Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 74(2): 121-130, 2021 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-958 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Kun Zhu, Lijie Liu, Shanshan Li, Bo Li, Majid Khayatnezhad, Abdul Shakoor (2021) Morphological method and molecular marker determine genetic diversity and population structure in Allochrusa. Caryologia 74(2): 121-130. doi: 10.36253/caryologia-958 Received: June 02, 2020 Accepted: March 05, 2021 Published: October 08, 2021 Copyright: © 2021 Kun Zhu, Lijie Liu, Shanshan Li, Bo Li, Majid Khayat- nezhad, Abdul Shakoor. This is an open access, peer-reviewed article published by Firenze University Press (http://www.fupress.com/caryologia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distri- bution, and reproduction in any medi- um, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. Morphological method and molecular marker determine genetic diversity and population structure in Allochrusa Kun Zhu1,*, Lijie Liu1, Shanshan Li1, Bo Li1, Majid Khayatnezhad2, Abdul Shakoor3,4 1 Key Laboratory of Resistance Gene Engineering and Preservation of Biodiversity in Cold Areas, College of Life Science, Agriculture and Forestry, Qiqihar University, Qiqihar Hei- longjiang Province 161006, China 2 Department of Environmental Sciences and Engineering, Ardabil Branch, Islamic Azad University, Ardabil, Iran 3 College of Environment and Planning, Henan University, Kaifeng, 475004, Henan, China 4 Key Laboratory of Geospatial Technology for the Middle and Lower Yellow River Regions, Ministry of Education, Kaifeng 475004, Henan, China *Corresponding author. E-mail: zhukunqd@163.com; abdul_shakoor954@yahoo.com Abstract. The Caryophyllaceae family is complex. Several attempts have been car- ried out in the past to study Caryophyllaceae members. This study mainly focused on Allochrusa Bunge to determine its genetic structure and used ISSR markers, ITS, and rps16 data to classify and differentiate Allochrusa species. We collected 122 Allochrusa specimens. Our analysis included morphological and molecular method approaches. Morphometry analysis indicated that floral characters could assist in the identifica- tion of Allochrusa species. A. persica (Boiss.) Boiss. and A. versicolor Fisch. & C.A.Mey. showed affinity to each other. A. bungei Boiss. formed a separate group. Analysis of molecular variance showed significant genetic differentiation in Allochrusa (p= 0.001). The majority of genetic variation was among the Allochrusa population. We recorded minimum gene flow (Nm=0.176) between Allochrusa species. Besides this, isolation by distance occurs in Allochrusa members, as shown in the Mantel test result (r = 0.01, p = 0.0002). STRUCTURE analysis revealed three genetic groups. It is evident that A. persica, A. versicolor, and A. bungei differ genetically from each other. Our current findings have implications in plant systematics and biodiversity management. Keywords: Allochrusa, ISSR–Analysis, network, population structure, species delimi- tation. INTRODUCTION Caryophyllaceae contains 88 genera and 2,200 species. The Caryophyl- laceae family is subdivided into three subfamilies, ie. Caryophylloideae, Alsi- noideae, and Paronychioideae (Greenberg and Donoguhe 2011; Pirani et al. 2014; Hernandez-Ledesma et al. 2015). The Caryophyllaceae has a worldwide distribution, and this family is diverse. The Mediterranean region is consid- 122 Kun Zhu et al. ered a hot spot or center of diversity for Caryophyllaceae (Harbaugh et al. 2010; Greenberg and Donoguhe 2011). Allochrusa Bunge has about eight species distributed in Turkey, Central Asia, Afghanistan, Caucasus, Trans- caucasia, and Iran (Boissier 1867; Schischkin 1936; Cul- len 1967; Schiman-Czeika 1988). According to Flora Ori- entalis by Bunge (Boissier 1867: 559), Allochrusa includes three species in Iran [A. versicolor Boissier (1867: 559), A. bungei Boissier (1867: 560), A. persica Boissier (1867: 560)]. Schischkin (1936) classified Acanthophyllum C.A.Mey. into two subgenera [Euacanthophyllum (Bois- sier, 1867: 561) Schischkin (1936: 783) and Allochrusa (Bunge in Boissier, 1867: 