The ITS sequence has proven to be valuable in systematic studies [3, 11, 16]). As part of the rDNA sequence, ITS2 can fold into variable secondary structures depending on its primary sequence, which has been proven useful for resolving closely related taxa phylogenies since they are relatively fast-evolving sequences [11, 28]). Based on the structural parameters of the predicted secondary structure (geometrical features, bond energies, base composition) , the ITS2 folded form provide a new source of phylogenetic information which has been used as supplement evidence in sequence alignment for phylogenetic reconstructions [9, 11, 30]). The visible morphological markers of structure contained a remarkable signature sequence, e.g., the bulge and apical portion shared among specific taxa. In the Juglandaceae family, except for the accessions that did not fold the standard structure, the common core 3-helices and 4-helices structures of ITS2 were observed in several taxa; the U-U mismatch at the 5' sequence and the highly 5'-GGUGGU-3’ conserved sequence hallmarks at the 3' sequence were also present, both of which are putatively important for ribosomal RNA processing .
In the previous works of the evolution of the Juglandaceae family, the accepted clades were (Juglans-(Cyclocarya + Pterocarya)) and (Carya + Annamocarya) [12, 15]). The folded ITS2 secondary structure is the standard structure popular among all genera, except for C. cathayensis and C. tonkinensi, which constructed the unique three-helices structure; Platycarya strobilacea folded the 2-helices structure. We downloaded two sequences of Platycarya strobilacea, the only species included in the genus, and both constructed the excludable 2-helices structure with free energy higher than the standard structure. Therefore, the Platycarya genus was highly specialized in the Juglandaceae family and coincided with Manning's classification .
The identified marker sites could be considered diagnostic since they were specific to certain groups of species; for example, the U-U positional homologous marker site included in the helix B was identified as a diagnostic marker shared across the Juglans, Cardiocaryon, Trachycaryon section, as well as the Pterocarya and Cyclocarya genera. Simultaneously, the C-U mismatch base pair was matched in the Rhysocaryon section and the Carya, Annamocarya, Rhoiptelea, and Engelhardia genera.
Within the genus, the different clades were concordant with the geographical distribution. The taxa of the Rhysocaryon section were divided into tropical and temperate region populations. We observed that J. hindsii and J. californica folded the standard 3-helices and 4-helices structures, but other black walnut species only folded the 3-helices structure. The phenomenon was explained through the clustering analyses of NCS (chloroplast DNA noncoding spacer) + matK by Aradhya . The explanations included the historical gene introgression from the ancestral J. cinera species and black walnut species derived from an independent ancestor in Northwestern America .
Data from the ITS2 NJ analyses confirmed that the Cardiocaryon-Trachycaryon clade was monophyletic. This result was congruent with results of the distribution of fossils, which revealed that butternuts might have originated and radiated independently from high Northern latitudes while leaving small refuges of disjunction populations in Eastern Asia and North America of geo-climatic change . In the Cardiocaryon-Trachycaryon clade, included J. hopeiensis which the morphology closely resembles that of J. Regia, yet the nut characters are similar to that of J. mandshurica. J. hopeiensis was considered a subspecies of J. mandshurica  or an interspecific hybrid between J. Regia and J. mandshurica . So far, it was argued to the J. hopeiensis taxonomic status. In the N.J. analysis, the bootstrap (47%) confirmed the subspecies relationship between J. mandshurica and J. hopeiensis, as did secondary structure features. These results were similar to those of , who used the RAPD marker, and the data clustered J. hopeiensis into the Cardiocaryon section. The structure of ITS2 typically supported the Cardiocaryon-Trachycaryon monophyletic clade, i.e., the marker site U-U mismatch and the D helix with one bulge in the 4-helices structure. The accessions collected in China (coded), including wild walnut located in Ili county, the cultivar (including senility trees and variety), considered J. Regia frequently, and the J. sigillata native to Yunnan province. In our results, the three groups clustered as a monomorphic clade composed the Juglans section. They did not diverge into a new species, thus matching the result of China's ecotype distributions from a morphology study .
The structures of the numbered accessions were all examples of standard folding. The typical accessions were collected from Ili county (collection location: latitude 43° 28'N, longitude 82° 08'E) and were considered wild walnut. From the results of Xi , the Ili county wild walnut was a remnant of the later Tertiary, and the wild walnut were the immediate ancestors to the cultivar in Central Asia. From the result of Chang , the temperate mixed conifer-broadleaf forest during the Oligocene was widely distributed in the Tianshan Mountain area Juglans existed in it. N.J. analyses positioned the wild walnut and mixed cultivar together. Therefore, the monomorphic clade result could not validate the direct ancestral relationship to the present cultivar variety. Meanwhile, the monomorphic analysis supported the results of Aradhya , which stated that the Juglans section was an ancient lineage, and the entire lineage of the Juglans section perhaps evolved in isolation in Eurasia from ancestral forms because of the scarcity of stratigraphic data from the places, where the historical distribution of ancestral forms and the domestication were known to have existed.
The palynology evidence in China supported the results of Aradhya , which stated the Juglans section evolved in isolation in Eurasia and from ancestry forms, such as in Southern and Southwestern China; J. Regia pollen existed during the later Eocene to early Oligocene; and Qaidam Basin and Tarim Basin discovered J.regia fossil pollen grains dating from the early Oligocene period . In the Tuotuo River area in the Tanggula Mountains, Juglans pollinates during the later Eocene to the early Oligocene were reported by Duan . The J. sigillata, with many primitive nut characteristics, is widely distributed in Yunnan and Tibet province. Comparing the J. sigillata position to the surrounding of cultivar and wild walnut (bootstrap 94%) showed that the J. sigillata species might be more closely related to the ancestor of the Juglans section. In the Carya genus, four populations that were clustered into two groups were distributed in the Old World (margin bootstrap 97%) (C.cathayensis + C.tonkinensis) and two in the New World (bootstrap 69%) (C. glabra + C. myristiciformis). Compared to the structures of the two Carya clades' accessions, the accession from the New world folded the lowest free energy structures were unique two-helix structures. In contrast, the accession from the Old world folded the special 3-helices structure and did not have the obvious U-U mismatch at the 5' sequence.
In conclusion, the ITS2 sequence was a double-edged tool for comparing low levels of taxonomic phylogeny relationships. The phylogeny tree from the sequence alignment and parsimony-informative sites, another is the conserved ITS2 typical secondary structure prevalent in the Juglandaceae family, and further comparisons of the structure are the powerful supplement to the sequence alignment results. The following conclusions can be formed from this work. (1) In the Juglandaceae, the 3-helices and 4-helices structures are popular among all genera except the Platycarya genus. (2) The ITS2 structure results were arrested for the disjunction of the Cardiocaryon Dode (Asian butternuts) and the Trachycaryon Dode (American butternut). The clustering and structural features of the butternuts completely supported the close disjunction relationship between Cardiocaryon and the Trachycaryon. (3) The structure of J. hopeiensis supplied the direct evidence of the formal characteristics for taxonomic classification and therefore strengthened the clustering results to the Cardiocaryon section.
The ITS2 sequence of walnut in the Supplemental Table will be submitted on NCBI, the submission will be completed before final acceptance, and the accession numbers will be supplied once available.