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41Новости систематики высших растений 2019
Novitates Systematicae Plantarum Vascularium 50: 47-79 'Ц ISSN 0568-5443
Caelestium, genus novum (Polygonaceae, Polygoneae): evidence bssed on the results of molecular phylogenetic analyses of tribe Polygoneae, established with consideration of the secondary structure of the ITS nrDNA regions
Caelestium, genus novum (Polygonaceae, Polygoneae): обоснование, представленное по результатам молекулярно-филогенетических анализов трибы Polygoneae, предпринятых с учетом вторичной структуры локусов ITS1 и ITS2 ядерной рибосомной ДНК
O. V. Yurtseva1, E. V. Mavrodiev2
1 Lomonosov Moscow State University Faculty of Biology, Department of Higher Plants Leninskiye Gory, 1, Bld. 12, 119991, Moscow, Russia [email protected]
2 University of Florida
Florida Museum of Natural History Gainesville, Florida, 32611, USA [email protected]
https://doi.org/10.31111/novitates/2019.50.47
O. В. Юрцева1, Е. В. Мавродиев2
1 Московский государственный университет имени М. В. Ломоносова
Биологический факультет, кафедра высших растений Ленинские горы, 1, стр. 12, Москва, 119991, Россия [email protected]
2 Университет Флориды Флоридский музей естественной истории Гейнсвилл, Флорида, 32611, США [email protected]
Abstract. The genus Bactria, recently described to house Atraphaxis ovczinnikovii from the Pamirs and Bactria lazkovii from the Central Tien Shan, may also include Polygonum tianschanicum from the Eastern Tien Shan. In order to re-circumscribe Bactria and to clarify the place of Polygonum tianschanicum in tribe Polygoneae, we performed Maximum Likelihood and Bayesian analyses of combined regions of the plastid genome and ITS1-2 regions of nrDNA for 58 species of tribe Polygoneae, with special attention to the secondary structure of pre-rRNA of the ITS1 and ITS2 loci. In all our analyses, Bactria lazkovii and Polygonum tianschanicum formed a highly supported clade, sister to Bactria ovczinnikovii in the plastid trees, but separate from the latter, as well as from the remaining genera in the ITS-based trees. Details of the secondary structure of pre-rRNA of the ITS1 and ITS2 loci also confirmed the close relationships of Bactria lazkovii and Polygonum tianschanicum, quite different from Bactria ovczinnikovii. Based on the molecular analyses, details of the secondary structure of pre-rRNA of the ITS1 and ITS2 loci, fine morphological distinctions, and distributional data, we propose the new genus Caelestium Yurtseva et Mavrodiev, presumably of hybrid origin, to include C. lazkovii and C. tianschanicum.
Keywords: Polygoneae, Atraphaxis, Bactria, Caelestium, new genus, incongruence, molecular analyses, pseudogene, rpl32-trnL(UAG), secondary structure of pre-rRNA, trnL-trnF, Pamirs, Tien Shan.
Аннотация. Род Bactria, недавно установленный по результатам молекулярного и морфологического исследования трибы Polygoneae для Atraphaxis ovczinnikovii с Южного Памира и Bactria lazkovii с Центрального Тянь-Шаня, может включать также морфологически близкий Polygonum tianschanicum, редкий эндемик Восточного Тянь-Шаня, который ранее не был исследован с молекулярно-филогенетической точки зрения. Чтобы уточнить состав рода Bactria и выяснить филогенетическое положение Polygonum tianschanicum в трибе Polygoneae, мы выполнили молекулярно-филогенетические анализы (метод максимального правдоподобия и Байесов анализ) объединенных участков пластидного генома и участка ITS ядерной рибосомной ДНК для 58 видов Polygoneae, с особым вниманием ко вторичной структуре пре-рРНК локусов ITS1 и ITS2. Во всех наших анализах Bactria lazkovii и Polygonum tianschanicum образовали хорошо поддержанную кладу, сестринскую к Bactria ovczinnikovii в пластидных деревьях, но обособленную от последнего вида (как и от прочих родов Polygoneae) в анализе ITS. Детали вторичной структуры пре-рРНК локусов ITS1 и ITS2 также показали тесное родство Bactria lazkovii и Polygonum tianschanicum, равно как и их существенные отличия от Bactria ovczinnikovii. Результаты молекулярных анализов пластидных и ядерных участков, различия вторичной структуры пре-рРНК локусов ITS1 и ITS2, морфологические отличия и географическое распространение позволяют выделить новый для науки род Caeles-tium Yurtseva et Mavrodiev. Род имеет предположительно гибридогенное происхождение и включает два узколокальных эндемика Тянь-Шаня: C. lazkovii (= Bactria lazkovii) из Кыргызстана и C. tianschanicum (= Polygonum tianschanicum) из
Поступила в редакцию | Submitted: 26.07.2019
Принята к публикации | Accepted: 14.12.2019
Северо-Восточного Китая. Полученные результаты обсуждаются в контексте уточненного состава трибы Polygoneae, который определяется на основании интерпретации результатов молекулярно-филогенетических анализов.
Ключевые слова: Polygoneae, Atraphaxis, Bactria, Caelestium, новый род, неконгруэнтность, молекулярный анализ, псев-доген, фШ—тЬ*™0', вторичная структура пре-рРНК, trnL-trnF, Памир, Тянь-Шань.
Introduction
Atraphaxis L. and Polygonum L. established by C. Linnaeus (1753) are perhaps the most problematic taxa of tribe Polygoneae (Polygonaceae Juss., Polygono-ideae); their relationships and circumscriptions are still not exactly clarified, despite several attempts (Ronse De Craene et al., 2004; Yurtseva et al., 2010, 2012a, b, 2016, 2017; Schuster et al., 2011b, 2015). S. Tavakkoli et al. (2015) recently transferred Polygonum sect. Avi-cularia Meisn. "§" Spinescentia Boiss. (Boissier, 1879: 1027) to Atraphaxis as A. sect. Polygonoides S. Tavakkoli, Kaz. Osaloo et Mozaff., but O. V. Yurtseva et al. (2017) accepted it as the genus Persepolium Yurtseva et Mavrodiev, which is sister to Atraphaxis. A new genus Bactria Yurtseva et Mavrodiev was established recently to include Atraphaxis ovczinnikovii (Czukav.) Yurtse-va from the Pamirs (Yurtseva et al., 2014: 43), originally described in Polygonum (Czukavina, 1962: 64), and Bactria lazkovii Yurtseva et Mavrodiev from the Tien Shan (Yurtseva et al., 2016: 43). These two species formed a monophyletic Bactria sister to the clade (Atraphaxis s. str. + Persepolium) in plastid trees, but showed paraphyly in ITS-based trees (Yurtseva et al., 2016), which requires further research and reappraisal.
Polygonum tianschanicum Chang Y. Yang (Yang, 1983) from Xinjiang, China, strongly resembles Bactria lazkovii in its habit, perianth, fruit and pollen morphology, and at the first approach may be treated as part of Bactria. We fully agree with C. Y. Yang (2010) who correctly considered Polygonum popovii Borodina (1989: 104) from China as a synonym of P. tianschanicum. Based solely on perianth and pollen morphology, P. popovii was recently transferred to Atraphaxis as A. popovii (Borodina) Yurtseva (Yurtseva et al., 2014). In this study we aimed to clarify the phylogenetic placement of Polygonum tianschanicum within Polygoneae.
Because P. tianschanicum and Bactria ovczinniko-vii (Czukav.) Yurtseva et Mavrodiev were originally described in Polygonum, in our analyses we used selected members of all intrageneric taxa currently accepted in the genus numbering ca. 130 species. Traditionally, four "main" sections were recognized in Polygonum in a "moderately narrow" circumscription (e. g., Dammer, 1893; Hedberg, 1946; Haraldson, 1978), namely: P. sect. Polygonum (= Avicularia) with ca. 100 species (Meisner, 1826: 43, 85), P. sect. Tephis (Adans.) Meisn.
