Accurate species identification is essential to assess biodiversity and species richness in ecosystems threatened by rapid and recent environmental changes, such as warming in most Antarctic waters. The Lepidonotothen species complex comprises demersal notothenioid fishes which inhabit the shelf areas of the Antarctic Peninsula, the Scotia Arc and sub-Antarctic islands with a circum-Antarctic distribution. Species determination in this group has often been problematic. In particular, whether Lepidonotothen squamifrons and Lepidonotothen kempi are valid as separate species has been questioned. In this study, we analysed the genetic variation among four nominal southern polar species within this complex (L. kempi, L. squamifrons, Nototheniops larseni, Nototheniops nudifrons) by means of three different markers (ND2 and tRNA mitochondrial genes and a panel of 16 nuclear microsatellites). We tested whether individuals morphologically assigned to L. kempi showed genetic separation from L. squamifrons. Our analyses indicated a lack of differentiation between L. kempi and L. squamifrons. However, a genetically distinct population was found for L. squamifrons at the Shag Rocks islands near South Georgia. Antarctic and sub-Antarctic islands are known to be home to many cryptic species and further studies will elucidate if the genetically differentiated population we found potentially originated from this context and can be considered as an incipient species. Our analysis contributes to further characterize the species composition of the most abundant fish suborder in the Southern Ocean, which is amongst the regions most threatened by climate change.

Mitochondrial markers: A 1,247 bp long mitochondrial fragment containing the entire ND2 gene and the upstream (trnM) and downstream (trnW and trnA) tRNAs was amplified with the primers GLN 5'-CTACCTGAAGAGATCAAAAC-3' and ASN 5'-CGCGTTTAGCTGTTAACTAA-3' following Kocher et al. (1995). The mitochondrial sequences were trimmed, assembled and cut to isolate the portion of ND2 (1041 bp) from the tRNAs. The sequence was revised by eye when necessary, by two operators using the software MacVector ver. 12.6 (Rastogi, 2000). Molecular validation with ND2 of morphological species identification was performed by querying every sample against the GenBank database with the Blastn and Blastp algorithms (Altschul et al., 1990). To check for presence of stop codons, nucleotide sequences were translated into aminoacids with the ExPASy translate tool (Gasteiger, 2003), resulting in a length of 347 amino acids. The sequences of the trnA, trnM and trnW were manually aligned together with all orthologs available for notothenioids in GenBank.

Microsatellite markers: We genotyped 20 microsatellite loci that have been previously characterized in different notothenioid species. Eleven loci linked to expressed sequence tags (ESTs) were isolated from N. nudifrons and described in Papetti et al. (2016): Ln22268, Ln23194, Ln35217, Ln36100, Ln36156, Ln40551, Ln41281, Ln42016, Ln42233, Ln45257, Ln45589. In the original study, these loci were successfully amplified also in L. squamifrons and N. larseni. Seven further EST-linked loci were isolated from the species Chionodraco hamatus and described in Molecular Ecology Resources Primer Development Consortium et al. (2011): Ch126, Ch623, Ch1968, Ch2788, Ch3603, Ch3866, Ch5817. Finally, two loci were isolated from the species Chaenocephalus aceratus and described in Susana et al. (2007): Ca35 and Ca48. Amplification conditions followed the original primer notes (Molecular Ecology Resources Primer Development Consortium et al., 2011; Papetti et al., 2016; Susana et al., 2007). Fragment lengths were estimated on an Applied Biosystems 3130 XL automated sequencer (Life Technologies, USA; using ROX500 as size standard). Allele scoring was independently performed by two operators using the software Genemarker ver. 2.6.3 (SoftGenetics LLC), and the Excel macro Flexibin was used for allele binning (Amos et al., 2007). The final dataset was refined by eye. Since microsatellite alleles can suffer from size shifts among different genotyping runs (Davison & Chiba, 2003; Lahood et al., 2002), we used samples replicates and the software Allelogram ver. 2.2 (Morin et al., 2009) to normalize allele size. The input files for subsequent analyses were generated with Create ver. 1.38 (Coombs et al., 2008) or PGDSpider ver. 2.1.1.5 (Lischer & Excoffier, 2012).

