Next-generation sequencing, phylogenetic signal and comparative mitogenomic analyses in Metacrangonyctidae (Amphipoda: Crustacea)
© Pons et al.; licensee BioMed Central Ltd. 2014
Received: 5 March 2014
Accepted: 26 June 2014
Published: 6 July 2014
Comparative mitochondrial genomic analyses are rare among crustaceans below the family or genus level. The obliged subterranean crustacean amphipods of the family Metacrangonyctidae, found from the Hispaniola (Antilles) to the Middle East, including the Canary Islands and the peri-Mediterranean region, have an evolutionary history and peculiar biogeography that can respond to Tethyan vicariance. Indeed, recent phylogenetic analysis using all protein-coding mitochondrial sequences and one nuclear ribosomal gene have lent support to this hypothesis (Bauzà-Ribot et al. 2012).
We present the analyses of mitochondrial genome sequences of 21 metacrangonyctids in the genera Metacrangonyx and Longipodacrangonyx, covering the entire geographical range of the family. Most mitogenomes were attained by next-generation sequencing techniques using long-PCR fragments sequenced by Roche FLX/454 or GS Junior pyro-sequencing, obtaining a coverage depth per nucleotide of up to 281×. All mitogenomes were AT-rich and included the usual 37 genes of the metazoan mitochondrial genome, but showed a unique derived gene order not matched in any other amphipod mitogenome. We compare and discuss features such as strand bias, phylogenetic informativeness, non-synonymous/synonymous substitution rates and other mitogenomic characteristics, including ribosomal and transfer RNAs annotation and structure.
Next-generation sequencing of pooled long-PCR amplicons can help to rapidly generate mitogenomic information of a high number of related species to be used in phylogenetic and genomic evolutionary studies. The mitogenomes of the Metacrangonyctidae have the usual characteristics of the metazoan mitogenomes (circular molecules of 15,000-16,000 bp, coding for 13 protein genes, 22 tRNAs and two ribosomal genes) and show a conserved gene order with several rearrangements with respect to the presumed Pancrustacean ground pattern. Strand nucleotide bias appears to be reversed with respect to the condition displayed in the majority of crustacean mitogenomes since metacrangonyctids show a GC-skew at the (+) and (-) strands; this feature has been reported also in the few mitogenomes of Isopoda (Peracarida) known thus far. The features of the rRNAs, tRNAs and sequence motifs of the control region of the Metacrangonyctidae are similar to those of the few crustaceans studied at present.
The metazoan mitochondrial genome (mitogenome) usually consists of a single compact circular DNA with a highly conserved gene content. It harbours the coding capacity for 13 proteins of four complexes of the respiratory chain, two ribosomal RNAs, and 22 genes coding for the tRNA set, including two gene copies for each leucine and serine tRNAs . A non-coding region (control region) of variable length is also typically present (called d-loop in vertebrates and AT-rich non-coding region in arthropods); this region provides the site for the initiation of transcription and the initiation of replication of one or both strands [2–4].
A wealth of data on DNA sequence and gene organization of metazoan mitogenomes has been gathered in the last decades, with about 4000 complete mitochondrial genomes already deposited in DNA sequence databases (RefSeq release 64), of which two thirds correspond to vertebrates . Data gathering has been fuelled by the advancements performed in “next generation sequencing” techniques and the need of generating robust phylogenetic information for evolutionary studies at all taxonomic levels, from deep metazoan evolution , relationships among vertebrate orders [7, 8], or even to resolve species-level phylogenies after rapid radiation processes [9, 10].
Up to now (May 2014) 134 crustacean mitogenomes have been completely sequenced, 107 of them corresponding to species in the class Malacostraca (NCBI RefSeq release 64 database). Within the malacostracan peracarid order Amphipoda, the sequences of species within the genera Parhyale (Hyalidae), Caprella (Caprellidae), Onisimus (Lysianassidae), Gondogeneia (Pontogeneiidae), Gammarus (Gammaridae), Eulimnogammarus (Eulimnogammaridae), Pseudoniphargus (Pseudoniphargidae), Bahadzia (Hadziidae) and Metacrangonyx (Metacrangonyctidae) have been reported or are deposited in sequence databases ( and references therein; NCBI RefSeq database).
