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J
SE Journal of Systematics
and Evolution doi: 10.1111/jse.12721
Research Article
Targeted sequencing supports morphology and embryo
features in resolving the classification of Cyperaceae tribe
Fuireneae s.l.
Julian R. Starr
1
*, Pedro Jiménez‐Mejías
2,3
, Alexandre R. Zuntini
4
, Étienne Léveillé‐Bourret
5
, Ilias Semmouri
6
,
Muthama Muasya
7
, William J. Baker
4
, Grace E. Brewer
4
, Niroshini Epitawalage
4
, Isabel Fairlie
4,8
, Félix Forest
4
,
Izai A. B. Sabino Kikuchi
4,9
, Lisa Pokorny
4
, and Isabel Larridon
4,10
1
Department of Biology, University of Ottawa, Ottawa, ON K1N 6N5, Canada
2
Departamento de Biología (Botánica), Facultad de Ciencias Biológicas, Universidad Autónoma de Madrid, C/Darwin, 2, Madrid 28049, Spain
3
Centro de Investigación en Biodiversidad y Cambio Global (CIBC‐UAM), Universidad Autónoma de Madrid, Madrid 28049, Spain
4
Royal Botanic Gardens, Kew, Richmond, Surrey TW9 3AE, UK
5
Institut de recherche en biologie végétale (IRBV), Université de Montréal, Montréal, QC H1X 2B2, Canada
6
Laboratory of Environmental Toxicology and Aquatic Ecology, Faculty of Bioscience Engineering, Ghent University, Ghent 9000, Belgium
7
Department of Biological Sciences, Bolus Herbarium, University of Cape Town, Private Bag X3, Rondebosch, Cape Town 7701, South Africa
8
Department of Animal and Plant Sciences, University of Sheffield, Alfred Denny Building, Western Bank, Sheffield S10 2TN, UK
9
Hortus botanicus Leiden, Universiteit Leiden, PO Box 9500, Leiden 2300 RA, The Netherlands
10
Systematic and Evolutionary Botany Lab, Department of Biology, Ghent University, K.L. Ledeganckstraat 35, Gent 9000, Belgium
Current address: Lisa Pokorny, Centre for Plant Biotechnology and Genomics (CBGP, UPM‐INIA), 28223 Pozuelo de Alarcón, Madrid, Spain.
*Author for correspondence. E‐mail: jstarr@uottawa.ca
Received 31 August 2020; Accepted 3 December 2020; Article first published online 14 January 2021
Abstract Molecular phylogenetic studies based on Sanger sequences have shown that Cyperaceae tribe Fuireneae s.l. is
paraphyletic. However, taxonomic sampling in these studies has been poor, topologies have been inconsistent, and
support for the backbone of trees has been weak. Moreover, uncertainty still surrounds the morphological limits of
Schoenoplectiella, a genus of mainly small, amphicarpic annuals that was recently segregated from Schoenoplectus.
Consequently, despite ample evidence from molecular analyses that Fuireneae s.l. might consist of two to four tribal
lineages, no taxonomic changes have yet been made. Here, we use the Angiosperms353 enrichment panel for targeted
sequencing to (i) clarify the relationships of Fuireneae s.l. with the related tribes Abildgaardieae, Eleocharideae, and
Cypereae; (ii) define the limits of Fuireneae s.s., and (iii) test the monophyly of Fuireneae s.l. genera with emphasis on
Schoenoplectus and Schoenoplectiella. Using more than a third of Fuireneae s.l. diversity, our phylogenomic analyses
strongly support six genera and four major Fuireneae s.l. clades that we recognize as tribes: Bolboschoeneae stat.nov.,
Fuireneae s.s., Schoenoplecteae, and Pseudoschoeneae tr. nov. These results are consistent with morphological,
micromorphological (nutlet epidermal cell shape), and embryo differences detected for each tribe. At the generic level,
most sub‐Saharan African perennials currently treated in Schoenoplectus are transferred to Schoenoplectiella.Our
targeted sequencing results show that these species are nested in Schoenoplectiella, and their treatment here is
consistent with micromorphological and embryo characters shared by all Schoenoplectiella species. Keys to recognized
tribes and genera are provided.
Key words: Angiosperms353, classification, Cyperaceae, Fuireneae, targeted sequencing, taxon limits, taxonomy.
1 Introduction
Since the first molecular phylogenies of sedges (Cyperaceae)
over 20 years ago, our knowledge of relationships within the
family has improved at an ever‐astonishing pace. Initially,
these analyses made use of just a few plastid markers (e.g.,
rbcL,ndhF,trnL‐F; Muasya et al., 1998; Yen & Olmstead, 2000)
and nuclear markers (e.g., ITS; Starr et al., 1999), but they
immediately confirmed the suspicions of many authors on
the basis of differences in macro‐and micromorphological
July 2021
|
Volume 59
|
Issue 4
|
809–832 © 2021 The Authors. Journal of Systematics and Evolution published
by John Wiley & Sons Australia, Ltd on behalf of Institute of Botany,
Chinese Academy of Sciences
This is an open access article under the terms of the Creative Commons Attribution‐NonCommercial License, which permits use,
distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.
features (e.g., Schuyler, 1971; Wilson, 1981; Goetghe-
beur, 1986; Bruhl, 1995), including embryo types (Van der
Veken, 1965; Goetghebeur, 1986), that many generic and
tribal circumscriptions within the family did not reflect their
evolutionary history. Unfortunately, these early molecular
analyses did not have a major impact on family classification
due to poor taxonomic sampling or topologies that were
either insufficiently resolved or inadequately supported to
make lasting taxonomic changes (e.g., Simpson et al., 2003).
However, as taxonomic sampling increased and more
molecular markers were developed, statistical support for
trees improved, and a series of new genera (e.g.,
Zameioscirpus Dhooge & Goetghebeur, Dhooge et al., 2003;
Calliscirpus C.N.Gilmour, J.R.Starr & Naczi, Gilmour et al., 2013;
Afroscirpoides García‐Madrid & Muasya, García‐Madrid
et al., 2015) was described. Changes to tribal (Léveillé‐
Bourret et al., 2018; Léveillé‐Bourret & Starr, 2019;
Semmouri et al., 2019; Larridon et al., 2021a), generic
(Léveillé‐Bourret et al., 2020), and subgeneric (Villaverde
et al., 2020; Roalson et al., 2021) classifications in the family
are now taking place at a rapid pace, owing in part to
advances in sequencing technology. However, one major
problem has yet to be resolved: the status and classification
of tribe Fuireneae Rchb. ex Fenzl.
Fuireneae s.l. is a cosmopolitan tribe consisting of 153
species assigned to six genera: Bolboschoenus (Asch.) Palla,
Fuirena Rottb., Actinoscirpus (Ohwi) R.W.Haines & Lye,
Pseudoschoenus (C.B.Clarke) Oteng‐Yeb., Schoenoplectus
(Rchb.) Palla, and Schoenoplectiella Lye (Figs. 1, 2). They
are found in tropical to temperate wetlands where they
purify water and provide essential food and habitat for
wildlife(Fassett,1957;Smith&Coops,1991;Kimetal.,2013;
Mishra et al., 2015; Naczi et al., 2018), mitigate flooding, and
even protect coastal shorelines from erosion (Albert
et al., 2013). They are also useful to humans as food,
building materials, and medicines (Simpson & Inglis, 2001);
however, many are among the world's worst weeds,
especially in rice fields (e.g., Bolboschoenus maritimus (L.)
Palla, Schoenoplectiella mucronata (L.) J.Jung & H.K.Choi;
Bryson & Carter, 2008).
Like many sedge genera, all six currently placed in
Fuireneae are segregates of Scirpus L. s.l., a heteroge-
neous entity now corresponding to c. 50 natural genera
and over 250 species (Koyama, 1958; Goetghebeur, 1998).
Among the segregates of Scirpus s.l., Fuireneae s.l. species
are particularly interesting, because they share a series of
morphological and embryo features that seem to mark
them as a natural group. All Fuireneae s.l. species have
terete spikelets with spirally arranged glumes, bisexual
flowers with or without perianth parts, and embryos with a
distinctive mushroom‐like (fungiform) shape. As embryo
characters often provide key features for circumscribing
sedge tribes and genera (e.g., Léveillé‐Bourret &
Starr, 2019; Semmouri et al., 2019), Goetghebeur (1998)
considered these distinctive embryos of the
Bolboschoenus‐or Schoenoplectus‐type as evidence that
Fuireneae s.l. could represent a specialized offshoot within
the family. Nevertheless, from the first molecular
phylogeny of Cyperaceae (Muasya et al., 1998), it was
not only clear that Scirpus s.l. consisted of numerous,
often distantly related genera, as many had suggested
(e.g., Van der Veken, 1965; Schuyler, 1971; Wilson, 1981),
but even Fuireneae s.l. could consist of multiple
independent lineages.
Molecular studies have since retrieved four distinct
lineages within Fuireneae s.l.: (i) a clade comprising species
from the subcosmopolitan genus Bolboschoenus (15 species;
Govaerts et al., 2020); (ii) a clade consisting of the largely
American and African, tropical to warm temperate genus
Fuirena (55 species; Govaerts et al., 2020); (iii) a clade with
the monotypic, (sub)tropical, south Asian–Australasian genus
Actinoscirpus as sister to the cosmopolitan, mainly temperate
genus Schoenoplectus (27 species, hereafter the Schoeno-
plectus Clade; Govaerts et al., 2020), and (iv) the monotypic,
South African endemic genus Pseudoschoenus as sister to the
cosmopolitan, mainly tropical genus Schoenoplectiella (52
species, hereafter the Schoenoplectiella Clade; Govaerts
et al., 2020), which is a segregate of Schoenoplectus
(Lye, 2003). Among the four larger, widespread genera,
Bolboschoenus and Fuirena are easily defined morphologically
and they have always been monophyletic in previous
molecular studies (Muasya et al., 2009a; Escudero &
Hipp, 2013; Hinchliff& Roalson, 2013; Glon et al., 2017;
Semmouri et al., 2019). In contrast, the separation and
relationships of Schoenoplectus and Schoenoplectiella have
not been so clear.
As Scirpus s.l. was progressively dissolved, the focus for
large genera like Schoenoplectus was to provide an
infrageneric classification that reflected evolutionary rela-
tionships while dividing the genus into workable groups
(Oteng‐Yeboah, 1974a; Raynal, 1976a, 1976b; Smith &
Hayasaka, 2001). Authors were basically unanimous that
either three or four major taxonomic divisions adopted from
Scirpus s.l. could be made within Schoenoplectus, either at
the subgeneric level, as subgenera Schoenoplectus,Mala-
cogeton (Ohwi) Oteng‐Yeboah, and Actaeogeton (Reichenb.)
Oteng‐Yeboah, or at the sectional level, as sections
Schoenoplectus (=Scirpus section Pterolepis Pfeiff.), Mala-
cogeton (Ohwi) S.G.Smith & Hayasaka, Actaeogeton
(Reichenb.) J.Raynal, and Supini (Cherm.) J.Raynal
(Raynal, 1976a; Smith & Hayasaka, 2001; Smith & Hay-
asaka, 2002). Moreover, it was clear that when treated as
sections, taxa could be divided into two distinct groups: (i)
sections Actaeogeton and Supini were typically densely
tufted plants with sculptured, black nutlets and entire glume
apices, whereas (ii) sections Schoenoplectus and Malacogeton
were rhizomatous plants with smooth, yellow to dark brown
nutlets and glumes entire to emarginate (Raynal, 1976b;
Smith & Hayasaka, 2001, 2002). Moreover, embryo features
supported this division with the embryo scutellum of
sections Actaeogeton and Supini being umbonate or pileate
like a mushroom cap versus a turbinate to rhomboid
scutellum in sections Schoenoplectus and Malacogeton (Van
der Veken, 1965). When the molecular analysis of Muasya
et al. (1998) placed two small, tropical annuals, S. articulata
(L.) Palla and S. junceus (Willd.) J.Raynal, in a clade separate
from the type for Schoenoplectus,S. lacustris (L.) Palla, Lye
(2003) decided to segregate 26 species into Schoenoplec-
tiella. This new genus was roughly meant to elevate
Schoenoplectus section Supini to generic status, a section
defined by Raynal (1976a) to contain the small, tufted
annuals of Schoenoplectus whose bristles were reduced or
J. Syst. Evol. 59(4): 809–832, 2021 www.jse.ac.cn
810 Starr et al.
absent. In addition, the species of section Supini also stood
out, because beyond sharing other features such as rugulose
to highly sculpted nutlets, most displayed amphicarpy, a rare
condition in Cyperaceae where pistillate flowers at culm
bases develop larger nutlets than the nutlets produced in
aerial inflorescences (Raynal, 1976a; Bruhl, 1994). Nonethe-
less, in a molecular analysis of Korean Scirpus s.l., Jung & Choi
(2010) expanded the genus to include some species from
Scirpus section Actaeogeton, which does not display
amphicarpy, after results suggested they formed a clade
with Schoenoplectiella species separate from Schoenoplectus
s.s. (i.e., sections Schoenoplectus and Malacogeton). Hayasaka
(2012) subsequently redefined Schoenoplectiella to include all
species from sections Actaeogeton and Supini, which resulted
in a genus composed of small, amphicarpic annuals and
large, single‐fruit‐type perennials. Unfortunately, this merger
J. Syst. Evol. 59(4): 809–832, 2021www.jse.ac.cn
Fig. 1. Morphological diversity of Fuireneae s.l. A,Bolboschoenus maritimus (L.) Palla (Asturias, Spain). B,Fuirena pubescens (Poir.)
