Gene tree conflict between genomic and functional partitions impacts phylogenetic inference in Zygophylloideae

  1. Pieter de Wet van der Merwe
  2. Michael D. Pirie
  3. Dirk U. Bellstedt

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Abstract

The Zygophyllaceae subfamily Zygophylloideae includes seven genera and around 180 species distributed worldwide particularly in arid areas where they show adaptations to drought including C 4 and CAM photosynthesis. Phylogenetic relationships of Zygophylloideae have been analysed in several studies using Sanger sequenced markers, but none have been able to resolve all deeper relationships between genera in the subfamily. Here, we use ion semi-conductor sequencing of chloroplast and nuclear ribosomal regions to analyse these relationships and assess why they have proved so challenging to resolve. We sequenced 39 chloroplast genes and nuclear ribosomal 18S, 5.8S, and 26S genes and internal transcribed spacers (ITS) for key species of Zygophyllum sensu stricto, Roepera, Tetraena, Fagonia , Zygophyllum stapffii and monotypic Augea capensis ; and Tribulus of the subfamily Tribuloideae as outgroup. We aligned these with data from published chloroplast genomes followed by phylogenetic analyses using Maximum Likelihood and Bayesian approaches, summarising phylogenetic results separately for non-coding introns and intergenic spacers, and for genes that are, or are not, involved in photosynthesis. We combined the chloroplast data with sequences of three highly variable noncoding chloroplast regions used in previous studies of greater numbers of taxa, and similarly the nuclear ribosomal data with more densely sampled ITS sequences and assessed whether adding such data resulted in improved support for consistent topologies. We used a chloroplast sequence matrix to infer a dated phylogeny. Adding data did not always improve phylogenetic resolution which was apparently impacted by non-informative variation in chloroplast photosynthesis related genes as well as by conflicting chloroplast and nuclear signals. Chloroplast genes not related to photosynthesis provide robust support for a topology that is consistent with independent data and may therefore represent a useful species tree hypothesis. The cause of the gene tree conflicts remains unclear: potential explanations include functional constraints in photosynthesis related genes and introgressive hybridization and/or incomplete lineage sorting during an ancient rapid divergence. Further testing will require a broader sampling of lineages with different photosynthetic modes and multiple informative, independent, and selectively neutral gene trees.

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  1. Peer Community in Evolutionary Biology

    Dear authors,

    Your manuscript has been evaluated by three reviewers. The reviewers’ reports are generally positive, but they all point out important issues that should be addressed before the work can be recommended.

    Importantly, there is a discrepancy between the proposed causes of discordance and phylogenetic conflicts and the absence of formal analyses testing these claims. This issue was emphasized by both Reviewer #1 and Reviewer #3 and should be carefully addressed in both the revised manuscript and the response letter.

    In particular, the title and discussion currently place strong emphasis on convergence and/or selection as explanations for the observed phylogenetic conflicts, but these results are not clearly presented and these interpretations are not yet sufficiently supported by the presented analyses. At present, all chloroplast datasets (39C, 18C, and 21C) contain several weakly supported nodes, making the reported discrepancies not strongly convincing. Moreover, it is expected that the 39C and 18C datasets would produce more similar topologies, as the 18C dataset contains more informative sites than the 21C dataset. So this result could not be used as a support for the convergence/selection hypothesis. Therefore, claims regarding selection or convergence should ideally be supported by additional analyses demonstrating that the 18C and 21C datasets support different nodes with strong statistical support. Otherwise, the observed differences may simply reflect stochastic noise. Alternatively, the title and discussion should be revised to present more cautious interpretations.

    Following Reviewer #3’s comments, I also found that the Results section could be improved. Some very short subsections could likely be merged or removed (e.g., “Nuclear ITS cassette gene sequencing and alignment” and “ITS gene sequencing and alignment”).

    Additionally, it is very important that you deposit the raw sequence data in public databases such as EBI ENA (https://www.ebi.ac.uk/ena/) or NCBI GenBank for assembled sequences, and ENA or NCBI SRA for the raw NGS reads. Even if the data are currently available in another public repository, they are not easily discoverable by other researchers and are not accessible through BLAST searches or SRA Explorer queries, for example. Moreover, as part of PCI policy, all analyses should be fully reproducible. You will therefore also need to provide the scripts and datasets used in the analyses, including phylogenetic trees, in a publicly accessible repository.

    Finally, Reviewers #2 and #3 provided many detailed comments and suggestions that should help improve the manuscript.

    Please respond to all comments raised by the reviewers. If you choose not to implement certain suggestions, please provide a clear justification in your response letter.

