Unusual conservation of mitochondrial gene order in Crassostrea oysters: evidence for recent speciation in Asia

BMC Evolutionary Biology Unusual conservation of mitochondrial gene order in Crassostrea oysters: evidence for recent speciation in Asia Jianfeng Ren 1 4 Xiao Liu 1 Feng Jiang 2 3 Ximing Guo 0 Bin Liu 1 2 0 Haskin Shellfish Research Laboratory, Institute of Marine and Coastal Sciences, Rutgers University , NJ 08349 , USA 1 Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences , Qingdao 266071 , China 2 Center of Systematic Genomics, Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences , Urumqi 830011 , China 3 Current Address: Institute of Zoology, Chinese Academy of Sciences , Beijing 100101 , China 4 Current Address: Institute of Genetics and Development Biology, Chinese Academy of Sciences , Beijing 100101 , China Background: Oysters are morphologically plastic and hence difficult subjects for taxonomic and evolutionary studies. It is long been suspected, based on the extraordinary species diversity observed, that Asia Pacific is the epicenter of oyster speciation. To understand the species diversity and its evolutionary history, we collected five Crassostrea species from Asia and sequenced their complete mitochondrial (mt) genomes in addition to two newly released Asian oysters (C. iredalei and Saccostrea mordax) for a comprehensive analysis. Results: The six Asian Crassostrea mt genomes ranged from 18,226 to 22,446 bp in size, and all coded for 39 genes (12 proteins, 2 rRNAs and 25 tRNAs) on the same strand. Their genomes contained a split of the rrnL gene and duplication of trnM, trnK and trnQ genes. They shared the same gene order that differed from an Atlantic sister species by as many as nine tRNA changes (6 transpositions and 3 duplications) and even differed significantly from S. mordax in protein-coding genes. Phylogenetic analysis indicates that the six Asian Crassostrea species emerged between 3 and 43 Myr ago, while the Atlantic species evolved 83 Myr ago. Conclusions: The complete conservation of gene order in the six Asian Crassostrea species over 43 Myr is highly unusual given the remarkable rate of rearrangements in their sister species and other bivalves. It provides strong evidence for the recent speciation of the six Crassostrea species in Asia. It further indicates that changes in mt gene order may not be strictly a function of time but subject to other constraints that are presently not well understood. - Background Mitochondrial (mt) DNA is widely used for phylogenetic analysis because of its unique architecture, inheritance and small size. Metazoan mtDNA is nearly always a circular molecule except for some cnidarians [1]. It contains the same 37 genes, specifying 13 proteins of the respiratory chain [cytochrome c oxidase subunits I-III (cox1-cox3), apocytochrome b (cob), ATP synthase subunits 6 and 8 (atp6 and atp8), and NADH dehydrogenase subunits 1-6 and 4L (nad1-6, nad4L)], 2 ribosomal RNAs and 22 transfer RNAs. Although there are exceptions, most mtDNAs range in size from 14 to 17 kb. Typically, there are few intergenic nucleotides except for a single large non-coding region generally thought to contain elements that control the initiation of replication and transcription [2]. Size variation in mtDNA is usually due to the different length of the non-coding regions. Gene order is generally conserved in most metazoan taxa although some groups show considerable variation. Variations in both mtDNA sequence and gene order have been used for phylogenetic analysis. Because mtDNA is fast evolving and nucleotide mutations may return to an early state, mtDNA sequences may not allow deep phylogenetic reconstruction. Gene order, on the other hand, has very small probability of back-mutation and may be particularly useful for high level phylogenetic analysis. Although the mechanism of mtDNA rearrangement is poorly understood, mt gene order has been increasingly used for phylogenetic studies [3-6]. As the second species-rich phylum of the animal kingdom after Arthropoda, Mollusca exhibits tremendous variation in their mt genomes. Seven bivalve lineages (Mytilidae, Unionidae, Margaritiferidae, Hyriidae, Donacidae, Solenidae, and Veneridae) have been found to have an unusual mode of inheritance for mtDNA, termed doubly uniparental inheritance (DUI) [7]. Some pulmonate gastropods have unusual tRNAs lacking the T-stem or the D-stem, similar to nematode mt tRNAs [8]. The atp6 and atp8 genes are separated in the scaphopods and two groups of gastropods (Patellogastropoda and Heterobranchia). In addition, several mt genes are duplicated in cephalopods Watasenia scintillans and Todarodes pacificus [9]. Unlike the general conservation in gene order in most other metazoan groups, most molluscan mt genomes reported so far contain considerable rearrangements especially in Bivalvia and Scaphopoda [10]. At the same time, the phylogeny of molluscs is poorly studied. Phylogenetic relationships among major molluscan groups are not well understood. The species identity and classification of some most common molluscs remain questionable. Oysters are bivalve molluscs widely distributed in world oceans. They are benthic, sessile filter-feeders with important roles in estuary ecology. Some species support major fishery and aquaculture industries worldwide. Despite the abundance, ecological and economic significance of oysters, we know little about their species diversity and evolutionary history. Classification of oysters remains a challenge partly due to the lack of well-defined morphological characters. Shell morphology, the main character used in oyster classification, is known to be plastic and subject to environmental variation. Much of the oyster classification to date is based on shell characteristics and has resulted in considerable errors and confusion. The difficulty of oyster classification is particularly pronounced in China and other parts of Asia where a large number of species are sympatric [11,12]. About thirty species have been recorded along the coast of China [13]. The presence of a large number of oyster species has led many to believe that Asia Pacific is the epicenter of oyster speciation, but the inability to reliably identifying them has hindered our understanding of oyster evolution. Among the extent oysters, five Crassostrea oysters, namely C. gigas, C. angulata, C. sikamea, C. hongkongensis and C. ariakensis are commonly found in China and other parts of Asia and yet, they have been difficult to identify by shell morphology alone. Recently, some confusion in the oyster identification have been resolved using DNA sequence data [12,14-20], and the five Crassostrea oysters can be reliably identified [21]. Still, we know little about the evolutionary history of the Crassostrea species and how they relate to each other and the other oysters. For example, it is not clear whether C. angulata should be considered as an independent species and which species is the closest relative of (...truncated)