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.