In a previous work we sequenced the mitochondrial genomes of several Metacrangonyctid taxa by both classic and next-generation sequencing techniques to resolve, in combination with nuclear ribosomal sequences, the phylogenetic relationships within this family and to establish a time frame for its diversification . The Metacrangonyctidae is a phylogenetically enigmatic amphipod lineage composed of stygobiont (occurring only in subterranean waters) species with an extreme disjunct geographic distribution . Our phylogenetic reconstruction suggested that the major lineages of Metacrangonyctidae diversified during the Cretaceous, c. 96–83 million years ago (mya), and that the diversification of an insular clade was compatible with vicariance by plate tectonics . In the present study we aim to analyse in more detail the mitochondrial DNA sequences of Metacrangonyctidae, their genome organization and evolution and compare them to other amphipodan mitogenomes. Aside of two species of Caprella[14, 15], the few known amphipod mitogenomes derive from species placed in distant genera and families. Here we analyse 21 metacrangonyctid mitochondrial complete or nearly complete genome sequences (including the 20 mitogenomes reported in  plus one previously obtained by us, Metacrangonyx longipes from Mallorca ), to explore their phylogenetic signal and to perform a comparative intra-familiar genomic analysis. We have compared them also with the mitogenomic features displayed by other amphipod families. We have paid particular attention to the role played by tRNAs and the secondary structure of the small/large ribosomal RNAs and their nucleotide substitution patterns, since few data are available for these genes in crustaceans.
Results and discussion
Metacrangonyctidae mitochondrial genome information
Average read length
A + T
M. longipes (Mallorca)
M. longipes (Menorca)
“L. stocki” (Tafraut)
“M. boutini boutini”
M. goulmimensis (Erfoud)
M. goulmimensis (Ousroutou)
M. goulmimensis (Zouala)
“M. nicoleae tamri” (Tamri)
“L. stocki” (Tiznit)
“M. nicoleae tamri” (Aksri)
Base composition and AT- and GC-skews
Metazoan mitogenomes show a marked strand bias in nucleotide composition, which is thought to be due to exposure to different mutational pressures during replication, transcription or during both processes . Most malacostracan mitogenomes exhibit a negative GC-skew for genes coded in the (+) strand and positive values for genes of the (-) strand . The Isopoda (which, as the Amphipoda, belong to the superorder Peracarida) seem to be an exception to this rule since their mitogenomes show a reversed pattern where most genes of the (+) strand have a positive GC-skew; i.e. more G than C. Nevertheless, we have found that metacrangonyctid amphipod mitogenomes show GC-skew positive values at both (+) and (-) strands (Figure 2c), with the exception of cob and nad6 genes. AT-skew values are in turn negative for all protein-coding genes but atp8 (Figure 2b), with genes coded on the (+) strand showing lower overall values than those coded on the (-) strand. The reversed strand bias pattern of isopodan mitogenomes and in general any other metazoan strand bias have been explained advocating to the occurrence of an inversion of the control region. This inversion presumably included the replication origin at the base of Isopoda, changing the mutational pressure leading to strand-bias [24–26]. The control region is placed between the rrnS (- strand) and trnY (- strand) genes in the amphipod completed mitogenomes (with the exception of Caprella, that displays two A + T-rich regions at non-conserved positions, and metacrangonyctids). The segment assigned as the control region in metacrangonyctids is flanked also by rrnS (as in all amphipods except in Caprella) but trnS2 (UCN), followed by the cob gene are at the other side. It can be deduced that both trnS2 (UCN) and cob have suffered a reverse transposition (i.e. transposition plus strand switch) respect to the hypothetical arrangement displayed by other amphipods suggesting that this could have caused an inversion of the control region in the Metacrangonyctidae lineage. In the isopods Eophreatoicus sp. and Ligia oceanica, that show a similar strand bias pattern as metacrangonyctids, the control region appears between the trnQ and trnI genes (both coded at the (+) strand in L. oceanica while trnI is at the (-) strand in Eophreatoicus sp.). In any case, the strand bias pattern of metacrangonyctid mitogenomes is