Biodiversity Data Journal :
Research Article
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Corresponding author: Dilzara Aghayeva (a_dilzara@yahoo.com)
Academic editor: Anatoliy Khapugin
Received: 24 Nov 2020 | Accepted: 07 Jan 2021 | Published: 27 Jan 2021
© 2021 Parvin Aghayeva, Salvatore Cozzolino, Donata Cafasso, Valida Ali-zade, Silvia Fineschi, Dilzara Aghayeva
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.
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
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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 rpoB, 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 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.
accD, asterids, Azerbaijan, barcoding identification, Caucasus, floristic surveys, ITS, native plants, orchids, rpoB
DNA barcoding in botany has rapidly spread as a reliable tool for the accurate identification of plant species or genus (
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 (
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 (
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 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 (
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
Sequence editing and alignment were performed by using BioEdit v.7.2.0 (
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.
Herbarium of the Institute of Botany, ANAS (BAK)
Dryad Data Repository - doi: 10.5061/dryad.2ngf1vhmw
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 rpoB (88.8% samples) than for ITS (83.3% samples) markers (Tables
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 |
x |
x |
x |
x |
x |
x |
G4 |
x |
x |
x |
x |
x |
x |
G5 |
x |
x |
x |
x |
x |
x |
G6 |
x |
x |
x |
x |
x |
x |
G7 |
x |
x |
x |
x |
x |
x |
G8 |
NO |
NO |
x |
x |
x |
NO |
G9 |
x |
x |
x |
x |
x |
x |
G10 |
x |
x |
x |
x |
x |
x |
G11 |
x |
x |
x |
x |
x |
x |
G12 |
x |
x |
x |
x |
x |
x |
G13 |
NO |
NO |
x |
x |
x |
NO |
G14 |
x |
x |
x |
x |
x |
x |
G15 |
x |
x |
x |
x |
x |
x |
G16 |
x |
x |
x |
x |
x |
x |
G17 |
x |
x |
x |
x |
x |
x |
G18 |
x |
x |
x |
x |
x |
x |
G19 |
x |
x |
x |
x |
x |
x |
G27 |
x |
x |
x |
x |
x |
x |
G28 |
NO |
NO |
NO |
NO |
NO |
NO |
G29 |
x |
x |
x |
x |
x |
x |
G30 |
NO |
NO |
NO |
NO |
NO |
NO |
G31 |
x |
x |
x |
x |
x |
x |
G32 |
x |
x |
x |
x |
x |
x |
G33 |
NO |
NO |
x |
NO |
x |
NO |
G34 |
NO |
NO |
x |
NO |
x |
NO |
G35 |
x |
x |
x |
x |
x |
x |
G36 |
x |
x |
x |
x |
x |
x |
G37 |
x |
x |
x |
x |
x |
x |
G38 |
x |
x |
x |
x |
x |
x |
Orchis purpurea Herbarium 1 |
x |
x |
x |
x |
x |
x |
Orchis purpurea Herbarium 2 |
x |
x |
x |
x |
x |
x |
Orchis simia Herbarium 80873 |
x |
x |
x |
x |
x |
x |
Orchis simia Herbarium 80876 |
x |
x |
x |
x |
x |
x |
Orchis mascula Herbarium 80801 |
x |
x |
x |
x |
x |
x |
Orchis mascula Herbarium 80797 |
x |
x |
x |
x |
x |
x |
30/36 83.3% |
32/36 88.8% |
30/36 83.3% |
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 |
A1 |
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 |
A9 |
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 |
A15 |
x |
x |
x |
x |
x |
x |
Centaurea trinervia Herbarium 18066 |
x |
x |
x |
x |
x |
x |
Centaurea trinervia Herbarium 18067 |
x |
x |
x |
x |
x |
x |
Psephellus hymenolepis Herbarium 22220 |
x |
x |
x |
x |
x |
x |
Psephellus daghestanicus Herbarium 22262 |
x |
x |
x |
x |
x |
x |
Psephellus dealbatus Herbarium 22213 |
x |
x |
x |
x |
x |
x |
Psephellus intergrifolius Herbarium 18082 |
x |
x |
x |
x |
x |
x |
Psephellus xantocephalus Herbarium 18471 |
x |
x |
x |
x |
x |
x |
Psephellus transcaucasicus Herbarium 22234 |
x |
x |
x |
x |
x |
x |
Psephellus transcaucasicus Herbarium 22256 |
x |
x |
x |
x |
x |
x |
Pyrethrum carneum Herbarium 22357 |
x |
x |
x |
x |
x |
x |
Taraxacum officinale Herbarium 24510 |
x |
x |
x |
x |
x |
x |
Senecio vernalis Herbarium |
x |
x |
x |
x |
x |
x |
Bellis perennis Herbarium 170015 |
x |
x |
x |
x |
x |
x |
Centaurea cheiranthifolius Herbarium |
x |
x |
x |
x |
x |
x |
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.
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
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
Neighbour-joining phylogenetic tree, based on ITS sequences of selected orchids. All sequences have been deposited in the Dryad Data Repository - doi: 10.5061/dryad.2ngf1vhmw
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.
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
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.2ngf1vhmw
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.2ngf1vhmw
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
Orchid species resolution for each barcode region, based on an all-to-all Blast analysis.
