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. 2021 Jan 16;27(1):53–68. doi: 10.1007/s12298-020-00920-0

Molecular characterization of endangered Iranian terrestrial orchids using ISSR markers and association with floral and tuber-related phenotypic traits

Soheila Gholami 1, Yavar Vafaee 1,2,✉, Farzad Nazari 1, Abdolbaset Ghorbani 3
PMCID: PMC7873147  PMID: 33627962

Abstract

We aimed to study the genetic diversity, population structure, and phylogeny of Iranian orchids using inter-simple sequence repeat (ISSR) markers to find markers associated with phenotypic traits. Based on the phenotypic analysis, the inflorescence length and the flower number of studied accessions ranged from 3.92 to 27.13 cm and 5 to 50, respectively. On the other hand, the tuber length ranged from 1.80 to 9.35 cm. A total of 310 reproducible ISSR fragments with a size range of 150 to 3000 bp were amplified. ISSR primers provided an average polymorphism information content of 0.391, varied from 0.488 for UBC-876 to 0.351 for UBC-842. Os.J population showed the lowest genetic diversity (H = 0.057 and I = 0.075), while Oyst.JA population showed the highest genetic diversity (H = 0.114 and I = 0.158). At species level, the average coefficient of genetic differentiation (GST) ranged from 0.265 for Orchis simia to 0.587 for Himantoglossum affine. Gene flow (Nm) varied from 1.38 (O. simia) to 0.756 (Anacamptis collina). The UPGMA genetic similarity dendrogram using Jaccard coefficients (r = 0.973) revealed six main clusters. Based on the Bayesian clustering method, the highest probability of the data was achieved when accessions were divided into eight groups. Floral and tuber-related phenotypic traits represented high correlations together, and they were associated with some ISSR bands based on the multiple association analysis. Altogether, ISSR markers proved to be useful for discrimination and clarification of the relationships among species and populations collected from geographically different locations. Furthermore, it could identify the polymorphism among accessions within each population and species.

Supplementary information

The online version of this article (10.1007/s12298-020-00920-0) contains supplementary material, which is available to authorized users.

Keywords: Endangered orchids, Genetic diversity, ISSR, Marker association, Phylogeny

Introduction

As the second-largest family within the angiosperms, Orchidaceae comprises over 28,000 accepted species representing more than 8% of all flowering plant species on a world scale (Zhang et al. 2018). Orchids are economically important to medicine, food, and horticulture industries. In this regard, the dried tubers of terrestrial orchid species are consumed in the Minor Asia and the Middle East due to their high glucomannan content, which gives special rheological features to a traditional beverage known as “Salep” and special ice creams (Ghorbani et al. 2014a; Kasparek and Grimm 1999). Turkey is the main market for the dried tubers of terrestrial orchids, and it has been estimated that about 40–50 million plants are gathered annually. This has led to endangering and even extinction of some orchid species in Anatolia (Molnár et al. 2017; Ghorbani et al. 2014a). The depletion of wild orchid resources in Turkey has shifted traders to other countries in the region like Iran (Ghorbani et al. 2014a, b). The Orchidaceae family in Iran is represented by 46 species and subspecies, of which 36 species produce underground fleshy tubers (Ghahraman 1989; Renz 1978). Among them, 19 species and subspecies are indiscriminately exploited, leading to defragmentation and threatening several species (de Boer et al. 2017; Ghorbani et al. 2014a). Besides, significant changes in climate and microclimate of terrestrial Iranian orchids’ niches and also anthropogenic activities intensified their scarcity and reduced population sizes (Nosrati et al. 2011; Kreutz and Spencer 2011).

Defining suitable and effective conservation strategies for endangered and threatened species needs clarifying their pattern and complexity of phylogeny and diversity at accession, population, and species levels (Warghat et al. 2013; Ebadi et al. 2019; Roy et al. 2017). The level of inter- and intra-population genetic diversity is generally exploited as an indirect index of species response to evolutionary forces (Ilves et al. 2016; Bhattacharyya and van Staden 2018). Higher geographical distances between plant populations decrease their connectivity, resulting in lower gene flow between fragments, which finally reduces the fitness and survival rate of remnant populations. Therefore, obtaining knowledge on phenotypic, phytochemical, and especially molecular diversity within and among populations or species may provide pivotal information for visualizing phylogeny, preserving germplasm, and developing breeding programs (Vafaee et al. 2017; Giri et al. 2017).

Inter-simple sequence repeats (ISSR) (a modified form of simple sequence repeat, SSR marker system) are more advantageous over other molecular markers like random amplification of polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), and SSR as they does not require previous information of the genome, variations in primer length, motif, and anchor are possible, and they are highly polymorphic and informative (Reddy et al. 2002). Furthermore, the association of ISSR loci with important phenotypic traits could be employed to identify elite populations through marker-assisted selection (MAS) (Giri et al. 2017). These associations can serve as molecular signatures that facilitate defining and implementation of conservation and breeding scenarios for sustainable utilization of plant genetic resources (Bhattacharyya et al. 2015).

Little information is available on genetic diversity and differentiation of populations and phylogeny of Iranian terrestrial orchid species. In an only available study, Ghorbani et al. (2017) determined the identity of wild orchid species by analyzing boiled and dried tuber samples traded in the main export market hubs in Iran using DNA-metabarcodes (nrITS, trnL-F spacer, and matK sequences) at the genus and species levels. Even though, DNA barcodes are becoming more important tools in plant genetics, fingerprinting techniques are still utilized for genetic identification and development of plant resource's identity (Reddy et al. 2002). On the other hand, there is no information on horticulturally important morphological characteristics of Iranian terrestrial orchids. Any available data on phenotypic traits can be useful in domestication of these worthwhile species. Therefore, the present research attempts to (1) evaluate floral and tuber related important traits and their variation, (2) study genetic diversity and population structure using 14 ISSR markers, and (3) understand associations of ISSR loci with evaluated phenotypic traits over 97 accessions belonging to 16 Iranian orchid populations.

