferences i.e. Id est IUCN International union for conservation of nature LGM Last glacial maximum MCMC Markov chain Monte Carlo algorithms ML Maximum likelihood MLE Marginal likelihood estimation MRCA Most recent common ancestor MSC Multispecies coalescent models Mya Million year ago NA Data not available NORs Nucleolar organized region PCR Polymerase chain reaction PP Bayesian posterior probability PTP Multirate Poisson tree processes rDNA Ribosomal genes SC Secondary constriction SexC Presence of sexual chromosomes STEM Estimation using maximum likelihood Tr2 Trinomial distribution model θ = 4Neμ Population size τ Divergence time
IX
RESUMEN
Eupsophus es un género de ranas de hojarasca, endémicas del Sur de Chile y Argentina. La taxonomía y sistemática de este grupo ha sido controversial desde sus inicios. Actualmente son 10 las especies reconocidas para este género, las cuales clásicamente son divididas en dos grupos: El grupo roseus que comprende ocho especies (2n=30) y el grupo vertebralis compuesto por dos especies, E. vertebralis y E. emiliopugini (2n=28). Dentro del grupo vetebralis, la distribución geográfica, las características morfológicas y las variaciones genéticas permanecen confusas y sin hipótesis filogenéticas acerca de las relaciones entre sus linajes. Además, la comparación de los cariotipos entre ambas especies, utilizando citogenética clásica (tinción Giemsa), evidenció diferencias en el número fundamental de cromosomas (FN) entre los cariotipos de E. vertebralis (FN= 54) y E. emiliopugini (FN= 56). Sin embargo, la consistencia de estos reportes aún no ha sido explorada a nivel intraespecífico, así como tampoco mediante otras técnicas de citogenética molecular.
Los objetivos abordados en este trabajo de tesis fueron, por una parte, evaluar las relaciones filogenéticas, los tiempos de divergencia, y
X determinar los límites de especies para E. vertebralis y E. emiliopugini. Por otra parte, se propuso determinar la presencia o ausencia de patrones citogenéticos especie específicos para el grupo vertebralis y probar posibles hipótesis acerca de re-arreglos cromosómicos, considerando individuos de diferentes poblaciones y sexos.
Para los análisis moleculares, se secuenciaron tres marcadores mitocondriales (D-loop, cytb y coI) y dos nucleares (CrybA1 y pomc) para 91 individuos del grupo vertebralis, colectados de 19 localidades diferentes. Luego, se realizaron análisis filogenéticos de Máxima Verosimilitud (ML) e Inferencia Bayesiana (IB) y análisis de delimitación de especies unilocus y multilocus coalescente. Por otro lado, para los análisis citogenéticos, se emplearon técnicas clásicas (Tinción Giemsa, Bandeo C y Ag-NORs) y moleculares (hibridación in situ fluorescente FISH, usando zonas teloméricas y ribosomales) sobre placas metafásicas de 23 individuos del grupo vertebralis proveniente de 15 localidades diferentes.
Las reconstrucciones filogenéticas de ML e IB, recuperaron la monofilia del grupo vertebralis (Bootstrap: 100%; Probabilidad a posterior Bayesiana PP: 1.0), no así de sus especies (Bootstrap: < 50; PP: < 80) exhibiendo un patrón polifilético dentro del grupo. Los resultados de los seis análisis de delimitación de especies fueron incongruentes entre ellos, recuperando desde una a 11 especies putativas (Máximo
XI índice de congruencia para una especie= 0.59; Mínimo índice de congruencia para once especies= 0.18). El tiempo de divergencia entre E. emiliopugini y E. vertebralis (0.040 Mya) indicaría que la historia evolutiva de este grupo fue moldeada por periodos interglaciares del Pleistoceno tardío.
Por otro lado, las diferencias en FN entre los cariotipos de ambas especies, fueron corroborados mediante la tinción de Giemnsa y Bandeos C (E. vertebralis FN= 54; E. emiliopugini FN=56). Los resultados de FISH, usando la sonda ribosomal 28S rDNA, confirmaron la señal obtenida para los NOR activos (de acuerdo con la tinción Ag-NORs) en E. vertebralis y en E. emiliopugini. Aún más, estos resultados mostraron un polimorfismo en relación al número y a la posición de los genes 28S rDNA (cuatro señales) y de los NORs (dos señales) activos para un espécimen de E. emiliopugini, colectado en la localidad de Puyehue. Esta variación cromosómica a nivel intraespecífico, podría explicarse por la ocurrencia de arreglos cromosómicos o procesos de hibridación. De los resultados de FISH con sonda telomérica, no se encontraron señales fluorescentes intersticiales, pero si teloméricas terminales para ambas especies.
En definitiva, los resultados genético moleculares sugieren baja diferenciación genética y diversificación reciente para el grupo E. vertebralis. Además, los resultados citogenéticos corroboraron que la diferencia en el FN de los cariotipos de E. emiliopugini y E. vertebralis, es
XII conservada a nivel intraespecífico, respectivamente. Sin embargo, diferencias intraespecíficas para la posición de NORs en E. emiliopugini fueron encontradas (solo una muestra analizada fue polimórfica). Por lo tanto, los resultados encontrados no brindan evidencia suficiente que soporte a E. emiliopugini y E. vertebralis como una o como dos especies. Por consiguiente, el proceso de especiación de los linajes de E. vertebralis and E. emiliopugini es discutido bajo un marco integrativo, considerando relaciones filogenéticas, historia evolutiva, antecedentes geográficos, citogenéticos y etológicos (cantos). Finalmente son mencionados nuevos linajes con importancia para conservación.
XIII ABSTRACT
Eupsophus is a genus of ground frogs, endemic from Southern Chile and Argentina. The taxonomy and systematic of the genus have been controversial since its founding. Currently, Eupsophus is composed by 10 species, which have been match up into two groups: The roseus group that comprise eight species (2n=30), and the vertebralis group with two species E. vertebralis, and E. emiliopugini (2n=28). Within the vertebralis group, geographic distribution, morphologic and genetic variation remain unclear and without hypotheses about the relationships between these lineages. Moreover, morphology karyotype description made by Giemsa stained, evidenced differences on the fundamental number (FN) between E. vertebralis (FN= 54) and E. emiliopugini (FN= 56). Nevertheless, the consistency of this report at intraspecific levels and with others cytogenetics techniques remain unexplored.
Thus, the goals of this thesis were, on the one hand, to evaluated phylogenetic relationships, diversification times, and to determine species limits of Eupsophus vertebralis and E. emiliopugini. On the other hand, it was proposed to determine the presences or absences of specie specific cytogenetics patterns for the vertebralis group, and test
XIV hypothesis about chromosomal rearrangements, considering individuals from different localities and sexes.
For the molecular analyses, three mitochondrial (D-loop, cytb, and coI) and two nuclear (crybA1, and pomc) markers, from 91 individuals of the vertebralis group collected in 19 localities, were sequenced. Then, it was performed phylogenetics analyses with Maximum Likelihood (ML) and Bayesian Inference (IB), and species delimitation analyses using unilocus and multilocus coalescent approaches. For its part, for the cytogenetic analyses it was employed conventional (Giemsa staining, C- banding and, Ag-NOR) and cytomolecular (fluorescence in situ hybridization FISH, using telomeric and 28S ribosomal probes) techniques, on metaphasic plates of 23 individuals of the vertebralis group from 15 different localities.
ML and IB phylogenetic reconstructions recovered the monophyly of the vertebralis group (Bootstrap: 100%; Bayesian posterior Probability PP: 1.0), but not the reciprocal monophyly of its species (Bootstrap: < 50; PP: < 80), exhibiting a polyphyletic pattern within the vertebralis group. Incongruence among the six species delimitation analyses carried out, detecting from one to eleven species (Max. congruence index= 0.59, for one putative species; Min. congruence index= 0.18, for eleven putative species). Also, the divergence time between E. emiliopugini and E.
XV vertebralis (0.040 Mya) indicates an evolutionary history probably molded by the interglacial periods of Late Pleistocene.
On the other hand, FN differences between E. vertebralis and E. emiliopugini were corroborated through Giemsa staining and C-banding (FN= 54 and 56, respectively). FISH with 28S rDNA probes confirmed the active NORs signal for E. vertebralis and E. emiliopugini (in accordance with the Ag-NORs staining results). Nevertheless, we found polymorphisms relative to the numbers and location of 28S rDNA (four signals) and active NORs (two signals) in one specimen of E. emiliopugini from Puyehue locality. This intraspecific chromosomal variation could be explained by chromosomal rearrangements or derived from hybridization process. FISH using telomeric probe over spreads from both species detected no interstitial fluorescent signals, but clearly stained telomeric regions.
The molecular results of this study, suggest low genetic differentiation and the early diversification for the vertebralis group. Also, the cytogenetics results corroborate that the difference in FN between the karyotype of E. vertebralis and E. emiliopugini is conserved at interspecific level. Nonetheless, intraspecific differences for the NORs position were found in one sample of E. emliopugini from Puyehue locality. Therefore, the results do not provide sufficient evidence to support E. emiliopugini and E. vertebralis as one or two species. Hence, the speciation process of
XVI E. vertebralis and E. emiliopugini lineages are discussed under an integrative framework, considering the phylogenetic relationships, evolutionary history, geographic antecedents, cytogenetic, and ethologic (calls) data. Finally, new concerning lineage for conservation efforts are proposed.
XVII VII. GENERAL INTRODUCTION
1. RESEARCH CONTEXT
1.1.- DETERMINATION OF BIODIVERSITY ON EARTH
Biodiversity is a central component of the systems that support life on the planet (Savage, 1995; Dirzo & Raven, 2003; Lausch et al., 2016). From the operational point of view, the notion of biodiversity encompasses several different levels of biological organization, from the species’ make up genetic to ecosystems, in which the species is the most significant unit (Hey, 2001; Coyne & Orr, 2004; De Queiroz, 2007). Species are used for comparisons in almost all biological fields; therefore, its accurate identification is a critical issue in fields as ecology, evolutionary, and conservation biology (Sites & Marshall, 2003; Balakrishnan, 2005; Fujita et al., 2012; Luo et al., 2018). Even more, we do not have the capacity to protect and conserve organisms that we cannot identify. Further, our attempts to understand the consequences of degradation and climate changes are negatively compromised if we are not able to achieve successful identifications at different levels of biodiversity (Mace, 2004),
1 including important lineages on intraspecific levels. In fact, biodiversity hotspots are selected on the basis of the species they possess, conservation schemes are assessed on how many species are preserved, and conservation legislation and politics are focused on species preservation (Agapow et al., 2004). Consequently, enhancing or diminishing the biodiversity attention increase or reduce the benefits we can derive from them.
Extensive is the literature that exposes the different species concepts and their biases (Sokal et al., 1970; Wheeler & Meier, 2000; De Queiroz, 2007; Aldhebiani, 2018). Despite there is no agreement yet about a species concept that unifies all the criteria, it is broadly accepted that species are dynamic entities which are connected by “gray zones” throughout the continuous evolutionary process (De Queiroz, 2007). Thus, at the time to distinguish biological categories and make accurate taxonomy proposals, the challenge for biologists is to overcome the subjectivity that surround the problem of species concept (Sites & Marshall, 2004).
Systematic biology is the scientific discipline that seeks to infer the links between the lineages of individuals from their ancestor-descendant relationships (Ax, 1999). Allowing researchers to understand the
2 evolutionary history of the lineages under study, through the inference of the process that promotes speciation (Carstens et al., 2013; Fujita et al., 2012; Sites & Marshall, 2003). In order to identify these processes, biologists must first recognize and delimit nascent evolutionary lineages (Sites & Marshall, 2004; Wiens, 2007; Carstens & Dewey, 2010). Therefore, the main objectives in systematics have been: i) to discover monophyletic groups (clades) and relationships within them at all hierarchical levels above species; and ii) discover lineages (i.e. species) at lower levels (Wheeler & Meier, 2000). Here, under an operational framework, the species is considered according to its taxon connotation, which refers to the practical application of species category, how was exposed by Bock et al. (2004). Nevertheless, the task of unequivocally recognize biodiversity at different levels, it become extremely complex when the systematic relationships are controversial, plagued by synonymies, lack of type material for the species already described, the existence of cryptic species, and if the geographic range of the nominal species is unknown, subjective, or uncertain (Agapow et al., 2004; Fujita et al., 2012).
1.2.- SPECIES DELIMITATION
3 The procedure by which biological diversity is identified at species level is known as “species delimitation” (Carstens et al., 2013). There are several methodological approaches for species delimitation, which can be subdivided in non-based and based tree methods, and can be implemented using different kinds of data such as, morphological, molecular and ethological, among others (Sites & Marshall, 2003, 2004; Camargo & Sites, 2013).
Despite the taxonomic practice has been an active research field, during the last two decades, species delimitation methods using molecular data has become a trended topic in systematic research (Sites & Marshall, 2003; Camargo & Sites, 2013). This has led to the development of new empirical methods. (Sites & Marshall, 2003; Pons et al., 2006; Knowles & Carstens, 2007; Wiens, 2007; Carstens & Dewey, 2010; Carstens et al., 2013). These new methods evaluate lineage composition in a phylogenetic framework, and can be implemented under coalescent models using unilocus or multilocus datasets (Kubatko et al., 2009; Carstens & Dewey, 2010; Carstens et al., 2013; Fujisawa & Barraclough, 2013). These approaches estimate phylogeny while allowing for the action of population-level processes, such as genetic drift in combination with migration, expansion, population divergence or the combinations of these processes (Maddison & Knowles, 2006; Liu et al., 2009).
4 On the one hand, unilcous species delimitation approaches use sequence information itself as the primary information source for establishing group membership and defining species boundaries (Pons et al., 2006; O’Meara, 2010; Fujisawa & Barraclough, 2013). On the other hand, multilocus methods, operated under multispecies coalescent models (MSC), use concordance of gene trees across multiple loci as evidence for existence of multiple species, but do not rely on reciprocal monophyly. Thus, MSC take into account species phylogeny, random fluctuations in the coalescent process, uncertainties in the gene tree topology and branch lengths (Zhang & Yang, 2011; Fujita et al., 2012). Also involve population size parameters (θ), divergence time parameters (τ), a priori assignment of evolutionary models per locus and individuals for defining species categories (Kingman, 1982; Kubatko et al., 2009; Liu et al., 2009; Heled & Drummond, 2010; Harrington & Near, 2012; Rannala, 2015).
Robust examples of multilocus species delimitation methods under MSC, are Bayesian methods such as BPP (Yang & Ziheng, 2015) and BEAST (Liu et al., 2008). These methods implement Markov Chain Monte Carlo (MCMC) algorithms to calculate the median over gene tree topologies and branch lengths, and over the parameters. Then, the analyses considered a stochastic process between the recent lineages and their most recent common ancestor (MRCA), responding to the coalescent
5 models (Xu & Yang, 2016). That is how, coalescent techniques to delimit species allows researchers to infer divergence dynamics between lineages, and delimit nascent evolutionary lineages, in order to identify species, and understand processes that promote speciation (Sites & Marshall, 2003; Wiens, 2007; Fujita et al., 2012; Carstens et al., 2013).
Despite the above, the results of the species delimitation methods could will not congruent between different approaches, or could will not concordant with the biology of the species, resulting in an overestimation of the candidate species (Luo et al., 2018). Therefore, some authors proposed the use of genomic-based species delimitation results as a hypothesis, which has to be corroborated with other kinds of evidence and can change with new discoveries (Fujita et al., 2012; Sukumaran & Knowles, 2017). In fact, the field of taxonomy is approaching to the use of different lines of data that allows researchers to make a more accurate species delimitation (Padial et al., 2010; Pante et al., 2015; e.g. Rojas et al., 2018). Thus, before to make a new taxonomic proposal, it is necessary to considered several kinds of data, such as geographic, physiologic, cytogenetic, molecular, ethological among others (Padial et al., 2010; Puillandre et al., 2012; Pante et al., 2015), to ensure the species delimitation according with the biology of the groups in question. So, the use of multiple and complementary sources of evidence for distinguishing and delimit species it has been named as “integrative taxonomy” (Dayrat,
6 2005; Will et al., 2005), and provides a stable and objective background to delimit species (Fujita et al, 2012).
1.3.- PLEISTOCENE HISTORY OF SOUTHERN CHILE
During the Pleistocene, the biogeographic history at southern South America has been influenced by different and complex topography, accompanied and molded by at least four glaciations periods: i) the most extensive Andean Glaciation (1.1 Mya); ii) the Coldest Pleistocene Glaciation (0.7 Mya); iii) the Last Southern Patagonian Glaciation or Santa María Glaciation (0.18 Mya); and iv) the Last Glacial Maximum (LGM) or Llanquihue Glaciation (0.020 – 0.014 Mya) (Paskoff, 1977; Rabassa & Clapperton, 1990; Denton et al., 1999; Hulton et al., 2002; McCulloch et al., 2000; Rabassa et al., 2011; Sánchez-García & Castresana, 2012). These glaciation periods would have generated climatic changes that affected the physical and biological environments (Villagrán & Armesto, 2005); which, in conjunction with the geologic and hydric systems of Southern Chile, would have molded the distribution, influenced diversification, and speciation events of Patagonian biodiversity (e.g. Zemlak et al., 2008 Cosacov et al., 2010; González-Ittig et al., 2010). There are several studies that show phylogeographic patterns influenced by Pleistocene glaciation. Particularly in Chile, we have examples of terrestrial vertebrates, among
7 them, lizards of the genus Liolaemus (Victoriano et al., 2008; Muñoz- Mendoza et al., 2017), terrestrials mammals (Lessa, et al., 2010), and marine fauna such as dolphins (Pérez-Alvarez et al., 2016), whales (Attard et al., 2015), and fishes (Ruzzante et al., 2006; Zemlak et al., 2008; Vera-Escalona et al., 2019).
The amphibians of Chile, present high levels of endemism, in fact, 70% of the amphibian species present in Chile are endemic (Lobos et al., 2014). Amphibian species have proved to be a good model for studies in the evolutionary field, because they have restricted dispersal capabilities, which tend to promote differentiation (Duellman, 1999; Prohl et al., 2010). Moreover they are highly sensitive to climatic changes, recognized as bioindicators, owing to complex life histories, permeable skin, and exposed eggs (Fitzpatrick et al., 2009). For example, into the frog Eupsophus calcaratus, Nuñez et al. (2011) described lineage diversity and phylogeographic patterns, associated with Pleistocene glacial retreats. Uncertainties remain about the evolutionary history, and species limits in several amphibian endemic species of Chile. Some examples are the frogs of genera Batrachyla, Alsodes, and Eupsophus (Blotto et al., 2013).
1.4.- FROGS OF THE GENUS Eupsophus
8 The genus Eupsophus (Fitzinger 1843), is a genus of ground frogs whose taxonomy and systematics have been controversial and discussed to this day. Currently, this genus is composed of 10 species (Suárez- Villota et al., 2018a), with a broad distribution in the Valdivian rainforest, and temperate Nothofagus forest of Southern Chile and Argentina (Formas, 1978; Ibarra-Vidal et al., 2004). Most species of this genus have endotrophic-nidicolous tadpoles, which develop in water housed (except E. altor, Nuñez et al., 2012) on small cavities in the ground, or holes at the end of flooded tunnels (Úbeda & Nuñez, 2006). From the taxonomic point of view, Eupsophus is frequently partitioned into two groups on the basis of different chromosome numbers, and advertisement calls (Table 1): first, the roseus group is composed by E. calcaratus, E. contulmoensis, E. septentrionalis, E. nahuelbutensis, E. insularis, E. migueli, E. roseus, and E. altor. These species have diploid chromosome number 2n=30 (Formas, 1978; Formas, 1980; Veloso et al., 2005; Formas et al., 2008; Nuñez et al., 2012), vocalizations with one note per call (described for five species in Formas & Brieva, 1994), and a body size 34-42 mm (snout-cloaca distance in adults; Nuñez, 2003). Second, the vertebralis group is composed by E. vertebralis (Grandison, 1961), and E. emiliopugini (Formas, 1989), both with 2n=28 (Formas, 1991), and with four to six, and two notes per call, respectively (Formas & Vera, 1980; Formas, 1989; Formas & Brieva, 1994). The individuals of these species have a body size 50-59 mm (snout-cloaca distance in adults; Nuñez, 2003).
9
In the last decades, the studies that deal with the phylogenetic relationships within the roseus group are those of Veloso et al. (2005), Correa et al. (2006), Formas et al. (2008), Nuñez et al. (2012), Blotto et al. (2013), Correa et al. (2017), and Suárez-Villota et al. (2018a). Comparing the results, and the proposal from only the latest two articles (Correa et al., 2017 and Suárez-Villota et al., 2018a), it become clear the existences of controversies around the genus Eupsophus and their lineages. Correa et al., (2017) reduce the roseus group to four species (E. insularis, E. migueli, E. roseus, E. calcaratus); while Suárez-Villota et al., (2018a) revitalize the classic taxonomic proposal of eight species for the roseus group (ANNEX I). The results of both works were based on different species delimitation analyses.
10 Table 1: Classification of Eupsophus groups according to their karyotypes and advertisement calls. 2N= diploid chromosome numbers, FN= fundamental number, BC= Bibrachial chromosomes, SexC= presence of sex chromosomes, NA= data not available. Notes per call with (*) indicated discrepancies in their measures on different works.
Species name 2N FN BC SexC Notes per call
The roseus group
Eupsophus roseus 30 46 16 yes 1*
Eupsophus migueli 30 45.46 15.14 yes 1* Eupsophus altor 30 44 NA no 1 Eupsophus nahuelbutensis 30 NA NA NA NA
Eupsophus contulmoensis 30 46 16 NA 1 Eupsophus calcaratus 30 46 16 NA 1 Eupsophus septentrionalis 30 46 16 yes NA Eupsophus insularis 30 45.46 15.14 yes 1 The vertebralis group
Eupsophus vertebralis 28 54 26 NA 4 to 6 * Eupsophus emiliopuguini 28 56 28 NA 2
11 1.5.- CYTOGENETIC WITHIN THE GENUS Eupsophus
It has been described that cytological techniques are capable to revealed chromosome evolution, infer species relationships, corroborate suggestion of new species, and differentiate cryptic species successfully in anuran groups (Bruschi et al., 2012; Bruschi et al., 2014; Bakloushinskaya, 2017). Nevertheless, the role of chromosomal rearrangements in speciation process, need to be addressed by experimental methods according to the group in question (Rieseber, 2001), mostly because anurans could present individuals or populations variations in the chromosomal positions of certain molecular markers (e.g. Vitelli et al., 1982; Cuevas, 2008). Thus, the combination of classical and molecular cytogenetic techniques represents appropriated approaches to perform comparative analyses between the genome of amphibian species, and provided insights into patterns of lineage evolution within phylogenetically related groups (Hillis, 1991; Barth et al., 2014; Bruschi et al., 2014). Classical Chromosomal banding such as Giemsa stain, C- Banding, and Nucleolus organizer regions stain (NORs) have been widely used in karyological descriptions. On the same way, molecular cytogenetic techniques based on Fluorescence in situ hybridization (FISH) have improved the chromosomal comparisons. Some of the most used cytogenetic molecular markers in evolutionary, and comparative
12 investigations are Ribosomal RNA gene (rDNA) and telomeric sequences (Bolzán, 2012; Gazoni, 2012; Bruschi et al., 2014).
