Hydrozoans display the most morphological diversity within the phylum Cnidaria. While recent molecular studies have provided some insights into their evolutionary history, sister group relationships remain mostly unresolved, particularly at mid-taxonomic levels. Specifically, within Hydroidolina, the most speciose hydrozoan subclass, the relationships and sometimes integrity of orders are highly unsettled. Here we obtained the near complete mitochondrial sequence of twenty-six hydroidolinan

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... ozoan species from a range of sources (DNA and RNA-seq data, long-range PCR). Our analyses confirm previous inference of the evolution of mtDNA in Hydrozoa while introducing a novel genome organization. Using RNA-seq data, we propose a mechanism for the expression of mitochondrial mRNA in Hydroidolina that can be extrapolated to the other medusozoan taxa. Phylogenetic analyses using the full set of mitochondrial gene sequences provide some insights into the order-level relationships within Hydroidolina, including siphonophores as the first diverging clade, a well-supported clade comprised of Leptothecata-Filifera III-IV, and a second clade comprised of Aplanulata-Capitata s.s.-Filifera I-II. Finally, we describe our relatively inexpensive and accessible multiplexing strategy to sequence long-range PCR amplicons that can be adapted to most highthroughput sequencing platforms. PeerJ reviewing PDF | (2015:07:5908:1:0:NEW 22 Oct 2015) Manuscript to be reviewed 1 Phylogenetic analysis of higher-level relationships within Hydroidolina (Cnidaria: Hydrozoa) using 2 mitochondrial genome data and insight into their mitochondrial transcription 3 Manuscript to be reviewed 21 Abstract 22 Hydrozoans display the most morphological diversity within the phylum Cnidaria. While recent molecular studies 23 have provided some insights into their evolutionary history, sister group relationships remain mostly unresolved, 24 particularly at mid-taxonomic levels. Specifically, within Hydroidolina, the most speciose hydrozoan subclass, the 25 relationships and sometimes integrity of orders are highly unsettled. Here we obtained the near complete 26 mitochondrial sequence of twenty-six hydroidolinan hydrozoan species from a range of sources (DNA and RNA-seq 27 data, long-range PCR). Our analyses confirm previous inference of the evolution of mtDNA in Hydrozoa while 28 introducing a novel genome organization. Using RNA-seq data, we propose a mechanism for the expression of 29 mitochondrial mRNA in Hydroidolina that can be extrapolated to the other medusozoan taxa. Phylogenetic analyses 30 using the full set of mitochondrial gene sequences provide some insights into the order-level relationships within 31 Hydroidolina, including siphonophores as the first diverging clade, a well-supported clade comprised of 32 Leptothecata-Filifera III-IV, and a second clade comprised of Aplanulata-Capitata s.s.-Filifera I-II. Finally, we 33 describe our relatively inexpensive and accessible multiplexing strategy to sequence long-range PCR amplicons that 34 can be adapted to most high-throughput sequencing platforms. 35 PeerJ reviewing PDF | (