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\begin{document}

\title{Roegneria yenchiana: a new species in the Triticeae (Poaceae) from the
Hengduan Mountain Region}



\author[1]{Li-Na Sha}%
\author[2]{Xiao Liang}%
\author[2]{Xin-Yi Zhang}%
\author[2]{Shan Gao}%
\author[2]{Yue Zhang}%
\author[2]{Hong Zhou}%
\author[2]{Xing Fan}%
\affil[1]{Sichuan Agricultural University}%
\affil[2]{Sichuan Agricultural University - Chengdu Campus}%


\vspace{-1em}



  
  \date{May 24, 2024}


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\selectlanguage{english}
\begin{abstract}
Roegneria yenchiana sp. nov. (Triticeae) is described as a new species
collected from Shangri-la of Yunnan Province in China based on
morphological, cytological, and molecular data. It is morphologically
characterized by one spikelet per node, rectangular glums, awns flanked
by two short mucros in lemmas, which is distinguished from other species
of Roegneria. The genomic in situ hybridization results indicate that R.
yenchiana is an allotetraploid, and its genomic constitution is StY.
Phylogenetic analyses based on multiple loci suggested that R. yenchiana
is closely related to Pseudoroegneria and Roegneria, and the
Pseudoroegneria served as the maternal donors during its polyploid
speciation.%
\end{abstract}%



\sloppy


\textbf{1 Introduction}

\emph{Roegneria} C, Koch was erected and circumscribed by Koch (1848)
according to \emph{Roegneria caucasica} C. Koch as the type species.
Nevski (1934) played a major role in popularizing the
genus\emph{Roegneria} , which he recognized distinct from
\emph{Agropyron}Gaertner as an independent genus and described 49
species of\emph{Roegneria} . Tzvelev (1976) and Chen et al., (2006)
included\emph{Roegneria} in \emph{Elymus} because of their many commonly
morphological characters, e.g., plants usually tufted; lemma
lanceolate-oblong, rounded abaxially, 5-veined, veins connivent at apex.
Despite the criteria used, there is morphological disagreement over
circumscription of \emph{Elymus} and \emph{Roegneria} ,
and\emph{Roegneria} is distinguished from \emph{Elymus} by single
spikelet per rachis node with lanceolate glumes and short and broader
palea as well as glume retention after caryopsis abscission. Based on
these morphological disagreements as a guide to resolve taxonomic
treatment, Baum et al. (1991) recognized only one genus. Cytogenetics
and molecular phylogeny provided additional support for the
\emph{Roegneria} as an independent genus (Lu et al., 1988; Yen et al.,
2011; Fan et al., 2013), corresponding to genome system of
classification in Triticeae. Since then, \emph{Roegneria} is a large
genus in the wheat tribe, Triticeae (Poaceae), which includes
approximately 120 species that are of Asian origin and distributed in
the Qinghai--Tibetan Plateau, Central Asia, East Asia, West Asia and
South-east Europe (Baum et al., 1995; Yen \& Yang, 2011). The largest
number is found mainly in the mountains of the Qinghai--Tibetan Plateau,
with 33 species being recorded. Most species of \emph{Roegneria} can
usually be recognized in the field by a combination of characters: (a)
lack of underground rhizomes; (b) spikes often slightly curved to erect,
with arrangement of spikelets nearly parallel to and appressed to rachis
and only one per node, with long rachis internodes; (c) spikelets
functionally disarticulating above the glumes (Baum et al., 1995).
Cytologically, all the species of\emph{Roegneria} are allotetraploids
with \textbf{StY} genomes (Yen \& Yang, 2011). The \textbf{St} genome is
derived from\emph{Pseudoroegneria} (Nevski) \selectlanguage{ngerman}Á. Löve (Löve, 1984; Wang et
al., 1994). It is unknown where the \textbf{Y} genome originates,
although it is a fundamental \emph{Roegneria} genome (Yen \& Yang,
2011). Dewey (1984) considered that the \textbf{Y} genome has its origin
in Central Asia or the Himalaya region, and may be extinct. Extensive
cytogenetic and molecular studies have suggested that the
\textbf{St}(\emph{Pseudoroegneria} ), \textbf{W} {[}\emph{Australopyrum}
(Tzvelev) Á. Löve{]}, \textbf{V} {[}\emph{Dasypyrum villosum} (L.)
Candargy{]}, and \textbf{Xp} {[}\emph{Peridictyon} O. Seberg, S.
Frederiksen \& C. Baden{]} genomes are potential donors of the
\textbf{Y} genome (Liu et al., 2006; Sun \& Komatsuda, 2010; Fan et al.,
2013; Lei et al., 2022; Liu et al., 2022).