Kunth (Ávila, Spain). C,Fuirena hirsuta (P.J.Bergius) P.L.Forbes (Cape Region, South Africa). D,F. umbellata Rottb. (Pernambuco, Brazil).
E,Actinoscirpus grossus (L.f.) Goetgh. & D.A.Simpson (Phuket, Thailand). F,Schoenoplectus torreyi (Olney) Palla (Québec, Canada). G,S.
americanus (Pers.) Volkart (Arizona, USA). H,S. tabernaemontani (C.C.Gmel.) Palla (Minnesota, USA). I,S. subulatus (Vahl) Lye
(Australia). J,Pseudoschoenus inanis (Thunb.) Oteng‐Yeb. (Cape Region, South Africa). K,Schoenoplectus corymbosus (Roth ex Roem. &
Schult.) J.Raynal (Huelva, Spain). L,S. corymbosus rooting pseudo‐viviparous inflorescence (Huelva, Spain). M,Schoenoplectus confusus
(N.E.Br.) Lye (Trans‐Nzoia, Kenya). N,Schoenoplectiella smithii (A.Gray) Hayas. (Québec, Canada). O,S. senegalensis (Steud.) Lye
(Botswana). Photos A–Eand J–Lby Modesto Luceño. Fby Jean Marc Vallières. Gby Julian Starr. Hby Peter M. Dziuk (reproduced with
permission from www.minnesotawildflowers.info). Iby Russell Barrett; Mby Marcial Escudero; Nby Marie‐Ève Garon‐Labrecque; Oby
Jane Browning.
811Targeted sequencing of Fuireneae s.l.
meant that all the characters used to separate Schoenoplec-
tiella from Schoenoplectus were overlapping. Moreover,
discontinuous characters could not be found for the
separation of Schoenoplectiella sections Actaeogeton and
Schoenoplectiella (=Supini), even though they were still
assumed to be natural groups.
Although our understanding of Fuireneae s.l. has pro-
gressed enormously over the past 50 years, and a pattern
pointing toward a grade of multiple major clades has
emerged, the most recent molecular analyses are equivocal
about the way to proceed from a taxonomic point of view.
Recent molecular studies of Fuireneae s.l. have retrieved the
tribe as a grade of three (Semmouri et al., 2019) or four
clades (Muasya et al., 2009a; Escudero & Hipp, 2013; Hinchliff
& Roalson, 2013; Glon et al., 2017) branching offafter an
Abildgaardieae–Eleocharideae Clade and paraphyletic with
respect to tribe Cypereae. Depending on the markers used,
analyses place either Bolboschoenus (Glon et al., 2017; Spalink
et al., 2018) or Fuirena (Glon et al., 2017) as branching first,
and the Schoenoplectiella Clade (Escudero & Hipp, 2013;
Shiels et al., 2014) or the Schoenoplectus Clade (Glon
et al., 2017; Spalink et al., 2018) as sister to Cypereae or
sister to each other (Semmouri et al., 2019). Early molecular
analyses have also suggested that Schoenoplectiella (Muasya
et al., 1998; Simpson et al., 2007) or Fuirena (Muasya
et al., 2009a) could be sister to tribe Eleocharideae Goetgh.
or that Bolboschoenus could be sister to tribe Abildgaardieae
Lye (Muasya et al., 2009a). These relationships were not
entirely implausible, given the early cladistic analyses of
sedge morphology by Goetghebeur (1986) and Bruhl (1995),
J. Syst. Evol. 59(4): 809–832, 2021 www.jse.ac.cn
Fig. 2. Ecological diversity of Fuireneae s.l. A,Bolboschoenus maritimus (L.) Palla (Minnesota, USA). B,Fuirena sp. (KwaZulu‐
Natal, South Africa). C,Schoenoplectus torreyi (Olney) Palla (Minnesota, USA). D,Schoenoplectus americanus (Pers.) Volkart
(Arizona, USA). E,Pseudoschoenus inanis (Thunb.) Oteng‐Yeb. (Cape Region, South Africa). F,Schoenoplectiella smithii (A.Gray)
Hayas. (Québec, Canada). Photos Aand Cby Peter M. Dziuk (reproduced with permission from www.minnesotawildflowers.
info); Band Eby Modesto Luceño; Dby Julian Starr; Fby Étienne Léveillé‐Bourret.
812 Starr et al.
but support for major clade relationships in molecular
analyses was poor and taxonomic sampling was limited. No
previous molecular phylogenetic study that has generated
novel data has included more than 25 species of Fuireneae
s.l., and no study has included more than five plastid markers
(matK,ndhF,rbcL,rps16,trnL‐F) and two nuclear markers
(ETS‐1f, ITS; Semmouri et al., 2019), with the exception of the
supermatrix analysis of Hinchliffand Roalson (23 loci;
Hinchliff& Roalson, 2013), where the relationships of the
major Fuireneae s.l. clades were completely unresolved.
Moreover, the limits of Schoenoplectus and Schoenoplectiella
remain unresolved, because molecular studies have often
been regional in scope (e.g., Japan, Yano & Hoshino, 2005a;
Korea, Jung & Choi, 2010), and even when geographic
sampling was wider (e.g. Shiels et al., 2014), a lack of critical
species of African Schoenoplectus (e.g., Schoenoplectus
rhodesicus (Podlech) Lye, S. paludicola (Kunth) Palla ex
J.Raynal; Smith & Hayasaka, 2001; Browning, 2012) or low
branch support (Muasya et al., 2009a) has meant that a
satisfactory separation for these two genera has yet to be
achieved. An overview of the species of Schoenoplectus and
Schoenoplectiella, their distribution range, and inclusion in
previous molecular studies is detailed in Table S1.
In this study, we use the Angiosperms353 enrichment panel
(Johnson et al., 2019) for targeted sequencing of an in‐depth
sampling of tribe Fuireneae s.l. (52 species, all genera) and
putatively related tribes Eleocharideae, Abildgaardieae, and
Cypereae. Additionally, off‐target nrDNA sequence data were
recovered from the generated raw reads and complemented
with ITS sequence data available on GenBank to help resolve the
monophyly of Schoenoplectus and Schoenoplectiella.Weinclude
critical tropical African Schoenoplectus,andweuseembryo
morphology and macro‐and micromorphological characters to
understand Fuireneae s.l. relationships and to circumscribe the
natural tribes and genera currently treated within Fuireneae s.l.
2 Material and Methods
2.1 Taxon sampling for the targeted sequencing study
Sequence data for a total of 99 accessions were analyzed in
this study, including an in‐depth sampling of (i) tribe
Fuireneae s.l., 62 accessions representing 52 species (c.
30%), and (ii) related tribes Abildgaardieae, Eleocharideae
and Cypereae (36 species), with Dulichium arundinaceum (L.)
Britton (tribe Dulichieae W.Schultze‐Motel) as the outgroup.
Most samples were taken from herbarium specimens;
however, some silica gel‐dried samples and Kew DNA bank
samples are also included (Table S2).
2.2 DNA extraction, genomic library preparation,
hybridization, and sequencing
Molecular work was carried out at the Sackler Phylogenomics
Laboratory, within the Jodrell Laboratory at the Royal Botanic
Gardens, Kew (Richmond, Surrey, UK). Genomic DNA was
extracted from leaf tissue obtained from herbarium specimens
or from samples collected and stored on silica gel using either
a standard CTAB approach (Doyle & Doyle, 1987) or a CTAB
protocol based on the study of Beck et al. (2012) that was
modified for optimal simultaneous extraction of 96–192
samples (i.e., one or two plates) from suboptimal (i.e.,
herbarium) tissue (see Data Sheet S1 in Larridon et al., 2020).
Eighteen accessions were sourced from the Kew DNA Bank
(http://dnabank.science.kew.org/) (Table S2). CTAB‐extracted
samples were purified using an Agencourt AMPure XP Bead
Clean‐up (Beckman Coulter, Indianapolis, IN, USA). All DNA
extracts were quantified using a Quantus™Fluorometer
(Promega Corporation, Madison, WI, USA) and then run on
a 1% agarose gel to assess the average fragment size. Samples
with very low concentration (not visible on a 1% agarose gel)
were assessed on an Agilent Technologies 4200 TapeStation
System using Genomic DNA ScreenTape (Santa Clara, CA,
USA). DNA extracts with average fragment sizes above 350 bp
were sonicated using a Covaris M220 Focused‐ultrasonicator™
(Covaris, Woburn, MA, USA), following the manufacturer's
protocol, to obtain an average fragment size of 350 bp.
Genomic library preparation, quality control, hybridization
with the Angiosperms353 probes (Johnson et al., 2019; Arbor
Biosciences, Ann Arbor, MI, USA), enrichment, and sequencing
were performed following the protocols outlined in the study
of Larridon et al. (2020). Raw reads for all accessions are
available from the NCBI GenBank Sequence Read Archive
(SRA) under Bioproject numbers: PRJNA553989 (http://www.
ncbi.nlm.nih.gov/bioproject/PRJNA553989), PRJNA649146
(http://www.ncbi.nlm.nih.gov/bioproject/PRJNA649146), and
PRJNA668802 (http://www.ncbi.nlm.nih.gov/bioproject/PRJN
A668802) and from the European Nucleotide Archive (ENA)
under EMBL Project numbers: PRJEB39590 (https://www.ebi.
ac.uk/ena/browser/view/PRJEB39590) and PRJEB41806
(https://www.ebi.ac.uk/ena/browser/view/PRJEB41806).
2.3 Read processing and sequence assembly
Bioinformatics settings follow Larridon et al. (2021b). Raw reads
were trimmed to remove adapter sequences and portions of low
quality with Trimmomatic v.0.39 (Bolger et al., 2014) according to
the following settings: LEADING:30 TRAILING:30 SLIDING-
WINDOW:4:2:30 MINLEN:36. HybPiper v.1.3.1 (Johnson et al., 2016)
was used to process the quality‐checked, trimmed reads, with all
settings at default, except minimum coverage, which was set to
4x. Paired and unpaired reads from all accessions were mapped
to targets with BLASTx (Altschul et al., 1990) using the
Angiosperms353 target loci amino acid (AA) sequences (see
Data Sheet S3 in Larridon et al., 2020). Mapped reads were
subsequently assembled into contigs with SPAdes v.3.13.1
(Bankevich et al., 2012). Exonerate v.2.2 (Slater & Birney, 2005)
was then used to align the assembled contigs to their associated
target sequence and to remove intronic regions (exons data set).
The HybPiper script intronerate was used to generate a
supercontigs data set. HybPiper flags potential paralogs when
multiple contigs map well to a single reference sequence. As few
random paralog warnings were raised, no sequence was
excluded.
The consensus sequences for each gene were then used to
generate the two nuclear data sets including all accessions
(i.e., exons data set and supercontigs data set) for those
genes with more than 10 sequences. Contigs were aligned
using MAFFT v.7 (Katoh & Standley, 2013) with the “–auto”
option. The alignments were trimmed in phyutility (Smith &
Dunn, 2008) to remove sites missing in 60% of the samples.
The number of potentially parsimony‐informative sites was
calculated using AMAS (Borowiec, 2016) for each contig
alignment, before and after trimming.
J. Syst. Evol. 59(4): 809–832, 2021www.jse.ac.cn
813Targeted sequencing of Fuireneae s.l.
2.4 Phylogenomic analyses of the targeted sequencing data
Phylogenomic analyses also follow Larridon et al. (2021b).