Results of classical cytogenetic approaches have found differences in fundamental number (FN), sex chromosome structure, C-banding pattern, and nucleolus pair morphology between the species of the roseus group (2n=30) (Formas, 1978; Formas, 1991; Formas et al., 2008; Veloso et al., 2005; Nunez et al., 2012). On the other hand, the species of vertebralis group species (2n=28), present a secondary constriction on pair 5, and lack sex chromosomes. Further, the pair 13 is metacentric for E. emiliopugini (FN=56), and telocentric for E. vertebralis (FN=54) (Formas, 1991). Formas (1991) exposed an evolutionary hypothesis to explain the chromosomal differences on pair 13 between E. vertebralis and E. emiliopugini. The author proposed a pericentric inversions more than loss or win of genetic material. However, more cytogenetic researches for the genus Eupsophus, specifically for the vertebralis group, are necessary to test these hypotheses.
In order to perform a refined perspectives on cytogenetic features of the species of the vertebralis group, and to contribute with evidence that help to interpret the relationships within the vertebralis group lineages, we proposed the use of classical and molecular cytogenetic bandings, to
13 obtain patterns that can shed light on the evolution of the karyotypes of these two species.
1.6.- PHYLOGENETICS RELATIONSHIP OF THE vertebralis GROUP
Previous works (Formas, 1991; Formas & Brieva, 1994; Pyron & Wiens, 2011; Blotto et al., 2013; Suárez-Villota et al., 2018a; Suárez- Villota, et al., 2018b) employing cytological, ethological or molecular characters, have not presented phylogenetics proposals focusing on the vertebralis group. For example, Pyron & Wiens (2011), reject the monophyly of Eupsophus genus and their groups (The roseus and the vertebralis group); they include Batrachyla antartandica, B. taeinata, and Hylorina sylvatica within the clade of Eupsophus, E. calcaratus (from the roseus group) is recovered as sister taxa of E. emiliopugini (from the vertebralis group), and include E. verterbalis (from the vertebralis group) whitin the clade of the roseus group. In contrast, Blotto et al. (2013) and Suárez-Villota et al. (2018a, 2018b) recovered the genus Eupsophus and their species groups (the roseus and the vertebralis groups) as monophyletic groups. All of these studies include a low number of individuals of the vertebralis group, which turns out not to be representative of the range of distribution and the polymorphism of the group. Moreover, these studies have not allowed to detail the
14 phylogenetic relationships and evolutionary history of the species of the vertebralis group, since they could not make inferences about the intraspecific relationships of the group.
The previously background leaves uncertain about the relationships between the lineages and the evolutionary history of E. verterbalis and E. emiliopugini. Such uncertainties have to be taken into attention and further analyzed, due to these both species inhabit in currently fragmented and intervened areas. Furthermore, E. vertebralis has been listed as near threatened by the “Red list of threatened species” (IUCN, 2019) and vulnerable by the “Reglamento para la Clasificación de Especies Silvestres según Estado de Conservación” (RCE, 2019). In addition, there are inconsistencies with respect to the distribution range of both species: First, the distribution of Eupsophus vertebralis has been reported from 37ºS to 40ºS by Formas (1992), or approximately from 40ºS to 44ºS by IUCN (2019) in Chile, with a single locality in Puerto Blest, Río Negro, Argentina. Second, E. emiliopugini has a further south distribution comprising from 41ºS to 45ºS (Úbeda, 1998), or from 40ºS to 45ºS by IUCN (2019) in Chile, with a single location in Lago Puelo, Chubut, Argentina. Moreover, Correa & Durán (2019) describe one locality of sympatry, and other two localities with close distribution between the species of the vertebralis group, but they do not describe how the species were determined.
15
According with the previous antecedents, both species would have an overlapping distribution; despite it has been described theoretically as allopatric (Formas, 1992; Figure 1). Nevertheless, the discrepancies around the distribution ranges of these species, could be due to their individuals are morphologically similar (Figure 1) and have ample phenotypic variation (Correa et al., 2017, Correa & Durán 2019), making difficult their identification on fieldworks. Besides, low genetic differentiation was reported between mitogenomes of E. vertebralis and E. emiliopugini (both genomes share 94.5% identity with 879 variable sites; Suárez-Villota et al., 2018b ANNEX II).
16
Figure 1: Morphological similarities and distribution range of Eupsophus vertebralis and E. emiliopugini. Photography of one E. vertebralis specimen in orange circle, and of one specimen of E. emiliopugini in blue circle are shown. Chilean distribution range according to IUCN (2019), for E. vertebralis it is marked with orange, and for E. emiliopugini it is in blue. Overlapping distribution for these both species is marked with light orange color. Green areas correspond with National Chilean Parks.
17 2. HYPOTHESIS AND OBJECTIVES
2.1.- HYPOTHESIS
Cytogenetic, morphologic, ethologic and geographic antecedents for Eupsophus vertebralis and Eupsophus emiliopugini, support the proposal of these both species as reciprocally monophyletic lineages.
2.2.- GENERAL OBJECTIVE
Evaluate the delimitation of Eupsophus vertebralis and E. emiliopugini, trough multilocus coalescent approaches and karyotype characterization.
18 2.3.- SPECIFIC OBJECTIVES
2.3.1.- Specific objectives: CHAPTER 1
Perform phylogenetic reconstructions for the vertebralis group, using DNA sequences and considering samples of its wide range of distribution. Specify with statistical rigor the limits of Eupsophus vertebralis and E. emiliopugini, using mitochondrial and nuclear sequences under a coalescent framework.
Determine the divergence (stem) and diversification (crown) time for the vertebralis group.
2.3.2.- Specific objectives: CHAPTER 2
Characterize the karyotypes of Eupsophus vertebralis and E. emiliopugini, using conventional cytogenetics techniques.
Evaluate the presence of chromosomic differences between Eupsophus vertebralis and E. emiliopugini, trough fluorescent in situ hybridization.
19
ANNEX I
20
ANNEX II
21
CHAPTER 1
MOLECULAR PHYLOGENY, SPECIES DELIMITATION, AND TIMING OF DIVERGENCE OF THE vertebralis GROUP (Anura: Alsodidae: Eupsophus) IN SOUTHERN CHILE
ABSTRACT
The alsodid ground frog genus Eupsophus comprised ten species. Two of them Eupsophus vertebralis and E. emiliopugini belonging to the vertebralis group (2n=28), exhibited clear genetic and morphological differences with the others eight species (the roseus group, 2n=30). Nevertheless, the geographic distribution, morphology, and genetic variation within the vertebralis group remain unclear. In this work, we aimed to determine species limits of Eupsophus vertebralis and E. emiliopugini and give a first inside about their evolutionary history. For this, we carried out a phylogeographic analyses, using multilocus coalescent approaches and divergence time estimations. Five molecular markers, three mitochondrial (d-loop, cytb, coI) and two nuclear (crybA1 and pomc), from 91 individuals of 19 localities were amplified.
22 Phylogenetic reconstructions (Maximum Likelihood and Bayesian analysis) recovered the vertebralis group as monophyletic (Bootstrap: 100%; Bayesian posterior probability: 1.0), nevertheless within the vertebralis group a polyphyletic pattern was observed. Incongruence among the six species delimitation analyses (two unilocus and four multilocus approaches) was detected, these analyses indicated from one to eleven species. The divergence times recovered for the vertebralis group are coincident with cycles of deglaciation during the Late Pleistocene in Patagonia. The results obtained do not allow a new taxonomic proposal and are discussed around the low genetic differentiation and the recent diversification among the lineages of the vertebralis group. Comments about the evolutionary history of the vertebralis group molded by Quaternary changes and seemingly divergent lineage within the vertebralis group are also displayed.
Keywords: Ground frogs, Patagonia, Polyphyly, Multilocus coalescent, Pleistocene glaciations
1. INTRODUCTION
The resolution of species limits and their relationships is a fundamental task to make an accurate assessment of the biodiversity on Earth, as well as to perform consistent conservation policies (Mayr, 1982; Wiens & Servedio, 2000; Agapow et al., 2004; De Queiroz, 2005; De
23 Queiroz, 2007; Lausch et al., 2016; Pecl et al., 2017) Also, this issue is a main topic in systematic biology, and it is primordial to any other field in biological research (Sites & Marshall, 2003; Wiens, 2007; Fujita et al., 2012; Luo et al., 2018). Example of the above is the extensive literature focused on the problems that surround species concepts and species delimitation methods (e.g. Sokal & Crovello, 1970; Sites & Marshall, 2004; Carstens et al., 2013; Rannala, 2015; Luo et al., 2018). In this sense, species delimitation, i.e. the act of identifying species-level biological diversity (Carstens et al., 2013), is challenging due to the inability to distinguish several or even all properties that define species according to the different species concepts (de Queiroz, 2007; Fujita et al., 2012; Carstens et al., 2013). Examples of these properties include intrinsic reproductive incompatibility, phenotypic distinctiveness, monophyly and ecological uniqueness (Shaffer & Thomson, 2007; De Queiroz, 2007; Gratton et al., 2016). Species delimitation can be even more difficult for cryptic and/or allopatric species, recently diverged lineages, and actively radiating groups, due to incomplete separation or secondary introgression, plus sampling deficiencies, and different taxonomic practices (Carstens & Knowles, 2007; De Queiroz, 2007; Fujita et al., 2012).
Nowadays genetic data have become more accessible for research, which has led to an explosion in the number and variety of
24 methodological approaches in the field of species delimitation (Wiens, 2007; Carstens et al., 2013; Flot, 2015; Luo et al., 2018). The new approaches developed lately, provide a genealogy based, and statistically grounded test of alternative hypotheses of species delimitation (Knowles & Carstens, 2007; Wiens, 2007; Yang & Rannala, 2010; Carstens & Dewey, 2010; Carstens et al., 2013), and can be implemented using unilocus or multilocus datasets (Carstens et al., 2013; Fujisawa & Barraclough, 2013). Multilocus species delimitation approaches can be performed incorporating Multispecies Coalescent Models (MSC) (Degnan & Rosenberg, 2009; Fujita et al., 2012; Rannala, 2015; Xu & Yang, 2016), which provide a robust framework for identifying distinct evolutionary lineages and cryptic and/or allopatric species. This is because MSCs involves (i) population size parameters (i.e. Population sizes (θs) for extant species and common ancestors), (ii) parameters for divergence times (τ), (iii) coalescent models specifying the distribution of gene trees at different loci, and (iv) a priori assignment of evolutionary models per locus and individuals for defining species categories (Kingman, 1982; Kubatko, et al, 2009; Liu et al, 2009; Harrington & Near, 2012; Rannala, 2015). Thus, species delimitation coalescent methods use concordance of gene-trees across multiple loci as evidence for existence of multiple species and do not rely on reciprocal monophyly (Degnan & Rosenberg, 2009; Heled & Drummond, 2010; Fujita et al., 2012; Rannala, 2015). So, these approach make it possible to infer divergence dynamics between lineages and delimit nascent evolutionary lineages in order to identify
25 species and understand processes that promote speciation (Sites & Marshall, 2003; Wiens, 2007; Fujita et al., 2012; Carstens et al., 2013). However, these results should be taken as hypothesis with the aimed to validated them with other sources of data such as ecological, morphological, cytological among others. (Puillandre et al., 2012; Sukuman & Knowles, 2017; Luo et al., 2018).
In South America, the geographic history of Patagonia has included intermittent periods of glaciation such as i) the most extensive Andean Glaciation (1.1 Mya); ii) the Coldest Pleistocene Glaciation (0.7 Mya); iii) the Last Southern Patagonian Glaciation (0.18 Mya); and iv) the Last Glacial Maximum or Llanquihue Glaciation (0.020 – 0.014 Mya) (Paskoff, 1977; Rabassa & Clapperton, 1990; McCulloch et al., 2000; Hulton et al., 2002; Rabassa, 2011) This fact has led to complex geography on this area (Rabassa & Clapperton, 1990; Denton et al., 1999; Hulton et al., 2002; García, 2012), and has molded the distribution and influenced diversification and speciation events of Patagonian biodiversity (Zemlak et al., 2008; Cosacov, et al., 2010; González-Ittig et al., 2010). This allows researchers to assess and to test hypotheses about the evolutionary processes underlying the formation of different endemic taxa of southern Chile (e.g. Ruzzante et al., 2006; Nuñez et al., 2011; Blotto et al., 2013). Amphibian species are an excellent group for this kind of studies. They have restricted dispersal capabilities, which tend to promote
26 differentiation (Duellman, 1999; Prohl et al., 2010), and are highly sensitive to climatic changes owing to complex life histories, permeable skin, and exposed eggs (Fitzpatrick et al., 2009).
The genus Eupsophus is composed currently by 10 species of ground frogs distributed throughout temperate Nothofagus forests in Chile and Argentina (Frost, 2013). The systematics status of this genus has remained complex for decades (e.g. Formas, 1978; Formas et al., 1992; Veloso et al., 2005; Correa et al., 2006; Formas et al., 2008; Pyron & Wiens, 2011; Blotto et al., 2013; Correa et al., 2017; Suárez-Villota et al., 2018a). The genus Eupsophus is currently divided into two groups (Formas, 1991; Nuñez et al., 2012). First, the roseus group composed of E. migueli, E. insularis, E. roseus, E. calcaratus, E. contulmoensis, E. nahuelbutensis, E. altor, and E. septentrionalis (Suárez-Villota et al., 2018a). These species have 30 chromosomes, and the individuals of this group have body sizes of 34 - 42 mm (snout-vent distance). Second, the vertebralis group (taxonomic details in ANNEX III) composed of E. vertebralis (Grandison, 1961) and E. emiliopugini (Formas, 1989). Both of these species have 28 chromosomes, and the individuals of this group have body sizes of 50 - 59 mm (snout-vent distance). Regarding the conservation status, E. vertebralis is categorized as vulnerable, while E. emiliopugini as least concern (RCE, 2019).
27 On the one hand, antecedents that support the species E. vertebralis and E. emiliopugini as different entities are based on: i) different morphology of pair 13 in the karyotype of both species (Formas, 1991), and ii) differences in the advertisement call between them (Formas & Bireva, 1994). It should be noted that these cytogenetic and ethologic studies were made with few individuals and localities. On the other hand, there is uncertainty about the geographical limits between E. vertebralis and E. emiliopugini. Formas (1991) indicates that both species are allopatrically distributed in Southern Chile. Nevertheless in the recent literature (Blotto et al., 2013; Correa & Durán, 2019; IUCN, 2019), the distribution of these species has been listed as partially sympatric, despite until today sympatry have not been phylogenetically validated. This incongruence may be due to the fact that both species are very difficult to distinguish in the field owing to their morphological similarities.
Besides, previous systematic reviews for the vertebralis group are not concordant in their results (Pyron & Wiens, 2011 compared to Blotto et al., 2013 and Suárez-Villota et al., 2018a). Pyron & Wiens (2011) did not recover the monophyly of the roseus and the vertebralis groups, contrary to the results of Blotto et al. (2013) and Suárez-Villota et al. (2018a), in which it was recover the monophyly of both groups of Eupsophus. It should be pointed out that these works (Pyron & Wiens,
28 2011; Blotto et al., 2013; Suárez-Villota, et al 2018a) are the only studies that inferred the phylogeny of the vertebralis group, and have been carried out with very few variants (e.g. four individual for the verterbalis group by Blotto et al., 2013). Thus, these works do not represent the entire distribution area or polymorphism of the group. Hence, the evolutionary history within the vertebralis group remains poorly knows.
Here, we conducted a comprehensive molecular phylogeny of the vertebralis group; carried out with a broad dataset covering most of distribution range of E. vertebralis and E. emiliopugini. We use three mitochondrial markers and two nuclear ones to assess phylogenetic relationships hypothesis, species delimitation, and divergence time estimations. We discuss questions about the accuracy of the species delimitation methods here used, and how these methods should be applied to this group of study. Also, this work contributes with new information for the vertebralis group, and proposes some lineages of interest for conservation.
2. MATERIALS AND METHODS
2.1.- SAMPLES AND ETHIC STATEMENTS
29 Biological samples were obtained via collections carried out throughout the distribution area of Eupsophus vertebralis and E. emiliopugini. We followed the recommendations of the Bioethics and Biosecurity Committee of the Universidad Austral de Chile (UACh, Resolutions No. 236/2015 and 61/15), and Servicio Agrícola y Ganadero Chile (Permission No. 9244/2015). The Corporación Nacional Forestal Chile, allows to collect buccal swabs samples of Eupsophus species from wild protected areas (Permit No. 11/2016. -CPP/ MDM/jcr/ 29.02.2016). A total of 91 individual DNA samples (58 = E. vertebralis and 33 = E. emiliopugini) were obtained from the 19 localities shown in Figure 1 and in Supplementary Table 1. The individuals were determinated by an expert herpetologist in fieldwork, and then corroborated by the groupings recovered by the phylogenetic analyses.
2.2.- GENETIC DATA
Whole DNA was obtained from buccal swabs (Broquet et al., 2007) and from liver tissue using the Walsh et al. (1991) protocol for extraction with 5% Chelex. DNA concentration and integrity were measured by spectrophotometry using a NanoDrop 1000, and by electrophoresis with 1% agarose gels. Using Polymerase Chain Reaction (PCR), we amplified the partial sequences of three mitochondrial molecular markers: Cytochrome b (Cytb; Degnan & Moritz, 1992), Cytochrome oxidase
30 subunit I (coI; Folmer et al., 1994), and Control region (d-loop; Goebel et al., 1999); and two nuclear genes: Crystallin B (crybA1; Dolman & Phillips, 2004), and Proopiomelanocortin (pomc; Gamble et al., 2008). Each PCR mixture contained 100 ng of DNA, 0.5 mM of each respective primer, 0.5 mM of dNTPs, 0.75 mM of MgCl2, 2.5 µL of reaction buffer, 0.2 units of Taq DNA Polymerase (Taq Platinum, Cat. No. 10966, Invitrogen), and 19.5 µL of ultrapure water. Thus, the PCR reactions had a total volume of 25 µL. Amplification success was verified using electrophoresis with 1% agarose gels run in 1X TAE buffer. The sequencing process was conducted in Macrogen Inc. (South Korea). Electropherograms were observed and edited using the software Geneious v10.1.3 (Kearse et al., 2012).
2.3.- PHYLOGENETIC TREE RECONSTRUCTION
Previous to phylogenetic analyses, sequences were aligned with the same software using MAFFT algorithm (Katoh et al., 2005) under the iterative method of global pairwise alignment (G-INS-i). Default settings were maintained for all parameters involved. Best-fit partitioning schemes and models of nucleotide substitution were selected using the Bayesian information criterion (BIC) (Schwarz, 1978) implemented in Partition Finder v2.2.0 (Lanfear et al., 2017). Phylogenetic hypotheses were constructed under Maximum likelihood (ML) and Bayesian inference (BI), using the 91 sequences generated in this study for the
31 vertebralis group, plus eight sequences representing each species of the roseus group (taken from Suárez-Villota et al., 2018a), and one sequence of Alsodes valdiviensis as outgroup. As input, we use: firstly, the dataset with three mitochondrial markers concatenated (mitochondrial matrix; useful for phylogenetics comparations and necessary for unilocus species delimitation analyses); secondly, two nuclear markers concatenated (nuclear matrix; useful for phylogenetics comparations); thirdly, five molecular markers concatenated (concatenated matrix: necessary for multilocus species delimitation analyses); and finally each molecular marker separately (necessary for species delimitation methods, see below). All these matrices were tested independently.
Maximum likelihood analyses were performed using Garli v2.0 (Bazinet et al., 2014), and statistical support for the nodes was estimated by nonparametric bootstrapping (Felsenstein, 1985) with 200 pseudo replicates. Bootstrap support values of >70% were considered as statistically significant support for a clade present in the tree (Hillis & Bull, 1993). Bayesian inference (BI) analyses were performed using MrBayes v3.04 (Ronquist & Huelsenbeck, 2003). Four Markov Chain Monte Carlo (MCMC) were performed from a random start tree with 10 x 107 generations, and sampling every 1,000 generations. Maximum clade credibility trees were constructed with MrBayes after discarding the initial burn-in of 20%. Both analyses were performed four times to confirm the results. Posteriori probability values of >0.95 were
32 considered as statistically significant support for clades (Huelsenbeck & Rannala, 2004).
2.4.- SPECIES DELIMITATION
To determine species limits into the vertebralis group, we apply unilocus and multilocus approaches. The first inquest was unilocus species delimitation analyses. For this approaches we used two methods: 1) General Mixed Yule Coalescent model (GMYC; Fujisawa & Barraclough, 2013) and 2) Multirate Poisson Tree Processes (mPTP; Kapli et al., 2017). Here we considered as independent loci the mitochondrial matrix and each one of the nuclear genes (pomc, crybA1). Therefore, we performed independent analyses with those three datasets. GMYC consider a neutral coalescent model in joint with a Yule stochastic model for infer species boundaries. This method can be performed with single or multiple threshold models, using the maximum likelihood approach (Fujisawa & Barraclough, 2013). As inputs we used a dated tree for mitochondrial, for pomc and for crybA1 dataset (see section below for dating setup). The GMYC analyses were performing at web service (https://species.h- its.org/gmyc/) only with single-threshold model (sGMYC); because it has been described that single-threshold overcomes the multi-threshold (Fujisawa & Barraclough, 2013; Talavera,et al., 2013). The mPTP method determines the transition points between intra and interspecific branching events. Under the postulates, that the number of substitutions
33 between species is higher than that within species. Also, the algorithm implemented in mPTP incorporate the potential divergence in intraspecific level of divergence, making them a better algorithm than PTP (Kapli et al., 2017). For the mPTP analyses we used the tree obtained from MrBayes as input for the web service mPTP Exelixis laboratory (https://mptp.h-its.org/#/tree).
