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Characteristics and evolution of satellite DNAsequences in bivalve mollusks

E. Šatović, T. Vojvoda Zeljko & M. Plohl

To cite this article: E. Šatović, T. Vojvoda Zeljko & M. Plohl (2018) Characteristics and evolution ofsatellite DNA sequences in bivalve mollusks, The European Zoological Journal, 85:1, 95-104

To link to this article: https://doi.org/10.1080/24750263.2018.1443164

© 2018 The Author(s). Published by InformaUK Limited, trading as Taylor & FrancisGroup.

Published online: 20 Mar 2018.

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Characteristics and evolution of satellite DNA sequences in bivalvemollusks

E. ŠATOVIĆ, T. VOJVODA ZELJKO, & M. PLOHL

Division of Molecular Biology, Ruđer Bošković Institute, Zagreb, Croatia

(Received 20 December 2017; accepted 13 February 2018)

AbstractMollusks of the class Bivalvia have attracted attention because of the extraordinarily important roles they play in marineecosystems and in aquaculture. Data obtained from genetic studies performed on these species are accumulating rapidly,particularly in recent years when several genomic and transcriptomic studies have been carried out, or are in progress. Despitethis, knowledge concerning satellite DNAs, tandemly repeated non-coding genomic sequences important for comprehendinggenomic architecture and function as a whole, is fragmentary and limited to a relatively small number of mollusk species. Here,we present an overview of the studied satellite DNAs and their characteristics in bivalve mollusks, and discuss the implications ofthese results. In addition to the general features common for these sequences, bivalve satellite DNAs show some distinctspecificities which may be intriguing for the broad scientific community involved in unravelling repetitive genome components.The most striking are low genomic contribution, diversity of sequence families, extremely old ancestry, links with mobileelements, and unusual methylation patterns. Although current results were obtained in classical studies on individual speciesand their satellite DNA families, it can be postulated that they defined fundamental characteristics of these sequences in bivalvespecies generally, and will be further explored in detail by future satellitome and other high-throughput studies.

Keywords: Bivalves, mollusks, satellite DNA, repetitive DNA sequences, evolution

Introduction

The class Bivalvia consists of 9200 species found inmarine and freshwater habitats throughout theworld. They play an important role in the ecosystemsthey inhabit and serve as support to many molluskand other communities, participating in the transferof minerals and organic matter to benthic habitats.As a result of filtration during the feeding process,they can accumulate various xenobiotic substances,which make them also organisms of choice for envir-onmental monitoring (Gosling 2003). In accordancewith their extreme ecological and commercial impor-tance in marine ecosystems and aquaculture, interestin genetic research on these organisms is growingsteadily, and has increased particularly in recentyears. Linkage maps, transcriptomics and proteo-mics studies have been carried out to reveal thegenetic and molecular basis of traits of interest tothe bivalve farming industry, mainly disease suscept-ibility, tolerance to environmental stress, and

processes connected to metabolism and growth(Saavedra & Bachère 2006). Previous research onthese organisms was largely the result of interest inspecific gene functions, while recent studies havebeen moving towards genome-wide analyses.Genome sequencing projects have so far been per-formed only for the pearl oyster Pinctada fucata(Gould, 1850) (Takeuchi et al. 2012), the pacificoyster Crassostrea gigas (Thunberg, 1793) (Zhanget al. 2012) and the filter-feeder mussel Mytilus gal-loprovincialis Lamarck, 1819 (Murgarella et al.2016), and sequenced and assembled genomes ofother bivalve species can be anticipated in the nearfuture.Eukaryotic genomes contain a significant portion

of non-coding DNA represented by a large amountof different classes of repetitive sequences, foundeither interspersed or organised in tandem (López-Flores & Garrido-Ramos 2012; Biscotti et al. 2015).Satellite DNAs (SatDNAs) are the most abundant

Correspondence: M. Plohl, Ruđer Bošković Institute, Bijenička 54, 10000 Zagreb, Croatia. Tel: +385 1 4561 083. Fax: +385 1 4561 177. Email: plohl@irb.hr

The European Zoological Journal, 2018, 95–104Vol. 85, No. 1, https://doi.org/10.1080/24750263.2018.1443164

© 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), whichpermits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Published online 20 Mar 2018

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class of tandem repeats, usually arranged as longarrays associated with heterochromatin. Recentresults have assigned significant functions to thisextremely diverse group of sequences. Briefly, theyare involved in modelling the genome landscape inboth structural and functional aspects, and theirevolution contributes to raising reproductive barriersin speciation (reviewed by Garrido-Ramos 2017).