not only more similar to the condition found in the Isopoda than to amphipods, but also to other non-peracarid crustaceans such as Hutchinsoniella macracantha (Cephalocarida); Tigriopus californicus, T. japonicus, Lepeophtheirus salmonis, Calanus hyperboreous (Copepoda); Argulus americanus (Branchiura) and some decapods (Procambarus claarkii, P. fallax, Corallianassa couitierei, Nihonotrypaea japonica, N. thermophila, Cambaroides similis, Homarus gammarus). All these species share with metacrangonyctids the display of a positive GC-skew in genes coded in the (+) strand (Additional file 1). This suggests that the reversal of the ordinary strand bias has occurred independently multiple times, and not only in very distant metazoans  but also within the Crustacea, even within members of the same taxonomic order. This is presumably due to the fixation of different independent ancestral inversions of the same block of mitochondrial genes with respect to the control region, or vice versa .Nucleotide composition per codon site showed a sharp contrast between third and first/second positions, as expected (Figure 2a). Third codon positions displayed a high AT-content with similar values at both strands (AT = 81.30% on (+) strand; 80% on (-) strand), and a large variation across species. In contrast, AT-content at the first and second codon positions were lower and differed in genes coded on different strands, in particular the first codon positions. AT skew was close to zero at first codon positions, second codon positions showed a T nucleotide-enrichment (about -0.4 AT skew value on average) in genes of both strands, whereas third codon positions showed intermediate negative AT skews. GC skew per codon position was positive for first codon positions, slightly negative or close to zero for second codon positions, showing a substantial variation for third codon positions (Figure 2c).
Amino acid frequencies and codon usage
Protein coding gene phylogenetic informativeness
Non-synonymous/synonymous substitution rates
Start and stop codons
Most PCGs displayed ATN start codons, with ATG and ATT as the most frequent (Additional file 3: Table S1). The rest of start codons found are considered as canonical for invertebrate mitochondrial PCGs, such as TTG, which is conserved in all metacrangonyctid mitogenomes except in M. remyi that shows the non-canonical CTG. In addition, the canonical start codon GTG is present in the atp8 gene of two species (M. remyi and M. repens) instead of the ATN displayed in the rest of metacrangonyctids. The TAA complete or incomplete TAa or Taa stop codons are usually the norm in metacrangonyctid mitogenomes, although TAG appears as the stop codon for the gene nad3 in the majority of species (Additional file 3: Table S1). Incomplete stop codons are believed to be completed by post-transcriptional polyadenylation .
A total of eighteen different sequence spacers (isA-isR) were inferred to occur across the studied mitogenomes. Their length varied between 1 and 246 bp, although most of them were short 1–3 bp spacers (Additional file 3: Table S2). Six genome spacers appear to be conserved since they are placed in the same position in almost all species, suggesting an ancestral common origin although the primary sequences are not conserved. Two of these spacers are intergenic sequences separating PCGs: isH has 1 or 3 bp and is situated between cox3 and nad3 and isL, placed between nad6 and nad1, varies from 2 to 246 bp. The mitogenome of M. goulmimensis (Zouala) has the longest isL due to 22 perfect repeats of the motive AAATTTATTT flanked by non-repetitive regions of 14 and 12 bp, respectively, while the spacer in the mitogenome of M. goulmimensis (Erfoud) forms a palindrome capable of forming a stem of 26 bp. In turn, the isL spacer of “M. notenboomi” is 73 bp long, of which 51 bp possibly derive from a duplication of the 3′ end of the gene nad1 (88% similarity). All other genomic spacers are located between tRNA genes. The mitogenome of M. spinicaudatus shows a unique internal spacer (isN) of 173 bp between trnR and trnF genes; it could have originated from duplication since it shows a 73% similarity with gene trnD. The long inverted repeats present in mitogenomic spacers have been interpreted in some cases as an extra origin of replication ( and references therein). In other cases, spacers could be just remnants of a duplication process produced by slipped strand mispairing or imprecise termination during replication .
