nrDNA-ITS Variation , Structure , and Molecular Phylogeny of Abies beshanzuensis ( Pinaceae ) , a Critically Endangered Species

Abies beshanzuensis, an extremely endangered species, attracts serious concerns on genetic diversity recovery and population protection. However, little genetic information was known about it till now. In this study, intra-/intergenomic ITS variation, secondary structure, and molecular phylogeny of A.beshanzuensis were determined by nrDNA-ITS marker, and results indicated that, ITS region featured rich polymorphism in ITS1 and highly conservative in 5.8S/ITS2 among intra-/inter-genome, implying rich genetic diversities and the occurrence of non-concerted evolution in ITS region. Predicated 5.8S structure possessed Helices-Loops with 3 motifs (M1/M2/M3) and 5 helices B4-B8, and ITS2 could form conservative Clover-like structure with 5 ribotypes, manifesting genetically structural generalities and individualities. Thirteen ITS pseudogenes were also identified from 76 samples (or clones). Molecular phylogeny revealed that, A.beshanzuensis shared genetically closer relationships with A.fanjingshanensis, A.chensiensis, A.yuanbaoshanensis among genus Abies. The available original data provided rich genetic information on ITS sequence, structure and molecular phylogeny of A.beshanzuensis, which not only updated the databases of ITS sequences as well as secondary structures essential for phylogenetic reference and DNA barcoding analyses, but also contributed to deep exploration of population diversities, origin and evolution as well theoretical direction for population recovery and protection of A.beshanzuensis.


Introduction
As the second largest genus in Pinaceae, Abies is an ecologically significant for climate and economically important for timer.It comprises approximately 48 species, and discontinuously distributes from central and north America, eastern Asia to the Mediterranean area of Europe and southwestern Asia [1].In China, approximately 28 Abies species (including varieties and imported species of A.firma), distribute from North to South China.Particularly, several rare, endangered species Abies beshanzuensis (Zhejiang), A. fanjingshanensis (Guizhou), A. yuanbaoshanensis (Guangxi) and A. ziyuanensis (Guangxi) have been attaching great attention from researchers and governments at all levels [2].
Abies beshanzuensis M.H.Wu (A. beshanzuensis), a critically endangered, endemic, ancient relic species, was putatively migrated from North to South at high altitude during Quaternary glacial period [3]. A. beshanzuensis only distributes in the extremely narrow Fengyang Mountain at the altitude of 1700 meters in Qingyuan county, China.Currently, only 4 naturally wild trees (one young and other 3 ancient) were discovered in their natural habitats, and approximately 2000 artificial trees were derived from these 3 ancient trees by direct seed breeding or xenostock grafting [4].The extremely small population of individuals was possibly due to the following factors: 1) Extremely low hybridization rate due to imperfectly identical flowering period between male and female gametes; 2) Hybridization restriction from extremely limited individuals with island-isolated distribution in natural habitats; 3) Drastic interspecific survival competition pressure and fragile adaptation ability to ecological condition; 4) Possibly artificial destruction in the early or middle of the last century.However, the extremely small population of individuals would raise serious question of population diversities and recovery, ecological protection and sustainable development of A. beshanzuensis.
DNA molecular marker, played dominant roles in genetic diversities evaluation, phylogeny reconstruction, species identification and classification, and were successfully applied to Abies plants such as RFLP [5], AFLP and SSR [6], nrDNA-ITS [7] and/or combined/individual nuclear and organellar DNA [1,[8][9][10] or nuclear microsatellites [11].And these studies opened the door to deep exploring the origin and evolution, genetic diversities and phylogeny of genus Abies.Currently, an increasing data focused on the nrDNA-ITS analysis of genetic background, but there were still few reports on ITS variation and structure of Abies, particularly for A.beshanzuensis.
Compared with angiospermae, gymnosperms have much longer and more complex ITS sequence, particularly for Pinaceae plants [7,17,19].Though thousands of ITS regions have been sequenced and deposited in NCBI database for many species, knowledges about their secondary structures were still limited [12,13,16,20,[21][22][23][24].To our knowledge, there were no reports on ITS sequence and structure from A.beshanzuensis till now.rDNA-ITS variation of intraspecies were discovered in diverse species and high levels of intragenomic and intraspecific polymorphism attribute to formation of non-functional ITS (i.e.ITS pseudogenes) and of non-concerted evolution [25][26][27].ITS secondary structures not only represented genetically structural information but also attributed to identification of ITS pseudogenes.The presence of ITS pseudogenes would cause the corresponding nrDNA pre-transcripts not to be correctly processed and maturated [12].Thus, it's believed if ITS pseudogenes would be introduced into the phylogenetic analysis, it possibly produces erroneous hypothesis about phylogeny [16,20,21,28].However, previous most studies on Pinaceae plants failed to care about the presence of ITS pseudogenes in phylogenies analysis.
The present study aimed to investigate nrDNA-ITS variation, structure, pseudogenes, non-concerted evolution and molecular phylogeny of A.beshanzuensis, which would contribute to deep exploration of genetic diversities, origin and evolution, biogeographical distribution as well as population recovery and protection of A.beshanzuensis.