SatDNA sequences are usually AT rich, and theirabundance in genomes shows a wide range of varia-tion, with values from less than 0.5% to more than50%. Due to the phenomenon of concerted evolu-tion, sequence variability among monomers within asatDNA family is low, usually 2–3% (reviewed byPlohl et al. 2012; Garrido-Ramos 2017). Althoughmany unrelated satellites can populate genomes andgenomic satDNA profiles evolve rapidly, their DNAsequences can remain conserved over long evolu-tionary periods (reviewed by Plohl et al. 2008,2012). Among diverse families, satDNA monomerlength can vary significantly, but 150 to 210-bp-longmonomers predominate. They are considered to beevolutionarily favoured (Heslop-Harrison &Schwarzacher 2013), as they correspond to thenucleosomal length (Henikoff et al. 2001). SatDNAmonomers sometimes contain about 30 bp longsequence segments that exhibit reduced variabilitywith respect to the rest of the monomer sequence.Such conserved segments can facilitate recombina-tion between satDNA monomers and sequencehomogenisation (Kuhn et al. 2009) or have someother functional roles, for example, in DNA–proteininteractions. One example is the CENP-B protein,which facilitates centromere formation and plays animportant role in the assembly of centromere-speci-fic structures upon recognising and binding theCENP-B box within the higher primate alpha-satel-lite sequence (Masumoto et al. 1989). SatelliteDNAs have also shown strong relationships withmobile elements, in addition to being located inclose proximity to each other in certain genomicregions. For example, tandem repeats are detectedas structural components of some mobile elementsor satellite repeats can be derived from parts ofmobile elements (reviewed by Meštrović et al. 2015).

The commonly used method employed in thedetection of satellite sequences is digestion of geno-mic DNA with restriction endonucleases, followedby electrophoretic separation. A characteristic ladderpattern is the result of variability in the restrictionsite within satellite monomers organised in an array.Alternatively, construction of partial genomiclibraries followed by colony-lift hybridisation withfragmented total genomic DNA is sometimes used

as a strategy in detecting repetitive genomic frag-ments, including satDNAs (e.g. Biscotti et al. 2007).Difficulties in sequencing and assembly of arrays

composed of highly similar tandem repeats haveresulted in the fact that satDNA sequences are ser-iously underrepresented in genome sequencing out-puts. With respect to bivalve species, genomicprojects are no exception, and have not yielded com-prehensive information regarding satDNAs.Predictions are that 202 Mb (36% of the genome)of Crassostrea gigas genome is enriched in repetitivesequences (Zhang et al. 2012), but over 62% of thedetected repeats could not be assigned to knowncategories. In the case of the oyster Pinctada fucata(Takeuchi et al. 2012), about 7.9% of the genome isoccupied by tandem repetitive elements, amongwhich 3.7% represents microsatellite sequences. InMytilus galloprovincialis (Murgarella et al. 2016) itwas found that repetitive elements make up 36.13%of the assembly (more than 80% being unclassified),with the focus being mostly on transposable ele-ments (TEs).Recent methodological advancements in next-gen-

eration sequencing (NGS) and data processingopened up the possibility of obtaining detailedinsight into the repetitive composition (repeatome),particularly satDNAs (satellitome) of a genome(Ruiz-Ruano et al. 2016; Pita et al. 2017; Silvaet al. 2017). However, until newly arising methodsstart to be more intensively employed, conventionalmethods remain the main source of informationabout tandemly repeated sequences in bivalvegenomes.So far, 48 species belonging to all main bivalve

clades have been investigated, yielding 26 differentsatDNAs, with Mytilidae, Ostreidae and Veneridaebeing the most explored families so far (Figure 1).The accumulated results point to some peculiaritiesin bivalve mollusk satDNA structure and organisa-tion, specifically a relatively low genomic content,and extreme evolutionary ancestry and dispersal ofsome of them, as well as linkage with certain types ofmobile elements. In order to summarise the resultsobtained until now, we present an overview of themost prominent observations on content, features,genomic localisation and species distribution ofsatDNAs in bivalve mollusk species. Their maincharacteristics can also be found in Table I.

SatDNAs in Pectinidae

The Antarctic scallop Adamussium colbecki (Smith,1902) was found to contain a tandemly repeatedsequence, detected upon restriction with the

96 E. Šatović et al.

endonuclease BglII (Canapa et al. 2000). The mono-mer length of this satellite DNA, named pACS, is~170 bp. Its AT composition ranges from 59.2 to73%, with the presence of adenine and thyminestretches in the monomer sequence. The degree ofsimilarity between monomers varies between 57 and95%, and this satellite constitutes about 0.2% of thetotal DNA of A. colbecki. Sequence motifs similar toCENP-B box and CDEIII were found within themonomer sequence, suggesting that A. colbecki satel-lite DNA possesses some of the features associatedwith organisation and activity of the centromere.Subsequent research has shown that it is indeedlocated in the centromeric region of several A. col-becki chromosomes (Odierna et al. 2006).