559). Schischkin (1936: 799)] included two sections in the subgenus. Four Allochrusa species were reported in Iran by Schiman-Czeika (1988). Acanthophyllum Meyer plant species are shrubs and perennial. The majority of Acanthophyllum occurs in Iran and Central Asian countries (Ghaffari 2004; Pirani et al. 2014; Mahmoudi Shamsabad et al. 2020). The Car- yophyllaceae family is a complex taxonomical family. Therefore given the taxonomic complexity in Caryophyl- laceae, some studies were conducted to resolve taxonom- ical and classification issues. For instance, phylogenetic data on Acanthophyllum supports the notion of inclusion of Allochrusa within Acanthophyllum (Pirani et al. 2014). However, traditional taxonomical and morphological characters are dissimilar between Acanthophyllum and Allochrusa. Henceforth, Allochrusa is classified as a sep- arate genus (Pirani et al. 2014). According to Madhani et al. (2018) the Acanthophyl- lum clade includes Allochrusa, Gypsopgila herniarioides, and Allochrusa species. They revealed that both mark- ers (ITS) and the chloroplast gene rps16 does not allow Allochrusa to differentiate from Acanthophyllum. The species of the genus Allochrusa were considered once as members of Acanthophyllum subgenus. Allochrusa (Schis- chkin 1936) and molecular phylogenetic studies by Mad- hani et al. (2018) corroborate the taxonomic treatment performed by Pirani et al. (2014) and contradict the treat- ment by Hernandez-Ledesma et al. (2015), where it was recognized provisionally at the generic level. According to this concept, it is necessary to resurrect the generic name Acanthophyllum for some taxa treated as Allochru- sa in recent taxonomic surveys (Madhani et al., 2018). Morphological characters such as leaves, f lower arrangement, or inflorescence are crucial characters to identify Allochrusa species (Boissier 1867; Schischkin 1936; Cullen 1967; Schiman-Czeika 1988). Plant leaves are narrow and spiny. Corymbose inflorescence, calyx tubular, petals 5, ovules 4‒5, and seed are reniform and curved in Allochrusa (Boissier 1867; Schischkin 1936; Cullen 1967; Schiman-Czeika 1988). Based on morpho- logical characters, new species, ie. Allochrusa lutea Falat. & Mahmoodi was recorded in Iran (Mahmoodi and Falatoury 2016). This species is limited to the North- Western part of Iran. A. lutea differs from A. persica in stem length and flower symmetry and shape (Mahmoodi and Falatoury 2016). Advent in molecular biology has paved our under- standing to characterize genetic diversity and population structure in plant species (Shakoor et al. 2021). Molecu- lar markers played a vital role in conservation biology and plant genetic resources (Erbano et al. 2015; Esfan- dani-Bozchaloyi and Sheidai 2018 ). Molecular mark- ers, including Inter Simple Sequence Repeats (ISSR) and ITS phylogenetic studies on the Caryophyllaceae family, showed the significance of molecular methods to resolve the genetic and evolutionary relationship within the members of Caryophyllaceae (Greenberg and Donoguhe 2011; Korkmaz and Yildirim 2015). Allochrusa lutea is restricted to the Zanjan prov- ince, while its closest relative species (A. Persica) occurs in NW Iran. The altitudinal range is 1300–1600 m a.s.l. A. lutea grows on low montane steppe life zone in open, disturbed, and dry areas with a high percentage of Scree on the ground (Mahmoodi and Falatoury 2016). A. persica has been reported from Iran, East Azerbaijan Province. A. bungei: TURKEY: Kars, Kaĝziman, Tuzluça, 13 km west of Tuzluça, 1060 m; IRAN: East Azarbayejan, between Marand and Jolfa. A. versicolor: IRAN: East Azarbayejan: 42–55 km W Marand toward Evowghli, 1000 m; Marand- Khoy; West Azarbayejan: 60 km after Makou to Dasht-e Zanganeh, 900 m; Khoy road of Marand; ca. 10 km from Gharaziaeddin to Marand, 8 km from Babolabad, 982 m; Maku, Kulus Bulaghi; between Maku & Khoy, Evaghli, 1100 m.Three species of Allochrusa versicolor, A. bungei, and A. persica are found in Iran. These species have almost similar morphological features. It is difficult to identify and separate these species on the basis of traditional tax- onomy and morphology. Therefore, due