Origin and Diversification of the Genera Aratinga, Eupsittula, and Psittacara (Aves: Psittacidae)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This is a thorough investigation of the historical biogeography of several genera of New World parrots. The species sampling is good and the illustrations are clear and to the point. However, there is one major drawback which is that the sequence data are limited to two mitochondrial genes (1610 bp). This makes the phylogenetic results too uncertain for detailed biogeographic analysis.

A comprehensive phylogeny of the parrots and parakeets was published in 2023, and is cited by the authors as reference no. 55. This study was based on 1000s of nuclear markers and taxa were equally well, or even better, sampled than in the present study. I have compared the phylogenies of this study with that of Smith et al. (2023) and found numerous differences in topology, both at species level and among genera, that likely affect inferences of historical biogeography.

Thus, I recommend that the authors redo their study but use the topology of the study by Smith et al. (2023) as their basis. This would still be a very useful study worthy of publication.

Author Response

Regarding review of our submission, below is the list of responses to your comments/queries. As you will notice, all suggestions were attended and duly replied. We hope that this contribution is now in an acceptable form for publication in Diversity. We thank your consideration and peer review which will undoubtedly end in better communication. We will gladly consider further suggestions. 

Comment:

This is a thorough investigation of the historical biogeography of several genera of New World parrots. The species sampling is good and the illustrations are clear and to the point.

However, there is one major drawback which is that the sequence data are limited to two mitochondrial genes (1610 bp). This makes the phylogenetic results too uncertain for detailed biogeographic analysis. A comprehensive phylogeny of the parrots and parakeets was published in 2023, and is cited by the authors as reference no. 55. This study was based on 1000s of nuclear markers and taxa were equally well, or even better, sampled than in the present study. I have compared the phylogenies of this study with that of Smith et al. (2023) and found numerous differences in topology, both at species level and among genera, that likely affect inferences of historical biogeography. Thus, I recommend that the authors redo their study but use the topology of the study by Smith et al. (2023) as their basis. This would still be a very useful study worthy of publication.

Answer:

In the work titled “Phylogenomic Analysis of the Parrots of the World Distinguishes Artifactual from Biological Sources of Gene Tree Discordance”, Smith et al. (2023) examine the impact of heterogenous data quality among terminals and subclades in establishing evolutionary relationships in parrots (Order: Psittaciformes). They estimated trees using concatenated nuclear ultraconserved elements (UCEs) under Maximum likelihood method and gene trees using ASTRAL-III, a multispecies coalescent tool for estimating an unrooted species tree given a set of unrooted gene trees. Their aim was to assess evolutionary relationships and the sensitivity of those relationships to the information content available in the DNA sequence alignment.

Why the sensitivity to the information content available in the DNA sequence alignment? Of the 382 parrot species analyzed, 121 were museum specimens (skin), which present low-quality DNA, that is, degraded DNA with loss of regions and showing short sequences.

UCEs are highly conserved regions (95-100%) of organismal genomes shared among evolutionary distant taxa. It is likely that UCEs may be regulatory sequences and/or enhancers of genetic expression, knowing in molecular biology as elements (not genes), although this has not been demonstrated, and their locations (loci) are identical in several vertebrate genomes. Alignment of UCEs loci for phylogenetic analysis must be performed under strict filtering conditions so that no loci are missing from any species.

The authors obtained 3242 loci (UCEs) corresponding to sequences of 100-1572 bp, however, they say that there were between 18 to 3086 missing loci per taxon with a mean of +/- SD of 198 +/- 327. Alignments were made with IQ-TREE2. A phylogenetic tree was constructed with the concatenated UCEs (loci) of each species, including those species with missing loci, using ML method. There were missing loci in both the skin samples (museum) and the fresh tissue samples. In addition, they constructed gene trees from locus alignments where heterozygous sites had ambiguity code in IQ-TREE2. That is, they obtained UCE sequences with a signal of presence of heterozygosity, which suggests the presence of fragments that do not correspond to ultraconserved elements that must be single-copy. It is important to note that phylogenomic analyses based on coding sequences use orthologous single-copy genes.

