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Population Genomics of Nymphon australe Hodgson, 1902 (Pycnogonida, Nymphonidae) in the Western Antarctic.

Introduction

The benthic communities of the Southern Ocean are vastly different from those found in other regions of the world because of the ocean's geological and thermal isolation (Peck et al., 2006). The onset of the Antarctic Polar Front (APF) about 41 million years ago (mya) isolated the region, including the benthic fauna, through a number of geological processes (Scher and Martin, 2006). The APF is postulated to be responsible for isolating Southern Ocean biota and for the high rates of endemism for organisms unable to cross the differential in temperature and salinity (Clarke et ai, 2005; Thornhill et al., 2008; Kaiser et al., 2013). Although oceanic fronts have the potential to act as barriers to dispersal, oceanic currents also have the potential to act as dispersal vectors (Scheltema, 1986; Clarke et ai, 2005; Thornhill et ai, 2008; Galaska et al., 2017a). The Antarctic Circumpolar Current (ACC) has long been a force credited with the dissemination of larval and/or adult life stages, resulting in homogeneous populations around the continent of Antarctica (Arntz et ai, 1994; Clarke and Johnston, 2003). Along with the ACC, coastal currents, Antarctic deep-water currents, the Ross Sea Gyre, and the Weddell Sea Gyre, for example, have a significant impact on organisms inhabiting the Antarctic Shelf (Riesgo et al, 2015).

Antarctic glaciation was initiated during the Cenozoic when atmospheric C02 decreased (DeConto and Pollard, 2003). Glacial activity limited the availability of habitable space on the Antarctic Shelf (Thatje et ai, 2005). Glacial coverage is not static, rendering habitat necessary for the survival of benthic organisms unreliable over a geologic time-scale (Berger, 1988; Thatje et ai, 2005). For this reason, benthic organisms have had potential paths to persist through periods of glacial maxima. Eurybathic species could seek refuge in the depths or within deglaciated areas (Thatje et al, 2005). During periods of high glacial cover, subsets of organismal populations become isolated from one another, whereas times of glacial minima allow for the mixing of populations that are no longer separated (Thatje et al., 2005). Alternating periods of isolation and coalescence of taxa under fluctuating environmental conditions led to the condition dubbed the "diversity pump" (Clarke and Crame, 1992). The diversity pump functions at two scales: long-term climatic fluctuations influence the success of particular physiologies and ecologies for organisms, and shorter cycles influence their distribution (Clarke and Crame, 1992).

Pycnogonids (sea spiders) are globally distributed marine arthropods and are a ubiquitous component of the Southern Ocean benthic community. Sea spiders in the Southern Ocean are speciose, with more than 192 described species, 108 of which are endemic to the region (Munilla and Soler-Membrives, 2009). The brooding reproductive strategies of pycnogonids and the particularly slow ambulatory capabilities of adults would suggest limited dispersal capabilities (Poulin and Feral, 1996). However, many species are reported to be circumpolar, based simply on presence/absence data (Munilla and Soler-Membrives, 2009). Recently, some pycnogonid species previously thought to have circumpolar distributions have been shown to be a cryptic species complex (Krabbe et al, 2010; Weis et al, 2014; Domel et al, 2017). Nymphon australe Hodgson, 1902 is the most commonly found species of pycnogonids in the Southern Ocean, and it has, as do nearly all pycnogonids, a brooded, lecithotrophic larval protonymphon stage (Arango et al, 2011 ; Brenneis et al, 2017). The species is noted for high phenotypic plasticity, and previous studies also found high genetic diversity within the species (Child and Cairns, 1995; Mahon et al, 2008; Arango et al, 2011; Soler-Membrives et al, 2017). For example, phenotypically variable or plastic traits include the number of chelae teeth per finger (36-65), a short or long propodus, and vestigial or absent auxiliary claws; but every character can vary, even in individuals sampled from the same trawl (Child and Cairns, 1995).

Most recently, Soler-Membrives et al. (2017) found N. australe to have a circumpolar distribution and geographically structured haplotypes by using mitochondrial DNA sequences from a partial fragment of cytochrome c oxidase sub-unit I (COI).

