trnL-F Sequences Research Articles

Intergrative Taxonomic Study of the Frullania parvistipula Complex with a Modern Circumscription of the Section Trachycolea (Frullaniaceae, Marchantiphyta).

Frullania (subg. Trachycolea) sect. Trachycolea has been studied using integrative taxonomy methods and utilizing sampling from almost all areas of distribution of the species previously referred to this section. A phylogenetic analysis based on nuclear ribosomal ITS1-2 and chloroplast trnL-F sequence data and a morphological study reveal a wide range of morphological variability within specimens that has largely disguised the overall taxonomic diversity. Frullania parvistipula, previously regarded as a widespread species, has been found to represent a group of separate species within different sections of F. subg. Trachycolea: F. caucasica and F. conistipula in F. sect. Trachycolea, F. parvistipula in F. sect. Australes, and F. fukuzawana in F. sect. Integristipulae II. Illustrations of the type specimens of F. conistipula, F. fukuzawana, and F. parvistipula, as well as illustrations of the sequenced specimens belonging to two of the discussed species (F. conistipula and F. parvistipula), are provided. The morphological differences separating the highly similar F. caucasica, F. conistipula, F. fukuzawana, F. koponenii, and F. parvistipula are discussed. A dichotomous key is presented for accepted species. New combinations are provided for two taxa.

Campanula bergomensis (Campanulaceae), a new species from Bergamo Prealps (Northern Italy)

A new species of Campanula (Campanulaceae), C. bergomensis sp. nov., is described from the Bergamo Prealps (Orobic Prealps, Lombardy, Northern Italy), based on both morphological and molecular evidence. The new species was considered in the past as an isolated population of C. cespitosa, which presents an eastern Alpine distribution. C. bergomensis is morphologically well distinguishable from C. cespitosa on the number of flowers in the racemose inflorescence, the corolla shape, and the whitish-yellow pollen surface with many spinulae. Genetically, the presence of an insertion of 81-bp in the trnL-F sequences is very characteristic. Further studies are needed to better define the phylogenetic relationship among the three closely related species, C. bergomensis, C. cespitosa and C. cochleariifolia. C. bergomensis inhabits dolomitic debris cones at low elevations. The species is range-restricted and is severely threatened by human activities. Therefore, it is urgent to adopt protection and conservation measures for the new species.

First Report of Karelinia caspia (Pall.) Less as a new host of Orobanche cumana Wallr. in Xinjiang, China.

Sunflower broomrape (Orobanche cumana Wallr.) is a holoparasitic plant species which mainly parasitizes a few species of the Asteraceae in the wild and is exclusively found growing on sunflower in agricultural fields (Fernández-Martínez et al. 2015). O. cumana is a serious threat to sunflower production in Xinjiang and Inner Mongolia (Shi et al. 2015). Karelinia caspia (Pall.) Less. (Asteraceae) is an ecologically important plant species occurring across the desert ecosystems of Russia, Central Asia, and northwest China. It plays an important role in reducing wind erosion and desertification (Xu et al. 2018). During the 2018 and 2019 growing seasons, sunflower broomrape was observed parasitizing K. caspia in non-cultivated areas adjacent to sunflower fields near Beitun city (87°51'E, 47°15'N) in Xinjiang, China. Sunflower broomrape plants were identified morphologically as O. cumana according to Pujadas-Salvà and Velasco (2000). The host plants were identified morphologically as K. caspia according to Lin et al (1979). The ribosomal DNA internal transcribed spacer (ITS) and the trnL-F region of the parasite were amplified by PCR using primer pairs ITS1/ITS4 and trnL-FF/trnL-FR, respectively (Taberlet et al. 1991; Anderson et al. 2004). The ITS sequence of the parasite (Accession No. MT795725.1) showed 100% identity (675bp out of 689bp) to that of O. cernua var. cumana (KC811228.1). The trnl-F sequence of the parasite (Accession No. ON843707) showed 98% identity (675 of 689 bp) to O.cernua var. cumana (KT387722.1). Multi-locus phylogenetic analysis of the two sequences showed clustering with sunflower broomrape. The ITS region of the parasite and host was were amplified by PCR using the primer pair ITS1F/ITS4R (Taberlet et al.1991), and the ITS sequences of the host (Accession No. MT791995.1) showed 99.86% identity (728bp of 802bp) to that of K. caspia (LN607483.1). Rhizotron and pot experiments were carried out to assess the parasitic relationship between O. cumana and K. caspia. In the rhizotron experiment, 2-week-old seedlings of K. caspia were inoculated with sterilized 400 O. cumana seeds in a 15-cm petri dish filled with a sponge overlaid with glass fiber filter paper. The parasitic state of O. cumana was observed 9 days after inoculation. In another trial, seeds of K. caspia were sowed in 2-L and 4-L pots containing sand-vermiculite-compost (1:1:1 v:v:v). These pots were artificially inoculated with 50 mg of O. cumana seeds per 1 kg of substrate. After 20 and 70 days, corresponding to the early parasitic and flowering stages, respectively, of O. cumana, K. caspia plants were uprooted from the media and washed carefully. The parasitic relationship was confirmed by the attachment position of the broomrape to the K. caspia root. To our knowledge, this is the first report of O. cumana parasitizing K. caspia in Xinjiang, China. This phenomenon means that sunflower broomrape can raise up seed on a newly recognized host. Weed eradication in and near sunflower fields is a key measure to control sunflower broomrape.

