A new natural hybrid of Iris (Iridaceae) from Chongqing, China

Abstract A newly discovered natural hybrid, Iris×ampliflora Y.E. Xiao, F.Y. Yu & X.F. Chen (Iridaceae: subgenus LimnirissectionLophiris) from Chongqing, China, is described and illustrated. This hybrid is morphologically similar to I.japonica Thunb. and I.wattii Baker, but can be distinguished by its giant leaves and large purple flowers. Phylogenetic trees based on cpDNA data support the separation of I.×ampliflora from other closely related species in the section Lophiris. According to its morphological features, molecular systematic evidence and chromosome data, we speculate that I.×ampliflora [31 chromosomes] likely is a new hybrid between I.japonica [2n = 32] and I.wattii [2n = 30].


Introduction
Iris L. is the largest genus in family Iridaceae with up to 280 species that are mainly distributed in temperate regions of the Northern Hemisphere (Goldblatt and Manning 2008). The characteristics of iris flowers are petaloid-style branches, two obvious perianth whorls, and floral tubes with nectaries (Goldblatt and Manning 2008). Irises produce showy flowers and are well-known and popular ornamental plants worldwide. To gain more cultivars for gardens, many crosses between Iris species have been attempted and achieved success by artificial hybridization (Hu and Xiao 2012;Yu et al. 2017). Natural hybrids between sibling species do occur in the genus Iris, for example in some species within subgen. Limniris in North America (Burke et al. 1998). However, there is no natural hybrid of Iris reported in East Asia.
The outer sepals of irises are equipped with raised beards, crests, signal patches, and midveins, which probably have significance for pollination (Sapir et al. 2005;Guan et al. 2009;Xiao et al. 2019). The sepal crest is an important character in the taxonomic delineation of higher ranks of Iris. Dykes (1913) first placed six rhizomatous species in sect. Evansia. Lawrence (1953) changed sect. Evansia to the subsection level. Rodionenko (1987) elevated subsect. Evansia to subgen. Crossiris. Mathew (1981) elevated subsect. Evansia of Lawrence to section level, namely sect. Lophiris, placing the section within subgen. Limniris. Sect. Lophiris contains 11 species, most of which are distributed in Eastern Asia, apart from three species in North America (I. cristata Solander, I. lacustris Nuttall, and I. tenuis S. Watson) (British Iris Society Species Group 1997; Zhao et al. 2000). Mathew's classification was used in this study.
During field work in Chongqing, we found an interesting specimen originally from the Qingyang Town in Fuling District, Chongqing City, China. Our observations show that it is morphologically similar to I. japonica Thunb. and I. wattii Baker with a yellow and irregularly toothed crest on the outer segments. However, its morphological features differ markedly from those of all known species in sect. Lophiris described by Mathew (1981). We have observed the situation concerning seed set since 2014 when the species was initially introduced to the conservation nursery of Shanghai Botanical Garden. It is sterile with no seed production, which indicates its hybrid origin.
This study was undertaken to assess the status and parentage of the new hybrid by morphological surveys, phylogenetic and chromosome data. The Qingyang collection is a large evergreen plant with large attractive flowers, and it can adapt to warm, wet, and full-sun or partly shady environmental conditions. It has potential uses in breeding and landscaping. We formally publish its description here with the aim of better understanding and utilization of the remarkable morphological divergence of Iris species in subgenus Limniris sect. Lophiris.

Morphological surveys
Living specimens and vouchers of the new hybrid were examined and compared with four species of sect. Lophiris (I. confusa Sealy, I. japonica Thunb., I. tectorum Maxim. and I. wattii Baker) in Southern China using measurements and descriptions of the main characteristics. Species from the conservation nursery of Shanghai Botanical Garden from mid-March and late-April in 2019 were compared. Eight or ten randomly chosen individuals of each taxon were used for the morphometric surveys (Table 1). Meanwhile, herbarium sheets (CDBI, CSH, IMC, IBSC, LBG, KUN, PE) and type descriptions of I. confusa, I. japonica, I. milesii Baker ex Foster, I. tectorum and I. wattii were compared with the new hybrid.

Phylogenetic analyses
We collected samples of five species / hybrid (I. confusa, I. japonica, I. tectorum, I. wattii, and Qingyang collection) of sect. Lophiris, and three species (I. anguifuga Y.T. Zhao, I. henryi Baker and I. proantha Diels) of section Limniris and Iris domestica (L.) Goldblatt & Mabb. to construct phylogenetic trees based on cpDNA data. The samples of the new hybrid were collected from the type locality. Other iris samples were collected from the conservation nursery of the Shanghai Botanical Garden.
Total genomic DNA was extracted from each sample (30 mg dried leaves) using a DNA Plant Kit (Tiangen, Shanghai, China) according to manufacturer's instructions. The extracted DNAs were dissolved in 100 μl TE buffer for storage. We amplified and sequenced part of the matK gene (Wilson 2004) and ndhF gene (Shaw et al. 2007) ( Table 2). Each PCR mixture (50 μl) contained ddH 2 O, 1 × buffer (Mg 2+ CCCCCTA(CT)ATATTTGATACCTTCTCC free), 2.5 mM MgCl 2 , 2.5 mM each dNTP, 0.5 μM each primer, 2 U Taq polymerase (Sangon, Shanghai, China), and 20 ng DNA. The PCR reactions were conducted on a Mastercycler pro Thermal Cycler (Eppendorf, Hamburg, Germany). The procedure was performed with initial denaturing for 5 min at 94 °C followed by 35 cycles of 30 sec at 94 °C (denaturing), 30 sec at 50 °C (annealing), and 45 sec at 72 °C (elongation), and final extension for 10 min at 72 °C. After checking products by electrophoresis on a 1.2% agarose gel, the purified products were bi-directionally sequenced by standard methods on the ABI 3731 automated sequencer (Applied Biosystems, Foster City, CA, USA). The sequences of these irises have been deposited in GenBank (see accession numbers in Appendix 1: Table A1. For the cpDNA datasets, the CLUSTALW program combined with manual adjustment was used for multiple alignments of all sequences (Thompson et al. 1994). We used three phylogenetic methods (i.e., Bayesian inference, maximum parsimony, and maximum likelihood) to analyze the alignments. We conducted the Bayesian inference analysis with MrBayes v.3.2.6 (Ronquist and Huelsenbeck 2003), and the maximum parsimony and maximum likelihood analyses using PAUP v.4.0b10 (Swofford 2002).