High phenotypic plasticity, as well as the structured nature of the COI data, necessitates utilization of a high-resolution genomic technique and an increased sampling effort to accurately assess the diversity and population connectivity of N. australe in the Southern Ocean. A single uniparentally (maternally) inherited marker does not provide enough resolution to detect N. australe admixture of populations. Further, Soler-Membrives et al. (2017) did not use 16S and internal transcribed spacer ribosomal RNA fragments or microsatellite markers because they did not show sufficient sequence variation. The use of restriction site-associated DNA sequencing (RADseq) to discover single nucleotide polymorphisms (SNPs) and to genotype nonmodel organisms is cost effective, easily generates hundreds to thousands of SNPs, and provides the ability to differentiate populations with confidence (Andrews et al, 2016). With the greatly improved resolution generated by our RADseq analyses, we hypothesize that we can distinguish between one homogenous population, geographically distinct populations, or cryptic species of N. australe throughout the Western Antarctic, as potential explanations for the observed pattern of circumpolarity of one N. australe species.

Materials and Methods

Sample collection

Specimens of Nymphon australe Hodgson, 1902 were collected via Blake trawls during two research expeditions aboard the RVIB Nathaniel B. Palmer (NBP12-10) and on the ASRV Laurence M. Gould (LMG13-12). Sampling sites included locations in the Ross, Bellingshausen, Amundsen, and western Weddel 1 Seas and along the Western Antarctic Peninsula, covering an over-water distance of more than 5000 km (Fig. 1). Upon collection, specimens were sorted to morphospecies and preserved either at -80 [degrees]C or in ~95% ethanol. Subsequent identification to species was done upon return of samples to our home institution. For this investigation, a total of 92 individuals were included from throughout the sampled range, including 39 from the Western Antarctic Peninsula, 2 from the Bellingshausen Sea, 23 from the Amundsen Sea, and 28 from the Ross Sea (Fig. 1 ; Table Al).

Molecular methods

Genomic DNA extractions were performed using the QIA-GEN (Hilden, Germany) DNeasy Blood and Tissue Kit, following the manufacturer's protocol. The 2b-RAD protocol was utilized because it uses type IIB restriction enzymes, which cleave the DNA into short, uniform fragments, allowing for even sequencing coverage across the genome (Wang etal, 2012). With 2b-RAD, a subset of restriction sites can be targeted by using modified oligonucleotide adaptors that reduce genome complexity, thus decreasing marker density and allowing more individuals to be multiplexed per lane (Wang etal, 2012). Samples were prepped using the 2b-RAD protocol (Wang etal, 2012), with the Alf] restriction enzyme. A 1/8 reauction scheme was selected on the basis of the estimated genome size of about 500 megabases (Mb). Approximate genome size was determined from previously sized sea spider genomes ranging between 205 and 743 Mb (Libertini and Krapp, 2007). The 1/8 reduction scheme was accomplished with the addition of adaptors 5ILL-NC and 3ILL-NG. Immediately following the 2b-RAD protocol, gel purification was performed using a QIA-GEN QIAquick Gel Extraction Kit. All samples had been incorporated with unique barcode combinations during the protocol and were pooled in equal concentrations prior to sequencing (Wang et al., 2012). The prepared samples were then sequenced at the HudsonAlpha Institute for Biotechnology (Huntsville, Alabama) on an Illumina (San Diego, CA) HiSeq 2500 platform, using v4 chemistry to generate 50-bp single-end reads.

Data analyses

Raw sequence reads were de-multiplexed by individual, on the basis of unique barcode combinations used in 2b-RAD preparation. Resulting FASTQ files (Illumina) were filtered to remove any samples that did not contain the Alf I restriction enzyme and were subsequently truncated to the uniform 36-bp amplicon with the AlfIExtract.pl 2.0 script from Meyer (2016). Loci were removed from further analyses if they did not have [greater than or equal to]15X coverage, <1% variance if homozygous, >25% variance if heterozygous, presence in [greater than or equal to]10% sample sites (collection latitude and longitude; Table Al), and [greater than or equal to]70% of individuals within sample sites, with the denovo_map.pl 2.0 script and populations program from Stacks (Catchen et ai. 2011, 2013).

The data set was evaluated as follows under the assumption that all N. australe individuals collected were one species. To estimate the most likely number of populations (K). we used the adegenet. version 2.0.1. package (Jombart, 2008; Jombart and Ahmed, 2011) in R 3.3.2 (R Core Team, 2016). Adegenet estimates K, or the presumed number of populations, by evaluating Bayesian information criterion values informed by retained principal components. Principal Component Analyses (PCAs) are useful to spatially compare allele frequencies of loci among all individuals. The retained PCAs were used to perform a discriminant analysis of principle components (DAPC) within adegenet. The DAPC recovers maximum genetic variation between clusters, while minimizing genetic variation within clusters (Jombart, 2008; Jombart and Ahmed, 2011).

