The challenge of delineating species when they are hidden
Systematics and geographical distribution of Galba species, a group of cryptic and worldwide freshwater snails
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 , 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 , 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.  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 . Here, Alda et al.  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.
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 De Queiroz, K. (2007). Species concepts and species delimitation. Systematic Biology, 56(6), 879–886. doi: 10.1080/10635150701701083
 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
 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
 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
 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
Fabien Condamine (2019) The challenge of delineating species when they are hidden. Peer Community in Evolutionary Biology, 100089. 10.24072/pci.evolbiol.100089
Reviewed by Christelle Fraïsse, 16 Nov 2019
Evaluation round #1
DOI or URL of the preprint: https://doi.org/10.1101/647867
Version of the preprint: 1
Author's Reply, 01 Nov 2019
Decision by Fabien Condamine, 19 Jul 2019
Thank you for soliciting the Peer Community in Evolutionary Biology to assess your study.
We have received the feedback of three reviewers for your preprint study. You will see that the three referees are positive about the paper but they also bring up very interesting and useful comments as well as suggestions that I am sure will improve the study. Overall, I agree with the reviewers that the study has many merits and that the findings are interesting. I also think the approach proposed here is original and may be useful for further studies in taxonomy and systematics of difficult groups, especially for invertebrate clades. That being said, the study should be more rooted in the concept of integrative taxonomy (e.g. Dayrat 2005 – Biol. J. Linn. Soc.). In addition, the study suffers from some methodological and conceptual issues. I think the main issues concern the phylogeny and dating analyses, but because these results are the cornerstone for the interpretation of species delimitation, the corresponding results may be inconclusive as it stands. The referees also felt that the manuscript suffers from a lack of clarity in several parts of the text, especially in the Methods and the interpretations (Results and Discussion).
To summarize, I have identified six major points raised by the reviewers that you would need to carefully address. This includes the following: (1) Running phylogenetic analyses removing the third position of mitochondrial coding genes from the alignment; (2) Running a second dating analysis using relaxed clocks and compare the results obtained with a strict clock; (3) Performing a multivariate analysis on some quantitative trait(s) and calculating a distance between clusters on this morphometric space; (4) Strengthening the analyses of ancestral state reconstructions, perhaps with the use of other models (e.g. maximum-likelihood models like Dispersal-Extinction-Cladogenesis; Ree & Smith 2008 – Syst. Biol.) and by including the uncertainty around the node estimates; (5) Clarifying the rationale used for distinguishing cryptic species and the evolutionary scenarios tested; (6) Taking into account all the comments that aim at improving the text, the hypotheses, and the figures.
Based on the referees’ comments and my reading, I believe the manuscript will benefit from a revision and a second round of reviews. If you chose to resubmit a revised paper, please make a point-by-point reply to the comments (like for a traditional journal). For the moment, I do not recommend the study in PCi Evol. Biol. but if the revision is thorough (satisfies the reviewers) and the results still support the conclusions, I will be supportive for the paper as being recommended.
Dr. Fabien Condamine, recommender for PCi Evol. Biol.
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