Here, we described a new species of \emph{Roegneria} that was discovered
from the Hengduan Mountain Region during an expedition to the
Qinghai-Tibetan plateau in 2021 to collect germplasm of various members
of the Triticeae. The new species was discovered in stony slope of Haba
Snow Mountain with about 3200m and 2500m altitude. A few weeks later, it
was collected in stony slope of Yulong Snow Mountain with approximately
3200m altitude. The two localities are more than 90 km apart. This study
conducted on morphological observations, sampling of multiple
accessions, genomic in situ hybridization (GISH), and phylogenetic
reconstruction methods. Our goal was to determine whether this was the
case as a new species within \emph{Roegneria} and, if so, to determine
the genomic constitution of the new species and provide it with an
appropriate name.

\textbf{2 Materials and Methods}

\textbf{2.1 Plant sampling}

Three accessions of the new species were sampled, with each accession
representing one sample point based on distinct locality and/or altitude
(Table S1). Twelve \textbf{StY} genome species from the
genus\emph{Roegneria} and 33 diploid taxa representing 19 basic genomes
in Triticeae were included in this study, and their DNA sequences for
phylogenetic analyses were obtained from published data (Huang et al.,
2002; Mason-Gamer, 2004; Fan et al., 2013; Lei et al., 2022) (Table S1).
The plants and voucher specimens are deposited at Herbarium of Triticeae
Research Institute, Sichuan Agricultural University, China (SAUTI).

\textbf{2.2 Morphological observation}

Morphological features including underground rhizomes, stalks, cauline
internodes, leaf auricles and blades, glumes, lemmas, paleas, awns, and
anthers were observed. At least 20 measurements on fresh material were
performed on each morphological variable by using a stereomicroscope
(Olympus SZX7, Tokyo, Japan) with a digital camera.

\textbf{2.3 Genomic in situ hybridization (GISH)}

Roots of the new species were collected from germinating seeds and adult
plants, treated with nitrous oxide for 2.5 h and fixed in 90\% glacial
acetic acid for 5 min. Chromosome preparations were performed using drop
methods according to Tan et al. (2021). The genomic DNA was isolated
from fresh leaves by the CTAB method and then labelled with the DIG-Nick
Translation Kit (Roche, Indianapolis, IN, USA). \emph{Roegneria
ciliaris} with the \textbf{StY} genomic constitution was used as a
probe. The hybridization procedure, detection and visualization methods
follow those described by Tan et al. (2021). Images were captured with
an Olympus BX51 fluorescence microscope (Japan).

\textbf{2.4 Phylogenetic analysis}

Phylogenetic analysis is routinely applied to illustrate evolutionary
and taxonomic questions. We carried out phylogenetic analysis for the
new species and its affinitive species within the Triticeae based on
three unlinked single-copy nuclear genes (\emph{Acc1} , plastid
Acetyl-CoA carboxylase; \emph{DMC1} , disrupted meiotic
cDNA;\emph{GBSSI} , Granule-Bound Starch Synthase I) and three
chloroplast regions {[}\emph{trn} L-F, \emph{trn} L (UAA)-\emph{trn} F
(GAA);\emph{mat} K, maturase coding gene; \emph{rbc} L, ribulose-1,
5-bisphosphate carboxylase/oxygenase{]}. Prior to phylogenetic analysis,
The \emph{Acc1} , \emph{DMC1} , \emph{GBSSI} , \emph{trn} L-F,
\emph{mat} K, and \emph{rbc} L sequences were amplified by polymerase
chain reaction (PCR) using the primers listed in Table S2 under cycling
conditions reported previously (Sha et al., 2016; Sha et al., 2017), and
PCR products were cloned into the pMD18-T vector (TaKaRa, Dalian, China)
following the manufacture's instruction. At least 10 random independent
clones were selected for commercially sequencing. For each gene
fragment, in cases when multiple identical sequences resulted from
cloned PCR products of each accession, only one sequence was included in
the data matrix.