The Landscape and Ecological Associations 1.6.0 (LEA) package (Frichot and Francois, 2015) in R implements sparse nonnegative matrix factorization least squares optimizations to estimate population structure and visualize the results (Frichot et al, 2014). Estimation of K is evaluated using the minimum cross-entropy criterion. Once K is determined, admixture coefficient analyses were plotted in LEA to assess the genetic mixing of populations.

The summary statistics, Mantel test, and pairwise fixation index ([F.sub.ST]) values were calculated with samples separated into geographic regions (localities in Table Al; Wier and Cockerham, 1984). Summary statistics of SNP loci were calculated in HIERFSTAT. version 0.4.22 (Goudet, 2005). Mantel tests were performed to compare distance between geographic regions and pairwise [F.sub.ST] values to test for isolation by distance. Pairwise [F.sub.ST] values were calculated in HIERFSTAT, and distances between geographic regions were measured with the ruler tool in Google Earth Pro (Google, 2017). One distance was measured between the east coast of the Antarctic Peninsula and the Ross Sea by going around the continent as the ACC travels, and a second distance was measured over land between the east coast of the Antarctic Peninsula and the Ross Sea to represent the distance for a trans-Antarctic seaway (Barnes and Hillenbrand, 2010). Genetic differentiation was assessed for all individuals, and then pairwise comparisons were made on the basis of geographic regions.

Results

After application of final filtering steps to raw sequencing reads, 61 of the initial 92 individuals remained in our data set, and 3086 SNP loci were retained. Individuals retained after SNP loci filtering collected from locations surrounding the Antarctic Peninsula numbered 23; in the Ross Sea, 26; and in the Amundsen Sea, 12. Individuals were removed from the data set if they no longer retained informative loci after filtering. Raw sequencing reads were deposited in the National Center for Biotechnology Information GenBank Sequence Read Archive (SRP130-364). Calculated with adegenet, the DAPC resulted in K = 2, Densities of individuals were plotted for varying discriminant functions, resulting in two clusters (Fig. 2A).

Estimates of K based on the cross-entropy criterion indicated that K = 2 had the highest likelihood in LEA (Fig. 2B). Both DAPC and the cross-entropy criterion analyses clustered individuals into the same two discrete populations. Two ancestral populations were consistently recovered, so K = 2 was used for all further analyses.

Admixture analysis yielded similar proportions of the two ancestral populations for the individuals collected from the Antarctic Peninsula and the Ross Sea and a distinctive difference in proportions from individuals collected from the Amundsen Sea. The Antarctic Peninsula and Ross Sea individuals were dominated by one ancestral population, and the Amundsen Sea individuals were dominated by the other ancestral population (Fig. Al).

Mantel tests were also executed to determine whether isolation by distance was occurring in this data set, but we did not find a significant relationship between genetic structure (represented by corrected [F.sub.ST] values for samples from each geographic region, calculated with HIERFSTAT) and distance between each region (measured with the ruler tool in Google Earth Pro) (Fig. A2; Rousset. 1997; Spong and Creel, 2001). The regression of the ACC and the trans-Antarctic seaway measured by Mantel tests was not significant ([R.sup.2] = 0.117, P = 0.506 and [R.sup.2] = 0.037, P = 0.713, respectively; Fig. A2). Additional Mantel tests were conducted between collection sites within the east and west coasts of the Antarctic Peninsula, the Amundsen Sea, and the Ross Sea. and none were significant ([R.sup.2] = 0.104. P = 0.532; [R.sup.2] = 0.437, P = 0.153; [R.sup.2] = 0.037, P = 0.589; [R.sup.2] = 0.052, P = 0.321, respectively; Fig. A3).