First Report of Orobanche cumana on Coleus in Xinjiang, China.

Coleus (Plectranthus scutellarioides [L.] R.Br., [syn.: Solenostemon scutellarioides], Lamiaceae) is a popular ornamental plant for its colorful and showy foliage, and widely planted as a garden plant, and a medicinal herb in some countries, including India, Indonesia, Mexico (Zhu et al. 2015). In March 2022, parasitism of broomrape, on coleus plants was found in a greenhouse (86° 3' 36" E, 44° 18' 36" N, 500 m elevation) at Shihezi University, Xinjiang, China. A few plants (6%) were parasitized with 2.5 emerged broomrape shoots per host plant. The host-parasite connection was confirmed by microscopy. Morphological characteristics of the host were consistent with coleus described by Cao et al. (2023). The broomrapes were: stem simple and slender, slightly bulbous at the base, glandular-pubescent; inflorescence usually many-flowered, lax, dense in the upper third; bracts 8 to 10 mm long, ovate-lanceolate; calyx segments free, entire, seldom bifid with markedly unequal subulate teeth; corolla markedly curvate, dorsal line inflected, white at the base, bluish violet in the upper part; stamens adaxial with filaments 6 to 7 mm long; abaxial stamens with filaments 7 to 10 mm long; gynoecium 7 to 10 mm long; ovary 4 to 5 mm long, glabrous; style with short glandular hairs; stigma white, keyed to sunflower broomrape (Orobanche cumana Wallr.) (Pujadas-Salvà and Velasco 2000). Total genomic DNA of this parasite flowers was extracted and the trnL-F gene and ribosomal DNA internal transcribed spacer (ITS) region were amplified using the primer pairs C/F and ITS1/ITS4, respectively (Taberlet et al. 1991; Anderson et al. 2004). Sequences of ITS (655 bp) and trnL-F (901 bp) were obtained (GenBank ON491818 and ON843707). BLAST analysis showed the ITS sequence was identical to that of sunflower broomrape (MK567978.1), also the trnL-F sequence matched that of sunflower broomrape (MW809408.1, identity 100%). Multi-locus phylogenetic analyses of the two sequences showed this parasite is clustered with sunflower broomrape. Together, morphological and molecular evidences confirmed the parasite on coleus plants was sunflower broomrape, a root holoparasitic plant with a narrow host range, which mainly posed a devastating threat to sunflower planting industry (Fernández-Martínez et al. 2015). To verify that coleus sunflower broomrape parasitic association, seedlings of this host were planted in 1.5-L pots containing compost-vermiculite-sand mixture (1:1:1 v:v:v) and sunflower broomrape seeds (50 mg seeds per 1 kg, soil). Three coleus seedlings, transplanted into pots without sunflower broomrape seeds, served as control. Ninety-six days later, the infected plants were smaller, their leaf color was observed to a lighter green than those of control plants and were similar to the broomrape-infected coleus plants observed in the greenhouse. The coleus roots with sunflower broomrape were carefully washed with running water, 10 to 15 emerged broomrape shoots and 14 to 22 underground attachments were observed on the coleus roots. The parasite grew well in coleus roots, from germination, attachment to host roots, and tubercles development. At the tubercle stage, the endophyte of sunflower broomrape had connected with the vascular bundle of the coleus root, confirming the sunflower broomrape-coleus connection. To the best of our knowledge, this was the first report of sunflower broomrape parasitizing coleus in Xinjiang, China. This indicates that sunflower broomrape can be propagated and survived by coleus, in fields or greenhouses with sunflower broomrape. To limit the spread of sunflower broomrape, preventive field management is needed for the coleus farmlands and greenhouse where the root holoparasite is prevalent.