Chromosome number analyses
To determine the chromosome number of the new hybrid from somatic cells. Root tips (1-1.5 cm in length) were collected and washed with distilled water and immersed in 0.002 mol/L 8-hydroxyquinoline with dark pretreatment for 2-2.5 h. These roots then were fixed in Carnoy solution (volume ratio: 95% ethanol: acetic acid = 3:1) at 4 °C for 2-3 h. The fixed roots were dissociated in 5 mol/L hydrochloric acid for 8-10 min and washed with distilled water, then stained with carbol fuchsin and squashed on glass slides. Finally, the samples were observed and photographed using a Motic BA400 optical microscope. More than 20 cells were observed to determine the number of chromosomes for each specimen examined.

Morphological comparisons
The Qingyang collection is morphologically similar to the species of section Lophiris, I. japonica which has 5-10 branches of the flowering stem, a yellow crest on the outer sepals, and is the most common species of Iris in Southern China (Zhao et al. 2000). However, the Qingyang collection has several characteristics that distinguish it from I. japonica, I. confusa, I. milesii, I. wattii and I. tectorum, including plant larger flowers, larger leaves, and other features as described in Table 1. The flowering stem of this specimen has 7-10 branches and an aerial stem (mean length = 27.0 ± 2.7 cm, n = 10) (Fig. 1). Compared with other species in sect. Lophiris, the Qingyang collection has larger leaves (mean length = 74.2 ± 6.4 cm, n = 10; mean width = 6.6 ± 0.8 cm, n = 10) and larger purple flowers (mean diameter = 12.5 ± 0.5 cm, n = 10) (Fig. 2).

Phylogenetics
Among eight individuals of different species / hybrid, there were 318 variable sites, 187 singleton variable sites, and 131 parsimony informative sites across 4779 bp aligned positions of two cpDNA fragments. There were 22 and 39 mutations between the sequences of I. japonica / I. wattii and the new hybrid, respectively. In the molecular tree based on cpDNA data, the sampled new hybrid was resolved as sister to the sample of I. japonica (Fig. 3). In Bayesian, maximum parsimony, and maximum likelihood trees based on cpDNA data, this specimen clustered into a clade with I. japonica (Fig. 3). The inter-clade sequence difference (I. japonica vs. I. wattii, 0.11) was 22 times greater than the intra-clade sequence differences (the new hybrid vs. I. japonica, 0.05).

Chromosome number
Cytological study indicated that the Qingyang collection has 31 chromosomes (Fig. 4). No evidence of chromosome pairing could be examined and it is unknown if it functions as a diploid.
Preliminary conservation status. Only one population of I. × ampliflora was found by our investigation in Qingyang Region and there are risks of disturbance by human activities. I. × ampliflora is currently cultivated in the conservation nurseries of Shanghai Botanical Garden and the Flower Fragrance Horticulture Limited Company in Chongqing. Other
Furthermore, the parents of I. × ampliflora can be deduced according to morphological features. The hybrid has aerial stems (mean length = 27.0 ± 2.7 cm, n = 10). Without an aerial stem, I. tectorum cannot be the paternal parent. Iris wattii and I. confusa both have aerial stems (I. wattii, mean length = 67.2 ± 19.9 cm, n = 10; I. confusa, mean length = 30.3 ± 9.3 cm, n = 10). However, the leaf surface of I. × ampliflora has no waxy coat; it is dull and ruffled similar to that of I. wattii. The leaf surfaces of I. confusa and I. japonica have a waxy coat, glossy and smooth. Thus, compared with I. confusa, I. wattii is possibly more likely to be the paternal parent of I. × ampliflora.
Though I. japonica and I. × ampliflora are clustered into one clade, there are 22 mutations between the sequences of these two species. Compared with leaves (length 45-51 cm and width 3-4.5 cm) and flowers (diam. 4-5 cm) of I. japonica, I. × ampliflora has larger leaves (length 68-80 cm and width 6-7 cm) and larger purple flowers (diam. 11-13 cm). It cannot be determined which population of I. japonica it is derived from for the maternal parent since intraspecific chromosome numbers are variable. Thus, the sequence divergence could not reflect the real evolutionary distance between I. japonica and I. × ampliflora. The evolution of I. × ampliflora is in need of further study. Figure A1. The photo of holotype of Iris × ampliflora.