DOI: 10.5061/dryad.wpzgmsbqv
title: Data for: Species identification and population genetics of the Antarctic fish genera Lepidonotothen and Nototheniops (Perciformes, Notothenioidei)

description: 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.
description: <strong>Mitochondrial markers</strong>: A 1,247 bp
 long mitochondrial fragment containing the entire <em>ND2</em>
 gene and the upstream (<em>trnM</em>) and downstream
 (<em>trnW</em> and <em>trnA</em>) 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 <em>ND2</em> (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
 <em>ND2</em> 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
 <em>trnA</em>, <em>trnM</em> and
 <em>trnW</em> were manually aligned together with all
 orthologs available for notothenioids in GenBank.
 <strong>Microsatellite markers</strong>: 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 <em>N. nudifrons</em> 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
 <em>L. squamifrons</em> and <em>N. larseni</em>.
 Seven further EST-linked loci were isolated from the species
 <em>Chionodraco hamatus</em> 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 <em>Chaenocephalus aceratus</em> 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).
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