ITS sequence and length variation of A.beshanzuensis
Great variation occurred on nucleotides sequence and length in ITS region in A.beshanzuensis (Table S1).The available data indicated that intra-/intergenomic ITS length ranged from 1411 (A.beshanzuensis A2-2) to 1755bp (A.beshanzuensis A4-2), in which ITS1 sizes ranged from 1006bp to 1350bp, and the sizes of 5.8S and ITS2 were 162bp and 243bp with some exceptions, respectively.
Apparently, variations on ITS sizes and nucleotides sequence for A.beshanzuensis mainly occurred in ITS1 region, which implied that ITS1, not 5.8S and ITS2, was responsible for most of variation of length and sequences.In contrast to ITS length of around 1700bp for most Abies species, longer ITS sizes were observed in A.grandis (2215bp, EF057690.1)(North America), A.concolor (2212bp, EF057692.1)(North America) and A.religiosa (2180bp, EF063714.1)(Central America).These species with longer ITS sizes featured highly similar morphological traits, and were regarded as evolutional Abies species [29].Early data supported an "out of America" migration for the origin of an eastern Asian and western North America disjunct species pairs in section amabilis [1].
Subrepeats were present in ITS region in A.beshanzuensis.The available data supported 1-3 small subrepeats (SSR) (5'-GGCCACCCTAGTC-3') and the unique 298bp large subrepeats (LSR) in ITS1 region, implying these subrepeats largely contributed to ITS1 length variations among Abies species [7,17].Previous reports displayed SSR sequences appeared in all Pinaceae while LSR sequences were only present in Abies [17] and in Picea [18].Moreover, it's believed that, GC content in ITS1 region was relevant to its sequence length and subrepeats number [17].Therefore, the sizes, GC contents, subrepeats and nucleotides variation of ITS regions provided basic genetic information for the origin and evolution of Abies plants [1].

Secondary structure predication of 5.8S and ITS2
The 5.8S region of A.beshanzuensis could form typical "Helices-Loops" structure with 3 highly conserved motifs (M1/M2/M3) and helices (B4-B8) [21,31].Of 3 motifs in all tested samples (Figure with similar open loop at similar position as well as protruding loop on the left/right of helices Ⅲ (Figure 2).This study supported slight differences of ITS2 structure with some exceptions in A.beshanzuensis (particularly in intra-individuals), and considerable differences among genus Abies.
Generally, ITS2 structures represented evolutional and functional conservation as well as genetically structural diversities and uniqueness.Additionally, we failed to determine ITS1 structures of A.beshanzuensis, mainly due to that ITS1 regions had different number of shorter subrepeats (SSR)/longer subrepeat (LSR) sequences and high nucleotides variation rate, which would result in formation of extremely complex secondary structures [17]，moreover, there lacked of effective methods to evaluate ITS1 structural differences.