The second inquest was multilocus species delimitation analyses. We implemented four methods based on coalescence, these included 1) Species Tree Estimation using Maximum likelihood (STEM; Kubatko et al., 2009); 2) Bayesian Phylogenetics and Phylogeography program (BPP; Yang, 2015); 3) Multilocus species delimitation using trinomial distribution model (Tr2; Tomochika Fujisawa et al., 2016); and 4) Bayes factor delimitation (BFD; Grummer et al., 2014). For all analyses we used the concatenated matrix, excepting for the STEM, in which we used phylogenetic gene trees (see section above). Because all four analyses require assign individuals to species groups defined a priori, we assigned individuals to different delimitation alternatives considering from one to six species in different combinations. These alternative species delimitation scenarios were defined based on phylogenetic results and in similarities of its geographical distribution. The nine best-evaluated scenarios are described in Supplementary Table 2.
The STEM analyses, according to the ML criteria, allowed us to
34 obtain estimations of phylogenetic species trees starting from genes trees. Gene trees were obtained using Garli v2.0, and polytomies were resolved using internode branch lengths of 1.0 x 10-8 in Mesquite v2.75 (Maddison & Maddison, 2011). Because population size (θ = 4Neμ) is a value set by the user in the STEM, we calculated it following recommendations from Harrington and Near (2012). ML punctuations for each species tree were assessed using STEM v2.0 (Kubatko et al., 2009), and these punctuations were evaluated using the theoretical approach described by Carstens and Dewey (2010). The BPP analysis allows the user to compare different MSC models with or without a fixed start tree (Rannala & Yang, 2003; Yang & Rannala, 2010). We used A11 model (without fixed tree) performed with the Bayesian Phylogenetics and Phylogeography software v.2.2 (Yang, 2015). Here it is requiring as prior the population size (θ) and species divergence time (τ). We set these according to Yang (2015). As a guide tree, we used the topology of the tree obtained previously from the IB phylogenetic analyses. The MCMC analyses begin with different starts points however each was run with 20,000 generations and 10,000 as the burn-in. Each analysis was running four times to confirm consistency among results. The Tr2 analysis delimits species groups from unlinked multilocus gene trees by measuring congruencies and incongruences between the species tree topology. This analysis was performed following Fujisawa et al. (2016); the population size (θ) calculated as before, and the rest of parameters were set to defaults. The BFD analysis is a model selection tool, in which it is possible to test different hypothesis. The
35 hypotheses are compared through the calculation of Bayes factor from Marginal likelihood estimations (MLE) (Kass & Raftery, 1995). In order to assess species delimitation analysis, each individual is assigned to a putative species before run the analysis. Thus, runs with different assignments to putative species correspond to different test hypotheses. The running conditions are described in the section below.
Finally, to assess which methods yielded the most congruent results, we calculated the taxonomic index of congruence (Ctax) between pairs of species delimitation analyses (Miralles & Vences, 2013). Ctax is calculated between two species delimitation analyses as the ratio of the total number of speciation events, congruently supported by both methods, to the total number of pairwise species boundaries cumulatively supported by the two methods (Miralles & Vences, 2013).
2.5.- TIME OF DIVERSIFICATION
To estimate the divergence time between lineages, we employed the concatenated matrix. We used the software BEAST v.2.4 (Bouckaert et al., 2014) to run the analysis. With this program, first we calculated differences in MLE scores for different molecular clock models and tree prior settings (Drummond & Rambaut, 2007; Li & Drummond, 2012;). Then these MLE scores were compared calculating pairwise Bayes Factor (BF) values (See Supplementary Table 3; Leaché et al., 2014). Thus,
36 according to MLE/BF scores, the conditions implemented for calculated the divergence times and BFD were Lognormal Clock model and Birth Death Tree prior. Also, we use the nucleotide substitution models previously calculated with Partition Finder, and the nucleotide substitution rates reported by Irisarri et al. (2012) and Suárez-Villota et al. (2018a) (Supplementary Table 4, and Supplementary Table 5, respectively). MCMC in BEAST were running for 4 x 107 generations, sampling every 1000 generations and discarding the samples trees prior to the plateau phase as burn-in (20%) using the software Tracer v.1.7 (Rambaut et al., 2018) The maximum credibility tree was obtained using the software Tree annotator v.1.8.3 (Rambaut & Drummond, 2016) with all default configuration options. Additional BEAST analyses were carried out with the same conditions using the following unilocus datasets: i) mitochondrial matrix, ii) pomc and iii) crybA1. The resulting dated trees were used as inputs in the GMYC analysis (see above).
3. RESULTS
3.1.- PHYLOGENETIC PATTERNS INTO THE vertebralis GROUP
For a total of 100 samples (91 samples for the vertebralis group, plus 9 samples for the outgroup), we aligned the five DNA markers obtaining a total of 2,677 nucleotide sites and 572 segregating sites. Three of these markers corresponded to mitochondrial dataset, with a total of 1,934
37 nucleotide sites and 516 segregating sites (Supplementary Table 4). Partitioning strategy and evolutionary models obtained in Partition Finder are indicated in Supplementary Table 4.
Overall, the phylogenetic analyses (ML, IB) recovered the monophyly of the vertebralis group with the nuclear, mitochondrial (Supplementary Figure 1), and concatenated matrices (Bootstrap: 100, Posterior Probability PP: 1; Figure 2). Nevertheless, the analyses did not recover the reciprocal monophyly of the E. vertebralis and E. emiliopugini. We found incongruence in tree topology of phylogenetic reconstruction between matrices (nuclear, mitochondrial, and concatenated matrix), and between ML and IB analyses. For the concatenated matrix in IB analysis we found E. vertebralis as monophyletic group with low support (PP: 0.75), and E. emiliopugini as polyphyletic group. On the contrary, for the concatenated matrix in ML analyses, the best tree recovered E. emiliopugini as monophyletic group and E. vertebralis as polyphyletic one; nonetheless the consensus support (Bootstrap) did not recover this topology (Bootstrap < 0.50; Figure 2).
Further, we found E. emiliopugini variants from Cordillera del Sarao (thereafter named as E. emiliopugini Sarao lineage) and variants from Pucatrihue (thereafter named as E. emiliopugini Pucatrihue lineage) as
38 monophyletic groups; considering their locality of bellowing in ML and IB analyses using concatenated matrix (Sarao lineage Clade SA: Bootstrap: 72.2, PP: 0.55; Pucatrihue lineage Clade PU: Bootstrap 72, PP: 1; see Figure 2). Besides, four out of five variants from Lago Huillinco were recovered together in the same group with high support (Clade HI: Bootstrap: 97.7, PP: 0.99; Figure 2) been Lago Huillinco group paraphyletic. Within E. vertebralis we found two groups with high support (Clade ME: Bootstrap: 84, PP: 0.98; Clade LP: Bootstrap: 90, PP: 1; Figure 2), but these groups are also paraphyletic if we consider the locality of origin of its individuals.
Otherwise, mitonuclear discordance was recovered in phylogenetic ML and IB analyses. For nuclear matrix, both species were recovered as polyphyletic with low support between their relationships (Bootstrap: <50; PP: <0.95; Supplementary Figure 1). One interesting observation here is that the variants of E. emiliopugini from the locality of Los Mañios were recovered in the same group with the variants of E. vertebralis from Lago Pellaifa (Bootstrap: <50, PP: 0.9; Supplementary Figure 1). The same grouping was not recovered in the trees performed with the mitochondrial matrix in ML and IB analyses. Here it was found E. vertebralis as monophyletic group (Bootstrap: 92, PP: 0.95) and E. emiliopugini as polyphyletic group (Supplementary Figure 1).
39 3.2.- SPECIES DELIMITATION ANALYSES
The results of the unilocus (sGMYC and mPTP) and the multilocus analyses (STEM, BP&P, Tr2, and BFD) were not congruent. Depending on the analysis, we found from one to eleven species within the vertebralis group. In particular on the unilocus analyses, sGMYC using the mitochondrial matrix (sGMYC(m)) recovered eleven species (confidence interval 9-12): six for E. vertebralis and five for E. emiliopugini (Figure 3). While, using the pomc and crybA1 matrix recovered just one species (confidence interval: 1-3 and 1-9, respectively). For mPTP one species was recovered independently of the dataset used (Figure 3).
Among multilocus analyses we found from one to six species. BPP analysis was the most conservative recovering only one species, merging Eupsophus vertebralis with E. emiliopugini (Supplementary Table 2). STEM analysis recovered four species, corresponding to: E. vertebralis, E. emiliopugini, E. emiliopugini Sarao lineage (Clade SA) and E. emiliopugini Pucatrihue lineage (Clade PU) (Supplementary Table 6). BFD analysis recovered tree species corresponding to: E. vertebralis, E. emiliopugini, and E. emiliopugini Sarao lineage. Nevertheless, this scenario has not statistically significant more probable than the others four possible scenarios, which recovered four and six species (BF < 20, see
40 Supplementary Table 3). Finally, Tr2 analysis recovered six species, two for E. vertebralis and four for E. emiliopguni. (Supplementary Table 3;
Figure 3). According to Ctax, the most congruent species delimitation result was one species (Ctax: 0.59), obtained by BPP and unilocus analyses except by sGMYC(m). In contrast the most incongruent species delimitation result was found for sGMYC(m) (Ctax: 0.18; Table 1).
3.3.- DIVERSIFICATION TIME
The estimates of divergence times performed in BEAST had 221 ESSs value for posterior statistic and Likelihood mean = -11731.0842. The divergence (stem age) of the vertebralis group from the roseus group dates back to the Pleistocene at 0.81583 Mya (0.53913 - 1.12891 Mya, 95% Highest Posterior Density interval, HPD). Within the vertebralis group, E. vertebralis and E. emiliopugini lineage diverged at 0.04092 Mya (0.02705 -0.0503 Mya HPD). The crown age for E. vertebralis lineage start at [0.01918 (0.01259 – 0.02792 HPD)] and the crown age of E. emiliopugini lineage begin at [0.02786 (0.01819 – 0.04073 HPD)]. Also, the crown age of E. emilipugini Pucatrihue lineage dated at 0.01508 Mya (0.00624 - 0.02604 Mya HPD; Figure 3) follow by Sarao lineage was dated at 0.01015 Mya (0.00326 - 0.01699 Mya HPD)
41 4.- DISCUSSION
4.1.- PHYLOGENETIC RELATIONSHIPS AND DIVERGENCE TIMES IN THE vertebralis GROUP
Here, we present a phylogenetic reconstruction of the vertebralis group, using a wide dataset considering most of its distribution range. In agreement with previous analyses (Blotto et al., 2013; Suárez-Villota et al., 2018a), all the phylogenetic analyses of this study, recovered the vertebralis group as monophyletic (Bootstrap: 100, PP: 1; Figure 2). Within the vertebralis group, topologic discordances among phylogenetic trees constructed with different datasets (nuclear, mitochondrial and concatenated matrix) and methods (ML and IB) were detected. All the trees recovered in this study indicated a predominant polyphyletic pattern with short branches within the variants of the vertebalis group (Figure 2; Supplementary Figure 1). Consequently, these results suggest low genetic differentiation among the vertebalis group (Knowles & Carstens, 2007), probably as consequence of their recent differentiation (Patton & Smith, 1994; Hudson & Coyne, 2002; Fujita et al., 2012).
This assumption finds support in the literature. First, low genetic differentiation within the vertebralis group was reported by Suárez- Villota et al. (2018b), who constructed phylogenies using the
42 mitochondrial genomes of E. vertebralis and E. emiliopugini (both genomes share 94.5% identity with 879 variable sites). Second, it has been described that reciprocal monophyly is more difficult to recover when a multilocus dataset is used, due to single nuclear locus take more time in becoming reciprocal monophyletic than single mitochondrial locus (Hudson & Coyne, 2002; Knowles & Carstens, 2007).
The nuclear tree recovered a paraphyletic pattern, where variants of E. emiliopugini from Los Mañios, and variants of E. vertebralis from Lago Pellaifa were recovered in a monophyletic group, which is not present in the mitochondrial tree (Supplementary Figure 1). There are several explanations for mitonuclear discordances like introgressive hybridization (Anderson, 1953; Petit & Excoffier, 2009), incomplete lineage sorting (Pollard et al., 2006; Syring et al., 2007; Degnan & Rosenberg, 2009), selection or distinct mutation rates, demographic asymmetries, or human actions that facilitate secondary contact (Toews & Brelsford, 2012).
The evolutionary history of the genus Eupsophus and others endemic amphibians of Patagonia, is firmly related to the late Pleistocene glaciation (Nuñez et al., 2011; Blotto et al., 2013). That is also inferable from the divergence times results found in this research for the vertebralis group. Despite that the literature reveals several methodological and sampling biases that can negatively influence the
43 inferences of divergence times (Arbogast et al., 2002; Rannala & Yang, 2003; Hoareau, 2016), we perform this analysis as a first insight into the evolutionary history of the vertebralis group. The stem age founded by Suárez-Villota et al. (2018a) between the vertebralis group and the E. roseus group [0.79 Mya (0.68– 0.90)], is in agreement with the range of stem age found here [0.81 Mya (0.53 - 1.12)]. These ranges are coincident with the transition period between the Andean Glaciation (1.1 Mya) and the Coldest Pleitocene Glaciation (0.70Mya). In addition, the stem age between E. emiliopugini lineage and E. vertebralis lineage was dated at 0.040 Mya, been earlier than the stem age reported by Suárez-Villota et al., 2018a [0.023(0.014-0.032) Mya]. This result would indicate that the divergence of the vertebralis group were mostly influenced by the interglacial period before the Last Glacial Maximum (0.020 Mya; McCulloch et al., 2000; Hulton et al., 2002).
Later, the crown age of E. emiliopugini lineage (south distribution; 0.027 Mya) is suggested that occurred earlier than the crown age of E. vertebralis lineage (with north distribution; 0.019 Mya), dated from the start of LGM period. Within E. emiliopugini lineages, the crown age of Pucatrihue lineage was the earliest dated at 0.015 Mya, followed by Sarao lineage at 0.010 Mya. Therefore, Cordillera del Sarao and Pucatrihue localities were colonized after the LGM, despite this was a non-glaciated area during the LMG (McChulloch et al., 2000). The deglaciation period of LGM started at 0,017 Mya, from north to south direction, reaching the ice
44 its current distribution at 0.014 Mya (Hulton, et al., 2002; Mendelova, et al 2017). Hence, the lineage divergence and posterior diversification within E. vertebralis and E. emiliopugini could be have influenced by the ice retreat.
Thus, the results of this study suggest that the diversification within the vertebralis group was influenced by the LGM, mainly by the deglaciation and post glaciation periods (from 0.017 Mya to present). In fact, the interglacial periods are characterized by the presence of Valdivian rainforest and Patagonian forest (Astorga & Pino, 2011) which are habitat associated to Eupsophus species (Nuñez, 2003). Probably, during glaciation and deglaciation periods these habitats suffered contractions and expansions, influencing the pattern of distribution of the species that lived there. Leon-Paniagua & Guevara (2019) proposed that the poorly differentiation between closely related lineage, is an evolutionary consequence of the influence that the Quaternary climate together with complex topography changes had on the distributional pattern. This hypothesis can be applying to the results of this study, whereat the vertebralis group show signals of low genetic differentiation in joint with times of divergences influenced by the glacial cycles of the late Pleistocene. Other examples of the influence of the climatic oscillations during the Pleistocene in Patagonia biodiversity, are those reported by Ruzzante et al. (2006) Lessa et al. (2010); Vera-Escalona et al. (2019).
45
4.2.- SPECIES DELIMITATION ANALYSES OF THE vertebralis GROUP
According to unilocus analyses (sGMYC, mPTP), a single species within the vertebralis group was inferred, merging Eupsophus vertebralis with E. emiliopugini (Table 1, Figure 3); except for sGMYC(m), which recovered 11 candidate species. It seems that these results were biased by the presence or absences of clades on the input phylogenetic tree used (i.e. mitochondrial phylogenetic tree and nuclear pomc and cryb phylogenetic trees). In fact, it is described that these unilocus analyses can be biased by reciprocal monophyly (Fujisawa & Barraclough, 2013; Kapli et al., 2017). Particularly, GMYC methods consider that distinct genetic clusters delimited as different species are separated by longer internal branches, and present reciprocal monophyly (Fujisawa & Barraclough, 2013; Zhang et al., 2013). This bias can explain why with different loci, sGMYC analyses yield to two different results. In contrast to sGMYC, mPTP assume that each found substitution has a small probability of generating a divergence event. Therefore, branching events are expected to be more frequent within species than between species (Kapli et al., 2017). This may be the explanation of why the results obtained by mPTP are more conservative than results obtained by sGMYC with the same mitochondrial input tree.
Regarding to multilocus analyses, we obtained different results in
46 all the methods used (Tr2, STEM, BFD, BPP). Here we recovered from one (BPP) to six (Tr2) species (Supplementary Table 2, 3 and 6; Figure 3). It has been suggested that the results inconsistency among multilocus species delimitation analyses is due to different parameters, theoretical models, and statistical powers among approaches (Carstens et al., 2013; Jacobs et al., 2018). This type of incongruence have been widely reported in the literature both with empirical and simulated data (Harrington & Near, 2012; Jacobs et al., 2018; Mutanen et al., 2016; Stokkan et al., 2018). This makes sense for the findings here exposed because it was applied different kinds of approaches considering Likelihood and Bayesian approximations, each one with different inputs, priors and models. For example, STEM use gen trees as input, while BPP use the sequence alignment; also STEM and BPP require theta priors, not so BFD. Further, these methods based on MSC model, considered speciation as a punctual act, without taking account that speciation can be a continuous process (Freudenstein et al., 2016; Sukumaran & Knowles, 2017), such as Birth- Dead model used in BFD. Thus, species delimitation under MSC model are not capable to distinguish between the limits among genetic structure of population and structure of species (Sukumaran & Knowles, 2017). At this point, it seems that researchers are under the bias of what we are considering as species, versus others operational taxonomic units that can be identified by MSC delimitation methods (Luo et al., 2018).
The results above present high rates of incongruence, which is
47 evidenced by the low value of Ctax (0.59) reached by the most congruent scenarios. Therefore, in this case, this index would be accounting for the existing incongruence, beyond finding the most plausible scenario. Thus, despite we follow some of the advices of Carstens (2013), we “failed at species delimitation” for the vertebralis group, and we highlight that for this case of study, the evolutionary history and biology of the species goes far beyond the statistical models used (Rannala, 2015). Consequently, the genetic approaches used in this study should be taken as a hypothesis and complemented with evidence from other sources (Padial et al., 2010; Puillandre et al., 2012; Pante et al., 2015; Sukumaran & Knowles, 2017; Rojas et al., 2018). Hence, I refrain to merge E. vertebralis with E. emiliopugini and to make a new taxonomic proposal.
Moreover, other antecedents that distinguish both species separately are: firstly, the cytogenetic evidence, that shown differences between both species on the chromosomes morphology in pair 13, been telocentric for E. vertebralis, and metacentric for E. emiliopugini (Formas, 1991); secondly differences in advertisement calls, founded four to six notes per call for E. vertebralis and two notes per call for E. emiliopugini (Formas & Brieva, 1994). Nevertheless, these results have to be taken carefully, because they were performed with very few individuals and localities. Despite that advertisement calls (Passmore, 1981) and karyotypic (Barth, 2014) features are characters useful to distinguish between amphibian species, have been reported some cases with
48 intraspecific variation in ethological (i.e. advertisement calls; Rodriguez et al., 2010; Turin et al., 2018) and cytogenetic features (Cuevas, 2008). In fact, the measures of the advertisement calls for E. vertebralis, differ between the research of Formas & Vera (1980) and Penna & Veloso (1990). Therefore, extended cytogenetic and ethologic studies will be very useful to clarify the evidence that currently distinguishes between E. vertebralis and E. emiliopugini.
Finally, it calls the attention that within E. emiliopugini some groups were recovered as putative species by more than one analysis. That was the case of Sarao lineage (recovered by Tr2, STEM, BFD, sGMYC(m)), Pucatrihue lineage (recovered by Tr2, STEM, sGMYC(m)) and Lago Huillinco lineage (recovered by Tr2, sGMYC(m)) (Supplementary Table 1, 2 & 6; Figure 3). Also, these three groups diverged earlier than other lineages within E. emiliopugini (Figure 3) and come from currently isolated location. These results lead us to infer that they represent differentiated evolving lineages (Freudenstein et al., 2016; Sukumaran & Knowles, 2017; Luo et al., 2018). Therefore, conservation on these lineages should be considered by public policies, especially because the great intervention and degradation that these localities have today.
4.3.- FIELDWORKS REMARKS
It is necessary to state that the gray marks in the sampling localities
49 map (See Figure 1), are due to the fact that despite the intensive efforts over time, no specimens were found in those places, historically inhabited by E. vertebralis and E. emiliopugini. This fact confines the species in question within the indicated localities, and not to the wide distribution reported by IUCN. So, the preservation of the current areas of distribution has a higher value to conserve the important lineages and the species that compound the vertebralis group. Mainly we suggest taking into consideration those areas with increasingly impoverished native forests of Coast range and Andes Mountains.
5. CONCLUSION
In this research, the species delimitation analyses failed in recovered to E. emiliopugini and E. vertebralis as two species, probably due to they are recently diverged and poorly genetically differentiated lineages. Furthermore, the findings make explicit that species delimitation results have to be taken as a hypothesis; and then, have to be discussed at the light of other kinds of evidence to make an accurate proposal according to the biological features of the studied model. The incongruences found among species delimitations analyses deprive of describing only one species for the vertebralis group. Therefore, considering previous the cytogenetic and the ethologic evidence, the current taxonomic status for E. vertebralis and E. emiliopugini as two independent species it is maintained.
50
ACKNOWLEDGMENTS
I thank to Engr. Nicolás González for his field assistance. Founded by Fondecyt 3160328 to EYS-V, and CONICYT grants 22180766 to CAQ.