(Meisner, 1857: 84) with two species, P. sect. Pseudo-mollia Boiss. (Boissier, 1879: 1027) with two species, and P. sect. Duravia S. Watson (1873: 665) with ca. 20 species. N. N. Tzvelev (1987: 78) added P. sect. Plebeja Tzvelev for P. plebeium R. Br. Polygonella Michx. (Michaux, 1803: 240) was traditionally accepted as a separate genus numbering 11 species (e. g., Meisner, 1857; Bentham, Hooker, 1880; Dammer, 1893; Horton, 1963; Freeman, 2005), but today it is also treated as part of Polygonum (Ronse De Craene et al., 2004; Schuster et al., 2011b, 2015). Hereinafter we attributed all the listed sections of Polygonum and Polygonella to the genus Polygonum s. l. in its "moderately narrow" circumscription, and treat the Old World sections as Polygonum s. str.
As a result of Maximum Parsimony (MP) analysis based on 51 morphological characters of 25 species of Polygonum a priori accepted in a wide circumscription, L.-P. Ronse De Craene et al. (2004) recognized P. sect. Polygonum with subsect. Polygonum and subsect. Tephis (Adans.) Ronse Decr. et S. P. Hong; P. sect. Pseudomol-lia; and P. sect. Duravia with subsect. Duravia (S. Watson) Ronse Decr. et S. P. Hong and subsect. Polygonella (Michx.) Ronse Decr. et S. P. Hong.
Our phylogenetic analyses of nrDNA ITS 1-2 data of Polygonum s. l. (Yurtseva et al., 2010, 2012b) confirmed two major clades combining, first, mostly Eurasian species of P. sect. Polygonum (incl. P. sect. Pseu-domollia); second, the North American P. sect. Duravia and P. sect. Polygonella. These results were generally confirmed by T. M. Schuster et al. (2011b, 2015).
Our preliminary study of the ITS sequence alignment of Polygoneae (Yurtseva et al., 2010, 2016) showed that many taxa of Polygoneae have extensive indels in the ITS1 and ITS2 regions, which cause some difficulties in the alignment of the sequence data. These difficulties, however, can be overcome by comparing the secondary structures of pre-RNA of the rDNA loci, since it is a way to align sequence regions more precisely and to clarify the relations of organisms of different taxonomic ranks more accurately (e. g., Denduangboripant, Cronk, 2001; Gottschling et al., 2001; Goertzen et al., 2003; Wang et al., 2007; Tippery, Les, 2008; Milyutina et al., 2010; Edg-er et al., 2014; Giudicelli et al., 2017).
Despite intensive modern molecular-phylogenetic studies of tribe Polygoneae (Schuster et al., 2011a, b, 2015), not much attention has been paid to the incon-gruences of the plastid- and ITS-based trees. Sometimes
the ITS sequence data were just merged with the plas-tid ones after exclusion of poorly aligned regions (e. g., Schuster et al., 2015), that obscured the difference in phylogenetic signals.
The main goals of our present study were: (1) to update the ITS phylogeny of tribe Polygoneae, but with special attention to the secondary structure of pre-RNA of the ITS1 and ITS2 regions, which has never been done before; (2) to compare the ITS and plastid topologies in more detail; (3) to clarify the circumscriptions of the genera Bactria and Polygonum s. l., with special attention to the pre-RNA secondary structure of the ITS1 and ITS2 regions.
Material and methods Morphological study
The morphological study was based on the type and verified specimens of Bactria ovczinnikovii, B. lazkovii, and Polygonum tianschanicum (= P. popovii) deposited in the Herbaria of the Komarov Botanical Institute of RAS, St. Petersburg, Russia (LE) and Lomonosov Moscow State University, Moscow, Russia (MW).
Sampling for molecular study
Our study involved selected members of the taxa of Polygoneae analyzed by T. M. Schuster et al. (2011b), although they took Polygonella as part of Polygonum s. l.: Atraphaxis (10 species), Duma T. M. Schust. (3), Fal-lopia Adans. (3), Polygonella (8), Polygonum sect. Polygonum (11), P. sect. Duravia (6), P. sect. Pseudomollia (1), and P. sect. Plebeja (1), Muehlenbeckia Meisn. (2), Reynoutria Houtt. (2), and Knorringia (Czukav.) Tzvelev (1). The list of taxa was supplemented by members of Persepolium (5), Bactria (2), Polygonum tianschanicum (1), and P. afromontanum Greenway (1), one of two members of Polygonum sect. Tephis. Oxyria digyna Hill (Rumiceae) was selected as an outgroup (Sanchez et al., 2011; Schuster et al., 2011a, b). The names of taxa and GenBank sequences (http://www.ncbi.nlm.nih.gov) are presented in Appendix (see the journal's website: www.binran.ru/journals/novitates/).
In total, 31 sequences of the cpDNA rpl32-tmL(UAG) region representing 30 species were analysed: one sequence was generated for this study, 13 sequences were obtained from our previous studies (Yurtseva et al., 2016), and 17 sequences were downloaded from GenBank. In total, 48 sequences of the cpDNA trnL intron(UAA) + trnL-trnF IGS region were analysed: two sequences were elaborated for this study, 14 sequences were obtained from our previous studies, and the remaining ones were downloaded from GenBank. Finally, the combined plastid alignment includes 60 accessions of 58 species of Polygoneae.
The same set of taxa (60 accessions of 58 species) was used for the analysis of nrDNA ITS1-5.8S-ITS2 regions. In total, 60 ITS sequences have been analysed; two of them were sequenced for this study, 31 sequences have been obtained earlier (Yurtseva et al., 2010, 2012b), and 27 sequences were downloaded from GenBank.
DNA isolation, amplification and DNA sequencing
Methods of DNA extractions and amplification, as well as the strategies of the phylogenetic analyses were described in O. V. Yurtseva et al. (2016). The nrDNA ITS region for most of the taxa was amplified using external primers and, in some cases, internal primers (Table 1). Due to the substantial problems with reading of heterogeneous nrDNA ITS sequences of Bactria laz-kovii, in this study we designed various primers as well as PCR profiles. Specifically, we used internal primers ITS2-BaN, ITS2-BaPSE, ITS3-BaN and ITS3-BaPSE and combined them with external primers ITS1, E1128f for amplification of the18S-ITS1 region, and with B, R2, R3, R4, R5 for amplification of the ITS2-26S region. In the case of Polygonum tianschanicum, we used the RCA technique (Brockington et al., 2008) to improve the quality of weak DNA extraction.
Identification of putative pseudogenes
To distinguish the putative pseudogenic ITS region, we examined nucleotide divergence, insertion-deletion events, the secondary structures themselves, as well as the methylation-induced substitution patterns (e. g., Buckler, Holtsford, 1996; Buckler et al., 1997; Mayol, Rosselloo, 2001; Hughes et al., 2002; Bailey et al., 2003; Harpke, Peterson, 2006; Bayly, Ladiges, 2007; Ochieng et al., 2007; Zheng et al., 2008). The indel events and nucleotide substitutions in the 5.8S region were used as a primary criterion, while the lower GC-content, the destruction of the secondary structure, and pattern of substitutions of the ITS1-5.8S-ITS2 region were used as additional criteria for ITS pseudogene identification. Pairwise comparisons used to detect pseudogenic sequences were conducted separately for the regions with different levels of variation (ITS1, 5.8S, ITS2), as C. D. Bailey et al. (2003) recommended. The nucleotide composition of the sequences and p-distance were calculated using MEGA V.6.0 (Tamura et al., 2013) using Tamura three-parameter distance (Kimura, 1980) with a gap treated as a complete deletion.
Strategies of sequence alignment
All sequences have been edited using BioEdit 7.0 (Hall, 1999). As before (Yurtseva et al., 2016), the combined trnL-trnF and rpl32-trreL(UAG) sequences were
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aligned using MAFFT (Katoh et al., 2002; Katoh, Standley, 2013), and then adjusted manually. We followed MAFFT's L-INS-i alignment strategy (Yurtseva et al., 2016), with the default settings for gap opening penalty and offset value strategy (Katoh et al., 2002; Katoh, Standley, 2013).
Because the explored ITS1 and ITS2 loci contained GC-rich repeats, forming several helices in the secondary structure, we used two identical ITS data sets aligned with two different alignment strategies: MAFFT's L-INS-i alignment strategy mentioned above, and accurate manual adjustment of the MAFFT-aligned matrix according to the general architecture of the helices recognized in the secondary structure of pre-rRNA of the ITS1 and ITS2 loci (see below).