These authors determined how the concatenated UCEs and gene trees topology changed by filtering individuals/taxa (excluding genera and species) based on two metrics that reflect data quality: The deviation in number of parsimony informative sites (dPIS), which can indicate which individuals have fewer or most PIS than expected for a given locus, and the number of missing loci for each individual (Taxa). They found that many of the cases of nonmonophyly of genera were driven by low-quality samples (sequences). Thus, as can be seen in the Supplementary File Figures S1-S27, Smith et al. (2023) filtered out low-quality samples in their alignments to demonstrate artefactual sources of tree discordance, ultimately ruling out several parrot genera and species. For example, in Arini tribe the genera Rhynchopsitta, Cyanopsittaand Orthopsittaca were discarded, which logically changes the topology of the tree. The dating tree was made with the Maximum likelihood tree estimated in IQ-TREE2 with concatenated alignments and all individuals (Taxa), that is, with missing loci.

The objective of our work differs from that of Smith et al. (2023). The result of the construction of any molecular phylogenetic tree is a hypothesis. We make a proposal with sequences that, being in mitochondrial DNA share the same evolutionary history, and have been described as the ideal molecular marker (Avise, 2000). We consider that the hypotheses that are built based on this information cannot simply be ignored by the perception that they are less impressive in the data set. mtDNA is haploid and transmitted to offspring through the maternal line and exhibits clonal expansion, increasing variance in the population, while nuclear genes recombine by sex in each generation. So, the two genomes generate variability through different mechanisms. The mtDNA variations distinguish species and are extensive between populations of the same species and influence their interaction with nuclear genome. The different mtDNA haplotypes have been maintained and selected in nature due to mito-nuclear interactions (Lechuga et al. 2020). We used concatenated sequences of cytochrome c oxidase subunit I (COI) and NADH dehydrogenase subunit 2 (ND2) (in total 1610 characters) two highly conserved mitochondrial genes.

Our data set was 94.4% (17/18) of the genera recognized in the Arini tribe, with which we built phylogenetic trees using Maximum likelihood (ML) and bayesian inference (BI) methods, and reported a consensus tree.

To show the value, the weight and security of the mtDNA used in our analyses, we note the following: in Smith et al. (2023) analyses, clades with poorly supported relationships are shown, surprisingly matching the weakly supported clades in our analysis. On the other hand, see the relationship of Ara and Primolius genera, in Smith et al. (2023) the support value is BP = 95, and in our analysis is PP = 0.98 and BP = 71. The uncertainty of the C. carolinensis relationships within the Aratinga genus remains; in Smith et al. (2023) it is supported by a value of BP = 88, and in our work, it is of PP = 0.77 and BP = 78. We identified other relationships widely supported in Smith et al. (2023), as in our analyses. For example, in Psittacara, where topologies differed because of the placement of taxa such as P. brevipes, the concatenated UCEs tree (Smith et al. 2023) had the taxon nested within the geographic and phenotypic species complex of P. holochlorus, which was consistent with our results, whereas in the genes tree (Smith et al 2023) the taxon was outside of that complex. Smith et al. (2023) conclude that it is more likely that the genes tree topology is aberrant for that subclade because in agreement with dPIS predicted scores, there were 303 to 44 loci favoring the concatenated (UCEs) over the genes tree, respectively. In our discussion we include these comparisons that support the accuracy and topology of the tree built with mtDNA under ML and BI.

Bayesian analyses do not assume large samples, typically smaller data set can be analyzed without losing power while retaining precision. Bayesian inference allows prior experience (“the prior”). Both the prior distribution and the data are used to get the final result. The prior information may be especially important when there is not much data. Therefore, the resulting tree under Bayesian inference from our work shows acceptable support.

Smith et al. (2023) finally filtered low quality samples (different genera and species) in their alignments to demonstrate the artefactual sources of concatenated and gene trees discordance. Moreover, they conclude: “Collectively, our results show that neither the concatenated nor the species (genes) tree is likely to be completely correct.”

For the above reasons, we consider that it is not necessary to redo our study (our phylogenetic analyzes are reliable) and it is worthy of publication.