The two data sets (exons and supercontigs) were analyzed
using two approaches: a multi‐species summary coalescent
approach and a concatenated maximum likelihood approach.
For the multi‐species summary coalescent approach, indi-
vidual gene trees were constructed using RAxML v.8
(Stamatakis, 2014), applying GTRCAT and 100 bootstrap
replicates, followed by a slow ML optimization with the
“‐fa”option. Branches with support equal to 10 or lower
were collapsed using Newick Utilities (Junier &
Zdobnov, 2010). Each gene tree set was analyzed in
TreeShrink (Mai & Mirarab, 2018) to remove excessively
long branches. The resulting trees were used as input to infer
species trees in ASTRAL‐III v.5.5.11 (Zhang et al., 2018), a
summary method that is statistically consistent under the
Multiple Species Coalescent (MSC). The option “‐t2”was
used to output quartet support values. For the concatenated
maximum likelihood approach, gene alignments were first
concatenated using AMAS, cleaned by removing the outliers
identified by TreeShrink, and analyzed in IQ‐TREE 2.1.0 (Minh
et al., 2020) with mode set to “MFP+MERGE”and 10000
replicates of ultrafast bootstrap replications (Hoang
et al., 2018). Tree images were plotted in R (R Core
Team, 2020) using the packages ape (Paradis & Schliep, 2018),
ggimage (Yu, 2019a), ggtree (Yu et al., 2017), treeio
(Yu, 2019b), and their dependencies.
2.5 Phylogenetic analyses of nrDNA data
Off‐target nrDNA sequences were recovered from the
paired and unpaired trimmed reads of the targeted
sequencing data using GetOrganelle (Jin et al., 2018),
following the recommended parameters for nrDNA re-
covery (‐R20‐k 21,45,65,85,105). The two largest sequences
were annotated on the basis of two Poaceae complete
cistrons (GenBank accession numbers: KT281166 and
KY826229), given the absence of complete sequences for
Cyperaceae. Using these newly annotated sequences as a
query, the regions between 26S and 18S were blasted and
those with the complete fragment were kept. A second
blast round included the samples with fragmentary
recovery, which were then manually curated in Geneious
10.2.6 (https://www.geneious.com). We obtained nrDNA
sequence data for 80 of the 99 accessions included in the
targeted sequencing study (Table S3). ITS data for a further
58 accessions were obtained from GenBank (Table S3). The
retrieved off‐target nrDNA sequence data, together with
the obtained ITS data, were combined into an alignment in
PhyDE 0.9971 (Müller et al., 2010) and aligned using MAFFT
on the CIPRES portal (http://www.phylo.org/; Miller
et al., 2010), after which the alignment was checked
manually following nucleotide homology criteria, as
summarized by Morrison et al. (2015). Equally, a reduced
sampling alignment of 62 accessions was prepared to
answer the key question of this nrDNA study, that is, the
monophyly of Schoenoplectus and Schoenoplectiella.Thefull
sampling alignment of 137 accessions (Data S1) and the
reduced sampling alignment of 62 accessions (Data S2)
were analyzed on the CIPRES portal using RAxML 8.2.10
(Stamatakis, 2014) with the model set to GTR (gamma).
2.6 Embryo morphology
Embryo characteristics are widely acknowledged to be
among the most phylogenetically informative features for
sedges at the tribal and generic levels (Goetghebeur, 1998;
Semmouri et al., 2019). Twenty‐nine embryographs repre-
senting the six Fuireneae s.l. genera first brought together
for the study of Semmouri et al. (2019) were reinvestigated
for characters delineating the four Fuireneae s.l. clades (Data
S3). These embryographs representatively cover all genera
and most infrageneric groupings of the tribe Fuireneae s.l.
(except Schoenoplectus section Malacogeton).
As important differences in size appear to occur among
Fuireneae s.l. embryos, all embryographs were measured for
length and width at their widest point using ImageJ
(Rasband, 1997). To further quantify shape variation, we
used geometric morphometrics to test whether the four
major clades identified in phylogenetic analyses differed in
embryo shape. A total of 17 landmarks and 70 pseudoland-
marks representing all shape and size variations found in
Fuireneae s.l. embryos were digitized on embryos using
tpsDig v2.17 (Rohlf, 2015). Landmark positions are described
and illustrated in Fig. S1. Size and shape variables were
generated by Generalized Procrustes Analysis using the R
v.3.6.3 (R Core Team, 2020) package geomorph (Adams
et al., 2020). Differentiation between the four major clades in
size+shape was tested by MANOVA and by linear discrim-
inant analysis in the R package MASS (Venables &
Ripley, 2002). Finally, the average embryo shape of each
clade was computed from group centroids to visually
represent salient embryological differences between clades.
2.7 Micromorphology of nutlet epidermal cells
The nutlet epidermal cell shape has been shown to be an
effective tool for separating genera and infrageneric taxa in
Fuireneae s.l. (e.g., Schuyler, 1971; Haines & Lye, 1983;
Gordon‐Gray, 1995; Hayasaka, 2012; Elkordy et al., 2020).
A literature survey of existing data was performed to
characterize cell shape in all genera. To clarify the use of
terms for the cell shape, the ratio of the length to the width
of a representative cell was taken for each taxon when both
ends of a cell were clearly visible. The cell shape was
characterized as follows: (i) cells <2 times longer than wide
were described as isodiametric when roughly round,
hexagonal, or quadrate; (ii) cells 2 to 4 times longer than
wide were described as oblong when sides were nearly
parallel or elliptic when not; (iii) cells >4to<8 times longer
than wide were characterized as elongated, and (iv) cells ≥8
to <21 times longer than wide were described as linear. The
morphology of anticlinal cell walls (linear, undulate, sinuous)
was described when clearly visible. However, many published
images of Fuireneae nutlet epidermal cells do not have the
periclinal cell wall removed. This means that the morphology
of anticlinal cell walls at their suture with periclinal walls
could differ from the morphology observed when the
periclinal wall is removed (cf. Figs. 1B, 1D to 1F, 1H in
Browning et al., 1996). Consequently, this character was
described, but not used in making taxonomic decisions.
Scanning electron microscopy (SEM) of the nutlet
epidermal cell shape of Pseudoschoenus inanis and Schoeno-
plectus scirpoides (Schrad.) Browning (section Schoeno-
plectus) was also performed, as data were lacking for this
J. Syst. Evol. 59(4): 809–832, 2021 www.jse.ac.cn
814 Starr et al.
genus and species. Micrographs of fruit epidermal cells were
obtained at the Electron Microscope Unit at the University of
Cape Town. Nutlets were removed from specimens housed
at the Bolus Herbarium (BOL; Thiers, continuously updated),
sputter‐coated, and images were made using Tescan MIRA3
equipment.
3 Results
3.1 Quality of the targeted sequencing data
Recovery statistics are available in Table S4 and Fig. S2. The
average percentage recovery was 50% (4%–78% range). For
the exon data set, the alignment length per gene ranged
from 99 to 4341 bp long, with a mean length of 928 bp
before trimming, and from 6 to 2320 bp long, with a mean
length of 311 bp after trimming (Tables S5A, S6A). For the
supercontigs data set, the alignment length per gene ranged
from 285 to 27 545 bp long, with a mean length of 6323 bp
before trimming, and from 2 to 3269 bp long, with mean
length of 488 bp after trimming (Tables S5B, S6B). Therefore,
the amount of data retrieved in the supercontigs data set
was much larger when comparing the length of the contigs,
166 431 bp vs. 104 349 bp for the exons data set (Table S6),
and the total number of parsimony‐informative sites (PIS)
was also higher (c. 2.2×more PIS) in the supercontigs vs. the
exons data set. Nonetheless, before trimming, the relative
number of PIS was c. 0.3 PIS/bp for both the exons and
supercontigs data sets. However, as a result of the chosen
trimming strategy, it was c. 0.4 and 0.5 PIS/bp, respectively,
meaning that the contig length is positively correlated with
the number of PIS.
3.2 Relationships in tribe Fuireneae s.l. as inferred from
targeted sequencing data
As the topologies obtained from all analyses were very
similar, we only provide a summary of the relationships
shown in the tree resulting from the multi‐species summary
coalescent approach on the supercontigs data set (Fig. 3).
The results of the other analyses are presented in
Figs. S3–S5. We retrieved a topology showing an
Abildgaardieae–Eleocharideae Clade as sister to a Fuireneae
s.l. grade, leading to a monophyletic tribe Cypereae. Within
the Fuireneae s.l. grade, Bolboschoenus is sister to the
remaining Fuireneae s.l. grade lineages plus Cypereae,
followed by a monophyletic Fuirena, the Schoenoplectus
Clade, and the Schoenoplectiella Clade. The nodes in the
backbone of the phylogeny and for each major clade are well
supported with high local posterior probabilities (LPP) and
generally high gene tree concordance (Fig. 3).
3.3 Relationships in Fuirena
Within Fuirena, two well‐supported clades are visible (Fig. 3):
(i) the Pentasticha Clade comprising all species with three‐
angled stems that roughly corresponds to subg. Pentasticha
with the addition of some anomalous species with a variable
perianth morphology, and (ii) the Fuirena Clade that
comprises all species with a relatively stable 3 +3 perianth
placed in subg. Fuirena. The Fuirena Clade is subdivided into
two well‐supported sister subclades: (i) the Umbellata
Subclade with the type F. umbellata Rottb. and some closely
related species with highly compound, corymbiform inflor-
escences (F. robusta Kunth, F. camptotricha C.Wright), and (ii)
the Squarrosa subclade comprising all other species of subg.
Fuirena with generally depauperate inflorescences reduced
to a single or two to three glomerules of spikelets.
3.4 Exploring the monophyly of Schoenoplectus and
Schoenoplectiella using nrDNA
The full sampling alignment included 137 accessions and had
a total aligned length of 15 625 characters (Data S1), whereas
the reduced sampling alignment included 62 accessions and
12 401 characters (Data S2). The phylogenetic hypothesis
obtained for the full sampling alignment is available in Fig.
S6. It shows an Abildgaardieae–Eleocharideae Clade as sister
to a Fuireneae s.l. grade +Cypereae Clade. Within the
Fuireneae s.l. grade, the four major lineages are retrieved
as monophyletic: (i) Bolboschoenus; (ii) Fuirena; (iii) the
Schoenoplectus Clade (Actinoscirpus +Schoenoplectus s.s.),
and (iv) the Schoenoplectiella Clade (Pseudoschoenus +
Schoenoplectiella s.l., which includes a group of largely
African Schoenoplectus species nested in Schoenoplectiella).
However, nodes in the backbone of the grade are not well
supported, resulting in uncertain relationships among these
four major clades.
The phylogenetic hypothesis obtained for the reduced
sampling alignment is available in Fig. 4. It shows two well‐
supported clades: (i) the Schoenoplectus Clade (Actino-
scirpus +Schoenoplectus s.s.), and (ii) the Schoenoplectiella
Clade (Pseudoschoenus +Schoenoplectiella s.l.). Within Schoe-
noplectus, sections Malacogeton and Schoenoplectus are both
retrieved as monophyletic. Within Schoenoplectiella, section
Schoenoplectiella is retrieved as paraphyletic with respect to
a grade of African Schoenoplectus species transferred below
to Schoenoplectiella (indicated as “new group”in Fig. 4; see
Section 5). The crown clade consists of a monophyletic
Schoenoplectiella section Actaeogeton.
Several species in the ITS tree were not monophyletic
(Schoenoplectus triqueter,S. mucronata). However, this was
not treated in the Discussion (Section 4), because multiple
samples for these species could not be included in our
targeted sequencing analysis (ITS taken from Genbank), the
bootstrap support for the key branch separating S.
mucronata samples was low (59%), and two of the three
samples of Schoenoplectus triqueter were found in the same
clade as S. tabernaemontani, a species with which it is known
to form hybrids (e.g., Smith, 2002).
3.5 Embryo morphology
All members of Fuireneae s.l. possess embryos with a more
or less mushroom‐shaped or top‐shaped outline, with leaf
primordia in a basal position and a lateral displacement of
the root cap (Fig. 5). While rare in the family, fungiform
embryos are also typical for tribe Eleocharideae and are
sometimes found in some of the largest Abildgaardieae
embryos, suggesting that this special morphology is a
plesiomorphy for Fuireneae s.l. (Semmouri et al., 2019).