Modelling of the secondary structure of pre-rRNA of the ITS regions
We modelled the secondary structure of pre-rRNA of the ITS1 and ITS2 loci using program MFOLD (Zuker, 2003), version 3.6 (http://mfold.rna.albany. edu/). The 5' and 3' ends of the ITS1 and ITS2 regions were determined using the available ITS sequences of Polygonaceae from GenBank. For each sequence, the optimal structure and a set of suboptimal ones were determined. The choice of the secondary structure model relied on the analysis of several species for each genus. Generally, the thermodynamically optimal model was chosen. However, in a few cases (Polygonum minimum S. Watson, Duma florulenta (Meisn.) T. M. Schust., Polygonella basiramea (Small) G. L. Nesom et V. M. Bates) the subsequent analysis considered thermodynamically suboptimal models, which bore the most resemblance to the models of closely related taxa.
Results
Combined plastid topology
The Maximum Likelihood (ML) and Bayesian (BI) analyses (Figs. 1; 2) resulted in similar topologies. Knorringia sibirica (Laxm.) Tzvelev is a sister to the remaining members of Polygoneae, which are subdivided in two major clades. The first one (the RFM-clade sensu Schuster et al., 2011b) includes mono-phyletic Reynoutria, Fallopia, and Muehlenbeckia. The sister clade (the APD-clade sensu Schuster et al., 2011b), includes Polygonum s. l. and a highly supported clade (Bactria (Atraphaxis + Persepolium)). The Duma clade is a non-supported sister of the latter.
Polygonum s. l. (as outlined above) is monophyletic and highly supported. It combines the highly supported New World clade with subclades Polygonella
and Polygonum sect. Duravia, and the highly supported Old World clade Polygonum s. str. with two subclades. Within Polygonum s. str., the subclade which includes P. aviculare L., the nomenclatural type of the section, was recognized as P. sect. Polygonum. Besides the species of this section distributed worldwide, it includes also P. molliiforme (the type of P. sect. Pseudomollia) from Iran and Tajikistan, and P. plebeium (the type of P. sect. Plebeja) distributed in Southeast Asia, Australasia and Africa. The sister subclade includes P. afromontanum (one of two members of P. sect. Tephis) from East Africa, which is nested among the remaining members of P. sect. Polygonum distributed in Central Asia, so this subclade was recognized here as P. sect. Tephis.
The strongly supported (ML/BI = 0.86/0.99) clade Bactria including B. ovczinnikovii is a sister of a highly supported subclade (Polygonum tianschanicum plus Bactria lazkovii), which is recognized here as the new genus Caelestium.
Bactria lazkovii differs from B. ovczinnikovii by having 43 nucleotide substitutions, 6 one-nucleotide and 8 larger (5-28 bp) indels on the combined 3-loci plastid alignment 1218 bp long. The p-distance between Cae-lestium (Bactria lazkovii and Polygonum tianschanicum) and other genera varies from 9.3 % (for Bactria ov-czinnikovii) to 16.5 % (for Fallopia), and these values are the maximum among the estimates of total difference over sequence pairs between genera compared in tribe Polygoneae (Table 2). Bactria lazkovii differs from Polygonum tianschanicum with 9 one-nucleotide substitutions, 3 one-nucleotide and 3 non-single nucleotide (4 and 28 nt long) indels of the combined 3-loci plas-tid alignment 1324 bp long. For this group, the within-group average p-distance is 14.9 %.
Table 2. The number of differences per site over Sequence Pairs between selected genera of Polygoneae based on 21 sequences of combined plastid regions (trnL intron + trnL-trnF IGS and rpl32-tmL(UAG) IGS), excluding all positions that contain gaps and missing data. A total of 1218 positions were analyzed in MEGA 6.0 (Tamura et al., 2013)
ITS topology based on the MAFFT alignment strategy
The ML and BI analyses of the MAFFT-based ITS matrix demonstrated that Knorringia sibirica is a sister to the rest of Polygoneae, which contain several highly supported clades, although their relationships are not well resolved (Figs. 3; 4).
Both ML and BI analyses confirmed the RFM-clade, including Reynoutria, Fallopia, and Muehlenbeckia, but Fallopia is monophyletic only on the BI tree. Duma appeared as part of the RFM-clade (ML tree in Fig. 3), or as part of the APD-clade, including Atraphaxis, Polygonum, and Duma (BI tree in Fig. 4); however, the position of Duma is not supported in either case.
Polygonum s. l. is monophyletic, but non-supported on the BI tree (Fig. 4), and paraphyletic on the ML tree (Fig. 3). It includes the same highly supported Old World clade Polygonum s. str. and the highly supported New World clade with subclades Polygonella and Polygonum sect. Duravia. The Old World clade Polygonum s. str. combines two subclades. Like in plastid trees, the subclade P. sect. Polygonum unites part of P. sect. Polygonum (incl. P. aviculare), and the subclade P. sect. Tephis unites P. afromontanum (P. sect. Tephis) and the rest of P. sect. Polygonum from Central Asia. Polygonum molliiforme (P. sect. Pseudomollia) and P. plebeium (P. sect. Plebeja) fall into the latter subclade, in contrast to their positions in P. sect. Polygonum on the plastid trees (Figs. 1; 2).
Atraphaxis and Persepolium are highly supported, although their combined clade is not supported. Bactria is polyphyletic: B. ovczinnikovii is a highly supported sister of the clade (Atraphaxis + Persepolium), while the highly supported subclade Caelestium (Bactria lazkovii + Polygonum tianschanicum) is nested in Polygonum s. l. on the ML tree (Fig. 3), or joins Polygonum s. l. on the BI tree without support (Fig. 4).
ITS topologies based on the MAFFT alignment, manually adjusted according to pre-rRNA secondary structure of the ITS1 and ITS2 loci of Polygoneae
The ML and BI analyses based on the MAFFT alignment, manually adjusted according to the secondary structure of pre-rRNA of the ITS1 and ITS2 loci of Polygoneae (the secondary-structure based alignment, SSBA hereinafter) show that Polygoneae include the same several clades (Figs. 5; 6), with their relationships being not fully resolved, as in the previous analyses (Figs. 3; 4).
Polygonum s. l. is polyphyletic. The clade Polygo-num s. str. is nested separately from the North American clade (Polygonella + Polygonum sect. Duravia); the latter is part of the grade that also includes the RFM-clade and Duma. Polygonella and Polygonum sect. Du-
1 2 3 4 5 6
[1] Fallopia
[2] Reynoutria 0.080
[3] Muehlenbeckia 0.067 0.037
[4] Atraphaxis 0.104 0.060 0.057
[5] Bactria 0.097 0.052 0.053 0.029
[6] Caelestium 0.165 0.127 0.125 0.107 0.093
[7] Polygonum s. l. 0.107 0.091 0.085 0.093 0.085 0.161
APD Clade 0.989
Atraphaxis spinosa Atraphaxis fischeri Atraphaxis frutescens Atraphaxis badghysi Atraphaxis teretifolia Atraphaxis atraphaxiformis 1 Atraphaxis toktogulica Atraphaxis atraphaxiformis 2 Atraphaxis pyrifolia Atraphaxis seravschanica Atraphaxis ariana Persepolium dumosum Persepolium spinosum Persepolium khajeh-jamalii Persepolium aridum Persepolium salicornioides Bactria ovczinnikovii 1 Bactria ovczinnikovii 2 Bactria lazkovii Polygonum tianschanicum Duma coccoloboides Duma horrida Duma florulenta Polygonum plebeium Polygonum molliiforme Polygonum equisetiforme Polygonum ramosissimum Polygonum arenastrum Polygonum arenarium Polygonum aviculare Polygonum luzuloides Polygonum boreale Polygonum paronychioides Polygonum thymifolium Polygonum cognatum Polygonum afromontanum Polygonum alpestre Polygonum pinicola Polygonella ciliata Polygonella basiramia Polygonella polygama Polygonella myriophylla Polygonella macrophylla Polygonella articulata Polygonella robusta Polygonum douglasii Polygonum engelmannii Polygonum austiniae Polygonum vagans Polygonum minimum Polygonum cascadense Fallopia convolvulus Fallopia dumetorum Fallopia baldschuanica Muehlenbeckia australis Muehlenbeckia platyclada Reynoutria sachalinensis Reynoutria japonica Knorringia sibirica Oxyria digyna
Atraphaxis (SE and Central Europe, SW and Central Asia)
Persepolium (SW Asia: Zagros)
Bactria
(Central Asia: Pamir) Caelestium (Central Asia: Tien Shan)
Duma (Australia)
Polygonum sect.