Materials and methods

Plant material

Sixteen geographically isolated populations, including a total of 97 accessions from eight Iranian terrestrial orchid species including Dactylorhiza umbrosa, H. affine, Orchis mascula, O. simia, O. coriophora were studied (Fig. 1). The details on population origin, code, accession number, and geographical information the studied populations are represented in Table 1. The botanical identification of orchid species was authenticated by Dr. Karel Kreutz (Department of Botany, Centre for Biodiversity Naturalis, Leiden, Netherlands) and Eng. Hossein Maroofi (Agriculture and Natural Resources Research Center, Sanandaj, Iran) and the voucher specimens were preserved at the herbarium of Horticultural Sciences and Engineering Department, University of Kurdistan, Iran. The flowering accessions from each population in the same phenological age (during May–June 2018) were selected for morphological evaluation. For ISSR analysis, young leaf samples were collected, labeled, and frozen in liquid nitrogen and were kept at −80 °C until DNA extraction.

Fig. 1.

Fig. 1

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Studied terrestrial orchid species. a Orchis simia, b Dactylorhiza umbrosa, c Anacamptis collina, d Orchis coriophora, e Ophrys schulzei, f Ophrys straussii, g Orchis mascula, h Himantoglossum affine

Table 1.

Information on species, Origin, sampling size, population code and location and of the sixteen Iranian terrestrial orchid populations

Species name Origin No. Pop. Code Latitude (N) Longitude (E) Altitude (M)
Dactylorhiza umbrosa Sanandaj, Kurdistan 6 Du.S 46°37′48.5″ 35°15′35.0″ 1937
Dactylorhiza umbrosa Dehgolan, Kurdistan 5 Du.D 47°09′32.9″ 35°15′55.1″ 2042
Dactylorhiza umbrosa Marivan, Kurdistan 6 Du.M 46°01′47.8″ 35°35′47.5″ 1351
Himantoglossum affine Javanrood, Kermanshah 6 Ha.JA 46°26′38.9″ 34°53′17.6″ 1644
Himantoglossum affine Javanrood, Kermanshah 7 Ha.JB 46°24′18.4′ 34°51′39.8′ 1339
Orchis mascula Paveh, Kermanshah 5 Om.Pa 46°19′31.8″ 34°58′42.1″ 1860
Orchis simia Paveh, Kermanshah 5 Os.Pa 46°25′44.9″ 34°53′18.0″ 1644
Orchis simia Javanrood, Kermanshah 5 Os.J 46°24′45.7″ 34°55′58.9″ 1767
Orchis coriophora Salian, Kurdistan 7 Ocor.S 46°38′26.6″ 35°15′19.6″ 1737
Orchis coriophora Javanrood, Kermanshah 8 Ocor.J 46°26′41.1″ 34°53′7.3″ 1280
Anacamptis collina Kerend, Kermanshah 8 Acol.K 46°7′31.2″ 34°37′3.79″ 2045
Anacamptis collina Dalahoo, Kermanshah 5 Acol.D 46°10′56.3″ 34°33′45.8″ 2149
Ophrys straussii Javanrood, Kermanshah 7 Oyst.JA 46°26′33.9′ 34°53′18.8′ 1690
Ophrys straussii Javanrood, Kermanshah 5 Oyst.JB 46°27′1.74′ 34°53′10.5′ 1710
Ophrys schulzei Javanrood, Kermanshah 6 Oysc.JA 46°25′53.0″ 34°53′10.2″ 1470
Ophrys schulzei Javanrood, Kermanshah 6 Oysc.JB 46°26′57.0″ 34°53′18.2″ 1390
Total 97
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Genomic DNA extraction

The genomic DNA was isolated from approximately 100 mg frozen leaf samples of target species using the cetyltrimethyl ammonium bromide (CTAB) method as described by Doyle and Doyle (1987) with minor modifications. The quantity and quality of the purified DNA samples were assessed using both UV spectrophotometer and agarose (1.5%) gel electrophoresis procedures. The concentration and purity of the extracted DNA proved to be optimal for further PCR reactions.

PCR optimization and ISSR analysis

For ISSR reactions, in the first step, two accessions belonging to each population were selected to have an initial assessment of each primer by identifying plants with consistent band profiles and excluding primers generating low quality and ambiguous bands. Out of 20 studied ISSR primers, 14 primers with consistent and clear band profiles were selected for the final assessments. PCR amplification reactions performed in a final volume of 10 µl based on the protocol employed by Yan et al. (2019). The amplified DNA fragments were then run on 1.5% agarose gel (SinaGene, Iran) at 85 V in 1× TAE running buffer for 2–3 h. To visualize PCR products, the gels were stained with ethidium bromide solution (0.0015% (w/v), and were then photographed under UV light using a Bio-Rad Gel Documentation System (Gel Doc 2000 model, Bio-Rad, Hercules, CA, USA). Ambiguous and faint bands were excluded from scoring, and only sharp and distinguishable ones were selected for analyses where they scored as 1 and 0 for the presence and absence, respectively. The size of PCR products was assessed using a 1 kb molecular ladder (250–3500 bp, SinaGene, Iran).

Morphological evaluation

The collected terrestrial orchid accessions were assessed for 16 morphological characteristics, including ten floral and six tuber-related phenotypic traits (Table 2). For each population, multiple visits were conducted during tuber maturation, flowering, and capsule ripening and drying. Characteristics like inflorescence length (InLe), flowering stalk length (FlStLe), flowering stalk diameter (FlDi), tuber width (TuWi), and tuber length (TuLe) were determined using a digital caliper. On the other hand, tuber fresh weight (TuFrWe), and tuber dry weight (TuFrDr) were determined by a digital balance with precision of 0.1 mg. To evaluate qualitative traits including inflorescence shape (InSh), flower predominant color (FlPrCo), and tuber shape (TuSh), rating and coding were used according to the recommended guidelines and descriptors (Sheehan and Sheehan 1994; Dressler 1982).

Table 2.