Multiple sequence alignment was conducted using ClustalX (Thompson et
al., 1999), with default parameters and additional manual edits to
minimize gaps. Phylogenetic analyses were conducted using Maximum
likelihood (ML) and Bayesian inference (BI). ML analysis was performed
using RAxML v8.2.8 under the GTR + GAMMA model on the XSEDE
supercomputer at the CIPRES Science Gateway platform (Miller et al.,
2010). Analyses included inference of the `best tree' and generation of
1,000 bootstrap replicates to obtain node support measures. BI analysis
was conducted with MrBayes v3.2.7a under the same evolutionary model and
supercomputer platform (Miller et al., 2010) as ML analysis. Four MCMC
(Markov Chain Monte Carlo) chains were run for 2,000,000 generations.
Trees were sampled every 1,000 generation until reaching the convergence
parameters (standard deviation less than 0.01). The first 25\% of
generated trees representing the burn-in phase were discarded, and the
remaining trees were used to construct the 50\%-majority rule consensus
trees. The statistical confidence in nodes was evaluated by posterior
probabilities (PP). PP-value less than 90\% was not included in figures.

\textbf{3 Results}

\textbf{3.1 Morphological characters}

\emph{Roegneria yenchiana} is morphologically recognized as a species
of\emph{Roegneria} by the typical characters that have traditionally one
spikelets per node and spikelets with functionally disarticulating above
the glumes. This species is distinguished from other species
of\emph{Roegneria} by its rudimentary spikelets at the tortuous
inflorescence base, rectangular glumes, and awns flanked by two short
mucros in lemmas. \emph{Roegneria ciliaris} has the \textbf{StY} genomes
and is morphologically the most similar species to \emph{R.
yenchiana}occurring in the Hengduan Mountain region (Lu, 1992). Despite
this,\emph{R. ciliaris} has not been observed near to the place
where\emph{R. yenchiana} grows. \emph{Roegneria yenchiana} can be
distinguished from \emph{R. ciliaris} in several traits, including glume
length, ciliates in lemma margin, lemma back, and the paleas length.

\textbf{3.2 GISH}

GISH was carried out with the \textbf{StY} genome species
(from\emph{Roegneria ciliaris} ) being used as a probe. GISH results
showed that \emph{R. yenchiana} has 28 chromosomes, and all the
chromosomes displayed \textbf{StY} genome hybridization signal along
their entire chromosomal length (Figure 2E). Thus, the genomic
constitution of\emph{R. yenchiana} is \textbf{StStYY} .

\textbf{3.3 Phylogenetic analyses}

Three unlinked low-copy nuclear gene (\emph{Acc} 1, \emph{DMC} 1,
and\emph{GBSSI} ) and three chloroplast region (\emph{trn} L-F,
\emph{mat} K, and \emph{rbc} L) sequences were separately amplified and
sequenced for all the accessions of \emph{R. yenchiana} . They were then
analyzed with published \textbf{St} - and \textbf{Y} -type sequences
from twelve species of \emph{Roegneria} and those from 33 diploid taxa
representing 19 basic genomes in Triticeae. Consequently, six
homoeologous sequences representing two distinct types (\textbf{St} -
and \textbf{Y} -type) of each nuclear gene and three sequences of each
chloroplast region were detected from all the accession of \emph{R.
yenchiana} . Four datasets, including \emph{Acc1} data, \emph{DMC1}
data, \emph{GBSSI} data, and combined chloroplast (\emph{trn} L-F +
\emph{mat} K + \emph{rbc} L) data, were used to conduct separately
alignments and phylogenetic analyses.

