Biodiversity Data Journal 9: e61333 'e @)
doi: 10.3897/BDJ.9.e61333 open access
Research Article

DNA barcoding of native Caucasus herbal plants:
potentials and limitations in complex groups and
implications for phylogeographic patterns

Parvin Aghayeva?, Salvatore Cozzolino’, Donata Cafasso§, Valida Ali-zade?,
Silvia Fineschil, Dilzara Aghayeva*

¢ Institute of Botany, Azerbaijan National Academy of Sciences, Baku, Azerbaijan
§ Department of Biology, University of Naples Federico Il, Complesso Universitario di Monte S. Angelo, Napoli, Italy
| CNR - Istituto di Scienze del Patrimonio Culturale, Sesto Fiorentino, Italy

Corresponding author: Dilzara Aghayeva (a_dilzara@yahoo.com)

Academic editor: Anatoliy Khapugin

Received: 24 Nov 2020 | Accepted: 07 Jan 2021 | Published: 27 Jan 2021

Citation: Aghayeva P, Cozzolino S, Cafasso D, Ali-zade V, Fineschi S, Aghayeva D (2021) DNA barcoding of

native Caucasus herbal plants: potentials and limitations in complex groups and implications for
phylogeographic patterns. Biodiversity Data Journal 9: e61333. https://doi.org/10.3897/BDJ.9.e61333

Abstract

DNA barcoding has rapidly become a useful complementary tool in floristic investigations
particularly for identifying specimens that lack diagnostic characters. Here, we assess the
capability of three DNA barcode markers (chloroplast rooB, accD and nuclear ITS) for
correct species assignment in a floristic survey on the Caucasus. We focused on two
herbal groups with potential for ornamental applications, namely orchids and asterids. On
these two plant groups, we tested whether our selection of barcode markers allows
identification of the “barcoding gap” in sequence identity and to distinguish between
monophyletic species when employing distance-based methods. All markers successfully
amplified most specimens, but we found that the rate of species-level resolution amongst
selected markers largely varied in the two plant groups. Overall, for both lineages, plastid
markers had a species-level assignment success rate lower than the nuclear ITS marker.
The latter confirmed, in orchids, both the existence of a barcoding gap and that all
accessions of the same species clustered together in monophyletic groups. Further, it also
allowed the detection of a phylogeographic signal.The ITS marker resulted in its being the
best performing barcode for asterids; however, none of the three tested markers showed

© Aghayeva P et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC
BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are
credited.

2 Aghayeva P et al

high discriminatory ability. Even if ITS were revealed as the most promising plant barcode
marker, we argue that the ability of this barcode for species assignment is strongly
dependent on the evolutionary history of the investigated plant lineage.

Keywords

accD, asterids, Azerbaijan, barcoding identification, Caucasus, floristic surveys, /TS, native
plants, orchids, rooB

Introduction

DNA barcoding in botany has rapidly spread as a reliable tool for the accurate identification
of plant species or genus (Hebert et al. 2004), as well as for determining the origin of
plants and their derivatives (Galimberti et al. 2019, Saravanan et al. 2019). Several studies
highlighted the potential ecological applications of DNA barcoding in biodiversity
assessments of both existing and past communities (Valentini et al. 2009). It was largely
utilised in studies on local floras and plant communities for identifying specimens that are
hard to recognise by morphological characters or that lack diagnostic floral characters
(such as rarely blooming species or species with a short blooming period and/or brief
juvenile stages) including identification of cryptic species (Xu et al. 2018). DNA barcoding
allows potentially higher levels of species discrimination, particularly at regional floristic
level; in fact, a geographically-restricted context usually contains fewer closely-related
species than a comprehensive taxonomic treatment (Kress et al. 2009). At a local scale,
the approach is particularly reliable when combined with the development of localised
barcoding libraries for determining the identity of unknown samples (Chase and Fay 2009,
Kress et al. 2009). Accordingly, the capacity of DNA barcoding in resolving species in local
floras has been tested in many plant groups, including species-rich tropical communities
(Ebihara et al. 2010, Burgess et al. 2011, Costion et al. 2016). These studies have also
demonstrated that combined chloroplast and nuclear markers provided additional
discriminatory power and increased percentage of success in species-level assignment,
compared to the more traditional two-locus (robcL and matK) barcode (Vijayan and Tsou
2010). Due to the high rate of nucleotide substitution, the relatively easy amplification and
the large sequence data already available, the internal transcribed spacer (ITS) regions of
the nuclear ribosomal cistron (18S-5.8S-26S) have been very successful at species-level
discrimination across flowering plants (Li et al. 2011, Feng et al. 2016, Hosseinzadeh-
Colagar et al. 2016a). Nuclear barcodes are particularly useful for cases of recent
hybridisation or ongoing introgression, because they can recover different allelic variants
from a sample (Chase and Fay 2009). Thus, nuclear markers have been usually combined
with (haploid) plastid markers in most DNA barcoding studies (Hosseinzadeh-Colagar et al.
2016b, Castro et al. 2015). Indeed, the adaptation of a multi-locus barcoding system, with
at least two markers, each representing a distinct DNA source as nuclear and organellar
genome, could contribute to the gathering of independent evidence of the species
attribution and accessions relationships from independent gene trees (Moore 1995, Hu et
al. 2015). Finally, barcode markers may also eventually show consistent intra-specific

DNA barcoding of native Caucasus herbal plants: potentials and limitations ... 3

variability (Hollingsworth et al. 2011). In that case and with a sampling representative of
species distribution, haplotypic structure within a species can allow allocation of an
individual to a geographic region and identify potential phylogeographic routes (Huemer
and Hebert 2011).

The Caucasus represents one of the twenty-six biodiversity hotspot areas worldwide and
has been the subject of botanical investigation since the beginning of the last century
(Grossheim 1949, Karjagin 1928, Tutayuk et al. 1961). Recent research confirmed the
Caucasus as part of the European flora (Bohn et al. 2007); indeed, many European plant
lineages have close relatives in this region, including several domesticated plant species.
The Caucasian flora represents a wonderful source of new food and medicinal plants and
of new ornamentals with high adaptation potential in European gardens. Herbaceous
monocots and dicots, particularly ornamental ones, are very numerous in the flora of the
Caucasus and are characterised by low maintenance requirements. Indeed, these plants
display high tolerance to environmental stresses as required for ornamental plants in the
Mediterranean regions (Heywood 2003, Gray and Brady 2016).

Here, we employed DNA barcoding with the aim of investigating and quantifying plant
diversity in the Quba and Qusar districts of Azerbaijan Caucasus. DNA-based methods are
being increasingly used in floristic analyses, because they are not limited by taxonomic
hindrances, such as: missing morphological features at any life stage (Wells and Stevens
2008, Ebihara et al. 2010); absence of distinctive identification characters in young or
immature plants; and homoplasy of some characters (Vences et al. 2005). Nevertheless,
the approach has some limitations when applied in the same critical groups as herbal
species, particularly in the ability of species discrimination when closely-related species are
examined (Chase and Fay 2009, Hubert and Hanner 2015). Here, we focused on two
lineages, orchids and asterids, which are particularly interesting as they contain many
ornamental species. We tested the potential of DNA barcodes for identifying unknown plant
specimens and for identifying phylogenetic/phylogeographic relatedness with allied species
and populations of other geographic origins. For this aim, we chose a combination of
nuclear and plastid barcodes (ITS and chloroplast rp0oB, accD), because DNA barcoding is
particularly challenging when hybridisation might occur in conjunction with potential plastid
capture or when lineage sorting has not yet been completed because of recent, rapid
radiation (Fazekas et al. 2009, Chen et al. 2010), as expected in orchids and asterids. In
particular, we tested whether the selection of barcode markers allows: i) the identification of
the “barcoding gap” (Meyer and Paulay 2005) i.e. that the variation of the nucleotide
sequences within species is lower than the differences amongst species and ii) the
distinction between species, based on monophyletic clustering in distance-based
neighbour-joining (NJ) trees (Hebert et al. 2004).

