Orange is the new white: taxonomic revision of Antarctic Tritonia species (Gastropoda: Nudibranchia)

Among nudibranch molluscs, the family Tritoniidae gathers taxa with unclear phylogenetic position, such as some species of the genus Tritonia Cuvier, 1798. Currently, 35 valid species belong to this genus and only three of them are found in the Southern Ocean, namely T. challengeriana Bergh, 1884, T. dantarti Ballesteros & Avila, 2006, and T. vorax (Odhner, 1926). In this study, we shed light on the long-term discussed systematics and taxonomy of Antarctic Tritonia species using morpho-anatomical and molecular techniques. Samples from the Weddell Sea and Bouvet Island were dissected and prepared for scanning electron microscopy. The three molecular markers COI, 16S, and H3 were sequenced and analysed through maximum likelihood and Bayesian methods. The phylogenetic analyses and species delimitation tests clearly distinguished two species, T. challengeriana and T. dantarti, being widely-spread in the Southern Ocean, and endemic to Bouvet Island, respectively. Coloration seemed to be an unreliable character to differentiate among species since molecular data revealed both species can either have orange or white colour-morphotypes. This variability could be explained by pigment sequestration from the soft coral species they feed on. Morphological analyses reveal differences between Antarctic and Magellanic specimens of T. challengeriana, thus, we suggest the resurrection of T. antarctica Martens & Pfeffer, 1886 to encompass exclusively the Antarctic species. To progress further, additional molecular data from Magellanic specimens are required to definitely resolve their taxonomy and systematics.


INTRODUCTION 33
The organisms composing Antarctic benthic fauna tend to present long life cycles, slow growth rates due to slow 34 metabolism, and direct development; and this is particularly true for molluscs (Peck et al. 2006; Moles et al. 2017). All 35 these common characteristics seem to be the consequence of the peculiar characteristics of the Southern Ocean (SO), e.g. 36 low temperatures, relative stability in the frequency of physical disturbance, and pronounced seasonality (Dayton et al. (Goodheart et al. 2015). One of these taxa is the family Tritoniidae, among which the genus Tritonia 71 from the Magellan area, and this led to synonymize again T. antarctica with T. challengeriana (Schrödl 2003). According 72 to Schrödl (2003Schrödl ( , 2009, there are also other described species that are no longer valid and are considered synonyms of 73 T. challengeriana, i.e. Microlophus poirieri Rochebrune & Mabille, 1889, T. poirieri Odhner (1926), and T. australis 74 (Berg, 1898). The specimens collected for these studies were often limited to a single individual and thus these 75 identifications might be unreliable (Wägele, 1995;Schrödl, 2003Schrödl, , 2009Shields et al. 2009). Furthermore, until now, no 76 molecular data are available for any of these species when given the wide range of distribution that T. challengeriana 77 seems to present, the implementation of molecular tools could prove helpful to solve this phylogenetic conundrum. Here, 78 we aim to combine molecular techniques, used here for the first time in this species complex, with detailed morpho-79 anatomical analysis to shed light into the long-term discussed systematics and taxonomy of Antarctic Tritonia species.

MATERIAL AND METHODS 81
Sample collection

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The best-fit model of evolution (GTR + Г + I; Yang 1996) was chosen using the Akaike information criterion (AIC;

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For each gene a maximum-likelihood (ML) analysis was conducted, the final result was given by a concatenated 115 alignment of all three genes. ML analyses were conducted using RAxML 8.1.2 (Stamatakis 2014), using a GTR model 116 of sequence evolution with corrections for a discrete gamma distribution and invariable sites (GTR + Г + I; Yang 1996) 117 was specified for each gene partition, and 500 independent searchers were conducted. Nodal support was estimated 118 through bootstrap algorithm (500 replicates) using the GTR-CAT model (Stamatakis et al. 2008). The Bayesian inference 119 (BI) was performed on the concatenated alignment of the three genes using MrBayes 3.2.5 (Ronquist et al. 2011). Two 120 runs were conducted in MrBayes for 10 million generations, sampling every 2,000th generation, using random starting 6 trees. A 25% of the runs were discarded as burn-in after checking for stationarity with Tracer 1.7 ).