to complexity in identification, we only used ISSR markers to identify/ separate these species. The phylogenetic approach has been used on other accessions, and no unedited sequences were produced. Our approach integrated morphological and molecular methods to analyze Allochrusa species. MATERIALS AND METHODS Plants collection 122 plant samples were collected. Overall, seven natural populations were sampled. Five to eight speci- mens from each plant population were recorded. Further details about the plant location are provided (Table 1, 123Morphological method and molecular marker determine genetic diversity and population structure in Allochrusa Figure 1). We carefully identified the plant species, i.e., Allochrusa versicolor, A. bungei, and A. persica according to previous identification protocols (Boissier 1867; Schis- chkin 1936; Cullen 1967; Schiman-Czeika 1988). Dr. Shahram Mehri helped in plant collections. Plant sam- ples were deposited in the Islamic Azad University her- barium. We examined 38 morphological characters (10 qualitative, 28 quantitative). The details of morphologi- cal characters are provided (Table 2). Plant morphology analysis Before morphometric analysis, we transformed data. Mean and variance was coded as 0 and 1. To measure the similarity among plant individuals, we followed Euclidean distance (Podani 2000). Multidimensional scaling (MDS) and Unweighted Pair-Group Method with Arithmetic Mean (UPGMA) method to group the plant species (Podani 2000). Principal component anal- ysis (PCA) to find the variation in the morphological characters of Allochrusa plant species. These analyses were done in the PAST software, version 2.17. (Hammer et al. 2001). Phylogenetic reconstruction Two different nuclear and chloroplastidial DNA markers (ITS an rps16 respectively) were prelimi- Table 1. Location and herbarium accession numbers of of A. bungei, A. persica and A. versicolor Sp Pop Locality Latitude Longitude Altitude (m) Voucher no. A. bungei 1 East Azerbaijan,Tabriz to Sperkhan to Sahand 36°43’20.25” 48°20’32.07” 1450-2000 PAMH 3455 A. bungei 2 East Azerbaijan, Nematabad, near Tabriz 36°44’22.38” 48°14’35.88” 1400 PAMH 7896 A. bungei 3 East Azerbaijan between Marand and Jolfa 36°65’86 48°38’65” 1800 PAMH 6899 A. versicolor 4 East Azerbaijan, Marand-Khoy 36°36’39 48°83’93” 1300 PAMH 4187 A. versicolor 5 West Azerbaijan, 10 km from Gharaziaeddin to Marand, 8 km from Babolabad 36°87’77 48°90’10” 955 PAMH 4629 A. persica 6 East Azerbaijan,Tabriz to Sperkhan to Sahand 36°19’22 48°34’88” 1500 PAMH 4567 A. persica 7 East Azerbaijan, Tabriz, Nematabad 36°30’97 48°90’10” 1200 PAMH 6309 Table 2. Morphological characters of A. bungei, A. persica and A. versicolor populations. No Characters No Characters 1 Plant height (mm) 20 Fruit length (mm) 2 Length of stem leaves petiole (mm) 21 Bract length (mm) 3 Length of stem leaves (mm) 22 Bract width (mm) 4 Width of stem leaves (mm) 23 Bract length / Bract width (mm) 5 Length of stem leaves / Width of stem leaves(mm) 24 Pedicel length (mm) 6 Width of stem leaves/ Length of stem leaves (mm) 25 Peduncle length (mm) 7 Number of segment stem leaves (mm) 26 Style length (mm) 8 Length of basal leaves petiole (mm) 27 Stamen filament length (mm) 9 Length of basal leaves (mm) 28 Number of flowers per inflorescence 10 Width of basal leaves (mm) 29 Phyllotaxy 11 Length of basal leaves / Width of basal leaves (mm) 30 Vegetation-forms 12 Width of basal leaves / Length of basal leaves (mm) 31 Leave shape 13 Number of segment basal leaves 32 Plant color 14 Calyx length (mm) 33 Shape of segments cauline leaves 15 Calyx width (mm) 34 Shape of calyx 16 Calyx length/ Calyx width (mm) 35 Calyx apex 17 Petal length (mm) 36 Petal shape 18 Petal width (mm) 37 Leaf tips 19 Petal length / Petal width (mm) 38 Shape of segments basal leaves 124 Kun Zhu et al. nary used to represent the phylogenetic relatedness of Allochrusa versicolor, A. bungei and A. persica. For this purpose, no new sequences were produced, and we used accessions available in GenBank (see the supplementary material S1). The phylogenetic inference was based on three different