Material and methods

Study area. Qusar and Quba districts are located between 500—4466 m above sea level in
the in the south macro-slope of the Greater Caucasus and north-eastern part of Azerbaijan.
These districts spread along various altitudinal zonations (foothills, low, middle and high

4 Aghayeva P et al

mountain zones, subalpine, alpine habitats) and represent the richest floristic part of the
country. The climate of the districts in summer is dry in the meadows and moderately hot in
the foothills, whereas it is cold and very humid in the highlands and winter is usually cold.
In the past couple of decades, increasing anthropogenic impacts, along with climate
change, has contributed to the ecosystem degradation in these two districts.

Sampling. Approximately 500 ornamental herb specimens were collected during a floristic
sampling campaign from 2012-2018 and were identified by means of morphological traits
as belonging to 229 taxa, which are detailed as: 23 orders, 39 families and 129 genera.
Morphologic identification was performed either by visual analysis or by using a dissection
microscope, based on reliable diagnostic characters. Available checklists and recent
literature on local floras (Karjagin 1928, Karjagin 1950, Aghayeva et al. 2018, Alizade et al.
2019, Tutayuk et al. 1961) were utilised as reference. The species status was further
checked in the “World Flora Online” (http:/\www.worldfloraonline.org). Within this floristic
survey, we selected altogether 54 accessions which were not clearly classified according to
distinctive morphological characters. Thirty out of fifty-four accessions were roughly
classified as Orchids and twenty-four accessions were roughly classified as Asterids. A
small portion of leaf was preserved in silica gel and a barcode approach was performed as
described below. All sampled specimens were collected in a herbarium within the
Herbarium of the Institute of Botany, ANAS (BAK). We also had access to twenty
herbarium vouchers of orchids and asterids, previously sampled from the same region and
collected a small portion of dry specimens for performing the same barcode analysis.

DNA isolation, amplification and sequencing. Dried leaves from both field collection and
herbarium samples were ground in a Tissue-lyser (Qiagen) and total DNA was extracted
using GenElute™ Plant Genomic DNA Miniprep Kit (Sigma) following the manufacturer’s
instructions. The nuclear ribosomal DNA (internal transcribed spacer regions ITS1 and
ITS2) was amplified with primers described by Aceto et al. 1999). For plastid barcode
analysis, the two coding regions ro0oB (RNA polymerase subunit) and accD (acetyl-CoA
carboxylase subunit) were amplified with specific primers (Sequences available at
http:/Awww.kew.org; barcoding/protocol.htlm). All PCRs were performed in a final reaction
volume of 25 ul using about 10 ng of template DNA, 200 mM of each dNTP, 10 pmol of
each of the two primers, 1 Tag buffer (50 mM KCI, 10 mM Tris—HCI pH 9.0), 1.5 mM MgCl
2 and 0.3 U of Tag polymerase (Sigma). Amplification of all barcodes was performed using
the following protocol: initial denaturation at 94°C for 3 min, 35 cycles of denaturation at
94°C for 30 s, annealing at 53°C for 45 s and extension at 72°C for 1 min, followed by a
final extension at 72°C for 7 min and final hold at 4°C. Amplification products were
visualised on a 1.5% agarose gel and photographed after ethidium bromide staining. All
successfully amplified DNA fragments were purified using the Clean Sweep PCR
Purification Kit (Life Technology), following the manufacturer’s instructions and then
sequenced in both directions using a modification of the Sanger dideoxy method as
implemented in a double-stranded DNA cycle sequencing system with fluorescent dyes.
Sequence reactions were then run on a 3130 Automated sequence system (Applied
Biosystem).

DNA barcoding of native Caucasus herbal plants: potentials and limitations ... 5

Sequence editing and alignment were performed by using BioEdit v.7.2.0 (Hall 2018). The
species discrimination ability of each barcode marker was evaluated using GenBank
(http://www.ncbi.nim.nih.gov), a public available nucleotide sequences database. For
species assessment, the database was screened for the presence of each of the marker
sequences at the species or genus level relative to our dataset, using BLAST
(https://blast.ncbi.nim.nih.gov/Blast.cgi). We considered as correct assignment when the
query sequence has at least 99.5% of identical sites to the reference sequences in the
database and when the top Bit-Score obtained in the GenBank matched the name of a
single species. When the closest reference sequence scored lower than 99.5%, the result
was considered as incomplete identification and imputable to the absence of the specific
reference sequence in the database. Instead, when multiple reference sequences (i.e. from
different species) shared the same top Bit-Score to the query sequence, the result was
considered as incomplete identification due to insufficient discrimination power of the
selected barcode.

Generated sequences and closest reference sequences (i.e. those identified by using
BLAST and assigned to the same species) were aligned by using the MUSCLE
programme in Mega X. For each barcode marker, a distance-based neighbour-joining (NJ)
tree was then built with the Maximum Composite Likelihood model, uniform rates amongst
sites and pairwise deletion in the gaps, for giving a graphic representation of the genetic
distances within and amongst species.

Data resources

Herbarium of the Institute of Botany, ANAS (BAK)

Dryad Data Repository - doi: 10.5061/dryad.2ngfivhmw

Results

In total, we examined 24 fresh samples and 14 herbarium vouchers for asterids and 30
fresh samples and six herbarium vouchers for orchids, respectively. We successfully
amplified and sequenced all asterids, whereas two collected samples of the orchids
dataset did not amplify with any marker and four other samples failed amplification across
the three gene regions. Sequence recovery was slightly higher for plastid rooB (88.8%
samples) than for ITS (83.3% samples) markers (Tables 1, 2). All herbarium material from
both plant lineages was successfully amplified and sequenced with selected barcode
markers.

6 Aghayeva P et al

Table 1.

Sequence recovery for the three selected barcode regions from unknown (G1-G38) and Herbarium
orchid samples.

Sample ITS rpoB accD

amplification sequencing amplification sequencing amplification sequencing

G1 x x x x x x
G3 Xx Xx Xx Xx x Xx
G4 x x x x x x
G5 Xx Xx x Xx Xx Xx
G6 x x Xx x Xx Xx
G7 x x x x x Xx
G8 NO NO x x x NO
G9 Xx x x Xx Xx Xx
G10 x x Xx x x Xx
G11 x x x x Xx Xx
G12 x x x x Xx Xx
G13 NO NO x Xx Xx NO
G14 x x x x Xx Xx
G15 x Xx x x Xx Xx
G16 Xx Xx Xx x Xx Xx
G17 x x x x x Xx
G18 Xx Xx Xx Xx Xx x
G19 Xx Xx x Xx Xx x
G27 x x x x Xx Xx
G28 NO NO NO NO NO NO
G29 Xx x x x Xx x
G30 NO NO NO NO NO NO
G31 Xx Xx Xx Xx Xx x
G32 Xx Xx x Xx x Xx
G33 NO NO Xx NO Xx NO
G34 NO NO Xx NO Xx NO
G35 Xx Xx Xx Xx x x
G36 Xx x Xx x Xx x
G37 x x Xx Xx Xx x
G38 Xx Xx x x Xx x
Orchis purpurea Herbarium 1 x x x x x x

Orchis purpurea Herbarium 2 x x x x x x

DNA barcoding of native Caucasus herbal plants: potentials and limitations ... 7

Sample ITS rpoB accD

amplification sequencing amplification sequencing amplification sequencing

Orchis simia Herbarium 80873 x x x x x x
Orchis simia Herbarium 80876 x x x x x x
Orchis mascula Herbarium x x x x x x
80801

Orchis mascula Herbarium x x x x x x
80797

30/36 83.3% 32/36 88.8% 30/36 83.3%

Table 2.