51 TABLES
Table 1: Taxonomic index of congruence (Ctax) among species delimitation analyses. Descriptive statistics for each pair of species delimitation approaches (Ctax) are indicated. The analyses single-threshold General Mixed Yule Coalescent model (sGMYC) for mitochondrial (m) and nuclear markers (n), multi-rate Poisson Tree Processes (mPTP) for mitochondrial (m) and nuclear markers (n), Tree Estimation using Maximum likelihood (STEM), Bayesian Species Delimitation (BPP), Multilocus Species Delimitation using a Trinomial Distribution Model
(Tr2), and Bayes factor delimitation (BFD) are shown. Also Mean Ctax and number of species (sp.) recovered for each approached is specified.
Ctax sGMYC sGMYC mPTP mPTP Mean STEM BPP Tr2 BFD sp. (m) (n) (m) (n) Ctax
sGMYC(m) 0.18 11 sGMYC(n) 0.1 - 0.59* 1 mPTP(m) 0.1 1 - 0.59* 1 mPTP(n) 0.1 1 1 - 0.59* 1 STEM 0.3 0.33 0.33 0.33 - 0.40 4 BP&P 0.1 1 1 1 0.33 - 0.59 1 Tr2 0.4 0.20 0.20 0.0 0.6 0.20 - 0.36 6 BFD 0.2 0.50 0.50 0.50 0.6 0.50 0.4 - 0.46 3 * Most congruent analyses
52 FIGURES
Figure 1: Maps of sampling locations for the vertebralis group. Circles in the map represent the prospected localities for this study. Orange circles (1-11) represent localities of Eupsophus vertebralis, and blue circles (12-19) localities of Eupsophus emiliopugini. Gray circles indicated prospected areas without findings of individuals of the vertebralis group. The name of the localities and the number of individuals collected per localities are detailed in the Supplementary Table 1.
53
54 Figure 2: Phylogenetic relationships among the vertebralis group. The tree corresponds to the result of Bayesian analyses, reconstructed using concatenated nuclear and mitochondrial data set. Numbers in the tree represent bootstrap scores of Maximum likelihood analyses and Bayesian posterior probabilities, respectively. The tips correspond to the species abbreviation (EE: E. emiliopugini, EV: E. vertebralis), follow by abbreviation of the sampling localities listed in Supplementary Table 1. As outgroup was used: E. calcaratus, E. roseus. E. insularis, E. migueli, E. altor, E. septentrionalis, E. nahuelbutensis, E. contulmoensis and Alsodes valdivienses.
55
56 Figure 3: Phylogenetic dated tree by BEAST and results of species delimitation analyses for vertebralis group. Numbers on the branches indicated the mean divergence times in million years, and 95% credible intervals are indicated in parenthesis. The tips correspond to the species and sampling localities abbreviation (EE: Eupsophus emiliopugini, EV: E. vertebralis, and the localities are listed in Supplementary Table 1). Bars on the right side of the images indicate the species limits recovered by multilocus coalescent analyses Tr2, STEM, BFD, BPP, and unilocus coalescent analyses with mitochondrial (sGMYC(m), mPTP) and nuclear (sGMYC(n), mPTP) data sets. Limits within Eupsophus vertebralis, and E. emiliopugini are indicated with different colors. Note: In sGMYC(m), the putative specie recovered for Lago Huillinco (LH) include five individuals.
57 SUPPLEMENTARY TABLES
Supplementary Table 1: Sampling locations of Eupsophus vertebralis (EV) and E. emiliopugini (EE). Number map (Nº Map) is corresponding with the numbers in Figure 1. Sample size (N), coordinates, species name according to Frost 2018, and Tips corresponding with the phylogenetics trees (Figure 2 and Supplementary Figure 1) are also showed.
Nº Sampling N Latitude Longitude Species Tips Map localities 1 Tolhuaca 1 -38.206666 -71.820291 E. vertebralis EVTO 2 Lago Pellaifa 7 -39.610365 -71.985451 E. vertebralis EVLP 3 Colihual Alto 1 -39.411666 -73.111975 E. vertebralis EVCA 4 Mehuín 3 -39.434674 -73.202896 E. vertebralis EVME 5 ChanChan 1 -39.58 -73.223763 E. vertebralis EVCC 6 Bonifacio 1 -39.705046 -73.370553 E. vertebralis EVBO 6 Las Huellas 2 -39.708055 -73.388333 E. vertebralis EVLH 6 Oncol 1 -39.698333 -73.327222 E. vertebralis EVON 6 Pilolcua 2 -39.694605 -73.341959 E. vertebralis EVPL 6 Punucapa 1 -39.767222 -73.261666 E. vertebralis EVPU 7 Llancahue 7 -39.839166 -73.13 E. vertebralis EVLL 8 Reumén 9 -39.951944 -72.903333 E. vertebralis EVRE 9 Pupunahue 4 -39.799964 -72.907103 E. vertebralis EVPP 10 Chamil 3 -40.010697 -73.107963 E. vertebralis EVCH 11 Cordillera 5 -40.103333 -73.454444 E. vertebralis EVCP
58 Pelada 12 Los mañios 2 -40.332556 -72.32542 E. emiliopugini EELM 13 Pucatrihue 3 -40.57641 -73.703042 E. emiliopugini EEPU 14 Antillanca 1 -40.793455 -72.314823 E. emiliopugini EEAN 14 Puyehue 1 -40.744483 -72.273087 E. emiliopugini EEPY Cordillera 15 9 -41.164166 -73.727222 E. emiliopugini EESA del Sarao P. Alerce 16 6 -41.592928 -72.59005 E. emiliopugini EEPA Andino 17 Huinay 2 -42.353739 -72.434066 E. emiliopugini EEHU 18 Chaitén 4 -42.905122 -72.716669 E. emiliopugini EECT 19 Huillinco 5 -42.67316 -74.008008 E. emiliopugini EEHI
59
Supplementary Table 2: Species delimitation scenarios and results obtained for Tr2 and BPP. Species delimitation scenarios are specified in the second column using tips name (See Supplementary Table 1 for abbreviations). S = number of delimitation scenario. Sp. Number of species delimited by each scenario. For Tr2 lowest score indicates the best delimited scenario, and for BPP higher Posterior probability indicates the best delimited scenario.
S Species delimitation scenario sp. Tr2 BPP 1 EEEV (E. emiliopugini joint to E. verterbralis) 1 298032,34 0.8245* 2 EE/EV 2 75054,4 0.78142 3 EE/EESA/EV 3 71926,87 0.69973 4 EE/EESA/EEHI/EV 4 69462,29 0.79285 5 EE/EESA/EEHI/EEPU/EV 5 68094,92 0.37908 6 EEPU/EE/EESA/EV 4 70465,35 0.41745 7 EEPU/EE/EV 3 74716,54 0.78878 8 EE/EEHI/EV 3 72829,99 0.79953 9 EE/EESA/EEPU/EEHI/EV/EVLP 6 66097,19* 0.38559 * Indicates the best estimated species delimitation scenario for each analysis.
60 Supplementary Table 3: Bayes factor estimates for Molecular Clock/Tree priors and Species delimitation scenarios. Marginal likelihood (MLE) and Bayes Factor values (BF) for Molecular clock/Tree priors and Species delimitation scenarios (S) are shown. Values using Path sampling (PS), and Stepping Stone sampling (SS) are indicated. Species delimitation scenarios are described in Supplementary Table 2, species number (sp) for each delimitation scenario is indicated.
Molecular Clock/Tree prior PS SS BF PS BF SS scenario STRICK/BIRTH -11.276 -11.276 341.3474279 387.1860432 STRICK/YULE -11.505 -11.505 800.6930348 846.7807238 RANDOM/BIRTH -11.252 -11.251 293.0364993 338.6574516 RANDOM/YULE -11.264 -11.258 317.2898399 351.3128687 LOGNormal/Birth* -11.105 -11.082 0 0 LOGNormal/Yule -11.239 -11.239 268.4807826 314.8674587
Species delimitation scenarios S sp. PS SS BF PS BF SS 3* 3 -11904,15578 -11892,20073 0 0 9 6 -11906,77689 -11907,46397 2,621106906 15,2632397 7 3 -11918,7694 -11913,49195 14,61361763 21,29121541 8 4 -11915,95667 -11915,19924 11,80088705 22,99850648 4 4 -11914,47574 -11915,87479 10,31995565 23,67406153 6 4 -11924,2355 -11916,67967 20,07972478 24,47894029 5 5 -11943,10741 -11936,14502 38,95163493 43,94428679 2 2 -11948,19604 -11949,49938 44,04026434 57,29865226 1 1 -12111,34003 -12088,94693 207,1842548 196,7462021 BF > 20 indicates statistically significant differences between scenarios * Indicates the best estimated species delimitation scenario.
61 Supplementary Table 4: Total nucleotide sites, partition schemes and nucleotide substitution models for DNA sequences used in this study. Total nucleotide sites for each sequence, and their partition schemes with the corresponding evolutionary models of nucleotide substitution, calculated in Partition Finder.
Sequence Total sites Codon Partition Best Model d-loop 542 d-loop HKY+G cytb 756 Pos1 y 3: cytb K80+I+G Pos2: cytb GTR+G coI 639 Pos1: coI GTR+G Pos2: coI K80+I Pos3: coI F81 crybA1 225 crybA1 HKY pomc 523 Pos1 y 3: pomc F81+G Pos2: pomc K80+I+G
62 Supplementary Table 5: Nucleotide substitution rates used for DNA sequences of vertebralis group in this study. The rates are presented correspondingly for each molecular marker (M.M.).
Nucleotide M.M. Reference Group substitution rates Suárez-Villota et d-loop 0.166 Eupsophus al., 2018a Suárez-Villota et cytb 0.2729 Eupsophus al., 2018a Irrisarri et al., coI 0.291037 Neobatrachya 2012 pomc 0.374114 Irisarri et al., 2012 Neobatrachya Suárez-Villota et crybA1 0.1129 Eupsophus al., 2018a
63 Supplementary Table 6: Likelihood scores and Akaike’s information criterion (AIC) results for STEM analysis. Description of delimitation scenarios (S), Species number (sp.), Log-likelihood of the species tree (−lnL), number of parameters (k), AIC difference (Δi), relative likelihood of model given the data (L), and the model probabilities (wi) are indicated. See Carstens and Dewey 2010.
L(Model S. sp. -lnL k *k 2*-lnL AIC wi Data) 6* 4 46635,39 5 0 93270,78 93280,78 1 1 3 3 47171,07 4 8 94342,14 94350,14 6,173E-233 6,173E-233 7 3 47181,89 4 8 94363,79 94371,79 1,226E-237 1,226E-237 1 1 53898,32 2 4 107796,64 107800,64 0 0 2 2 47669,68 3 6 95339,37 95345,37 0 0 4 4 49668,69 5 10 99337,38 99347,38 0 0 5 4 48534,33 5 10 97068,66 97078,66 0 0 8 3 49883,24 4 8 99766,49 99774,49 0 0 9 6 48613,76 7 14 97227,53 97241,53 0 0 * Indicates the best estimated species delimitation scenario
64 SUPPLEMENTARY FIGURE
65 Supplementary Figure 1: Phylogenetic relationships among species of the vertebralis group. A. Best phylogenetic tree from Bayesian inference analyses, carried out with nuclear data set. B. Best phylogenetic tree from Bayesian inference analyses, carried out with mitochondrial data set. Bayesian posterior probabilities and bootstrap support from Maximum likelihood analyses are shown to the left of each tree.
66 6. REFERENCES
Agapow, P. M., Bininda-Emonds, O. R. P., Crandall, K. A., Gittleman, J. L., Mace, G. M., Marshall, J. C. & Purvis, A. (2004). The impact of species concept on biodiversity studies. Quarterly Review of Biology, 79, 161– 179. Anderson, E. (1953). Introgressive hybridization. Biological Reviews, 28, 280–307. Arbogast, B. S., Edwards, S. V., Wakeley, J., Beerli, P. & Slowinski, J. B. (2002). Estimating divergence times from molecular data on phylogenetic and population genetic timescales. Annual Review of Ecology and Systematics, 33, 707–740. Astorga, G. & Pino, M. (2011). Fossil leaves from the last interglacial in Central-Southern Chile: Inferences regarding the vegetation and paleoclimate. Geologica Acta, 9, 45–54. Barth, A., Vences, M., Solé, M. & Costa, M. A. (2014). Molecular cytogenetics and phylogenetic analysis of Brazilian leaf frog species of the genera Phyllomedusa and Phasmahyla (Hylidae: Phyllomedusinae). Canadian Journal of Zoology, 92, 795–802. Bazinet, A. L., Zwickl, D. J. & Cummings, M. P. (2014). A gateway for phylogenetic analysis powered by grid computing featuring GARLI 2.0. Systematic Biology, 63, 812–818. Blotto, B. L., Nuñez, J. J., Basso, N. G., Úbeda, C. A., Wheeler, W. C. & Faivovich, J. (2013). Phylogenetic relationships of a Patagonian frog
67 radiation, the Alsodes+Eupsophus clade (Anura: Alsodidae), with comments on the supposed paraphyly of Eupsophus. Cladistics, 29, 113–131. Bouckaert, R., Heled, J., Kühnert, D., Vaughan, T., Wu, C.-H., Xie, D., … Drummond, A. J. (2014). BEAST 2: A software platform for Bayesian evolutionary analysis. PLOS Computational Biology, 10, e1003537. Broquet, T., Berset-Braendli, L., Emaresi, G. & Fumagalli, L. (2007). Buccal swabs allow efficient and reliable microsatellite genotyping in amphibians. Conservation Genetics, 8, 509–511. Carstens, B. C. & Dewey, T. A. (2010). Species delimitation using a combined coalescent and information-theoretic approach: an example from north American Myotis bats. Systematic Biology, 59, 1– 15. Carstens, B. C., Pelletier, T. A., Reid, N. M. & Satler, J. D. (2013). How to fail at species delimitation. Molecular Ecology, 22, 4369–4383. Correa, C. & Durán, F. (2019) Taxonomy, systematics, and geographic distribution of ground frogs (Alsodidae, Eupsophus): A comprehensive synthesis of the last six decades of research. Zookeys, 863, 107–152. Correa, C., Vásquez, D., Castro-Carrasco, C., Zúñiga-Reinoso, Á., Ortiz, J. C. & Palma, R. E. (2017). Species delimitation in frogs from South American temperate forests: the case of Eupsophus, a taxonomically complex genus with high phenotypic variation. PLOS ONE, 12, e0181026.
68 Correa, C., Veloso, A., Iturra, P. & Mendez, A. M. (2006). Phylogenetic relationships of Chilean leptodactylids: a molecular approach based on mitochondrial genes 12S and 16S. Revista Chilena de Historia Natural, 79, 435–450. Cosacov, A., Sérsic, A. N., Sosa, V., Johnson, L. A. & Cocucci, A. A. (2010). Multiple periglacial refugia in the Patagonian steppe and post-glacial colonization of the Andes: The phylogeography of Calceolaria polyrhiza. Journal of Biogeography, 37, 1463–1477. Cuevas, C. C. (2008). Intraspecific chromosomic divergence in Batrachyla antartandica (Ceratophryidae) from Southern Chile: further evidences of cytogenetics diversity in frogs. International Journal of Cytology, Cytosystematics and Cytogenetics, 6, 10–18. de Queiroz, K. (2005). A unified concept of species and its consequences for the future of taxonomy. Proceedings of the California Academy of Sciences, 56, 196–215. de Queiroz, K. (2007). Species concepts and species delimitation. Systematic Biology, 56, 879–886. Degnan, J. H. & Rosenberg, N. A. (2009). Gene tree discordance, phylogenetic inference and the multispecies coalescent. Trends in Ecology and Evolution, 24, 332–340. Degnan, S. M. & Moritz, C. (1992). Phylogeography of mitochondrial DNA in two species of white-eyes in Australia. The Auk, 109, 800–811. Denton, G. H., Lowell, T. V., Heusser, C. J., Schlüchter, C., Andersen, B. G.,
69 Heusser, L. E., … Marchant, D. R. (1999). Geomorphology, stratigraphy, and radiocarbon chronology of Llanquihue drift in the area of the southern Lake District, Seno Reloncavi, and Isla Grande de Chiloe, Chile. Geografiska Annaler, Series A: Physical Geography, 81, 167–229. Dolman, G. & Phillips, B. (2004). Single copy nuclear DNA markers characterized for comparative phylogeography in Australian wet tropics rainforest skinks. Molecular Ecology Notes, 4, 185–187. Drummond, A. & Rambaut, A. (2007). BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evolutionary Biology, 7, 1–8. Duellman, W. D. (1999). Patterns of Distribution in Amphibians: A Global Perspective. Systematic Biology, 51, 980–981.
Felsenstein, J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution, 39, 783–791. Fitzpatrick, S. W., Brasileiro, C. A., Haddad, C. F. B. & Zamudio, K. R. (2009). Geographical variation in genetic structure of an Atlantic Coastal Forest frog reveals regional differences in habitat stability. Molecular Ecology, 18, 2877–2896. Flot, J. F. (2015). Species delimitation’s coming of age. Systematic Biology, 64, 897–899. Folmer, O., Black, M., Hoeh, W., Lutz, R. & Vrijenhoek, R. (1994). DNA primers for amplification of mitochondrial cytochrome oxidase subunit I from diverse metazoan invertebrates. Molecular Marine
70 Biology and Biotechnology, 3, 294–299. Formas, J. R. (1978). A new species of leptodactylid frog (Eupsophus) from the coastal range in Southern Chile. Studies on Neotropical Fauna and Environment, 13, 1–9. Formas, J. R. (1991). The karyotypes of the Chilean frogs Eupsophus emiliopugini and E. vertebralis (Amphibia:Anura:Leptodactylidae). Proceedings of the Biological Society of Washington, 104, 7–11. Formas, J. R. & Brieva, L. (1994). Advertisement calls and relationships of Chilean frogs Eupsophus contulmoensis and E. insularis (Amphibia: Anura: Leptodactylidae). Proceedings of the Biological Society of Washington, 107, 391–397. Formas, J. R., Lacrampe, S. & Brieva, L. (1992). Allozymic and morphological differentiation among three south American frogs, genus Eupsophus. Comparative Biochemistry and Physiology, 102, 57– 60. Formas, J. R., Nuñez, J. J. & Cuevas, C. (2008). Identidad de la rana austral chilena Eupsophus coppigeri (Amphibia, Anura, Neobatrachia): evidencias morfológicas, cromosómicas y moleculares. Revista Chilena de Historia Natural, 81, 3–20. Formas, J. R. & Vera, M. A. (1980). Reproductive Patterns of Eupsophus roseus and E. vittatus. Journal of Herpetology, 14, 11.
Freudenstein, J. V., Broe, M. B., Folk, R. A. & Sinn, B. T. (2016). Biodiversity and the species concept - lineage are not enough. Systematic Biology,
71 66, 644-656. Frost, D. R. (2013). Amphibian species of the world: an online reference. Version 5.6. Electronic database Fujisawa, T., Aswad A. & Barraclough, T. G. (2016). A rapid and scalable method for multilocus species delimitation using Bayesian model comparison and rooted triplets. Systematic Biology, 65, 759–771. Fujisawa, T. & Barraclough, T. G. (2013). Delimiting species using single- locus data and the Generalized Mixed Yule Coalescent approach: a revised method and evaluation on simulated data sets. Systematic Biology, 62, 707–724. Fujita, M. K., Leache, A. D., Burbrink, F. T., McGuire, J. A. & Moritz, C. (2012). Coalescent-based species delimitation in an integrative taxonomy. Trends in Ecology & Evolution, 27, 480–488. Gamble, T., Bauer, A. M., Greenbaum, E. & Jackman, T. R. (2008). Out of the blue: a novel, trans-Atlantic clade of geckos (Gekkota, Squamata). Zoologica Scripta, 37, 355–366. García, J. L. (2012). Late pleistocene ice fluctuations and glacial geomorphology of the Archipiélago de Chiloé, Southern Chile. Geografiska Annaler, Series A: Physical Geography, 94, 459–479. Goebel, M., Donnelly, J. M. & Atz, M. E. (1999). PCR primers and amplification methods for 12S ribosomal DNA, the control region, cytochrome oxidase I, and cytochrome b in bufonids and other frogs, and an overview of PCR primers which have amplified DNA in amphibians successfully. Molecular Phylogenetics and Evolution, 11,
72 163–199. González-Ittig, R. E., Rossi-Fraire, H. J., Cantoni, G. E., Herrero, E. R., Benedetti, R., Gallardo, M. H. & Gardenal, C. N. (2010). Population genetic structure of long-tailed pygmy rice rats (Oligoryzomys longicaudatus) from Argentina and Chile based on the mitochondrial control region. Canadian Journal of Zoology, 88, 23–35. Gratton, P., Trucchi, E., Trasatti, A., Riccarducci, G., Marta, S., Allegrucci, G., … Sbordoni, V. (2016). Testing classical species properties with contemporary data: How “bad Species” in the Brassy Ringlets (Erebia tyndarus complex, Lepidoptera) turned good. Systematic Biology, 65, 292–303. Grummer, J. A., Bryson, R. W. & Reeder, T. W. (2014). Species delimitation using Bayes Factors: Simulations and application to the sceloporus scalaris species group (Squamata: Phrynosomatidae). Systematic Biology, 63, 119–133. Harrington, R. C. & Near, T. J. (2012). Phylogenetic and coalescent strategies of species delimitation in snubnose darters (Percidae: Etheostoma). Systematic Biology, 61, 63–69. Heled, J. & Drummond, A. J. (2010). Bayesian inference of species trees from multilocus data. Molecular Biology and Evolution, 27, 570–580. Hillis, D. M. & Bull, J. J. (1993). An empirical test of Bootstrapping as a method for assessing confidence in phylogenetic analysis. Systematic biology, 42, 182–192. Hoareau, T. B. (2016). Late glacial demographic expansion motivates a
73 clock overhaul for population genetics. Systematic Biology, 65, 449– 464. Hudson, R. R. & Coyne, J. A. (2002). Mathematical consequences of the genealogical species concept. Evolution, 56, 1557–1565. Huelsenbeck, J. P. & Rannala, B. (2004). Frequentist properties of bayesian posterior probabilities of phylogenetic trees under simple and complex substitution models. Systematic Biology, 53, 904–913. Hulton, N. R. J., Purves, R. S., McCulloch, R. D., Sugden, D. E. & Bentley, M. J. (2002). The Last Glacial Maximum and deglaciation in southern South America. Quaternary Science Reviews, 22, 751–757. Irisarri, I., Mauro, D. S., Abascal, F., Ohler, A., Vences, M. & Zardoya, R. (2012). The origin of modern frogs (Neobatrachia) was accompanied by acceleration in mitochondrial and nuclear substitution rates. BMC Genomics, 13, 626. IUCN. (2019). The IUCN Red List of Threatened Species. Electronic database Jacobs, S. J., Kristofferson, C., Uribe-Convers, S., Latvis, M. & Tank, D. C. (2018). Incongruence in molecular species delimitation schemes: What to do when adding more data is difficult. Molecular Ecology, 27, 2397–2413. Kapli, P., Lutteropp, S., Zhang, J., Kobert, K., Pavlidis, P., Stamatakis, A. & Flouri, T. (2017). Multi-rate Poisson tree processes for single-locus species delimitation under maximum likelihood and Markov chain Monte Carlo. Bioinformatics, 33, 1630–1638.