Polygonum
(Worldwide)
Polygonum sect. Tephis
(Eurasia and Africa)
Polygonella (N America: East)
Polygonum sect. Duravia (N America: West)
RFM Clade (SE Asia, Australia, Central and South America)
Outgroups
Fig. 1. ML phylogeny of Polygoneae based on the MAFFT-alignment of the plastid dataset: trnL intron + trnL-trnF IGS and rpl32-trnL(UAG) IGS (log likelihood: -11015.339832 ). Numbers above or below branches indicate the aLRT support values equal to or greater than 0.8.
0.975
0.997
0.991
APD Clade 1.000
.Atraphaxis spinosa . Atraphaxis fischeri .Atraphaxis frutescens Atraphaxis badghysi Atraphaxis teretifolia Atraphaxis seravschanica Atraphaxis pyrifolia Atraphaxis atraphaxiformis 1 Atraphaxis toktogulica Atraphaxis atraphaxiformis 2 .Atraphaxis ariana Persepolium spinosum . Persepolium dumosum . Persepolium khajeh jamalii . Persepolium aridum
Persepolium salicornioides . Bactria ovczinnikovii 1 . Bactria ovczinnikovii 2 . Bactria lazkovii Polygonum tianschanicum Duma coccoloboides . Duma horrida subsp horrida Duma florulenta Polygonum luzuloides Polygonum aviculare Polygonum boreale . Polygonum molliiforme Polygonum plebeium Polygonum arenastrum Polygonum ramosissimum Polygonum arenarium Polygonum equisetiforme . Polygonum thymifolium Polygonum paronychioides Polygonum cognatum . Polygonum afromontanum . Polygonum alpestre Polygonum pinicola Polygonella basiramia Polygonella ciliata . Polygonella polygama Polygonella myriophylla Polygonella macrophylla Polygonella articulata . Polygonella robusta Polygonum douglasii Polygonum engelmannii Polygonum austiniae Polygonum vagans Polygonum minimum . Polygonum cascadense . Fallopia dumetorum Fallopia convolvulus Fallopia baldschuanica Muehlenbeckia platyclada . Muehlenbeckia australis . Reynoutria sachalinensis . Reynoutria japonica Knorringia sibirica Oxyria digyna
Atraphaxis (SE and Central Europe, SW and Central Asia)
Persepolium (SW Asia: Zagros)
Bactria
(Central Asia: Pamir) Caelestium (Central Asia: Tien Shan)
Duma (Australia)
Polygonum sect.
Polygonum
(Worldwide)
Polygonum sect. Tephis
(Eurasia and Africa)
Polygonella (N America: East)
Polygonum sect. Duravia (N America: West)
RFM Clade (SE Asia, Australia, Central and South America)
Outgroups
Fig. 2. Bayesian phylogeny of Polygoneae based on the MAFFT-alignment of the plastid dataset: trnL intron + trnL-trnF IGS and rpl32-trreL(UAG) IGS. Numbers above branches indicate the posterior probabilities from BI analysis equal to or greater than 0.9.
0.894
Atraphaxis ariana Atraphaxis badghysi Atraphaxis seravchanica Atraphaxis atraphaxiformis 1 Atraphaxis atraphaxiformis 2 Atraphaxis frutescens Atraphaxis pyrifolia Atraphaxis spinosa Atraphaxis fischeri Atraphaxis teretifolia Atraphaxis toktogulica Persepolium salicornioides Persepolium khajeh-jamalii Persepolium spinosum Persepolium dumosum Persepolium aridum Bactria ovczinnikovii 1 Bactria ovczinnikovii 2 Polygonum afromontanum Polygonum thymifolium Polygonum paronychioides Polygonum cognatum Polygonum plebeium Polygonum alpestre Polygonum molliiforme Polygonum arenarium Polygonum boreale Polygonum equisetiforme Polygonum luzuloides Polygonum aviculare Polygonum arenastrum Polygonum ramosissimum Bactria lazkovii Polygonum tianschanicum Polygonella ciliata Polygonella basiramia Polygonella gracilis Polygonella polygama Polygonella macrophylla Polygonella myriophylla Polygonella articulata Polygonella robusta Polygonum douglasii Polygonum engelmannii Polygonum austiniae Polygonum vagans Polygonum cascadense Polygonum minimum Reynoutria sachalinensis Reynoutria japonica Fallopia baldschuanica Muehlenbeckia platyclada Muehlenbeckia australis Fallopia dumetorum Fallopia convolvulus Duma coccoloboides Duma horrida Duma florulenta Knorringia sibirica Oxyria dygina
Atraphaxis (SE and Central Europe, SW and Central Asia)
Persepolium (SW Asia: Zagros)
Bactria
(Central Asia: Pamir)
Polygonum sect. Tephis
(Eurasia and Africa)
Polygonum sect.
Polygonum
(Worldwide)
Caelestium (Central Asia: Tien Shan)
Polygonella (N America: East)
Polygonum sect. Duravia (N America: West)
RFM Clade (SE Asia, Australia, Central and South America)
Duma (Australia)
Outgroups
Fig. 3. ML phylogeny of Polygoneae based on the MAFFT-alignment of the ITS1-2 loci nrDNA (log likelihood: -6641.832866). Numbers above or below branches indicate the aLRT support values equal to or greater than 0.8.
Duma horrida Duma coccoloboides Duma florulenta Atraphaxis atraphaxiformis 1 Atraphaxis atraphaxiformis 2 Atraphaxis frutescens Atraphaxis ariana Atraphaxis badghysi Atraphaxis seravchanica Atraphaxis pyrifolia Atraphaxis spinosa Atraphaxis fischeri Atraphaxis teretifolia Atraphaxis toktogulica Persepolium salicornioides Persepolium khajeh-jamalii Persepolium spinosum Persepolium aridum Persepolium dumosum Bactria ovczinnikovii 1 Bactria ovczinnikovii 2 Polygonum plebeium Polygonum alpestre Polygonum afromontanum Polygonum cognatum Polygonum molliiforme Polygonum paronychioides Polygonum thymifolium Polygonum arenarium Polygonum boreale Polygonum luzuloides Polygonum equisetiforme Polygonum aviculare Polygonum arenastrum Polygonum ramosissimum Polygonella basiramia Polygonella ciliata Polygonella gracilis Polygonella polygama Polygonella macrophylla Polygonella myriophylla Polygonella articulata Polygonella robusta Polygonum douglasii Polygonum engelmannii Polygonum austiniae Polygonum vagans Polygonum minimum Polygonum cascadense Bactria lazkovii Polygonum tianschanicum Fallopia convolvulus Fallopia dumetorum Fallopia baldschuanica Muehlenbeckia australis Muehlenbeckia platyclada Reynoutria japonica Reynoutria sachalinensis Knorringia sibirica Oxyria dygina
Duma (Australia)
Atraphaxis (SE and Central Europe, SW and Central Asia)
Persepolium (SW Asia: Zagros)
Bactria
(Central Asia: Pamir)
Polygonum sect. Tephis
(Eurasia and Africa)
Polygonum sect.