    Best regards,

    Benoit Nabholz

  2. Peer Community in Evolutionary Biology

    The manuscript presents new sequencing data for six species representing six genera of Zygophylloideae, which is a meaningful contribution to the study of this group. Given the wide, near-global distribution of Zygophylloideae, expanding molecular resources across its diversity is both necessary and timely. The effort to generate chloroplast and nuclear ribosomal data for key taxa is commendable and provides a useful foundation for future phylogenetic and evolutionary studies. In this regard, the manuscript is suitable for publication. However, to maximize its value to the broader research community, the authors should ensure that all raw sequencing data are deposited in a publicly accessible database (e.g., NCBI SRA or similar). Considering the geographic breadth of the group, facilitating data sharing will also support collaborative, global-scale efforts to resolve its evolutionary history.

    At the same time, there is a mismatch between the scope implied in parts of the manuscript and the analyses actually conducted. While the study discusses issues such as discordance between chloroplast and nuclear signals, rapid diversification, and possible convergence or selection, it does not include explicit analytical tests addressing these processes (e.g., formal assessments of gene tree discordance, tests for incomplete lineage sorting, or analyses of convergent evolution). As such, these interpretations remain largely speculative. I recommend that the title and framing of the manuscript be revised to focus more explicitly on phylogenetic reconstruction and observed patterns of conflict, rather than on broader evolutionary processes that are not directly tested. This adjustment would better align the claims with the evidence presented and improve the overall clarity and rigor of the study.

  3. Peer Community in Evolutionary Biology

    Dear editor and authors:

    The study is well organized and presents a range of complementary strategies for phylogenetic inference. The Discussion effectively captures the main conclusions, and the Results are appropriately addressed. I have only a few suggestions:

     

    Title and abstract
    Does the title clearly reflect the content of the article?
    [ X] Yes
    Does the abstract present the main findings of the study?
    [X ] No: There is only one sentence about the findings and many about the methods. Please consider adding more information about the results.

    Introduction
    Are the research questions/hypotheses/predictions clearly presented?
    [X ] Yes
    Does the introduction build on relevant research in the field?
    [ X] Yes
     

    Materials and methods
    Are the methods and analyses sufficiently detailed to allow replication by other researchers?
    [ X] No: Need to add information about  how many bootstrap replicates were used in the maximum likelihood analysis.

    Results
    Are the results described and interpreted correctly?
    [ X] Yes.

    Discussion
    Have the authors appropriately emphasized the strengths and limitations of their study/theory/methods/argument?
    [ X] Yes
    Are the conclusions adequately supported by the results (without overstating the implications of the findings)?
    [X ] Yes
     
    __________________________
     
    Additional coments:

    1 - Add the author’s name of the species and genera when it is cited for the first time in the text.  Examples in the introduction:

     The flowering plant family Zygophyllaceae has a world-wide distribution. They can be trees, such as species of the genus Balanites; shrubs such as the well-known creosote bush, Larrea tridentata, from the USA; or annual herbs, such as Tribulus terrestris. The family includes examples of tropical rainforest trees in South America, such as Bulnesia and Guaiacum which are known for their particularly hard wood.

    However, they also included Zygophyllum stapffii from the coastal areas of the Namib desert in Namibia and Angola, retrieving this species as sister to Augea capensis and thereby implying paraphyly of Zygophyllum s.s..

    Augea capensis plus Zygophyllum stapffii / Z. orbiculatum (the latter treated as conspecific by Bellstedt et al., 2008);

    C4 photosynthesis evolved in the subfamily Zygophylloideae in Tetraena simplex and independently in clades of

    _____________

     

    2 - Please provide references for statements that fall outside the scope of the present study, such as in:

    INTRODUCTION:

    The flowering plant family Zygophyllaceae has a world-wide distribution. They can be trees, such as species of the genus Balanites; shrubs such as the well-known creosote bush, Larrea tridentata, from the USA; or annual herbs, such as Tribulus terrestris. The family includes examples of tropical rainforest trees in South America, such as Bulnesia and Guaiacum which are known for their particularly hard wood. However, Zygophyllaceae are more typically characterised by adaptations to drought tolerance that have allowed the family to radiate into all arid areas of the world.

    DISCUSSION:

    The species with the widest geographical distribution in the Zygophylloideae (from southern- to northern Africa including the Azores) is Tetraena simplex. It is an annual prostrate herb with a tumbleweed seed distribution mechanism and C4 photosynthesis. Roepera cordifolia that exhibits CAM photosynthesis also has a large range and has dispersed into the hyper-arid southern Namib desert in Namibia. Tribulus terrestris, another example of C4, in the Tribuloideae, is distributed almost worldwide

    _________________

    3 - Add number of species for Tetraena:

    According to their redefinition, the subfamily Zygophylloideae consisted of the genera Zygophyllum L. (including ca. 150 species), Fagonia L. (30 species) and the monotypic genera Augea Thunb. and Tetraena Maxim.