Sequence recovery for the three selected barcode regions from unknown (P1-A15) and Herbarium
asterid samples.

Sample ITS rpoB accD

amplification sequencing amplification sequencing amplification sequencing

P1 x x x x x x
P2 x x x x x x
P3 x x x x x x
P4 x x x x x x
P5 x x x x x x
P6 x x x x x x
P7 x x x x x x
P8 x x x x x x
P9 x x x x x x
Al x x x x x x
A2 x x x x x x
A3 x x x x x x
A4 x x x x x x
A5 x x x x x x
A6 x x x x x x
A7 x x x x x x
A8& x x x x x x
AQ x x x x x x
A10 x x x x x x
A11 x x x x x x
A12 x x x x x x
A13 x x x x x x

A14 x x x x x x

8 Aghayeva P et al

Sample ITS rpoB accD
amplification sequencing amplification sequencing amplification sequencing
A15 x x x x x x

Centaurea trinervia x x x x x x
Herbarium 18066

Centaurea trinervia x x x x x x
Herbarium 18067

Psephellus hymenolepis x x x x x x
Herbarium 22220

Psephellus daghestanicus x x x x x x
Herbarium 22262

Psephellus dealbatus x x x x x x
Herbarium 22213

Psephellus intergrifolius x x x x x x
Herbarium 18082

Psephellus xantocephalus x x x x x x
Herbarium 18471

Psephellus transcaucasicus x x x x x x
Herbarium 22234

Psephellus transcaucasicus x x x x x x
Herbarium 22256

Pyrethrum carneum x x x x x x
Herbarium 22357

Taraxacum officinale x x x x x x
Herbarium 24510

Senecio vernalis x x x x x x
Herbarium
Bellis perennis x x x x x x

Herbarium 170015

Centaurea cheiranthifolius x x x x x x
Herbarium

38/38 100% 38/38 100% 38/38 100%

Local intraspecific variation for plastid barcodes was detected when multiple records were
examined. In orchids, more than one haplotype for accD were detected in O. purpurea and
O. militaris (Fig. 1) and different haplotypes for rooB were detected in O. mascula and A.
pyramidalis (Fig. 2). ITS base variation was detected in O. mascula and an ITS paralogy
was detected in O. purpurea (Fig. 3).

In asterids, variation for plastid accD was detected within genera (Psephellus,
Leucanthemum), but not within species, with the notable exception of two haplotypes found
in Bellis perennis (Fig. 4). No intraspecific and only very low interspecific variation (i.e.
within genera) was detected for rpoB (Fig. 5). ITS variation within species was only
detected between herbarium and wild-collected Senecio vernalis (Fig. 6).

DNA barcoding of native Caucasus herbal plants: potentials and limitations ...

Orchis purpurea- herbarium1

Orchis purpurea- herbarium2

G38
G37
G35
G31
-—— G27
—— G32
G6
G29
G11
G36
G5
G4
G10
Orchis mascula-herbarium-80801

Orchis mascula-herbarium-80797

Dactylorhiza viridis-MN200382.1
Orchis simia-herbarium-80876

Gi7

Orchis simia-herbarium-80873

Ophrys sphegodes-AP018717.1

Ophrys fusca-AP018716.1

G15

Cephalanthera damasonium-NC 041179.1

Cephalanthera longifolia-NC 030704.1
Cephalanthera rubra-NC 041181.1
Cephalanthera longibracteata-MH590346.1

G3

Platanthera mandarinorum-NC 046805.1
Dactylorhiza majalis-NC 044644.1
G7

G9

G12

G14

Platanthera mandarinorum-MN200370.1

Dactylorhiza majalis-MK984209.1
Platanthera chlorantha-NC 044626.1
iz Platanthera chlorantha-MK937914.1
Gymnadenia conopsea-NC 046820.1

Gymnadenia conopsea-MN200391.1

G18

| Gymnadenia nigra-AM883516.1
Gymnadenia borealis-AM883512.1

Dactylorhiza viridis-AM883526.1

Dactylorhiza euxina-AM883518.1
Dactylorhiza incarnata-AM88351 1.1
Dactylorhiza iberica-AM883509.1
G19

Dactylorhiza saccifera-AM883527.1

Platanthera japonica-MN631092.1

+ + + + 1
0.0200 0.0150 0.0100 .0060 0.0000

Figure 1. EES]

Neighbour-joining phylogenetic tree, based on accD sequences of selected orchids. All
sequences have been deposited in the Dryad Data Repository - doi: 10.5061/dryad.

2ngf1vhmw

10 Aghayeva P et al

Gymnadenia nigra-AM883624.1
Dactylorhiza incamata subsp. ochroleuca-AM883629.1
Dactylorhiza majalis-MK984209.1

Dactylorhiza romana-AM883634.1

Dactylorhiza incarnata subsp. pulchella-AM883618.1
+ Dactylorhiza euxina-AM883626.1

Dactylorhiza viridis var. coreana-MN200382.1

+ Dactylorhiza iberica-AM883616.1

Dactylorhiza majalis-AM883621.1

Dactylorhiza majalis-NC 044644.1

Dactylorhiza fuchsii-AM883620.1

G18

Gymnadenia conopsea-NC046820.1

Gymnadenia conopsea-MIN200391.1

Gymnadenia borealis-AM883619.1

Gymnadenia conopsea-AM883628.1

G19

Dactylorhiza incarnata-AM882622

Dactylorhiza alpestris-AM883617.1

Dactylorhiza foliosa-AM883615.1

Dactylorhiza foliosa-GQ248762.1
Platanthera chlorantha-NC044626.1
Platanthera chlorantha-MK937914.1
G16
Gi
Platanthera mandarinorum-NC046805
Platanthera mandarinorum-MIN200370
Platanthera japonica-N631092.1
Platanthera japonica-NC037440.1
Cephalanthera rubra-MH590347.1
Cephalanthera longibracteata-MH590346.1
Cephalanthera longibracteata-NC 041180.1
Cephalanthera rubra-041181.1
G15
Cephalanthera damasonium-NC 041179.1
Cephalanthera damasonium-MH590345.1
Cephalanthera humilis-KU551265.1
Cephalanthera longifolia-KU551263.1
Git

Orchis mascula-herbarium-80801

Orchis mascula-herbarium-80797

G10
G3
G4
G27
G29
G31
G32
G35
G36
Ophrys sphegodes-AP018717.1
il Ophrys araneola~-GQ248828.1
Ophrys fusca-AP018716.1
— GI7
G7
th oe
G14
G12
G13
G8
G37
G38
G5
G6
Orchis simia-herbarium-80873
Orchis simia-herbarium-80876
Orchis purpurea-herbarium
Orchis purpurea-herbarium(2)
|

0.040 0,030 0.020 0.010 0.000

Figure 2. EES)

Neighbour-joining phylogenetic tree, based on rpoB sequences of selected orchids. All
sequences have been deposited in the Dryad Data Repository - doi: 10.5061/dryad.

2ngf1vhmw

DNA barcoding of native Caucasus herbal plants: potentials and limitations ...