The Current Diversity and Distribution of the Simple Thalloid Genus Apopellia (Marchantiophyta): Evidence from an Integrative Taxonomic Study

An integrative study of expanded sampling of Apopellia species, including the topotype of Apopellia megaspora, made it possible to clarify the taxonomic position and distribution of the species of the genus. The ITS1-2 and trnL-F sequence data were obtained for 36 molecularly tested specimens, including the topotype Apopellia megaspora, that together with data previously deposited in GenBank, support the treatment of Apopellia as a separate genus and A. alpicola as a distinct species, as well as radically change the idea on the distribution of the species of the genus. It is shown that A. megaspora is an American-Asian species with single records in Europe, whereas A. alpicola is a West-American-Eurasian species widespread in western North America and occurring scattered in Eurasia. Both species occur in the mountains of western North America and south Siberia. A. endiviifolia is widespread in Europe, scattered in Asia and so far not confirmed for America. The expanded sampling of Apopellia spp. allows us to clarify the morphological features of the species of the genus, and microphotographs illustrate the more-important morphological features.

Inflorescences of Cuscuta (Convolvulaceae): Diversity, evolution and relationships with breeding systems and fruit dehiscence modes.

Cuscuta (dodder) includes ca. 200 species of plant obligate stem parasites with enormous ecological and economical significance. Inflorescences have been historically used in Cuscuta for species descriptions and identification keys, but no comprehensive study exists to date. The main objectives of this study were to survey the diversity and evolution of inflorescences and to uncover their possible form-function relationships. The inflorescence architecture of 132 Cuscuta taxa was analysed using herbarium specimens and eight species were grown to study their inflorescence development. Inflorescence traits were mapped into a genus phylogeny obtained from a combined analysis of nuclear ITS and plastid trnL-F sequences. To test the hypothesis that inflorescence architecture is connected to sexual reproduction, correlations between inflorescence traits (using Principal Components), sexual reproductive traits (pollen/ovule ratios, corolla length and diameter), fruit charaters (fruit length and width), and the modes of dehiscence were analyzed. Based on their development, three major types of inflorescences were observed: "Cuscuta type", a simple, monochasial scorpioid cyme; "Monogynella type", a compound monochasial scorpioid cymes with the longest primary axes having prolonged vegetative growth and giving the appearance of thyrses; and "Grammica type", a compound monochasial scorpiod cymes with up to five orders of axes. Maximum likelihood analyses suggested Monogynella as the ancestral type, while Cuscuta and Grammica were derived. Overall, the total length of axes exhibited a reduction trend throughout the genus evolution, but it was not correlated with the pedicels length. Inflorescences with similar architectures may exhibit contrasting pollen-ovule ratios. Positive significant correlations were noted between the size of the flower traits and pollen-ovule ratios. Several modes of dehiscence had statistically significant different total axes lengths, suggesting that the infructescence architecture is connected to the modes of dehiscence in Cuscuta and therefore seed dispersal.