The resulting data set and output file from Stacks revealed that only 2.5% of all SNPs had alignment stacks between the two "populations" of what we assumed was a single species based on morphology, knowledge of phenotypic plasticity of the species, and collection sites. The output file contained 3086 SNP loci, but only 78 loci had alignment stacks between the two "populations," with fixed alleles at 8 loci. When individuals from "population" 1 were analyzed, 2580 loci were coded for. When individuals from "population" 2 were analyzed with Stacks, 1095 loci were coded for. Furthermore, HIERFSTAT was used to calculate a Cavalli-Sforza and Edwards chord genetic distance between the two groups, which amounted to 0.334, demonstrating divergence greater than what would be expected within a single species (Frankham et ai, 2010; Krabbe et ai, 2010; Harder et ai, 2016). Only SNP alignments between populations (not between species) should be analyzed together. The two "populations" were first analyzed as one species with two populations, until we realized they were two different species, and then they were analyzed separately. Due to this divergence, the two "populations" were considered two putative species and analyzed as such from this point forward.

The two putative species were stratified geographically, with the 12 individuals of putative species 1 collected within the Amundsen Sea and the remaining 49 individuals of putative species 2 surrounding the Antarctic Peninsula and in the Ross Sea (Fig. 1). Each putative species was individually analyzed, as described above, in an attempt to identify population structures within each putative species. Adegenet and LEA identified both putative species to have K = 1 and K = 1 within each locality (Fig. A4). Admixture analyses and PCAs are not informative with K = 1 and thus were not conducted. Instead. [F.sub.ST] statistics were calculated for each putative species and supported low population structuring within each group. The [F.sub.ST] calculated among putative species 1 sample sites (collection latitude and longitude; Table Al) is 0.0591 (P [less than or equal to] 0.01), and the [F.sub.ST] calculated for putative species 2 sample sites is 0.0304 (P = 0.027). Pairwise [F.sub.ST] values among sample sites in the Antarctic Peninsula and Ross Sea regions were lower, but with significant P-values (Table 1). As could be expected, pairwise [F.sub.ST] values were higher and had significant P-values when either the Antarctic Peninsula or the Ross Sea individuals were compared to the Amundsen Sea individuals (Table 1). A subsequent screening of these individuals found no distinct morphological characteristics separating the two groupings, identified all individuals as N. australe, and thus supported the notion of two cryptic species within the N. australe individuals included in this study.

Discussion

In this study, two geographically structured Nymphon species, thought initially to both be Nymphon australe, were recovered in the Western Antarctic. Nymphon australe Hodgson, 1902 has been previously described as a circumpolar species with presence/absence data (Munilla and Soler-Membrives, 2009); and more recently, molecular studies have found it to be a geographically structured circumpolar species, by using a coarser molecular data set (COI; Soler-Membrives et ai, 2017). Also using the coarser molecular data set (COI and 16S), Mahon et ai (2008) supported the possibility of two previously unrecognized Nymphon species initially identified (morphologically) as N. australe in the Antarctic Peninsula region.

A possible explanation for undetected N. australe speciation is that most sea spiders collected from the Southern Ocean are identified as N. australe, even though it has long been recognized as a species with wide variability of morphology (Child and Cairns, 1995). Difficulty in species delineation within the Nymphonidae stems from variability of morphology and few physical traits useful to taxonomic analyses (Hedgpeth. 1947. 1955; Arnaud and Bamber, 1987;Arango, 2003). For example, a specimen presented as Colossendeis sp. was later found, with the sequenced mitochondrial genome of Colossendeis megalonyx, to be misidentified, and the identification was corrected to a nymphonid species (Dietz et ai, 2011). The two Nymphon species delineated with COI and 16S by Mahon et ai (2008) identified both Nymphon species 1 and species 2 as N. australe when observed according to morphological traits alone.

Previous findings (Munilla and Soler-Membrives, 2009; Soler-Membrives et ai, 2017) of circumpolarity were not supported by the genome-wide SNP data set produced in this study. However, this is not entirely unexpected because SNP data provide a much higher level of molecular resolution and genetic subdivision than had been previously noted. Importantly, the Soler-Membrives et al. (2017) study did not sample the Bellingshausen and Amundsen Seas, which is where our data found the first N. australe lineage. Inspection of mitochondrial markers has resulted in the discovery of cryptic or unrecognized species that overturns circumpolarity findings for many other Antarctic invertebrates, such as amphipods (Held, 2003; Held and Wagele, 2005; Havermans et ai. 2011), isopods (Raupach and Wagele, 2006), bivalves (Linse etal, 2007), crinoids (Wilson et al, 2007), and cephalopods (Allcock et al, 2011). Possible cryptic speciation was also uncovered in other pycnogonid groups previously considered circumpolar (Krabbe et al, 2010; Domel et al, 2017).