Descriptive statistics for the sixteen phenotypic traits over 97 terrestrial orchid accessions

No. Trait Abbreviation Unit Min Max Mean SD CV%
1 Inflorescence length InfLe cm 3.92 27.13 12.54 5.17 41.3
2 Inflorescence shape InfSh code 1.00 9.00 4.93 2.89 58.5
3 Flowering stalk length FlStLe cm 11.60 57.00 25.14 9.66 38.43
4 Flowering stalk diameter FlStDi code 0.45 1.48 0.833 0.282 33.85
5 Flower predominant color FlPrCc code 0.00 5.00 2.70 1.70 63.2
6 Flowering duration FlDu day 29.00 64.00 41.54 10.13 24.38
7 Flower number FlNu number 5.00 50.00 23.79 13.13 55.20
8 Capsule number CaNu number 2.00 34.00 12.38 8.62 69.66
9 Empty capsule number EmCaNu number 3.00 27.00 11.38 6.83 60.01
10 Percentage of fruit set PeFrSe % 28.00 86.21 49.23 14.08 28.60
11 Tuber shape TuSh code 1.00 11.00 6.46 3.20 49.6
12 Tuber number TuNu number 1.00 3.00 1.98 0.33 17.00
13 Tuber width TuWi cm 1.02 8.60 3.10 1.43 46.38
14 Tuber length TuLe cm 1.80 9.35 4.39 1.78 40.57
15 Tuber fresh weight TuFrWe g 4.52 28.63 10.68 5.51 51.57
16 Tuber dry weight TuDrWe g 0.12 4.43 1.63 0.76 47.02
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Data and statistical analysis

The descriptive statistics, including mean, minimum, and maximum values, standard deviation (SD) and coefficient of variation (CV%) for phenotypic traits were calculated using SPSS software version 17.0 (SPSS, Chicago, IL, USA). The Spearman correlation values was obtained using PAST software (Hammer et al. 2001).

Similarity matrix (based on the Dice’s coefficient of similarity) and clustering dendrogram of studied accessions were constructed using SIMQUAL and SAHN functions (implemented in NTSYS software version 2.20), respectively (Rohlf 2000). To draw dendrogram, un-weighted paired group methods with arithmetic mean (UPGMA) was employed. The effectiveness of ISSR primers was calculated by estimating the total number of polymorphic bands, polymorphism information content (PIC), effective multiplex ratio (EMR), marker index (MI), and resolving power (RP) parameters (Powell et al. 1996). The genetic diversity parameters of studied orchid accessions such as number of different alleles (Na), number of effective alleles (Ne), Shannon index (I), Nei’s gene diversity (H), and percentage of polymorphic loci (Pp%) were calculated by GenAlEx software ver. 6.5 (Peakall and Smouse 2006). The principal coordinate analysis (PCoA) was also performed using GenAlEx software based on the variance–covariance matrix, and subsequently, a biplot was constructed using the first two main coordinates. The phylogeny of studied terrestrial orchid species was revealed based on the similarity matrix using TFGPA software version 1.32 (Miller 1997).

The Bayesian model implemented in the STRUCTURE software package 2.3.4 was used for analysis of population structure (Falush et al. 2007; Pritchard et al. 2000). This model identifies subpopulations, assigns their individuals, and estimates the allele frequency. The structural analysis was conducted by 15 independent runs per each K with a cluster number ranging from K = 1 to K = 10. For each run, a 50,000 burn-in length was set with 5000 Markov Chain Monte Carlo (MCMC) replications using the admixture model and independent allele frequency. To calculate the best K value, both the log-probability of the data (LnP(D)) and the delta K values were determined with the online program Structure Harvester (Earl and vonHoldt 2011).

For the identification of informative loci associated with the evaluated phenotypic traits, a multiple regression analysis (MRA) was used to estimate the associations between ISSR data (as independent variables) and floral- and tuber-related phenotypic characteristics (as dependent variables). This was achieved based on stepping criteria using SPSS software. The MRA was performed employing models proposed by Virk et al. (1996). ISSR markers with statistically significant value of regression were regarded to be associated with the corresponding phenotypic trait. The selection of independent variables was performed based on F-values with the probability of 0.045 and 0.099 for entry and removal, respectively. Furthermore, the studied ISSR markers were individually examined with linear curve fitting through linear models to confirm the significance of standardized beta coefficients (β-statistics) for each marker determined by MRA.

Result and discussion

Morphological analysis

So far, the Iranian terrestrial orchids have not been evaluated for their ecologically and economically important phenotypic traits. The knowledge on morphological traits would be pivotal for defining conservational strategies to protect Iranian terrestrial orchids as well as to exploit superior individuals to use in breeding and improvement programs. The evaluated accessions represented a considerable variability regarding the floral and tuber-related traits (Table 2). In this regard, the CV values for floral phenotypic traits were ranged from 36.78% (for flowering duration) to 69.66% (for capsule number) while for tuber-related traits it varied from 17% (for tuber number) to 51.57% (for tuber fresh weight), respectively. Erzurumlu et al. (2018) evaluated 56 accessions belonged to 16 terrestrial orchid species using 15 morphological traits and found a high diversity in the evaluated traits. The high variation in phenotypic traits observed in our study, especially for economically valuable floral and tuber-related phenotypic traits, could uncover the potential of studied orchid species for different purposes, including ornamental and food industries. In the present study, the highest tuber width and length (5.25 and 8.31 cm, respectively), and tuber fresh and dry weights (28.63 and 3.84 g, respectively) were recorded for Ha.Ja5, while the smallest tuber with the lowest fresh and dry weight was observed in Ocor.S4 (O. coriophora, Salian population) (Supplementary Information 1). In this regard, the species with big tubers and high tuber dry matter including H. affine, O. mascula, and D. umbrosa (Fig. 2) can be the best choice for immediate domestication programs and commercial production of Salep powder. This would decrease the harvesting and exploitation pressure on the species with smaller tubers like O. schulzei, O. straussii, O. coriophora and A. collina, which as a wake-up call for their conservation has been already advised (Ghorbani et al. 2014a, b).

Fig. 2.