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Bootstrap support (BS) and posterior probabilities (PP) were thereafter mapped onto the optimal tree from the independent 123 searches. The tree was rooted using four selected Proctonotoidea species as sister group to the rest of the Dendronotoidea 124 species included in this study (see Goodheart et al. 2015).    hours and later rinsed with distilled water in ultrasound baths. The reproductive system was depicted, and the penial 139 papilla extracted, and critical point dried prior to mounting on stubs with carbon sticky-tabs, as for the radulae and jaws, 140 for scanning electron microscopy (SEM). The stubs were carbon-coated, and images were taken using a J-7100F Jeol 141 scanning electron microscope at the UB Scientific and Technological Centers (CCiT-UB).

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The description of the 50 specimens collected allowed us to classify them into the two known species Tritonia 144 challengeriana and T. dantarti, which have been studied in detail here for their morphology and anatomy, as well as for             198 Schrödl 1996) to Ancud Bay (Schrödl 1996), South Georgia (Odhner 1926 (Odhner 1926;Schrödl 1996). Living specimens present a whitish to 225 brownish colouration, with white or opaque white reticulations on the notal surface. Preserved specimens can be whitish, 226 yellowish or pinkish and their notum can be more or less smooth. This species differs from T. challengeriana by having 10 less number of gills, extremely large and strong jaws, which cause an elevated mediodorsal protuberance in between the 228 rhinophores, and the lack of oral lips, with a higher jaws:body length ratio than T. challengeriana (Table 1)

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dantarti presents lesser teeth rows and a monocuspidated rachidian tooth, while T. vorax presents a higher number of 264 rows with a tricuspidated rachidian tooth. Additionally, the jaws:body length ratio is higher in T. vorax (Table 1).

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The ABGD analyses additionally supported the taxonomic classification of T. challengeriana and T. dantarti with an 289 intraspecific variation of 1.7 and 1.9 % on average, respectively; whereas their interspecific variation ranged from 12 to 290 14 %. Intraspecific variation within other Tritonia species considered in this study range from 0 to 7 %, while their 291 interspecific variations ranges approx. 9.1-25.7 % (Table 2). We have chosen to not consider in this species delimitation 292 tests, the unidentified Tritonia spp. (Sup. Material 2) due to a possible misinterpretation of the specimens, that may belong 293 to the genus Marionia (Fig. 4). The GMYC analysis also recovers two distinct species groups belonging to T. The specimens analysed in this study from the high Antarctic belonged to the only current valid species Tritonia 298 challengeriana, while the specimens from Bouvet Island belonged to T. dantarti. Phylogenetic analyses and species 299 delimitation tests recovered these two species with a strong support (Fig. 4), including the specimens of T. challengeriana  Antarctic regions (Figs. 5-6). These results were supported for our molecular analyses. Besides this, no other clear 306 diagnostic characters were found in the morpho-anatomical analyses to allow the discrimination among these two species.

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For instance, shape and body measurements, the number of velar processes, the shape and number of gills, the radular 308 formula, and the shape of the jaws are not quite discernible between T. dantarti and T. challengeriana. In fact, both 309 species overlap in the range of the aforementioned characters (Table 1)

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showing that the glands were present on the dorsal surface of the specimens from the Magellanic area, even if sporadically 316 and in a lower number. Our specimens seem to be similar to the T. antarctica described by Wägele (1995). Pictures of 317 living specimens from the Magellanic region ( Fig. 6A-C) do not show visible knobs, which are easily detectable on 318 specimens from Antarctica (Fig. 6D-F). Unfortunately, we cannot confirm the validity of T. antarctica, since there are

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These defences include chemicals (natural products), which can be either de novo synthesized by the own slug or gathered 335 from their prey (i.e. kleptochemistry). An example of kleptochemistry in Antarctica is found in Tritoniella belli Eliot,