approaches; Maximum parsimony (MP), Maximum likelihood (ML), and the Bayesian. Maximum parsimony (MP) analysis was done in PAUP (Swofford 2002). The heuristic search option was used for each of the two single region datasets, using tree bisection– reconnection (TBR) branch swapping, with 1,000 repli- cates of the random addition sequence. Uninformative characters were excluded from the analysis. Branch sup- port values were calculated using a full heuristic search with 1,000 bootstrap replicates (Felsenstein 2005), each with a simple addition sequence. The Combinability of these two datasets was assessed by use of the partition homogeneity test (the incongruence length difference test (ILD) of Farris et al. (1995) as implemented in PAUP (Swofford 2002). The test was conducted with invariant characters excluded (Felsenstein 2005), using the heuris- tic search option involving 100 replicates of the random addition sequence and TBR branch swapping with 1,000 homogeneity replicates. The maximum number of trees was set to 500. The model of sequence evolution for each dataset was selected by use of the software MrModeltest v. 2.3 (Kumar et al. 2016) as implemented in MrMTgui based on the Akaike information criterion (AIC) (Edgar 2004). All datasets were analyzed as a single partition with the Kimura 2-parameters + G model by Bayes- ian inference (BI) using the software MrBayes version 3.12(Ronquist and Huelsenbeck 2003) . Posteriors on the model parameters were estimated from the data using the default priors. The analysis was performed with 4 million generations, using Markov chain Monte Carlo search. MrBayes performed two simultaneous analyses starting from different random trees (Nruns = 2) each with four Markov Chains trees sampled every 100 gen- erations. No new sequences were produced. We down- loaded the ITS and rps16 data on Allochrusa species from National Center for Biotechnology Information. Accession numbers obtained from NCBI are provided in Appendix. Acanthophyllum mucronatum and Acan- thophyllum cerastioides (D.Don) Madhani & Zarre were used as outgroup taxa. Molecular marker assay (ISSR) We extracted DNA from the fresh leaves of plants. Plant DNA was extracted according to a previous proto- Figure 1. Location of Allochrusa species in map. 125Morphological method and molecular marker determine genetic diversity and population structure in Allochrusa col (Esfandani-Bozchaloyi et al. 2019). The plant leaves samples were dried with the aid of silica gel. Twenty-two ISSR primers from the University of British Columbia were initially chosen for the ISSR assay. However, we selected 10 primers that could amplify the DNA and yielded clear bands (Table 3). The ISSR marker had a 16-18 bp nucleotide repeat sequence. DNA amplifica- tion was done through PCR. A 25μl volume contain- ing 10 mMTris-HCl buffer at pH 8; 50 mMKCl; 1.5 mM MgCl2; 0.2 mM of each dNTP (Bioron, Germany); 0.2 μM of single primer, 20 ng of genomic DNA; and 3 U of Taq DNA polymerase were subjected to PCR reactions (Bioron, Germany). The PCR was carried out in Techne thermocycler (Germany). The initial denaturation stage of 5 minutes is 94°C. The initial denaturation step was followed by 36 cycles of 1 minute at 95°C, 1 minute at 50-52°C and 1 minute at 72°C. The final extension stage of 5-10 min at 72°C completed the reaction. The qual- ity of the amplified product was checked on 1% agarose gel. Ethidium bromide was used to dye the gel. We used a 100 bp molecular size ladder to compare the fragment size of the PCR product. We conducted genetic diversity, gene diversity (H), Shannon information index (I), number of effective alleles, and percentage of polymorphism analysis while following previous protocols (Weising et al. 2005; Free- land et al. 2011). Neighbor-joining (NJ) algorithm (Sai- tou and Nei 1987) was used to detect the evolutionary relationship between plant populations. We also per- formed network computation, i.e., TCS (Clement et al. 2002), to construct the Allochrusa plant population net- work. TCS analysis was done in the PopART (Population Analysis with Reticulate Trees) (Clement et al. 2002). The Mantel test was