Phylogenetic analysis of Ziziphora (Lamiaceae) using nuclear (nrITS) and plastid (trnL-F) DNA: suggestions for new arrangements and status changes

In this study, Ziziphora taxa grown in Turkey along with those grown in different parts of the world were analyzed with respect to morphological revision and molecular systematic analyses using nuclear ribosomal ITS (nrITS) sequences and the chloroplast (trnL-F) sequences. Both nrITS and trnL-F sequences clearly suggested Ziziphora was a monophyletic genus. Morphological revision mostly confirmed the previous systematic position of the taxa examined, while for some taxa, morphological uncertainties combined with DNA sequence data suggested rearrangements and new taxa. With this respect, some of the unidentified Z. clenopodioides specimens were suggested as potentially 2 new subspecies while the status of Z. clenopodioides subsp. filicaulis was suggested to change from subspecies to species. Interestingly, four Z. tenuior ecotypes from the eastern, central and southern parts of Turkey displayed different nrITS sequences and hence they were suggested as four different new subspecies of Z. tenuior. This is the first report presenting a revised status of Ziziphora L. taxa in Turkey based on molecular systematic analysis, with respect to its related members in different parts of the world.

Genetic evaluation and germplasm identification analysis on ITS2, trnL-F, and psbA-trnH of alfalfa varieties germplasm resources.

In this study, genetic diversity and germplasm identification of 28 alfalfa germplasm cultivars materials were evaluated by analyzing their internal transcribed spacer 2 (ITS2), trnL-F, and psbA-trnH sequences to provide the innovative reference of alfalfa varieties genetic diversity and identify research. The results showed that the fragment average length of ITS2, trnL-F, and psbA-trnH sorting sequences were 455.7 bp, 230.3 bp, and 345.6 bp, respectively. The ITS2 sequence was too conservative to reflect the individual differences between intercultivars and intracultivars in the preliminary experiment. Furthermore, trnL-F and psbA-trnH sequence differences were relatively small between intercultivars but significant between intracultivars. Alfalfa cultivars were divided into four groups by sequence similarity clustering. Alfalfa cultivars trnL-F and psbA-trnH sequences have apparent differences, showing that chloroplast conservative sequences were independent evolution. Compared with trnL-F and psbA-trnH sequences of alfalfa cultivars, psbA-trnH sequence has abundant variation sites and can better reflect the differences between cultivars than the trnL-F sequence. Therefore, the psbA-trnH sequence can identify different alfalfa cultivars and establish the DNA sequence fingerprint.

First Report of Cirsium arvense (Canada Thistle) as a New Host of Orobanche cumana in Xinjiang, China.

