DOI: 10.5061/dryad.b2rbnzsdj
title: The evolution of hemocyanin genes in Tectipleura - a multitude of conserved introns in highly diverse gastropods

description: Background: Hemocyanin is the oxygen transporter of most molluscs. Thus,
 it is an essential protein of these animals which needs to be adapted
 perfectly to their environments. In Tectipleura, which is a very large and
 diverse gastropod group with &gt;27,000 species living in all kinds of
 habitats, several hemocyanin genes have already been identified. They
 evolved independently from each other within different lineages due to
 multiple gene duplications and represent potential adaptations to
 different environments or lifestyles. The aim of this study is to explore
 the evolution of these genes by analyzing their exon-intron architectures
 for characteristic features indicating adaptations. Results: We
 reconstructed gene architectures of ten hemocyanin genes of four species
 of Tectipleura: (i) Aplysia californica (ii) Lymnaea stagnalis (iii) Cornu
 aspersum and (iv) Helix pomatia . Their hemocyanin genes comprise 53
 introns each, which is conspicuously more than in known hemocyanin genes
 of Cephalopoda (9-11), Vetigastropoda (15) and Caenogastropoda (28-33).
 The gene structures of Tectipleura hemocyanins are identical in terms of
 number and locations of the introns with exception of only one hemocyanin
 of Lymnaea stagnalis that comprises one additional intron. Deeper analyses
 reveal that introns which vary between gene structures of different
 molluscan lineages most probably evolved more recently through independent
 intron gains. Conclusions: The strong conservation of the large number of
 introns in hemocyanin genes in Tectipleura for over 200 million years
 suggests a selective pressure on the gene structure. While we have not
 found characteristic positions or sequence motifs of introns that are
 conserved, it may be simply the great number of introns that offers
 increased possibilities of gene regulation and thus may facilitate habitat
 shifts, adaptive radiation and speciation. This hypothesis is supported by
 the increased number of introns within hemocyanin genes of Pomacea
 canaliculata which evolved independently from those of Tectipleura. This
 species belongs to Caenogastropoda, the sister group of Heterobranchia
 (where Tectipleura belong to) which is also very diverse and comprises
 species living in different habitats. Thus, our study provides first
 evidence that a multitude of introns may contribute to adaptive gene
 diversity of animals.
description: <strong>Animal sampling and DNA
 isolation</strong> Adult snails
 of <em>Cornu aspersum</em> and <em>Helix
 pomatia </em>were obtained from a commercial dealer (Wiener
 Schneckenmanufaktur e.U., Vienna, Austria). Three individuals of each
 species were anesthetized on ice for 20 minutes and subsequently
 sacrificed. Hepatopancreatic tissue was isolated on an ice cooled aluminum
 plate. Tissue aliquots were stored in RNAlater™ (Invitrogen by Thermo
 Fisher Scientific, Waltham, MA, USA) at −80 °C. Samples were homogenized
 with a Precellys® homogenizer (Bertin Instruments, Montigny-le-Bretonneux,
 France). Subsequently, DNA was isolated applying the DNeasy Plant Mini Kit
 (Qiagen, Hilden, Germany). DNA integrity was checked on a 0.8% agarose gel
 (Biozym Scientific GmbH, Hessisch Oldendorf, Germany) and quantified via
 Nanodrop (ThermoFisher). <strong>Compiling exon-intron
 structures of hemocyanin genes of <i>Cornu aspersum
 </i>and<i> Helix pomatia</i></strong>
 DNA
 samples from one adult snail of <i>C. aspersum </i>and
 <i>H. pomatia </i>were sent to StarSeq (Mainz, Germany) for
 NGS (Illumina Next Seq500) and library preparation. Bioinformatics were
 performed using Geneious 9.1.8. Sequencing adapters were removed and raw
 reads were quality trimmed. Processed genomic data were mapped to coding
 sequences of three known hemocyanins of <i>C. aspersum</i>
 (<i>CaH αD</i>: MH485355, <i>CaH αN</i>: MH485356,
 <i>CaH β</i>: MH485357) and <i>H. pomatia</i>
 (<i>HpH αD</i>: MH485358, <i>HpH αN</i>: MH485359,
 <i>HpH β</i>: MH485360). Sequence sections which were not
 covered by genomic NGS data or which were incongruous to cDNA sequences
 were used to separate nucleotide sequences in different parts which
 represent segments of different exons. Exon sequences were completed by
 repetitive mappings of genomic data to these sequence parts until non-cDNA
 sequences were assembled. At least ten base pairs of the 3’ and 5’ ends of
 each intron were assembled to assure that flanking sequences differ from
 those of neighboring cDNA sequences and therefore represent
 introns. <strong>PCR confirmation of
 ambiguous hemocyanin sequence sections</strong>
 For exon-intron borders which had a low assembly quality or which
 deviated from the Chambon’s / GT-AG rule (Breathnach et al., 1978; Jacob
 & Gallinaro, 1989) in hemocyanin genes of <i>H.
 pomatia</i> and <i>C. aspersum</i>, gene-specific
 primers (additional file 8, tab. S3 (A)) were designed (CLC main
 workbench, Version 6.9) and respective gene regions were confirmed via
 Long Distance (LD) PCR for <i>H. pomatia</i>. Long Fragments
 were PCR-amplified applying the Platinum™ SuperFi™ Green PCR Master Mix
 (Invitrogen by Thermo Fisher Scientific, Waltham, MA, USA) whereas shorter
 fragments were generated using the Advantage® 2 Polymerase Mix (Takara Bio
 Europe, Saint-Germain-en-Laye, France) (for PCR parameters see additional
 file 8, tab. S4). PCR products were visualized on a 0.8% agarose gel
 (Biozym, Hessisch Oldendorf, Germany). PCR products were cleaned up
 directly using the PCR clean-up kit (Qiagen, Hilden, Germany) or
 gene-specific bands were cut out and purified with the QIAquick Gel
 Extraction kit (Qiagen, Hilden, Germany). If possible, clean gene-specific
 products were sequenced directly by Microsynth (Balgach, Switzerland)
 using the same primers for sequencing as applied for LD PCR (additional
 file 8, tab. S3 (A)). Otherwise, they were cloned using the TOPO™ XL-2
 Complete PCR Cloning Kit (Invitrogen by Thermo Fisher Scientific, Waltham,
 MA, USA) for long fragments or the TOPO® TA Cloning® Kit (Invitrogen by
 Thermo Fisher Scientific, Waltham, MA, USA) for shorter fragments. Three
 plasmids of one or two individuals were purified using the QIAprep Spin
 Miniprep Kit (Qiagen, Hilden, Germany) and sequenced via Sanger sequencing
 by Microsynth (Balgach, Switzerland) (sequencing primers in additional
 file 8, tab. S3 (B)).