performed in the PAST program ( Hammer et al. 2001) to know the correlation between geographical and genetic distances between Allochrusa plant population. We investigated the genetic differen- tiation between plant populations through the AMOVA test (Analysis of molecular variance) in GenAlex 6.4 (Peakall and Smouse 2006). The data was iterated1000 times to infer the statistical significance. To unveil Allochrusa plant population genetic structure, we did genetic structure analysis through a Bayesian-based model in STRUCTURE software (Pritchard et al. 2000). Under the correlated allele frequency model, we used the admixture ancestry model. We ran twenty times Markov chain Monte Carlo simulation to get the reliable results of K. Besides this, the Evanno test (Evanno et al. 2005) was done to discern correct values of K. Since our prime aim was to describe the genetic structure of the Allochrusa plant population. Therefore, gene flow analy- sis was carried out in PopGene version 1.32 (Yeh et al. 1999). RESULTS Morphometry Our clustering analysis showed the same results. UPGMA cluster results were generated based on mor- phological characters (Figure 2). Morphological char- acters failed to separate A. versicolor (2) and A. persica (3). The principal component results explained the mor- phological variation within species. Overall first three Table 3. Details about the banding pattern revealed by ISSR prim- ers. Primers Primers sequence (5’-3’) ISSR-1 DBDACACACACACACACA ISSR-2 GGATGGATGGATGGAT ISSR-3 GACAGACAGACAGACA ISSR-4 AGAGAGAGAGAGAGAGYT ISSR-5 ACACACACACACACACC ISSR-6 GAGAGAGAGAGAGAGARC ISSR-7 CTCTCTCTCTCTCTCTG ISSR-8 CACACACACACACACAG ISSR-9 GTGTGTGTGTGTGTGTYG ISSR-10 CACACACACACACACARG Figure 2. UPGMA dendrogram of Allochrusa. Abbreviations: 1-3. A. bungei (1); A. versicolor (2); A. persica (3). 126 Kun Zhu et al. components explained the majority of variation (74%) in Allochrusa species. Among three components, the first component described 55% of the total variation. Floral characters such as calyx teeth, petals, and limb shape showed a positive correlation (> 0.70). The second PCA component explained the variation in ovary shape, seed morphology. A. bungei, A. persica, and A. versicolor had morphological differences. Phylogenetic tree The reconstructed phylogenetic tree is shown (Figure 3). Acanthophyllum mucronatum and Acanthophyllum cerastioides constituted in a single clade, while other spe- cies were in two separate clades. ITS and rps16 data set supported separation of A. versicolor, A. Persica and, A. Bungei with high bootstrap value (> 0.98) (Figure 3). The results show that Allochrusa species are monophyletic. ISSR and genetic diversity We conducted detailed genetic diversity and other genetic parameters on the ISSR generated data (Table 4). A. versicolor showed high polymorphism (57.53%), gene diversity (0.33), and Shannon information index (0.30). A. persica plant population had low polymorphism and Shannon information index (0.15). Analysis of molecular variance showed population differentiation in Allochrusa (p= 0.001). Seventy-three percentage of genetic varia- tion was among the Allochrusa population. Comparative less genetic variation, i.e., 27%, was reported within the population (Table 5). FsT pairwise analysis showed that Allochrusa members are genetically dissimilar. Mini- mum gene flow occurs (Nm=0.176) between Allochrusa species. A. versicolor and A. persica were genetically related (0.88). These species are more closely related to each other. A. versicolor and A. persica can exchange genetic material and hybrid with each other. The Mantel test result indicated a positive corre- lation (r = 0.01, p = 0.0002) between genetic and geo- graphical distances among Allochrusa taxa. TCS network analysis and clustering results showed a similar clustering pattern (Figure 4 A, B). ISSR molec- ular primer demonstrated its utility to divide Allochrous species into different groups or clades, as evident in the WARD tree (Figure 4 A). It is evident that A. persica, A. versicolor, and A. bungei differ genetically from each other (different segment colors) (Figure 5). STRUCTURE