In the scallop Pecten maximus (Linnaeus, 1758), sixdifferent repetitive DNA sequences have beendescribed (Biscotti et al. 2007). Three repetitive

DNAs have monomer lengths of about 40 bp (families2, 3 and 4), while another three have monomer lengthsof 156 bp (family 5), 178 bp (family 1) and 326 bp(family 6). Family 1 represents about 0.13% of thegenome. Family 5 monomers are characterised by har-bouring successive repeats of one base, in particular G,or GG, AA, TT, CC and AGGGmotifs, and having avery low copy number, 0.003%. The longest repeatunit, family 6, consists of two consecutive 163-bp sub-repeats, exhibiting 75% similarity to each other. Theauthors have also found that it possesses a certain levelof similarity with the DTE satellite from the speciesDonax trunculus Linnaeus, 1758 (Plohl & Cornudella1996; see below). Fluorescent in-situ hybridisation(FISH) has shown that family 2 was concentrated ona single chromosome pair, while family 1 showedmanydiscrete hybridisation sites varying in size, indicating itspresence at multiple chromosomal locations. Family 5

Figure 1. Bivalve species explored from the aspect of satDNAs, their classification and age estimation of the main clades (taken from Bieleret al. 2014), together with satDNA families reported in these species.

Satellite DNA in bivalves 97

shows six to eight major sites with some more diffusehybridisation signals, in agreement with its lower geno-mic abundance.

PjHhaI satDNA was described in Pecten jacobaeus(Linnaeus, 1758) upon HhaI digestion of genomicDNA (Petraccioli et al. 2015). This satellite is wide-spread not only among scallops but also in otherbivalve species, including P. maximus, A. colbecki,Aequipecten opercularis (Linnaeus, 1758) andChamelea gallina (Linnaeus, 1758). In P. jacobaeusthe monomer length is 178 bp, and similarity amongmonomers is between 92.1 and 95.5%. Sequencesobtained from other species ranged from 173 to 181bp, and showed high similarity to those from P. jaco-baeus (89.3–93.6%). FISH experiments localised thePjHhaI satDNA to restricted regions of all the biva-lents of P. jacobaeus, while for other bivalve species

hybridisation signals on the chromosomes were scar-cely visible. Quantitative analyses showed that thePjHhaI satDNA accounts for ~20% of the genomeof P. jacobaeus and P. maximus. In other bivalve spe-cies, dot blot hybridisation signals were barely detect-able, making it impossible to determine precisely thevery small amount of this satellite DNA. Petraccioliet al. (2015) also found that in the genomes of C.gigas and Capitella teleta Blake, Grassle andEckelbarger, 2009 sequences similar to the PjHhaIsatDNA exist, flanked by TEs.

SatDNAs in Mytilidae

Following genomic DNA restriction with ApaIendonuclease, three different satDNAs were initiallydetected in Mytilus edulis Linnaeus, 1758 (Ruiz-Lara

Table I. General features of satDNA sequences presented in the text.

Monomerlength (bp)

AT content(%)

Similarity amongmonomers (%)

Abundance(%)

Conservedmotifs

Connection to mobileelements

PectinidaepACS ~170 59–73 57–95 0.2 +Family 1 178 89.9 0.13Family 2 ~40 90Family 3 ~40Family 4 ~40Family 5 156 0.003Family 6 326 75 +PjHhaI 178 92–95 20 +ApaI 1 170–175 + +ApaI 2 159–162 + +ApaI 3 88–167 + +MytilidaeApaI 1 173 55 91 0.10–0.79 + +ApaI 2 161 65 91 0.85–1.66 + +ApaI 3 166 55 91 0.01–0.12 + +Mg1 169 4.1 - +Mg2 260 2.7 - +Mg3 70 1 - +DonacidaeDTHS1 136–167 ~55 ≤ 95 0.043DTHS2 136–167 ~55 ≤ 95 0.081DTHS3 136–167 ~55 ≤ 95 0.035DTHS4 136–167 ~55 ≤ 95 0.008DTE 155 89 0.09–0.23 + +DTF1 169 62 ≥ 83 0.1 +DTF2 155,169 37.5 92–100 2MactridaeSSU 315 56 97 1.3–4 +OstreidaeCg170/HindIII 166 60 79–94 1–4 +BclI 150 50 90–96.6 + +VeneridaephBglII400 400 60 98Broadly distributed

satDNAsBIV160 158–166 61 46–100 (interspecies) 0.1–2 + +DTHS3 138–164 63 48–75 (interspecies) +