Fig. 2

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Tuber shape and morphology of studied terrestrial orchid species. a Orchis coriophora, b Anacamptis collina, c Himantoglossum affine, d Orchis mascula, e Orchis simia, f Ophrys Schulzei, g Ophrys straussii, h Dactylorhiza umbrosa

Based on the obtained results, the flowering duration was ranged from 29 days for Os.J5 accession (O. simia, Javanrood population) to 64 days for Du.D1 accession (D. umbrosa, Dehgolan population) (Supplementary Information 1). In this regard, the accessions belonging to D. umbrosa, H. affine, O. coriophora, and A. collina represented the flowering duration more than 40 days. Therefore, providing suitable climatic and edaphic conditions, the flowering duration of some terrestrial orchid species can be comparable with the epiphytic ornamental orchids that are among the most traded cut and pot flowers in the world. Nevertheless, despite other promising ornamental features, including high number of flowers, long inflorescence, and attractive flower color, terrestrial orchids have not yet been introduced as pot or cut flowers due to their problematic propagation. In this connection, in vitro flowering has been proposed to propagate recalcitrant orchids (Teixeira da Silva et al. 2014) and could also be tried for accessions with superior flower features identified in the present study. Moreover, the diverse range of flower color can be considered as a potential feature in breeding programs for the development of new interspecific (Pellegrino et al. 2000) and even intergenic hybrids (Kishor and Sharma 2009) with novel floricultural characteristics.

One of the most important traits evaluated in the present study was the percentage of fruit set (PeFrSe), which is the number of flowers with successful pollination and fertilization generating capsule bearing viable seeds in ratio to total flowers. This variable was ranged from 28% for Os.J4 accession (O. simia, Javanrood population) to 82.96% for Ocor.J6 (O. coriophora, Javanrood population) (Table 2 and Supplementary Information 1). Based on the results, the accessions within D. umbrosa and O. coiophora had reported PeFrSe value higher than 70%. In comparison, PeFrSe was lower than 50% in all accessions of H. affine, O. mascula, O. simia, A. collina, and most accessions within O. schulzei and O. straussii. The low rate of fruit formation in O. simia, Ophrys scolopax, and Platanthera biofolia collected from Iran has also been reported by Nosrati et al. (2011) in both natural and control groups (pollinators prevented). One of the probable reasons for lower fruit formation of these species can be the lack of pollinators or their activities that are required for pollination and seed formation (Schiestl and Schluter 2009). Seed formation is vital for the long-term survival of terrestrial orchid populations as it increases the genetic variability and the capability to adapt under new habitats and dynamic environmental conditions (Rasmussen et al. 2015). Therefore, urgent conservational activities are needed to conserve remnant populations of endangered terrestrial orchid species with low fruit formation rates. These conservation programs may concentrate on available and straightforward procedures such as artificial hand pollination, which enhance pollination success and, in turn, increase the percentage of fruit formation (Swarts and Dixon 2017; Balilashaki et al. 2015).

Pearson correlation coefficients were calculated to resolve the strength of associations among the studied phenotypic traits. Based on the results, there were significantly positive or negative correlations among the studied phenotypic traits and we only discuss the horticulturally and ecologically important correlations (Fig. 3). Inflorescence (InfLe) and flowering stalk length (FlStLe) were positively correlated with tuber features, including tuber length (TuLe), tuber width (TuWi), tuber fresh weight (TuFrWe), and tuber dry weight (TuDrWe). It means that terrestrial orchid species with bigger and heavier tubers had longer inflorescences. This may attribute to the higher content of polysaccharide carbohydrates including glucomannan and starch in species with bigger tubers, which can support stronger vegetative and reproductive growth in the new season. However, the impact of other variables such as soil texture and structure, symbiosis with mycorrhizal fungi, and climactic and micro-climactic conditions should also be considered. The fruit set percentage was positively correlated with flower number (r = 0.36) and flowering duration (r = 0.57). Sonkoly et al. (2016) and Nosrati et al. (2011) stated that low fruit formation in terrestrial orchid species might be compensated by higher number flowers in which the chance of flowers to be pollinated and reproduced is increased. In this regard, it has been proposed that floral display attributes in particular flower number are probably among the most important features for insect-mediated pollination and capsule formation in both rewarding (nectariferous) and deceptive (nectarless) orchid species (Kindlmann and Jersáková 2006). On the other hand, the fruit set percentage represented a positive correlation with flowering duration (r = 0.36). This correlation shows that terrestrial orchid plants with extended flowering periods need more time to ripen and release the dried seed in nature. The strength of phenotypic correlations demonstrates the link among the examined traits originating from the combined impact of the genotype and the environmental correlation analysis (Khadivi 2018) On the other hand, correlation analysis not only helps to define and operate appropriate conservation strategies in endangered and rare plant species but also it can be helpful for the domestication of potential economic wild species (Reiter et al. 2016).

Fig. 3.

Fig. 3

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Bivariate correlations among the evaluated phenotypic traits in the accessions of terrestrial orchid species. For explanation of the trait abbreviation, see Table 2. The boxed circles are significant at the 0.05 level. The red circles show negative correlations

Molecular analysis

ISSR banding profile

For analysis of diversity and phylogeny of terrestrial orchid accessions, 14 primers were employed to study the ISSR banding profiles. A total of 310 reproducible fragments ranged from 150 to 3000 bp with a mean of 22.5 fragments per primer were amplified. Out of these fragments, 308 fragments (99.35%) were polymorphic (Table 3). The number of scorable fragments ranged from 12 for UBC-835 to 25 for UBC-823, UBC-824, and UBC-825 primers. ISSR primers exhibited a narrow range of informativeness with an average polymorphism information content (PIC) of 0.391 varied from 0.488 for UBC-876 primer to 0.351 for UBC-842 primer. The marker index (MI) of the primers ranged from 4.14 (UBC-835) to 11.22 (UBC-876). On the other hand, a mean RP (the primer's ability to differentiate among the individuals) value of 10.29 with a range of 7.77 for UBC-853 primer to 12.78 for UBC-878 primer was obtained. Finally, the mean effective multiplex ratio (EMR) was 21.18 and it varied from 8.33 (by UBC-853 primer) to a maximum of 25 (by UBC-823, UBC-824, and UBC-825 primers).

Table 3.