analysis revealed three genetic groups. Yellow and blue segments indicated individuals of A. bungei and A. versi- color. On the other hand, the green color segment high- lighted A. persica specimens (Figure 5). Allochrous show genetic variability within taxa due to introgression (hybridization) between different spe- Figure 3. Maximum Likelihood phylogram based on the com- bined ITS – rps16 dataset, with Acanthophyllum mucronatum and Acanthophyllum cerastioides as outgroups. Abrreviations: a1 = A. versicolor; b1= A. persica; c1-c3= A. bungei; d1= Acanthophyllum mucronatum; e1-e2= Acanthophyllum cerastioides; Numbers above branches: Maximum likelihood bootstrap support values, numbers below branches: Bayesian posterior probabilities. Table 4. Genetic diversity parameters based on ISSR data Allochru- sa species. (N = number of samples, Ne = number of effective alleles, I= Shannon information index, He = gene diversity, UHe = unbiased gene diversity, P%= percentage of polymorphism). Species N Na Ne I He UHe %P A. bungei 5.000 0.336 1.034 0.23 0.25 0.19 51.83% A. versicolor 4.000 0.344 1.042 0.30 0.33 0.20 57.53% A. persica 5.000 0.369 1.011 0.15 0.22 0.22 42.15% N = number of samples, Ne = number of effective alleles, I= Shan- non information index, He = gene diversity, UHe = unbiased gene diversity, P%= percentage of polymorphism. Table 5. Analysis of molecular variance result. Source df SS MS Est. Var. % ΦPT Among Pops 27 1501.364 95.789 18.154 73% 73% Within Pops 139 334.443 3.905 2.888 27% Total 166 1955.807 20.060 100% ΦPT: proportion of the total genetic variance among individuals (p < 0.001). 127Morphological method and molecular marker determine genetic diversity and population structure in Allochrusa cies. Henceforth, we performed Horizontal gene trans- fer (HGT) analysis on ISSR and ITS data of the studied Allochrous species (Figure 6). We obtained two introgres- sion events between A. persica and A. versicolor, and the same events happened between A. persica and A. bungei. Allochrous species revealed 0.2-0.3 observed heterozygo- sity (Ho) value. In addition to this, inbreeding depres- sion showed high values (FIS = 0.3-0.7). DISCUSSION We used traditional taxonomical and molecular methods to understand genetic and population struc- ture in Allochrusa. The current climate change scenario and biodiversity threats have emphasized the need to conduct genetic diversity studies. Given the progress in molecular tools, several investigations have been done to analyze population structure in plants (Pirani et al. 2014; Erbano et al. 2015; Esfandani-Bozchaloyi et al. 2017; Esfandani-Bozchaloyi et al. 2018; Shakoor et al. 2021). Current morphological findings showed the impor- tance of floral characters to explain the variation and difference among Allochrusa species. PCA analysis highlighted the significance of calyx teeth, petals, and limbs to identify the Allochrusa species. In Iran, a new Allochrusa was reported based on f loral characters (Mahmoodi and Falatoury 2016). Past and current eco- logical and taxonomical investigations have successfully implemented morphological characteristics to study plant species (Neal et al. 1998; Borba et al. 2002; Mahmoo- di and Falatoury 2016; Chen et al. 2020). However, the rationale for choosing molecular tools to study Allochru- sa was the overlapping of morphological characters in Allochrusa. Besides using ISSR markers, we also assessed the evolutionary relationship among Allochrusa mem- bers. Our results revealed genetic differentiation among studied species. A. persica and A. versicolor had a close genetic affinity between them. Genetic association and relationship studies were conducted in Caryophyllaceae ( Fior et al. 2006; Pirani et al. 2014; Madhani et al. 2018). These studies recommended the use of ITS, cpDNA, and matk to classify Caryophyllaceae plant individuals. Genetic diversity is a central theme in plant adaptabil- ity to cope with changing environments (Tomasello et al. 2015). Our analysis showed genetic diversity was low within the same individuals; however, comparative high genetic differentiation existed between different