Polygonum
(Worldwide)
Polygonella (N America: East)
Polygonum sect. Duravia (N America: West)
Caelestium (Central Asia: Tien Shan)
RFM Clade (SE Asia, Australia, Central and South America)
Outgroups
Fig. 4. Bayesian phylogeny of Polygoneae based on the MAFFT-alignment of the ITS1-2 loci nrDNA. Numbers above or below branches indicate posterior probabilities from BI analysis equal to or greater than 0.9.
ravia are both monophyletic and highly supported. The clade Polygonum s. str. is highly supported only in ML analysis (Fig. 5). It combines two strongly supported subclades. The first includes the members of P. sect. Polygonum (incl. P. aviculare) distributed worldwide. The second subclade recognized as P. sect. Tephis combines P. afromontanum (P. sect. Tephis) and the members of P. sect. Polygonum from Central Asia. Polygonum mollii-forme (P. sect. Pseudomollia) and P. plebeium (P. sect. Plebeja) appear again in the same subclade, in contrast to their positions in the subclade P. sect. Polygonum on the plastid trees (Figs. 1; 2).
The clades Atraphaxis and Persepolium are highly supported, however, the clade (Atraphaxis + Persepolium) has no support. Bactria is clearly polyphyletic (Figs. 5; 6). Only on the BI tree it is associated with Atraphaxis and Persepolium (Fig. 6). The position of Caelestium is not resolved, but in either case it is not combined with any genus of Polygoneae, in contrast to the sistership with Bactria ovczinnikovii on the plastid trees.
Locus ITS1 and its pre-rRNA secondary structure
Since the phylogenetic analysis based on ITS region did not show a resolved topology and the positions of some taxa were not determined, we compared the pre-rRNA secondary structure of their ITS1 and ITS2 loci.
The pre-rRNA secondary structure of the ITS1 has a central loop with several inner and outer helices formed by inverted repeats with high GC content. Maximum (five) outer helices are present in pre-rRNA secondary structure of the ITS1 of Reynoutria (Fig. 7: A), Fal-lopia, Muehlenbeckia, Duma (Fig. 7: G), Atraphaxis ariana (Grig.) T. M. Schust. et Reveal (Fig. 7: B) (and also A. badghysi Kult., A. radkanensis S. Tavakkoli, Kaz. Osaloo et Mozaff. absent from the analyses). Most species of Atraphaxis (Fig. 7: C), as well as Persepolium and Bactria ovczinnikovii (Fig. 7: D), have helix II, but lack helix III. In contrast, Bactria lazkovii and Polygonum tianschanicum recognized here as the members of the new genus Caelestium, and the members of Polygonum s. l. lack helix II, but have helix III (Fig. 7: E, F, H, I).
The outer helix I (12 nt) is formed by 5'-CUGC-3' and 5'-GCAG-3' inverted repeats.
The outer helix II (22-41 nt) is variable, but present only in part of Polygoneae (Fig. 8; Table 3). Atraphaxis ariana, Persepolium, and Bactria ovczinnikovii have a similar length (32-37 nt) and composition of helix II (Fig. 8: A-D). In all of these taxa, the conserved part of the stem is formed by inverted repeats (5'-GGCGGGGG-3' and 5'-UCCCCCCC-3') shown in a frame in Fig. 8. The differences are in several one-nucleotide substitutions and one-nucleotide deletions. Persepolium (Fig. 8: A) and some of Atraphaxis species
(Fig. 8: D) have additional GC pairs at the base of the stem. Duma florulenta (Fig. 8: E) has the shortest helix II (22 nt). It has the conserved part of the stem as short as that in Reynoutria and Muehlenbeckia, but this conserved part is even shorter in Fallopia (Table 3). The members of the RFM-clade have the longest helix II (41 nt) due to additional GC-rich inverted repeats in the apical part of the stem, but lack base pairs in the proximal part (Fig. 8: F).
The outer helix III (17-25 nt) (Fig. 9; Table 3) has a stem formed by inverted repeats 5'-GCCCGGG-(nN)-CCCGGGC-3'. Reynoutria, Muehlenbeckia, Fallopia, and Duma have the identical composition of helix III (Fig. 9: A). The same, or almost the same, composition of helix III was found in some species of Polygonella and in the Polygonum species, which fall within the subclade Polygonum sect. Tephis on the ITS-based trees and grow in Central Asia and Africa (Fig. 9: E, H; Table 3): P. mol-liiforme (P. sect. Pseudomollia), P. plebeium (P. sect. Plebeja), P. afromontanum (P. sect. Tephis), P. cognatum Meisn., P. thymifolium Jaub. et Spach (among other Central Asian members of P. sect. Polygonum).
The members of Polygonum sect. Duravia (P. minimum S. Watson, P. douglasii Greene) and P. sect. Polygonum (P. aviculare, P. boreale Small) have 1-2 extra base pairs in the outer position and lack one of nucleo-tids in the right arm of helix III (Fig. 9: F, G; Table 3). Besides, the members of P. sect. Polygonum (P. aviculare, P. boreale) have the largest terminal loop (Fig. 9: F) of helix III.
Atraphaxis ariana (and also A. badghysi, A. radkanensis) have an extra base pair (A-U) in the outer position (Fig. 9: B) of helix III. Polygonum tianschani-cum and Bactria lazkovii share the shortest helix III (17 nt), which lacks one base pair (C-C) in the stem (Fig. 9: C-D), and can be produced from helix III of any taxon. Both have 5 nucleotids in the terminal loop, like some species of Polygonum s. str. (e. g., P. plebeium, P. boreale) and Polygonella (Table 3).
The outer helix IV (22-23 nt) is the most conserved (Table 3). The stem is normally formed by 9 bp. The motif GGCTY-(4-7n)-GYGYCAAGGAA noted by J. S. Liu and C. L. Schardl (1994) as conserved across plants is present in helix IV as 5'-GGCGC-(GGA(U/C)U)-GCGCCAAGGAC-3' in all members of Polygoneae. Although the composition of helix IV is almost the same in most of Polygoneae, one-nucleotide substitutions are present in some non-related taxa. Some members of Polygonella and Polygonum sect. Duravia (P. minimum) lack one nucleotide in the left arm of the stem. Duma florulentha (Meisn.) T. M. Schust. and Polygonum douglasii have an extra base pair in the stem, but smaller terminal loop.
Outer helix V is the most variable in Polygoneae (36-45 nt) (Table 3). Duma is close to the members of the RFM-clade by the structure of helix V. The North American Polygonella and Polygonum sect. Duravia slightly differ from each other and from the Old World Polygonum s. str. in the structure of helix V. The same applies to the members of the subclades P. sect. Tephis and P. sect. Polygonum (Table 3) as they appeared on the ITS-based trees (Table 3; Figs. 3-6). Atraphaxis, Bactria ovczinnikovii and Persepolium share the length (40 nt) and structure of helix V, though some species of Atraphaxis have extra base pairs in the inner position. Bactria lazkovii and Polygonum tianschanicum have unique structure of helix V, distinct in the proximal part of the stem.
To summarize, Bactria ovczinnikovii, Persepolium, and most of Atraphaxis species have the same set of helices of similar length in the pre-rRNA secondary structure of the ITS1 (Table 3). Polygonum sect. Duravia and Polygonella differ from the rest of Polygonum s. l. by the length and structure of helix V. The subclades P. sect. Polygo-num and P. sect. Tephis, as they appeared in the results of the ITS analyses, differ in the length and structure of helices III and V. Duma is close to the members of the RFM-clade in the whole secondary structure of pre-RNA of the ITS1, and in the structure of helices II-V. Polygonum tianschanicum and Bactria lazkovii resemble Polygonum s. l. in the general pre-rRNA secondary structure of the ITS1 locus (Fig. 7: E, F, H, I), but differ in having the unique structures of helices III and V.
Locus ITS2 and its pre-rRNA secondary structure
The pre-rRNA secondary structure of the ITS2 region has a central loop and four outer helices (Fig. 10). Helices I and II have a common stem, and helices III and IV are branched from the central loop. Helices I and IV are the most variable, while helices II and III are more conserved (Table 4).
The outer helix I 30-43 nt long includes two conserved inverted repeats in the stem base (5'-CGCCCC-3' and 5'-GGGGCG-3', 5'-CCCCCC-3' and 5'-GGGGGG-3), but is rather polymorphic in the apical part (Table 4). It is shortest in the RFM-clade and Duma, in some members of Polygonum sect. Duravia and Polygonella, e. g., P. robusta (Small) G. L. Nesom et V. M. Bates (= Thy-sanella robusta Small) (30-32 nt), and Polygonum tianschanicum (33 nt). Helix I is longest in P. afromontanum, P. cognatum (P. sect. Tephis), and Bactria lazkovii (43 nt). In some aspects, the structure of helix I of B. lazkovii and Polygonum tianschanicum is similar to that of Polygonella (Table 4). In contrast, Atraphaxis, Persepolium and Bac-tria ovczinnikovii share the distinct length (38-40 nt) and structure of helix I.