74 Kass, R. E. & Raftery, A. E. (1995). Bayes Factors. Journal of the American Statistical Association, 90, 773–95. Katoh, K., Kuma, K., Toh, H. & Miyata, T. (2005). MAFFT version 5: improvement in accuracy of multiple sequence alignment. Nucleic Acids Research, 33, 511–518. Kearse, M., Moir, R., Wilson, A., Stones-Havas, S., Cheung, M., Sturrock, S., … Drummond, A. (2012). Geneious Basic: An integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics, 28, 1647–1649. Kingman, J. (1982). Genealogy Populations. Journal of Applied Probability, 19, 27–43. Knowles, L. & Carstens, B. C. (2007). Delimiting species without monophyletic gene trees. Systematic Biology, 56, 887–895. Kubatko, L. S., Carstens, B. C. & Knowles, L. L. (2009). STEM: species tree estimation using maximum likelihood for gene trees under coalescence. Bioinformatics, 25(7), 971–973. Lanfear, R., Frandsen, P. B., Wright, A. M., Senfeld, T. & Calcott, B. (2017). Partitionfinder 2: new methods for selecting partitioned models of evolution for molecular and morphological phylogenetic analyses. Molecular Biology and Evolution, 34, 772–773. Lausch, A., Bannehr, L., Beckmann, M., Boehm, C., Feilhauer, H., Hacker, J. M., … Cord, A. F. (2016). Linking earth observation and taxonomic, structural and functional biodiversity: Local to ecosystem perspectives. Ecological Indicators, 70, 317–339.
75 Leaché, A. D., Fujita, M. K., Minin, V. N. & Bouckaert, R. R. (2014). Species delimitation using genome-wide SNP Data. Systematic Biology, 63, 534–542. León-Paniagua, L. & Guevara, L. (2019). Quaternary range-shifts of arboreal rodents of the genus Habromys (Cricetidae, Neotominae) in Mesoamerica and their evolutionary consequences. Mammalian Biology, 94, 4–10. Lessa, E. P., D’ElÍa, G. & Pardiñas, U. F. J. (2010). Genetic footprints of late Quaternary climate change in the diversity of Patagonian-Fueguian rodents. Molecular Ecology, 19, 3031–3037. Li, W. L. & Drummond, A. J. (2012). Model averaging and Bayes Factor calculation of relaxed molecular clocks in Bayesian phylogenetics. Molecular biology and Evolution, 29, 751–761 Liu, L., Yu, L., Kubatko, L., Pearl, D. K. & Edwards, S. V. (2009). Coalescent methods for estimating phylogenetic trees. Molecular Phylogenetics and Evolution, 53, 320–328. Luo, A., Ling, C., Ho, S. Y. W. & Zhu, C.-D. (2018). Comparison of methods for molecular species delimitation across a range of speciation scenarios. Systematic Biology, 0, 1–13.
Maddison, W. P. & Maddison, D. R. (2011). Mesquite: A modular system for evolutionary analysis. Version 2.75. Available at http://mesquiteproject.org Mayr, E. (1982). Part II. Chapter 11. The Causation of Evolution: Natural
76 Selection. In: Mayr, E. (Ed), The Growth of Biological thought, Diversity, Evolution, and Inheritance (pp. 477 534). Harvard University Press. United States. McCulloch, R. D., Sugden, D. E., Purves, R. S., Clapperton, C. M., Hulton, N. R. J. & Bentley, M. J. (2000). Climatic inferences from glacial and palaeoecological evidence at the last glacial termination, southern South America. Journal of Quaternary Science, 15, 409–417. Mendelova, M., Hein, A. S., McCulloch, R. & Davies, B. (2017). The Last Glacial Maximum and deglaciation in central Patagonia, 44°S–49°S. Cuadernos de Investigación Geográfica, 43, 719. Miralles, A. & Vences, M. (2013). New metrics for comparison of taxonomies reveal striking discrepancies among species delimitation methods in Madascincus lizards. PLOS ONE, 8, e68242 Nuñez, J. J. (2003). Taxonomía y sistemática de las ranas del género Eupsophus (Leptodactylidae). Tesis Doctoral, Universidad Austral de Chile, Valdivia. Nuñez, J. J., Rabanal, E. F., & formas, J. R. (2012). Description of a new species of Eupsophus (Amphibia: Neobatrachia) from the Valdivian Coastal range, Southern Chile: an integrative taxonomic approach. . Zootaxa, 3305, 53–68. Nuñez, J. J., Wood, N. K., Rabanal, F. E., Fontanella, F. M. & Sites Jr., J. W. (2011). Amphibian phylogeography in the Antipodes: Refugia and postglacial colonization explain mitochondrial haplotype distribution in the Patagonian frog Eupsophus calcaratus (Cycloramphidae).
77 Molecular Phylogenetics and Evolution, 58, 343–352. Padial, J. M., Miralles, A., De la Riva, I. & Vences, M. (2010). The integrative future of taxonomy. Frontiers in Zoology, 7, 16. Pante, E., Schoelinck, C. & Puillandre, N. (2015). From integrative taxonomy to species description: One step beyond. Systematic Biology, 64, 152–160. Paskoff, R. P. (1977). Quaternary of Chile: The state of research. Quaternary Research, 8, 2–31. Passmore, L. N. (1981). The relevance of the specific mate recognition concept to anuran reproductive biology. Monitore Zoologico Italiano, 15, 93–108. Patton, J. L. & Smith, M. F. (1994). Paraphyly, polyphyly, and the nature of species boundaries in Pocket Gophers (Genus Thomomys). Systematic Biology, 43, 11–26. Pecl, G. T., Araújo, M. B., Bell, J. D., Blanchard, J., Bonebrake, T. C., Chen, I. C., … Williams, S. E. (2017). Biodiversity redistribution under climate change: Impacts on ecosystems and human well-being. Science, 355, eaai914. Penna, M. & Veloso, A. (1990). Vocal diversity in frogs of the South American temperate forest. Journal of Herpetology, 24, 23–33. Petit, R. J. & Excoffier, L. (2009). Gene flow and species delimitation. Trends in Ecology & Evolution, 24, 386–393. Pollard, D. A., Iyer, V. N., Moses, A. M. & Eisen, M. B. (2006). Widespread discordance of gene trees with species tree in Drosophila: Evidence
78 for incomplete lineage sorting. PLOS Genetics, 2, e173. Prohl, H., Ron, S. R. & Ryan, M. J. (2010). Ecological and genetic divergence between two lineages of Middle American túngara frogs Physalaemus (=Engystomops) pustulosus. BMC Evolutionary Biology, 10, 146. Puillandre, N., Modica, M. V., Zhang, Y., Sirovich, L., Boisselier, M. -C., Cruaud, C., Holford, M. & Samadi, S. (2012). Large-scale species delimitation method for hyperdiverse groups. Molecular Ecology, 21, 2671–2691. Pyron, R. A. & Wiens, J. J. (2011). A large-scale phylogeny of Amphibia including over 2800 species, and a revised classification of extant frogs, salamanders, and caecilians. Molecular Phylogenetics and Evolution, 61, 543–583. Rabassa, J. (2011). Late Cenozoic glaciations in Patagonia and Tierra del Fuego: an update review. Developments in Quaternary Sciences, 11, 151–204. Rabassa, J. & Clapperton, C. M. (1990). Quaternary glaciations in the southern hemisphere: An overview. Quaternary Science Reviews, 9, 299–304. Rambaut, A. & Drummond, A. J. (2016). TreeAnnotator version 1.8.3. Available at http://beast.bio.ed.ac.uk. Rambaut, A., Drummond, A. J., Xie, D., Baele, G. & Suchard, M. A. (2018). Posterior summarization in Bayesian phylogenetics using Tracer 1.7. Systematic Biology, 67, 901–904. Rannala, B. (2015). The art and science of species delimitation. Current
79 Zoology, 61, 846–853. Rannala, B. & Yang, Z. (2003). Bayes estimation of species divergence times and ancestral population sizes using DNA sequences from multiple loci. Genetics, 164, 1645–1656. RCE (2019). Decreto supremo N° 25 de 2011, Reglamento para la clasificación de Especies Silvestres según Estado de Conservación. Ministerio de Medio Ambiente, Santiago de Chile. Rodriguez, A., de la Nuez, D. & Alonso, R. (2010). Intraspecific variation in the advertisement call of the Cloud-forest frog Eleutherodactylus glamyrus (Anura: Eleutherodactylidae). Journal of Herpetology, 44, 457–466. Rojas, R. R., Fouquet, A., Ron, S. R., Hernández-Ruz, E. J., Melo-Sampaio, P. R., Chaparro, J. C., … Hrbek, T. (2018). A Pan-Amazonian species delimitation: High species diversity within the genus Amazophrynella (Anura: Bufonidae). PeerJ, 6, e4941. Ronquist, F. & Huelsenbeck, J. P. (2003). MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics, 19, 1572–1574. Ruzzante, D. E., Walde, S. J., Cussac, V. E., Dalebout, M. L., Seibert, J., Ortubay, S. & Habit, E. (2006). Phylogeography of the Percichthyidae (Pisces) in Patagonia: Roles of orogeny, glaciation, and volcanism. Molecular Ecology, 15, 2949–2968. Schwarz, G. (1978). Estimating the Dimension of a Model. The Annals of Statistics, 6, 461–464.
80 Shaffer, H. B. & Thomson, R. C. (2007). Delimiting species in recent radiations. Systematic Biology, 56, 896–906. Sites, J. W. & Marshall, J. C. (2003). Delimiting species: a renaissance issue in systematic biology. Trends in Ecology & Evolution, 18, 462–470. Sites, J. W. & Marshall, J. C. (2004). Operational criteria for delimitng species. Annual Review of Ecology, Evolution and Systematics, 35, 199– 227. Sokal, R. R., Crovello, T. J., The, S., Naturalist, A. & Apr, N. M. (1970). The biological species concept: a critical evaluation. The American Naturalist, 104, 127–153. Stokkan, M., Jurado-Rivera, J. A., Oromí, P., Juan, C., Jaume, D. & Pons, J. (2018). Species delimitation and mitogenome phylogenetics in the subterranean genus Pseudoniphargus (Crustacea: Amphipoda). Molecular Phylogenetics and Evolution, 127, 988–999. Suárez-Villota, E. Y., Quercia, C. A., Díaz, L. M., Vera-Sovier, V. & Nuñez, J. J. (2018a). Speciation in a biodiversity hotspot: Phylogenetic relationships, species delimitation, and divergence times of Patagonian ground frogs from the Eupsophus roseus group (Alsodidae). PLOS ONE, 13, e0204968. Suárez-Villota, E. Y., Quercia, C. A. & Nuñez, J. J. (2018b). Mitochondrial genomes of the South American frogs Eupsophus vertebralis and E. emiliopugini (Neobatrachia: Alsodidae) and their phylogenetic relationships . Journal of Genomics, 6, 98–102. Sukumaran, J. & Knowles, L. L. (2017). Multispecies coalescent delimits
81 structure, not species. Proceedings of the National Academy of Sciences, 114(7), 1607–1612. Syring, J., Farrell, K., Businský, R., Cronn, R. & Liston, A. (2007). Widespread genealogical nonmonophyly in species of Pinus subgenus Strobus. Systematic Biology, 56, 163–181. Talavera, G., Dincǎ, V. & Vila, R. (2013). Factors affecting species delimitations with the GMYC model: Insights from a butterfly survey. Methods in Ecology and Evolution, 4, 1101–1110. Toews, D. P. L. & Brelsford, A. (2012). The biogeography of mitochondrial and nuclear discordance in animals. Molecular Ecology, 21, 3907– 3930. Turin, R. A., Nali, R. C. & Prado, C. P. (2018) Intraspecific call variation in a Neotropical gladiator frog with a complex advertisement call. Amphibia-Reptilia, 39, 31–39. Veloso, A., Celis-Diez, L. J., Guerrero, P. C., Mendez, A. M., Iturra, P. & Simonetti J. A. (2005). Description of a new Eupsophus species (Amphibia, Leptodactylidae) from the remnants of Maulino forest, Central Chile. Herpetological Journal, 15, 159–165. Vera-Escalona, I., Habit, E. & Ruzzante, D. E. (2019). Invasive species and postglacial colonization: Their effects on the genetic diversity of a Patagonian fish. Proceedings of the Royal Society B: Biological Sciences, 286, 20182567. Walsh, P. S., Metzger, D. A. & Higuchi, R. (1991). Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic
82 material. Biotechniques, 10, 506–513. Wiens, J. J. (2007). Species delimitation: new approaches for discovering diversity. Systematic Biology, 56, 875–878. Wiens, J. J. & Servedio, M. R. (2000). Species delimitation in systematics: Inferring diagnostic differences between species. Proceedings of the Royal Society B: Biological Sciences, 267, 631–636. Xu, B. & Yang, Z. (2016). Challenges in species tree estimation under the multispecies coalescent model. Genetics, 204, 1353–1368. Yang, Z. (2015). The BPP program for species tree estimation and species delimitation. Current Zoology, 61, 854–865. Yang, Z. & Rannala, B. (2010). Bayesian species delimitation using multilocus sequence data, 107, 9264–9269. Zemlak, T. S., Habit, E. M., Walde, S. J., Battini, M. A., Adams, E. D. M. & Ruzzante, D. E. (2008). Across the southern Andes on fin: Glacial refugia, drainage reversals and a secondary contact zone revealed by the phylogeographical signal of Galaxias platei in Patagonia. Molecular Ecology, 17, 5049–5061. Zhang, J., Kapli, P., Pavlidis, P. & Stamatakis, A. (2013). A general species delimitation method with applications to phylogenetic placements. Bioinformatics, 29, 2869–2876.
83 CHAPTER 2
COMPARATIVE CYTOGENETICS OF TWO GROUND FROGS OF GENUS EUPSOPHUS: E. emiliopugini AND E. vertebralis (Alsodidae); WITH COMMENT ON INTRASPECIFIC POLYMORPHISM IN NORS
ABSTRACT
South American frogs of the genus Eupsophus comprise 10 species. Two of them, Eupsophus vertebralis and E. emiliopugini belonging to the vertebralis group, exhibit 2n=28, while the rest of species contain 30 chromosomes and belong to the roseus group. Fundamental number differences between the species of the vertebralis group have been described; nevertheless, the consistency of this report has been unexplored intraspecifically, neither with other chromosome markers. In this work, cytogenetic analyses for the vertebralis group were carried out based on conventional (Giemsa staining, C-banding and, Ag-NOR) and cytomolecular (fluorescence in situ hybridization using telomeric and 28S ribosomal probes) techniques. We corroborate fundamental number differences between Eupsophus vertebralis and E. emiliopugini through
84 Giemsa staining and C-banding (FN= 54 and 56, respectively). No interstitial fluorescent signals, but clearly stained telomeric regions were detected by FISH using telomeric probe over spreads from both species. FISH with 28S rDNA probes confirmed the active NOR signal on pair 5 visualized by silver nitrate staining for both, Eupsophus vertebralis and E. emiliopugini. Nevertheless, an E. emiliopugini individual from Puyehue locality exhibited 28S ribosomal signals on pairs 4 and 5. Interestingly, only one chromosome of each pair showed Ag-NOR positive signals, showing a nucleolar dominance pattern. This interpopulation chromosomal variation could be explained by chromosomal rearrangements or derived from species hybridization process. Since, nucleolar dominance is a peculiar phenomenon in hybrids, the last option receives greater support. We present a comparative cytogenetic analysis between Eupsophus vertebralis and E. emiliopugini and point to plausible explanations about the hybrid nature of a E. emiliopugini sample. Further analyses and samples will be necessary to test this hybridization hypothesis.
Keywords: Karyotypes, FISH, Patagonian frogs, intraspecific variations, nucleolar dominance.
1. INTRODUCTION
85 The Alsodidae family is the most species-rich of the Southern Chile and Argentina endemic frogs group, including nearly 31 of the slightly more than 50 species of that region and representing by Alsodes and Eupsophus genera (Frost, 2017). The genus Eupsophus is currently understood by 10 species (Suárez-Villota et al., 2018a) and it is endemic from the Valdivian rainforest and temperate Nothofagus forest of Southern Chile (Formas, 1978; Ibarra-Vidal et al., 2004). The evolutionary history of the Eupsophus species has been molded and influenced by the Pleistocene cycling events of Southern Chile, which has been associated with restricted distribution of some species, isolation, and speciation processes (Nuñez, 2003; Suárez-Villota et al., 2018a).
Based on ethologic (advertisement calls; Formas & Brieva, 1994), morphologic (body size; Nuñez, 2003), molecular (allozymes and DNA sequences; Formas et al., 1992; Blotto et al., 2013;), and cytogenetics (Formas, 1991; Veloso et al., 2005) analyses, Eupsophus is divided into two groups the roseus group, and the vertebralis group. The roseus group is composed by eight species (E. calcaratus, E. contulmoensis, E. septentrionalis, E. nahuelbutensis, E. insularis, E. migueli, E. roseus, E. altor; Suárez-Villota et al., 2018a) exhibiting the same diploid number 2n=30 with some species specific characteristics (e.g. fundamental number, sex chromosomes, secondary constriction location; Iturra & Veloso, 1988; Veloso et al., 2005; Nuñez et al., 2012). On the other hand, the vertebralis group, composed by Eupsophus vertebralis and E. emiliopugini, exhibit the
86 same diploid number 2n=28, do not have sex chromosomes, and present a secondary constriction in pair 5 (Formas, 1991). Nevertheless, thirteen pair is metacentric in E. emiliopugini and telocentric E. vertebralis karyotypes, differing in their fundamental numbers (FN=56 and FN=54, respectively).
Formas (1991), exposes a chromosome model of evolution to explain the morphological difference of the 13 pair between both species of the vertebralis group. The proposed mechanism of change is a chromosome rearrangement (King, 1994) as pericentric inversions, more than win or loss of genetic material. According to the author the mechanism could have been given in two ways: the first alternative is a pericentric inversion in telocentric 13 pair of E. vertebralis, which shifted the centromere to the metacentric position of E. emiliopugini. The alternative event is the addition of heterochromatic segments in the centromeric region of the telocentric thirteen pair in E. vertebralis, which lead to a metacentric pair in E. emiliopugini. The author considers more plausible the first alternative because both 13 pairs, telocentric and metacentric, have the same size. Formas (1991) also points out that the direction of the most probable change was E. vertebralis to E. emiliopugini, based on the possession of telocentric chromosomes of E. vertebralis that is considered a condition ancestry in anuran amphibians (Morescalchi, 1973).
87 Cytogenetics studies in the vertebralis group have used classical cytogenetics in order to establish the karyotypes and to analyze chromosome morphology. Since classical and molecular cytogenetic approaches can be help to understand mechanisms of chromosomal differentiation and to test hypotheses about their chromosomal evolution, we apply these approaches in the species of the vertebralis group. Thus, the aim of this study was to describe the karyotypes of Eupsophus vertebralis and E. emiliopugini using a combination of cytogenetic techniques and considering specimens from almost all the distribution range. In this sense, we analyze at population level the NOR position in E. vertebralis and E. emiliopugini karyotypes, using Ag-NOR banding and fluorescent in situ hybridization (FISH) with 28S rDNA probe. Using FISH with telomeric probe and C-Banding, we seek interstitial telomeric signals, which could suggest chromosomal rearrangements in both species. This comparative cytogenetic analysis provides a refined description of the vertebralis group karyotypes, leaves new questions regarding the chromosomal patterns, and gives lights on how to address them in future studies.
2. MATERIALS AND METHODS
2.1.- SAMPLES COLLECTION AND CYTOLOGICAL PREPARATIONS
Cytologic preparations were obtained from 14 and 9 individuals of
88 each Eupsophus vertebralis and E. emiliopugini species, respectively (Supplementary Table 1). These individuals were collected according to permit of Servicio Agrícola y Ganadero (No. 9244/2015) from 15 locations in Southern Chile (Figure 1). Mitotic plates were obtained from intestine cell suspension. For this purpose, we applied 30 µL/gr of 0.1% colchicine (Sigma-Aldrich) on the abdominal cavity of each individual. After 12 hours, the individuals were euthanized with benzocaine gas inversion, according to recommendations of the Bioethics and Biosecurity Committee of the Universidad Austral de Chile (UACh, Resolutions No. 236/2015 and 61/15). Immediately after euthanized, the gut cells were extracted and prepared according to Schmid (1978) protocol, with some modifications. Then, the specimens were saved in the herpetological collection of Instituto de Ciencias Marinas y Limnológicas, UACh (vouchers number and molecular code from CHAPTER 1 are in Supplementary Table 1.).
2.2.- CLASSICAL CYTOGENETIC TECHNIQUES
Mitotic plates were staining with 10% GIEMSA to karyotype determination. Active nucleolar organizer regions (NORs) were detected using silver nitrate staining (Ag-NOR) according to (Howell & Black, 1980). This chromosomal material was analyzed in Digital Lab Trinocular microscope, photographed with AmScope camera using IQ capture software.
89
To identify constitutive heterochromatic regions, we carried out C- banding protocol using formamide for DNA denaturation, according to Fernández et al., (2002). In this case, mitotic plates were counterstained with DAPI (4’,6-diamino-2-phenylindole; 1 μg/mL) and mounted with Vectashield antifade. Subsequently, metaphases were visualized through a BX61 Olympus microscope, and captured with adequate filter using a DP70 Olympus digital camera with Pro MC Image software. Images were overlaid and contrast enhanced using Adobe Photoshop CS6.