Refernces

Altschul, S. F., Gish, W., Miller, W., Myers, E. W., & Lipman, D. J. (1990). Basic local alignment search tool. Journal of Molecular Biology, 215, 403–410. https://doi.org/10.1016/S0022-2836(05)80360-2

Amos, W., Hoffman, J. I., Frodsham, A., Zhang, L., Best, S., & Hill, A. V. S. (2007). Automated binning of microsatellite alleles: problems and solutions. Molecular Ecology Notes, 7, 10–14. https://doi.org/10.1111/j.1471-8286.2006.01560.x

Coombs, J. A., Letcher, B. H., & Nislow, K. H. (2008). Create: a software to create input files from diploid genotypic data for 52 genetic software programs. Molecular Ecology Resources, 8, 578–580. https://doi.org/10.1111/j.1471-8286.2007.02036.x

Davison, A., & Chiba, S. (2003). Laboratory temperature variation is a previously unrecognized source of genotyping error during capillary electrophoresis. Molecular Ecology Notes, 3, 321–323. https://doi.org/10.1046/j.1471-8286.2003.00418.x

Gasteiger, E. (2003). ExPASy: the proteomics server for in-depth protein knowledge and analysis. Nucleic Acids Research, 31, 3784–3788. https://doi.org/10.1093/nar/gkg563

Kocher, T. D., Conroy, J. A., McKaye, K. R., Stauffer, J. R., & Lockwood, S. F. (1995). Evolution of NADH dehydrogenase subunit 2 in east African cichlid fish. Molecular Phylogenetics and Evolution, 4, 420–432. https://doi.org/10.1006/mpev.1995.1039

Lahood, E. S., Moran, P., Olsen, J., Stewart Grant, W., & Park, L. K. (2002). Microsatellite allele ladders in two species of Pacific salmon: preparation and field-test results. Molecular Ecology Notes, 2, 187–190. https://doi.org/10.1046/j.1471-8286.2002.00174.x

Lischer, H. E. L., & Excoffier, L. (2012). PGDSpider: an automated data conversion tool for connecting population genetics and genomics programs. Bioinformatics, 28, 298–299. https://doi.org/10.1093/bioinformatics/btr642

Molecular Ecology Resources Primer Development Consortium, Agostini, C., Agudelo, P. A., Bâ, K., Barber, P. A., Bisol, P. M., … Zulaiha, A. R. (2011). Permanent Genetic Resources added to Molecular Ecology Resources Database 1 October 2010-30 November 2010. Molecular Ecology Resources, 11, 418–421. https://doi.org/10.1111/j.1755-0998.2010.02970.x

Morin, P. A., Manaster, C., Mesnick, S. L., & Holland, R. (2009). Normalization and binning of historical and multi-source microsatellite data: overcoming the problems of allele size shift with allelogram. Molecular Ecology Resources, 9, 1451–1455. https://doi.org/10.1111/j.1755-0998.2009.02672.x

Papetti, C., Windisch, H. S., La Mesa, M., Lucassen, M., Marshall, C., & Lamare, M. D. (2016). Non-Antarctic notothenioids: past phylogenetic history and contemporary phylogeographic implications in the face of environmental changes. Marine Genomics, 25, 1–9. https://doi.org/10.1016/j.margen.2015.11.007

Rastogi, P. A. (2000). MacVector: integrated sequence analysis for the Macintosh. In Bioinformatics Methods and Protocols (pp. 47–69). https://doi.org/10.1385/1-59259-192-2:47

Susana, E., Papetti, C., Barbisan, F., Bortolotto, E., Buccoli, S., Patarnello, T., & Zane, L. (2007). Isolation and characterization of eight microsatellite loci in the icefish Chaenocephalus aceratus (Perciformes, Notothenioidei, Channichthyidae). Molecular Ecology Notes, 7, 791–793. https://doi.org/10.1111/j.1471-8286.2007.01703.x

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