Nonetheless, subtle differences can be found among the
different clades, and these differences are supported
by geometric morphometrics. The MANOVA based on the
first three principal components of embryo size +shape
(representing 70% of the total variation) found significant
J. Syst. Evol. 59(4): 809–832, 2021www.jse.ac.cn
815Targeted sequencing of Fuireneae s.l.
differences between the four clades (P<0.0001), and a linear
discriminant analysis on the full size +shape data showed a
good separation of the clades and no overlap (Fig. 6).
Bolboschoenus has the largest embryos in Fuireneae s.l.
(Table 1), with at least two species possessing embryos over
a millimeter in length and more than half a millimeter in
width. The embryo of Bolboschoenus is fungiform, with a
broadened, rhomboid scutellum (Figs. 5, 6). The root cap is
well differentiated, in a lateral position, and separated from
the scutellum by a notch. The first leaf is well developed,
J. Syst. Evol. 59(4): 809–832, 2021 www.jse.ac.cn
Fig. 3. Phylogenetic reconstruction of relationships in tribe Fuireneae s.l. and related tribes based on analysis of the
supercontigs data set. Species tree inferred in ASTRAL from RAxML gene trees. Numbers on branches represent local
posterior probabilities (LPPs) and pie charts at nodes correspond to quartet support with blue for agreeing genes, red for
disagreeing genes, and gray for uninformative genes.
816 Starr et al.
J. Syst. Evol. 59(4): 809–832, 2021www.jse.ac.cn
Fig. 4. Continued
817Targeted sequencing of Fuireneae s.l.
second and third leaf primordia are present in a basal
orientation. The germ pore is slit‐like and in a parallel
orientation, relative to the first plumular leaf primordium. As
stated in Semmouri et al. (2019), these character states
correspond most closely to the Bolboschoenus‐type embryo,
as defined by the typology of Goetghebeur (1986), and
variant 1 of the Scirpus‐type embryo of Van der Veken (1965),
as noted by Pignotti (2003).
In length, the embryos of Fuirena are the smallest (Table 1),
more than three standard deviations smaller in length than
the mean of all other Fuireneae s.l. embryos, except
Actinoscirpus (2.25 standard deviations), and from one to
over six standard deviations narrower than other Fuireneae
s.l. genera at their widest point. Fuirena embryos always
displayed length to width ratios below 1.1, a consequence of
their roughly turbinate shape, and their horizontally
broadened scutellum comprised 40%–60% of the total length
of the embryo. In contrast, the embryos of Actinoscirpus,
Pseudoschoenus,Schoenoplectus, and Schoenoplectiella fre-
quently had length to width ratios above 1.1 (up to 2.0) due
to their more elongate fungiform outline and their scutellum
comprised only 30%–50% of the total length of the embryo.
Moreover, in these genera, the left (or upper) coleoptile lip is
strongly outgrown (less so in Fuirena) and a second leaf
primordium is always present (absent or poorly developed in
Fuirena species). Given the numerous morphological differ-
ences of Fuirena embryos noted here, we consider these
differences sufficient to recognize the Fuirena embryo as a
distinct type within the typology scheme of Goetghebeur
(1986; i.e., Fuirena‐type).
Although Actinoscirpus,Pseudoschoenus,Schoenoplectus,
and Schoenoplectiella possess very similar embryos (Figs. 5, 6),
two groups can be distinguished, which we designate here as
Schoenoplectus‐type I and Schoenoplectus‐type II given
their similarity. Whereas the scutellum in Actinoscirpus and
Schoenoplectus s.s. is turbinate to rhomboid in shape, with
straight to convex lower margins, the scutellum of
Pseudoschoenus and Schoenoplectiella is often umbonate,
with a distinct knob at the apex, or strongly pileate, with the
margins clearly incurved (Schoenoplectus‐type II). These two
groups correspond to the taxonomically significant embryo
variants 2 and 3 of the Scirpus‐type embryo sensu Van der
Veken (1965) highlighted by Pignotti (2003). Of the nine
species of African Schoenoplectus nested within our
Schoenoplectiella Clade, Van der Veken (1965) includes
embryographs for three of these: Schoenoplectus rhodesicus,
S. paludicola, and S. muricinux. All three possess strongly
pileate, Schoenoplectus‐type II embryos (Figs. 5, 6), and are
more consistent in size with species of Schoenoplectiella than
Schoenoplectus (Table 1). In summary, four groups of
embryos are distinguished within Fuireneae s.l. and each
corresponds to one of the four major clades retrieved in
molecular studies, here recognized as tribes. While no
embryo of Schoenoplectus section Malacogeton has been
studied, it seems rather probable that these species also
display a Schoenoplectus‐type I embryo since this is the
embryo displayed by both Actinoscirpus and Schoenoplectus
section Schoenoplectus.
3.6 Nutlet epidermal cell micromorphology
Nutlet epidermal cell shape was isodiametric in all species of
Bolboschoenus examined (eight species; length to width
range 1.00–1.67, mean =1.22; Tables 2, S7). Anticlinal cell
walls for Bolboschoenus ranged from straight to undulate.
Cell shape in Fuirena was more variable than in
Bolboschoenus (length to width range 1.01–7.23, mean =3.42;
Tables 2, S7). Among the studied species, three possessed
roughly isodiametric cells, whereas four displayed oblong to
elongated cells, often in transverse rows. The morphology of
anticlinal cell walls in Fuirena ranged from straight to
undulate.
Most species in the Schoenoplectus Clade (Schoeno-
plectus +Actinoscirpus; 12 species examined; Table S7;
Fig. 7) displayed isodiametric to oblong or elliptic cells
(length to width range 1.05–3.86, mean =2.15; Table 2),
except S. nipponicus, the only member of Schoenoplectus
section Malacogeton examined, which possessed elon-
gated cells (length to width 6.32; Tables 2, S7). Within the
Schoenoplectus Clade, anticlinal cell walls ranged from
straight or slightly undulate in most Schoenoplectus
section Schoenoplectus species, to highly sinuous in
Actinoscirpus and Schoenoplectus nipponicus (section
Malacogeton).
Cell shape for members of the Schoenoplectiella Clade was
nearly uniform (28 species examined; Table S7; Fig. 7). All
species possessed linear epidermal cells (length to width
range (8.00–)9.22–20.21, mean =13.87; Table 2) in longitu-
dinal rows, except Pseudoschoenus inanis, which displayed
isodiametric to oblong cells (length to width range 1.45–3.81,
mean =2.73; Table 2). Anticlinal cell walls in the clade ranged
from straight to slightly undulate in almost all species. The
linear cells of Schoenoplectiella are clearly associated with the
wavy, transversely ridged nutlets of annuals from Schoeno-
plectiella section Schoenoplectiella (Fig. 7L), but linear cells
are also seen in the large perennial species of Schoenoplec-
tiella from section Actaeogeton regardless of whether the
nutlet is sculpted (e.g., S. mucronata,S. juncoides) or smooth
(e.g., S. lineolata,S. wallichii) (see Table S7). Longitudinally
linear epidermal cells were also present in all the African
Schoenoplectus (8 species) nested within the Schoenoplectiella
J. Syst. Evol. 59(4): 809–832, 2021 www.jse.ac.cn
Fig. 4. Phylogenetic reconstruction of relationships in the Schoenoplectus and Schoenoplectiella Clades based on nrDNA data.
The Schoenoplectus Clade comprises the genera Actinoscirpus and Schoenoplectus, with Schoenoplectus divided into two
clades, sections Schoenoplectus and Malacogeton. The Schoenoplectiella Clade consists of the genera Pseudoschoenus and
Schoenoplectiella, with Schoenoplectiella section Schoenoplectiella paraphyletic with respect to a grade of largely African
Schoenoplectus species here transferred to Schoenoplectiella that is terminated by a monophyletic Schoenoplectiella section
Actaeogeton. The reconstruction is based on an RAxML analysis of the reduced sampling alignment with 62 accessions
obtained from nrDNA off‐target reads and ITS sequences available from GenBank. Branch thickness represents bootstrap
support (values are provided in Fig. S6).
818 Starr et al.
Clade (length to width range 15.25–19.56, mean =16.77; Table 2)
for which data was available regardless of whether pericarp
surface features were present (S. muricinux,S. muriculatus,S.
confusus, Browning, 1991a; Gordon‐Gray, 1995; S. dissachantha,
Hayasaka, 2009) or absent (S. brachyceras,S. corymbosus,
Browning, 1991b; Gordon‐Gray, 1995; S. paludicola,S. decipiens,
Browning,1990;Gordon‐Gray, 1995). Two further species of
African Schoenoplectus not included in our molecular analyses
possessed linear epidermal cells in longitudinal rows,
S. pulchellus (Browning, 1990) and S. heptangularis (Jiménez‐
Mejías & Cabezas, 2009). These species will be treated in
Schoenoplectiella (see Section 5).
J. Syst. Evol. 59(4): 809–832, 2021www.jse.ac.cn
Fig. 5. Embryographs of the genera of Fuireneae s.l. Bolboschoenus:A,B. fluviatilis (Torr.) Soják; B,B. maritimus (L.) Palla; C,B.
grandispicus (Steud.) Lewej. & Lobin; Fuirena:D,F. abnormalis C.B.Clarke; E,F. ciliaris (L.) Roxb.; F,F. incompleta Nees; G,F.
pachyrrhiza Ridl.; H,F. scirpoidea Michx.; I,F. stricta Steud.; J,F. trilobites C.B.Clarke; K,F. umbellata Rottb.; Actinoscirpus and
Schoenoplectus:L,A. grossus (L.f.) Goetgh. & D.A.Simpson; M,S. californicus (C.A.Mey.) Soják; N,S. lacustris (L.) Palla; O,S.
litoralis (Schrad.) Palla; Pseudoschoenus and Schoenoplectiella:P,P. inanis (Thunb.) Oteng‐Yeb; Q,S. dissachantha (S.T.Blake)
Lye; R,S. juncea (Willd.) (S); S,S. juncoides (Roxb.) Lye; T,S. laevis (S.T.Blake) Lye; U,S. lateriflora (J.F.Gmel.) Lye; V,S. lineolata
(Franch. & Sav.) J.Jung & H.K.Choi; W,S. mucronata (L.) J.Jung & H.K.Choi; X,S. praelongata (Poir.) Lye; Y,S. roylei (Nees) Lye;
Z,S. smithii (A.Gray) Hayas.; a,S. vohemarensis (Cherm.) Lye; b,S. paludicola (Kunth) Palla; c,S. rhodesicus (Podlech) Lye). Scale
bars 100 μm. See Data S3 for the voucher information of the studied embryographs.
819Targeted sequencing of Fuireneae s.l.
4 Discussion
4.1 The Fuireneae s.l. grade consists of six genera and four
tribes
As in earlier molecular phylogenetic studies (e.g., Shiels
et al., 2014; Spalink et al., 2016; Glon et al., 2017; Semmouri
et al., 2019), tribe Abildgaardieae +tribe Eleocharideae form
a clade that is sister to the Fuireneae Grade +tribe Cypereae.
Our results (Fig. 3) indicate that tribe Fuireneae is a grade of
six genera arranged into four major clades that are
successive sisters to tribe Cypereae: (i) the genus Bolbo-
schoenus; (ii) followed by Fuirena; (iii) the Schoenoplectus
Clade consisting of Actinoscirpus +Schoenoplectus s.s., and
(iv) the Schoenoplectiella Clade containing Pseudoschoenus,
Schoenoplectiella s.l., and a collection of largely African
Schoenoplectus species nested in Schoenoplectiella. Although
all previous molecular analyses have also found Fuireneae s.l.
to consist of a grade and have often recovered the same
major clades as in our analyses, topological relationships
among these major clades have been inconsistent and
support for the backbone of trees has been weak. Even in
Glon et al. (2017), who used seven markers and obtained the
same topology we infer here, the node separating
Bolboschoenus from Fuirena, as well as the node placing
the Schoenoplectiella Clade as sister to Cypereae, had very
low support in maximum parsimony, maximum likelihood,
and Bayesian analyses. Moreover, like all previous analyses
that used novel data, the analysis of Glon et al. (2017)
included only a fraction of tribal diversity (18 spp. or 12%;
others 10–25 spp.). This explains why no taxonomic changes
to the circumscription of Fuireneae s.l. have taken place to
date, despite ample evidence from previous molecular
analyses that the group was not natural.