On the other hand, our study focuses on the genera Aratinga, Eupsittula and Psittacara, which occupy the more northern distribution ranks of all the Arini tribe and have representative species in South America. We propose the early divergence of lineages in regions of South America, its migrations to north and speciation of some taxa in Central America. There are no alternative scenarios described by other authors, this is the novel thing of our study. There are no fossil records containing usable specimens for our analysis (consultation: www.fossilworks.org). Here we propose a species diversification process strongly ligated to geologic transformations. Mainly, geo-climatic events gave form to the Neotropics, the most important include the isolation and reconnection of South America, the uplift of the Andes, the extensive flood-basin system in the Amazonian Miocene, the formation of Orinoco and Amazon drainages, and the dry-wet climate cycles of the Pliocene-Pleistocene. Some phylogenetic studies with molecular data have demonstrated diversification of Neotropical genera prior to the Pleistocene (Brumfield et al. 2007; Brumfield and Edwards 2007; Burns & Naoki 2004; Barker 2007; Selvatti et al. 2022). In addition, other phylogenetic studies of plant and animal taxa showing that many neotropical sister species diverge in the Pleistocene (Richardson et al. 2001; Hughes and Eastwood 2006; Madriñán et al. 2013; Garzón-Orduña et al. 2014; Koenen et al. 2015; Byrne et al. 2016). Our results do not coincide with diversification due to the dry-wet climate cycles of the Pliocene-Pleistocene (Pleistocene refuges hypothesis) as proposed by Haffer (1969). In concordance with our results the diversification of the genera starts in Miocene; the uplift of the Andes and the extensive flood-basin system in the Amazonian Miocene are related, as it has been observed in Rubiaceae (Antonelli et al 2009), Alouatta (Cortés-Ortiz et al 2003), Amphibians (Santos et al. 2009) and bats (Lim 2007). In conclusion, the divergence of genera during the Miocene is related to geo-climatic events associated with the uplift of the central and northern portions of the Andes and the closure of the Isthmus of Panama. Although some species diverged during Pliocene-Pleistocene, apparently this divergence is related to in situ speciation.

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Reviewer 2 Report

Comments and Suggestions for Authors

In the article, the authors identify seven well-supported clades, some of which coincide with recognized genera (Rhynchopsitta, Psittacara, Eupsittula and Pyrrhura). Although the relationships between the clades show variable support, they remain consistent under different analyses. The results reveal a complex evolutionary history in the Arini tribe, with most of the genera analyzed originating during the Miocene (Ara, Orthopsittaca, Aratinga, Cyanopsitta, Guaruba, Leptosittaca, Enicognathus, Cyanoliseus, Eupsittula and Rhynchopsitta) and some in the Miocene-Pliocene (Psittacara and Pyrrhura). Biogeographical proposals indicate that the genera Rhynchopsitta and Conuropsis reached North America before the closing of the Isthmus of Panama, while the species P. finschi, P. brevipes, P. holochlorus and E. canicularis arrived after this event.

Given the high degree of diversity and wide distribution of the Arini tribe in the Neotropics, this study highlights the need for detailed analyses for each of the seven clades identified. The authors should continue to explore this group for further clarification by including additional data, such as molecular, ecological and morphological information, as well as considering species and subspecies within each group, in order to refine the results presented here and advance knowledge of the evolution of this tribe. Some small suggestions can be found in the pdf.

Comments for author File: Comments.pdf

Author Response

Regarding review of our submission, below is the list of responses to your comments/queries. As you will notice, all suggestions were attended and duly replied. We hope that this contribution is now in an acceptable form for publication in Diversity. We thank your consideration and peer review which will undoubtedly end in better communication. We will gladly consider further suggestions.

Comment:

In the article, the authors identify seven well-supported clades, some of which coincide with recognized genera (Rhynchopsitta, Psittacara, Eupsittula and Pyrrhura). Although the relationships between the clades show variable support, they remain consistent under different analyses. The results reveal a complex evolutionary history in the Arini tribe, with most of the genera analyzed originating during the Miocene (Ara, Orthopsittaca, Aratinga, Cyanopsitta, Guaruba, Leptosittaca, Enicognathus, Cyanoliseus, Eupsittula and Rhynchopsitta) and some in the Miocene-Pliocene (Psittacara and Pyrrhura). Biogeographical proposals indicate that the genera Rhynchopsitta and Conuropsis reached North America before the closing of the Isthmus of Panama, while the species P. finschi, P. brevipes, P. holochlorus and E. canicularis arrived after this event.

Given the high degree of diversity and wide distribution of the Arini tribe in the Neotropics, this study highlights the need for detailed analyses for each of the seven clades identified.

The authors should continue to explore this group for further clarification by including additional data, such as molecular, ecological and morphological information, as well as considering species and subspecies within each group, in order to refine the results presented here and advance knowledge of the evolution of this tribe. Some small suggestions can be found in the pdf.

Answer:

Given the conservation status of these species, we consider contributions in this area to be of great importance. However, the objectives, tools and results of this work limit the arguments in the area of conservation genetics. Recognizing the importance of conservation genetics (evolution-conservation), our working group has been addressed these issues in other papers.

