Growth, development and reproduction in animals are all limited by dietary nutrients. Expansion of an organism’s diet to sources not accessible to closely related species reduces food competition, and eases the constraints of nutrient-limited diets. Adult butterflies are herbivorous insects known to feed primarily on nectar from flowers, which is rich in sugars but poor in amino acids.  Only certain species in the genus Heliconius are known to also feed on pollen, which is especially rich in amino acids, and is known to prolong their lives by several months. The ability to digest pollen in Heliconius has been linked to specialized feeding behaviors (Krenn et al. 2009) and extra-oral digestion using enzymes, possibly including duplicated copies of cocoonase (Harpel et al. 2016; Smith et al. 2016 and 2018), a protease used by some moths to digest silk upon eclosion from their cocoons. In this reprint, Pinheiro de Castro and colleagues investigated the impact of artificial and natural diets on egg-laying ability, body weight, and cyanogenic glucoside abundance in adult Heliconius erato butterflies of both sexes. 

Previous studies (Dunlap-Pianka et al. 1981) in H. charithonia demonstrated that access to dietary pollen led to extended egg-laying ability among adult female butterflies compared to females deprived of pollen, and compared to Dryas iulia females which feed only on nectar. In the current study, Pinheiro de Castro et al. (2023) examine the impact of diet on both young and old H. erato, over a longer period of time than the earlier work, highlighting the importance of extending the time period over which effects are evaluated. In addition to extending egg-laying ability in older females, the authors found that pollen in the diet appeared to maintain older female body weight, presumably because the pollen contained nutrients depleted during egg-laying.

The authors then investigated the effects of nutrition on the production of cyanogenic glycoside defenses. Heliconius are aposematic butterflies that sequester cyanide-forming defense chemicals from food plants as larvae or synthesize these compounds de novo. The authors found the abundance of cyanogenic glycosides to be significantly greater in butterflies with access to pollen, but again only in older females.

Curiously, field studies of male and female H. charithonia butterflies found that females in the wild collected more pollen than males (Mendoza-Cuenca and Macías-Ordóñez 2005). Taken together, these new findings raise the intriguing possibility that females collect more pollen than males, in part, because pollen has a bigger impact on female survival and reproduction. A small limitation of the study is the use of wing length, rather than body weight, at the zero time point. But the trend is clear in both males and females, and it adds supporting detail to the efficacy of pollen feeding as an unusual strategy for increasing fertility and survival in Heliconius butterflies.

References
 
Dunlap-Pianka, Helen, Carol L. Boggs, Lawrence E. Gilbert. (1977) Ovarian dynamics in heliconiine butterflies: Programmed senescence versus eternal youth. Science, 197: 487-490, https://doi.org/10.1126/Science.197.4302.487
 
Pinheiro de Castro, Erika C., Josie McPherson, Glennis Julian, Anniina L. K. Mattila, Søren Bak, Stephen H. Montgomery, Chris Jiggins. (2023) Pollen-feeding delays reproductive senescence and maintains toxicity of Heliconius erato. bioRxiv, 2023.01.13.523799, ver. 4 peer-reviewed and recommended by Peer Community in Evolutionary Biology. https://doi.org/10.1101/2023.01.13.523799
 
Krenn, Harald W., Monika J. B. Eberhard, Stefan H. Eberhard, Anna-Laetitia Hikl, Werner Huber, Lawrence E. Gilbert (2009). Mechanical damage to pollen aids nutrient acquisition in Heliconius butterflies (Nymphalidae).  Arthropod-Plant Interactions, 3: 203–208. https://doi.org/10.1007/s11829-009-9074-7
 
Harpel, Desiree, Darron A. Cullen, Swidbert R. Ott, Chris D. Jiggins, James R. Walters (2015) Pollen feeding proteomics: Salivary proteins of the passion flower butterfly, Heliconius melpomene. Insect Biochemistry and Molecular Biology, 63: 7-13, https://doi.org/10.1016/j.ibmb.2015.04.004
 
Mendoza-Cuenca, Luis, Rogelio Macías-Ordóñez (2005) Foraging polymorphism in Heliconius charitonia (Lepidoptera: Nymphalidae): morphological constraints and behavioral compensation. Journal of Tropical Ecology, 21: 407-415. https://doi.org/10.1017/S0266467405002385
 