The features of \emph{Acc1} data, \emph{DMC1} data, \emph{GBSSI} data,
and combined chloroplast data were summarized in Table S3. The
aligned\emph{Acc1} sequences yielded a total of 1400 characters of which
411 variable characters and 201 were informative. ML analysis of
the\emph{Acc1} data yielded a single phylogenetic tree (-Lnlikelihood =
5890.5424). ML and Bayesian analyses of the \emph{Acc1} data recovered
the same topology. The tree illustrated in Figure 3 was the ML tree of
bootstrap support (BS) above and posterior probabilities (PP) below
branches. The \emph{Acc1} sequences of \emph{R. yenchiana} were
represented in two clades, corresponding to the two genomic types
(\textbf{St} and \textbf{Y} ). The \textbf{St} -type \emph{Acc1}
sequences of \emph{R. yenchiana} were in one monophyletic group and then
formed a paraphyletic grade with those St-type sequences from
other\emph{Roegneria} and \emph{Pseudoroegneria} . The \textbf{Y}
-type\emph{Acc1} sequences of \emph{R. yenchiana} and the sequence
of\emph{Roegneria} \emph{ciliaris} formed a paraphyletic grade. Of 959
total characters of the \emph{DMC1} data, 343 characters were variable
and 158 characters were informative. ML and Bayesian analyses of
the\emph{DMC1} data recovered the same topology. The ML tree
(-Lnlikelihood = 4907.2580) inferred from the \emph{DMC1} data showed
that the\textbf{St} -type and \textbf{Y} - type sequences from the new
species were split into two well supported clades (Figure S1).
The\textbf{St} -type \emph{DMC1} sequences of \emph{R. yenchiana} were
scattered in different group and clustered with those from different
species of \emph{Roegneria} . The \textbf{Y} -type \emph{DMC1} sequences
of \emph{R. yenchiana} formed one monophyletic group and grouped with
the sequence of \emph{Roegneria brevipes} . In the \emph{GBSSI} sequence
data matrix, of the 1104 total characters, 472 were variable and 277
were parsimony informative. ML and Bayesian analyses of the
\emph{GBSSI}data recovered the same topology. In ML tree (-Lnlikelihood
= 8424.8144) inferred from the \emph{GBSSI} data, the \textbf{St} -type
\emph{GBSSI}sequences of \emph{R. yenchiana} formed one monophyletic
group, and then clustered with one group including \emph{Roegneria
gmelinii} and\emph{Roegneria pendulinus}(Figure S2). The\textbf{Y} -type
\emph{GBSSI} sequences of \emph{R. yenchiana} were in one monophyletic
group and formed a paraphyletic grade with those\textbf{Y} -type
sequences from several species of \emph{Roegneria}(\emph{R.
semicostatus} , \emph{R. anthosachnoides} , and \emph{R. ciliaris} ).
The combined \emph{trn} L-F, \emph{mat} K, and \emph{rbc} L data yielded
a total of 3089 characters of which 306 were variable characters and 132
were informative. ML and Bayesian analyses of the combined chloroplast
data recovered the same topology. In ML tree (-Lnlikelihood = 7088.8370)
inferred from the combined chloroplast data, all the accessions of
\emph{R. yenchiana} formed one monophyletic group, and this group was
clustered with the species from \emph{Roegneria}
and\emph{Pseudoroegneria} (Figure S3).

\textbf{3.4 Taxonomic treatment}

\emph{Roegneria yenchiana} \textbf{X. Fan et L. N. Sha, \emph{sp. nov}}
. (Figures 1 and 2)

TYPE: CHINA, \textbf{Yunnan} , Shangri-la, Tiger Leaping Gorge, in stony
slope of Haba Snow Mountain, 3200m, 11 November 2021, X. Fan \& L.N. Sha
20211127 (holotype: SAUTI; isotype: SAUTI).

3.4.1 Diagnosis

\emph{Roegneria yenchiana} is morphologically the most similar species
to \emph{R. ciliaris} (\textbf{StY} ) but is distinguished from \emph{R.
ciliaris} by its lower glume length (3-5 mm vs. 7-8 mm), ciliates in
lemma margin (absent vs. about 1 mm), lemma back (scabrous or pubescent
on lower parts vs. hispid), and the paleas length (slightly shorter than
lemma vs. 2/3 the length of lemma).