Ee
eiewer tt
_

i

Lean eer
=
nasi
cacao
| esses
sidtatasriean
Siok Seal til
Sedesaaedfal
iancnealol

05

ew
Orchis purpurea. 294103-NAP
Occhis sia 294108.NAP

Orchis sia KYS64050 1

Orchis seria hears 80876
Occhi seni nerbarum- 80873
cs

(Orch purpurea herbarium

(Orch purpurea-berbarum 2
‘Orch purpures-AY264862.1
Orch sma KUGG7260.1

Orch simi KU981682 1

‘Orchis seria KUSSIEEO.1

Orchis senia KUBBT691 1

Occhis semia-KUG3 1600.1

[> Ochs masculs- Z04086-NAP

rchis macula AYI51379.1

(Orchis mascula KUI31688 1

‘chs maseula-KUS31687 1
Orchis mascula-KUGE7ST.1
(fc mascula-KU831686 1
ot
(rehis maseila-herbaium-80804,
Orehis maseia-herbarim 80787
cro
Orcs mascula-KUE31685 +
‘Orch mascula-KUS31684 1
(Orch mascula-KU831683 1

ore

Deetylottiza maculata MT170743.1

]Daevomza acuta Horse

DDactyothiza marata AY7OAO7S 1

| oacvenicamacutoaores7s +

Ip cin mca rer
onyice mcinteaveena
Oneonas mat a7

Dactyorhiza maculata AYEOO45S 1

Dactloriza maculata AS99462 1
Dactyorhza maculata.00022868 1
— Gyradenia conopsea-NE86449 1

Gyrnadeniaconopses-00022868.1

Gymnadenia conopsea-00351281 1
Gymnaderta conopsea-JA7BR210 1
| cymnadena conopsea 5124081
ymmaderia conopsea JQ788207.1
‘Gymmnadenia conopsea-10768208 1
ore

‘Oymmnadenia conopses-234008.NAP
Gymaderia conopsea 4140251

‘Gymnadeniaconopsea-F17517581

‘Gyrmaceria conopsea-FJTSt7S7 1

‘Gyrmacera conopseaIGTE8205 1

{Gyrmadeniaconopsea 12730084 1

\Gymnadenia conopsea.JQ7E8204 1

‘Gymnadenia conopsea-AYTO4975 1
Plotanthera chorantha-AY704574 1
Patanherachlcantha-KUS31699.1
4 Patanterachlrantha KU9G1702
Patantherachlorantha-KU881700 1

Potanthera chlorantha:KU93170%.1

Platantherachlorantha-KTS36767.1

ct

16

Platarthera chlorareha.794118.NAP

Platanthera chlorantha-KYS12507.1

Patarthera chlorantha 007623 4

Platarthera chloartha-WT179753.1

Potanthera chlorartha-K460085 1

Platanthera chlorantha-MF948973.1

Platanthera chirantha NIT179736 1

Platanthera chlorartha-KY007624 1
(Ophuys sphegodes:KU931703 1
a7

Optuys sphegodes-KU931720:1

‘Oph sphegodes-KU831706 1
COphrys sphegases AVEQ097S 1
Ophrys sphegodes-AVOTA542 1
Cptrys sphegodes-ANO73255.1
‘Ophnys sphegodes-AMTH173941

‘Anacarmts pyraridaks-AY384670 1

/Anacarits pyramids 20406 1-NAP

seacoast Tes
jo
a
or

seacatspri TYTET4 1
|asenets ones
[insane 17244
[icone oronise
|e rin
pease ps N70

ors

Cephatanthera damasoniun-AFE21080.1

‘Cephatanthera damasonium-AY 145448 1

‘Cephatanthera damasonivm-AYB3027 1

Cephalanthera damasoniue-UT17Q737 1

Figure 3. EES

Neighbour-joining
sequences have
2ngf1vhmw

phylogenetic tree, based on ITS sequences of selected orchids. All
been deposited in the Dryad Data Repository - doi: 10.5061/dryad.

11

12 Aghayeva P et al

Senecio vulgaris-MK654722.1
Senecio vulgaris-MH746728.1
Senecio vulgaris-NC 046693.1

Bellis perennis-herbarium-170015

Senecio roseiflorus-MH483948.1

Senecio moorei-MH483949.1

Senecio keniophytum-MH483946.1

Senecio purtschelleri-MH483947.1
Senecio schweinfurthii-MH483950.1

Senecio vernalis-herbarium

A13
Al4

Aster hypoleucus-NC 046503 1

Aster hypoleucus-MK290824.1

Aster hersileoides-NC 042944. 1

Aster hersileoides-MK290823.1
Aster tataricus-NC 042913.1
Aster tataricus-MH669275.1

AT
AB

Al

AB
Leucanthemum vulgare-NC047460.1

Leucanthemum vulgare-MN989913.1

Leucanthemum virgatum-NC 0474611

Leucanthemum virgatum-MN996243.1

Al2

Pyrethrum carneum-herbarium-22357
Tanacetum coccineum-MT104463.1
Tanacetum coccineum-NC 047308.1
Tanacetum cinerariifolium-NC 047309.1
Tanacetum cinerariifolium-MT 104464.1

A4

A15

Centaurea diffusa-KJ690264.1

Centaurea cheiranthifolius-herbarium

Centaurea trinervia-herbarium-18067

Centaurea trinervia-herbarium-18066
Psephellus transcaucasica-herbarium-22256
Psephellus transcaucasicus-herbarium-22234
Psephellus xanthocefalus-herbarium-18471
Psephellus integrifolius-herbarium-18082
Psephellus dealbatus-herbarium-22213
Psephellus daghestanicus-herbarium-22262

Psephellus hymenolepsis-herbarium-22220

Pg
P8
PT
Pb
P5
P4
P3
Pt
P2
an
| ;araxacum officinale-herbarium-24510
10
Ag
Taraxacum officinale-KX198561.1
Taraxacum officinale-KU361241.1
Taraxacum kok-saghyz-KX198560.1
Taraxacum brevicorniculatum-KX198559.1
Taraxacum amplum-KX499525.1
Taraxacum obtusifrons-KX499524.1
Taraxacum sp-KX499523.1
Taraxacum mongolicum-KU736961.1
: + + : : 1
0.0250 0.0200 0.0150 0.0100 0.0050 0.0000

Figure 4. EES]

Neighbour-joining phylogenetic tree, based on accD sequences of selected asterids. All
sequences have been deposited in the Dryad Data Repository - doi: 10.5061/dryad.

2ngf1vhmw

DNA barcoding of native Caucasus herbal plants: potentials and limitations ...

Senecio roseiflorus-MH483948.1
Senecio keniophytum-MH483946.1
Senecio vulgaris-MKE54722.1
Senecio vulgaris-NC 0466931
Senecio vulgaris-MH746728.1
Senecio vernalis-herbarium
AI3

Senecio polypodivides-EU385492.1

Senecio integerrimus-KP126897.1

Senecio purtschelleri-MH483947.1

Senecio moorei-MH483949.1

Senecio schweinfurthii-MH483950.1

At4

Aster hypoleucus-NC 046503.1

Aster hypoleucus-MK290824.1

Aster altaicus-KX352465.1

Aster tataricus-NC 042913.1

Aster hersileoides-NC 042944 1

Aster hersilecides-MK290823.1
AtO
‘Taraxacum mongolicum-KU736961.1

Taraxacum platycarpum-KU736960.1

Taraxacum brevicorniculatum-KX198559.1

Taraxacum kok-saghyz-KX198560.1

'— Taraxacum officinale-KU361241.1

x)

Att

Taraxacum officinale-herbarium-24510

Taraxacum officinale-KX198561.1
Taraxacum amplum-KX499525.1
Taraxacum obtusifrons-KX499524.1
Taraxacum sp.-KX499523,1
Centaurea diffusa-KJ690264.1
ro Centaurea nigra-FJ395675.1

Centaurea melitensis-£U385427.1

Centaurea cheiranthifolius-herbarium
Centaurea trinervia-herbarium-18066
Psephellus transcaucasica-herbarium-22256

Psephellus transcaucasicus-herbarium-22234

Psephellus xanthocephalus-herbarium-18471
Psephellus integrifolius-herbarium-18082
Psephellus daghestanicus-herbarium-22262
P9

P7

Ups
P3
Pt
P2
P4
P6
P8
Psephellus hymenolepis-herbarium-22220

Psephellus dealbatus-herbarium-22213

Centaurea trinervia-herbarium-18067
Tanacetum cinerariffolium-MT104464.1
Tanacetum coccineum-MT104463.1

Tanacetum cinerariifolium-NC 047309.1
Tanacetum coccineum-NC 0473081

Pyrethrum carneum-herbarium-22357

At2

AIS

Bellis perennis-herbarium-170015
A8

AT

Leucanthemum vulgare-FJ395654.1

Leucanthemum virgatum-NC 047461.1

Leucanthemum virgatum-MN996243.1

Leucanthemum maximum-NC 046827.1

Leucanthemum maximum-MN518843.1

At

a2

3

a4

AS
AB
Leucanthemum vulgare-NC047460.1

Leucanthemum vulgare-MN989913.1

0.0260 0200 ‘ao1so oo1oo o.ons0 ‘o.o000

Figure 5. EES

Neighbour-joining phylogenetic tree, based on rpoB sequences of selected asterids. All
sequences have been deposited in the Dryad Data Repository - doi: 10.5061/dryad.