The non-coding unassigned region located between the rrnS and trnS2 (UCN) genes in all mitogenomes show the expected characteristics of control regions such as a high A-T content, presence of a secondary structure with T-rich loops, plus repetitive elements and palindromes . This region displays also the lowest GC-skew, a feature indicative of the presence of the origin of replication . The complete control region was obtained for eight mitogenomes and showed an AT-richness in the range 85.5-100% and lengths between 25 and 963 bp. Although variable in size and sequence, all control regions showed five common features ; namely: i) a TATA motif followed by ii) a 14–15 poly-T stretch; iii) a variable region (absent in some mitogenomes) capable of forming one or several stem-loop structures; iv) a 10–12 poly-A stretch and v) a GANT motif embedded in the trnSUCN gene (Additional file 4). The variable region of M. goulmimensis (Ousroutou) comprises one motif of 216 bp followed by the sequence in inverted orientation (97% sequence identity lacking indels, Additional file 4). M. spinicaudatus has flanking repeats with inverted orientation of 93 bp (84% sequence identity without indels, not shown). Seemingly, “M. boveei” shows a long inverted repeat of 378 bp (100% identity and 11 indels, Additional file 4). In some mitogenomes these palindromes could have in part originated from a short tandem repeat, such as in the mitochondrial genome of M. goulmimensis (Ousroutou) where five monomers of 24 bp show a 74.2% identity (motif T7A7TA9). Two of the mitogenomes (M. longipes Menorca and M. goulmimensis Zouala) showed minimal and similar control regions with the motif (T)14–15(A)13–15 as they lack the variable region and show a secondary structure identical to the one deduced for M. longipes (Mallorca)  (Additional file 4). Similar structures have been described in the control regions of the isopods Armadillium vulgare and A. pelagicum.
RrnS and rrnLstructure
The rrnL sequences were similarly AT-rich (76.5% on average) and the inferred secondary structures showed the five canonical domains (I-II, IV-VI) displayed in all metazoans and absence of domain III as in all arthropods  (Figure 8). The metacrangonyctid rrnL multiple alignment comprised a total of 1105 positions, of which 475 were conserved (43.0%). Domains I and II were the most variable, showing only 30 and 25% identical positions, respectively, while domains IV, V and VI were more conserved (52-54% conserved positions) (Figure 8). A similar conservation pattern is shown in neuropterid insects 16S RNAs .
The analysed metacrangonyctid mitochondrial genomes have a conserved gene order with a diagnostic translocation of the trnS2 (UCN) and cob genes. This gene order differs from the pancrustacean gene arrangement and is unique among amphipods. In addition, PCGs show a reversed strand mutational bias pattern with GC-skew positive values at both strands except for two genes (cob and nad6), while codon usage seems to be influenced by base composition and strand mutational bias. The atp8 gene displays the highest non-synonymous/synonymous rate ratio, being the more phylogenetically informative per position due to the frequent occurrence of non-synonymous changes at first and second codon positions. Purifying selection appears to have been stronger on genes of the cytochrome oxidase respiratory complex, in particular the cox1 gene as shown for other mitogenomes. tRNA genes show a mutation dynamics similar to other metazoans, with frequent compensatory mutations at stems. Aberrant secondary structures lacking the D-stem have been determined in several metacrangonyctid tRNAs. AT-rich control regions, albeit quite variable in length, show common features and sequence motifs that can be related to their possible role as the replication origin. The rrnS and rrnL secondary structures of a reference Metacrangonyx mitogenome have been modelled based on the structures determined elsewhere in Artemia and sequence conservation within Metacrangonyctidae mapped on the obtained structures. These structures are similar to those shown in other arthropods, where conservation is concentrated at certain segment domains. To our knowledge these are the first rrnS and rrnL secondary structures determined for a peracarid and second for a crustacean.
