Henricia Henricia Henricia Henricia Henricia, 1840

Henricia Henricia Henricia Henricia Henricia View in CoL genetic clade morphological

,,,,,; the group’ group’ group’ group’ group’ characters of perforata ‘ perforata ‘ ‘ perforata perforata ‘ ‘ perforata morphological re-examination after 853327 KY KY 853353 View Materials 853328 KY KY 853372 View Materials 853385 KY examination of identification 853321 853318 853316 853315 853314 based on final and KY KY KY KY KY E Clade E Clade E Clade Clade E E Clade identification this study in; initial) obtained group perforata group perforata group perforata . perforata group temperature for sequence sp and. Henricia Henricia Henricia Henricia Henricia depth longitude, number accession not measured 4.2 2.7 2.0 x 16 latitude, GenBank;, x determined 184.5 1457.0 320.0 x 13.0, code; genes S not 41.22087 − − 39.75628 − 29.70060 11.13556 5.16472 locality (COI and 16, nd sequence staff.

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Table

2

PA

04016

PA

04021 04116

PA

SMNH Tussøyna, For each revealed specimens of * Identification the amplification reactions was checked on a 1% agarose gel and then, amplification products were treated enzymatically with Exonuclease-I and Fast-SAP (shrimp alkaline phosphatase; both from Fermentas, Thermo Scientific). These products were then sequenced along both strands in 10 µL reactions with 0.165 µM primer, 1× Sequencing Buffer and 0.5 µL Big Dye v. 3.1 PreMix (Applied Biosystems, Thermo Scientific) following a standard thermocycling protocol. Sequencing products were separated on an ABI3130xl sequencer and bases were called using Sequence Analysis 6 software (both from Applied Biosystems, Thermo Scientific).

PHYLOGENETIC ANALYSES

Data from the different genes were aligned and analyzed as separate data sets. DNA sequence quality was checked from the sequence electropherograms and the data were input to MEGA7 software ( Kumar, Stecher & Tamura, 2016). Both sequenced strands were aligned using the clustal W algorithm in MEGA7 and any ambiguous bases were checked with sequence from the complementary strand when creating a consensus sequence for each individual. Best models for use in phylogenetic analyses were chosen according to the Bayesian Information Criterion (BIC) using jModelTest2 ( Darriba et al., 2012; http://jmodeltest.org/). Phylogenetic analyses were performed using the maximum likelihood algorithms implemented in MEGA7 and PhyML 3.0 ( Guindon et al., 2010; http://www.atgc-montpellier.fr/phyml/) using the HKY+G model, for both data sets. Five hundred replicates were performed to estimate bootstrap values. In addition, we also analyzed the data sets using the algorithm implemented in RAxML 8.2.7 ( Stamatakis, 2014) with the plugin available in Geneious v. 10.2 (Biomatters Limited; www.geneious.com). In this case, the GTR+G model was used and 1000 bootstraps were performed. For outgroups, we chose sequences from distantly related asteroids in the genus Echinaster available in GenBank [16S: Echinaster sentus (Say, 1825) DQ 297088.1; COI: Echinaster spinulosus Verrill, 1829 GAVE 01107285.1]. MEGA7 was also used to calculate genetic distances between the clades identified in the phylogenetic analyses. In these calculations, the Kimura 2 parameter evolutionary model was used. The sequences obtained in this study are available in GenBank with accession numbers KY853246 View Materials – KY853395 View Materials ( Table 2).