Geographie structuring of the postulated two different TV. australe species is supported in Western Antarctic waters because sample locations coincide with the separation of individuals by SNP loci (Fig. 1). Genetic connectivity is also supported by [F.sub.ST] values between individuals sampled from the Ross Sea and the Antarctic Peninsula, encompassing a distance of more than 5000 km (Table 1). The long-distance genetic connectivity excluding the Amundsen Sea can potentially be explained by the fact that on the western side of the continent, the ACC travels past the Ross Gyre and does not reach the continental shelf again until the peninsula (Tynan, 1998). Rafting on other organisms, such as a food source, provides sea spiders a potential way to travel long distances along currents (Fraser et al, 2010, 2013; Leese et al, 2010; Nikula et al, 2010). This phylogeographic pattern was uncovered with RADseq techniques applied to a benthic brittle star population also sampled from Western Antarctica (Galaska et al, 2017b).

An alternative hypothesis that could instead explain the phylogeographic patterns uncovered for N. australe included in this study, as well as other Southern Ocean species of brittle star (Galaska et al, 2017b), bivalve (Linse et al, 2007), and octopus (Allcock et al, 2011). is a connection between the Ross and Weddell Seas. Geological data suggest that the West Antarctic ice sheet collapse and formation of trans-Antarctic seaways has occurred in the past and may occur in the future (Naish et al, 2009; Pollard and DeConto, 2009). Trans-Antarctic passages connecting the Ross and Weddell Seas have existed in the recent past (125,000 years ago to 1.1 mya), facilitating organismal dispersal (Barnes and Hillenbrand. 2010). If dispersal occurred by means of a trans-Antarctic passage, putative species 2 would have dispersed over a distance of 3050 km to connect the individuals present in both the Antarctic Peninsula and the Ross Sea.

In this study, an overall homogenous population was not recovered for N. australe, indicating some restriction of genetic connectivity. However, similar to previous studies (Manon etal, 2008; Arango etal, 2011; Soler-Membrives etal, 2017), restriction to gene flow is not absolute, as putative species 2 may exhibit an extensive dispersal. Nymphon australe has a brooding, lecithotrophic larval protonymphon stage that limits dispersal and is not eurybathic. Thus, we are uncertain whether long-distance genetic connectivity is due to glacial activity, the ACC, other currents, the ability to raft on other organisms, or other unknown methods of mobility (Moon et al, 2017). We are also uncertain whether putative species 2 may be exhibiting a phylogenetic pattern representative of a past connection of the Ross and Weddell Seas. Additionally, the influence of genetic connectivity through introgression or hybridization and selective effects on these species cannot be ruled out.

Discovery of cryptic species is paramount to the true understanding of species distributions. Such understanding contributes to comprehension of the planet's past changes in climate and geology. Knowledge of how past conditions impacted gene flow can contribute to predictions for future reactions to major environmental fluctuations in a rapidly changing environment, such as in polar regions afflicted by polar wanning amplification (Taylor et al, 2013). This study demonstrates the ability of RAD-based SNP markers to detect geographically stratified, cryptic species missed by other lower-resolution markers. Also, this work points to the importance of geographic sampling and the continuing need to sample the full range of organisms with high-resolution markers.

Acknowledgments

We thank the National Science Foundation (NSF ANT-1043670 to ARM, NSF ANT-1043745, and OPP-0132032 to KMH) for the funding to collect the specimens and perform the research. This research was made possible with assistance from the captains and crews of NBP12-10, LMB13-12, LMG04-14. and LMG06-05. Additional support for this work to EEC was provided by the Central Michigan University College of Science and Engineering through an Earth and Ecosystem Sciences PhD program fellowship. This is Molette Biology Laboratory contribution no. 77 and Auburn University Marine Biology Program contribution no. 174.

Data Accessibility

Raw reads for 2b restriction site-associated DNA single-nucleotide polymorphism data are deposited in the National Center for Biotechnology Information GenBank Sequence Read Archive (SRP130-364).