Smith, Gilbert, Aide Macias-Muñoz, John Kelly, Carter Butts, Rachel Martin, Adriana D. Briscoe (2018) Evolutionary and structural analyses uncover a role for solvent interactions in the diversification of cocoonases in butterflies. Proceedings of the Royal Society B, 285: 20172037. https://doi.org/10.1098/rspb.2017.2037 
 
Smith, Gilbert, Aide Macias-Muñoz, Adriana D. Briscoe (2016) Gene duplication and gene expression changes play a role in the evolution of candidate pollen-feeding genes in Heliconius butterflies. Genome Biology and Evolution, 8: 2581-2596. https://doi.org/10.1093/gbe/evw180

Duplications, mutations and recombination may be considered the main sources of genomic variation and evolution. In addition, sexual recombination is essential in purging deleterious mutations and allowing advantageous allelic combinations to occur (Glémin et al. 2019). However, in parthenogenetic asexual organisms, variation cannot be explained by sexual recombination, and other mechanisms must account for it. Although it is known that transposable elements (TE) may influence on genome structure and gene expression patterns, their role as a primary source of genomic variation and rapid adaptability has received less attention. An important role of TE on adaptive genome evolution has been documented for fungal phytopathogens (Faino et al. 2016), suggesting that TE activity might explain the evolutionary dynamics of this type of organisms.
The phytopathogen nematode Meloidogyne incognita is one of the worst agricultural pests in warm climates (Savary et al. 2019). This species, as well as other root-knot nematodes (RKN), shows a wide geographical distribution range infecting diverse groups of plants. Although allopolyploidy may have played an important role on the wide adaptation of this phytopathogen, it may not explain by itself the rapid changes required to overcome plant resistance in a few generations. Paradoxically, M. incognita reproduces asexually via mitotic parthenogenesis (Trudgill and Blok 2001; Castagnone-Sereno and Danchin 2014) and only few single nucleotide variations were identified between different host races isolates (Koutsovoulos et al. 2020). Therefore, this is an interesting model to explore other sources of genomic variation such the TE activity and its role on the success and adaptability of this phytopathogen.
To address these questions, Kozlowski et al. (2020) estimated the TE mobility across 12 geographical isolates that presented phenotypic variations in Meloidogyne incognita, concluding that recent activity of TE in both genic and regulatory regions might have given rise to relevant functional differences between genomes. This was the first estimation of TE activity as a mechanism probably involved in genome plasticity of this root-knot nematode. This study also shed light on evolutionary mechanisms of asexual organisms with an allopolyploid origin. These authors re-annotated the 185 Mb triploid genome of M. incognita for TE content analysis using stringent filters (Kozlowski 2020a), and estimated activity by their distribution using a population genomics approach including isolates from different crops and locations. Canonical TE represented around 4.7% of the M. incognita genome of which mostly correspond to TIR (Terminal Inverted Repeats) and MITEs (Miniature Inverted repeat Transposable Elements) followed by Maverick DNA transposons and LTR (Long Terminal Repeats) retrotransposons. The result that most TE found were represented by DNA transposons is similar to the previous studies with the nematode species model Caenorhabditis elegans (Bessereau 2006; Kozlowski 2020b) and other nematodes as well. Canonical TE annotations were highly similar to their consensus sequences containing transposition machinery when TE are autonomous, whereas no genes involved in transposition were found in non-autonomous ones. These findings suggest recent activity of TE in the M. incognita genome. Other relevant result was the significant variation in TE presence frequencies found in more than 3,500 loci across isolates, following a bimodal distribution within isolates. However, variation in TE frequencies was low to moderate between isolates recapitulating the phylogenetic signal of isolates DNA sequences polymorphisms. A detailed analysis of TE frequencies across isolates allowed identifying polymorphic TE loci, some of which might be neo-insertions mostly of TIRs and MITEs (Kozlowski 2020c). Interestingly, the two thirds of the fixed neo-insertions were located in coding regions or in regulatory regions impacting expression of specific genes in M. incognita. Future research on proteomics is needed to evaluate the functional impact that these insertions have on adaptive evolution in M. incognita. In this line, this pioneer research of Kozlowski et al. (2020) is a first step that is also relevant to remark the role that allopolyploidy and reproduction have had on shaping nematode genomes.