In our analyses, which include nearly a third of Fuireneae
s.l. diversity and numerous generic and infrageneric types
(Table S2), statistical support is strong for the backbone of
trees and for all four Fuireneae s.l. clades and their six
genera. Consequently, we can firmly reject the monophyly of
Fuireneae s.l. (Muasya et al., 1998, 2009a; Simpson
et al., 2007) or the idea that Bolboschoenus cannot be
separated from Schoenoplectus s.s. (e.g., Lye, 1971a; Haines &
Lye, 1983; Tucker, 1987; Strong, 1994). Branch support (in all
analyses) and branch lengths (in the concatenated maximum
likelihood analyses) clearly indicate that Fuireneae s.l. forms
a grade of successive sisters terminating with tribe Cypereae.
Moreover, morphology, anatomy, embryo features (Goet-
ghebeur & Simpson, 1991; Tatanov, 2007), and even fungal
parasites (Savile, 1972; Léveillé‐Bourret et al., 2021) suggest a
distant relationship between Bolboschoenus and Schoeno-
plectus s.s. Likewise, the genus Fuirena is monophyletic and
very strongly supported as in previous molecular analyses
(e.g., Hinchliffet al., 2010; Spalink et al., 2016; Glon
et al., 2017). This is not surprising as many characters, such
as terminal racemose or paniculate inflorescences, nodose
leafy stems, and peculiar perianth‐segments (often unguicu-
late in form) supported it as an independent genus
(Kral, 1978; Haines & Lye, 1983; Goetghebeur, 1998) and
potentially a tribe (Haines & Lye, 1983; Tatanov, 2007). Our
molecular analyses further support the recognition of the
monotypic genera Actinoscirpus and Pseudoschoenus as each
taxon possesses numerous molecular autapomorphies,
which is consistent with a series of morphological,
anatomical, and embryo features that suggest they represent
genera worthy of taxonomic recognition (Oteng‐
Yeboah, 1974b; Goetghebeur, 1986; Goetghebeur &
Simpson, 1991). Their generic status is further supported by
the fact they are each sister to strongly supported and
morphologically distinct clades that we here recognize as
monophyletic genera, Schoenoplectus s.s. and Schoenoplec-
tiella s.l. Although these genera share numerous overlapping
characters that can make identification difficult, we confirm
J. Syst. Evol. 59(4): 809–832, 2021 www.jse.ac.cn
Fig. 6. Linear discriminant analysis of embryo size +shape,
with each major lineage represented by a convex hull with a
distinct color. Embryographs represent the average shape of
each major lineage as calculated from group centroids,
illustrating the main embryological characters used to
delineate the groups.
Table 1 Size differences among embryos of Fuireneae s.l. genera
Genus Species (n) Length, (mean ±SD) Width (mean ±SD) Ratio (L:W)
Bolboschoenus 3 987.4 ±302.4 649.2 ±188.6 1.52
Fuirena 8 272.4 ±60.0 288.3 ±56.4 0.94
Actinoscirpus 1 407.6 ±n/a 347.1 ±n/a 1.17
Schoenoplectus s.s. 3 676.2 ±96.1 622.2 ±145.2 1.09
Pseudoschoenus 1 716.4 ±n/a 352.2 ±n/a 2.03
Schoenoplectiella 10 478.5 ±134.3 424.4 ±149.0 1.13
Schoenoplectus →Schoenoplectiella 3 579.8 ±70.3 483.2 ±44.0 1.20
Embryographs were measured for length and width at their widest point using ImageJ. Measurements in μm.
820 Starr et al.
that each possesses distinct combinations of embryo and
micromorphological characters that support their separation.
In fact, all four major clades in the Fuireneae s.l. grade can be
distinguished by a combination of embryo and micro-
morphological features, and when this is evaluated within
the context of considerable molecular and morphological
support, it suggests that each major Fuireneae s.l. clade is
best treated as a distinct tribe.
All four major Fuireneae s.l. clades can be distinguished by
embryo morphology, a feature acknowledged as being
among the most phylogenetically informative characters in
sedges at the tribal (e.g., Goetghebeur, 1986; Léveillé‐
Bourret & Starr, 2019; Semmouri et al., 2019), generic (e.g.,
Gilmour et al., 2013; Léveillé‐Bourret et al., 2015, 2020), or
even infrageneric (Larridon et al., 2011) levels. In the case of
taxa currently treated in Fuireneae s.l., Van der Veken (1965)
recognized three variants of his Schoenoplectus‐type embryo
that had taxonomic significance (Pignotti, 2003). All three of
these variants correspond to natural groups in our analyses,
and we here recognize a fourth variant to account for the
small embryos of Fuirena (on average 135.2 μm shorter and
58.8 μm narrower than Actinoscirpus, the next smallest
Fuireneae s.l. embryo; Table 1) where a second leaf
primordium is either absent or poorly developed and the
hypocotyl is only slightly longer than the scutellum.
Bolboschoenus is unique in having the largest embryos in
the tribe (on average 987.4 μm long, 649.2 μm wide; Table 1),
and in possessing a plumule where a third primordial leaf
develops and a notch is present below the root cap (i.e.,
variant 1 sensu Van der Veken, 1965; Bolboschoenus‐type
sensu Goetghebeur, 1986; Semmouri et al., 2019). Interest-
ingly, the close relationships between the genera Actino-
scirpus and Schoenoplectus s.s. (Schoenoplectus Clade), and
between Pseudoschoenus and Schoenoplectiella s.l. (including
tropical, largely African Schoenoplectus; the Schoenoplec-
tiella Clade), are also supported by embryo morphology
(Fig. 5). Whereas the scutellum in Actinoscirpus and
Schoenoplectus s.s. is turbinate to rhomboid in shape (variant
2sensu Van der Veken, 1965), species in the Schoenoplec-
tiella Clade possess an umbonate or strongly pileate
scutellum in the form of a mushroom cap with incurved
margins (variant 3 sensu Van der Veken, 1965).
In part, these relationships are further supported by the
shape of epidermal nutlet cells (Fig. 7). Whereas all species in
the Schoenoplectus Clade possess epidermal nutlet cells that
are isodiametric to oblong or elliptic, except Schoenoplectus
nipponicus (elongated), the cells in every species of the
Schoenoplectiella Clade are linear, with the exception of
Pseudoschoenus inanis (isodiametric to oblong). Cell shape
thus clearly separates species in the Schoenoplectus Clade
from Schoenoplectiella, where cells in longitudinal rows can
be as much as 20 times longer than wide. In fact, among
Fuireneae s.l. species as a whole, only the elongated cells of
Fuirena ciliaris (7.23 times longer than wide) can even come
close to the species of Schoenoplectiella with the least linear
of cells, S. articulata (8.00 times longer than wide), and in
this case, there is a clear morphological difference: the
elongated cells of F. ciliaris are in transverse rows, whereas
the linear cells of S. articulata—and of Schoenoplectiella as a
whole—are longitudinally arranged. It is also important to
note that although linear epidermal cells are clearly
associated with the many small annual members of
Schoenoplectiella whose nutlets are typically dark at maturity
and display distinct, wavy rows of transversely orientated
ridges (roughly equivalent to section Schoenoplectiella), even
smooth nutlets in some large perennial species of
Schoenoplectus possess these linear cells (Haines &
Lye, 1983; Gordon‐Gray, 1995). Although Van der Veken
(1965) was unaware of this cell shape character, he
circumscribed Scirpus section Actaeogeton (=Schoenoplec-
tiella section Actaeogeton) to include not only its traditionally
large perennials, but also the small annuals of Scirpus section
Supini (=Schoenoplectiella section Schoenoplectiella) because
they all shared strikingly pileate embryos.
Not only can we confirm that all the Schoenoplectus
species nested within Schoenoplectiella s.l. in our analysis
possess these distinctively shaped pileate embryos when
data is available (i.e., Schoenoplectus rhodesicus,S. paludicola,
S. muricinux (C.B.Clarke) J.Raynal), they also possess
longitudinally linear epidermal cells regardless of whether
the surface is rugose (S. muricinux) or smooth (S. paludicola)
(Gordon‐Gray, 1995). The pattern holds even when embryo
data is lacking. For the tropical, largely African Schoeno-
plectus species nested in Schoenoplectiella s.l. that lack
embryo data, but whose epidermal cell shape is known, all
species possess longitudinally linear cells regardless of nutlet
pericarp texture (smooth in S. corymbosus (Roth ex Roem. &
Schult.) J.Raynal, S. brachyceras (Hochst. ex A.Rich.) Lye,
S. decipiens (Nees) J.Raynal; vs. rugose in S. confusus
(N.E.Br.) Lye and S. muriculatus (Kük.) Browning; Haines &
Lye, 1983; Browning, 1991a, 1991b; Gordon‐Gray, 1995).
Because embryo and cell shape appear to be such
consistently important taxonomic characters, we are con-
fident that Schoenoplectus species not included in our
J. Syst. Evol. 59(4): 809–832, 2021www.jse.ac.cn
Table 2 Ratios of fruit epidermal cell length to width
Genus Taxa (n) Ratio, range Ratio, mean ±SD
Bolboschoenus 8 1.00–1.67 1.22 ±0.23
Fuirena 7 1.01–7.23 3.42 ±2.19
Actinoscirpus 1 2.86 n/a
Schoenoplectus s.s. 12 1.05–3.86(−6.32) 2.15 ±1.35
Pseudoschoenus 1 1.45–3.81 2.73 ±1.19
Schoenoplectiella 11 (8.00–)9.22–20.21 13.87 ±3.66
Schoenoplectus →Schoenoplectiella 5 15.25–19.56 16.77 ±2.51
The largest measurement was always taken as length. More than one cell was measured for Pseudoschoenus due to variability.
821Targeted sequencing of Fuireneae s.l.
J. Syst. Evol. 59(4): 809–832, 2021 www.jse.ac.cn
Fig. 7. Nutlets and glumes of species of the Schoenoplectus Clade and the Schoenoplectiella Clade. A, Scanning electron microscopy
(SEM) micrograph of a nutlet, including perianth bristles of Schoenoplectus scirpoides (Schrad.) Browning (section Schoenoplectus). B,
light microscopy image of a nutlet, including perianth bristles of Schoenoplectus acutus (Muhl. ex Bigelow) Á.Löve & D.Löve (section
Schoenoplectus). C, light microscopy image of a nutlet, including perianth bristles of Schoenoplectus torreyi (Olney) Palla (section
Malacogeton). D, SEM micrograph of the nutlet surface of S. scirpoides.E, SEM micrograph of the nutlet surface of Schoenoplectus
lacustris (L.) Palla (section Schoenoplectus). F, SEM micrograph of the nutlet surface of Schoenoplectus nipponicus (Makino) Soják
(section Malacogeton). G, SEM micrograph of the nutlet, including perianth bristles and stamen filaments of Pseudoschoenus inanis
(Thunb.) Oteng‐Yeb. (Schoenoplectiella Clade). H, SEM micrograph of the nutlet of Schoenoplectus corymbosus (Roth ex Roem. &
Schult.) J.Raynal (nested in Schoenoplectiella s.l.). I, SEM micrograph of the nutlet of Schoenoplectus muricinux (C.B.Clarke) J.Raynal
(nested in Schoenoplectiella s.l.). J, SEM micrograph of the nutlet surface of P. inanis.K, SEM micrograph of the nutlet surface of S.
corymbosus.L, SEM micrograph of the nutlet surface of S. muricinux.M,glumeofS. acutus.N,glumeofSchoenoplectus heterochaetus
(Chase) Soják (section Schoenoplectus). O,glumeofS. torreyi (section Malacogeton). P,glumeofS. corymbosus.Q,glumeof
Schoenoplectiella smithii (A.Gray) Hayas (section Actaeogeton). A–C,G–I,andM–Q,scalebar=1 mm; D–Fand J–L,scalebar=50 μm.
Samples in Eand Fhave been treated and the upper periclinal walls removed, displaying the cell lumen with the anticlinal walls entirely
exposed. A,G,H,J,Knewly generated for this study by Muthama Muasya; B,C,M–O,Q, reproduced with permission from www.
minnesotawildflowers.info; D,I,Lreproduced with permission by Jane Browning (originally published in Gordon‐Gray, 1995); E,F
reproduced with permission from Hayasaka (2012); Pnewly generated for this study by P. Jiménez‐Mejías.
822 Starr et al.
molecular analyses with one or more of these characters are
most likely nested in Schoenoplectiella and should be
transferred to it. For example, although we did not include
Schoenoplectus heptangularis Cabezas & Jim.Mejías and S.
pulchellus (Kunth) J.Raynal in our molecular analyses, these
species possess longitudinally linear epidermal nutlet cells.