Padilla-Jacobo, G.; Monterrubio-Rico, T.C.; Camacho, H.C.; Zavala-Páramo, M.G. Use of phylogenetic analysis to identify evolutionarily significant units for the Orange-fronted parakeet (Eupsittula canicularis) in Mexico. Ornitología Neotropical 2016, 26, 325−335.

Padilla-Jacobo, G.; Monterrubio-Rico, T.; Cano-Camacho, H.; Zavala-Páramo, M.G. Demographic history of the Orange-fronted parakeet (Eupsittula canicularis) in Mexico. Ornitología Neotropical 2018, 29, 323−336.

Padilla-Jacobo, G.; Monterrubio-Rico, T.C.; Cano-Camacho, H.; Zavala-Páramo, M.G. Genealogical relationship inference to identify areas of intensive poaching of the Orange-fronted Parakeet (Eupsittula canicularis). BMC Zoology 2021, 6, 1−12.

Cano-Zavala, E. T., Monterrubio-Rico, T. C., Zavala-Páramo, M. G., Cano-Camacho, H., & Padilla-Jacobo, G. (2023). Genetic diversity and structure of the White-fronted Parrot (Amazona albifrons) in Mexico. Ornitología Neotropical, 33(2), 192-201. 

Padilla-Jacobo, G., Monterrubio-Rico, T. C., Cano-Camacho, H., & Zavala-Páramo, M. G. (2024). Genetic Diversity of the Lilac-Crowned Parrot (Amazona finschi), a Species Endemic to Mexico. Diversity, 16(8), 435.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This is another contribution to the evolution of parrots based on DNA analysis. The methodology, results presentation and interpretations/hypothesis are univesally accepeted. No problems regarding the whole work. However, there are some minor issues which may increase readibilty/digestibility/clarity of the paper. 

1) In the 'Introduction' it would be good to briefly outline the parrot diversity in Mexico, the most northern country in Neotropics, where the parrots occur. Most species are, however, rare or mariginal.

2) Throughout the text, and specifically in Table 1, only Latin names of parrots species are given. I would suggest to include here also their common names (Editor's decision). In this table also non-parrot species are included. I would suggest to put them in brackets, while parrot species occurring in Maxico (and Central America) in bold case. 

3) Symbols: A, B, C ...are used to denotes many different aspects (species, clades, nodes, regions) in ryc., tab. and text. There is a bit confusion. Perhaps it would be better to use alternative symbols (small cases: a, b, c...., numbers: 1, 2, 3, etc.).

4) Line 222-224, should also include the exact numbers, not only percentages.    

Author Response

Regarding review of our submission, below is the list of responses to your comments/queries. As you will notice, all suggestions were attended and duly replied. We hope that this contribution is now in an acceptable form for publication in Diversity. We thank your consideration and peer review which will undoubtedly end in better communication. We will gladly consider further suggestions.

This is another contribution to the evolution of parrots based on DNA analysis. The methodology, results presentation and interpretations/hypothesis are univesally accepeted. No problems regarding the whole work. However, there are some minor issues which may increase readibilty/digestibility/clarity of the paper.

Comment:

1) In the 'Introduction' it would be good to briefly outline the parrot diversity in Mexico, the most northern country in Neotropics, where the parrots occur. Most species are, however, rare or mariginal.

Answer:

Ok, a statement with the required information was added (Lines 46-51).

Comment:

2) Throughout the text, and specifically in Table 1, only Latin names of parrots species are given. I would suggest to include here also their common names (Editor's decision). In this table also non-parrot species are included. I would suggest to put them in brackets, while parrot species occurring in Maxico (and Central America) in bold case.

Answer:

We agree. But common names were not included in Table 1 because it greatly increases the size of the table. However, we added the common names in Supplementary Table 2, highlighted in bold the species with distribution in Mexico and Central America, and used parenthesis in the common names of species that do not belong to the family Psittacidae.

Comment:

3) Symbols: A, B, C ...are used to denotes many different aspects (species, clades, nodes, regions) in ryc., tab. And text. There is a bit confusion. Perhaps it would be better to use alternative symbols (small cases: a, b, c...., numbers: 1, 2, 3, etc.).

Answer:

1. In Table 2 we replaced the letters (A, B, C...) with abbreviations (e.g. Pleu = P. leucophthalmus; Pwag = P. wagleri; etc.)

Comment:

4) Line 222-224, should also include the exact numbers, not only percentages.

Answer:

Ok, It was corrected (Lines 232-234).