References

[1] Bessereau J-L. 2006. Transposons in C. elegans. WormBook. 10.1895/wormbook.1.70.1
[2] Castagnone-Sereno P, Danchin EGJ. 2014. Parasitic success without sex - the nematode experience. J. Evol. Biol. 27:1323-1333. 10.1111/jeb.12337
[3] Faino L, Seidl MF, Shi-Kunne X, Pauper M, Berg GCM van den, Wittenberg AHJ, Thomma BPHJ. 2016. Transposons passively and actively contribute to evolution of the two-speed genome of a fungal pathogen. Genome Res. 26:1091-1100. 10.1101/gr.204974.116
[4] Glémin S, François CM, Galtier N. 2019. Genome Evolution in Outcrossing vs. Selfing vs. Asexual Species. In: Anisimova M, editor. Evolutionary Genomics: Statistical and Computational Methods. Methods in Molecular Biology. New York, NY: Springer. p. 331-369. 10.1007/978-1-4939-9074-0_11
[5] Koutsovoulos GD, Marques E, Arguel M-J, Duret L, Machado ACZ, Carneiro RMDG, Kozlowski DK, Bailly-Bechet M, Castagnone-Sereno P, Albuquerque EVS, et al. 2020. Population genomics supports clonal reproduction and multiple independent gains and losses of parasitic abilities in the most devastating nematode pest. Evol. Appl. 13:442-457. 10.1111/eva.12881
[6] Kozlowski D. 2020a. Transposable Elements prediction and annotation in the M. incognita genome. Portail Data INRAE. 10.15454/EPTDOS
[7] Kozlowski D. 2020b. Transposable Elements prediction and annotation in the C. elegans genome. Portail Data INRAE. 10.15454/LQCIW0
[8] Kozlowski D. 2020c. TE polymorphisms detection and analysis with PopoolationTE2. Portail Data INRAE. 10.15454/EWJCT8
[9] Kozlowski DK, Hassanaly-Goulamhoussen R, Da Rocha M, Koutsovoulos GD, Bailly-Bechet M, Danchin EG (2020) Transposable Elements are an evolutionary force shaping genomic plasticity in the parthenogenetic root-knot nematode Meloidogyne incognita. bioRxiv, 2020.04.30.069948, ver. 4 peer-reviewed and recommended by PCI Evolutionary Biology. 10.1101/2020.04.30.069948
[10] Savary S, Willocquet L, Pethybridge SJ, Esker P, McRoberts N, Nelson A. 2019. The global burden of pathogens and pests on major food crops. Nat. Ecol. Evol. 3:430-439. 10.1038/s41559-018-0793-y
[11] Trudgill DL, Blok VC. 2001. Apomictic, polyphagous root-knot nematodes: exceptionally successful and damaging biotrophic root pathogens. Annu Rev Phytopathol 39:53-77. 10.1146/annurev.phyto.39.1.53