2ngfilvhmw

14 Aghayeva P et al

P2
P4
P9
P5
Pt
Centaurea nogmovii-JX274510-Russia
P7
Ps

Centaurea cabardensis-JX274509.1-Russia

Centaurea cheiranthifolius-herbarium

Centaurea trinervia-AJ304790

Centaurea trinervia-herbarium-18066

Centaurea trinervia-herbarium-18067

P6
| Psephellus hadimensis-KX158189.1
P3

Psephellus xanthocephalus-herbarium-18471

Psephellus daghestanicus-herbarium-22262
Psephellus dealbatus-herbarium-22213
Psephellus hymenolepis-herbarium-22220
Psephellus integrfolius-herbarium-18082
Psephellus transcaucasica-herbarium-22256

Psephellus transcaucasicus-herbarium-22234

Psephellus xanthocephalus-AY829445,1
‘Taraxacum officinale-JQ230979.1
‘Taraxacum officinalie-herbarium-24510
AN
Taraxacum officinale-AY548211.1
Taraxacum officinale-KT249883.1-Germany
Taraxacum officinale-KY860926.1
Taraxacum officinale-KT249884. 1-Germany
Taraxacum officinale-AB766235.1-France
Taraxacum officinale-MG519306.1

T.officinale-AJ633290.1-Romania

Taraxacum alpinum-AJ633289.1-Romania

Ag

Leontodon hispidus-AF528485.1

AIO

Leontodon hispidus-KX643619 1

Leontodon hispidus-DQ451770.1

Leontodon hispidus-KT249914.1-Romania
Leontodan hispidus-DQ451771.1

Leontodon hispidus-JF801910.1

‘Senecio vernalis-KT249846.1-Germany

‘Senecio vernalis-KT249850.1-Germany

‘Senecio vernalis-AJ400806.1-Germany

Senecio vernalis-herbarium

AI3

Senecio vernalis-T249848.1-Germany

‘— Senecio vernalis-JN789911.1-Israel
ANS
AT

Bellis pusilla-KP175223.1-Italy

AS

‘— Bellis margaritifolia-AF492846.1-Italy

— At4

Symphyotrichum novae-angliae-GU818492.1-USA
‘Symphyotrichum novae-angliae-JQ360398.1

Pyrethrum carneum-herbarium-22357

Tanacetum coccineum-KY397500.1
AN2

Tanacetum coccineum-AB608333.1

Bellis perennis-herbarium-170015

Bellis perennis-KX446803.1

-— Leucanthemum vulgare-EF577215.1-

Leucanthemum gallaecicum-MK481560.1-Spain

2

Leucanthemum pyrenaicum-MK481548.1-Spain
At

A3

Leucanthemum cacuminis-MK481415.1-Spain
Leucanthemum gaudinii-MK481547 1-Slovakia
Leucanthemum ligusticum-MK481412.1-taly
Leucanthemum plurifiorum-MK481410.1-Spain
Leucanthemum vulgare-MK481420.1-France

Leucanthemum vulgare-MK481551.1-Germany

0750 100 0.050 ‘o.o00

Figure 6. EES

Neighbour-joining phylogenetic tree, based on ITS sequences of selected asterids. All
sequences have been deposited in the Dryad Data Repository - doi: 10.5061/dryad.

2ngf1lvhmw

DNA barcoding of native Caucasus herbal plants: potentials and limitations ... 15

Species discrimination ability using BLAST differs for each barcode marker and for the two
plant groups. For orchids, ITS provided the highest species resolution (22 out of 26) (Table
3, Suppl. material 1), while both accD (1 out of 24) (Table 3, Suppl. material 2) and rpoB (3
out of 25) largely failed (Table 3, Suppl. material 3). For asterids, ITS (15 out of 24) (Table
4, Suppl. material 4) and rpoB (15 out of 24) (Table 4, Suppl. material 5) gave intermediate
values for species resolution, while accD completely failed (Table 4, Suppl. material 6), as
most species had identical sequences. Further, for asterids, there were several
discrepancies on species assignment depending on the employed marker even when the
top Bit-Score obtained in the GenBank matched a single species (in bold in Table 4).

Table 3.

Orchid species resolution for each barcode region, based on an all-to-all Blast analysis.
Noll: more than one reference sequence at top Bit-Score (at least 99.5%)

NO/I: all reference sequences at top Bit-score lower than 99.5%

Sample ITS accD rpoB

G1 Nol] Nol] (Platanthera chlorantha)
G3 (Orchis militaris) Nol] No F1

G4 (Orchis militaris) Nol] No F1

G5 (Orchis adenocheila) Nol! No [2]

G6 (Orchis simia) No!) No [2]

G7 (Anacamptis pyramidalis) Nol! No [2]

G8 No [2]

G9 (Anacamptis pyramidalis) Nol! No/!

G10 (Orchis mascula) No [1] No/!

G11 (Orchis mascula) No [1 No?!

G12 (Anacamptis pyramidalis) Nol! NO!

G13 No F]

G14 (Anacamptis pyramidalis) Nol! No/!

G15 Not] Nol] Nol!

G16 No!) No!) (Platanthera chlorantha)
G17 (Ophrys sphegodes) Nol! No/!

G18 (Gymnadenia conopsea) No!) (Gymnadenia conopsea)
G19 No!) (Dactylorhiza saccifera) No!)

G27 (Orchis militaris) NO! NO?!

G29 (Orchis militaris) Nol] No F1

G31 (Orchis militaris) NO?! NOF!

G32 (Orchis militaris) Nol! NO!

G35 (Orchis militaris) Nol! No/!

16

Sample
G36
G37
G38

Table 4.

Aghayeva P et al

ITS accD
(Orchis militaris) No!)
(Orchis adenocheila) NO!2]
(Orchis adenocheila) NOI]

rpoB

NO I
NO?!
NO!2]

Asterid species resolution for each barcode region, based on an all-to-all Blast analysis.

No!"l: more than one reference sequence at top Bit-Score (at least 99.5%)

NO/I: all reference sequences at top Bit-score lower than 99.5%

Sample
P1
P2
P3
P4
P5
P6
P7
P8
P9
A1
A2
A3
A4
A5
AG
A7
A8
AQ
A10
A11
A12
A13
A14
A15

ITS

(Centaurea nogmovii)
No F!

(Psephellus hadimensis)
(Centaurea nogmovii)
(Centaurea nogmovii)
(Psephellus hadimensis)
(Centaurea nogmovii)
(Centaurea nogmovii)
(Centaurea nogmovii)
Nol]

No [1

No [']

NO!

No!)