Cirsium arvense (Canada thistle) is a perennial herb native to Eurasia that has been introduced to temperate regions of the world where it is considered one of the serious weeds for arable and pastoral agriculture (Schröder et al. 1993). C. arvense reproduces both clonally and sexually. The weed is highly competitive, causes yield reductions in crops such as wheat, alfalfa, sugarbeet, and can reduce forage availability and production (Wilson 1981). Canada thistle is also a harbour for plant pathogens such as plant-parasitic nematodes (Tenuta et al. 2014). Sunflower broomrape (Orobanche cumana Wallr.) is a holoparasitic plant species with a restricted range of hosts both in the wild, where it mainly parasitizes a few species of the Asteraceae, and in agricultural fields, where it is exclusively found growing on sunflower (Fernández-Martínez et al. 2015). O. cumana infection can cause up to 80% of the yield loss in sunflower, which is a serious threat for sunflower production in Xinjiang and Inner Mongolia, China (Parker 2009). In July 2019, broomrape was observed parasitizing C. arvense in the greenhouse used for sunflower resistance identification (Shihezi, 86° 3' 36" E, 44° 18' 36" N, 500 m elevation) in Xinjiang, China. Fifty percent of the plants were parasitized by broomrape in the greenhouse and the host had an average of 1-2 broomrape shoots per plant. For molecular analysis, total genomic DNA was extracted from the flowers of broomrape and the rps2, rbcL, trnL-F genes, and ribosomal DNA internal transcribed spacer (ITS) region were amplified by PCR using the primer pairs rps2F/rps2R, rbcLF/rbcLR, C/F, ITS1/ITS4, respectively (Park et al. 2007; Manen et al. 2004; Taberlet et al. 1991; Anderson et al. 2004). The ITS (659bp), rps2 (451 bp), trnL-F (914 bp), and rbcL (961 bp) gene sequences of the broomrape were deposited in GenBank, the accession numbers are MT856745, MW809407, MW809408, and MW809409. The results of BLAST analysis showed that ITS sequence shared 100% similarity with O. cumana (659/659 nucleotide identity, MK567978), the rps2 sequence shared 99% similarity with O. cumana (449/451 nucleotide identity, KT387722), trnL-F sequence shared 99% similarity with O. cumana (907/911 nucleotide identity, MT027325), rbcL sequence shared 99% similarity with O. cumana (956/964 nucleotide identity, MK577840). The morphological characteristics such as stem, inflorescence, corolla, bracts, calyx, stamens, gynoecium are consistent with O. cumana described by Pujadas-Salvá and Velasco (2000). Morphological and molecular identification strongly support that the broomrape parasitic on C. arvense belonged to the O. cumana. Greenhouse pot experiments were carried out to assess the parasitic relationship between sunflower broomrape and C. arvense (Fernández-Martínez et al. 2000). In January 2020, C. arvense roots were harvested from an extant field of C. arvense in the greenhouse at Shihezi University (Supplementary Figure S1A). The soil was dug to 30-40 cm depth and C. arvense roots were removed and carefully washed in water. The healthy and living C. arvense roots were selected and cut into 10-11 cm pieces. Four C. arvense root pieces were grown (buried at a depth of 10-12 cm) in 8-L pots containing a mixture of sand-vermiculite-compost (1:1:1 v:v:v) and O. cumana seeds (50 mg of O. cumana seeds per 1 kg of the substrate) with 5 replicates. Three non-infected plants were grown and evaluated in parallel. Approximately 80 days after planting, at the flowering stage of the O. cumana, C. arvense plants were uprooted from the soil. Compared to non-infected plants, the hosts' symptoms were slow growth, leaf wilting, and chlorosis, and similiar to the broomrape-infected C. arvense plants observed in the greenhouse field. The roots of C. arvense and broomrape were carefully washed in water and observed the parasitism of O. cumana. The infection was confirmed by observation of the attachment of the O. cumana to the C. arvense roots (Supplementary Figure S1D). To the best of our knowledge, this is the first report of O. cumana parasitizing C. arvense in Xinjiang, China. C. arvense as a new host of O. cumana indicates that sunflower broomrape can also propagate and survive in a host such as Canada thistle grown in sunflower fields. This finding suggests that it may be more difficult to control sunflower broomrape by rotation. In the next study, the contaminated area and the degree of parasitism of broomrape on C. arvense in the field will be investigated, and better-integrated control methods for controlling O. cumana will be designed.

Genetic diversity and phylogenetic analyses of Vitex agnus castus L. populations using ISSR-PCR and chloroplast DNA trnL intron and trnL-F sequences

In this study, genetic diversity and phylogenetic analysis of some Vitex agnus-castus L. populations were conducted based on ISSR-PCR and chloroplast DNA trnL intron, and trnL-F sequence analyses. Vitex agnus-castus populations were detected in Aydın province, Turkey. Fresh leaf samples from the populations were collected and brought to the laboratory for genomic DNA isolation. 15 ISSR primers were used to determine the genetic diversity ofe Vitex agnus-castus populations. A total of 138 bands were obtained in ISSR analysis, 85 of which were polymorphic and 53 were monomorphic. Polymorphism rate was determined as 61.59 %. trnC, trnD, trnE, and trnF primers were used for PCR amplification of the chloroplast trnL intron and trnL-F region. A total of 138 bands were obtained by ISSR analysis. For trnL intron analyses, nucleotide lengths of 13 populations were between 508 and 516. The average nucleotide composition consisted of 38.5 % T, 18.3 % C, 27.5 % A and 15.7 % G. In trnL-F assays, the nucleotide lengths of the 13 populations ranged from 330 to 353. The average nucleotide composition consisted of 29.4 % T, 18.1 % C, 32.9 % A and 19.6 % G. The results of the phylogenetic trees constructed using some trnL intron and trnL-F sequences of Vitex doniana, Vitex trifolia, Vitex triflora, Vitex turczaninowii, Vitex queenslandica, Vitex axillariflora, Vitex rotundifolia and Vitex negundo species obtained from NCBI were compared. As a result of the study, polymorphisms were obtained at a rate of 61.59% from the ISSR analysis. In addition, the phylogenetic relationship between chloroplast trnL intron and trnL-F sequences of Vitex agnus-castus populations along with the other species was revealed.