    __________

    4 - The expansion was in number of species of Zygophyllum? Give more details. In the previous studies it was recovered as monophyletic? It is no clear:

    Beier et al. (2003) expanded the taxon sampling considerably and, based on non-coding chloroplast trnL intron sequences and a morphological character dataset, retrieved a polyphyletic genus Zygophyllum.

    ____________

    5 - For consistency with formal scientific style, consider replacing “the rest” with a more precise expression such as “the remaining” or “other members” when referring to Zygophyllum sensu stricto.

    2008); and the rest of Zygophyllum sensu stricto. Relationships between these clades remained unsupported and/or inconsistent.

    _________

    6 - Consider replacing “it” with “this gene,” as the latter is clearer:

    It is important to test whether it and potentially other genes in the chloroplast genome might show convergent signals that impact phylogenetic inference in plant groups in which both C3 and C4 photosynthesis occurs.

    ____________

    7 - Consider starting a new sentence at “Evolution towards…”

    As CAM metabolism involves a third, very different photosynthetic mechanism for photosynthetic processes to occur in plants exposed to aridity, and is not facilitated by a Kranz type morphology as in C4 plants but rather an intracellular adaptation, evolution towards this type of photosynthesis apparently requires mutation and expression of a different repertoire of genes, including rbcL, to those in plants that possess C3 or C4 photosynthetic mechanisms (Berry et al., 2013).

    _________________

    8 - The sentence would benefit from improved parallel structure. Consider revising “with comparisons … and assess …” to a consistent verbal form (e.g., “compare … compare … and assess …”) to improve clarity and readability:

    We compare phylogenetic signals both between genomes and between functional groups within them (photosynthetic genes versus non-photosynthetic genes), with comparisons to a supermatrix with denser taxon sampling, and assess the impact on results with the aim of inferring a robust and resolved phylogenetic hypothesis for the subfamily.

    ________________

    9 - Please add the full genus name in the figure captions to improve clarity and facilitate reader comprehension. Example: Z.: Zygophyllum

    ____________________

    10 - The branch support of T. tubinatum + T. mongolica is lacking in Figure 3.

    _____________________

    11 - Consider whether it is necessary to include a separate subsection in the Results when it contains only a single sentence.

    Exemple 1:

    ITS gene sequencing and alignment 

    The amplification of the ITS region (ITS1-5.8SRNA-ITS2), using Sanger sequencing, sequence assembly and alignment was successful for the 59 taxa included in the study.

     

    Exemple 2:

    Conflicts between phylogenies generated from chloroplast and nuclear gene matrices 

    Chloroplast and the nuclear genes tree topologies within the subfamily Zygophylloideae (Figures 3 and 5) showed conflict with regard the position of both the Asian Zygophyllum clade and Tetraena.

     

  5. Peer Community in Evolutionary Biology

    The phylogeny of Zygophylloideae is challenging to reconstruct because this subfamily has a worldwide distribution, making comprehensive taxon sampling difficult. Moreover, previous molecular systematics studies have relied on a limited number of loci and have struggled to provide a robust and well-resolved phylogenetic hypothesis.

    In this study, the authors assemble a relatively modest dataset in terms of the number of species, but that encompasses the major lineages of the subfamily. The sequencing effort is also substantially greater than in previous studies, allowing the authors to conduct an extensive set of phylogenetic analyses based on multiple dataset combinations and to investigate incongruence among them in detail. Conflicts emerge not only between chloroplast and nuclear loci but also, more surprisingly, among chloroplast loci. Given the largely non-recombining nature of the chloroplast genome, its loci are generally expected to share the same genealogical history, making incomplete lineage sorting or hybridization unlikely explanations for conflicts among these loci and raising questions about the underlying causes of the observed incongruence. The authors show that genes involved in photosynthesis yield a different and less well-supported topology than genes not involved in photosynthesis. More surprisingly, combining both groups of loci results in lower phylogenetic support, highlighting the impact of conflicting phylogenetic signals and illustrating that adding more data does not necessarily improve phylogenetic resolution.

    Although this study remains relatively modest in terms of both taxonomic sampling and the number of loci analysed, it represents a valuable contribution to resolving the phylogeny of Zygophylloideae. Beyond providing new insights into the evolutionary relationships within the subfamily, it offers an interesting perspective on the nature and potential origins of phylogenetic incongruence which could stimulate further research.

    References

    Pieter de Wet van der Merwe, Michael D. Pirie, Dirk U. Bellstedt (2026) Gene tree conflict between genomic and functional partitions impacts phylogenetic inference in Zygophylloideaee. bioRxiv, ver.4 peer-reviewed and recommended by PCI Evolutionary Biology https://doi.org/10.64898/2026.01.16.699981

  6. Version published to 10.64898/2026.01.16.699981 on bioRxiv