Phylogenetic trees can be used to extract information about the process of diversification that has generated them. The most common approach to conduct this inference is to rely on a likelihood, defined here as the probability of generating a dated tree T given a diversification model (e.g. a birth-death model), and then use standard maximum likelihood. This idea has been explored extensively in the context of the so-called diversification studies, with many variants for the models and for the questions being asked (diversification rates shifting at certain time points or in the ancestors of particular subclades, trait-dependent diversification rates, etc).
However, all this assumes that the dated tree T is known without error. In practice, trees (that is, both the tree topology and the divergence times) are inferred based on DNA sequences, possibly combined with fossil information for calibrating and informing the divergence times. Molecular dating is a delicate exercise, however, and much more so in fact than reconstructing the tree topology. In particular, a mis-specificied model for the relaxed molecular clock, or a mis-specifiied prior, can have a substantial impact on the estimation of divergence dates - which in turn could severely mislead the inference about the underlying diversification process. This thus raises the following question: would that be possible to conduct inference and testing of diversification models without having to go through the dangerous step of molecular dating?
In his article ""Probabilities of tree topologies with temporal constraints and diversification shifts"" [1], Gilles Didier introduces a recursive method for computing the probability of a tree topology under some diversification model of interest, without knowledge of the exact dates, but only interval constraints on the dates of some of the nodes of the tree. Such interval constraints, which are derived from fossil knowledge, are typically used for molecular dating: they provide the calibrations for the relaxed clock analysis. Thus, what is essentially proposed by Gilles Didier is to use them in combination with the tree topology only, thus bypassing the need to estimates divergence times first, before fitting a diversification model to a phylogenetic tree.
This article, which is primarily a mathematical and algorithmic contribution, is then complemented with several applications: testing for a diversification shift in a given subclade of the phylogeny, just based on the (undated) tree topology, with interval constraints on some of its internal nodes; but also, computing the age distribution of each node and sampling on the joint distribution on node ages, conditional on the interval constraints. The test for the presence of a diversification shift is particularly interesting: an application to simulated data (and without any interval constraint in that case) suggests that the method based on the undated tree performs about as well as the classical method based on a dated tree, and this, even granting the classical approach a perfect knowledge of the dates - given that, in practice, one in fact relies on potentially biased estimates. Finally, an application to a well-known example (rate shifts in cetacean phylogeny) is presented.
This article thus represents a particularly meaningful contribution to the methodology for diversification studies; but also, for molecular dating itself: it is a well known problem in molecular dating that computing and sampling from the conditional distributions on node ages, given fossil constraints, and more generally understanding and visualizing how interval constraints on some nodes of the tree impact the distribution at other nodes, is a particularly difficult exercise. For that reason, the algorithmic routines presented in the present article will be useful in this context as well.

References

[1] Didier, G. (2020) Probabilities of tree topologies with temporal constraints and diversification shifts. bioRxiv, 376756, ver. 4 peer-reviewed and recommended by PCI Evolutionary Biology. doi: 10.1101/376756

The science of naming species (taxonomy) has been renewed with the developments of molecular sequencing, digitization of museum specimens, and novel analytical tools. However, naming species can be highly subjective, sometimes considered as an art [1], because it is based on human-based criteria that vary among taxonomists. Nonetheless, taxonomists often argue that species names are hypotheses, which are therefore testable and refutable as new evidence is provided. This challenge comes with a more and more recognized and critical need for rigorously delineated species not only for producing accurate species inventories, but more importantly many questions in evolutionary biology (e.g. speciation), ecology (e.g. ecosystem structure and functioning), conservation biology (e.g. targeting priorities) or biogeography (e.g. diversification processes) depend in part on those species inventories and our knowledge of species [2-3]. Inaccurate species boundaries or diversity estimates may lead us to deliver biased answers to those questions, exactly as phylogenetic trees must be reconstructed rigorously and analyzed critically because they are a first step toward discussing broader questions [2-3]. In this context, biological diversity needs to be studied from multiple and complementary perspectives requiring the collaboration of morphologists, molecular biologists, biogeographers, and modelers [4-5]. Integrative taxonomy has been proposed as a solution to tackle the challenge of delimiting species [2], especially in highly diverse and undocumented groups of organisms.
In an elegant study that harbors all the characteristics of an integrative approach, Alda et al. [6] tackle the delimitation of species within the snail genus Galba (Lymnaeidae). Snails of this genus represent a peculiar case study for species delineation with a long and convoluted taxonomic history in which previous works recognized a number of species ranging from 4 to 30. The confusion is likely due to a loose morphology (labile shell features and high plasticity), which makes the identification and naming of species very unstable and likely subjective. An integrative taxonomic approach was needed. After two decades of taxon sampling and visits of type localities, the authors present an impressively dense taxon sampling at a global scale for the genus, which includes all described species. When it comes to delineate species, taxon sampling is often the key if we want to embrace the genetic and morphological diversity. Molecular data was obtained for several types of markers (microsatellites and DNA sequences for four genes), which were combined to morphology of shell and of internal organs, and to geographic distribution. All the data are thoroughly analyzed with cutting-edge methods starting from Bayesian phylogenetic reconstructions using multispecies coalescent models, followed by models of species delimitation based on the molecular specimen-level phylogeny, and then Bayesian divergence time estimates. They also used probabilistic models of ancestral state estimation to infer the ancestral phenotypic state of the Galba ancestors.
Their numerous phylogenetic and delimitation analyses allow to redefine the species boundaries that indicate that the genus Galba comprises six species. Interestingly, four of these species are morphologically cryptic and likely constitute species with extensive genetic diversity and widespread geographic distribution. The other two species have more geographically restricted distributions and exhibit an alternative morphology that is more phylogenetically derived than the cryptic one. Although further genomic studies would be required to strengthen some species status, this novel delimitation of Galba species has important implications for our understanding of convergence and morphological stasis, or the role for stabilizing selection in amphibious habitats; topics that are rarely addressed with invertebrate groups. For instance, in terms of macroevolutionary history, it is striking that an invertebrate clade of that age (22 million years ago) has only given birth to six species today. Including 30 (ancient taxonomy) or 6 (integrative taxonomy) species in a similar amount of evolutionary time does not tell us the same story when studying the diversification processes [7]. Here, Alda et al. [6] present a convincing case study that should foster similar studies following their approach, which will provide stimulating perspectives for testing the concepts of species and their effects on evolutionary biology.