No [2]

(Bellis pusilla)

(Bellis pusilla)
(Hypochaeris radicata)
(Leontodon hispidus)

NO [1]

(Tanacetum coccineum)
(Senecio vernalis)
(Symphyotrichum novae-angliae)

NOI

accD
No [1]
NO [1]
No 1]
No [1]
No [1]
No [1]
NO [1]
NO [1]
No [1]
No [1]
No [1]
No [1]
No ']
No [']
NO [1]
No [21
No [I
Nol]
Nol!)
No [I
Nol")
No!"]
Nol]
Nol!)

rpoB

(Carthamus tinctorius)
(Carthamus tinctorius)
(Carthamus tinctorius)
(Carthamus tinctorius)
(Carthamus tinctorius)
(Carthamus tinctorius)
(Carthamus tinctorius)
(Carthamus tinctorius)
(Carthamus tinctorius)
(Leucanthemum vulgare)
(Leucanthemum vulgare)
(Leucanthemum vulgare)
(Leucanthemum vulgare)
(Leucanthemum vulgare)
(Leucanthemum vulgare)
Nol]

No!

Nol!

Nol]

Nol!

No!)

No!)

No!)

Nol]

DNA barcoding of native Caucasus herbal plants: potentials and limitations ... 17

Table 5.

Orchid species resolution for each barcode region, based on the NJ tree (i.e. monophyletic species)

Sample ITS accD rpoB

G1 (Platanthera chlorantha) NO (Platanthera chlorantha)
G3 (Orchis militaris) NO NO

G4 (Orchis militaris) NO NO

G5 (Orchis adenocheila) NO NO

G6 (Orchis simia) NO NO

G7 (Anacamptis pyramidalis) NO NO

G9 (Anacamptis pyramidalis) NO NO

G10 (Orchis mascula) NO (Orchis mascula)

G11 (Orchis mascula) NO NO

G12 (Anacamptis pyramidalis) NO NO

G14 (Anacamptis pyramidalis) NO NO

G15 NO (Cephalanthera sp.) NO (Cephalanthera sp.) NO (Cephalanthera sp.)
G16 (Platanthera chlorantha) NO (Platanthera chlorantha)
G17 (Ophrys sphegodes) (Orchis simia) NO (Ophrys sp.)

G18 (Gymnadenia conopsea) (Gymnadenia conopsea) (Gymnadenia conopsea)
G19 (Dactylorhiza maculata) NO (Dactylorhiza sp.) NO (Dactylorhiza sp.)
G27 Orchis militaris NO NO

G29 (Orchis militaris) NO NO

G31 (Orchis militaris) NO NO

G32 (Orchis militaris) NO NO

G35 (Orchis militaris) NO NO

G36 (Orchis militaris) NO NO

G37 (Orchis adenocheila) (Orchis purpurea) NO

G38 (Orchis adenocheila) (Orchis purpurea) NO

G13 NO

G8 NO

18 Aghayeva P et al
Table 6.
Asterid species resolution for each barcode region, based on the NJ tree (i.e. monophyletic
species).
Sample ITS accD rpoB
P1 NO (Centaurea sp.) NO (Psephellus sp.) NO (Psephellus sp.)
P2 NO NO (Psephellus sp.) NO (Psephellus sp.)
P3 (Psephellus hadimensis) NO (Psephellus sp.) NO (Psephellus sp.)
P4 NO (Centaurea sp.) NO (Psephellus sp.) NO (Psephellus sp.)
P5 NO (Centaurea sp.) NO (Psephellus sp.) NO (Psephellus sp.)
P6 (Psephellus hadimensis) NO (Psephellus sp.) NO (Psephellus sp.)
P7 (Centaurea nogmoviil) NO (Psephellus sp.) NO (Psephellus sp.)
P8 (Centaurea nogmovii) NO (Psephellus sp.) NO (Psephellus sp.)
P9 NO (Centaurea sp.) NO (Psephellus sp.) NO (Psephellus sp.)
A1 NO (Leucanthemum sp.) NO (Leucanthemum sp.) (Leucanthemum vulgare)
A2 NO (Leucanthemum sp.) NO (Leucanthemum sp.) (Leucanthemum vulgare)
A3 NO (Leucanthemum sp.) NO (Leucanthemum sp.) (Leucanthemum vulgare)
A4 NO NO (Leucanthemum vulgare)
A5 NO NO (Leucanthemum sp.) (Leucanthemum vulgare)
A6 NO (Leucanthemum sp.) NO (Leucanthemum sp.) (Leucanthemum vulgare)
A7 (Bellis pusilla) NO (Bellis perennis)
A8 NO (Bellis sp.) NO (Bellis perennis)
AQ NO (Taraxacum sp.) NO (Taraxacum sp.) NO (Taraxacum sp.)
A10 (Leontodon hispidus) NO NO
A11 (Taraxacum officinale) (Taraxacum Officinale) NO (Taraxacum sp.)
A12 (Tanacetum coccineum) NO NO
A13 (Senecio vernalis) NO (Senecio sp.) NO (Senecio sp.)
A14 NO NO (Aster sp.) (Aster hypoleucus)
A15 NO NO (Bellis perennis)

ITS showed the highest discriminatory power also when evaluating genetic distances
within and between species by NJ tree. This was evident in orchids: more than 90% of the
sequences collected in this study had inter-specific diversity higher than intra-specific
diversity, indicating that the ITS sequences had clear species boundaries and _ all
accessions of the same species clustered in a monophyletic group (Table 5). Instead, in
asterids, the discriminatory power of ITS marker was higher when discriminating amongst
genera, but comparable with plastid markers when referring to species assignment (Table
6). When geographic origins of Genbank available sequences were plotted on the NJ tree,
the ITS marker showed the phylogeographic signal for orchids (Fig. 7, Suppl. material 7),

DNA barcoding of native Caucasus herbal plants: potentials and limitations ... 19

less for asterids (Fig. 8, Suppl. material 8). No phylogeographic variation was detected with
plastid barcodes (data not shown).

Discussion

We have tested the potential of barcode markers on a selection of herbal groups that are
traditionally difficult to be morphologically identified since discriminant flower traits are not
always available. Typically, a species discrimination is successful when the following
conditions are met: i) all individual barcode sequences are not shared by any other species
in the dataset; ii) genetic variation within species is lower than amongst species (i.e. the
barcoding gap); iii) all individuals of a species cluster together in a monophyletic group
when employing distance-based neighbour-joining (NJ) tree, at least at a local scale.
Preliminary analyses of available information in public databases (GenBank) and literature
data (Jin et al. 2014, Gao et al. 2010) confirmed the low level of species resolution when
using traditional rocL and matK barcodes in these two selected herbal groups. For this
reason, we preferred testing complementary barcode markers, such as chloroplast rpoB,
accD and nuclear ITS that are expected to have higher discriminatory power, particularly in
annual/rapidly evolving herbaceous groups as the ones we were focused on (Chen et al.
2010, Gao et al. 2010). We chose these barcodes because of the sequence availability in
public databases or, in the absence of available sequences, because of the level of
interspecific variability detected with the same markers in related plant groups (Gigot et al.
2007, Dong et al. 2012).

We found that the selected barcodes successfully amplified and sequenced all asterids
and almost all orchids (likely depending on the quality of dried samples, i.e. orchids have
thicker leaves than asterids), but we found that the rate of species-level resolution largely
varies amongst selected markers and plant groups. Overall, for both plant lineages, plastid
markers had a species discrimination success rate lower than nuclear ITS, which allowed
us, at least for orchids, to univocally discriminate most species. Sequence accessions of
each species clustered together in monophyletic groups confirming the existence of a
barcoding gap (Fig. 3). As already found in previous studies (Aceto et al. 1999, Cozzolino
et al. 2001), variation found in the ITS region allows determination of genetic divergence
amongst orchid species.

