4.3. Implications for classification, taxonomy and phylogeography
Our sequence data matrix recovered the same two clades found earlier (Pérez-Losada et al., 2009; Olesen et al., 2022), but we provide enhanced phylogenetic resolution by sequencing more markers for multiple new MOTUs, some of which may be putative species awaiting taxonomic description (Fig. 1). Importantly, most morphotypes are supported by molecular and morphological data in concert, testifying to the usefulness of combining these two data layers for phylogeny estimation and evaluation. Given the molecular diversity in the phylogeny and the morphological disparity of the larval forms, these two clades may represent high-level taxonomic units (e.g., families), but more study is required.
It is an inescapable fact that Facetotecta is in critical need of a novel higher-level classificatory scheme, as all species are currently placed in the single genus Hansenocaris. These new species are both morphologically and phylogenetically so diverse that it is highly unsatisfactory to retain them within the same genus. We expect that single-specimen culturing, live-imaging, amplification, and sequencing will be the key step towards a new classification of Facetotecta (Olesen et al., 2022). For planktotrophic specimens, which we are still unable to culture in vitro , this poses a significant challenge that may postpone detailed taxonomic descriptions. It is exceedingly difficult to morphologically separate Types A* and AE*, for example, into what are likely multiple “species” (or MOTUs) that together occupy a geographical range that extends from Japan and Taiwan to the Azores. It is highly unlikely that each Type constitutes one, big breeding population. Wide biogeographical ranges of morphotypes could occur in some planktotrophic y-nauplii, however, because of their long developmental times and putative long-distance dispersal capabilities. For example, completion of the larval development of the Arctic facetotectan Hansenocaris itoi requires three months (April through June: Kolbasov et al., 2021) and this species may have a wide distribution. Hansen (1899) identified y-nauplii that are virtually identical to H. itoi in the Baltic Sea (Kiel Bay), a place separated from the type locality in the White Sea by thousands of kilometers of open ocean. The lack of sequence data for the Baltic population, if it still exists, illustrates the importance of Protocol 1, in which molecular sequence data are linked to live image data.
In conclusion, the easy-to-follow and inexpensive Protocol 1 described herein allows the extraction of maximal molecular and morphological information from single y-larval specimens, although we expect that this method should work for any invertebrate taxon with free-living larvae. It should be used in future efforts to, for example, model life-history evolution in the Facetotecta or other groups. With a growing inventory of sequence data from cultured, imaged, and vouchered y-larva specimens/species from geographically diverse places, the evolution of Facetotecta, as well as a comprehensive new taxonomic scheme for them, may finally be within reach.
ACKNOWLEDGEMENTS
The authors extend their gratitude to Dr. Danny Eibye-Jacobsen (University of Copenhagen, Denmark) for assisting collections in Taiwan and Japan, sometimes under trying weather conditions. Members of the Coastal Ecology lab at Academia Sinica are also thanked for assistance with sampling in Taiwan. This work was supported by a double degree graduate grant from the Natural History Museum of Denmark and the Taiwan International Graduate Program to ND and a Villum Experiment grant to JO.
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FIGURE LEGENDS
Fig. 1. Multilocus phylogeny estimation for Facetotecta with single-specimen resolution. A Maximum-likelihood tree from an alignment spanning 6730bp and six loci with Ascothoracida chosen as outgroup, showing relationships of Facetotecta and its division into at least two major clades. B Putative life cycle of Facetotecta consisting of seven y-nauplius stages, one y-cyprid stage, one or more ypsigons, and as yet unknown adults. C Examples of morphological information extracted from Protocols 1, 2, and 3, represented by branch colors (red, grey, and yellow, respectively). Protocol 2 lumps different subprotocols that lack live image data, here illustrated by three examples.
Fig. 2. Comparison of primer amplification efficacy with two filter-free DNA extraction protocols for single-larva systematics. A Amplification success of all primers using GeneReleaser and Simplified DNeasy extraction kits. B Amplification success of all primers partitioned to locus using GeneReleaser and the Simplified DNeasy extraction kits. The large difference for 16S and H3 may be caused by the different primers used for each extraction protocol (Table 1S). C density plot of amplification success of all primers for both extraction kits combined, showing uniformly higher amplification rates for 12S, 18S, and COX1 and uniformly lower amplification rates for 16S and H3.
Fig. 3. Amplification success and efficacy of “legacy” primers . A Combined amplification success using both GeneReleaser and Simplified DNeasy extraction kits. B Summary of A but with amplification success partitioned to locus, hence the comparatively larger difference for 28S and 18S primers.
AUHTOR CONTRIBUTIONS
Niklas Dreyer (ND) designed and conceived the study. ND participated in sampling, co-designed and tested primers, extraction methods and protocols. ND performed all sequence, phylogenetic and statistical analyses, co-funded the project, designed graphical outputs, took specimen photos and wrote the first draft. Ferran Palero supervised the project, designed primers, assisted statistical analyses, participated in sampling and edited manuscript drafts. Mark J. Grygier (MJG) participated in sampling, co-developed protocols, originally developed the exuvium-voucher strategy and edited manuscript drafts. Alexandra S. Savchenko provided the valuable Azores-material and edited the manuscript drafts. Gregory A. Kolbasov provided the valuable White Sea-material and edited the manuscript drafts. Ryuji J. Machida co-supervised the project, designed DNA extraction methods, assisted primer testing, and edited manuscript drafts. Benny K. K. Chan supervised the project, led sampling in Taiwan and co-funded the project. Jørgen Olesen supervised the project, co-conceived the study, led sampling in Japan, participated in sampling in Taiwan, co-designed protocols, co-funded the project, assisted phylogenetic analyses, designed graphical outputs, took specimen photos, and edited manuscript drafts.
NIKLAS DREYER1,2,3,4, FERRAN P. PALERO5†*, MARK J. GRYGIER6,7, ALEXANDRA S. SAVCHENKO8, GREGORY A. KOLBASOV9, RYUJI J. MACHIDA1, BENNY K. K. CHAN1†* & JØRGEN OLESEN1†*
TABLES
Table 1. Amplification success of all primers with single larval Facetotecta specimens using Protocols 1 and 2.