Predication of ITS pseudogenes
According to identification methods for ITS pseudogenes [21,22], nucleotides sequences in 5.8S region were checked against the corresponding 3 motifs (M1: CGATGAAGAACGyAGC; M2: GAATTGCAGAAwyC; M3: TTTGAAyGCA) and 5 helices (B4-B8), the data disclosed that low level of mutations in M1 (16nt) for 4 samples: A2-2 (11th site of C→A), A3-3 (12th G→A), C4-2 (16th C→ T), C8-2 (1st C→T and 8th G →A), and that the 13th site of degenerate base was T not C in all samples.As for M3 (10nt), only single site variation (2nd T→C) was observed in sample A4-1, and the 7th site of degenerate base was T not C in all samples of A.beshanzuensis.Moreover, single nucleotide variation also appeared in sample A.spectabilis (2nd T→C) (JF416978.1).For M2 (14nt), all the samples had completely same sequences, and the degenerate bases WY in the 12th and 13th sites were T and C, respectively, which implied that M2 possibly play core role in the formation of 5.8S structure.In addition, several mutation sites were observed at non-motifs regions in  S1).Totally, ITS pseudogenes featured nucleotides mutations in 5.8S motifs, shorter ITS length, lower GC content, or unstable ITS2 structure, which contributed to extreme diversities and complex evolution of nrDNA [21,26,27].

Phylogenetic reconstruction based on complete ITS1-5.8S-ITS2 sequence
Phylogenetic tree of A.beshanzuensis was reconstructed based on complete ITS sequence (Figure 3).In this study, 13 identified ITS pseudogenes were excluded from 76 sequences.ITS sequences from 4 wild individuals of A.beshanzuensis were only considered for phylogeny of A.beshanzuensis, and single ITS sequence was selected from several clones of inter-/intra-individuals for each species [1,7].The phylogenetic tree revealed that, 28 samples were distinctly divided into 2 clades: the major one having 25 samples with ITS sizes of approximately 1700bp, and the minor one having 3 samples with ITS sizes of approximately 2200bp.Seemly, the ITS sizes played dominant role in phylogenetic analysis on Abies in this study.
In the major clade, A.fargesii (JF416972.Phylogenetic analysis represented sample A.beshanzuensis A1-1 was isolated from other 3 A.beshanzuensis samples (A2-1/A3-1/A4-2), suggesting sample A1 had genetically distant relationships with other 3 samples.Geographically, 3 wild ancient trees A1/A2/A3 clustered together and were approximately 50 meters away from sample A4.Moreover, all 4 wild individuals were in island-isolated distribution in their natural habitat.Thus, it was supposed that, the young tree A4, was possibly the hybrid offspring of ancient trees A1/A2/A3.This study supported A4 shared genetically close relationships with A2 and A3 and distant relationship with A1.
The phylogenetical relationships (Bootstrap:1000; Cutoff: 50%) for 28 samples were well supported with exceptions of one cluster in Subclade Ⅰ and 2 clusters in Subclade Ⅱ.Previous studies proposed ITS marker was robust for phylogenetic analysis at interspecific or lower taxonomic levels [32], and the available data implied ITS marker was applicable for phylogenetic analysis of A.beshanzuensis.The present study implied that, A.beshanzuensis shared genetically closer relationships with A.fanjingshanensis, A.chensiensis, A.yuanbaoshanensis among genus Abies.
Additionally, weak correlation between geographical distribution and genetic similarities appeared in several samples such as samples A.chensiensis a1 (Clades Ⅰ) and A.chensiensis b1 (Clades Ⅱ).The possible explanation was that, different ecological environments resulted in formation of varieties of A. chensiensis such as A. ernestii, A. ernestii var.salouenensis, A. recurvata [33], and introduction of Abies plants frequently occurred in both Nature Reserves, which possibly attributed to genetic variation for A.chensiensis a1 and b1.However, the true phylogenetic position of A.beshanzuensis as well as other species among genus Abies needed to be further valuated by data from diverse DNA markers, biochemical or morphological analysis.