Summary of ISSR primers used in the present study and the their extent of polymorphism

Primer Sequence (5′ → 3′) TB PB MB PPB% PIC EMR MI RP
UBC-807 AGAGAGAGAGAGAGAGT 23 23 0 100 0.426 23.00 9.79 9.77
UBC-812 GAGAGAGAGAGAGAGAA 23 23 0 100 0.440 23.00 10.12 10.2
UBC-823 TCTCTCTCTCTCTCTCC 25 25 0 100 0.419 25.00 10.47 10.53
UBC-824 TCTCTCTCTCTCTCTCG 25 25 0 100 0.399 25.00 9.97 9.19
UBC-825 ACACACACACACACACT 25 25 0 100 0.434 25.00 10.85 10.94
UBC-835 AGAGAGAGAGAGAGAGYC 12 10 2 83.33 0.497 8.33 4.14 12.78
UBC-840 GAGAGAGAGAGAGAGAYT 23 23 0 100 0.432 23.00 9.93 9.17
UBC-841 GAGAGAGAGAGAGAGYC 22 22 0 100 0.423 22.00 9.30 10.10
UBC-842 GAGAGAGAGAGAGAGAYG 21 21 0 100 0.391 21.00 8.21 8.78
UBC-853 ACACACACACACACACYT 21 21 0 100 0.419 21.00 8.79 7.77
UBC-861 ACCACCACCACCACCACC 23 23 0 100 0.419 23.00 9.63 11.19
UBC-873 GACAGACAGACAGACA 22 22 0 100 0.441 22.00 9.70 9.81
UBC-876 GATAGATAGACAGACA 23 23 0 100 0.488 23.00 11.22 11.87
nISSR1 CAGCAGCAGCAGCAG 22 22 0 100 0.457 22.00 10.05 12
Average _ 22.14 22 0.14 99.35 4.34 21.88 9.44 10.29
Total _ 310 308 2 – – – – –
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TB, total number of amplified bands; PB, polymorphic bands; MB, monomorphic bands; PPB%, the percentage of polymorphism; PIC, polymorphism information content; EMR, effective multiplex ratio; MI marker index; RP resolving power

In the genetic variation studies, the quantification of polymorphic loci is an important parameter. The observed polymorphism in the present study using ISSR markers was comparable with the results obtained in other orchid species, including Calanthe tsoongiana (Qian et al. 2013), Cattleya labiate (Pinheiro et al. 2012), and Habenaria edgeworthii (Giri et al. 2017), while it was higher than the polymorphism rate reported in Vanda coerulea (Manners et al. 2013).

The obtained PIC values were also in ranges calculated for other species within Orchidaceae using ISSR makers. For example, Fajardo et al. (2014) reported a mean PIC of 0.354 by analyzing genetic diversity and phylogeny of 25 accessions from five tropical orchid species. The PIC is an indicator of marker informativeness that measures the discriminating power of a locus by considering the number of expressing alleles and their relative frequencies (Shete et al. 2000). It has been estimated that about 50–100 loci are satisfactory to resolve genetic relationships and diversity among and within plant species (Fajardo et al. 2014). Moreover, the feasibility of ISSR markers was further supported by the Rp and MI values obtained for ISSR primers, which were generally high and in ranges reported in other orchid species (Bhattacharyya et al. 2015; Giri et al. 2017; George et al. 2009; Pinheiro et al. 2012; Qian et al. 2013; Warghat et al. 2013). As an important criteria, Rp, MI, and PIC provide a significant benchmark that can help to determine the effectiveness of the primers employed in the molecular genotyping techniques (Gholamian et al. 2019).

Genetic diversity and differentiation

Genetic diversity parameters at the population and species level are represented in Table 4. The Os.P population (O. simia) represented the lowest observed number of alleles (Na) (1.13), effective number of alleles (Ne) (1.09), Nei’s gene diversity (H) (0.057), Shannon’s information index (I) (0.086) and polymorphism percentage (Pp) (12.90%), while the highest Na (1.29) and Ne (1.18), H (0.114), I (0.158) and Pp (29.03%) values were obtained with Oyst.JA population (O. schulzei) (Table 4). The degree of genetic variation is characterized by variables including the contemporary dynamic of local populations, their fitness to the local climate, and historical events. The low rate of genetic variation at the population level in terrestrial orchids is due to the impact of genetic drift resulting from size decreasing and fragmentation of populations that limit gene flow among populations (Hens et al. 2017). In the case of Iranian terrestrial orchids, higher harvesting pressure and therefore more resource depletion could be the main reason for the reduced rate of diversity between some of the studied populations in the present study. It stems from the fact that the income of people in rural districts of Western Iran exclusively depends on the trading of medicinal plant products, particularly dried orchid tubers (Ghorbani et al. 2014a, b). The diversity indices at the population level obtained in the present research were lower than those reported in other orchid species, including Cattleya sp. (Leles et al. 2015) and Calanthe tsoongiana (Qian et al. 2013). However, in agreement with our data, the low rates of intrapopulation gene diversity have also been reported in other endangered epiphytic orchids like Paphiopedilum micranthum (Li et al. 2002) Habenaria edgeworthii (Giri et al. 2017), Dendrobium loddigesii (Cai et al. 2011).

Table 4.

The genetic diversity indices for terrestrial Iranian orchids at population and species level based on ISSR data