The outer helix II has a very conserved basal part of the stem 12 bp long, which is formed by inverted repeats (5'-GGCCCCCCGUGC-3' and 5'-G(C/U)GC-GGCCGGCC-3'), but the apical part of the stem and the loop vary in size and composition (Table 4). For example, Atraphaxis has additional base pairs flanking the terminal loop. Persepolium and Bactria ovczinnikovii have a very similar composition of helix II, but Persepo-lium lacks one nucleotide in the left arm and has two extra base pairs in the inner position. Bactria lazkovii and Polygonum tianschanicum have identical structure and length of helix II (38 nt), different from that of Bactria ovczinnikovii, with one extra base pair in the apical part of the stem and a compensatory substitution A-U ^ U-A in the outer position. Polygonella and Polygonum sect. Duravia are close to Duma and the members of the RFM-clade in the structure of helix II. The members of Polygonum s. str. demonstrate some differences, e. g., P. molliiforme (P. sect. Pseudomollia) has a much longer apical part of the stem.
The outer helix III is the longest (61-82 nt) and rather variable. The positions of inner loops in the stem distinguish the taxa of Polygoneae from each other. Polygonella differs from the rest of Polygonum s. l. in the structure of helix III. The sections of Polygonum s. l. demonstrate slight, but definite distinctions. Polygonum molliforme (P. sect. Pseudomollia) and P. plebeium (P. sect. Plebeja) have a distinct proximal part of helix III, but are close to the members of P. sect. Tephis in the apical part of the stem. Polygonum sect. Tephis shares the proximal part with P. sect. Polygonum. Bactria lazkovii and Polygonum tianschanicum are close to Atraphaxis, Persepolium, and Bactria ovczinnikovii in the structure of helix III (Table 4).
The outer helix IV is most variable; it strongly changes its secondary structure depending on thermo-dynamic conditions and is not reliable for comparing.
In summary, Duma is the most close to the members of the RFM-clade by the structure of helices I, II, and III. The New World Polygonella and Polygonum sect. Duravia demonstrate slight, but definite differences in the structure of helices I and III. The same applies to the subclades of the Old World Polygonum s. str. Bactria ovczinnikovii, Atraphaxis, and Persepolium have similar structure of helices I and III, but Atraphaxis has longer helix II. Bactria lazkovii and Polygonum tianschanicum (the genus Caelestium) have unique structure of helix II and share the structure of helix III with Bactria ovczinnikovii, Atraphaxis and Persepolium.
The "anomalous" ITS sequences of Bactria lazkovii and their pre-rRNA secondary structure
As a result of applying multiple sets of primers and PCR profiles, besides the putatively functional ITS(A),
0.955,-
0.9731-1
0.885 0.908
0.973
9 / 3|-
cE
0.951
0.924,-
rCE
0.941
0.990
Polygonum s. str.
0.962
—с
0.988
0.9941-
гСЕ —d
0.946
0.885
0.876
0.9871-
KÏE
с
0.960
0.965
0.987
Atraphaxis atraphaxiformis 1 Atraphaxis atraphaxiformis 2 Atraphaxis frutescens Atraphaxis seravchanica Atraphaxis pyrifolia Atraphaxis teretifolia Atraphaxis fischeri Atraphaxis spinosa Atraphaxis badghysi Atraphaxis ariana Atraphaxis toktogulica Persepolium salicornioides Persepolium khajeh-jamalii Persepolium spinosum Persepolium dumosum Persepolium aridum Polygonum thymifolium Polygonum paronychioides Polygonum alpestre Polygonum afromontanum Polygonum cognatum Polygonum molliiforme Polygonum plebeium Polygonum boreale Polygonum arenarium Polygonum luzuloides Polygonum arenastrum Polygonum aviculare Polygonum equisetiforme Polygonum ramosissimum Reynoutria sachalinensis Reynoutria japonica Fallopia baldschuanica Muehlenbeckia platyclada Muehlenbeckia australis Fallopia convolvulus Fallopia dumetorum Polygonella ciliata Polygonella basiramia Polygonella gracilis Polygonella polygama Polygonella macrophylla Polygonella myriophylla Polygonella robusta Polygonella articulata Polygonum engelmannii Polygonum douglasii Polygonum austiniae Polygonum vagans Polygonum cascadense Polygonum minimum Duma horrida Duma coccoloboides Duma florulenta Bactria lazkovii Polygonum tianschanicum Bactria ovczinnikovii 1
■ Bactria ovczinnikovii 2
■ Knorringia sibirica
■ Oxyria dygina
Atraphaxis (SE and Central Europe, SW and Central Asia)
Persepolium (SW Asia: Zagros)
Polygonum sect. Tephis
(Eurasia and Africa)
Polygonum sect.
Polygonum
(Worldwide)
RFM Clade (SE Asia, Australia, Central and South America)
Polygonella (N America: East)
Polygonum sect. Duravia (N America: West)
Duma (Australia)
Caelestium (Central Asia: Tien Shan) Bactria
(Central Asia: Pamir) Outgroups
Fig. 5. ML phylogeny of Polygoneae based on the pre-rRNA secondary structure on the related alignment of the ITS1 and ITS2 loci nrDNA (log likelihood: -6493.537870). Numbers above or below branches indicate the aLRT support values equal to or greater than 0.8.
0.999
1.00C
Atraphaxis ariana Atraphaxis badghysi Atraphaxis toktogulica Atraphaxis atraphaxiformis 1 Atraphaxis atraphaxiformis 2 Atraphaxis frutescens Atraphaxis seravchanica Atraphaxis pyrifolia Atraphaxis teretifolia Atraphaxis fischeri Atraphaxis spinosa Persepolium salicornioides Persepolium khajeh-jamalii Persepolium spinosum Persepolium aridum Persepolium dumosum Bactria ovczinnikovii 1 Bactria ovczinnikovii 2 Polygonella basiramia Polygonella ciliata Polygonella gracilis Polygonella polygama Polygonella macrophylla Polygonella myriophylla Polygonella robusta Polygonella articulata Polygonum douglasii Polygonum engelmannii Polygonum austiniae Polygonum vagans Polygonum minimum Polygonum cascadense Muehlenbeckia australis Muehlenbeckia platyclada Reynoutria sachalinensis Reynoutria japonica Fallopia baldschuanica Fallopia convolvulus Fallopia dumetorum Duma horrida Duma coccoloboides Duma florulenta Polygonum molliiforme Polygonum plebeium Polygonum alpestre Polygonum afromontanum Polygonum thymifolium Polygonum cognatum Polygonum paronychioides Polygonum arenarium Polygonum boreale Polygonum luzuloides Polygonum aviculare Polygonum arenastrum Polygonum equisetiforme Polygonum ramosissimum Polygonum tianschanicum Bactria lazkovii Knorringia sibirica Oxyria dygina
Atraphaxis (SE and Central Europe, SW and Central Asia)
Persepolium (SW Asia: Zagros)
Bactria
(Central Asia: Pamir)
Polygonella (N America: East)
Polygonum sect. Duravia (N America: West)
RFM Clade (SE Asia, Australia, Central and South America)
Duma (Australia)
Polygonum sect. Tephis
(Eurasia and Africa)
Polygonum sect.