2.3.- MOLECULAR CYTOGENETIC TECHNIQUES
The physical map of the rDNA genes was detected by FISH on mitotic plates from E. vertebralis (from Colihual Alto, and Reumén locatilities), and E. emiliopugini (from Puyehue, Cordillera del Sarao, and Parque Alerce Andino locatities) specimens. For this purpose, we amplified 28S rDNA fragment from E. vertebralis DNA, using 28SV (5´- AAGGTAGCCAAATGCCTCGTCATC-3´) and 28SJJ (5´- AGTAGGGTAAAACTAACCT-3´) primers (Bruschi et al., 2012; Hillis & Dixon, 1991). PCR was carried out according to the manufacture instructions for Taq Platinum DNA Polymerase (Cat. No. 10966, Invitrogen), at 55ºC of annealing temperature. The 28S probe was PCR- labeled with 11-digoxigenin dUTP (Sigma-Aldrich), hybridized according
90 to (Ferreira et al., 2011), and the hybridized signal was detected with an anti-digoxigenin antibody conjugated with rhodamine (Roche).
Telomere detection by FISH was carried out on metaphase chromosomes from E. vertebralis (from Tolhuaca, Reumén, and Colihual Alto), and E. emilipugini (from Puyehue, Parque Alerce Andino, and
Cordillera del Sarao) specimens. Universal telomeric probes (TTAGGG)n were PCR-generated and labeled with fluorescein-12-dUTP (Cat. No 11373242910, Roche) (Ijdo et al., 1991). Fluorescent in situ hybridization followed to Suárez-Villota et al. (2012) with modifications. Briefly, slides containing chromosomal material were aged by a thermal shock at 95ºC for 10 s, pretreated with a 10 mM HCl solution for 5 min, and treated with 5 mg/mL pepsin solution in 10 mM HCl, at 37º C for 1 min. Subsequently, slides were washed in 1% formaldehyde for 1 min, and two times in 2xSSC. Then, they were dehydrated with ethanol (50%, 70%, 99%), and denatured in 70% formamide/2xSSC, for 3 mins. Slides are dehydrated as above. Probes (100 ng) were mixed with a hybridization solution containing 50% formamide, 10% dextran sulphate, 0.1% SDS in 2xSSC (pH 7.0), denatured at 83ºC for 6 min, and 30 μL was applied onto each slide. Hybridization was carried out in a moist chamber at 37ºC for 72 h. Post-hybridization washes at 37ºC consisted of 50% formamide/2xSSC and 2xSSC for 3 min, followed by 3x5-min washes in 4xSSC/0.1% Tween 20.
91 Slides were mounted the with DAPI-Vectachield antifade for both 28S rDNA and telomeric FISH. Images were captured and treated as described previously for C-banding protocol.
3. RESULTS
3.1.- CLASSICAL CYTOGENETIC TECHNIQUES
For each of the species, we analyze 88 mitotic plates showing 2n=28 without the presence of sexual chromosomes. All the E. emiliopugini plates showed only chromosomes bi-armed with a FN=56. The pairs 1, 2, 8-14 were metacentrics, pair 7 is submetacentric, and pairs 3 to 6 are subtelocentrics. Secondary constriction (SC) was observed in the short arms of pair 5 in all E. emiliopugini specimens, except by the specimen of E. emiliopugini collected at Puyehue locality (thereafter referred as the Puyehue sample). This sample present one SC on one chromosome of the pair 4, and the other SC was present on one chromosome of pair 5.
All the E. vertebralis plates showed the same karyotype features than E. emiliopugini specimens, except for pair 13 which was telocentric, therefore present a FN=54 (Figure 2). Otherwise, heterochromatic regions revealed by C- banding for the specimens of both species, showed a predominantly centromeric distribution, and a pericentromeric banding in the long arms of chromosomes with SC. For all the metaphases plates
92 analyzed, between pairs 9 to 14 it was difficult to recover clearly the centromeric banding pattern (Figure 2). Ag-NOR staining revealed the active NORs sites coincident with the location of SC in all the analyzed metaphases of E. vertebralis, and E. emiliopugini. Except for Puyehue sample were the signal was in the centromeric region, and not in the SC on chromosome pair 4 (Figure 3), evidencing a polymorphism related to SC and active NORs regions.
3.2.- CYTOMOLECULAR TECHNIQUES
The signals obtained by FISH using 28S rDNA probe (28S-FISH) also revealed a polymorphism for Puyehue sample, related to the 28S rDNA location. For this sample, 28S-FISH shown four signals: two signals in the short arms of both chromosomes on pair 5, and the others two signals were located in the centromeric regions of both chromosomes on pair 4. One of two 28S-FISH signals for each chromosome pair (one for pair 4 and one for pair 5) was coincident with the location of active NOR site revealed by Ag-NOR for Puyehue sample (Figure 3). Instead, the others analyzed specimens of E. vertebralis and E. emiliopugini species, showed two 28S-FISH signals located in the short arms of both homologous on pair 5, coincident with the SC and Ag-NOR staining results. Otherwise, the telomeric probe revealed by FISH, hybridized with all the chromosome ends, without interstitial signals (Figure 4). This pattern was found on all specimens submitted to telomeric FISH, without locality variation.
93 4. DISCUSSION
4.1.- KARYOTYPES DESCRIPTION OF E. vertebralis AND E. emiliopugini SPECIES
We present the first comparative cytogenetic study using classical and cytomolecular techniques among specimens from different localities of E. vertebralis and E. emiliopugini species. According with previous analyses (Formas, 1989; Formas, 1991), both species exhibited 2n=28 and its pair 13 was polymorphic (Figures 2 and 3). Contrasting with Formas (1991), we place the largest submetacentric pair to third position, in order to consider decreasing size and chromosome morphology array (Figure 2a). We did not detect sex chromosome differentiation in the vertebralis group. This result is in accordance with the results findings of Formas (1991) (Figure 2b). So, we suggested that the absence of sexual chromosomes observed in the vertebralis group is a plesiomorphic condition, in accordance with the notion that sex chromosomes correspond to a derived condition in E. roseus group (King, 1991; Iturra & Veloso, 1988; Formas, 1991).
C-banding has been largely used in amphibians to compare karyotypes and to distinguish species with the same diploid number (Bogart, 1970; Cuevas & Formas, 2003; Nogueira et al., 2015; Sangpakdee et al., 2017; Targueta et al., 2018). On the other hand, homogeneous C-
94 banding pattern among related species has been associated to poorly genetic differentiation (Pellegrino & Trefaut, 1997; Lourenco & Recco- Pimentel, 1998; Bruschi et al., 2012) and enriched of repetitive elements, characteristic of amphibian chromosomes (Schmid, 1978; Bruschi et al., 2012; Zlotina et al., 2017). Therefore the absence of interspecific variations in heterochromatin banding found in this study (Figure 2b), could derive from the recent and low differentiation between E. vertebralis and E. emiliopugini species (see CHAPTER 1 and Suárez- Villota et al., 2018b ANNEX II).
4.2.- NUCLEOLAR ORGANIZER REGIONS POLYMORPHISM
Ag-NOR banding combined with fluorescent in situ hybridization (FISH) using rDNA probes allows characterizing the nucleolus organizer regions (NORs) (Wiley et al., 1989). NORs loci correspond to ribosomal genes (rDNA) coding for 18S rRNA, 5.8S rRNA and 28S rRNA (Preuss & Pikaard, 2007; McStay, 2016). Thus, while Ag-NOR staining detects active NORs, FISH check the total number of loci containing rDNA genes confirming the polymorphism (Zaleśna et al., 2017). For both species of the vertebralis group, excluding the Puyehue sample, we detected Ag-NOR signals on the short arms of pair 5 (Figure 3a; top and medium, see asterisks), colocalizing with the secondary constriction (SC; Figure 2, top and medium, see arrows), and with 28S rDNA FISH signal (Figure 3b, top and medium, red signal). Therefore, 28S rDNA locus was transcriptionally
95 active in both homologues from pair 5 for E. vertebralis and E. emiliopugini species. Thus, it was not possible to determine a species- specific pattern relative to NORs numbers and locations between these both species. A closely related example is found in the genus Alsodes, where Cuevas and Formas (2003) reported the same Ag-NOR banding for the karyotypes of three species of Alsodes, but in these cases the karyotypes differ in their C-banding pattern. On the contrary, NORs patterns, recovered by Ag-NOR staining in E. contulmoensis and E. migueli mitotic plates, exhibited species-specific characteristics (Veloso et al., 2005).
Intraspecific polymorphism in NORs was detected for one specimen of E. emiliopugini from Puyehue (Figure 3, down). We observed 28S FISH signals on pair 4 and 5 (four NOR clusters; Figure 3b, down), of which only one chromosome of each pair showed SC (Figure 2b, down) and Ag- NOR positive signal (Figure 3a, down). The absence of SC in one chromosome from one pair is a cytologic phenomenon known as differential amphiplasty (Navashin, 1928; Pikaard, 2000). This phenomena is understood as the morphological changes that can adopt homeologous chromosomes with SC from interspecific hybrids (Pikaard, 2000). Thus, differential amphiplasty is a karyological pattern directly associated with nucleolar dominance, an epigenetic phenomenon from hybrids (Pikaard, 2000).
96 Nucleolar dominance have been widely reported in plants and animals as a regulatory phenomenon of hybrids, that inactive NORs derived from one parental (Pontes et al., 2003). It has been proposed that the silencing mechanism of NORs regions it is due to an epigenetic switch (promoter methylation and histone modifications) in response to a dosage compensation phenomenon (Chen & Pikaard, 1997; Grummt & Pikaard, 2003; Lawrence et al., 2004; Preuss & Pikaard, 2007; Tucker et al., 2010). Examples of this phenomenon in diploid hybrids have been reported for plants (Pontes et al., 2003), mule (Kopp et al., 1986), rodents (Walker et al., 1999), and amphibians like Odontophrynus (Ruiz et al., 1980) and Xenopus (Honjo & Reeder, 1973).
Since this result showed a nucleolar dominance phenomenon on the Puyehue sample, where only two from four NOR clusters are active (Figure 3, down), we proposed different hypothesis to explain the origin of this specimen. Regarding its possible hybrid nature, we exclude the possibility of hybridization with one specimen of E. vertebralis, because we do not find any polymorphisms in SC, NORs or 28S rDNA genes position. Also, we discard specimens of the roseus group due to their different diploid number (2=30). In the case of hybridization between one specimen of the roseus group with one specimen of the vertebralis group, the karyotype of the hybrid would present 14 paired chromosomes plus one despaired chromosome (2n= 28+1). Therefore, the remainder alternative is a hybridization between one E. emiliopugini specimen with
97 NORs on pair 5, with other Eupsophus sp. specimen (2n=28) with NORs on pair 4 (Eupsophus sp.1 in Figure 5a.).
A second hybridization possibility could have been between two specimens of differents Eupsophus sp. with NORs position on pair 4 and on pair 5 (E. sp2 and E. sp3 in Figure 5.b.). Under these hybridization hypotheses, one NORs per parental was silenced and lost the SC, while the other one remains active in the Puyehue specimen (Figure 5). The molecular mechanisms by which active rDNA gene are selected the active rDNA gene in hybrids remain poorly known (Grummt & Pikaard, 2003; Preuss & Pikaard, 2007). The evidence suggest that the nucleolar dominance selection is independent of any parental sex effects, and it is unlikely that functional differences exist between ribosomes of either parent within hybrids of closely related species (Preuss & Pikaard, 2007).
These antecedents, allows us to infer that it is possible to found differential silencing for rDNA genes derived from each parent (i.e. in codominance condition). Other possible explanation to the nucleolar dominance found for Puyehue specimen can be addressed by chromosomal rearrangements (Schweizer & Loidl, 1987), or mobiles NORs (Schmid et al., 2017) hypotheses. Futures cytomolecular studies, considering others specimens from this locality, and using cytological preparations obtained from different tissues, could be available to test these hypotheses. It is worth mentioning that in phylogenetics
98 reconstructions the specimen of E. emiliopugini from Puyehue locality is recovered within E. emiliopugini group but with low statistical support (see CHAPTER 1).
4.3.- HYPOTHESIS ABOUT THE EVOLUTION OF PAIR 13
On the basis of previous karyotypic information, telomeric signals are conserved in vertebrates (Meyne et al., 1989) whereas its interstitial presence results from chromosomal rearrangements in animals (Ruiz- Herrera et al., 2002; Vitturi et al., 2002; Castiglia et al., 2006). For E. vertebralis and E. emiliopugini, telomeric probe analyzed by FISH did not recovered interstitial signals, therefore the chromosome translocation proposed by Formas (1991) to explain the differences on pair 13 between E. vertebralis and E. emiliopugini cannot be supported. Alternative, interstitial telomeric sequences can have been lost, as have been reported in other animals (Rogatcheva et al., 2002; Castiglia et al., 2006).
Another remained possibility proposed by Formas (1991) is the addition of heterochromatic segments in the centromeric region of telocentric pair 13 of E. vertebralis, leading to a metacentric pair 13 in E. emiliopugini. Nevertheless, in the C-banding results, it was not possible to observe a heterochromatic region in the short arms of metacentric pair 13 for E. emiliopugini. Therefore, we neither can corroborate this second hypothesis. However, the ancestral condition for karyotypes of Eupsophus
99 species includes the presence of telocentric chromosomes, such has been described by Veloso et al. (2005). Examples of this are E. migueli and species of the genus Alsodes. So, the presence of a metacentric chromosome in the karyotypes of E. emiliopugini would be an apomorphic character. Future cytogenetic investigations that develop a chromosomic probe of pair 13 to perform in situ hybridizations, could give valuable information to elucidate the evolution direction of the chromosomes of the par 13 of Eupsophus emiliopugini and E. vertebralis.
5. CONCLUSION
This work redescribes the karyotypes of E. vertebralis and E. emiliopugini, reporting additional karyotypes features such as C-banding pattern, NORs and telomeric signals locations. According to the results obtained here, it was not possible to found some species-specific patterns that can help to distinguish between E. emiliopugini and E. vertebralis. Except by the chromosome morphologic differences in pair 13, which were conserved at species level without population variations. Moreover, we reported an intraspecific polymorphism related to location and active NORs for one specimen of E. emiliopugini from Puyehue locality. We proposed some hypotheses for this phenomenon relative to chromosome rearrangements or a hybridization event. Future studies considering more individuals of E. emiliopugini from Puyehue locality, could be available to test some of these hypotheses. Finally, regarding the
100 morphologic difference of pair 13 between E. vertebralis and E. emiliopugini, the results of this study did not support the previous proposed hypotheses relative to chromosomal rearrangements or heterochromatin addition in the chromosomes of pair 13. Therefore, it is not possible to define the evolution direction of these both karyotypes. This last question could be answered by new studies that use molecular cytogenetic techniques with chromosome probes.
ACKNOWLEDGMENTS
I am grateful to Dr. Fausto Foresti for facilitated us his knowledge and laboratory dependencies (Laboratório de Biologia e Genética de Peixes, Botucatu, SP, Brasil), and also to Dr. Cristian Araya and R. Duilio Zerbinato for his laboratory assistance. The collaboration of Engr. Nicolás González in fieldworks was also very useful and grateful. Fondecyt 3160328 to EYS-V. and CONICYT grants 22180766 to CAQ funded this research.
101 FIGURES
Figure 1: Map depicting 15 collection localities of the vertebralis group specimens, in Southern Chile. E. vertebralis locations are represented by orange circles, and E. emiliopugini locations are shown with blue circles. Colorized sections on the map indicate water bodies. The numbers inside the circles corresponds with the next 15 localities: 1) Tolhuaca, 2) Lago Pellaifa, 3) Colihual Alto, 4) Chanchan, 5) Oncol, 6) Llancahue, 7) Chamil, 8) Cordillera Pelada, 9) Reumén, 10) Los Mañios, 11) Pucatrihue, 12) Puyehue, 13) Cordillera del Sarao, 14) Parque Alerce Andino, and 15) Huinay.
102
Figure 2: Classical Giemsa (a) and C- banding (b) of the vertebralis group. The top corresponds with E. emiliopugini karyotypes, the middle with E. vertebralis karyotype, and at the down is shown the karyotype of the E. emiliopugini specimen collected at Puyehue locality. Arrows indicate the location of secondary constrictions, and asterisk (*) indicates the telocentric pair of E. vertebralis.
103
Figure 3: Results of Ag-NOR staining (a), and FISH of 28S rDNA probe (b) for the vertebralis group. At the top are depict the E. emiliopugini karyotypes, at the middle are the E. vertebralis karyotypes and at the down section is shown the karyotype of E. emiliopugini specimen collected at Puyehue locality.
104
Figure 4: Telomeric signal obtained by FISH for the vertebralis group. It is shown in (a) Eupsophus emiliopugini, (b) E. vertebralis, and (c) E. emiliopugini from Puyehue locality. Note the absence of interstitial signals.
105
Figure 5: Proposed hypothesis to explain the nucleolar dominance phenomena observed in one specimen from Puyehue locality of E. emiliopugini species. Two possible hybridizations that’s origin E. emiliopugini Puyehue specimen. For the hypothesis (a) one parental corresponds to E. emiliopugini, the other one corresponds to Eupsophus sp. In (b). Eupsophus sp2 and Eupsophus sp3 correspond with to the hypothesized parental species. Satellite region, Secondary constriction and Centromere are indicated in (a). Also, the pair number is shown below each chromosome.
106 SUPPLEMENTARY TABLE
Supplementary Table 1: List of the specimens used for cytogenetic analyses in this study. It is shown the localities of sampling for both species. The voucher number of herpetological collections of Instituto de Ciencias Marinas y limnológicas, and the molecular code corresponding with the codes of CHAPTER 1 are also shown.
Nº Molecular Specie Locality Map Voucher Code E. vertebralis Tolhuaca 1 ICMLH439 EVTO1104 E. vertebralis Lago Pellaifa 2 ICMLH544 EVLP1350 E. vertebralis Lago Pellaifa 2 ICMLH545 EVLP1351 E. vertebralis Lago Pellaifa 2 ICMLH555 EVLP1361 E. vertebralis Colihual Alto 3 ICMLH500 EVCA1299 E. vertebralis Chanchan 4 ICMLH506 EVCC1305 E. vertebralis Oncol 5 ICMLH495 EVON1228 E. vertebralis Llancahue 6 ICMLH570 EVLA1376 E. vertebralis Llancahue 6 ICMLH509 EVLL1314 E. vertebralis Chamil 7 ICMLH557 EVCH1363 E. vertebralis Chamil 7 ICMLH558 EVCH1364 E. vertebralis Cordillera Pelada 8 ICMLH407 EVCP991 E. vertebralis Cordillera Pelada 8 ICMLH421 EVCP1027 E. vertebralis Reumen 9 ICMLH374 EVRE957 E. emiliopugini Los Mañios 10 ICMLH550 EELM1356
107 E. emiliopugini Los Mañios 10 ICMLH551 EELM1357 E. emiliopugini Pucatrihue 11 ICMLH414 EEPU1020 E. emiliopugini Puyehue 12 ICMLH499 EEPY1298 E. emiliopugini Cordillera del Sarao 13 ICMLH403 EESA987 E. emiliopugini Cordillera del Sarao 13 ICMLH404 EESA988 E. emiliopugini P. Alerce Andino 14 ICMLH467 EEPA1174 E. emiliopugini Huinay 15 ICMLH303 EEHU838 E. emiliopugini Huinay 15 ICMLH304 EEHU837
108 6. REFERENCES
Blotto, B. L., Nuñez, J. J., Basso, N. G. N. G., Úbeda, C. A., Wheeler, W. C. & Faivovich, J. (2013). Phylogenetic relationships of a Patagonian frog radiation, the Alsodes + Eupsophus clade (Anura: Alsodidae), with comments on the supposed paraphyly of Eupsophus. Cladistics, 29, 113–131. Bogart, J. P. (1970). Systematic problems in the amphibian family Leptodactylidae (Anura) as indicated by karyotypic analysis. Cytogenetics, 9, 369–383. Bruschi, D. P., Busin, C. S., Siqueira, S. & Recco-Pimentel, S. M. (2012). Cytogenetic analysis of two species in the Phyllomedusa hypochondrialis group (Anura, Hylidae). Hereditas, 149, 34–40. Castiglia, R., Garagna, S., Merico, V., Oguge, N. & Corti, M. (2006). Cytogenetics of a new cytotype of African Mus (subgenus Nannomys) minutoides (Rodentia, Muridae) from Kenya: C- and G- banding and distribution of (TTAGGG)n telomeric sequences. Chromosome Research, 14, 587–594. Chen, Z. J. & Pikaard, C. S. (1997). Epigenetic silencing of RNA polymerase I transcription: A role for DNA methylation and histone modification in nucleolar dominance. Genes and Development, 11, 2124–2136. Cuevas, C. C. & Formas, J. R. (2003). Cytogenetic analysis of four species of the genus Alsodes (Anura: Leptodactylidae) with comments about the karyological evolution of the genus. Hereditas, 138, 138–147.
109 Fernández, R., Barragán, M. J. L., Bullejos, M., Marchal, J. A., Díaz De La Guardia, R. & Sánchez Baca, A. (2002). New C-band protocol by heat denaturation in the presence of formamide. Hereditas, 137, 145–148. Ferreira, D. C., Oliveira, C. & Foresti, F. (2011). Chromosome mapping of retrotransposable elements Rex1 and Rex3 in three fish species in the subfamily hypoptopomatinae (Teleostei, Siluriformes, Loricariidae). Cytogenetic and Genome Research, 132, 64–70. Formas, J. R. (1978). A new species of leptodactylid frog (Eupsophus) from the coastal range in Southern Chile. Studies on Neotropical Fauna and Environment, 13, 1–9. Formas, J. R. (1991). The karyotypes of the chilean frogs Eupsophus emiliopugini and E. vertebralis (Amphibia: Anura: Leptodactylidae). Proceedings of the Biological Society of Washington, 104, 7–11. Formas, J. R., Lacrampe, S. & Brieva, L. (1992). Allozymic and Morphological Differentiation among three south American Frogs, genus Eupsophus. Comparative Biochemistry and Physiology, 102, 57– 60. Formas, J. R. & Brieva, L. (1994). Advertisement calls and relationships of chilean frogs Eupsophus contulmoensis and E. insularis (Amphibia: Anura: Leptodactylidae). Proceedings of The Biological Society of Washington, 107, 391–397. Formas, R. J. (1989). A new species of Eupsophus (Amphibia: Anura: Leptodactylidae) from Southern Chile. Proceedings of The Biological Society of Washington, 102, 568–576.