plant specimens of Allochrusa. Previous scientific data sug- gests that genetic diversity is linked with plant ability to endure against perturbation in the environment (Booy et al. 2000). A. persica showed a low level of genetic diver- sity in our analysis. The reason for such finding could be the small number of populations. Common logic sug- gests that population size correlates with genetic diversity (Leimu-Brown et al. 2006). Present results (Mantel test) Figure 6. Horizontal gene transfer (HGT) analysis based on ISSR and ITS data of Allochrusa species. (Dashed lines indicate introgres- sion vents). Figure 4. Species delimitation in Allochrusa species based on ISSR data. A = Ward dendrogram, B = TCS network. Figure 5. STRUCTURE plot of Allochrusa species. 128 Kun Zhu et al. about genetic and geographical distances indicated the distance isolation occurs in Allochrusa species. We detected high inbreeding depression showed high values in the Allochrusa population. High inbreed- ing depression reduces plant ability to survive against biotic and abiotic stress (Ramsey and Vaughton 1998). Inbreeding depression occurs due to reduced popula- tion size (Lonn and Prentice 2002). Inbreeding depres- sion analysis is critical in the biodiversity management sector (Neaves et al. 2015). Molecular markers provide in-depth analysis and several genetic diversity param- eters to describe inbreeding depression in plant species (Glemin et al. 2006). Current results showed limited gene f low in the Allochrusa population. Indeed, a low level of gene flow hinders the exchange of genetic material between spe- cies. It may pose survival threats to a small-sized plant population (Booy et al. 2000). Neighbor-joining and STRUCTURE indicated three groups of Allochrusa. Genetic variation among the three groups had the same pattern. Two hypotheses have been proposed in the past to explain the genetic variation pat- tern. Genetic diversity is maintained through gene flow; another explanation is connectivity among plant popula- tions (Dostalek et al. 2010). A. bungei and A. versicolor had similar macro and micromorphological similarities. Nonetheless, they are recognized as a separate taxon. The main differences noted in stem and calyx indumentum, pedicle size, the calyx teeth, petal apex, and limb shape were significant to separating the taxon. These findings are in accord- ance with Mahmoodi and Falatoury (2016). They also showed that A. lutea is close to A. persica morphologi- cally. A. persica and A. lutea are similar in habit, leaves shape. A. bungei is a subshrub, covered with glandular hairs. A. persica is perennial herbs with thick woody caudex, without distinctive glandular hairs, petals white with purple striate on the claw (Schischkin 1936; Schi- man- Czeika 1988). Our findings suggest the use of plant morphol- ogy features and molecular data to identify Allochrusa species. Species identification and differentiation is an essential task for systematic and evolutionary studies. We showed that molecular markers have resolving power to solve the plant systematics complex questions. Present results have applications in biodiversity and conserva- tion management. ACKNOWLEDGMENT This study was supported by the Basic Scientific Research Business Research Project of Provincial Col- leges and Universities in Heilongjiang Province (Project Number: 135409216). 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University of Alberta, Edmonton. APPENDIX/SUPPLEMENTARY DATA (S1) GenBank accession numbers and nrDNA ITS and cpDNA rps16 sequence data of Caryophyllaceae mem- bers. Acanthophyllum mucronatum: KF924652.1 (Madhani et al. 2018); MF401170.1 (Madhani et al. 2018). Acanthophyllum cerastioides: MF401122.1 (Madhani et al. 2018); MF401168.1 (Madhani et al. 2018). Allochrusa versicolor: AY936270.1 (Fior et al. 2006); KF924687.1 (Fior et al. 2006). Allochrusa bungei : KF924688.1 (Pirani et al. 2014); KF924634.1 Pirani et al. 2014). Allochrusa persica: MN310763.1 (Pirani et al. 2014); MN310916.1 (Pirani et al. 2014). Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Volume 74, Issue 1 - 2021 Firenze University Press