We sampled specimens assigned to species of Metacrangonyx and Longipodacrangonyx from freshwater wells and caves spanning almost the entire known geographic range of the family. Material used in analyses was collected under collection permits issued to authors by the corresponding local or governmental authorities (i.e. Conselleria d’Agricultura, Medi Ambient i Territori, Govern de les Illes Balears; Consejería de Medio Ambiente, Gobierno de Canarias; Dirección de Biodiversidad y Vida Silvestre, Secretaría de Estado de Medio Ambiente y Recursos Naturales, Dominican Republic). No specific permits were required for specimens collected in Morocco and Elba Island. Several Moroccan taxa included in our analyses are not formally described yet are quoted with a tentative Latinized binomen within inverted commas and not in italics to identify this feature. Sampling locations with their corresponding geographic coordinates are listed in Additional file 3: Table S4. Major phylogenetic lineages within the family were identified based on partial mitochondrial cytochrome oxidase 1 (cox1) DNA sequences .
Mitogenome amplification and sequencing
Long-range polymerase chain reaction (PCR) primers were designed on partial cox1 and rrnL sequences to amplify the entire mitogenomes as described in  (primer list in Additional file 3: Table S5). Alternatively, the mitogenomes of four species were amplified as a single long fragment with primers targeting the rrnL and rrnS genes. Mitochondrial genomes were amplified from 50 ng genomic DNA using Herculase™ II Fusion DNA polymerase (Agilent, Santa Clara, CA, USA) following manufacturer’s recommendations, except for DNA fragments comprising the AT-rich region that only amplified using an extension temperature of 60°C. The mitochondrial sequence of three species was obtained using standard protocols [12, 45]. The mitochondrial sequences of the remaining species were obtained by next generation sequencing using Roche FLX/454 or GS Junior technology. In a first approach, the long PCR amplicons were purified using Invitek columns (Invitek GMBH, Berlin, Germany), quantified on a Nanodrop spectrophotometer and 5 μg (300 ng/μL) used for FLX tagged library preparation. The individual libraries of seven species were pooled in equimolar ratios and analysed in parallel in a pyrosequencing reaction using the Roche FLX/454 giving a total number of 200,000 reads corresponding to the output of 1/8th lane of the Roche FLX/454 sequencer. Methods for tagging and library preparation were previously described in  and followed manufacturer’s instructions. The sequence data obtained was sorted according to their tag sequences and preliminary assembled into seven subsets using the Newbler assembler. For twelve additional species we explored the simpler and cost effective multiplex sequencing method without the need of individual tagging by barcodes . Firstly, we tested whether we correctly recover the previous mitochondrial sequences assembling from scratch in a pool of the total reads of the tagged library but after elimination of the species-specific sequence barcodes. This analysis demonstrated that mitochondrial sequence divergences were sufficiently high to reconstruct the whole mitogenome of each species without formation of chimerae. For these additional species, two batches of PCR amplicons representing six mitogenomes each were purified, quantified and pooled in equimolar ratios as above at a final concentration of 100 ng/ul per mitogenome. Each batch was sequenced as a single library in a Roche GS Junior giving a total number of about 100,000 reads per run. Sequences of cox1, cob and rrnL amplified from each species with universal primers and sequenced by standard Sanger method were included in the bioinformatic assembly of reads to confirm the correctness of assignment of each mitochondrial sequence (“bait” sequences as in ). Final assemblies from the FLX and GLS platforms were based on minimum sequence coverage of 59 ×.