Phylogenetics of Serjania (Sapindaceae-Paullinieae), with emphasis on fruit evolution and the description of a new species from Michoacán, Mexico

Serjania is among the largest Neotropical genera of Sapindaceae and comprises ∼240 species. Traditionally, the genus has been defined by its schizocarpic fruits separating into three distally winged mericarps. However, recent phylogenetic studies have revealed that fruit type is not consistent within the genus and that it also includes a few species having capsular fruits that were previously placed in other genera. A phylogenetic analysis is presented based on nuclear ITS and chloroplast trnL-F sequences. The ingroup consisted of 48 species broadly sampled from across the geographic range and taxonomic spectrum of Serjania and focusing on species with atypical fruits. An ancestral character state reconstruction of fruit type was performed and demonstrated that the ancestral fruit type of the genus is strongly supported to be a schizocarp, but there are at least five independent derivations to capsular fruits and at least one transition back from capsules to schizocarps. Also, transitions from winged to wingless mericarps have occurred at least twice. Infrageneric classification is problematic and all of the sections that were represented by more than one species come out as poly- or paraphyletic. Our limited sample of taxa precludes the possibility of a new classification at present and a broader phylogenetic sample of the genus will be needed to understand relationships and determine which lineages warrant formal recognition. In addition to the phylogenetic study, Serjania frutescens, a new capsular-fruited species from Michoacán, Mexico, is described, illustrated, and compared with its putative closest relatives. A key is provided to differentiate this species from other Mexican Serjania with capsular fruits, and new synonymies and lectotypifications associated with capsular-fruited Serjania tortuosa and S. sonorensis are established. Phylogenetic results suggest that these two are cryptic species and at least superficially indistinguishable by morphology.

Toxicodendron oligophyllum (Anacardiaceae), a new species from Fujian, China: evidence from morphological and molecular analyses

A new species of Anacardiaceae, Toxicodendron oligophyllum S. L. Tang, Liang Ma & S. P. Chen, from eastern Fujian Province, China, is described and illustrated. Morphologically, the leaf of T. oligophyllum includes only 3–5 leaflets which is the least number in all known species of Toxicodendron. Additionally, the length of inflorescence equal to which of the leaf can also be used as a well diagnostic characters to distinguish the new species from the other Toxicodendron. The phylogeny of Toxicodendron using the nuclear ribosomal internal transcribed spacer (nrITS) and chloroplast (trnL-F) sequences was reconstructed. The result showed the new species is sister to T. sylvestre (Sieb. & Zucc.) Kuntze.

Phylogenetic analysis of the genus Conringia Heist. Ex Fabr. (Brassicaceae) in Turkey based on nuclear (nrITS) and chloroplast (trnL-F) DNA sequences

In this study, phylogenetic analysis of Turkish Conringia (Brassicaceae) species was conducted based on nuclear ribosomal DNA (nrITS) and chloroplast DNA (trnL-F) sequences. In addition, the relationships between the sequences of some Brassicaceae family species retrieved from NCBI, and Conringia species were documented. All of the plant specimens were collected at their flowering and vegetation periods from different regions of Turkey, and brought to the laboratory. Total genomic DNA was extracted using the GeneMark kit. In PCR analyses, ITS4 and ITS5A primers were used for the amplification of the nrITS region, while the trnLe and trnLf primers were used for the cpDNA trnL-F region. The DNA sequences obtained were then edited using BioEdit and FinchTV, and analyzed using MEGA 6.0 software. Neighbor joining (NJ) and bootstrap trees were constructed in order to identify the relationships among Conringia taxa. The nrITS sequences ranged between 573 and 672 nucleotides, while the trnL-F sequences ranged between 346 and 764 nucleotides. The divergence values of nrITS sequences differed between 0.177 and 0.00 and divergence values of trnL-F sequences differed between 0.902 and 0.00. NJ tree generated using nrITS and trnL-F sequences consisted of two clades. In trees generated with both the nrITS and trnL-F sequences, C. orientalis, C. grandiflora and C. austriaca appeared within the same group. In addition, according to the phylogenetic analysis results obtained with other Brassicaceae species, it is revealed that the Conringia genus is polyphyletic.