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Appendix
Table Al

Specimens collected, with localities, latitudes, longitudes, depths,
and sample identifications

Species             Locality        Latitude

-                   Bellingshausen  70[degrees]48'42.81"S
-                   Bellingshausen  70[degrees]48'42.81"S
Putative species 1  Amundsen        72[degrees]28'95.08"S
Putative species 1  Amundsen        72[degrees]28'95.08"S
Putative species 1  Amundsen        72[degrees]46'83"S
-                   Amundsen        72[degrees]46'83"S
-                   Amundsen        72[degrees]46'83"S
-                   Amundsen        72[degrees]46'83"S
Putative species 1  Amundsen        73[degrees]43'29.16"S
Putative species 1  Amundsen        73[degrees]43'29.I6"S
Putative species 1  Amundsen        73[degrees]43'29.16"S
-                   Amundsen        73[degrees]43'29.16"S
Putative species 1  Amundsen        73[degrees]43'29.16"S
-                   Amundsen        72[degrees]12'15.15"S
-                   Amundsen        72[degrees]12T5.15"S
-                   Amundsen        72[degrees]12'15.15"S
-                   Amundsen        72[degrees]12'15.15"S
-                   Amundsen        72[degrees]12T5.15"S
Putative species 1  Amundsen        73[degrees]9'31.90"S
Putative species 1  Amundsen        73[degrees]9'31.90"S
Putative species 1  Amundsen        73[degrees]17'47.98"S
Putative species 1  Amundsen        73[degrees]17'47.98"S
Putative species 1  Amundsen        73[degrees]17'47.98"S
-                   Amundsen        73[degrees]9'31.90"S
-                   Amundsen        73[degrees]9'3I.90"S
Putative species 2  Ross            76[degrees]20'28.38"S
Putative species 2  Ross            76[degrees]14'42.94"S
Putative species 2  Ross            76[degrees]14'42.94"S
Putative species 2  Ross            76[degrees]14'42.94"S
Putative species 2  Ross            76[degrees]14'42.94"S
Putative species 2  Ross            76[degrees]14'42.94"S
Putative species 2  Ross            76[degrees]14'42.94"S
Putative species 2  Ross            76[degrees]14'42.94"S
Putative species 2  Ross            75[degrees]50'0.47"S
Putative species 2  Ross            75[degrees]50'0.47"S
Putative species 2  Ross            75[degrees]50'0.47"S
-                   Ross            75[degrees]50'0.47"S
-                   Ross            75[degrees]50'0.47"S
Putative species 2  Ross            76[degrees]54'13.68"S
Putative species 2  Ross            76[degrees]54'13.68"S
Putative species 2  Ross            75[degrees]50'0.47"S
Putative species 2  Ross            75[degrees]50'0.47"S
Putative species 2  Ross            75[degrees]50'0.47"S
Putative species 2  Ross            75[degrees]50'0.47"S
Putative species 2  Ross            74[degrees]41'0.14"S
Putative species 2  Ross            74[degrees]10'55.12"S
Putative species 2  Ross            74[degrees]10'55.12"S
Putative species 2  Ross            78[degrees]3'47.66"S
Putative species 2  Ross            76[degrees]14'42.94"S
Putative species 2  Ross            76[degrees]14'42.94"S
Putative species 2  Ross            76[degrees]14'42.94"S
Putative species 2  Ross            76[degrees]14'42.94"S
Putative species 2  Ross            76[degrees]14'42.94"S
Putative species 2  East Peninsula  64[degrees]2'6.60"S
Putative species 2  East Peninsula  64[degrees]2'6.60"S
Putative species 2  East Peninsula  64[degrees]2'6.60"S
Putative species 2  East Peninsula  64[degrees]2'6.60"S
-                   East Peninsula  64[degrees]2'6.60"S
Putative species 2  East Peninsula  64[degrees]2'6.60"S
Putative species 2  East Peninsula  64[degrees]2'6.60"S
Putative species 2  East Peninsula  64[degrees]08.357'S
Putative species 2  East Peninsula  63[degrees]41'8.82"S
Putative species 2  East Peninsula  63[degrees]41'8.82"S
-                   East Peninsula  63[degrees]41'8.82"S
Putative species 2  East Peninsula  63[degrees]41'8.82"S
Putative species 2  East Peninsula  63[degrees]45'13.38"S
Putative species 2  East Peninsula  63[degrees]45'13.38"S
Putative species 2  East Peninsula  63[degrees]45'13.38"S
Putative species 2  East Peninsula  63[degrees]45'13.38"S
-                   West Peninsula  64[degrees]24'40.32"S
-                   West Peninsula  64[degrees]24'40.32"S
-                   West Peninsula  64[degrees]24'40.32"S
-                   West Peninsula  64[degrees]24'40.32"S
-                   West Peninsula  64[degrees]24'40.32"S
-                   West Peninsula  64[degrees]24'40.32"S
-                   West Peninsula  64[degrees]24'40.32"S
-                   West Peninsula  64[degrees]24'40.32"S
-                   West Peninsula  63[degrees]48'19.95"S
Putative species 2  West Peninsula  63[degrees]48'19.95"S
-                   West Peninsula  63[degrees]48'19.95"S
-                   West Peninsula  63[degrees]48'19.95"S
Putative species 2  West Peninsula  65[degrees]1'15.12"S
Putative species 2  West Peninsula  64[degrees]38'24.00"S
Putative species 2  West Peninsula  64[degrees]38'24.00"S
Putative species 2  West Peninsula  64[degrees]38'24.00"S
-                   West Peninsula  64[degrees]38'24.00"S
-                   West Peninsula  64[degrees]38'24.00"S
Putative species 2  West Peninsula  64[degrees]38'24.00"S
-                   West Peninsula  64[degrees]38'24.00"S
Putative species 2  West Peninsula  64[degrees]38'24.0O"S
Putative species 2  West Peninsula  64[degrees]38'24.00"S
Putative species 2  West Peninsula  65[degrees]5'12.90"S