Population Na Ne H I Pp (%) GST Nm
Du.S 1.20 1.12 0.078 0.108 20.65
Du.D 1.21 1.13 0.088 0.118 21.29
Du.M 1.22 1.15 0.095 0.128 21.94
D. umbrosa 1.49 1.27 0.161 0.243 49.35 0.504 0.490
Ha.JA 1.27 1.15 0.101 0.140 27.42
Ha.JB 1.20 1.12 0.076 0.105 20.00
H. affine 1.52 1.35 0.195 0.286 52.26 0.587 0.351
Om.P 1.24 1.18 0.100 0.146 24.84
O. mascula 1.24 1.18 0.100 0.146 24.84 – –
Os.Pa 1.17 1.13 0.082 0.106 17.74
Os.J 1.13 1.09 0.057 0.075 12.90
O. simia 1.22 1.14 0.084 0.125 22.26 0.265 1.38
Ocor.S 1.2 1.13 0.083 0.114 20.00
Ocor.J 1.15 1.12 0.070 0.094 15.48
O. coriophora 1.42 1.28 0.161 0.236 42.58 0.558 0.394
Oysc.JA 1.2 1.13 0.084 0.113 20.00
Oysc.JB 1.18 1.12 0.075 0.101 18.39
O. schulzei 1.32 1.21 0.122 0.179 32.26 0.404 0.735
Oyst.JA 1.29 1.18 0.114 0.158 29.03
Oyst.JB 1.17 1.12 0.078 0.102 17.10
O. straussii 1.47 1.28 0.164 0.246 47.74 0.460 0.586
Acol.K 1.18 1.12 0.075 0.104 18.39
Acol.D 1.19 1.14 0.087 0.114 19.68
A. collina 1.31 1.20 0.120 0.177 31.94 0.398 0.756
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Na, Mean number of different alleles; Ne, Number of effective alleles; I, Shannon index; H, Genetic diversity; Pp%, Percentage of polymorphic loci; GST; Coefficients for genetic differentiation; Nm, Gene flow (estimate from GST (Nm = 0.5(1 − GST)/GST))

At the species level, the maximum and the minimum Na and Ne values were obtained for O. simia (1.22 and 1.14) and H. affine (1.52 and 1.35), respectively (Table 4). Similarly, the lowest H (0.084) and I (0.125) were obtained for O. simia. Besides, D. umbrosa and O. mascula represented the lowest (21.29%) and the highest (24.84%) Pp values, respectively. The average coefficient of genetic differentiation (GST) obtained was 0.453, ranged from 0.265 for O. simia to 0.558 for O. coriophora. Finally, gene flow (Nm) varied from a minimum of 1.38 for O. simia to a maximum of 0.756 for A. collina. An overall GST value of 0.453 was obtained for eight studied terrestrial orchid species indicating 45% and 55% of intra- and inter-species diversity, respectively. This finding was further confirmed by analysis of molecular variance (AMOVA), in which it represented a significant percentage of genetic differentiation among species (Table 5). Furthermore, the results of molecular variance (AMOVA) indicated a significant (P < 0.001) genetic difference within and among populations (Table 5). Of the total genetic variation, 76% and 24% was distributed among and within populations, respectively. This is in line with the AMOVA results obtained for Dactylorhiza hatagira (Warghat et al. 2013) and further shows the fragmentation of terrestrial orchid populations and their decreased connectivity and relationship.

Table 5.

Calculated AMOVA using ISSR markers in Iranian terrestrial orchid accessions at population and species levels

Source of variation df SS MS Est. var. %
Population level Among population 15 4444.64 296.30 46.56 76
Within population 81 1180.94 14.58 14.58 24
Total 96 5625.58 61.14 100
Species level Among species 7 3397.69 485.3 38.3 61
Within species 89 2227.88 25.0 25.032 39
Total 89 341.64 4.01 100
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Df, degree of freedom

The within-species diversity for some species like O. simia, O. schulzei, O. straussii, and A. collina was lower than values obtained for D. umbrosa, H. affine, and O. coriophora. The decrease in genetic diversity of a species affects population survival by minimizing individual adaptation to dynamic conditions (Giri et al. 2017). Therefore, according to Fajardo et al. (2014), the observed genetic variation at the population level (H = 0.086), which was lower than the species level (H = 0.138) may suggest the possibility of danger of extinction for given populations within vulnerable species including O. simia, O. schulzei, A. collina, and H. affine. On the other hand, Tian et al. (2018) observed low levels of genetic diversity at the species level in Cypripedium japonicom using ISSR and SCoT markers with a substantially high degree of genetic divergence. In Western regions of Iran, some terrestrial orchid species are more influenced by local harvester due to their higher quality of underground tubers. Other than overexploitation of Iranian orchids, the lack of pollinators or their decreased activity could also be other potential reasons for the lower genetic diversity of some studied species. Orchid species need specific pollinator such as solitary bees that their abundance and activity are strongly restricted by habitat fragmentation (Schiestl and Schluter 2009). However, the detailed investigations would be beneficial to understand the relationships of pollinators’ nature, abundance, and behavior with the level and pattern of genetic diversity in the target species, particularly vulnerable species such as O. simia and A. collina.

Clustering analysis

The UPGMA dendrogram of genetic similarity based on the ISSR data and generated from Jaccard coefficient (r = 0.973) revealed six main clusters (Fig. 4). The accessions belonging to O. coriophora, A. collina, H. affine, and D. umbrosa species were classified in II, III, IV and VI, respectively. On the other hand, O. simia with O. mascula, and O. schulzei with O. straussii have individually been grouped in I and V groups, respectively, where they were distinguished as sub-clusters. Furthermore, the geographically different populations within each species were classified as separate sub-clusters and accessions within each population were also grouped together. Principal Coordinate Analysis (PCoA) was additionally carried out to draw more information on the relationship between studied populations. In this regard, the first and the second principal coordinates (PCo1 and PCo2) were responsible for 39.40% and 30.76% of the total variation, respectively (71.16% of total variability). The PCoA biplot constructed using the first two principal coordinates further confirmed the UPGMA clustering results for populations where six groups separated mainly based on species taxonomy except for the I and VI groups (Supplementary Information 2). In fact, using the ISSR marker, O. schulzei populations were not separated from O. straussii counterparts even as sub-clusters. With more than 250 species, Ophrys is the most diverse genus among terrestrial orchids and their interspecific relationship has been proposed as the most confusing classification of the Orchidaceae family due to the diverse intra-species hybridization (Breitkopf et al. 2015; Soliva and Widmer 2003).

Fig. 4.