In orchids, ITS demonstrated a higher successful discrimination capability compared to
both plastid markers, whereas in asterids, both ITS and rpoB had a comparable
identification success (Table 4). accD completely fails in identifying asterids and most of
orchids for both BLAST and the nearest genetic distance method. The lower identification
success of plastid markers (particularly of accD) is largely due to its low discriminatory
power (different species with identical sequences) or because of missing available
reference sequences (Suppl. materials 2, 1, 3, 4, 5, 6, 7, 8).

20 Aghayeva P et al

eas

tchis mitans- Switzerland

Orchis mitars- aly
cor

ons

Orchis mitas- Spain
ox

Orchis pupurea- Denmark:
[7]! Orchis purpurea. Denmark
COrchis adenocheia-tran

ae

Orchis adenocheda-Azerbaljan
Orchis adenochella- ran
COrchis adenochella- ran

[|_| orehis adenocheia- tran

65

oar
Orchis purpurea. aly

‘rchis simi aly

| orcis simi. Turkey

‘Ochs simia-herbarum 80878
Orchissima-herbarum-80873

ca

1} Orchis pupurea-herbarium-1
Orchis purpurea-herbarium 2
Orchis pupuree- Spain
Orchissimia- Turkey

Orch simi ran

‘Occhissimia-ran

| ores sia tran
‘Occhi sia ran
(Orchis mascula-taly
Orchis mascula- Spain
(Orchis mascula- ran
JOrchis mascula- ran
‘Orchis mascula- Turkey
(Orchis masculatran
ont
Orchis masculacherbarium- 80801
Orchis mascula-herbarium-80757
10
Orehis mascuta- ran
Orchis mascula- ran
Orchis mascula- tran
oi
Daetyorhiza maculata: Denmark
Dactylortiza macuata-Russia
Dactloriza mactiata-Switzerland
Dactylorhiza macuata- Russia
| Dactyorhiza maculata- Oenmark
Dactyorhiza maculata-Russia
Dactyorhiza macuata- Begum
[ dactyortiza macuata- Belgium

Dactyorhiza macuata- Begum

+ Dactyomiza maculata-Belghum

Dactyorhiza maculata: Sweden

[— Gymnadenia conopsea- China

(Gymnadenia conopsea-France:

‘Gymnadenia conopsea- UK
‘Gymnaderia conopsea-Russia

(Gyrnadenia conopsea- Turkey

Gymnadena conopsea- Russia
Gyrnadena conopsea- Russia

ore

Gymnaderia conopsea aly
Gymnaseria conopsea- Germany
Gymmaseria conopsea. Czech Republic
Gymnadenia conopsea- Czech Republic
Gymnaderia conopsea: Russia
Gymnaderia conopsea- Russia

{Gyrmnadenia conopsea- Russia

madera conapes.Sutrertnd
Patanera chorea Sutin
| Piatanthera chlorantha-Iran

4 Patarnera coat ran

Platanthera chlorantha- Azerbaljan

Plotanthera chloranha- ran

} Platanthera chlorantha: Korea

|
ot
ors

Patanthera chorantha aly
Patathera chiorantha: Turkey
Patarthera chiorantha- Germany
Patathera chirartha- Denmark
Patathera chiorantha- China

Patathera chiorantha: China

Patanthera chiorartha- Denmark

Patanthera chiorantha- Germany
‘Ophrys sphegodes: tran
cu

‘Ophrys sphegodes- tran

COphrys sphegodes: tran
phys sphegodes-Spain
Optuys sphegodes- Switzertand
Ophrys sphegodes-Hungary
(phys sphegodes- Uk:

‘Anacamptispyramadaks- Spain

Anacamptis pyramidalis- aly

on
‘Anacamptis pyramidas-Dermark
on
co
or
/Anacampts pyramidas- Denmark
Anacampts pyramids: Turkey
Anacarpts pyramidal ran
| Anacamptis pyramidal Iran
‘Anacamptis pyramidals-ran
Anacamptis pyramidal an
- ots
Cephaanthera damasonium- Uk
Cephalantheradamasonium Austria

Cephalanthera damasonium. France

Cephalanthera damasorium-Derimark

n . : x L . =

Figure 7. EES

Neighbour-joining phylogenetic tree, based on /TS sequences of selected orchids with
geographic origins (green: Europe; red: Asia) as inferred from Suppl. material 7.

DNA barcoding of native Caucasus herbal plants: potentials and limitations ...

P9
P5
Pt
Centaurea nogmovii- Russia
PT
P8

Centaurea cabardensis-Russia

Centaurea cheiranthifolius-herbarium
Centaurea trinervia-Germany
Centaurea trinervia-herbarium-18066

Centaurea trinervia-herbarium-18067

P6
Psephellus hadimensis- Turkey

P3

Psephellus xanthocephalus-herbarium-18471
Psephellus daghestanicus-herbanum-22262

Psephellus dealbatus-herbarium-22213

Psephellus hymenolepis-herbarium-22220
Psephellus integrifolius-herbarium-18082
Psephellus transcaucasica-herbarium-22256

Psephellus transcaucasicus-herbarium-22234

Psephellus xanthocephalus-Spain
Taraxacum officinale-India
Taraxacum officinalie-herbarium-24510
ait
‘Taraxacum officinale-South Korea
Taraxacum officinale- Germany
Taraxacum officinale-USA
Taraxacum officinale- Germany
Taraxacum officinale- France

Taraxacum officinale- China

Taraxacum officinale - Romania

Taraxacum alpinum- Romania

ag

Leontodon hispidus- Austria

Ato

Leontodon hispidus- Slovakia

Leontodon hispidus- Austria
Leontodon hispidus- Romania
Leontodon hispidus- Austria
Leontodon hispidus- Germany
Senecio vernalis- Germany
Senecio vernalis- Germany
Senecio vernalis- Germany
‘Senecio vernalis-herbarium

Ata

Senecio vernalis- Germany

‘— Senecio vernalis-Israel
ANS
AT

Bellis pusilla- Italy

ag

__ Bellis margaritifolia- Italy

p— At4

Symphyotrichum novae-angliae- USA
‘Symphyotrichum novae-angliae- USA

Pyrethrum carneum-herbarium-22357

Tanacetum coccineum- Australia
Al2
Tanacetum coccineum-Iran

Bellis perennis-herbarium-170015

Bellis perennis- China

;— Leucanthemum vulgare- China

Leucanthemum gallaecicum- Spain

a2
Leucanthemum pyrenaicum- Spain
Al

a3

Leucanthemum cacuminis- Spain
Leucanthemum gaudinii- Slovakia
Leucanthemum ligusticum- Italy
Leucanthemum pluriflorum- Spain
Leucanthemum vulgare-France

Leucanthemum vulgare- Germany

AG
Lr A4
he Asia
B America
Australia
t fi f H HE
180 100 0.050 0.000

Figure 8. EES

Neighbour-joining phylogenetic tree based, on /TS sequences of selected asterids with
geographic origins (green: Europe; red: Asia) as inferred from Suppl. material 8.

22 Aghayeva P et al

In asterids, we also detected a discrepancy between species assignment with the query
sequences (i.e. at least 99.5% of identical sites to reference sequences) for different
barcodes. An example is given by the accession P6: ITS marker shared a top Bit-Score
(100%) with the Psephellus hadimensis reference sequence, while the rooB marker shared
a top Bit-Score (100%) with the Carthamus tinctorius reference sequence (Table 4).
Indeed, efficiency evaluation of correct assignment with DNA barcoding markers depends
both on how informative are the generated sequences and how many sequences of
representative groups are already available in public DNA databases. Ideally, the accuracy
of specimen identification is highly dependent on representation of databases in which
target species are represented by several individuals (Meyer and Paulay 2005) from
different geographic origins. However, such databases are often not sufficiently complete
and suited to exclude the risk of sequence matching due to missing data or of incorrect
estimation of the barcoding gap (Meyer and Paulay 2005), as we found here, particularly
for our plastid markers. In this perspective, the combined use of both unknown material
and well-identified herbarium specimens, as implemented in our study, may partially fulfil
such weaknesses (Kuzmina et al. 2017). Nevertheless, in case of discrepancy in the
species assignment with different DNA barcoding markers, we preferred the assignment,
based on those markers with larger bulk of reference sequences and/or that allow
accessions to cluster monophyletically with distance-based approaches.