Given this character and the fact that we included species
deeply nested in Schoenoplectiella to which they are
morphologically close (i.e., S. paludicola and S. decipiens for
S. pulchellus, Browning, 1990; S. brachyceras,S. corymbosus
and S. decipiens for S. heptangularis, Jiménez‐Mejías &
Cabezas, 2009), we are confident these species should be
transferred to Schoenoplectiella. Likewise, we are confident
that two further African species not in our analyses,
Schoenoplectus scirpoides and S. subulatus (Vahl) Lye, should
not be transferred to Schoenoplectiella because they clearly
show a morphological affinity to species in our analyses from
Schoenoplectus sect. Schoenoplectus (e.g., S. litoralis
(Schrad.) Palla; Browning et al., 1994). In addition, at least
one of these species, S. scirpoides (Fig. 7D), is known to
possess roughly isodiametric to oblong epidermal nutlet cells
similar to other species of Schoenoplectus like S. litoralis, its
close relative.
Although the circumscription of the widespread genera
Schoenoplectus and Schoenoplectiella has been difficult, this
was largely due to the fact that both taxonomic and molecular
studies were often regional in scope. The taxonomic studies of
Smith and Hayasaka (e.g., Smith & Hayasaka, 2001, 2002;
Hayasaka, 2012) focused on North American and East Asian
species; Luceño and Jiménez‐Mejías on the Iberian Peninsula
(Luceño & Jiménez‐Mejías, 2008); Pignotti and Mariotti on
Southwestern Europe (Pignotti, 2003; Pignotti & Mariotti, 2004);
Wilson on Australia (e.g., Wilson, 1981), and the studies of Haines
and Lye (e.g., Lye, 1971b, 2003; Haines & Lye, 1983), Raynal (e.g.,
Raynal,1976a,1976b)andBrowning (e.g., Browning, 1990, 2012;
Browning et al., 1995) on predominately African material.
Molecular studies show a similar pattern with analyses focused
on just Korean (Jung & Choi, 2010) or Japanese species (Yano &
Hoshino, 2005b), and even in those studies with a broader
geographic scope, poor taxonomic sampling, a lack of African
material or low branch support (e.g., Muasya et al., 2009a; Shiels
etal.,2014;Glonetal.,2017)meantthatthepatternsdiscovered
in this study were not detected before.
With the transfer of these African Schoenoplectus species
to Schoenoplectiella, we now have six, well‐circumscribed
genera arranged in a grade of four major Fuireneae s.l.
clades. Here, we choose to recognize these four major clades
as tribes as each is strongly supported by molecular
characters and each can be clearly defined by morphological,
micromorphological, and embryo characters (see tribal
diagnoses in Section 5).
The alternative strategy of expanding tribe Cypereae to
include all four clades or even just the Schoenoplectiella
Clade is not adopted because it would mean adding one to
three new embryo types to the circumscription of Cypereae,
a large and morphologically variable clade of c. 1130 species
that is best defined by its Cyperus‐type embryo (Muasya
et al., 2009b) or the highly similar but uncommon Ficinia‐type
embryo (Muasya et al., 2009b; Semmouri et al., 2019). Adding
any member of Fuireneae s.l. to Cypereae would make it
impossible to define Cypereae by any single embryological or
morphological character, creating a tribe defined only by
molecular synapomorphies. Although the embryo and nutlet
epidermal cell shape characters chosen to differentiate
Schoenoplecteae from Pseudoschoeneae are subtle, they
are stable and permit tribal and generic assignments to be
tested independently of molecular data.
The recent recircumscription of tribe Scirpeae also resulted
in a small group of genera whose only consistently shared
character was their Schoenus‐or Fimbristylis‐type embryos
(Léveillé‐Bourret & Starr, 2019). Macromorphological charac-
ters are typically highly homoplastic when studied at higher
taxonomic levels in sedges (Bruhl, 1995; Simpson, 1995;
Muasya et al., 2000), which means the most recalcitrant
clades may need to be defined by obscure but consistent
features, in addition to characters that may not be shared by
all the taxa in a group (i.e., polythetic) (Larridon et al., 2018;
Léveillé‐Bourret & Starr, 2019; this study).
4.2 The classification of Fuirena and the role of perianth
characters
The unguiculate tepals of Fuirena are one of its most striking
characteristics (Goetghebeur, 1998), and previous studies
and classifications have accordingly put a strong emphasis on
patterns of variation in perianth morphology (Vrijdaghs
et al., 2004). While most of the c. 55 species of Fuirena
have such tepaloid perianth parts, a few possess only
bristles, show an intergradation between tepals and bristles,
or lack any trace of a perianth (Muasya, 1998). These atypical
species have sometimes been interpreted as evidence
against recognizing Fuirena at the generic level (e.g.,
Koyama, 1958), although most previous authors concluded
that the combination of leafy culms, ciliate ligules, paniculi-
form inflorescences, "bristly" spikelets, and a unique embryo
morphology was strong evidence that Fuirena was distinct
from all other sedge genera (Kral, 1978; Haines & Lye, 1983;
Muasya, 1998). There have also been some early contro-
versies about whether the tepals in Fuirena represented the
ancestral condition for Cyperaceae, inherited from lilioid
ancestors (Raynal, 1973), or whether they were derived from
bristles (Koyama, 1958), but one commonality in all
infrageneric classifications has been to segregate species
with tepaloid perianth parts from those without (Clarke,
1901–02, 1908; Chermezon, 1936; Muasya, 1998).
Our molecular phylogenetic results generally uphold the
most recent classification of Fuirena proposed by Oteng‐
Yeboah (1974), which was based on a combination of
perianth and vegetative features. Subgenus Pentasticha
was established for species lacking a tepaloid perianth and
possessing 3‐angled culms, and it was further subdivided into
section Pseudoscirpus when bristles were present and
section Pseudoisolepis when the perianth was absent.
Species with a tepaloid perianth and 5‐angled or terete
culms were placed in subgenus Fuirena when cauline leaves
were bladed and subgenus Vaginaria when they were
reduced to sheaths. The first major split seen in our
phylogenetic analyses of Fuirena puts all species with
3‐angled culms and various perianth morphologies, roughly
equivalent to subgenus Pentasticha, in a natural group sister
to another clade containing species with 5‐angled or terete
culms and a perianth of 3 bristles +3 tepals that correspond
to subgenus Fuirena.
J. Syst. Evol. 59(4): 809–832, 2021www.jse.ac.cn
823Targeted sequencing of Fuireneae s.l.
Within the Pentasticha Clade, we retrieved a mixture of
species that either lacked a perianth (Fuirena pubescens
(Poir.) Kunth; section Pseudoisolepis), possessed a
variable number of bristles, tepals, or intermediate forms
(F. ecklonii Nees, F. coerulescens Steud.), or displayed a
perianth of bristles alone (F. incompl eta Nees, F. stricta
Steud.; section Pseudoscirpus). The deeply nested posi-
tion of section Pseudoscirpus refutes previous hypotheses
positing that a bristles‐only perianth could represent a
“transitional”form between Fuirena and other scirpoid
genera (Koyama, 1958). In contrast, our results suggest
tepaloid inner perianth parts to be the ancestral
condition in Fuirena, although character state reconstruc-
tions using a greater taxonomic sampling, including often
important infraspecific variation (Gordon‐Gray, 1995;
Muasya, 1998), would be needed to clarify major
morphological trends. Moreover, the polyphyly of
accessions identified as F. coerulescens in our analyses
would be consistent with known polymorphisms in
perianth and vegetative characters and the existence of
morphological “clusters”within this taxon (Forbes, 1997).
Molecular studies on F. coerulescens and other morpho-
logically variable species might uncover unsuspected
diversity within this poorly studied genus.
Morphological patterns are easier to interpret within the
Fuirena Clade (subgenus Fuirena; Fig. 3), which is subdivided
into two sister subclades with clear diagnostic characters.
The first subclade comprises the type F. umbellata and a few
closely related species possessing highly compound, cor-
ymbiform inflorescences, and frequently 5‐angled culms
(Muasya, 1998; Kral, 1978, 2002). The second subclade
comprises all other species of subgenus Fuirena, with
generally reduced inflorescences of 1–3 glomerules of
spikelets and terete culms (Gordon‐Gray, 1995; Muasya, 1998;
Kral, 2002). These two clades could merit taxonomic
recognition if further taxonomic sampling and phylogenetic
analyses continue to support the morphological pattern
seen here.
Although unrepresented in our molecular analyses, the
status of subgenus Vaginaria is doubtful given the existence
of a possible stabilized hybrid (F. longa Chapm.) between the
type of subgenus Vaginaria,F. scirpoidea Michx., and F.
breviseta (Coville) Coville, a member of subgenus Fuirena
(Kral, 1978, 2002). However, before a formal revision of the
infrageneric classification of Fuirena can be proposed, future
studies will need to expand taxonomic sampling to cover the
diversity seen in all its subgenera and to include as many
species as possible with anomalous perianths. This is the
necessary first step to better understand the morphological
patterns of perianth evolution in this most unusual of sedge
genera.
4.3 The infrageneric classification and delimitation of
Schoenoplectus and Schoenoplectiella
As one of the largest segregates of Scirpus with at least
50 species (Goetghebeur, 1998), an early focus for the
genus Schoenoplectus was to create an infrageneric
classification to divide species into manageable groups
(e.g., Oteng‐Yeboah, 1974a; Raynal, 1976a, 1976b; Smith &
Hayasaka, 2001). Authors were basically unanimous in
recognizing three or four infrageneric taxa adopted from
Scirpus, although there was no agreement as to whether
these groups should be treated at the subgeneric or
sectional level. Whereas Oteng‐Yeboah (1974a) divided
Schoenoplectus into subgenera Schoenoplectus,Actaeo-
geton,andMalacogeton,Smith&Hayasaka(2001,2002)
treated these taxa as sections in addition to a fourth,
section Supini, a collection of 24 species largely from
Africa and Madagascar that were dominated by amphi-
carpic annuals. Authors also recognized that these four
sections could be divided into two distinct groups.
Section Malacogeton, a collection of four species from
East Asia and eastern North America with long leaves and
unique foliar anatomy, appeared to be closely allied with
the species of the widespread section Schoenoplectus on
the basis of embryo morphology (Van der Veken, 1965).
Embryo morphology and morphological characters also
clearly linked sections Actaeogeton and Supini,evento
thepointwheresomeauthorsfelttheyshouldbetreated
as a single section (e.g., Beetle, 1942; Koyama, 1958; Van
der Veken, 1965; Lye, 2003). With the segregation of
Schoenoplectiella from Schoenoplectus,sectionsSchoeno-
plectus and Malacogeton continued as infrageneric taxa
within Schoenoplectus, whereas the remaining sections
were transferred to Schoenoplectiella as sections Actaeo-
geton and Schoenoplectiella (=Supini;Lye,2003;Hay-
asaka, 2012).
Within Schoenoplectus, our targeted sequencing and
nrDNA analyses support the recognition of sections
Schoenoplectus and Malacogeton as both of these
sections correspond to strongly supported clades. These
results are consistent with previous molecular analyses
(Shiels et al., 2014; Glon et al., 2017) and they agree with
the morphology and micromorphology of both sections.
Whereas section Malacogeton possesses glumes with
prominent veins, entire or obscurely emarginate apices
and tubers often terminating rhizomes, in section
Schoenoplectus, veins are absent on glumes (except basal
glumes), apices are clearly emarginate to deeply bifidin
the vast majority of the species, and tubers are absent
(Smith & Hayasaka, 2001; Hayasaka, 2012). Moreover, if
the elongated epidermal nutlet cells of Schoenoplectus
nipponicus with their highly sinuous anticlinal walls are
shared by all members of section Malacogeton,thisdiffers
significantly from the mostly isodiametric and hexagonal
cells with straight to slightly undulate anticlinal walls
seen in section Schoenoplectus (e.g., Schuyler, 1971;
Hayasaka, 2012; Table S7).