Fig. 4

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UPGMA dendrogram of genetic similarity based on ISSR data generated from Jaccard coefficients. Numbers above each node represents bootstrap value (1000 replicates)

Population structure

Bayesian analysis was performed to evaluate population structure and grouping and to determine mixed individuals using STRUCTURE software and admixture model (Evanno et al. 2005). The Bayesian clustering method assigns accessions to hypothetical populations. The most likely number of populations (K) is estimated based on the log probability for each K value, using delta K (ΔK) value, which is a secondary estimate of the change in Likelihood employed for accurate calculation of the real value of K. In the present study, the highest probability of the data was achieved when accessions were divided into eight groups (K = 8; Fig. 5). It means that K = 8 is optimum to estimate population structure and to calculate the membership matrix of each cluster. The I to VI clusters represent the accessions of D. umbrosa, H. affine, O. mascula, O. simia, and O. coriophora, respectively. The accessions of O. schulzei and O. straussii were placed together under VI and VII clusters. The last cluster of STRUCTURE bar plot (VIII) comprised A. collina individuals. The obtained Bayesian matrix clustering was in agreement with the AMOVA results, which revealed a higher level of inter-population variation compared to intra-population diversity. Due to high sensitivity to low degrees of genetic diversity, the ISSR technique is a powerful tool to study genetic diversity and structure of populations in various plant species (Reddy et al. 2002).

Fig. 5.

Fig. 5

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Population structure of 97 Iranian terrestrial orchids based on ISSR data analyzed by STRUCTURE software and Bayesian model (K = 8). a Bar plot of showing populations structure. Number of each horizontal lane is the individual code and each color shows a subpopulation. b The relationship between K and ΔK calculated by STRUCTURE harvester

Association between phenotypic and molecular traits

Multiple regression analysis (MRA) was performed to determine the associations between ISSR bands and phenotypic traits (Table 6). Floral and tuber-related phenotypic traits (as dependent variables) represented significantly high correlation values with some of ISSR markers (as independent variables). Out of 310 considered ISSR loci, 160 loci represented significant association values with floral and tuber-related phenotypic traits. UBC-873-7, UBC-812-23, UBC-823-14, and UBC-823-1 bands exhibited the highest associations with InLe showing negative beta coefficients for the first three bands (β = − 0.211, β = − 0.135 and β = − 5.711, respectively). FlStLe was significantly associated with UBC-823-15, UBC-823-8, UBC-841-18, and UBC-876-19 markers. Significant association values were also obtained between FlStDi with UBC-873-4, UBC-823-16, UBC-835-5, and UBC-841-14 markers where UBC-823-16 represented the highest β value (β = 0.974) among all evaluated ISSR markers. Four ISSR markers comprising nressl1-2, UBC-825-17, UBC-823-14, and UBC-842-2 were proved to be associated with FlDu. FlNu was correlated with UBC-812-23, UBC-807-20, UBC-861-7, and UBC-825-25 bands. Capsule Number (CaNu) showed the maximum associations with UBC-807-20, UBC-876-19, nressl1-16, and UBC-876-9 markers. Furthermore, EmCaNu was significantly associated with UBC-807-4, UBC-873-17, UBC-861-7, and UBC-825-21 ISSR markers. As on the most important trait evaluated in the present study, PeFrSe shown to be associated with nressl1-21, nressl1-2, UBC-842-10, and UBC-876-13 markers. Moreover, TuWi represented strong correlations with UBC-823-1, nressl1-2, nressl1-17 and UBC-861-10 markers. UBC-812-18, UBC-840-4, UBC-823-15 and UBC-853-20 represented the highest association values with TuLe. Finally, UBC-823-1 and nressl1-17 markers were significantly associated with TuFrWe and TuDrWe traits.

Table 6.

ISSR markers associated with phenotypic characteristics in terrestrial orchids as demonstrated by MRA

ISSR marker (band) r R2 β t value P value
Inflorescence length (IL) UBC-873-7 0.673a 0.454 − 0.211 − 4.908 0
UBC-812-23 0.846b 0.716 − 0.135 − 2.652 0.01
UBC-823-14 0.892c 0.796 − 0.298 − 5.711 0
UBC-823-1 0.914d 0.836 0.746 12.093 0
Flowering stalk length (FlStLe) UBC-823-15 0.726a 0.527 0.196 4.337 0
UBC-823-8 0.824b 0.678 0.502 11.209 0
UBC-841-18 0.865c 0.748 − 0.706 − 15.949 0
UBC-876-19 0.892d 0.796 0.48 10.541 0
Flowering stalk diameter (FlStDi) UBC-873-4 0.315a 0.099 0.761 7.11 0
UBC-823-16 0.483b 0.233 − 0.761 − 6.745 0
UBC-835-5 0.562c 0.315 0.243 2.875 0.005
UBC-841-14 0.597d 0.357 − 0.178 − 2.013 0.047
Flowering duration (FlDu) nressl1-2 0.766a 0.586 0.716 80.478 0
UBC-825-17 0.902b 0.813 0.08 20.469 0
UBC-823-14 0.962c 0.925 − 0.472 − 49.286 0
UBC-842-2 0.987d 0.974 0.349 39.468 0
Flower number (FlNu) UBC-812-23 0.901a 0.811 − 0.188 − 3.223 0.002
UBC-807-20 0.931b 0.867 − 0.255 − 6.138 0
UBC-861-7 0.941c 0.885 0.109 3.466 0.001
UBC-825-25 0.945d 0.893 − 0.062 − 2.072 0.042
Capsule number (CaNu) UBC-807-20 0.843a 0.71 − 0.191 − 5.913 0
UBC-876-19 0.896b 0.803 0.911 16.374 0
nressl1-16 0.938c 0.88 − 0.197 − 6.387 0
UBC-876-9 0.956d 0.913 − 0.091 − 4.22 0
Empty capsule number (EmCaNu) UBC-807-4 0.747a 0.557 0.122 3.463 0.001
UBC-873-17 0.895b 0.801 − 0.079 − 3.17 0.002
UBC-861-7 0.915c 0.837 0.216 7.592 0
UBC-825-21 0.928d 0.861 0.174 6.092 0
Percentage of fruit set (PeFrSe) nressl1-21 0.822a 0.676 0.991 22.262 0
nressl1-2 0.895b 0.801 0.288 6.89 0
UBC-842-10 0.920c 0.846 − 0.153 − 3.475 0.001
UBC-876-13 0.928d 0.86 0.114 2.733 0.008
Tuber number (TuNu) nressl1-14 0.527a 0.278 − 0.998 − 8.933 0
UBC-824-11 0.605b 0.366 0.422 5.903 0
UBC-807-13 0.642c 0.413 0.469 4.805 0
UBC-876-11 0.678d 0.46 − 0.335 − 4.506 0
Tuber width (TuWi) UBC-823-1 0.752a 0.565 0.515 10.305 0
nressl1-2 0.872b 0.76 0.43 11.332 0
nressl1-17 0.913c 0.834 0.256 7.119 0
UBC-861-10 0.930d 0.865 0.254 6.016 0
Tuber length (TuLe) UBC-812-18 0.668a 0.447 0.321 5.6 0
UBC-840-4 0.803b 0.644 0.402 7.861 0
UBC-823-15 0.846c 0.715 0.114 2.389 0.019
UBC-853-20 0.880d 0.774 − 0.252 − 6.22 0
Tuber fresh weight (TuFrWe) UBC-823-1 0.802a 0.643 1.268 10.326 0
nressl1-17 0.851b 0.725 0.504 10.15 0
UBC-841-13 0.883c 0.78 − 0.266 − 6.006 0
UBC-861-21 0.902d 0.814 − 0.428 − 7.799 0
Tuber dry weight (TuDrWe) UBC-823-1 0.674a 0.454 1.088 7.216 0
nressl1-17 0.764b 0.583 0.665 9.849 0
nressl1-16 0.811c 0.658 0.586 8.266 0
UBC-873-11 0.829d 0.688 0.258 3.743 0
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Values with the different alphabetical letter are significantly different