110 Frost, D. R. (2013). Amphibian species of the world: an online reference. Version 5.6. Electronic database. Grummt, I. & Pikaard, C. S. (2003). Epigenetic silencing of RNA polymerase I transcription. Nature Reviews Molecular Cell Biology, 4, 641–649. Hillis, D. M. & Dixon, M. T. (1991). Ribosomal DNA: Molecular evolution and phylogenetic inference. The Quarterly Review of Biology, 66, 411– 453. Honjo, T. & Reeder, R. H. (1973). Preferential transcription of Xenopus laevis ribosomal RNA in interspecies hybrids between Xenopus laevis and Xenopus mulleri. Journal of Molecular Biology, 80, 217–228. Howell, W. M. & Black, D. A. (1980). Controlled silver-staining of nucleolus organizer regions with a protective colloidal developer: a 1-step method. Experientia, 36, 1014–1015. Ibarra-Vidal, H., Ortiz, J. C., Torres-Pé rez, F., Ibarra-Vidal, F., Ortiz, J. C. & Torres-Perez, F. (2004). Eupsophus septentrionalis sp., nueva especie de Leptodactylidae (Amphibia) de Chile central. Boletín de La Sociedad Biológica de Concepción, 75, 91–112. Ijdo, J. W., Wells, R. A., Baldini, A. & Reeders, S. T. (1991). Improved telomere detection using a telomere repeat probe (TTAGGG)n generated by PCR. Nucleic Acids Research, 19, 4780. Iturra, P. & Veloso, A. (1988). Further evidence for early sex chromosome differentiation of Anuran species. Genetica, 78, 25–31. King, M. (1991). Chapter 15: The Evolution of Heterochromatin in the
111 Amphibian Genome. In: Green, D & Sessions, S (Eds), Amphibian cytogenetics and evolution, (pp. 359–391). Academic press. United States. King, R. B. (1994). The topology of coordination polyhedra and their rearrangements. Polyhedron, 13, 2005–2016. Kopp, E., Mayr, B. & Schleger, W. (1986). Species-specific non-expression in ribosomal RNA genes in a mammalian hybrid, the mule. Chromosoma, 94, 346–352. Lawrence, R. J., Earley, K., Pontes, O., Silva, M., Chen, Z. J., Neves, N., … Pikaard, C. S. (2004). A concerted DNA methylation/histone methylation switch regulates rRNA gene dosage control and nucleolar dominance. Molecular Cell, 13, 599–609. Lourenco, L. B. & Recco-Pimentel, S. M. (1998). Polymorphism of the nucleolus organizer regions (NORs) in Physalaemus petersi (Amphibia, Anura , Leptodactylidae) detected. Chromosome Research, 6, 621–628. McStay, B. (2016). Nucleolar organizer regions: genomic dark matter requiring illumination. Genes and Development, 30, 1598–1610. Meyne, J., Ratliff, R. L. & Moyzis, R. K. (1989). Conservation of the human telomere sequence (TTAGGG)n among vertebrates. Proceedings of the National Academy of Sciences of the United States of America, 86, 7049–53. Morescalchi, A. (1973). Amphibia. In: Chiarelli, A. B. & Capanna, E. (Eds), Cytotaxonomy and vertebrate evolution (pp. 233–347). Academic
112 Press. England. Navashin, M. S. (1928). Amphiplasty, a new caryological phenomenon. Zindukt Abstrarrtm Vererblehre, 1148–1152. Nogueira, L., Paim, F., Diniz, D., Solé, M., Affonso, P., Siqueira, S. & Sampaio, I. (2015). Cytogenetic analysis of Scinax auratus and Scinax eurydice (Anura, Hylidae) with emphasis on cytotaxonomy. Comparative Cytogenetics, 9, 227–236. Nuñez, J. J. (2003). Taxonomía y sistemática de las ranas del género Eupsophus (Leptodactylidae). Tesis Doctoral, Universidad Austral de Chile, Valdivia. Nuñez, J. J., Rabanal, F. E., formas, J. R. R., Nuñez, J. J., Rabanal, F. E., Formas, J. R. (2012). Description of a new species of Eupsophus (Amphibia: Neobatrachia) from the Valdivian Coastal range, Southern Chile: an integrative taxonomic approach. Zootaxa, 3305, 53–68. Pellegrino K. A., Kasahara, S., Trefaut, R. & Yonenaga-Yassuda, Y. (1997). Pericentric inversion events in karyotypic distinction of Brazilian lizards of genus Phyllopezus (Squamata, Gekkonidae) detected by chromosomal banding patterns. Hereditas, 127, 255 262. Pikaard, C. S. (2000). The epigenetics of nucleolar dominance. Trends in Genetics, 16, 495–500. Pontes, O., Lawrence, R. J., Neves, N., Silva, M., Lee, J.-H., Chen, Z. J., … Pikaard, C. S. (2003). Natural variation in nucleolar dominance reveals the relationship between nucleolus organizer chromatin topology and rRNA gene transcription in Arabidopsis. Proceedings of
113 the National Academy of Sciences, 100, 11418–11423. Preuss, S. & Pikaard, C. S. (2007). rRNA gene silencing and nucleolar dominance: Insights into a chromosome-scale epigenetic on/off switch. Biochimica et Biophysica Acta - Gene Structure and Expression, 1769, 383–392. Rogatcheva, M. B., Ono, T., Sonta, S., Oda, S. & Borodin, P. M. (2002). Robertsonian metacentrics of the house musk shrew (Suncus murinus, Insectivora, Soricidae) lose the telomeric sequences in the centromeric area. Genes & Genetic Systems, 75, 155–158. Ruiz-Herrera, A., García, F., Azzalin, C., Giulotto, E., Egozcue, J., Ponsà, M. & Garcia, M. (2002). Distribution of intrachromosomal telomeric sequences (ITS) on Macaca fascicularis (Primates) chromosomes and their implication for chromosome evolution. Human Genetics, 110, 578–586. Ruiz, I. R. G., Bonaldo, M. F. & Becak, W. (1980). In situ localization of ribosomal genes in a natural triploid of Odontophrynus. Journal of Heredity, 71, 55–57. Sangpakdee, W., Phimphan, S., Tengjaroenkul, B., Pinthong, K., Neeratanaphan, L., & Tanomtong, A. (2017). Cytogenetic study of three Microhylid species (Anura, Microhylidae) from Thailand. Cytologia, 82, 67–74. Schmid, M. (1978). Chromosome banding in Amphibia. Chromosoma, 66, 361–388. Schmid, M., Steinlein, C., Feichtinger, W. & Nanda, I. (2017). Chromosome
114 banding in amphibia. XXXV. Highly mobile nucleolus organizing regions in Craugastor fitzingeri (Anura, Craugastoridae). Cytogenetic and Genome Research, 152, 180–193. Schweizer, D. & Loidl, J. (1987). A Model for Heterochromatin Dispersion and the Evolution of C-Band Patterns. In: Stahl, A., Luciani, J. M., Vagner-Capodano, A. M. (Eds), Chromosomes Today, (pp. 61–74). Springer. Netherlands. Suárez-Villota, E. Y., Vargas,R. A., MArchant, J. E., Kohler, N., Nuñez, J. J., de la Fuente, R. R., … Gallardo, M. H. H. (2012). Distribution of repetitive DNAs and the hybrid origin of the red vizcacha rat (Octodontidae). Genome, 55, 105–117. Suárez-Villota, E. Y., Quercia, C. A., Díaz, L. M., Vera-Sovier, V. & Nuñez, J. J. (2018a). Speciation in a biodiversity hotspot: Phylogenetic relationships, species delimitation, and divergence times of Patagonian ground frogs from the Eupsophus roseus group (Alsodidae). PLOS ONE, 13, e0204968. Suárez-Villota, E. Y., Quercia, C. A. & Nuñez, J. J. (2018b). Mitochondrial genomes of the South American frogs Eupsophus vertebralis and E. emiliopugini (Neobatrachia: Alsodidae) and their phylogenetic relationships . Journal of Genomics, 6, 98–102. Targueta, C. P., Guerra, V., Gambale, P. G., Bastos, R. P., Silva, D. E. & Telles, M. P. (2018). Cytogenetics of two hylid frogs from Brazilian Cerrado. Genetics and Molecular Biology, 41, 814–819. Tucker, S., Vitins, A. & Pikaard, C. S. (2010). Nucleolar dominance and
115 ribosomal RNA gene silencing. Current Opinion in Cell Biology, 22, 351–356. Veloso, A., Celis-Diez, L. J., Guerrero, C. P., Méndez, A. M., Iturra, P. & Simonetti, J. A. (2005). Description of a new Eupsophus species (Amphibia, Leptodactylidae) from the remnants of Maulino forest Central Chile. Herpetological Journal, 15, 159–165. Vitturi, R., Libertini, A., Armetta, F., Sparacino, L. & Colomba, M. S. (2002). Chromosome analysis and FISH mapping of ribosomal DNA (rDNA), telomeric (TTAGGG), Genetica, 105, 189–194. Walker, L., Rojas, M., Flores, S., Spotorno, A. & Manriquez, G. R. (1999). Genomic compatibility between two phyllotine rodent species evaluated through their hybrids. Hereditas, 31, 227–238. Wiley, J. E., Little, M. L., Romano, M. A., Blount, D. A. & Cline, G. R. (1989). Polymorphism in the location of the 18S and 28S rRNA genes on the chromosomes of the diploid-tetraploid treefrogs Hyla chrysoscelis and H. versicolor. Chromosoma, 97, 481–487. Zaleśna, A., Florek, M., Rybacki, M. & Ogielska, M. (2017). Variability of NOR patterns in European water frogs of different genome composition and ploidy level. Comparative Cytogenetics, 11, 249–266. Zlotina, A., Dedukh, D. & Krasikova, A. (2017). Amphibian and avian karyotype evolution: Insights from lampbrush chromosome studies. Genes, 8. 311
116 X. GENERAL DISCUSSION AND CONCLUSIONS
The present thesis, comprised an extensive systematic study with an evolutionary perspective of two endemic frogs’ species of Southern Chile and Argentina: Eupsophus vertebralis and E. emiliopugini. The aims of this work were related to, assess the phylogenetic relationships, to determine dating times and species limits between Eupsophus vertebralis and E. emiliopugini, in addition to the species karyotypes characterization using molecular and classical cytogenetics markers.
Phylogenetic reconstructions recovered the monophyly of the vertebralis group regarding to the roseus group (Bootstrap: 100, PP: 1), but did not recovered the reciprocal monophyly of E. emiliopugini and E. vertebralis (Bootstrap: < 50, PP: < 0.5), showing a polyphyletic pattern with short branches among the lineages of the vertebrlais group, suggesting low genetic differentiation between both species (Knowles & Carstens, 2007). For its part, coalescent species delimitation analyses were incongruent in their results. Here it was found from one
(Congruence index: Ctax = 0.59) to eleven putative species (Ctax = 0.18) within the vertebralis group. These species delimitation results are coincident with different scales of genetic differentiation recovered by the phylogenetic reconstructions. For example, the 11 putative species obtained by GMYC analyses (employing mitochondrial dataset),
117 correspond to the 11 clades recovered in the phylogenetic tree used as input in this analysis. Therefore, according to the observed results, the species delimitation analyses on the vertebralis group would be recovering lineage structure more than species limits (Sukumaran & Knowles, 2017).
Nevertheless, some variants within E. emiliopugini (from Cordillera del Sarao and Pucatrihue localities) were recovered as monophyletic groups, and as putative species by phylogenetic reconstruction and species delimitation analyses, respectively. This suggests that Cordillera del Sarao and Pucatrihue E. emiliopugini variants represent differentiated evolving lineage (De Queiroz, 2007; Freudenstein et al., 2016; Sukumaran & Knowles, 2017; Luo et al., 2018) and therefore have to be consider for conservation efforts. In addition, it is worth mentioning that these variants inhabit one of the few territorial relics with native forest in the Coastal Mountain range.
The divergences time results showed that the lineages within the vertebralis group, are recently diverged (0.040 Mya) in comparison with the lineage of the E. roseus group (0.39 Mya; See ANNEX I). Furthermore, it was possible to infer that the crown ages of the vertebralis group were influenced by interglacial periods during late Pleistocene. The Last Glacial Maximum (LGM) occurred at 0.020 – 0.014 Mya in Southern Chile, and
118 started its deglaciation period at 0.017 Mya (Hulton et al., 2002), nearby the E. emiliopugini (0.027 Mya) and E. vertebralis (0.019 Mya) crown ages.
The karyotype description for both E. emiliopugini and E. vertebralis was performed, first, with the purpose to evaluate the presence of species specific features useful to differentiate between them (i.e. Nogueira et al., 2015; Sangpakdee et al., 2017). Second, with the aim to test the evolutionary hypothesis proposed by Formas (1991) to explain the differences in pair 13 between the karyotypes of E. vertebralis and E. emiliopugini. From the results of classical and cytomolecular analyses (i.e. C-banding, NORs position, telomeric signals), it was not possible to found any marked differences among the karyotypes of E. emiliopugini and E. vertebralis that help to distinguish between these both species. Except by the difference in the chromosome morphology of pair 13 for each species, as was reported previously by Formas (1991). Also, interstitial sequences were not recovered by telomeric FISH, and it was not observed heterochromatic patterns on pairs 13. Therefore, is not possible to corroborated the evolutionary hypothesis described by Formas (1991), regarding chromosomal rearrangements, or heterochromatin addition on pairs 13 of E. vertebralis species. Future researchs that addresses this last problem should consider the generation of a chromosomic probe of pair 13 to perform in situ hybridizations.
119 Interestingly, the presented cytogenetic research revealed a polymorphism related to secondary constriction (SC), active nucleolar organizer regions (NORs), and 28S rDNA genes locations in one specimen of E. emiliopugini collected at Puyehue locality. Here, it was detected 28S FISH signals on pair 4 and 5 (four NOR clusters), of which only one chromosome of each pair showed SC and Ag-NOR positive signal. This result suggests a nucleolar dominance phenomenon on the Puyehue specimen (Preuss & Pikaard, 2007; McStay, 2016), where only two from four NOR clusters are active.
Nucleolar dominance have been described broadly for polyploids and diploid interspecific hybrids (Wallace & Langridge, 1971; Honjo & Reeder, 1973; Kopp et al., 1986; Ruiz et al., 1980; Walker et al., 1999), and consisting in the inactivation of NORs derived from one parental (Pontes et al., 2003) due to an epigenetic switch on/off (promoter methylation and histone modifications) in response to a dosage compensation phenomenon (Chen & Pikaard, 1997; Grummt & Pikaard, 2003; Lawrence et al., 2004; Preuss & Pikaard, 2007; Tucker et al., 2010). However, the molecular mechanisms that “select” which NORs is switch off remain poorly known (Grummt & Pikaard, 2003; Preuss & Pikaard, 2007; McStay, 2016). Therefore, the possibility of a switch on/off in codominance condition (differential silencing for rDNA genes from each parent) could be a plausible explanation for the interspecific hybrid nature of Puyehue specimen. Also, other explanation could be chromosomal rearrangements
120 (Schweizer & Loidl, 1987), or mobile NORs (Schmid et al., 2017). Futures cytomolecular studies, considering other specimens from this locality, and using cytological preparations obtained from different tissues, could be available to test these hypotheses.
The polyphyletic pattern found by phylogenetic reconstructions, the incongruence between species delimitation results, the divergences times and the cytogenetic conserved patterns, suggest that E. vertebralis and E. emiliopugini are recent diverged, and poorly genetically differentiated lineages. In fact, Paniagua et al. (2007, 2019) proposed that Pleistocene climate fluctuations, could generate changes in the biodiversity distributions during the deglaciation periods, probably leading new contacts between groups at intra and interspecific level. This can result in poorly genetic differentiation between nearby related groups exposed at these fluctuations, as the case of the vertebralis group.
The congruence among the species delimitation analyses, suggest the presence of single species in the vertebralis group, nevertheless, there is other kind of evidences that distinguish both E. vertebralis and E. emiliougini as separately species. On the one hand, cytogenetic analyses from this study reveals that telocentric pair 13 in E. vertebralis karyotypes and metacentric pair 13 in E. emiliopugini karyotypes are conserved at species level without records of population variations. On the other hand, advertisement calls measures reported differences in the
121 notes per call between E. vertebralis and E. emiliopugini (Formas & Brieva, 1994). This last trait is particularly important in amphibians, due to its capability of reproduction is biased by the mate recognition guided by specifics features of its calls (Passmore, 1981). Therefore, despite that future studies can address better the differences in the advertisement calls between these both species, with the actuals evidence, and accordingly with an integrative framework (Padial et al., 2010), I refrain to merge E. vertebralis with E. emiliopugini, contrary, I support both species are maintained as separately and suggest that they are in a relatively recent and ongoing speciation process.
This research provides the first phylogenetic relationship inference in an extensive geographical scale within the vertebralis group, and contributes with the understanding of evolutionary history of endemic amphibian biodiversity at Southern Chile. Also, offer a clear example of incongruences among species delimitation results when the groups in question are in “gray zones” as the vertebralis group. This, demonstrate the necessity to use evidence from different approaches in an integrative framework at the time to defined species. Moreover, this study conferred new evidence for the karyotype description of E. verterbalis and E. emiliopugini species, and highlight a newly and interesting case of nucleolar dominance that need future studies.
122 XI. GENERAL REFERENCES
Agapow, P. M., Bininda-Emonds, O. R. P., Crandall, K. A., Gittleman, J. L., Mace, G. M., Marshall, J. C. & Purvis, A. (2004). The impact of species concept on biodiversity studies. Quarterly Review of Biology, 79, 162– 178.
Aldhebiani, A. Y. (2018). Species concept and speciation. Saudi Journal of Biological Sciences, 25, 437–440.
Attard, C. R. M., Jenner, K. C. S., Jenner, M.-N. M., Beheregaray, L. B., Moller, L. M., Gill, P. C., … Teske, P. R. (2015). Low genetic diversity in pygmy blue whales is due to climate-induced diversification rather than anthropogenic impacts. Biology Letters, 11, 20141037–20141037.
Ax, P. (1999). Multicellular Animals: Volumen II: The Phylogenetic System of the Metazo. Springer Science & Business Media. Germany
Bakloushinskaya, I. Y. (2017). Chromosomal rearrangements, genome reorganization, and speciation. Biology Bulletin, 43, 759–775.
Balakrishnan, R. (2005). Species concepts, species boundaries and species identification: a view from the tropics. Systematic Biology, 54, 689– 693.
Barth, A., Vences, M., Solé, M. & Costa, M. A. (2014). Molecular cytogenetics and phylogenetic analysis of Brazilian leaf frog species of the genera Phyllomedusa and Phasmahyla (Hylidae:
123 Phyllomedusinae). Canadian Journal of Zoology, 92, 795–802.
Blotto, B. L., Nuñez, J. J., Basso, N. G., Úbeda, C. A., Wheeler, W. C. & Faivovich, J. J. (2013). Phylogenetic relationships of a Patagonian frog radiation, the Alsodes + Eupsophus clade (Anura: Alsodidae), with comments on the supposed paraphyly of Eupsophus. Cladistics, 29, 113–131.
Bock, W. J. (2004). Species: the concept, category and taxon. Journal of Zooology and Evolution Research, 9, 178–190.
Bolzán, A. D. (2012). Chromosomal aberrations involving telomeres and interstitial telomeric sequences. Mutagenesis, 27, 1–15.
Bruschi, D. P., Busin, C. S., Siqueira, S. & Recco-Pimentel, S. M. (2012). Cytogenetic analysis of two species in the Phyllomedusa hypochondrialis group (Anura, Hylidae). Hereditas, 149, 34–40.
Bruschi, D. P., Rivera, M., Lima, A. P., Zúñiga, A. B. & Recco-Pimentel, S. M. (2014). Interstitial Telomeric Sequences (ITS) and major rDNA mapping reveal insights into the karyotypical evolution of Neotropical leaf frogs species (Phyllomedusa, Hylidae, Anura). Molecular Cytogenetics, 7, 22.
Camargo, A. & Sites, J. J. (2013). Chapter 9: Species Delimitation: A Decade After the Renaissance. In: Pavlinov, I. (Ed). The Species Problem: Ongoing Issues (pp. 225–247). InTech Open. England.
124 Carstens, B. C. & Dewey, T. A. (2010). Species delimitation using a combined coalescent and information-theoretic approach: an example from north American Myotis bats. Systematic Biology, 59, 400–414.
Carstens, B. C., Pelletier, T. A., Reid, N. M., & Satler, J. D. (2013). How to fail at species delimitation. Molecular Ecology, 22, 4369–4383.
Chen, Z. J. & Pikaard, C. S. (1997). Epigenetic silencing of RNA polymerase I transcription: A role for DNA methylation and histone modification in nucleolar dominance. Genes and Development, 11, 2124–2136.
Correa, C. & Durán, F. (2019) Taxonomy, systematics, and geographic distribution of ground frogs (Alsodidae, Eupsophus): a comprehensive synthesis of the last six decades of research. Zookeys, 863, 107–152.
Correa, C., Vásquez, D., Castro-Carrasco, C., Zúñiga-Reinoso, Á., Ortiz, J. C. & Palma, R. E. (2017). Species delimitation in frogs from South American temperate forests: the case of Eupsophus, a taxonomically complex genus with high phenotypic variation. PLOS ONE, 12, e0181026.
Correa, C., Veloso, A., Iturra, P. & Mendez, A. M. (2006). Phylogenetic relationships of Chilean leptodactylids: a molecular approach based on mitochondrial genes 12S and 16S. Revista Chilena de Historia Natural, 79, 435–450.
125 Cosacov, A., Sérsic, A. N., Sosa, V., Johnson, L. A. & Cocucci, A. A. (2010). Multiple periglacial refugia in the Patagonian steppe and post-glacial colonization of the Andes: The phylogeography of Calceolaria polyrhiza. Journal of Biogeography, 37, 1463–1477.
Coyne, J. A. & Orr, H. A. (2004). Chapter 2: Studying Speciation. In: Coyne, J. A. & Orr, H. A. Speciation (pp. 55–82). Sinauer Associates. United States.
