Thamnocephalidae, a family of Anostraca which is widely distributed on all continents of the world except Antarctica, currently consists of six genera and approximately 63 recognized species. The relationships among genera in Thamnocephalidae and the monophyly of Thamnocephalidae, determined using morphological characteristics or gene markers, remain controversial. In order to address the relationships within Thamnocephalidae, we sequenced Branchinella kugenumaensis mitogenomes and conducted a comparative analysis to reveal the divergence across mitogenomes of B. kugenumaensis. Using newly obtained mitogenomes together with available Anostracan genomic sequences, we present the most complete phylogenomic understanding of Anostraca to date. We observed high divergence across mitogenomes of B. kugenumaensis. Meanwhile, phylogenetic analyses based on both amino acids and nucleotides of the protein-coding genes (PCG) provide significant support for a non-monophyletic Thamnocephalidae within Anostraca, with Asian Branchinella more closely related to Streptocephalidae than Australian Branchinella. The phylogenetic relationships within Anostraca were recovered as follows: Branchinectidae + Chirocephalidae as the basal group of Anostraca and halophilic Artemiidae as a sister to the clade Thamnocephalidae + Streptocephalidae. Both Bayesian inference (BI)- and maximum likelihood (ML)-based analyses produced identical topologies.

It is still a challenge to reconstruct the deep phylogenetic relationships within spinicaudatans, and there are several different competing hypotheses regarding the interrelationships among Eocyzicidae, Cyzicidae s. s., Leptestheriidae, and Limnadiidae of the Suborder Spinicaudata. In order to explore the source of the inconsistencies, we focus on the sequence variation and the structure model of two rRNA genes based on extensive taxa sampling. The comparative sequence analysis revealed heterogeneity across species and the existence of conserved motifs in all spinicaudatan species. The level of intraspecific heterogeneity differed among species, which suggested that some species might have undergone a relaxed concerted evolution with respect to the 28S rRNA gene. The Bayesian analyses were performed on nuclear (28S rRNA, EF1α) and mitochondrial (16S rRNA, COI) genes. Further, we investigated compositional heterogeneity between lineages and assessed the potential for phylogenetic noise compared to signal in the combined data set. Reducing the non-phylogenetic signals and application of optimal rRNA model recovered a topology congruent with inference from the transcriptome data, whereby Limnadiidae was placed as a sister group to Leptestheriidae + Eocyzicidae with high support (topology I). Tests of alternative hypotheses provided implicit support for four competing topologies, and topology I was the best.

Conflicting phylogenetic signals are pervasive across genomes. The potential impact of such systematic biases may be reduced by phylogenetic approaches accommodating for heterogeneity or by the exclusive use of homoplastic sites in the datasets. Here, we present the complete mitogenome of Lynceus grossipedia as the first representative of the suborder Laevicaudata. We employed a phylogenomic approach on the mitogenomic datasets representing all major branchiopod groups to identify the presence of conflicts and concordance across the phylogeny. We found pervasive phylogenetic conflicts at the base of Diplostraca. The homogeneity of the substitution pattern tests and posterior predictive tests revealed a high degree of compositional heterogeneity among branchiopod mitogenomes at both the nucleotide and amino acid levels, which biased the phylogenetic inference. Our results suggest that Laevicaudata as the basal clade of Phyllopoda was most likely an artifact caused by compositional heterogeneity and conflicting phylogenetic signal. We demonstrated that the exclusive use of homoplastic site methods combining the application of site-heterogeneous models produced correct phylogenetic estimates of the higher-level relationships among branchiopods.