Mitogenome assembly, annotation and analyses
DNA sequence read quality filtering, contig assembly and gene annotation were performed as described in  with tRNA structures refined with tRNAscan-SE v1.21 (http://lowelab.ucsc.edu/tRNAscan-SE/) and checked using the MITOS webserver (http://mitos.bioinf.uni-leipzig.de/help.py) . The annotation of start and stop codons plus tRNAs secondary structures were accomplished after exhaustive comparisons among the obtained mitogenome sequences. Gene rearrangements with respect to other amphipod mitogenomes or to the hypothesized pancrustacean ancestral gene order were deduced using strong interval trees on the CREx webserver (http://pacosy.informatik.uni-leipzig.de/crex) . Nucleotide and amino acid composition plus codon usage profiles (Relative Synonymous Codon Usage RSCU) were estimated with MEGA v5.10 . AT and GC skew were estimated as follows: ATskew = (A-T)/(A + T) and GCskew = (G-C)/(G + C) . Effective number of codons (ENC) were determined taking into account background nucleotide composition as implemented in INCA v1.20 . DNA direct and inverted repeats in spacer regions were explored with the EMBOSS package v6.5 (http://emboss.sourceforge.net/)  with the tools einverted, palindrome and etandem. The phylogenetic informativeness (PI) of protein-coding genes was estimated using PhyDesign (http://phydesign.townsend.yale.edu/) . This method estimates maximum likelihood values per site on a tree topology derived from protein-coding sequences with each codon position considered as an independent partition . The tree topology obtained in  was used for the PhyDesign analysis. Non-synonymous/synonymous substitution rate analysis (dN/dS) of the protein-coding genes was performed with the basic codon substitution model  in the PAML v.4.7 software package . Nucleotide frequencies were calculated in the analysis from the average nucleotide frequencies at the three-codon positions (CodonFreq = 2).
RrnL and rrnS structures
Secondary RNA structures for the small (rrnS) and large (rrnL) ribosomal units where modelled based on the proposed rrnS structure of the crustacean Artemia franciscana (accession number X69067, structure at http://www.rna.ccbb.utexas.edu/RNA/Structures/b.16.m.A.franciscana.bpseq) and the rnnL structure of A. salina (X12965, http://www.rna.ccbb.utexas.edu/RNA/Structures/d.23.m.A.salina.bpseq; . Ribosomal sequences from metacrangonyctid and Artemia species were aligned using MAFFT v6.8  taking into account secondary structure (xinsi command). The aligned sequences were folded using as reference the secondary structures of the respective Artemia species using RNAsalsa v0.8.1 with default parameters . Folded structures were visualized and refined using the graphic tool VARNA v3.9 (Visualization Applet for RNA, http://varna.lri.fr/index.php?lang=en&css=varna&page=downloads) . Secondary structures were first obtained for major domains separately since global folding approaches artificially joined different domains. MFOLD (http://mfold.rna.albany.edu/?q=mfold/rna-folding-form)  was subsequently used to correct secondary structure discrepancies at sequence-conserved regions. In some cases, secondary structures showing suboptimal minimum free energy values were chosen, as they were more similar to those accepted for Artemia. Conservation profile of DNA sequences was implemented using the online program PRALINE (http://www.ibi.vu.nl/programs/pralinewww/)  using the standard progressive strategy.
Availability of supporting data
The data set supporting the results of this article is included within the article in Table 1.
This work was supported by Spanish MCINN grants CGL2009-08256 and CGL2012-33597 partially financed with EU FEDER funds. MMBR was supported by a Spanish FPI fellowship. Thanks are due to C. Boutin, N. Coineau, M. Messouli, M. Yacoubi-Khebiza, M. Boulanouar, J.A. Ottenwalder, J.A. Alcover, J.M. Bichain and A. Faille for providing samples or helping with sampling. F. Frati and F. Nardi kindly hosted MMBR at the Department of Evolutionary Biology of the University of Siena. The suggestions made by two anonymous reviewers considerably improved the final version of the manuscript.
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