Cuevas, C. C. (2008). Intraspecific chromosomic divergence in Batrachyla antartandica (Ceratophryidae) from Southern Chile: further evidences of cytogenetics diversity in frogs. International Journal of Cytology, Cytosystematics and Cytogenetics, 6, 10–18.
De Queiroz, K. (2007). Species concepts and species delimitation. Systematic Biology, 56, 879–886.
Denton, G. H., Lowell, T. V., Heusser, C. J., Schlüchter, C., Andersen, B. G., Heusser, L. E., … Marchant, D. R. (1999). Geomorphology, stratigraphy, and radiocarbon chronology of Llanquihue drift in the area of the southern Lake District, Seno Reloncavi, and Isla Grande de Chiloé, Chile. Geografiska Annaler, 81, 167–229.
Dayrat, B. (2005) Towards integrative taxonomy. Biological Journal of the Linnean Society, 85, 407–415.
Dirzo, R. & Raven, P. H. (2003). Global State of Biodiversity and Loss.
126 Annual Review of Environment and Resources, 28, 137–167.
Duellman, W. D. (1999). Patterns of Distribution in Amphibians: A Global Perspective. Systematic Biology, 51, 980–981.
Fitzpatrick, S. W., Brasileiro, C. A., Haddad, C. F. B. & Zamudio, K. R. (2009). Geographical variation in genetic structure of an Atlantic Coastal Forest frog reveals regional differences in habitat stability. Molecular Ecology, 18, 2877–2896.
Formas, J. R. (1978). A new species of leptodactylid frog (Eupsophus) from the coastal range in Southern Chile. Studies on Neotropical Fauna and Environment, 13, 1–9.
Formas, J. R. (1980). The chromosomes of E. calcaratus and the karyological evolution of the genus Eupsophus (Anura: Leptodactylidae). Experientia, 36, 1163–1164
Formas, R. J. (1989). A new species of Eupsophus (Amphibia: Anura: Leptodactylidae) from Southern Chile. Proceedings of the Biological Society of Washington, 102, 568–576.
Formas, J. R. (1991). The karyotypes of the Chilean frogs Eupsophus emiliopugini and E. vertebralis (Amphibia: Anura: Leptodactylidae). Proceedings of the Biological Society of Washington, 104, 7–11.
Formas, J. R. (1992). The tadpole of Eupsophus vertebralis (Anura: Leptodactylidae). Herpetologica, 48, 115–119.
127 Formas, J. R. & Brieva, L. (1994). Advertisement calls and relationships of Chilean frogs Eupsophus contulmoensis. Proceedings of The Biological Society of Washington, 107, 391–397.
Formas, J. R., Lacrampe, S. & Brieva, L. (1992). Allozymic and morphological differentiation among three south American frogs, genus Eupsophus. Comparative Biochemistry and Physiology 102, 57– 60.
Formas, J. R., Nuñez J. J. & Cuevas, C. (2008). Identidad de la rana austral Chilena Eupsophus coppigeri (Amphibia, Anura, Neobatrachia): evidencias morfológicas, cromosómicas y moleculares. Revista Chilena de Historia Natural, 81, 3–20.
Formas, J. R. & Vera, M. A. (1980). Reproductive Patterns of Eupsophus roseus and E. vittatus. Journal of Herpetology, 14, 11.
Freudenstein, J. V., Broe, M. B., Folk, R. A. & Sinn, B. T. (2016). Biodiversity and the species concept - lineage are not enough. Systematic Biology, 66, 644-656.
Fujisawa, T. & Barraclough, T. G. (2013). Delimiting species using single- locus data and the Generalized Mixed Yule Coalescent approach: a revised method and evaluation on simulated data sets. Systematic Biology, 62, 707–724.
Fujita, M. K., Leache, A. D., Burbrink, F. T., McGuire, J. A., Moritz, C., Leaché,
128 A. D., … Moritz, C. (2012). Coalescent-based species delimitation in an integrative taxonomy. Trends in Ecology & Evolution, 27, 480–488.
Gazoni, T. (2012). Cytogenetic analyses of eight species in the genus Leptodactylus Fitzinger, 1843 (Amphibia, Anura, Leptodactylidae ), including a new diploid number and a karyotype with multiple translocations. BMC Genetics, 13, 1–16.
González-Ittig, R. E., Rossi-Fraire, H. J., Cantoni, G. E., Herrero, E. R., Benedetti, R., Gallardo, M. H. & Gardenal, C. N. (2010). Population genetic structure of long-tailed pygmy rice rats (Oligoryzomys longicaudatus) from Argentina and Chile based on the mitochondrial control region. Canadian Journal of Zoology, 88, 23–35.
Grummt, I. & Pikaard, C. S. (2003). Epigenetic silencing of RNA polymerase I transcription. Nature Reviews Molecular Cell Biology, 4, 641–649.
Harrington, R. C. & Near, T. J. (2012). Phylogenetic and coalescent strategies of species delimitation in snubnose darters (Percidae: Etheostoma). Systematic Biology, 61, 63–79.
Heled, J. & Drummond, A. J. (2010). Bayesian inference of species trees from multilocus data. Molecular Biology and Evolution, 27, 570–580.
Hey, J. (2001). The mind of the species problem. Trends in Ecology and Evolution, 16, 326–329.
129 Hillis, D. M. (1991). Chapter 2: The phylogeny of amphibians: current knowledge and the role of cytogenetics. In: Green, D & Sessions, S (Eds), Amphibian cytogenetics and evolution, (pp. 7–31). Academic press. United States. Honjo, T. & Reeder, R. H. (1973). Preferential transcription of Xenopus laevis ribosomal RNA in interspecies hybrids between Xenopus laevis and Xenopus mulleri. Journal of Molecular Biology, 80, 217–228.
Hulton, N. R. J., Purves, R. S., McCulloch, R. D., Sugden, D. E. & Bentley, M. J. (2002). The Last Glacial Maximum and deglaciation in southern South America. Quaternary Science Reviews, 22, 751–757.
Ibarra-Vidal, H., Ortiz, J. C., Torres-Pé rez, F., Ibarra-Vidal, F., Ortiz, J. C. & Torres-Perez, F. (2004). Eupsophus septentrionalis sp., nueva especie de Leptodactylidae (Amphibia) de Chile central. Boletín de La Sociedad Biológica de Concepción, 75, 91–112.
IUCN. (2019). The IUCN Red List of Threatened Species. Electronic database.
Kingman, J. (1982). Genealogy Populations. Journal of Applied Probability, 19, 27–43.
Knowles, L. L. & Carstens, B. C. (2007). Delimiting species without monophyletic gene trees. Systematic Biology, 56, 887–895.
Kopp, E., Mayr, B. & Schleger, W. (1986). Species-specific non-expression
130 in ribosomal RNA genes in a mammalian hybrid, the mule. Chromosoma, 94, 346–352.
Kubatko, L. S., Carstens, B. C. & Knowles, L. L. (2009). STEM: species tree estimation using maximum likelihood for gene trees under coalescence. Bioinformatics, 25, 971–973.
Lausch, A., Bannehr, L., Beckmann, M., Boehm, C., Feilhauer, H., Hacker, J. M., … Cord, A. F. (2016). Linking earth observation and taxonomic, structural and functional biodiversity: Local to ecosystem perspectives. Ecological Indicators, 70, 317–339.
Lawrence, R. J., Earley, K., Pontes, O., Silva, M., Chen, Z. J., Neves, N., … Pikaard, C. S. (2004). A concerted DNA methylation/histone methylation switch regulates rRNA gene dosage control and nucleolar dominance. Molecular Cell, 13, 599–609.
León-Paniagua, L. & Guevara, L. (2019). Quaternary range-shifts of arboreal rodents of the genus Habromys (Cricetidae, Neotominae) in Mesoamerica and their evolutionary consequences. Mammalian Biology, 94, 4–10.
Léon-Paniagua, L., Navarro-Siguenza, A. G., Hernández-Baños, B. E. & Morales, J. C. (2007). Diversification of the arboreal mice of the genus Habromys (Rodentia: Cricetidae: Neotominae) in the Mesoamerican highlands. Molecular phylogenetics and evolution, 42 653 664.
131 Lessa, E. P., D’ElÍa, G. & Pardiñas, U. F. J. (2010). Genetic footprints of late Quaternary climate change in the diversity of Patagonian-Fueguian rodents. Molecular Ecology, 19, 3031–3037.
Liu, L., Pearl, D. K., Brumfield, R. T., Edwards, S. V. & Knowles, L. (2008). Estimating species trees using multiple-allele DNA sequence data. Evolution, 62, 2080–2091.
Liu, L., Yu, L., Kubatko, L., Pearl, D. K. & Edwards, S. V. (2009). Coalescent methods for estimating phylogenetic trees. Molecular Phylogenetics and Evolution, 53, 320–328.
Lobos, G., Vidal, M., Correa, C., Labra, A., Díaz-Páez, H., Charrier, A., Rabanal, F., Díaz, S. & Tala, C. (2014) Anfibios de Chile, un Desafío para la Conservación. Ministerio del Medio ambiente, Fundación Facultad de Ciencias Veterinarias y Pecuarias de la Universidad de Chile y Red Chilena de Herpetología, Santiago.
Luo, A., Ling, C., Ho, S. Y. W. & Zhu, C.D. (2018). Comparison of methods for molecular species delimitation across a range of speciation scenarios. Systematic Biology, 0, 1–13.
Mace, G. M. (2004). The role of taxonomy in species conservation. Philosophical Transactions of the Royal Society B: Biological Sciences, 359, 711–719.
Maddison, W. P. & Knowles, L. L. (2006). Inferring phylogeny despite
132 incomplete lineage sorting. Systematic Biology, 55, 21–30.
McCulloch, R. D., Sugden, D. E., Purves, R. S., Clapperton, C. M., Hulton, N. R. J., Bentley, M. J. & McCulloch, R. D. (2000). Climatic inferences from glacial and palaeoecological evidence at the last glacial termination, southern South America. Journal of Quaternary Science, 15, 409–417.
McStay, B. (2016). Nucleolar organizer regions: genomic dark matter requiring illumination. Genes and Development, 30, 1598–1610.
Muñoz-Mendoza, C., D’Elía, G., Panzera, A., Méndez T., M. A., Villalobos- Leiva, A., Sites, J. W. & Victoriano, P. F. (2017). Geography and past climate changes have shaped the evolution of a widespread lizard from the Chilean hotspot. Molecular Phylogenetics and Evolution, 116, 157–171.
Mutanen, M., Kivelä, S. M., Vos, R. A., Doorenweerd, C., Ratnasingham, S., Hausmann, A., … Godfray, H. C. J. (2016). Species-level para- and polyphyly in DNA barcode gene trees: Strong operational bias in European Lepidoptera. Systematic Biology, 65, 1024–1040.
Nogueira, L., Paim, F., Diniz, D., Solé, M., Affonso, P., Siqueira, S. & Sampaio, I. (2015). Cytogenetic analysis of Scinax auratus and Scinax eurydice (Anura, Hylidae) with emphasis on cytotaxonomy. Comparative Cytogenetics, 9, 227–236.
Nuñez, J. J. (2003). Taxonomía y sistemática de las ranas del género
133 Eupsophus (Leptodactylidae). Tesis Doctoral, Universidad Austral de Chile, Valdivia.
Nuñez, J. J., Rabanal, F. E. & formas, J. R. R. (2012). Description of a new species of Eupsophus (Amphibia: Neobatrachia) from the Valdivian Coastal range, Southern Chile: an integrative taxonomic approach. Zootaxa, 3305, 53–68.
Nuñez, J. J., Wood, K. N., Rabanal, E. F., Fontanelia, M. F. & Sites, W. J., (2011). Amphibian phylogeography in the Antipodes: Refugia and postglacial colonization explain mitochondrial haplotype distribution in the Patagonian frog Eupsophus calcaratus (Cycloramphidae). Molecular Phylogenetics and Evolution, 58, 343–352.
O’Meara, B. C. (2010). New heuristic methods for joint species delimitation and species tree inference. Systematic Biology, 59, 59–73.
Padial, J. M., Miralles, A., de la Riva, I. & Vences, M. (2010). The integrative future of taxonomy. Frontiers in Zoology, 7, 1-14
Pante, E., Schoelinck, C. & Puillandre, N. (2014) From integrative taxonomy to species description: One step beyond. Systematic Biology, 64, 152-160
Paskoff, R. P. (1977). Quaternary of Chile: The state of research. Quaternary Research, 8, 2–31.
Passmore, L. N. (1981). The relevance of the specific mate recognition
134 concept to anuran reproductive biology. Monitore Zoologico Italiano, 15, 93–108. Pérez-Alvarez, M. J., Olavarría, C., Moraga, R., Baker, C. S., Hamner, R. M. & Poulin, E. (2016). Historical dimensions of population structure in a continuously distributed marine species: The case of the endemic Chilean dolphin. Scientific Reports, 6, 1–11.
Pons, J., Barraclough, T. G. T. G., Gomez-Zurita, J., Cardoso, A., Duran, D. P. D. P., Hazell, S., … Vogler, A. P. (2006). Sequence-based species delimitation for the DNA taxonomy of undescribed insects. Systematic Biology, 55, 595–609.
Pontes, O., Lawrence, R. J., Neves, N., Silva, M., Lee, J.-H., Chen, Z. J., … Pikaard, C. S. (2003). Natural variation in nucleolar dominance reveals the relationship between nucleolus organizer chromatin topology and rRNA gene transcription in Arabidopsis. Proceedings of the National Academy of Sciences, 100, 11418–11423.
Preuss, S. & Pikaard, C. S. (2007). rRNA gene silencing and nucleolar dominance: Insights into a chromosome-scale epigenetic on/off switch. Biochimica et Biophysica Acta - Gene Structure and Expression, 1769, 383–392.
Prohl, H., Ron, S. R. & Ryan, M. J. (2010). Ecological and genetic divergence between two lineages of Middle American túngara frogs Physalaemus (=Engystomops) pustulosus. BMC Evolutionary Biology, 10, 146.
135 Puillandre, N., Modica, M. V., Zhang, Y., Sirovich, L., Boisselier, M. -C., Cruaud, C., Holford, M. & Samadi, S. (2012). Large-scale species delimitation method for hyperdiverse groups. Molecular Ecology, 21, 2671–2691.
Pyron, R. A. & Wiens, J. john. (2011). A large-scale phylogeny of Amphibia including over 2800 species, and a revised classification of extant frogs, salamanders, and caecilians. Molecular Phylogenetic and Evolution, 61, 543–583.
Rabassa, J. & Clapperton, C. M. (1990). Quaternary glaciations in the southern hemisphere: An overview. Quaternary Science Reviews, 9, 299–304.
Rabassa, J., Coronato, A. & Martínez, O. (2011). Late Cenozoic glaciations in Patagonia and Tierra del Fuego: An updated review. Biological Journal of the Linnean Society, 103, 316–335.
Rannala, B. (2015). The art and science of species delimitation. Current Zoology, 61, 846–853.
RCE (2019). Decreto supremo N° 25 de 2011, Reglamento para la clasificación de Especies Silvestres según Estado de Conservación. Ministerio de Medio ambiente, Santiago de Chile.
Rieseber, L. H. (2001). Chromosomal Rearrangements and Speciation. Trends in ecology and evolution, 16, 351–358.
136 Rojas, R.R., Fouquet, A., Ron, S. R., Hernández-Ruz, E. J., Melo-Sampaio, P. R., Chaparro, J. C., Vogt, R. C., …. Hrbek, R. (2018). A Pan-Amazonian species delimitation: high species diversity within the genus Amazophrynella (Anura: Bufonidae). PeerJ, 6, e4941.
Ruiz, I. R. G., Bonaldo, M. F. & Becak, W. (1980). In situ localization of ribosomal genes in a natural triploid of Odontophrynus. Journal of Heredity, 71, 55–57.
Ruzzante, D. E., Walde, S. J., Cussac, V. E., Dalebout, M. L., Seibert, J., Ortubay, S. & Habit, E. (2006). Phylogeography of the Percichthyidae (Pisces) in Patagonia: Roles of orogeny, glaciation, and volcanism. Molecular Ecology, 15, 2949–2968.
Sánchez-García, A., & Castresana, J. (2012). Impact of deep coalescence on the reliability of species tree inference from different types of DNA markers in mammals. PLOS ONE, 7, e30239.
Sangpakdee, W., Phimphan, S., Tengjaroenkul, B., Pinthong, K., Neeratanaphan, L. & Tanomtong, A. (2017). Cytogenetic Study of three Microhylid species (Anura, Microhylidae) from Thailand. Cytologia, 82, 67–74.
Savage, J. M. (1995). Systematics and the Biodiversity Crisis. BioScience, 45, 673–679.
Schmid, M., Steinlein, C., Feichtinger, W. & Nanda, I. (2017). Chromosome
137 banding in amphibia. XXXV. Highly mobile nucleolus organizing regions in Craugastor fitzingeri (Anura, Craugastoridae). Cytogenetic and Genome Research, 152, 180–193.
Schweizer, D. & Loidl, J. (1987). A Model for Heterochromatin Dispersion and the Evolution of C-Band Patterns. In: Stahl, A., Luciani, J. M., Vagner-Capodano, A. M. (Eds), Chromosomes Today, (pp. 61–74). Springer. Dordrecht, Netherlands. Sites, J. W. & Marshall, J. C. (2003). Delimiting species: a renaissance issue in systematic biology. Trends in Ecology and Evolution, 19, 462–470.
Sites, J. W. & Marshall, J. C. (2004). Operational criteria for delimiting species. Annual Review of Ecology, Evolution and Systematics, 35, 199– 227.
Sokal, R. R., Crovello, T. J., The, S., Naturalist, A. & Apr, N. M. (1970). The Biological Species Concept: A Critical Evaluation. The American Society of Naturalists, 104, 127–153.
Suárez-Villota, E. Y., Quercia, C. A., Díaz, L. M., Vera-Sovier, V. & Nuñez, J. J. (2018a). Speciation in a biodiversity hotspot: Phylogenetic relationships, species delimitation, and divergence times of Patagonian ground frogs from the Eupsophus roseus group (Alsodidae). PLOS ONE, 13, e0204968.
Suárez-Villota, E. Y., Quercia, C. A. & Nuñez, J. J. (2018b). Mitochondrial genomes of the South American frogs Eupsophus vertebralis and E.
138 emiliopugini (Neobatrachia: Alsodidae) and their phylogenetic relationships . Journal of Genomics, 6, 98–102.
Sukumaran, J. & Knowles, L. L. (2017). Multispecies coalescent delimits structure, not species. PNAS, 114, 1607–1612.
Tucker, S., Vitins, A. & Pikaard, C. S. (2010). Nucleolar dominance and ribosomal RNA gene silencing. Current Opinion in Cell Biology, 22, 351–356.
Úbeda, C. A. (1998). Batracofauna de los bosques templados patagónicos: un enfoque ecobiogeográfico. Tesis Doctoral, Universidad de Buenos Aires, Argentina, 1–353.
Úbeda, C. & Nuñez, J. (2006). New parental care behaviours in two telmatobiine genera from temperate Patagonian forests: Batrachyla and Eupsophus (Anura: Leptodactylidae). Amphibia-Reptilia, 27, 441– 444.
Veloso, A., Celis-Diez, L. J., Guerrero, C. P., Méndez, A. M., Iturra, P. & Simonetti, A. J. (2005). Description of a new Eupsophus species (Ampphibia, eptodactylidae) from the remnants of Maulino forest central Chile. Herpetological Journal, 15, 159–165.
Vera-Escalona, I., Habit, E. & Ruzzante, D. E. (2019). Invasive species and postglacial colonization: Their effects on the genetic diversity of a Patagonian fish. Proceedings of the Royal Society B, 286, 20182567.
139 Victoriano, P. F., Ortiz, J. C., Benavides, E., Adams, B. J. & Sites, J. W. (2008). Comparative phylogeography of codistributed species of Chilean Liolaemus (Squamata: Tropiduridae) from the central-southern Andean range. Molecular Ecology, 17, 2397–2416.
Villagrán, M. & Armesto, J. J. (2005). Fitogeografía histórica de la Cordillera de la Costa de Chile. Editorial Universitaria, Santiago Chile.
Vitelli, L., Batistoni, R., Andronico, F., Nardi, I. & Barsacchi-Pilone, G. (1982). Chromosomal localization of 18S + 28S and 5S ribosomal RNA genes in evolutionarily diverse anuran amphibians. Chromosoma, 84, 475–491.
Walker, L., Rojas, M., Flores, S., Spotorno, A. & Manriquez, G. R. (1999). Genomic compatibility between two phyllotine rodent species evaluated through their hybrids. Hereditas, 31, 227–238. Wallace, H. & Langridge, W. H. R. (1971). Differential amphiplasty and the control of ribosomal RNA synthesis, Heredity, 27, 1–13.
Ward, R. D., Skibinski, D. O. F. & Woodwark, M. (1992). Protein heterozygosity, protein structure, and taxonomic differentiation. Evolutionary Biology, 26, 73–159.
Will, K. W., Mishler, B. D. & Wheeler, Q. D. (2005). The perils of DNA barcoding and the need for integrative taxonomy. Systematic Biology, 54, 844–851.
140 Wheeler, Q. & Meier, R. (2000). Species Concepts and Phylogenetic Theory: A Debate. Columbia University Press.
Wiens, J. J. (2007). Species delimitation: new approaches for discovering diversity. Systematic Biology, 56, 875–878.
Xu, B. & Yang, Z. (2016). Challenges in species tree estimation under the multispecies coalescent model. Genetics, 204, 1353–1368.
Yang, Z., & Rannala, B. (2010). Bayesian species delimitation using multilocus sequence data. of the National Academy of Sciences, 107(20), 1–6.
Yang, Z. & Ziheng, Y. (2015). The BPP program for species tree estimation and species delimitation. Current Zoology, 61, 854–865.
Zemlak, T. S., Habit, E. M., Walde, S. J., Battini, M. A., Adams, E. D. M. & Ruzzante, D. E. (2008). Across the southern Andes on fin: Glacial refugia, drainage reversals and a secondary contact zone revealed by the phylogeographical signal of Galaxias platei in Patagonia. Molecular Ecology, 17, 5049–5061.
Zhang, C. & Yang, Z. (2011). Evaluation of a bayesian coalescent method of species delimitation. Systematic Biology, 60, 747–761.
141