3.4.2 Description

Perennial herb, cespitose; culms usually erect, 50-100 cm tall, purplish
at maturity. Leaf sheaths glabrous; ligule about 0.3-0.6 mm long,
truncate or lacerate; blades flat, 27-40 cm long, 4-6mm wide, glabrous.
Spikes linear, inclined to nodding, purplish at maturity, 10-26cm long
(excluding awns), 5-10 mm wide, 7-16 spikelets per spike; 1 spikelets
per node; rachises scabrous and margin shortly ciliolate; internodes
14-25 mm long; usually 1-4 rudimentary spikelets at the tortuous
inflorescence base. Spikelets 18-25 mm (excluding awns) long, with 6-10
florets, disarticulation above the glumes, beneath each floret. Glums
unequal, rectangular, oblique, margin membranous, usually lower glume
2/3 the length of upper glume, lower glum 3-5 mm, upper glume 5-8 mm,
3-5 prominent veins, sometimes setulose on upper veins, apex acuminate
and with 1 tooth. Lemmas oblong-lanceolate to oblong, scabrous,
pubescent on lower parts, 3 veins, prominent midvein, first lemma 9-12
mm, awned, awns 17-25 mm, reflexed, awns flanked by 2 short teeth with
about 0.5 mm long. Paleas oblong, slightly shorter than lemma, ciliolate
along keels in the distal 1/3, apex truncate. Chromosome number 2n = 4x
=28; genome constitution \textbf{StStYY} .

3.4.3 Phenology

Flowering: July-August and fruiting October-November.

3.4.4 Distribution, habitat, and name

\emph{Roegneria yenchiana} is known only from a few localities in the
northwest of Yunnan, China. It was found among bushes or on stony
mountain slopes between 2500 and 3500 m growing together with
\emph{Campeiostachys nutans} (Griseb.) J. L. Yang, B. R. Baum et C. Yen
and\emph{Campeiostachys dahurica} var. \emph{cylindrica} (Franch.) J. L.
Yang, B. R. Baum et C. Yen. This two species were previously included
into the genus \emph{Elymus} . According to the genome system of
classification, they were transferred into the
genus\emph{Campeiostachys} because of their \textbf{StYH} genome
constitutions.

\emph{Roegneria yenchiana} is named for commemorating Prof. Yen Chi, a
biosystematic scientist of the Triticeae from the Triticeae Research
Institute of Sichuan Agricultural University (SAU), China. He was
evaluated as an important person in the taxonomic research of the
Triticeae by Chairman Prof. Roland von Bothmer in eighth international
Triticeae symposium.

\textbf{4 Discussion}

The morphological characters of \emph{R. yenchiana} differ from all
species previously described in \emph{Roegneria} , especially in its
rudimentary spikelets at the inflorescence base, rectangular glumes, and
two short mucros in lemmas. GISH conformed that \emph{R. yenchicata} is
a tetraploid (2n=4x=28) with the \textbf{StY} genome constitution.
Phylogenetic analysis based on all the three unlinked single-copy
sequences showed that the \textbf{St} -genome homoeologous types
of\emph{R. yenchiana} were grouped with the sequences
of\emph{Pseudoroegneria} and \emph{Roegneria} , and the \textbf{Y}
-genome homoeologous types of \emph{R. yenchiana} were clustered with
the sequences of \emph{Roegneria} with high statistic supports (BS
\textgreater{} 70\% and PP \textgreater{} 0.9). In the phylogenetic tree
inferred from the combined chloroplast sequences, all the three
accessions of \emph{R. yenchiana} were grouped
with\emph{Pseudoroegneria} and \emph{Roegneria} (BS = 81\% and PP =
1.0)\emph{.} These results indicated that \emph{Pseudoroegneria}
and\emph{Roegneria} are closely related to \emph{R. yenchiana} .
Combined with GISH analysis, it can be concluded that the \textbf{St}
genome of\emph{R. yenchiana} is derived from the genus
\emph{Pseudoroegneria} , and the \emph{Pseudoroegneria} served as the
maternal donors during its polyploid speciation.