NO[1]: more than one reference sequence at top Bit-Score (at least 99.5%)
NO[2]: all reference sequences at top Bit-score lower than 99.5%
Sample |
ITS |
accD |
rpoB |
G1 |
NO[1] |
NO[1] |
(Platanthera chlorantha) |
G3 |
(Orchis militaris) |
NO[1] |
NO [2] |
G4 |
(Orchis militaris) |
NO[1] |
NO [2] |
G5 |
(Orchis adenocheila) |
NO[2] |
NO [2] |
G6 |
(Orchis simia) |
NO[1] |
NO [2] |
G7 |
(Anacamptis pyramidalis) |
NO[2] |
NO [2] |
G8 |
NO [2] |
||
G9 |
(Anacamptis pyramidalis) |
NO[2] |
NO[2] |
G10 |
(Orchis mascula) |
NO [1] |
NO[2] |
G11 |
(Orchis mascula) |
NO [1] |
NO[2] |
G12 |
(Anacamptis pyramidalis) |
NO[2] |
NO[2] |
G13 |
NO [2] |
||
G14 |
(Anacamptis pyramidalis) |
NO[2] |
NO[2] |
G15 |
NO[1] |
NO[1] |
NO[1] |
G16 |
NO[1] |
NO[1] |
(Platanthera chlorantha) |
G17 |
(Ophrys sphegodes) |
NO[2] |
NO[2] |
G18 |
(Gymnadenia conopsea) |
NO[1] |
(Gymnadenia conopsea) |
G19 |
NO[1] |
(Dactylorhiza saccifera) |
NO[1] |
G27 |
(Orchis militaris) |
NO[2] |
NO[2] |
G29 |
(Orchis militaris) |
NO[1] |
NO [2] |
G31 |
(Orchis militaris) |
NO[2] |
NO[2] |
G32 |
(Orchis militaris) |
NO[2] |
NO[2] |
G35 |
(Orchis militaris) |
NO[2] |
NO[2] |
G36 |
(Orchis militaris) |
NO[1] |
NO [2] |
G37 |
(Orchis adenocheila) |
NO[2] |
NO[2] |
G38 |
(Orchis adenocheila) |
NO[2] |
NO[2] |
Asterid species resolution for each barcode region, based on an all-to-all Blast analysis.
NO[1]: more than one reference sequence at top Bit-Score (at least 99.5%)
NO[2]: all reference sequences at top Bit-score lower than 99.5%
Sample |
ITS |
accD |
rpoB |
P1 |
(Centaurea nogmovii) |
NO [1] |
(Carthamus tinctorius) |
P2 |
NO [2] |
NO [1] |
(Carthamus tinctorius) |
P3 |
(Psephellus hadimensis) |
NO [1] |
(Carthamus tinctorius) |
P4 |
(Centaurea nogmovii) |
NO [1] |
(Carthamus tinctorius) |
P5 |
(Centaurea nogmovii) |
NO [1] |
(Carthamus tinctorius) |
P6 |
(Psephellus hadimensis) |
NO [1] |
(Carthamus tinctorius) |
P7 |
(Centaurea nogmovii) |
NO [1] |
(Carthamus tinctorius) |
P8 |
(Centaurea nogmovii) |
NO [1] |
(Carthamus tinctorius) |
P9 |
(Centaurea nogmovii) |
NO [1] |
(Carthamus tinctorius) |
A1 |
NO[1] |
NO [1] |
(Leucanthemum vulgare) |
A2 |
NO [2] |
NO [1] |
(Leucanthemum vulgare) |
A3 |
NO [1] |
NO [1] |
(Leucanthemum vulgare) |
A4 |
NO[2] |
NO [1] |
(Leucanthemum vulgare) |
A5 |
NO[1] |
NO [1] |
(Leucanthemum vulgare) |
A6 |
NO [2] |
NO [1] |
(Leucanthemum vulgare) |
A7 |
(Bellis pusilla) |
NO [2] |
NO[1] |
A8 |
(Bellis pusilla) |
NO [2] |
NO[1] |
A9 |
(Hypochaeris radicata) |
NO[1] |
NO[1] |
A10 |
(Leontodon hispidus) |
NO[1] |
NO[1] |
A11 |
NO [1] |
NO [2] |
NO[1] |
A12 |
(Tanacetum coccineum) |
NO[1] |
NO[1] |
A13 |
(Senecio vernalis) |
NO[1] |
NO[1] |
A14 |
(Symphyotrichum novae-angliae) |
NO[1] |
NO[1] |
A15 |
NO[2] |
NO[1] |
NO[1] |
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
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 |
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 nogmovii) |
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) |
A9 |
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) |
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 (
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.
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
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 rpoB marker shared a top Bit-Score (100%) with the Carthamus tinctorius reference sequence (Table
The discreteness of species boundaries, particularly in hybridising and/or fast-radiating lineages, may reduce the discriminatory power of barcode markers (
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 Irano-Turanian and Caucasus elements (the Irano-Turanian and Caucasus origin) and came into the Mediterranean basin during the 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 (
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 (
The authors thank reviewers for critical reading the former version of the manuscript and comments.
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.
None
BLAST identification for orchid
BLAST identification for orchid
BLAST identification for orchid
BLAST identification for asterid
BLAST identification for asterid
BLAST identification for asterid
BLAST match of ITS sequences for Orchidaceae
BLAST match of ITS of Asteraceae