98 E. Šatović et al.

et al. 1992), and were subsequently found in therelated species M. galloprovincialis, M. trossulusGould, 1850 and M. californianus Conrad, 1837(Martinez-Lage et al. 2002). ApaI satellite sequencesnamed 1, 2 and 3 exhibit monomer lengths of 173,161 and 166 bp, respectively. All are AT rich (55–65%), and sequence analysis in M. edulis showed ahigh degree of homology between monomers, 91%.Comparison of satellite profiles between the afore-mentioned species enabled the reconstruction ofphylogenetic relationships between them. It wasshown that M. californianus is the most diversifiedamong them, which is also consistent with morpho-logical observations, as well as with studies based onmitochondrial DNA. The amount of satellitesequences varies between species but satellite 2 isthe most represented in all of them, making up asmuch as 1.66% of the genome, while satellite 3 isleast represented (0.01–0.12%). Chromosomal loca-lisation of these satDNAs was performed for M.edulis, M. galloprovincialis and M. trossulus. Whilesatellite 2 exhibits a random distribution in smallgroups across all chromosomes, satellites 1 and 3are located in the subtelomeric areas of individualchromosomes (Martínez-Lage et al. 2002). In con-tinuation, these repetitive DNA sequences were ana-lysed in additional species belonging to Mytilida (M.chilensis Hupé, 1854, M. coruscus Gould, 1861, Pernacanaliculus (Gmelin, 1791), Aulacomya ater (Molina,1782), Choromytilus chorus (Molina, 1782), Septifervirgatus (Wiegmann, 1837), Lithophaga lithophaga(Linnaeus, 1758), Geukensia demissa (Dillwyn,1817)) as well as in a few other bivalves (Arca noae(Linnaeus, 1758), Pecten maximus, Mimachlamysvaria (Linnaeus, 1758)) by Martinez-Lage et al.(2005). These results showed that monomer lengthand sequence similarities correspond to thosereported for satellites from the four initially analysedMytilus species (Ruiz-Lara et al. 1992; Martínez-Lage et al. 2002). The genomic abundance of thesesatellites is significantly lower in the examined spe-cies of Mytilidae. Nevertheless, sequence alignmentsrevealed that all of these sequences contain regionsof similarity to tRNA A and B boxes, and the authorssuggest that they could be spread as tRNA-derivedpseudogenes (Martinez-Lage et al. 2005).

Another three repetitive sequences, with monomerlengths of 169, 260 and 70 bp, respectively, werereported in Mytilus galloprovincialis (Kourtidis et al.2006). They were named Mg1, Mg2 and Mg3, con-stituting 4.1, 2.7 and 1% of the M. galloprovincialisgenome, respectively. The Mg1 repetitive regionwith its flanking sequences showed significanthomology to the CvE, a member of the pearl familyof mobile elements described in the oyster

Crassostrea virginica (Gmelin, 1791) (Gaffney et al.2003). The central part of these elements is charac-terised by a short array of tandemly repeatedsequences, similar to satellite monomers. Pearl andother comparably structured elements were recentlyclassified as members of Helentrons, a rolling-circlenon-autonomous family of TEs (Thomas & Pritham2015). Being a putative TE, the structure describedby Kourtidis et al. (2006) was named MgE.Similarly, Mg2, and Mg3 repeats were found joinedto the previously described ApaI type 2 repeats(Ruiz-Lara et al. 1992; Martínez-Lage et al. 2002),in structural arrangements similar to MgE. Kourtidiset al. (2006) have reported that searches for con-served motifs, such as CENP-B and the yeastCDEIII sequence, yielded no significant hits. Inaddition, sequence alignments did not reveal anyother conserved segments.

