BackgroundComparative 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).ResultsWe 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.ConclusionsNext-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.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-566) contains supplementary material, which is available to authorized users.

Many continental subterranean water crustaceans ("stygobionts") display extreme disjunct distributions, where different species in the same genus are isolated on continents or islands separated by broad oceanic expanses. Despite their freshwater habitat, most of these taxa appear to be most closely related to typical marine groups ("thalassoid" origin). Among the hadzioids-thalassoid amphipods including the stygobiont families Hadziidae, Pseudoniphargidae, and Metacrangonyctidae-several genera are restricted to inland groundwaters ranging from the Caribbean region to the Mediterranean and Middle East, including interspersed oceanic islands. This distribution might have arisen from Tethyan vicariance triggered by the sequential occlusion of the former Tethys Sea, a vast circumtropical ocean existing from the Middle Jurassic up to 20 million years ago (mya). Previous studies have been based on morphological analyses or limited DNA sequence data, making it difficult to test this hypothesis. We used complete mitochondrial protein-coding gene sequences, mainly obtained by next-generation sequencing methods and a nuclear ribosomal gene to resolve the phylogeny and to establish a time frame for diversification of the family Metacrangonyctidae (Amphipoda). The results were consistent with the plate tectonics vicariance hypothesis, with major diversifications occurring between 96 and 83 mya.

Metazoan mitochondrial genomes usually consist of the same gene set, but some taxonomic groups show a considerable variety in gene order and nucleotide composition. The mitochondrial genomes of 37 crustaceans are currently known. Within the malacostracan superorder Peracarida, only three partial mitogenome sequences and the complete sequence of Ligia oceanica (Isopoda) are available.Frequent translocation events have changed the mitochondrial gene order in crustaceans, providing an opportunity to study the patterns and mechanisms of mitogenome rearrangement and to determine their impact on phylogenetic reconstructions. Here we report the first complete nucleotide sequence of an amphipod species, Metacrangonyx longipes, belonging to a phylogenetically enigmatic family occurring in continental subterranean waters. The genome has 14,113 base pairs (bp) and contains the usual 13 protein coding genes and two rRNA subunits, but only 21 out of the typical 22 tRNA genes of Metazoa. This is the shortest mitogenome described thus far for a crustacean and also one of the richest in AT (76.03%). The genome compactness results from a very small control region of 76 bp, the occurrence of frequent gene overlap, and the absence of large non-coding fragments. Six of the protein-coding genes have unusual start codons.Comparison of individual protein coding genes with the sequences known for other crustaceans suggests that nad2, nad6, nad4L and atp8 show the highest divergence rates. Metacrangonyx longipes shows a unique crustacean mitogenome gene order, differing even from the condition found in Parhyale hawaiiensis (Amphipoda), whose coding sequence has also been completed in the present study.

BackgroundMetacrangonyctidae (Amphipoda, Crustacea) is an enigmatic continental subterranean water family of marine origin (thalassoid). One of the species in the genus, Metacrangonyx longipes, is endemic to the Balearic islands of Mallorca and Menorca (W Mediterranean). It has been suggested that the origin and distribution of thalassoid crustaceans could be explained by one of two alternative hypotheses: (1) active colonization of inland freshwater aquifers by a marine ancestor, followed by an adaptative shift; or (2) passive colonization by stranding of ancestral marine populations in coastal aquifers during marine regressions. A comparison of phylogenies, phylogeographic patterns and age estimations of clades should discriminate in favour of one of these two proposals.ResultsPhylogenetic relationships within M. longipes based on three mitochondrial DNA (mtDNA) and one nuclear marker revealed five genetically divergent and geographically structured clades. Analyses of cytochrome oxidase subunit 1 (cox1) mtDNA data showed the occurrence of a high geographic population subdivision in both islands, with current gene flow occurring exclusively between sites located in close proximity. Molecular-clock estimations dated the origin of M. longipes previous to about 6 Ma, whereas major cladogenetic events within the species took place between 4.2 and 2.0 Ma.ConclusionsM. longipes displayed a surprisingly old and highly fragmented population structure, with major episodes of cladogenesis within the species roughly correlating with some of the major marine transgression-regression episodes that affected the region during the last 6 Ma. Eustatic changes (vicariant events) -not active range expansion of marine littoral ancestors colonizing desalinated habitats-explain the phylogeographic pattern observed in M. longipes.

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