Species delimitation of the northeastern Anatolian Symphytum (Boraginaceae) taxa

Symphytum is regarded one of the most complicated genera in terms of the classification of its members among the Boraginaceae. In addition to different infrageneric classification methods, several species complex or aggregates have been proposed to deal with the taxonomical problem of genus members. Symphytum asperum aggregate was first introduced by Kurtto, who proposed six taxa within this aggregate. However, according to further studies by different researchers based on morphological data, total number of species of the complex was variable. The number of species was reduced to three, comprising S. asperum, S. savvalense, and S. sylvaticum, after the phylogenetic and morphological studies of Tarıkahya-Hacıoğlu and Erik. However, the taxonomical status of some of these species (i.e., S. savvalense and S. sylvaticum, and S. sepulcrale), which was assigned as a member of this complex by Kurtto, has been regarded as unresolved. To solve this uncertainty, different species delimitation methods were used, including statistical parsimony network analysis (TCS), generalized mixed Yule coalescent (GMYC), and Bayesian Phylogenetics and Phylogeography (BPP) of the ITS, trnL-F and trnS-G sequence data. In addition to members of this complex, S. ibericum, which is phylogenetically nested within the S. asperum aggregate, was also used. The TCS and GMYC analyses demonstrated more complicated clusters, whereas high posterior probabilities of BPP clusters were more compatible with the morphological data. In accordance with the morphological approach of Tarıkahya-Hacıoğlu and Erik, the species delimitation analyses based on molecular data support the recognition of S. asperum, S. ibericum, S. savvalense, and S. sylvaticum as different species.

Finding Hidden Outliers to Promote the Consistency of Key Morphological Traits and Phylogeny in Dennstaedtiaceae

With the development of open science and technological innovation, using sharing data and molecular biology techniques in the study of taxonomy and systematics have become a crucial component of plants, which undoubtedly helps us discover more hidden outliers or deal with difficult taxa. In this paper, we take Dennstaedtia smithii as an example, based on sharing molecular database, virtual herbarium and plant photo bank, to clarify the outliers that have been hidden in Dennstaedtia and find the key morphological traits with consistent of molecular systematics. In molecular phylogenetic analyses, we used rbcL, rps4, psbA-trnH and trnL-F sequences from 5 new and 49 shared data; the results showed that Dennstaedtia smithii is nested within Microlepia rather than Dennstaedtia. We further studied the morphological characters based on the phylogeny result and found that D. smithii is distinguished from other species of Dennstaedtia by spore ornamentation and the unconnected of grooves between rachis and pinna rachis. According to morphological and molecular phylogenetic studies, our results supported that D. smithii should be a new member of Microlepia and renamed Microlepia smithii (Hook.) Y.H. Yan. Finding hidden outliers can promote the consistency of morphological and molecular phylogenetic results, and make the systematic classification more natural.

Rhododendron smokianum, a New Species from the Great Smoky Mountains

Abstract—Species delimitation in Rhododendron subsect. Caroliniana (Ericaceae) has been disputed in the past with one or three species accepted. Here we report a fourth species, R. smokianum, a narrow endemic from the Great Smoky Mountains (Tennessee/North Carolina, USA). It is characterized by short-tubed corollas and small, compact growth. We support our conclusion by phylogenetic analyses of ITS and plastid trnL-F sequence data. The new species is a specialist of open mafic rock slopes and maintains morphological and phenological differences to the related species even in cultivation.