Species             Longitude                 Depth (m)  Sample ID

-                    92[degrees]31'18.24" W   430        565
-                    92[degrees]31'I8.24"W    430        566
Putative species 1  104[degrees]33'77.10"W    591          5
Putative species 1  104[degrees]33'77.10"W    591          7
Putative species 1  104[degrees]33'23"W       496         13
-                   104[degrees]33'23"W       496         17
-                   104[degrees]33'23"W       496         18
-                   104[degrees]33'23"W       496         19
Putative species 1  103[degrees]37'01.16"W    699         34
Putative species 1  103[degrees]37'01.16"W    699         35
Putative species 1  103[degrees]37'01.16"W    699         36
-                   103[degrees]37'01.16"W    699         37
Putative species 1  I03[degrees]37'0I.16"W    699         52
-                   103[degrees]35'46.81"W    341         55
-                   103[degrees]35'46.81"W    341         56
-                   103[degrees]35'46.81"W    341         57
-                   103[degrees]35'46.81"W    341         58
-                   103[degrees]35'46.81"W    341         59
Putative species 1  129[degrees]53'41.85"W    516         66
Putative species 1  129[degrees]53'41.85"W    516         69
Putative species 1  129[degrees]11'32.80"W    655         90
Putative species 1  129[degrees]11'32.80"W    655         92
Putative species 1  I29[degrees]11'32.80"W    655         97
-                   129[degrees]53'41.85"W    516        589
-                   129[degrees]53'41.85"W    516        591
Putative species 2  170[degrees]51'1.78"W     764        205
Putative species 2  174[degrees]30'14.83"E    604        272
Putative species 2  174[degrees]30'14.83"E    604        273
Putative species 2  174[degrees]30'14.83"E    604        275
Putative species 2  174[degrees]30'14.83"E    604        276
Putative species 2  174[degrees]30'14.83"E    604        277
Putative species 2  174[degrees]30'14.83"E    604        278
Putative species 2  174[degrees]30'14.83"E    604        279
Putative species 2  166[degrees]30'19.78"E    552        332
Putative species 2  166[degrees]30'19.78"E    552        333
Putative species 2  166[degrees]30'19.78"E    552        334
-                   166[degrees]30'19.78"E    552        335
-                   166[degrees]30'19.78"E    552        336
Putative species 2  169[degrees]57'54.90"E    764        638
Putative species 2  169[degrees]57'54.90"E    764        639
Putative species 2  166[degrees]30'19.78"E    552        650
Putative species 2  166[degrees]30'19.78"E    552        651
Putative species 2  I66[degrees]30'19.78"E    552        652
Putative species 2  166[degrees]30'19.78"E    552        654
Putative species 2  168[degrees]28'0.19"E     513        656
Putative species 2  166[degrees]39'39.70"E    390        657
Putative species 2  166[degrees]39'39.70"E    390        658
Putative species 2  169[degrees]59'28.14"W    549        625
Putative species 2  I74[degrees]30'14.83"E    604        631
Putative species 2  174[degrees]30'14.83"E    604        632
Putative species 2  174[degrees]30'14.83"E    604        633
Putative species 2  174[degrees]30'14.83"E    604        634
Putative species 2  174[degrees]30'14.83"E    604        635
Putative species 2   56[degrees]43'41.70"W    290        427
Putative species 2   56[degrees]43'41.70"W    290        428
Putative species 2   56[degrees]43'41.70"W    290        429
Putative species 2   56[degrees]43'41.70"W    290        430
-                    56[degrees]43'41.70"W    290        431
Putative species 2   56[degrees]43'41.70"W    290        432
Putative species 2   56[degrees]43'41.70"W    290        435
Putative species 2   56[degrees]51.994'W        -        451
Putative species 2   56[degrees]51'32.40"W    400        464
Putative species 2   56[degrees]51'32.40"W    400        465
-                    56[degrees]51'32.40"W    400        466
Putative species 2   56[degrees]51'32.40"W    400        467
Putative species 2   55[degrees]41'2.34"W     334        484
Putative species 2   55[degrees]41'2.34"W     334        492
Putative species 2   55[degrees]41'2.34"W     334        495
Putative species 2   55[degrees]41'2.34"W     334        496
-                    61[degrees]57'47.40"W    664        402
-                    61[degrees]57'47.40"W    664        403
-                    61[degrees]57'47.40"W    664        404
-                    61[degrees]57'47.40"W    664        405
-                    61[degrees]57'47.40"W    664        412
-                    61[degrees]57'47.40"W    664        413
-                    61[degrees]57'47.40"W    664        414
-                    61[degrees]57'47.40"W    664        415
-                    60[degrees]28'44.70"W    428        422
Putative species 2   60[degrees]28'44.70"W    428        423
-                    60[degrees]28'44.70"W    428        424
-                    60[degrees]28'44.70"W    428        426
Putative species 2   64[degrees]25'30.12"W    312        546
Putative species 2   64[degrees]14'43.38"W    312        547
Putative species 2   64[degrees]14'43.38"W    312        548
Putative species 2   64[degrees]14'43.38"W    312        549
-                    64[degrees]14'43.38"W    312        550
-                    64[degrees]14'43.38"W    312        551
Putative species 2   64[degrees] 14'43.38" W  312        556
-                    64[degrees]14'43.38"W    312        559
Putative species 2   64[degrees]14'43.38"W    312        560
Putative species 2   64[degrees]14'43.38"W    312        561
Putative species 2   65[degrees]48'31.98"W    202        665