Polygonum
(Worldwide)
Caelestium (Central Asia: Tien Shan)
Outgroups
Fig. 6. Bayesian phylogeny of Polygoneae based on the pre-rRNA secondary structure and on the related alignment of the ITS1-2 loci nrDNA. Numbers above or below branches indicate posterior probabilities from BI analysis equal to or greater than 0.9.
the putatively pseudogenic ITS(B) was amplified from the same sample of Bactria lazkovii. External primers NNC-18S10 and C26 resulted in amplification of mostly heterogeneous ITS sequences. The most of combinations of selective internal primers for functional ITS (ITS2-BaN and ITS3-BaN) with universal external primers (Table 1), resulted in successful selective amplification of the fragments, including 3'-end of the 18S region, the ITS1-5.8S-ITS2 region, and 5'-end of the 26S region. Combinations of selective internal primer ITS2-BaPSE with external primers ITS1, E1128f were used for amplification of the 18S-ITS3-5.8S region, and combinations of selective internal primer ITS3-BaPSE with B, R2, R3, R4, R5 were used for amplification of the 5.8S-ITS2-26S region (Table 1), however only the combinations ITS1 with B, and ITS3-BaPSE with B and R2 resulted in successful amplification of the 5.8S-ITS2-26S region for the pseudogenic ITS.
In total, four identical sequences of putatively functional ITS(A) and five sequences of the highly variable pseudogenic ITS(B) (excluded from the phylogenetic analyses but used for further comparisons of the secondary structures) were successfully obtained.
The putatively pseudogenic ITS(B) of Bactria laz-kovii differs from the functional ITS(A) by the presence of 75 nucleotide substitutions, mainly transitions G^A (36 %) and C^T (33.3%) on the ITS1-2 region. As we originally estimated, on average, the GC-con-tent of the ITS1-2 region in Polygoneae is relatively high (65.5-70.1 %), especially in Fallopia, Reynoutria, and Muehlenbeckia. The pseudogenic ITS(B) of Bac-tria lazkovii has a lower GC-content (59.4 %) than its functional ITS(A) (68.8 %), or the ITS of other genera of Polygoneae, except for the ITS of Polygonella, which also has low (62.0 %) GC-content.
The functional ITS(A) of Bactria lazkovii shows 5.8-17.9 % range of p-distance from other genera on the ITS1-5.8S-ITS2 region (ITS1, 6.8-27.0 %; ITS2, 6.5-28.3 %; 5.8S, 0.8-3.2 %). These values correspond to the average level (5.3-22.9 %) of intergeneric divergence in Polygoneae on the same region (ITS1, 7.053.6 %; ITS2, 8.9-25.0 %; 5.8S, 0.3-3.5 %).
The pseudogenic ITS(B) of Bactria lazkovii has a much higher range of p-distance on the ITS1-2: 17.733.9 % (ITS1, 20.7-51.1 %; ITS2, 19.0-47.3 %; 5.8S, 10.5-15.6 %) from other genera of Polygoneae, and 17.9 % (ITS1, 17.6 %; ITS2, 23.2 %; 5.8S, 11.6 %) from the functional ITS(A) of B. lazkovii.
For the 5.8S gene, the range of p-distance between the functional ITS(A) of Bactria lazkovii and the ITS of other genera of Polygoneae (0.8-3.2 %) is comparable with the range of intergeneric (0.3-3.5 %) divergence in Polygoneae. In contrast, the pseudogenic ITS(B) of
Bactria lazkovii has a much higher level of intergeneric p-distance (10.5-15.6 %) for the 5.8S gene, which differs from the functional ITS(A) by 18 nucleotide substitutions and two one-nucleotide deletions.
In Bactria lazkovii, the pre-RNA secondary structure of the pseudogenic ITS1(B) does not form helix I, but forms helices II-V (Fig. 11: A; Table 3). In contrast to the functional ITS1(A) of the same sample (Fig. 7: E), its helix II (Fig. 11: A) is unique in Polygoneae: its basal conserved part is as short as in Duma, Reynoutria, and Muehlenbeckia (Fig. 8: F), but its apical part is as long as that of Atraphaxis, Persepolium, and Bactria ovczinnikovii (Fig. 8: A-D). Helix III of the ITS1 has 5 nucleotides in the terminal loop of both functional and pseudogenic variants, but the pseudogenic ITS1(B) has an extra nucleotide in the stem (18 vs 17), that can be viewed as a transitional state to its functional variant, which lacks the whole bp (Fig. 9: C, D; Table 3). In the pseudogenic ITS1(B), helix IV has an extra nucleotide in the stem (24 vs 23 nt), helix V is shorter (38 vs 40 nt) than that in the functional ITS1(A), but can be produced from the latter (Fig. 11: A; Table 3). Thus, the pre-rRNA secondary structure of the pseudogenic ITS1(B) of B. lazkovii can be produced from its functional ITS1(A), but has additional helix II unique in Polygoneae.
The pseudogenic ITS2 of B. lazkovii has the same four helices in the pre-rRNA secondary structure, like the ITS2 of the rest of Polygoneae, but due to numerous substitutions, the pseudogenic ITS2 of B. lazkovii differs in the size and composition of the helices from its own functional ITS2 (Fig. 11: B), these helices are absent from Table 4.
Discussion
Incongruence of the plastid and ITS trees and paraphyly of Bactria s. l.
Both BI and ML trees based on the ITS data (Figs. 3-6) and on the combined plastid matrix (Figs. 1; 2) recovered the same eight highly supported clades: the RFM-clade named by T. M. Schuster et al. (2011b), including Reynoutria, Fallopia, and Muehlenbeckia; the clade Duma; the clade Polygonum s. str.; the clade (Polygonella + Polygonum sect. Duravia); the clade Atraphaxis; the clade Persepolium; the clade Bactria ovczinnikovii (two accessions of the same species); and the clade Cae-lestium (Bactria lazkovii + Polygonum tianschanicum).
Our ML and BI plastid trees (Figs. 1; 2) are similar to those obtained by Schuster et al. (2011b, 2015) for the combined plastid plus ITS sequence data matrix, and in general agree with our previous plastid trees (Yurtseva et al., 2016). Our plastid data confirmed the RFM-clade and the APD-clade revealed by Schuster et al. (2011b, 2015).
Fig. 7. Pre-rRNA secondary structure of the ITS1 locus for some genera of tribe Polygoneae.
A — Reynoutria sachalinensis; B — Atraphaxis ariana; C — A. pyrifolia; D — Bactria ovczinnikovii; E — Caelestium lazkovii; F — C. tianschanicum; G — Duma florulenta; H — Polygonella polygama; I — Polygonum afromontanum. I-V — outer helices.
Fig. 8. Outer helix II of the pre-rRNA secondary structure of the ITS1 locus for some genera of tribe Polygoneae. A — Persepolium salicomioides; B — Atraphaxis ariana; C — Bactria ovczinnikovii; D — Atraphaxis pyrifolia; E — Duma florulenta; F — Reynoutria sachalinensis. The stem is formed by conserved poly-G and poly-C repeats (in frames). Arrows indicate nucleotide substitutions.
The APD-clade recovered by Schuster et al. (2011b, 2015), was confirmed only by the analyses of combined plastid data (Figs. 1; 2). It combines Atraphaxis, Duma, and Polygonum s. l. On the plastid trees, Duma is a weakly supported sister of the clade ((Atraphaxis + Persepolium) Bactria) (Figs. 1; 2). It is associated with the RFM-clade in the analyses of MAFFT-aligned matrix of the ITS (Figs. 3; 4), and forms a weak grade with the RFM-clade, Polygonella and Polygonum sect. Dura-via on the ITS SSBA-based ML and BI trees (Figs. 5; 6), that agrees with the similarity of the pre-RNA secondary structure of the ITS1 and ITS2 (Tables 3, 4).
All variants of analyses confirmed the clade combining Atraphaxis s. str., Persepolium, and Bactria ovczinnikovii, which corresponds to the Atraphaxis s. l. broadly treated by S. Tavakkoli et al. (2015). The highly supported clade that includes Atraphaxis and Persepolium constantly appears on our previous (Yurtseva et al., 2016: 8, fig. 2) and current plastid trees (Figs. 1; 2), and on morphology-based trees (Mavrodiev, Yurtseva, 2017; Yurtseva et al., 2017), but it is not supported on
the ITS-based trees (Figs. 3-6, see also Yurtseva et al., 2016: 7, fig. 1). Similarly, the clade ((Atraphaxis + Persepolium) + Bactria s. l.), circumscribed as Atraphaxis s. l. (Tavakkoli et al., 2015), is supported on the plastid trees (Figs. 1; 2), but not supported on the ITS ISSB-based trees (Figs. 5; 6). That argues for generic independence of Atraphaxis, Persepolium and Bactria, although these taxa demonstrate great similarity in the pre-RNA secondary structure of the ITS1 and ITS2 (Tables 3, 4).