SatDNAs in Donacidae

In the wedge clam, Donax trunculus, eight satelliteDNAs have been described so far, making it,together with Pecten maximus, one of the best-explored bivalve species from a repetitive DNApoint of view (Figure 1). Four described satelliteDNAs of D. trunculus were assigned to the DTHSfamily, detected after the restriction of genomicDNA with HindIII endonuclease (Plohl &Cornudella 1997). Monomer length of satDNAswithin this family ranges from 136 to 167 bp. Allfour satellites show low abundance: DTHS1 makesup 0.043% of the genome, DTHS2 0.081% andDTHS3 0.035%, while DTHS4 constitutes only0.008% of the genome. Despite the difference innucleotide sequence, members of the DTHS familyhave related oligonucleotide motifs repeated up to 15times within the satellite monomer. The main build-ing block is a TTAGGG motif and its variations.TTAGGG is a characteristic telomeric sequence invertebrates, and also builds chromosome ends of D.trunculus and some other sea invertebrates (Plohlet al. 2002).DTE satellite DNA with its 155-bp-long mono-

mer was detected after genomic restriction withEcoRV endonuclease (Plohl & Cornudella 1996).This satellite DNA contains two groups of mono-mers, DTE1 and DTE2, whose nucleotidesequences show 11% divergence and represent sub-families that can be clearly distinguished based ondiagnostic mutations. DTE1 constitutes 0.23% ofthe genome, and DTE2 0.09%. This satellite DNAshows similarity to the part of the aforementionedsatellite DNA found in P. maximus (Biscotti et al.2007) and to the HindIII satellite DNA of oysters

Satellite DNA in bivalves 99

(Wang et al. 2001; López-Flores et al. 2004) as wellas to the central repeats of the pearl element CvA(Gaffney et al. 2003).

Following HinfI-cleavage of D. trunculus genomicDNA, two different satellite DNAs were detected(Petrović & Plohl 2005). They were separated fromthe same electrophoretic band, with the only com-mon feature being a repeat length of 169 bp. One ofthe two satellites is DTF1, which is AT rich (62%)and constitutes 0.1% of the D. trunculus genome.DTF1 monomers are divided into two subgroupsexhibiting 17% nucleotide divergence, while mono-mers within each subgroup are highly conserved.Nucleotide diversity analysis of the repeats revealedunequal distribution of mutations, where all DTF1monomers contain a 30-bp segment showing extre-mely strong sequence conservation, probably as aconsequence of functional constraints on segmentsof this satDNA. A higher order repeat unit, DTRS,also exists, composed of a complete and a shortenedDTF1 monomer and the insertion of a 14-bp-longunrelated nucleotide sequence.

A second HinfI-retrieved satDNA is DTF2. Incontrast to other D. trunculus satellites, it has anunexpectedly high GC nucleotide content in themonomer (62.5%), and shows high abundance,2%. Another peculiarity of the DTF2 satellite ispartial methylation of monomer sequences.Nucleotide sequence analysis showed that DTF2monomers have low variability, with nucleotide dif-ferences uniformly distributed throughout the wholemonomer length. In addition to a 169-bp-longrepeat unit (DTF2L variant), a shorter variant of155 bp was found (DTF2S). FISH showed the pre-sence of this satellite on 14 out of 19 D. trunculuschromosomes, residing in telomeric and subtelo-meric regions and, interestingly, mostly not coincid-ing with the prominent GC-rich heterochromaticblocks (Petrović et al. 2009).

SatDNAs in Mactridae

García-Souto et al. (2017) reported the presence ofSSUsat in the genome of Spisula subtruncata (daCosta, 1778), with a monomer length of 315 bp.This satDNA is also present in two otherMactridae species, Spisula solida (Linnaeus, 1758)and Mactra stultorum (Linnaeus, 1758). TheSSUsat monomer sequence is highly conserved,does not show species-specific grouping, and exhi-bits only 3% of both intra- and inter-specific nucleo-tide sequence diversity. These repeats represent atleast 1.3% of the S. subtruncata genome, and itspresence in the other two species is significantlylower. Methylation levels for SSUsat are high

(3.38%), triplicating the mean of the S. subtruncatagenome. Methylation of the monomer sequence isinversely correlated with nucleotide diversity, asregions of highest methylation coincide with two ofthe three most conserved sequence segments.Interestingly, SSU satDNA located on one S. sub-truncata chromosome pair did not reveal any sign ofmethylation. Although exhibiting 44% of GCnucleotides itself, SSU satDNA is predominantlylocated at the GC-rich intercalary heterochromatinof S. subtruncata.