Species are labeled according to designation in adegenet (Jombart.
2008; Jombart and Ahmed, 2011) and Landscape and Ecological
Associations 1.6.0 (LEA) package (Frichot and Francois. 2015) analyses
or with a dash if filtered out before assignment to a particular
species.


E. E. COLLINS (1), M. P. GALASKA (2). K. M. HALANYCH (2), AND A. R. MAHON (1,*)

(1) Department of Biology, Central Michigan University, Mount Pleasant, Michigan; and (2) Department of Biological Sciences, Auburn University, Auburn, Alabama

Received 9 January 2018; Accepted 4 May 2018; Published online 4 June 2018.

(*) To whom correspondence should be addressed. E-mail: mahon2a@cmich.edu.

Abbreviations: 2b-RAD. 2b restriction site-associated DNA genotyping; ACC. Antarctic Circumpolar Current; APF. Antarctic Polar Front; COL cytochrome c oxidase subunit I; DAPC. discriminant analysis of principle components; FST. fixation index; K, number of populations; LEA, Landscape and Ecological Associations: Mb. megabases (unit of length for DNA fragments = 1 million nucleotides); mya. million years ago: PCA. Principal Component Analysis: RADseq. restriction site-associated DNA sequencing; SNP. single-nucleotide polymorphism.
Table 1

Pairwise fixation index ([F.sub.ST]) values and P-values presented by
locality and calculated with HIERFSTAT

                                                 East       West
[F.sub.ST] and P-values  Amundsen Sea  Ross Sea  Peninsula  Peninsula
Amundsen Sea              ...          0.132     0.116      0.112
Ross Sea                 <0.001        ...       0.031      0.030
East Peninsula           <0.001        0.007      ...       0.007
West Peninsula           <0.001        0.040     0.646       ...

[F.sub.ST] values were calculated with Weir and Cockerham's estimate
(1984) and are the values on the top right portion of the table.
P-values are reported on the bottom left portion of the table.
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Date:Jun 1, 2018
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