References

[1] Ohl, M. (2018). The art of naming. MIT Press.
[2] Dayrat, B. (2005). Towards integrative taxonomy. Biological Journal of the Linnean Society, 85(3), 407–415. doi: 10.1111/j.1095-8312.2005.00503.x
[3] De Queiroz, K. (2007). Species concepts and species delimitation. Systematic Biology, 56(6), 879–886. doi: 10.1080/10635150701701083
[4] Padial, J. M., Miralles, A., De la Riva, I., and Vences, M. (2010). The integrative future of taxonomy. Frontiers in Zoology, 7(1), 16. doi: 10.1186/1742-9994-7-16
[5] Schlick-Steiner, B. C., Steiner, F. M., Seifert, B., Stauffer, C., Christian, E., and Crozier, R. H. (2010). Integrative taxonomy: A multisource approach to exploring biodiversity. Annual Review of Entomology, 55(1), 421–438. doi: 10.1146/annurev-ento-112408-085432
[6] Alda, P. et al. (2019). Systematics and geographical distribution of Galba species, a group of cryptic and worldwide freshwater snails. BioRxiv, 647867, v3 peer-reviewed and recommended by PCI Evolutionary Biology. doi: 10.1101/647867
[7] Ruane, S., Bryson, R. W., Pyron, R. A., and Burbrink, F. T. (2014). Coalescent species delimitation in milksnakes (Genus Lampropeltis) and impacts on phylogenetic comparative analyses. Systematic Biology, 63(2), 231–250. doi: 10.1093/sysbio/syt099

Genome-wide data obtained from even a small number of individuals can provide unprecedented levels of detail about the evolutionary history of populations and species [1], determinants of genetic diversity [2], species boundaries and the process of speciation itself [3]. Loire and Galtier [4] present a clear example, using the emblematic Galápagos giant tortoise (Chelonoidis nigra), of how multi-species comparative population genomic approaches can provide valuable insights about population structure and species delimitation even when sample sizes are limited but the number of loci is large and distributed across the genome.

Galápagos giant tortoises are endemic to the Galápagos Islands and are currently recognized as an endangered, multi-species complex including both extant and extinct taxa. Taxonomic definitions are based on morphology, geographic isolation and population genetic evidence based on short DNA sequences of the mitochondrial genome (mtDNA) and/or a dozen or so nuclear microsatellite loci [5-8]. The species complex enjoys maximal protection. Population recoveries have been quite successful and spectacular conservation programs based on mitochondrial genes and microsatellites are ongoing. This includes for example individual translocations, breeding program, “hybrid” sterilization or removal, and resurrection of extinct lineages).

In 2013, Loire et al. [9] provided the first population genomic analyses based on genome scale data (~1000 coding loci derived from blood-transcriptomes) from five individuals, encompassing three putative “species”: Chelonnoidis becki, C. porteri and C. vandenburghi. Their results raised doubts about the validity/accuracy of the currently accepted designations of “genetic distinctiveness”. However, the implications for conservation and management have remained unnoticed.