SatDNAs in Ostreidae

Clabby et al. (1996) discovered the Cg170 satelliteDNA of 166 bp monomer length that occupies 1–4%of the Crassostrea gigas genome. The nucleotidesequence of this satellite is AT rich (60%) and theanalysis showed a high degree of homology betweenmonomers, 79–94%. Subsequent studies haveshown that these satellite sequences are located inthe centromeric areas of C. gigas chromosomes(Wang et al. 2001). The same satDNA was alsofound after HindIII restriction of genomic DNA,thereafter named HindIII satDNA, and wasdescribed in more detail in seven species belongingto the genera Ostrea and Crassostrea (Figure 1;López-Flores et al. 2004). Authors reported the con-sensus length of monomers from these species to be~166 bp. Species-specific differences among repeatswere taxonomically informative and enabled thereconstruction of phylogenetic relationships amongexamined taxa. In addition, two entries in Repbase(database of repetitive elements, Bao et al. 2015):SAT-1_CGi (Jurka 2012) and SATREP_CGi(Clabby et al. 1996), also belong to the same repeti-tive sequence. The presence of Cg170/HindIIIrepeats was also reported by Šatović et al. (2016)within structures that were assigned to DNA trans-posons of the Helitron superfamily, with varyingrepeat copy numbers in the internal array.After digestion with BclI enzyme, a 150-bp-long

repetitive DNA sequence was found in the genomeof the European flat oyster, Ostrea edulis Linnaeus,1758 (López-Flores et al. 2010). Its presence wasdetermined in five other species belonging to theorders of Ostrea and Crassostrea (Figure 1).Intraspecific variability is between 3.4% inCrassostrea angulata (Lamarck, 1819) and 10% inC. ariakensis (Fujita, 1913), with interspecific diver-gence being similar to intraspecific divergence. ATcomposition of this satDNA is 50%. In-situ hybridi-sation analysis showed many signals along all C.angulata chromosomes, exhibiting interspersed loca-lisation of BclI repeats. Together with this

100 E. Šatović et al.

organisational pattern, sequence blocks similar tobox A and B, characteristic for SINE elements,have been found in BclI repeats. The authors didnot determine the exact abundance of this repetitiveDNA, but they did report faint hybridisation signalsin both Southern and dot blot analysis, indicating alow representation of the BclI sequence in the gen-omes of Ostrea stentina Payraudeau, 1826, C. angu-lata and C. gigas, with respect to the genome of O.edulis.

SatDNAs in Veneridae

phBglII400 satDNA was described by Passamontiet al. (1998) in Ruditapes philippinarum (Adams &Reeve, 1850). This is an AT-rich satellite DNA andhas 400-bp-long monomers composed of two 200-bp subunits. Sequence analysis shows a high degreeof homology between monomers (~98%), and FISHanalysis showed that these satellite DNAs are foundmainly in pericentromeric chromosomal regions. Itspresence was also detected in the species Venerupisaurea (Gmelin, 1791) and Paphia undulata (Born,1778).

SatDNA families broadly distributed amongbivalve mollusks

Although broad interspecies distribution wasobserved for different mollusk satDNAs (for exam-ple, satellites in Pectinidae and Mytilidae, Figure 1),particularly interesting in this context is the BIV160satDNA. It was initially detected in nine species(Glycymeris glycymeris (Linnaeus, 1758), Mya are-naria (Linnaeus, 1758), Dosinia exoleta (Linnaeus,1758), Venus verrucosa (Linnaeus, 1758), Venerupispullastra (Montagu, 1803), Venerupis rhomboids(Pennant, 1777), Ruditapes decussatus (Linnaeus,1758), Ruditapes philippinarum and Nucula sp.Lamarck, 1799), belonging to all of the main sub-classes of bivalves (Protobranchia, Pteriomorphiaand Heteroconchia). According to its distribution,it was estimated to be about 540 million years old,with the longest evolutionary track record known sofar (Plohl et al. 2010). In the first survey it remainedundetected in D. trunculus, but its presence in thisspecies was subsequently confirmed by using specificprimers (Šatović et al. 2016). The average AT com-position of BIV160 satDNA is 61%, and the mono-mer length ranges from 158 to 166 bp. Isolatedmonomers showed three different distribution pat-terns: species-specific sets of variants (M. arenaria),intermixed variants without any species specificity(for example in R. decussatus), and monomer variantswhich can follow both the first and the second

pattern (R. philippinarum). BIV160 is the most abun-dant in R. decussatus, constituting 2% of the genome,with similarity among monomers ranging from 69 to97%. Within BIV160 monomers two conservedmotifs have been noted, which are also shared withthe HindIII satDNA (López- Flores et al. 2004),with DTE (Plohl & Cornudella 1996) and with theCvA mobile element (Gaffney et al. 2003).The similarity of BIV160 sequences and