Molecular data affirming a new occurrence of Encalypta streptocarpa (Encalyptaceae, Bryophyta) in Central China

Yi, Y.-J., Xiao, X.-X., Wu, L. & He, S. 2020. Molecular data affirming a new occurrence of Encalypta streptocarpa (Encalyptaceae, Bryophyta) in Central China. – Herzogia 33: 309 –318.Encalypta streptocarpa is herein newly reported from Central China, a rare occurrence of this species in Asia. A morphological study and phylogenetic analyses based on rps4 and trnL-F sequences affirm the taxonomic identity of this new population, which represents additional evidence of a Morocco-European-Asiatic disjunct distribution of the species. Photographic images of several noteworthy morphological characters and a full description of the species based on the Chinese specimen are provided.

<p align="left"><strong><em>Gyrocheilos taishanense</em>: A new species of Gesneriaceae from Guangdong, China</strong></p>

Gyrocheilos taishanense G.T. Wang, Yu Q. Chen & R.J. Wang (Gesneriaceae) from south China is described and illustrated with photographs. It is morphologically similar to G. microtrichum W.T.Wang, but it is distinguished by its glabrous or sparsely hairy pedicels, zygomorphic calyx with hairs in unequal length, and slightly bilobed adaxial corolla lips. Molecular phylogenetic analysis based on nuclear ITS and plastid trnL-F sequences strongly supported this new species as sister to a clade comprising the previously described Gyrocheilos species (100% posterior probability and bootstrap support). The new species is evaluated here as Least Concern (LC) according to the IUCN Red List Categories and Criteria.

Impatiens wuyiensis (Balsaminaceae), a new species from Fujian of Southeast China, based on morphological and molecular evidences

BackgroundSoutheast Asia, together with tropical Africa, Madagascar, South India and Sri Lanka, and the eastern Himalayas, are the five primary hotspots of species diversity of Impatiens (Balsaminaceae). China is also rich in Impatiens species, especially in the limestone karsts or ‘Danxia’ landforms. With zygomorphic flowers and diverse corolla morphology and color, the species in Impatiens are well-known for their ornamental use, but they are also notorious in taxonomy. During the preparation of revision of Impatiens in Zhejiang and adjacent regions, an unknown species was collected from Mt. Wuyi in Fujian Province, Southeast China.ResultsPhylogenetic analyses based on nuclear ITS, chloroplast atpB-rbcL and trnL-F sequences, together with micromorphology of pollen grains and seed coats, strongly supported the close relationship of the new species with Impatiens platysepala Y.L.Chen and I. chloroxantha Y.L.Chen. In turn, both molecular data and morphological characters also were sufficient to distinguish the new species from the other two counterparts.ConclusionsOur detailed morphological observations and molecular phylogenetic analyses support the recognition of Impatiens wuyiensis as a species new to science.

Molecular Genetic Diversity of Prunus armeniaca L. (Rosaceae) Genotypes by RAPD, ISSR-PCR, and Chloroplast DNA (cpDNA) trnL-F Sequences

ABSTRACT In this study, phylogenetic analysis of some Turkish Prunus armeniaca L. genotypes was conducted based on RAPD-PCR, ISSR-PCR, and chloroplast DNA (trnL-F) sequence analyses. Prunus armeniaca genotypes were obtained from Malatya province, and brought to the laboratory for genomic DNA isolation. Eleven RAPD and fifteen ISSR primers were used to determine molecular characterization of Prunus armeniaca genotypes. For the amplification of the trnL-F region, trne and trnf primers were used in Polymerase Chain Reaction (PCR). A total of 46 and 95 bands were obtained with RAPD and ISSR analysis, respectively. The trnL-F sequences varied from 398 to 403 nucleotides. Average nucleotide composition of trnL-F sequences was 31.0% T, 14.4% C, 37.4% A and 17.2% G. The genetic distance between the five apricot genotypes is 0.00. A maximum likelihood tree was generated to determine genetic relationships among Prunus armeniaca genotypes. Analysis of Prunus armeniaca genotypes based on RAPD, ISSR, and trnL-F sequences revealed that the rate of polymorphism obtained using RAPD and ISSR were higher than those obtained through the trnL-F sequences.