In 2017, Loire and Galtier [4] have re-appraised this issue using an original multi-species comparative population genomic analysis of their previous data set [9]. Based on a comparison of 53 animal species, they show that both the level of genome-wide neutral diversity (πS) and level of population structure estimated using the inbreeding coefficient (F) are much lower than would be expected from a sample covering multiple species. The observed values are more comparable to those typically reported at the “among population” level within a single species such as human (Homo sapiens). The authors go to great length to assess the sensitivity of their method to detect population structure (or lack thereof) and show that their results are robust to potential issues, such as contamination and sequencing errors that can occur with Next Generation Sequencing techniques; and biases related to the small sample size and sub-sampling of individuals. They conclude that published mtDNA and microsatellite-based assessment of population structure and species designations may be biased towards over-splitting.

This manuscript is a very good read as it shows the potential of the now relatively affordable genome-wide data for helping to both resolve and clarify population and species boundaries, illuminate demographic trends, evolutionary trajectories of isolated groups, patterns of connectivity among them, and test for evidence of local adaptation and even reproductive isolation. The comprehensive information provided by genome-wide data can critically inform and assist the development of the best strategies to preserve endangered populations and species. Loire and Galtier [4] make a strong case for applying genomic data to the Galápagos giant tortoises, which is likely to redirect conservation efforts more effectively and at lower cost. The case of the Galápagos giant tortoises is certainly a very emblematic example, which will find an echo in many other endangered species conservation programs.

References

[1] Li H and Durbin R. 2011. Inference of human population history from individual whole-genome sequences. Nature, 475: 493–496. doi: 10.1038/nature10231

[2] Romiguier J, Gayral P, Ballenghien M, Bernard A, Cahais V, Chenuil A, Chiari Y, Dernat R, Duret L, Faivre N, Loire E, Lourenco JM, Nabholz B, Roux C, Tsagkogeorga G, Weber AA-T, Weinert LA, Belkhir K, Bierne N, Glémin S and Galtier N. 2014. Comparative population genomics in animals uncovers the determinants of genetic diversity. Nature, 515: 261–263. doi: 10.1038/nature13685

[3] Roux C, Fraïsse C, Romiguier J, Anciaux Y, Galtier N and Bierne N. 2016. Shedding light on the grey zone of speciation along a continuum of genomic divergence. PLoS Biology, 14: e2000234. doi: 10.1371/journal.pbio.2000234

[4] Loire E and Galtier N. 2017. Lacking conservation genomics in the giant Galápagos tortoise. bioRxiv 101980, ver. 4 of September 26, 2017. doi: 10.1101/101980

[5] Beheregaray LB, Ciofi C, Caccone A, Gibbs JP and Powell JR. 2003. Genetic divergence, phylogeography and conservation units of giant tortoises from Santa Cruz and Pinzón, Galápagos Islands. Conservation Genetics, 4: 31–46. doi: 10.1023/A:1021864214375

[6] Ciofi C, Milinkovitch MC, Gibbs JP, Caccone A and Powell JR. 2002. Microsatellite analysis of genetic divergence among populations of giant Galápagos tortoises. Molecular Ecology, 11: 2265–2283. doi: 10.1046/j.1365-294X.2002.01617.x

[7] Garrick RC, Kajdacsi B, Russello MA, Benavides E, Hyseni C, Gibbs JP, Tapia W and Caccone A. 2015. Naturally rare versus newly rare: demographic inferences on two timescales inform conservation of Galápagos giant tortoises. Ecology and Evolution, 5: 676–694. doi: 10.1002/ece3.1388

[8] Poulakakis N, Edwards DL, Chiari Y, Garrick RC, Russello MA, Benavides E, Watkins-Colwell GJ, Glaberman S, Tapia W, Gibbs JP, Cayot LJ and Caccone A. 2015. Description of a new Galápagos giant tortoise species (Chelonoidis; Testudines: Testudinidae) from Cerro Fatal on Santa Cruz island. PLoS ONE, 10: e0138779. doi: 10.1371/journal.pone.0138779

[9] Loire E, Chiari Y, Bernard A, Cahais V, Romiguier J, Nabholz B, Lourenço JM and Galtier N. 2013. Population genomics of the endangered giant Galápagos tortoise. Genome Biology, 14: R136. doi: 10.1186/gb-2013-14-12-r136