\textbf{Acknowledgements}

This work was funded by the Second Tibetan Plateau Scientific Expedition
and Research Program (STEP) (No. 2019QZKK0303) and the National Natural
Science Foundation of China (Nos. 31870360 and 32171603).

\textbf{Data Accessibilty Statement}

The sequences used in this study have been deposited in The National
Center for Biotechnology Information (NCBI) database. GenBank accession
numbers are provided in supplementary file Table S1.

\textbf{Funding Information}

National Natural Science Foundation of China, Grant/Award Number:
31870360 \& 32171603; the Second Tibetan Plateau Scientific Expedition
and Research Program (STEP), Grant/Award Number: 2019QZKK0303.

\textbf{Competing Interests Statement}

The authors declare no conflicts of interest

\textbf{Author Contributions}

Li-Na Sha: Conceptualization (equal); writing original draft (lead);
writing review and editing (equal). Xiao Liang: Investigation (lead);
methodology (equal). Xin-Yi Zhang: Investigation (equal); methodology
(equal). Shan Gao: Investigation (equal); methodology (equal). Yue
Zhang: Investigation (equal); methodology (equal). Yong-Hong Zhou:
Conceptualization (equal); writing review and editing (equal). Xing Fan:
Conceptualization (equal); writing review and editing (equal); funding
acquisition (lead).

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\textbf{Figure Legends}

Figure 1. The holotype of the new species \emph{Roegneria yenchiana} X.
Fan et L. N. Sha.

Figure 2. Features of \emph{Roegneria yenchiana} . (A) Inflorescences;
(B) Glumes; (C) Lemmas; (D) Palea; (E) Genomic in situ hybridization
photographs of mitotic metaphase in \emph{Roegneria yenchiana} display
red fluorescence when probed with \emph{R. ciliaris} ,
indicating\emph{R. yenchiana} has the \textbf{StY} genomes.

Figure 3. Phylogenetic tree inferred from the \emph{Acc1} sequences
of\emph{Roegneria yenchiana} and its affinitive species within the
Triticeae. Numbers above nodes are bootstrap support values \selectlanguage{english}[?] 50\%, and
numbers below nodes are Bayesian posterior probability values \selectlanguage{english}[?] 90\%.
Polyploid species names are followed by GenBank numbers. The upper case
letters in parentheses indicate the genome type of the species.

\textbf{Supplement data}

Table S1. List of taxa used in this study.

Table S2. Names, sequences, and references of primers used in this
study.

Table S3. Features of the four matched data sets and their trees

Figure S1. Phylogenetic tree inferred from the \emph{DMC1} sequences
of\emph{Roegneria yenchiana} and its affinitive species within the
Triticeae. Numbers above nodes are bootstrap support values \selectlanguage{english}[?] 50\%, and
numbers below nodes are Bayesian posterior probability values \selectlanguage{english}[?] 90\%.
Polyploid species names are followed by GenBank numbers. The upper case
letters in parentheses indicate the genome type of the species.

Figure S2. Phylogenetic tree inferred from the \emph{GBSSI} sequences
of\emph{Roegneria yenchiana} and its affinitive species within the
Triticeae. Numbers above nodes are bootstrap support values \selectlanguage{english}[?] 50\%, and
numbers below nodes are Bayesian posterior probability values \selectlanguage{english}[?] 90\%.
Polyploid species names are followed by GenBank numbers. The upper case
letters in parentheses indicate the genome type of the species.

Figure S3. Phylogenetic tree inferred from the combined \emph{trn}
L-F,\emph{mat} K, and \emph{rbc} L sequences of \emph{Roegneria
yenchiana} and its affinitive species within the Triticeae. Numbers
above nodes are bootstrap support values \selectlanguage{english}[?] 50\%, and numbers below nodes
are Bayesian posterior probability values \selectlanguage{english}[?] 90\%. Polyploid species
names are followed by GenBank numbers. The upper case letters in
parentheses indicate the genome type of the species.\selectlanguage{english}
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