INTROGRESSION EVIDENCE AND PHYLOGENETIC RELATIONSHIPS AMONG THREE ( PARA ) MISGURNUS SPECIES AS REVEALED BY MITOCHONDRIAL AND NUCLEAR DNA MARKERS

The taxonomy of (Para)Misgurnus genera is still debated. We therefore used mitochondrial and nuclear DNA markers to analyze the phylogenetic relationships among Misgurnus anguillicaudatus, Paramisgurnus dabryanus and Misgurnus fossilis. Differing phylogenetic signals from mitochondrial and nuclear marker data suggest an introgression event in the history of M. anguillicaudatus and M. mohoity. No substantial genetic evidence was found that Paramisgurnus dabryanus should be classified as a separate genus.

Only a few studies have dealt with the phylogenetic relationships among the European and East-Asian Cobitidae (Šlechtová et al., 2008;Tang et al., 2008), and no study focusing exclusively on the phylogeny of European and East-Asian Misgurnus and Paramisgurnus genera has ever been published.As previous research has shown that incongruous results are obtained using mitochondrial and nuclear markers (Šlechtová et al., 2008), mitochondrial (cytochrome b (cyt b) and displacement loop (D-loop)) and nuclear (recombination activating gene 1 (RAG1)) markers were simultaneously used to study the evolutionary history of MF, MA and PD.All three genes have often been used in phylogenetic studies on Cypriniformes (Culling et al., 2006;Liu et al., 2003).

MATERIALS AND METHODS
Eight MF specimens, 3 Cobitis elongata (CE) and 2 Barbatula barbatula (BB), were obtained in 2007 from the River Sava, Croatia.Twelve MA, 14 PD and 3 M. sp.specimens were obtained in 2006 from fish markets in Wuhan, China.Specimens were determined according to Kottelat and Freyhof (2007).The remaining sequences were obtained from GenBank.Along with CE and BB, GenBank sequences of Gonorynchus abbreviatus, Leptobotia mantschurica and Cyprinus carpio were used as the second outgroup.
Sequences were aligned and edited by CLUS-TAL W (Thompson et al., 1997).909 to 940 bp of Dloop, 724 bp of RAG1 and 1140 bp of the complete cyt b gene were used for further analyses.Using the Akaike Information Criterion implemented in jMod-elTest 0.1.1 (Posada, 2008), HKY+I+G was chosen for RAG1 and F81 for cyt b and D-loop datasets as the best fitting evolution model.Two independent Bayesian searches were performed using MrBayes (Huelsenbeck et al., 2001) under the following settings: 4 chains; 10 6 generations; sampling frequency = 100 generations; burnin value = 10 3 .The remaining trees were used to build a consensus tree.MEGA (Tamura et al., 2007) and GIMP2 (www.gimp.org)were used for displaying and editing phylogenies.In order to be able to include GenBank data, phylogenetic analyses were performed separately for each amplified fragment.

RESULTS AND DISCUSSION
Apart from MF and PD forming a sister group in the former, Bayesian analyses of D-loop (Fig. 1. a) and cyt b (Fig. 1. b) datasets yielded highly congruent trees.Misgurnus was shown as paraphyletic with high posterior probability (PP) values.The RAG1 dataset phylogeny tree (Fig. 1. c) shows the entire (Para)Misgurnus as monophyletic.PP were high; none below 50%, the majority over 90%.

MF cluster
All datasets showed the MF cluster as very homogenous, with the exception of the MF sequence from Ukraine in the RAG1 dataset (Fig. 1. c).It is possible that some other cobitid was mistakenly classified as MF.

PD cluster
M. mizolepis sequences were dispersed within the PD cluster in all trees, suggesting it might be just a synonym for P. dabryanus.

MA/M. mohoity cluster
In all of the trees, the MA cluster was shown as closely related to M. mohoity (bipartitus).In the cyt b and D-loop trees, two newly sequenced M. sp.specimens clustered with M. bipartitus/mohoity specimens from the Chinese or Russian part of the Amur drainage, corroborating that mohoity and bipartitus are synonyms (http://fishbase.org).
As it was previously shown that introgressive hybridization occurs between Cyprinidae genera (Freyhof et al., 2005), and an introgression event has been proposed in the evolutionary history of Misgurnus (Šlechtová et al., 2008;Tang et al., 2008), it can be assumed that an introgression of mtDNA from Cobitis by hybridization has occurred in the common ancestor of M. anguillicaudatus and M. mohoity.This research suggests that the (Para) Misgurnus clade is comprised of five species.Two of these, Misgurnus anguillicaudatus, with a wide native range, and Misgurnus mohoity, predominantly found in the Amur ecoregion and Korea, underwent mitochondrial introgression.The remaining three species are Misgur- nus fossilis, confined to European waters; Misgurnus nikolskyi, mostly found in the Amur river drainage and Paramisgurnus dabryanus, with a wide native range from South China to South Korea.As the whole (Para)Misgurnus clade is relatively new,Tang et al. (2008) suggested a conservative approach to taxonomy, but more detailed research would be necessary to corroborate these results and possibly suggest merging Paramisgurnus into the Misgurnus genus.

Table 1 .
GenBank accession numbers of the specimens sequenced for the study