Plant samples
The young leaves from 24 samples were employed, including 13 individuals of A. beshanzuensis (4 wild trees and 9 artificial trees) and 11 other Abies plants in China (Table 2).All samples were completely cleaned by distilled water and stored in -80℃ freezer.Additionally, 26 ITS sequences from different Abies species were retrieved from NCBI database for comparative analysis (Table S1).5.8S secondary structure was predicted according to online software Mfold (RNA Folding Form) (http://unafold.rna.albany.edu/?q=mfold) and VARNA 3.9 (http://unafold.rna.albany.edu)with defaulted parameters (such as temperature and ionic condition etc.) [16]; ITS2 secondary structure and its minimum free energy were determined by direct folding or homologous modeling based on the online software with default setting, respectively (http://its2.bioapps.biozentrum.uni-wuerzburg.de)[24,35].

Figure 2 .
Figure 2. Ribotypes for ITS2 structure of A.beshanzuensis and other Abies species in this study (Notes: Ⅰ/Ⅱ/Ⅲ/Ⅳ for helices and the number in brackets for internal loop numbers in helices Ⅰ/Ⅱ/Ⅲ/Ⅳ, respectively; L/R for protruding loop present in the left/right of helices Ⅲ).
1), endemic to central China, featured distinctively morphological traits and individually formed one clade.And the remaining 24 samples shared another clade including 2 Subclades Ⅰ and Ⅱ. Thirteen samples in Subclade Ⅰ were originated from diverse areas such as East Asia (China, Japan, Korea) and Europe, while 11 samples in Subclade Ⅱ formed 2 clusters with one having 5 samples native to western north America, and the other having 6 samples native to China.Seemly, weak correlation in Subclade Ⅰ and strong correlation in Subclade Ⅱ occurred between genetic similarities and geographical distribution.

3. 3 .
ITS fragment clone, sequence analysis and alignmentTarget DNA fragment (ITS1-5.8S-ITS2)was directly extracted from agarose gel based on the instruction of DNA gel extraction kit (Cat.DP209-02, Tiangen Biotech, China); Target DNA fragment was ligated into vector pMDTM18-T (Code: D101A, Takara, China) and transformed into competent cells from bacterial strain E.coli (DH5 ɑ ) (Tiangen Biotech, China) according to manufacturer recommendations.Recombined plasmids were determined by colony PCR amplification from positive clones by primers ITS5/26S25R.And 4-5 identified clones from individual samples were randomly selected to investigate ITS polymorphism of intra-genome for some samples.Target DNA sequence of recombined plasmid was analyzed by bidirectional sequencing using ABI genetic analyzer (ABI-PRISM3730XL) (Lifei biotechnology, China) with sequencing primers M13F (TGTAAAACGACGGCCAGT) and M13R (CAGGAAACAGCTATGACC) in vector pMDTM18-T, and sequences edition and alignment were conducted by software BioEdit (7.0), respectively.Based on sequence annotation of Abies plants deposited in NCBI database (GenBank： DQ975365.1;EU196130.1),the 5'-/3'-end boundaries of complete ITS1-5.8S-ITS2region as well as respective ITS1, 5.8S, ITS2 regions were determined, respectively.Furthermore, the sequences and boundaries of 27 ITS retrieved from NCBI database were also performed for comparative analysis.ITS length, nucleotides variation, GC content and multiple sequence alignment were carried out by the softwares DNAstar and DNAMAN (8.0), respectively.

Table 1 .
Intragenomic ITS variation from 4 wild samples of A.beshanzuensis Notes: NC* for number of clones; HVR* for high nucleotides variance region.
and nucleotides variation in degenerate sites in 5.8S motifs implied base usage preference for A.beshanzuensis plants.Based on combined factors of nucleotides variances in 5.8S, GC content, ITS size, and minimum free energy of 5.8S/ITS2 structures [21], 9 ITS pseudogenes were identified in 35 samples (or clones) of A.beshanzuensis and 4 ITS pseudogenes in other Abies species (Table 5.8S.Possibly, these mutations in highly conservative 5.8 motifs would result in ITS functional inactivation (ITS pseudogenes),

Table 2 .
Geographical distribution of A.beshanzuensis and other Abies plants in this study