Within Schoenoplectiella, our targeted sequencing
analyses support two clades as well. One poorly
supported clade corresponds almost perfectly to section
Schoenoplectiella,includingthetypeforthegenus,S.
articulata (Hayasaka, 2003). However, the second
strongly supported clade contains a taxonomically
confusing collection of species with the type for section
Actaeogeton (Schoenoplectiella mucronata (L.) J.Jung &
H.K.Choi) as sister to a clade comprising all the largely
African Schoenoplectus species we transfer to Schoeno-
plectiella s.l. plus Schoenoplectiella proxima. As a small,
amphicarpous annual with rugose nutlets, Schoenoplec-
tiella proxima clearly belongs in section Schoenoplectiella
sensu Raynal (1976a) and Hayasaka (2012), but here it is
J. Syst. Evol. 59(4): 809–832, 2021 www.jse.ac.cn
824 Starr et al.
nested within a clade dominated by large perennials
(generally >30 cm, except S. rhodesicus)lackingamphi-
carpy. In addition, several of the African Schoenoplectus
speciesinthiscladealsoconfusethecurrentdivisionof
Schoenoplectiella into two sections. For example, because
Smith & Hayasaka (2001) could not assign Schoenoplectus
muriculatus and S. paludicola to any of their four sections,
they concluded that a worldwide sectional revision might
reveal more, and if all the species in this clade were
simply assigned to sect. Actaeogeton, the unusual, semi‐
terrestrial to aquatic Schoenoplectus rhodesicus
(Browning, 2012) would be the only species in Actaeo-
geton to possess nodose foliated stems (i.e., Actaeogeton
=stems nodeless, all leaves basal; Smith & Hay-
asaka, 2001; Hayasaka, 2012). Given that our nrDNA
analyses positioned Actaeogeton (eight species) within a
grade initiated by sect. Schoenoplectiella species and that
both sections are difficult to separate (e.g., Hay-
asaka, 2012), one solution to the infrageneric classifica-
tion of Schoenoplectiella would be to avoid sections
altogether. However, due to the unusual species noted
aboveandthepoorcladesupportseeninnrDNA
analyses, such a solution would be unsatisfactory at this
point. The infrageneric classification of Schoenoplectiella
will only be resolved when a significant increase in
taxonomic sampling is accompanied by further molecular
and morphological studies.
5 Taxonomic Treatment
5.1 Key to the tribes of the Fuireneae s.l. grade
1a. Embryo 176–305(–382 μm) long, with scutellum 39%–60%
of total embryo length; bracts sheathing, leaf‐like, rarely
cusp‐like; leaves with well‐developed blades, hairy at
least at the junction of blade and sheath, rarely glabrous
when the blade is reduced to a mucronate sheath
....................................................................... Fuireneae s.s.
1b. Embryo 315–1269 μmlong,when<380 μm, scutellum is
28%–32% of total embryo length; bracts sheathless, leaf‐like
or appearing to be a continuation of the stem; leaves well‐
developed or reduced to sheaths, glabrous........................2
2a. Lowermost primary bract leaf‐like with spikelets 10–40 mm
long; embryo with three primordial leaves, notch below the
root cap present.............................................. Bolboschoeneae
2b. Lowermost primary bract patent to erect, but stem‐like,
when leaf‐like, patent to reflexed with spikelets to 5 mm
long; embryo with two primordial leaves, notch below
root cap absent.........................................................................3
3a. Embryo scutellum turbinate to rhomboid; nutlet epidermal
cells isodiametric to oblong or elliptic, 1.0–3.9 times longer
than wide, rarely elongated, up to 6.3 times longer than
wide (Schoenoplectus sect. Malacogeton); nutlet surface
smooth; basal flowers absent ....................................................
........................................................................... Schoenoplecteae
3b. Embryo scutellum umbonate or distinctly pileate; nutlet
epidermal cells linear, (8.0–)9.2–20.2 times longer than
wide, rarely isodiametric to oblong, 1.5–3.8 times longer
than wide (Pseudoschoenus inanis); nutlet surface
smooth or transversely rugose; basal flowers sometimes
present ...................................................Pseudoschoeneae
5.2 Tribal diagnoses for the members of the Fuireneae s.l.
grade
1. Bolboschoeneae (Tatanov) J.R.Starr, stat. nov.
≡Schoenoplecteae subtribe Bolboschoeninae Tatanov, in
Novosti Sist. Vyssh. Rast. 39: 33 (2007).
Type: Bolboschoenus (Asch.) Palla
Diagnosis: Differs from all other Cyperaceae tribes by this
unique combination of characters: Perennials with long
rhizomes often forming hard ovoid tubers at tips and nodes.
Culms many‐noded, 3‐sided, thickened at the base. Leaves
well‐developed, basal and cauline, eligulate with blade often
reduced in lower leaves. Inflorescence terminal (in reduced
inflorescences, bract may be erect, but clearly leaf‐like),
sometimes pseudolateral, (compound) anthelate or capitate
with 1 to many spikelets. Inflorescence bracts leaf‐like,
patent, lowermost often suberect. Spikelets with many
spirally arranged, deciduous glumes, each subtending a
flower. Glumes puberulent, the apex entire to emarginate
or deeply 2‐fid, awned, or mucronate. Flowers bisexual,
perianth present, formed by 3–6 parts, shorter to longer than
the nutlet, bristle‐like, deciduous with nutlet. Stamens 3.
Styles 2 or 3. Style base persistent, barely thickened, if at all.
Nutlets obovate, dorsiventrally lenticular or trigonous,
surface smooth with epidermal cells more or less isodia-
metric, 1.0–1.7 times longer than wide. Embryo fungiform
with three primordial leaves and a notch below the root cap
(Bolboschoenus‐type).
Accepted genus: Bolboschoenus (Asch.) Palla (15 species;
temperate to tropical regions worldwide. Fresh to brackish
water habitats, saline shores, and marshes along coasts and
inland).
2. Fuireneae Rchb. ex Fenzl, Gen. Pl.: 116 (1836).
Type: Fuirena Rottb.
Diagnosis: Differs from all other Cyperaceae tribes by this
unique combination of characters: Annuals or rhizomatous
perennials. Culms many‐noded, rarely scapose, 3–5 sided or
terete, sometimes thickened at the base. Leaves well‐
developed, rarely a mucronate sheath, basal and cauline,
ligule tubular, membranous, with blade often reduced in lower
leaves. Inflorescence terminal, paniculate to capitate with few
to many spikelets, rarely pseudolateral. Inflorescence bracts
leaf‐like, sheathing, lowermost bract sometimes erect, rarely
short and scale‐like. Spikelets with many spirally or rarely
pentastichously arranged, deciduous glumes, each subtending
aflower. Glumes often pubescent, the apex entire and
mucronate to awned. Flowers bisexual, perianth present, as
long or shorter than nutlet, formed by 3 parts, or when 6 in 2
whorls, the inner parts scale‐like, the outer parts bristle‐like,
rarely all parts reduced or absent or only 1 scale developed,
deciduous with the nutlet. Stamens 1 to 3. Styles 3. Style base
persistent, barely thickened, if at all. Nutlets obovate,
triquetrous to trigonous, smooth or variously ornamented
with epidermal cells roughly isodiametric or oblong to
elongated, often in transverse rows, 1.0–
7.2 times longer
than wide. Embryo turbinate to weakly fungiform with a
horizontally broadened scutellum, first leaf primordium not
strongly outgrown, the second leaf primordium either absent
or poorly developed (Fuirena‐type).
Accepted genus: Fuirena Rottb. (55 species; tropical and
warm temperate regions worldwide, especially in the
J. Syst. Evol. 59(4): 809–832, 2021www.jse.ac.cn
825Targeted sequencing of Fuireneae s.l.
Americas and Africa. Open, humid localities, often at low
altitude).
3. Schoenoplecteae Lye, in Blyttia 29: 147 (1971)
Type: Schoenoplectus (Reichenb.) Palla.
Diagnosis: Differs from all other Cyperaceae tribes by this
unique combination of characters: Perennials with long
rhizomes sometimes ending in tubers at tips. Culms nodeless,
scapose, trigonous to terete, thickened at the base. Leaves
usually reduced to a sheath, sometimes developing a ligulate
blade, but rarely well‐developed. Inflorescence pseudo-
lateral, rarely clearly terminal, anthelate or capitate with
(1‐)few to many spikelets. Inflorescence bracts often large,
erect, stem‐like, rarely leaf‐like and patent to reflexed
(Actinoscirpus). Spikelets with many spirally arranged,
deciduous glumes, each subtending a flower. Glumes
puberulent to glabrous, the margins often ciliate or laciniate
distally, apex entire to emarginate or deeply 2‐fid, awned or
mucronate. Flowers bisexual. Perianth present, formed by
(–5–)6 parts, smooth to retrorsely scabrid, bristle‐like or
sometimes plumose, longer or shorter than nutlet, deciduous
with nutlet. Stamens 2 or 3. Styles 2 to 3. Style base not
thickened, persistent. Nutlets smooth, obovate, trigonous, or
dorsiventrally lenticular, yellow to dark brown when mature.
Nutlet epidermal cells isodiametric to oblong or elliptic,
rarely elongated, 1.1–3.9(–6.3) times longer than wide.
Embryo fungiform, scutellum turbinate to rhomboid in
shape, root cap lateral, first (well‐developed) and second
embryonic leaves basal (Schoenoplectus‐type I).
Accepted genera: Actinoscirpus (Ohwi) R.W.Haines &
Lye (1 species; Tropical and subtropical Asia from India east
to China and south to Northeast Australia. Common in
swampy areas and ditches at low altitudes), Schoeno-
plectus (Rchb.) Palla (16 spp.; predominantly temperate.
Common in fresh and brackish wetland habitats, such as in
marshes, lakes, and along streams. Often emergent, rarely
submerged).
Key to Schoenoplecteae genera
1a. Inflorescence terminal; proximal bracts leaf‐like, patent to
reflexed, forming an involucre at the base of the
inflorescence.....................................................Actinoscirpus
1b. Inflorescence pseudolateral; proximal bract culm‐like,
erect, other proximal bracts (if present) scale‐like and
much reduced.............................................Schoenoplectus
4. Pseudoschoeneae J.R.Starr, tr. nov.
Type: Pseudoschoenus (C.B.Clarke) Oteng‐Yeb.
Diagnosis: Differs from all other Cyperaceae tribes by this
unique combination of characters: Annuals or perennials,
tufted or with firm, short to creeping rhizomes. Culms
nodeless and scapose or 1(–8) noded above the base,
trigonous, terete, or rarely 7‐sided. Leaves are reduced to a
mucronate sheath, rarely with well‐developed blades,
ligulate, or rarely eligulate (Pseudoschoenus). Inflorescence
pseudolateral, rarely appearing terminal, anthelate or
capitate with one to many spikelets, rarely compound
paniculate with a conspicuously sinuous main axis (Pseudo-
schoenus). Inflorescence bracts culm‐like, erect, or patent
while fruiting, rarely short, rigid, and sheathing, but then
appearing as a continuation of the stem. Spikelets with many
spirally arranged, deciduous or persistent glumes, each
subtending a flower. Glume apex entire to apiculate. Flowers
bisexual, rarely polygamo‐dioecious. Perianth present or
absent, formed by 0–10 parts, smooth or retrorsely scabrid,
bristle‐like, as long as or longer than the nutlet, deciduous
with the nutlet. Stamens 2 or 3, rarely vestigial in female
flowers. Basal flowers often present in the axil of leaf
sheaths. Styles 2 or 3. Style base undifferentiated, rarely
distinct and somewhat thickened, persistent. Nutlets are
smooth or transversely rugose to distinctly ridged, obovate,
trigonous to planoconvex or biconvex, dark nearing black
when mature, sometimes brown. Nutlets from basal flowers
(when present) are much larger and bear an elongated
lignified style (amphicarpy). Nutlet epidermal cells linear,
(8.00–)9.22–20.21 times longer than wide, rarely isodiametric
to oblong, 1.45–3.81 times longer than wide (Pseudo-
schoenus). Embryo fungiform, scutellum umbonate or
distinctly pileate, root cap lateral, first (well‐developed) and
second embryonic leaves basal (Schoenoplectus‐type II).
Accepted genera: Pseudoschoenus (C.B.Clarke) Oteng‐Yeb.
(1 species; Southern Africa, along streams at higher
altitudes), Schoenoplectiella Lye. (63 species; temperate to
tropical regions worldwide. Common in freshwater bogs,
lake‐edges and along streams, often in seasonally wet
habitats or places with large fluctuations in water levels.
Terrestrial to emergent, sometimes submerged.)