Crassostrea gigas Cg170/HindIII repeats (Clabbyet al. 1996; López-Flores et al. 2004), the DTEsatellite (Plohl & Cornudella 1996), and the CvAelement (Gaffney et al. 2003) indicate extreme inter-connections between satDNAs in bivalve species,both in the evolutionary sense and in potential func-tional roles, particularly with regard to conservedmotifs retained in all of them.The previously mentioned DTHS3 satDNA, ori-

ginally discovered in D. trunculus (Plohl &Cornudella 1997), was further characterised withinthe class Bivalvia (Šatović & Plohl in press).Monomer variants of DTHS3 satDNA were com-pared in 12 clam species belonging to subclassesHeterodonta and Pteriomorphia (D. trunculus,Ruditapes decussatus, R. philippinarum, Sinonovaculaconstricta (Lamarck, 1818), Mercenaria mercenaria(Linnaeus, 1758), Meretrix lusoria (Röding, 1798),M. meretrix (Linnaeus, 1758), Spisula solidissima(Dillwyn, 1817), Crassostrea gigas, C. virginica,Mytilus galloprovincialis and Dreissena bugensis(Andrusov, 1897)). The results suggest thatDTHS3 and BIV160 behave quite similarly, bothin estimated age (516 MY in case of DTHS3) andin the presence of distribution patterns of monomervariants, which can be species specific, intermingledinterspecifically, or a combination of these two pat-terns. Sequence comparisons also invoked horizontaltransfer as a possible pathway by which DTHS3monomers may become shared between some dis-tant species belonging to different subclasses(Šatović & Plohl In press).

Integrating features common for bivalvemollusk satDNAs

One striking characteristic of satellite DNAs presentin bivalve genomes is their relatively low genomicabundance, compared to that found in many otherorganisms (Garrido-Ramos 2017). With the strongexception of the PjHhaI satDNA, which constitutes20% of the P. jacobaeus and P. maximus genomes(Petraccioli et al. 2015), the maximum genomeoccupancy by a certain satDNA is 1–4% for Cg170in C. gigas, being significantly lower for other satel-lites. What also characterises the satDNA profile of

Satellite DNA in bivalves 101

bivalve mollusks is the coexistence of many divergentsubgroups, often interrelated, with none of themexpanding into a dominant satDNA of the respectivespecies. An example is D. trunculus where eight dif-ferent satDNAs have been detected, none of themexceeding 2% (Petrović et al. 2009; Plohl et al.2010), or a family of six repeats detected in thescallop P. maximus (Biscotti et al. 2007).

Regarding AT content, bivalves show only a fewexceptions to the usual abundance of those nucleo-tides in the monomer sequence. BclI repeats of O.edulis (López-Flores et al. 2010) harbour 50% AT,while DTF2 of D. trunculus shows stronger GCenrichment, 62.5% (Petrović et al. 2009).

SatDNA physical mapping in bivalve mollusksencompasses all possible distribution patterns.The presence of BglII satDNA of A. colbecki andCg170 of C. gigas was found at centromeres(Canapa et al. 2000; Wang et al. 2001; Odiernaet al. 2006). Mytilus ApaI satellite 2 shows a ran-dom distribution across all chromosomes, whilesatellites 1 and 3 are located in the subtelomericareas (Martínez-Lage et al. 2002). Family 2 of P.maximus repeats (Biscotti et al. 2007) is present ona single chromosome pair, while family 1 showsmany hybridisation signals at multiple chromoso-mal locations. An interspersed pattern is also char-acteristic of BclI repeats of C. angulata (López-Flores et al. 2010). The distribution pattern ofsatDNAs is probably correlated with the mechan-ism of their dispersal; in other words, sequencesassociated with mobile elements or having the abil-ity to transpose might be expected to build arraysdispersed on many locations along the chromo-somes. Although this hypothesis still has to beconfirmed, there are indications of such connec-tions, for example in C. gigas (Šatović et al. 2016).

Conserved boxes and different sequence motifscan be found within monomer sequences of bivalvesatDNAs, and potential functional constraints havealready been mentioned.

It is interesting that different types of repetitivesequences, three belonging to satDNAs – DTE(Plohl & Cornudella 1996), BIV160 (Plohl et al.2010), HindIII (López-Flores et al. 2004) – andone belonging to the mobile element CvA (Gaffneyet al. 2003), share the same conserved boxes,although functional studies that would suggest invol-vement in similar functional interactions do not existyet. In this respect, it is no surprise that pACS satel-lite DNA, which contains motifs similar to theCENP-B box and CDEIII (Canapa et al. 2000),was found localised at the centromeres of A. colbeckichromosomes (Odierna et al. 2006). In addition to aputative centromeric function, the CENP-B box-like

motif could be related to the mobility of satDNAmonomers (Meštrović et al. 2013), based on thesimilarity between the CENP-B protein and transpo-sases of the pogo family of mobile elements (Kipling& Warburton 1997).Sequence alignments have revealed that ApaI-