The discreteness of species boundaries, particularly in hybridising and/or fast-radiating
lineages, may reduce the discriminatory power of barcode markers (Chase and Fay 2009,
Kress et al. 2009). For this reason, the combined use of plastid and nuclear markers allows
testing for hybridisation/reticulate evolution. In our case, we only detected a single case of
ITS paralogy (in O. purpurea). Overall, we did not detect a discordantspecies relationship
depending on the used barcode (nuclear or plastid) that could be a clear indication of
hybridisation/reticulate evolution. This points to the low plastid marker resolution amongst
closely-related species more likely due to their recent radiation (particularly in asterids). In
that case, if barcode markers are evolving slowly, relative to the speciation rate, there may
be insufficient nucleotide differences to distinguish recent species (Fazekas et al. 2009).

Barcode markers that univocally allow identifying species can also be used to reconstruct
main phylogeographic patterns, if they contain enough intra-specific variability. In such
cases, comparison of barcode sequences of plant specimens collected throughout their
geographical ranges may provide sufficient informative data for allocating individuals to a
well-defined geographic origin. Here, we also estimated whether nuclear and plastid
markers were sufficiently variable to provide insights into the historical phylogeography and
to detect the pattern of geographical distribution of infraspecific variation in Caucasian
orchids and asterids. In our case, both plastid markers almost fail in identifying geographic
origins of orchid and asterid accessions of different origins (identical barcode sequences)
while ITS, at least for orchids, displayed enough infraspecific variation leading to different
geographic rybotypes, potentially useful for tracking origins of plant materials.

Terrestrial orchids occurred both in the Caucasus and Europe. In particular, terrestrial
Orchidinae probably originate from lrano-Turanian and Caucasus elements (the Irano-
Turanian and Caucasus origin) and came into the Mediterranean basin during the

DNA barcoding of native Caucasus herbal plants: potentials and limitations ... 23

Messinian age where their radiation gave rise to one of the richest systems of vicariant
endemism between the two floristic regions. Some Mediterranean radiated lineages have
then secondarily recolonised the Caucasian region (Batemann et al. 2003). Interestingly,
for some orchid species (Orchis mascula, Platanthera chloranta, Anacamptis pyramidalis;
Fig. 8), ITS sequences clearly display such geographic variation (from west to east and
vice versa), while, for other species, almost no sequence variation occurs across all ranges
(Orchis militaris). In the former case, we suggest that this intraspecific variation represents
the signature of ancient phylogeographic routes, whereas in the latter, with no intraspecific
variation, we suspect recent post glacial phylogeographic migration erased the ancient
phylogeographic signal.

Conclusions

We found, for both lineages, plastid markers had a species-level assignment success rate
lower that nuclear ITS marker. Several processes, such as recent speciation events with
incomplete lineage sorting and retention of ancestral sequences, may cause a partial
failure of DNA barcodes to track species events. Indeed, the ITS sequence was successful
in orchids, but not in many asterids. We argue that, at least between the two herbal groups,
the diversification time marked the difference in barcode efficiency as the absence of a
barcoding gap amongst closely-related, recently-diverged species is quite common. While
orchids represent an old evolutionary lineage, with some groups radiating in the
Mediterranean and secondarily migrating to the Caucasus (Batemann et al. 2003),
diversification of asterid lineages is more recent and had its centre in the Caucasus and
surrounding west Asia (Barres et al. 2013). In contrast to orchids, many closely-related
asterids species occur within a geographically-restricted context, which makes difficult their
discrimination, particularly with plastid barcodes. Overall, our study suggests that the ITS
sequence can be potentially utilised as universal plant barcodes in herbal groups; at the
same time, it highlights that ITS sequence efficiency as barcode marker and its
discriminatory power are strongly dependent on the evolutionary history of the examined
plant group.

Acknowledgements

The authors thank reviewers for critical reading the former version of the manuscript and
comments.

Author contributions

Salvatore Cozzolino and Donata Cafasso performed the DNA extraction, all the
bioinformatics analyses and the molecular characterisation of the identified plants. Parvin
Aghayeva collected and provided the plant samples. Salvatore Cozzolino, Silvia Fineschi,
Dilzara Aghayeva and Valida Ali-zade supervised the work, provided funding and edited the
manuscript.

24 Aghayeva P et al

Conflicts of interest

None

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Supplementary materials

Suppl. material 1: Results of BLAST species identification test for orchid ITS EE}

Authors: Parvin Aghayeva, Salvatore Cozzolino, Donata Cafasso, Valida Ali-zade, Silvia
Fineschi, Dilzara Aghayeva

Data type: Top Bit-score

Brief description: BLAST identification for orchid

Download file (16.33 kb)

Suppl. material 2: Results of BLAST species identification test for orchid accD EE}

Authors: Parvin Aghayeva, Salvatore Cozzolino, Donata Cafasso, Valida Ali-zade, Silvia
Fineschi, Dilzara Aghayeva

Data type: Top Bit-score

Brief description: BLAST identification for orchid

Download file (15.50 kb)

Suppl. material 3: Results of BLAST species identification test for orchid rpoB EE)

Authors: Parvin Aghayeva, Salvatore Cozzolino, Donata Cafasso, Valida Ali-zade, Silvia
Fineschi, Dilzara Aghayeva

Data type: Top Bit-score

Brief description: BLAST identification for orchid

Download file (15.39 kb)

Suppl. material 4: Results of BLAST species identification test for asterid ITS EE}

Authors: Parvin Aghayeva, Salvatore Cozzolino, Donata Cafasso, Valida Ali-zade, Silvia
Fineschi, Dilzara Aghayeva
Data type: Top Bit-score

28 Aghayeva P et al

Brief description: BLAST identification for asterid
Download file (14.31 kb)

Suppl. material 5: Results of BLAST species identification test for asterid rpoB EE)

Authors: Parvin Aghayeva, Salvatore Cozzolino, Donata Cafasso, Valida Ali-zade, Silvia
Fineschi, Dilzara Aghayeva

Data type: Top Bit-score

Brief description: BLAST identification for asterid

Download file (15.39 kb)

Suppl. material 6: Results of BLAST species identification test for asterid accD ET]

Authors: Parvin Aghayeva, Salvatore Cozzolino, Donata Cafasso, Valida Ali-zade, Silvia
Fineschi, Dilzara Aghayeva

Data type: Top Bit-score
Brief description: BLAST identification for asterid
Download file (25.25 kb)

Suppl. material 7: Highest BLAST match of ITS sequences for the samples of
Orchidaceae examined in this study EE}

Authors: Parvin Aghayeva, Salvatore Cozzolino, Donata Cafasso, Valida Ali-zade, Silvia
Fineschi, Dilzara Aghayeva

Data type: BLAST match

Brief description: BLAST match of ITS sequences for Orchidaceae

Download file (293.50 kb)

Suppl. material 8: Highest BLAST match of ITS sequences for the samples of
Asteraceae examined in this study EU

Authors: Parvin Aghayeva, Salvatore Cozzolino, Donata Cafasso, Valida Ali-zade, Silvia
Fineschi, Dilzara Aghayeva

Data type: BLAST match

Brief description: BLAST match of ITS of Asteraceae

Download file (191.75 kb)