Dna

Loeffelholz, Jacob D., Mapalo, Marc A., Morris, Erin R. & Miller, William R., 2025, Tardigrades of North America: new records of occurrence, a morphological mystery, and phylogenetic analyses of Novechiniscus armadilloides (Schuster, 1975) (Tardigrada, Heterotardigrada, Echiniscoidea, Echiniscidae), Organisms Diversity & Evolution 25 (1), pp. 157-166 : 159-160

DNA barcoding and phylogenetic analyses

For DNA extraction, some specimens collected from St. George, Utah were placed into Eppendorf tubes filled with sterilized water, and extractions were done as outlined by Stec et al (2015). When possible, the remaining exoskeletons were collected from the tube and fixed on microscope slides in PVA mounting media. Three different gene sequences were sequenced in this study — 18S ribosomal RNA (rRNA), 28S rRNA, and cytochrome c oxidase subunit I (COI) genes. Gene amplification was done using the PCR

occurrence of Novechiniscus armadilloides in Utah, USA.

Sites where DNA barcodes of Nov. armadilloides were successfully obtained are indicated with GenBank accession numbers programs from Stec et al (2015) with 18S rRNA, 28S rRNA, and COI primers used in Momeni et al (2023) ( Table S1). Successfully amplified PCR products were purified and sent to the University of Missouri-Columbia Genomics Technology core for sequencing. Only one haplotype for each gene was detected, and their sequences are deposited in GenBank (Table S2).

To determine the relationship of Novechiniscus to extant echiniscoideans, phylogenetic reconstruction was done using 18S and 28S rRNA sequences. Sequences of other tardigrades were obtained from GenBank and together with Novechiniscus , the dataset consisted of 52 tardigrades, with the echiniscoidids used as an outgroup. When possible, each echiniscid genus was represented by two species and the samples selected had at least an 18S rRNA sequence that had a length of more than or equal to 700 bp. For the 28S rRNA, only overlapping sequences corresponding to one region of 28S were used. As a result of these selection processes, 52 sequences of 18S rRNA and 48 sequences of 28S rRNA were used (Table S2). Each of the rRNA sequences were individually aligned using MAFFT 7.5 ( Katoh & Standley, 2013) with the L-INS-i algorithm. The alignments were then visualized, and both ends were manually trimmed using Aliview 1.28 ( Larsson, 2014) which resulted in a final length of 1754 and 879 nucleotides for the 18S and 28S sequences, respectively. Both datasets were then concatenated using Seaview 5.0 ( Gouy et al., 2021) resulting in a dataset of 2633 nucleotides (Data S1).

The tree topology was reconstructed using maximum likelihood (ML) and Bayesian inference (BI). The ML tree was reconstructed using IQTree 1.6 ( Nguyen et al., 2015) with the matrix divided into two partitions corresponding to each of the rRNA sequences. The GTR + F + R5 and GTR + F + R3 models were used for the 18S and 28S partitions, respectively, which were obtained using the model selection in IQTree under the Akaike information criterion (AIC). Bootstrap analysis was done using 1000 replicates, and the consensus tree was obtained using the default setting. The BI tree was reconstructed using MrBayes 3.2 ( Ronquist et al., 2012) using the best model scheme obtained from Partitionfinder 2.1 ( Lanfear et al., 2017) under the AIC. The matrix was partitioned according to the different rRNA sequences, and the GTR model + Gamma + proportion of invariable site (nst = 6, rates = invgamma) for each partition was used. Additionally, the “statefreq,” “revmat,” “shape,” “pinvar,” and “tratio” were unlinked. The analysis was run for 60,000,000 generations sampling every 10,000 generations and with a 25% burn-in frequency. Two runs were simultaneously done with each having one cold and three heated chains. Convergence was assessed by checking that the average deviation of split frequencies of the two runs was less than 0.01, effective sample size values were greater than 200, and the potential scale reduction factor was approximately = 1. A 50% majority rule consensus tree was then obtained to summarize the resulting analysis.