repeats of Mytilus species contain segments showingsimilarity to RNA Pol III boxes A and B (Martínez-Lage et al. 2005), features characteristic of SINEelements. Degenerate sequences similar to boxes Aand B have been found within BclI repeats as well(López-Flores et al. 2010). Because the BclIsequence of O. edulis exhibits a length and inter-spersed distribution pattern which is expected forSINE elements, it is likely that the conserved boxesin those repeats could be connected to sequenceorigin and not a consequence of functionalinteractions.The connection of these sequences to mobile

elements goes even further, and might suggest atight interconnection particularly discernible inbivalves, although different forms of interconnec-tion were observed in other species as well (for areview, see Meštrović et al. 2015). As describedabove, sequence similarity expressed especiallythrough shared conserved boxes has been observedfor the CvA element of the pearl family (Gaffneyet al. 2003) and DTE, BIV160 and HindIII/Cg170repeats (Clabby et al. 1996; Plohl & Cornudella1996; López-Flores et al. 2004; Plohl et al. 2010).Crassostrea gigas HindIII/Cg170 repeats togetherwith their flanking sequences were identified asparts of putative TEs (Šatović et al. 2016). MgE,the putative mobile element characterised in M.galloprovincialis holds Mg1 repeats (Kourtidis et al.2006), while sequences showing similarity toPjHhaI sat in C. gigas and Capitella teleta were alsofound surrounded with structures belonging toTEs (Petraccioli et al. 2015). Additionally,DTHS3 satDNA in M. mercenaria, S. solidissima,C. virginica and M. galloprovincialis was foundembedded in structures that might be responsiblefor their mobility (Šatović & Plohl in press).Although scarce and far from being understood,

results concerning methylation profiles in repetitiveDNAs of bivalve species have disclosed some distinctfeatures. In the oyster C. gigas, repeat classes such asDNA transposons, helitrons, satellites, simple repeatsand tandem repeats all displayed average methylationlevels 2 times higher than the genome background(Wang et al. 2014). These authors noticed that ifthey divide the repeats into methylated and unmethy-lated, the divergence rate of the methylated group wassignificantly lower than that of the unmethylatedgroup. This observation is also in agreement with

102 E. Šatović et al.

data from SSU satDNA from S. subtruncata, whosemethylation is inversely correlated with nucleotidediversity in segments of monomer sequences(García-Souto et al. 2017). Methylated DTF2satDNA of D. trunculus shows uniform and highsequence conservation throughout the whole mono-mer sequence (Petrović et al. 2009).

Specificities in the methylation pattern of certaintypes of repetitive sequences in bivalves suggest role(s) alternative to mere DNA silencing. Furthermore,the existence of a single chromosome pair in S.subtruncata in which SSUsat is non-methylated(García-Souto et al. 2017) leads to the assumptionthat methylation processes in the repetitive part ofbivalve mollusk genomes are non-random and com-plex, and the significance of these patterns remainsto be revealed.

Concluding remarks

Research on bivalve mollusk satDNAs has broughtimportant information which has broadened ourgeneral views on satDNAs as a class of ubiquitousgenomic sequences. For bivalve mollusks, the moststriking features are (i) the low contribution of het-erochromatin and satDNAs in the genomes of theseancient organisms, including in the centromericregion of some species; (ii) in some cases, an extre-mely large palette of related monomer variantscomprising a library of a satDNA family; (iii) theextremely long ancestry of some of them; (iv) theability to easily link many of these sequences withcertain groups of mobile elements; and (v) unusualmethylation patterns. Despite the generally knowngenomic roles of satDNAs, at this moment it is notclear whether and how specific features of thesesequences present in bivalves influence the biologyof these organisms. Nevertheless, abundance,dynamics and differences between satDNAs makebivalve mollusks important organisms in exploringorganisational and functional aspects of satelli-tomes. We are sure that future research will bringa lot of additional information that will help tounravel roles of this specific form of noncodingDNA sequences.

Acknowledgements

We thank Dr Mary Sopta for critical reading andlanguage editing of the manuscript.

Disclosure statement

No potential conflict of interest was reported by theauthors.

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AbstractIntroductionSatDNAs in PectinidaeSatDNAs in MytilidaeSatDNAs in DonacidaeSatDNAs in MactridaeSatDNAs in OstreidaeSatDNAs in VeneridaeSatDNA families broadly distributed among bivalve mollusksIntegrating features common for bivalve mollusk satDNAsConcluding remarksAcknowledgementsDisclosure statementReferences