r, Pearson’s correlation coefficient; R2, the square of R multiple adjusted for the number of predictors in the model; β, standardized beta coefficients

In the present study, several ISSR bands showed association with more than one phenotypic characteristic. According to Culp et al. (1979), this pattern of association could be due to correlation between characteristic and/or pleiotropy impact mediated by linked quantitative trait loci (QTL) on different characters. Nevertheless, the generation of segregating populations and linkage maps could improve the knowledge of these relationships (Vafaee et al. 2017). Furthermore, an individual marker can comprise the tightly linked QTLs mediating morphologically different traits. For example, UBC-823-1 band was associated with InfLe, TuWi (β = 0.515), TuFrWe (β = 1.268) and TuDrWe (β = 1.088). On the other hand, nressl1-17 marker showed a strong association with TuWi, TuFrWe, and TuDrWe traits, which demonstrated a substantial correlation between these characters. In fact, in the present study, when an ISSR marker was associated with more than one phenotypic trait, those traits had high correlation values. The traits attributed to tuber, like tuber size and tuber weight were directly correlated and represented association with same markers. Accordingly, UBC-823-1 and nressl1-17 ISSR markers could be considered as informative molecular markers in breeding programs of terrestrial orchids for the domestication of superior populations within each species. In line with our results, significant associations between morphological traits, including plant height, leaf number, flower number, and tuber length with ISSR markers have also been reported in Habenaria edgeworthii (a threatened medicinal orchid of West Himalaya) (Giri et al. 2017). On the other side, the observed high association value between the percentage of fruit set with nressl1-21 marker (r = 0.822, R2 = 0.676, β = 0.991, t value = 22.262) can be useful to develop conservation strategies as some terrestrial orchid species enter flowering phase after 2–3 years. This marker can help to preselect plants with higher fruit set rates in nature, even in their early germination stages and plantlet growth.

Conclusion

The presence of variations in a terrestrial orchid species is crucially vital for preserving the evolutionary ability to live under dynamic climatic conditions and lower genetic variability is usually attributed to decreased fitness. Therefore, screening and preserving genetic diversity of the natural germplasm of wild terrestrial orchids are the main goals of conservation strategies. The ISSR primers employed in the current research considered to be appropriate for (1) discrimination of polymorphism among orchid individuals belonging to different populations and species, (2) differentiation of intraspecific populations and their inter- and intra-diversity levels, and (3) elucidation of interspecific phylogeny in endangered Iranian terrestrial orchids. These findings can be efficiently exploited to develop conservation strategies to rescue threatened populations within vulnerable species like O. simia, A. collina, and O. schulzei. Due to the specific ecological niche of Iranian terrestrial orchids, and also because of their habitat deterioration and population fragmentation mediated by anthropogenic activities, in situ and ex situ conservational strategies need to be conducted to attain efficient, long-term survival of species and sustainable collection practices. Furthermore, the vegetatively propagated materials or in vitro raised and acclimatized seedlings can successfully be reintroduced back into their natural or natural-like artificial habitats with desirable climatic and microclimatic conditions. Besides, marker-assisted selection offers more options to select promising individuals, populations, or species with special features for medicinal or horticultural purposes.

Supplementary information

Below is the link to the electronic supplementary material.

12298_2020_920_MOESM1_ESM.xlsx (21.9KB, xlsx)

Supplementary information 1. The values for the floral and tuber related traits in a collection of 97 terrestrial orchid accessions. (XLSX 21 kb)

12298_2020_920_MOESM2_ESM.docx (52.4KB, docx)

Supplementary information 2. The PCoA biplot of terrestrial orchid populations based on the two first principal coordinates. (DOCX 2459 kb)

Acknowledgement

This work was financially supported by the Iran National Science Foundation (Grant No. 95826350) and the University of Kurdistan.

Abbreviations

EMR

Effective multiplex ratio

ISSR

Inter simple sequence repeats

MI

Marker index

MAR

Multiple association analysis

MAS

Marker assisted selection

PCA

Principal component analysis

PCoA

Principal coordinate analysis

PIC

Polymorphism information content

UPGMA

Un-weighted paired group methods with arithmetic mean

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

12298_2020_920_MOESM1_ESM.xlsx (21.9KB, xlsx)

Supplementary information 1. The values for the floral and tuber related traits in a collection of 97 terrestrial orchid accessions. (XLSX 21 kb)

12298_2020_920_MOESM2_ESM.docx (52.4KB, docx)

Supplementary information 2. The PCoA biplot of terrestrial orchid populations based on the two first principal coordinates. (DOCX 2459 kb)

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