Key to Pseudoschoeneae genera
1a. Inflorescence paniculate or racemose, with a definite
sinuous main axis of well‐developed internodes
.....................................................................Pseudoschoenus
1b. Inflorescence anthelate or reduced to one or a cluster of
sessile spikelets, without a definite main axis due to
highly‐reduced internodes........................Schoenoplectiella
5.3 New combinations for species formerly placed in
Schoenoplectus
A recent study has merged several tropical African
Schoenoplectus with transversely rugose nutlets under the
name S. muricinux (Verloove et al., 2018). Although the
present study positions several of these taxa within the same
monophyletic group (e.g., S. confusus,S. muriculatus,
S. muricinux) it also places other broadly accepted taxa,
like S. decipiens (Browning, 1990; Gordon‐Gray, 1995) in the
same clade. Moreover, S. confusus var. rogersii, another
taxon in the synonymy of S. muricinux (Verloove et al., 2018),
is here shown to be sister to S. brachyceras, a widely
accepted taxon (Browning, 1992; Jiménez‐Mejías & Cabezas,
2009) also recognized as close to S. muricinux (Verloove
et al., 2018), but not placed in its synonymy. Although it is
clear that the limits of many of these taxa are not well
defined, we re‐adopt Browning's (Browning, 1991a) treat-
ment of S. muricinux and its allies until molecular, and
additional morphological and micromorphological data can
be applied to the problem.
Schoenoplectiella annamica (Raymond) J.R.Starr, comb. nov.
Scirpus annamicus Raymond, Naturaliste Canad. 84: 137
(1957). [basionym]
Schoenoplectus annamicus (Raymond) T.Koyama, Brittonia
31: 291 (1979).
J. Syst. Evol. 59(4): 809–832, 2021 www.jse.ac.cn
826 Starr et al.
Note: Koyama (1979) regarded this species as a member of
section Actaeogeton, similar to the North American S. hallii.
Distribution: Vietnam
Schoenoplectiella brachyceras (Hochst. ex A.Rich.) J.R.Starr &
Jim.Mejías, comb. nov.
Scirpus brachyceras Hochst. ex A.Rich., Tent. Fl. Abyss. 2:
496 (1850). [basionym]
Schoenoplectus brachyceras (Hochst. ex A.Rich.) Lye, Bot.
Not. 124: 290 (1971).
Note: Often considered conspecific with S. corymbosus,
previous work has demonstrated it is distinct from S.
corymbosus (Browning, 1992; Jiménez‐Mejías & Ca-
bezas, 2009). Samples of S. corymbosus and S. brachyceras
were not sisters in our analyses.
Distribution: Southern Africa north to Ethiopia
Schoenoplectiella confusa (N.E.Br.) J.R.Starr, comb. nov.
Scirpus confusus N.E.Br., Bull. Misc. Inform. Kew 1921: 300
(1921). [basionym]
Schoenoplectus confusus (N.E.Br.) Lye, Bot. Not. 124:
290 (1971).
Distribution: Southern Africa north to Ethiopia
Schoenoplectiella confusa subsp. natalitia (Browning)
J.R.Starr, comb. nov.
Schoenoplectus confusus subsp. natalitius Browning, S.
African J. Bot. 57: 258 (1991). [basyonym]
Distribution: South Africa
Schoenoplectiella corymbosa (Roth ex Roem. & Schult.)
J.R.Starr & Jim.Mejías, comb. nov.
Isolepis corymbosa Roth ex Roem. & Schult., Syst. Veg., ed.
15 bis 2: 110 (1817). [basionym]
Schoenoplectus corymbosus (Roth ex Roem. & Schult.)
J.Raynal in B.Peyre de Fabregues & J.P.Lebrun, Cat. Pl. Vasc.
Niger: 343 (1976).
Note: Proliferous inflorescences bearing plantlets at their
apex have been observed in this species (Fig. 1L). To date,
this characteristic has only been observed in Schoenoplec-
tiella species, but never in Schoenoplectus s.s. (Hay-
asaka, 2012).
Distribution: Southern Africa north to Spain and east to
India
Schoenoplectiella decipiens (Nees) J.R.Starr, comb. nov.
Isolepis decipiens Nees, Linnaea 10: 157 (1835). [basionym]
Schoenoplectus decipiens (Nees) J.Raynal, Adansonia, n.s.,
15: 540 (1976).
Distribution: Southern Africa to Madagascar
Schoenoplectiella heptangularis (Cabezas & Jim.Mejías)
J.R.Starr & Jim.Mejías, comb. nov.
Schoenoplectus heptangularis Cabezas & Jim.Mejías, Can-
dollea 64: 109 (2009). [basionym]
Distribution: Equatorial Guinea
Schoenoplectiella monocephala (J.Q.He) J.R.Starr, comb. nov.
Scirpus monocephalus J.Q.He, Acta Phytotax. Sin. 37: 291
(1999). [basionym]
Schoenoplectus monocephalus (J.Q.He) S.Yun Liang &
S.R.Zhang, Novon 20: 170 (2010).
Distribution: Eastern China
Schoenoplectiella muricinux (C.B.Clarke) J.R.Starr, comb. nov.
Scirpus muricinux C.B.Clarke, Bot. Jahrb. Syst. 38: 135
(1906). [basionym]
Schoenoplectus muricinux (C.B.Clarke) J.Raynal, Adansonia,
n.s., 15: 538 (1976).
Distribution: Southern Africa
Schoenoplectiella muriculata (Kük.) J.R.Starr, comb. nov.
Scirpus muriculatus Kük., Bot. Not. 1934: 75 (1934).
[basionym]
Schoenoplectus muriculatus (Kük.) Browning, S. African J.
Bot. 57: 254 (1991).
Distribution: Southern Africa
Schoenoplectiella paludicola (Kunth) J.R.Starr, comb. nov.
Scirpus paludicola Kunth, Enum. Pl. 2: 163 (1837).
[basionym]
Schoenoplectus paludicola (Kunth) Palla, Bot. Jahrb. Syst.
10: 299 (1888).
Distribution: Southern Africa
Schoenoplectiella pulchella (Kunth) J.R.Starr, comb. nov.
Ficinia pulchella Kunth, Enum. Pl. 2: 261 (1837). [basionym]
Scirpus pulchellus (Kunth) Boeckeler, Linnaea 36: 698
(1870).
Schoenoplectus pulchellus (Kunth) J.Raynal, Adansonia,
n.s., 15: 542 (1976).
Distribution: Southern Africa
Schoenoplectiella rogersii (N.E.Br.) J.R.Starr, comb. nov.
Scirpus rogersii N.E.Br., Bull. Misc. Inform. Kew 1921: 301
(1921). [basionym]
Schoenoplectus rogersii (N.E.Br.)Lye,Bot.Not.124:290(1971).
Schoenoplectus confusus var. rogersii (N.E.Br.) Lye, Nordic
J. Bot. 3: 242 (1983).
Distribution: Botswana north to Kenya
Schoenoplectiella rhodesica (Podlech) J.R.Starr, comb. nov.
Scirpus rhodesicus Podlech, Mitt. Bot. Staatssamml.
München 4: 117 (1961).
Schoenoplectus rhodesicus (Podlech) Lye, Nordic J. Bot. 3:
242 (1983).
Distribution: Zimbabwe north to Tanzania
Acknowledgements
This study was supported by the National Science and
Engineering Research Council (NSERC) of Canada Discovery
Grant to JRS (RGPIN 2018‐04115), enabling a 6‐week research
stay at the Royal Botanic Gardens, Kew, covering part of the
laboratory work and sequencing costs. The remaining costs
were covered by the Calleva Foundation and the Sackler
Trust through the Plant and Fungal Trees of Life programme
(https://www.kew.org/science/our‐science/projects/plant‐
and‐fungal‐trees‐of‐life), and a grant to IL from the Royal
Botanic Gardens, Kew to support monographic research
using targeted sequencing. We thank Prof. Eisuke Hayasaka
and the Journal of Japanese Botany for permission to
reproduce SEM micrographs. We also thank Russell Barrett,
Jane Browning, Marcial Escudero, Marie‐Ève Garon‐
Labrecque, Modesto Luceño, Jean Marc Vallières, and Peter
J. Syst. Evol. 59(4): 809–832, 2021www.jse.ac.cn
827Targeted sequencing of Fuireneae s.l.
M. Dziuk from www.minnesotawildflowers.info for permis-
sion to reproduce images for our figures.
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Supplementary Material
The following supplementary material is available online for
this article at http://onlinelibrary.wiley.com/doi/10.1111/jse.
12721/suppinfo:
Data S1. Alignment with 127 accessions obtained from nrDNA
off‐target reads and ITS sequences available from GenBank.
Data S2. Reduced sampling alignment of 62 accessions from
nrDNA off‐target reads and ITS sequences available from
GenBank.
Data S3. List of all taxa for which embryo data was obtained
for this study with their voucher information, literature
source, and geographic origin (when known).
Fig. S1. Landmark positions on the embryo of Bolbo-
schoenus maritimus. Type 1 and 2 landmarks are
symbolized by blue points with numbers, and they are
placed on (1) root cap lower margin, (2) root cap apex, (3)
root cap top margin, (4) junction of scutellum and
hypocotyl on the left side (local minimum in curvature),
(5) scutellum left side maximum width, (6) scutellum
apex, (7) scutellum right side maximum width, (8)
junction of scutellum and hypocotyl on the right side
(local minimum in curvature), (9) left coleoptile lip base
on abaxial face (aligned with landmark #11), (10) left
coleoptile lip apex, (11) junction of left coleoptile lip and
plumule, (12) plumular leaf 2 apex, (13) plumular leaf 1
base on the adaxial side, (14) plumular leaf 1 apex, (15)
junction of plumule and right coleoptile lip, (16) right
coleoptile lip apex, and (17) right coleoptile lip base on
the abaxial side (aligned with landmark 13). In addition,
seven pseudolandmarks are placed at an equal distance
along the green curves separating 10 pairs of landmarks:
4–5, 5–6, 6–7, 7–8, 9–10, 10–11, 13–14, 14–15, 15–16, and
16–17. This gives a total of 17 landmarks and 70
pseudolandmarks. The length of the scutellum was also
measured, as indicated.
Fig. S2. A heatmap of recovery of the Angiosperms353
probes for the accessions included in this study.
Fig. S3. Phylogenetic reconstruction of the relationships in
tribe Fuireneae s.l. and related tribes based on analysis of the
exons data set. Species tree inferred in ASTRAL from RAxML
gene trees. Numbers by branches represent local posterior
J. Syst. Evol. 59(4): 809–832, 2021www.jse.ac.cn
831Targeted sequencing of Fuireneae s.l.
probabilities (LPPs) and pie charts at nodes correspond to
quartet support with blue for agreeing genes, red for
disagreeing genes, and gray for uninformative genes.
Fig. S4. Phylogenetic reconstruction of the relationships in
tribe Fuireneae s.l. and related tribes based on analysis of the
exons data set. Concatenated IQ‐TREE analysis from RAxML
gene trees. Numbers by nodes represent bootstrap support.
Fig. S5. Phylogenetic reconstruction of the relationships in
tribe Fuireneae s.l. and related tribes based on analysis of the
supercontigs data set. Concatenated IQ‐TREE analysis from
RAxML gene trees. Numbers by nodes represent bootstrap
support.
Fig. S6. Phylogenetic reconstruction of the relationships
in tribe Fuireneae s.l. and related tribes based on a
RAxML analysis of the alignment with 137 accessions
obtained from nrDNA off‐target reads and ITS sequences
available from GenBank. Numbers by nodes represent
bootstrap support.
Table S1. Overview of the species of Schoenoplectus and
Schoenoplectiella, their classificationand distribution range
(Govaerts et al., 2020). *Available from GenBank, **se-
quenced for this study, ***sequenced in both.
Table S2. Voucher information for the accessions included
in the targeted sequencing study.Taxon names are in
accordance with Govaerts et al. (2020) and this study. The
voucher information includes collector, collector number,
herbarium codes according to Thiers (continuously
updated), and when available the specimen's herbarium
barcode. Origin information refers to whether DNA was
extracted from leaf samples taken in the herbarium or
stored in silica gel, or whether it originated from the Kew
DNA Bank (numbers are provided). Type species for
genera and sections are indicated under the column for
notes.
Table S3. Voucher information for accessions used in the ITS
analysis, including GenBank accession numbers for Sanger
sequencing data, or laboratory ID numbers (under “Number”
column) for data obtained from off‐target sequencing reads).
Table S4. Percentage recovery of the genes targeted by the
Angiosperms353 probes for the accessions included in this
study.
Table S5. AMAS statistics generated for the exons data set
(A), and supercontigs data set (B).
Table S6. AMAS statistics generated for the trimmed exons
dataset (A), and trimmed supercontigs data set (B).
Table S7. Overview of nutlet pericarp characters. Note
that for references followed by an asterisk “*”,species
were placed in broad groups for which a single
representative illustration was given. Species are not
listed from publications where illustrations were not
sufficiently resolved to determine nutlet epidermal cell
shape.
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832 Starr et al.