As we have mentioned above, and as we found before (Yurtseva et al., 2016), Bactria is monophyletic on the plastid trees (Figs. 1; 2), but non-monophyletic on all the ITS-based trees (Figs. 3-6). Although Bactria ovczinnikovii is associated with Atraphaxis and Persepolium on the plastid trees (Figs. 1; 2) and on most of the ITS-based trees, the clade Caelestium (Bactria lazko-vii + Polygonum tianschanicum) is separate from Bactria ovczinnikovii in all the ITS-based trees (Figs. 3-6).
According to the trees that resulted from the analysis of the MAFFT-aligned matrix of the ITS region (Figs. 3; 4), the clade Caelestium might be interpreted
Table 3. Helices II-V of the pre-RNA secondary structure of the ITS1 locus in tribe Polygoneae
Taxon Species Helix II Helix III Helix IV Helix V
loop loop loop loop
Reynoutria R. sachalinensis 6 12 5 1 3 3 4 2 2 5 5 3 - 2 0/1 6 0/1 6 3
Muehlenbeckia M. platyclada 6 12 5 1 3 3 4 2 2 5 5 3 - 2 0/1 6 0/1 6 3
Fallopia F. baldschuanica - 5 12/13 5 3 3 4 2 2 5 5 2 - 2 0/1 6 0/1 6 4
Duma D.flondentha 2 6 1 4 1 3 3 4 2 2 5+1 5-2 3 - 2 0/1 6 0/1 6-1 4+2
P. polygama - - - - 1 3 3 4 2 1/2 5 5 3 0/1 3 0/1 8 0/1 3 4+2
Polygonella P. gracilis - - - - 1 3/2 3 5 2 1/2 5 5 2 0/1 3+1 0/1 8 1/2 3 4
P. aiticulata - - - - ? ? ? ? 2 2 5 5 2 0/1 3 0/1 6 1/2 5 4
Polygonum P. minimum - - - - 1 3/2 3+2 4 2 1/2 5 5 5 0/1 2 0/1 8 0/1 4 4
P. sect. Duravia P. douglasii - - - - 1 3/2 3+1 4 2 2 5+1 5-2 5 0/1 2 0/1 8 0/1 4 4
P. sect. Pseudomollia P. molliiforme - - - - 1 3 3 4 2 2 5 5 3 - 2 0/1 2 0/1 10 4
P. sect. Plebeja P. plebeium - - - - 1 3/4 3 5 2 2 5 5 3 - - 0/1 2 0/1 10 4
P. afromontanum - - - - 1 3 3 4 2 2 5 5 3 - - 0/1 2 0/1 10 4
P. sect. Tephis P. cognatum - - - - 1 3 3 4 2 2 5 5 3 - - 0/1 2 0/1 10 4
P. thy mi folium. - - - - 1 3 3 4 2 2 5 5 2 - - 0/1 2 0/1 10 4
P. sect. Polygonum P. aviculare - - - - 1 3/2 3+2 5+3 2 2 5 5 3 - 2 0/1 4 1/2 7 4
P. boreale - - - - 1 3/2 3+2 5 2 2 5 5 3 - 2 0/1 4 1/2 7 4
C. tianschanicum - - - - 1 2 3 5 2 2 5 5 3 0/1 2+2 0/1 7 0/1 5 4
Caelestium C. lazkovii (A) - - - - 1 2 3 5 2 2 5 5 3 - 2 0/1 7 0/1 5 4
C. lazkovii (B) 1 6 5 3 1 3/2 3 5 2 2/3 5 5 3 - 2 0/3 4 1/2 5-1 4+2
Bactria B. ovczinnikovii 2 7/8 5 3 - - - - 2 2 5 5 2 - 2 0/1 8 0/1 4 4
Persepolium P. salicornioides 3 8 5-1 3+2 - - - - 2 2 5 5 2 - 2 0/1 8 0/1 4 4
A. ariana 2 8/7 5 3 1 3 3+1 4 2 2 5 5 3 - 2 0/1 8 0/1 4 4
Atraphaxis A. fnitescens 5 8 5 3 - - - - 2 2 5 5 3 - 2 0/1 8 0/1 4 4
A. pyrifolia 4 8 5-1 3+2 - - - - 2 2 5 5 3 0/1 2+2 0/1 8 0/1 4 4
o
^ Note. The nucleotides in the terminal loop of a helix are designated in bold. The rest numbers designate the number of base pairs in the stem; 0/1 — the number of
n> nucleotides in the left/right arm of the helix.
Ln
С с CCG
с с с и GCG
С G A U с и с и
G ■ U G -С G ■ С G ■ С
G -С G -С G ■ С GC
G ■ С G ■ С G ■ С G ■ С
С С С С
CG CG CG CG
CG CG С -G С -G
GC GC G ■ С GC
А В С D
АС
А А
С А С G
С А G С
и с U ■ G CG и с
С G С -G А ■ U С G
G ■ U G ■ U G ■ С G ■ U
G ■ С G -С G ■ С G ■ С
G ■ С G ■ С G ■ С G ■ С
С С С С С С
CG CG CG CG
CG CG CG CG
G ■ С GC G ■ С G -С
Е F G н
Fig. 9. Outer helix III of the pre-rRNA secondary structure of the ITS1 locus for some genera of Polygoneae. A — Reynoutria sachalinensis and Duma florulenta; B — Atra-phaxis ariana; C — Caelestium lazkovii; D — C. tianschanicum; E — Polygonum afromontanum; F — P. aviculare; G — P. doug-lasii; H — Polygonella polygama.
as a result of hybridization of Bactria with any lineage from Polygonum s. l. However, the ITS SSBA-based trees show the position of the clade Caelestium as a nonsupported sister of the group that combines the RFM-clade, Polygonum s. l., and Duma. The clade Caelestium is either grouped with the RFM-clade, Duma and the North American Polygonella and Polygonum sect. Dura-via (ML analysis, Fig. 5), or is weakly associated with the same taxa and all the clades of Polygonum s. l. (BI analysis, Fig. 6). Thus, the ITS secondary structure-based reconstructions provide even less information about the nature of Caelestium).
Such incongruence used to be technically interpreted as a result of ancient reticulation events, rapid radiation and "incomplete lineage sorting", higher divergence rate of the ITS1-2 regions in comparison to coding plastid regions, or other reasons (Rieseberg, Soltis, 1991; Doyle, 1992; Baldwin et al., 1995; Soltis,
Kuzoff, 1995; Wendel, Doyle, 1998; Holder et al., 2001; Alvarez, Wendel, 2003; Albach, Chase, 2004; Orthia et al., 2005; Fehrer et al., 2007; Degnan, Rosenberg, 2009; Mallet et al., 2016).
But why different alignment strategies of the same ITS matrix resulted in so different topologies? The pre-RNA secondary structure of the ITS1 and ITS2 loci clear up the reasons of these distinctions.
Alignment strategies and the pre-rRNA secondary structure of the ITS1 and ITS2 loci of Polygoneae
Both BI and ML trees based on the ITS SSBA (Figs. 5; 6) and on the "raw" MAFFT (Figs. 3; 4) alignment of the ITS region recovered the same eight highly supported clades listed above, although their relationships are not resolved. Similar unresolved topologies were obtained by T. M. Schuster et al. (2015) from the analyses of the ITS data set, or the combined plastid and nuclear data sets after deletion of poorly aligned regions. Due to the low support of the basal nodes (Figs. 3-6), the trees based on different alignment strategies may be tentatively treated as "softly" incongruent (Wendel, Doyle, 1998).
The ML tree based on the MAFFT-alignment of the ITS data set is almost congruent to the ML plastid tree and agrees with our previous results based on the same alignment strategy (Yurtseva et al., 2016), or the results by Schuster et al. (2011b, 2015) obtained for the combined plastid and nuclear data sets. Although the SSBA-based ML tree (Fig. 5) is generally incongruent to the ML plastid tree (Fig. 1), the ITS SSBA-based ML tree (Fig. 5) has a higher value of log likelihood than the MAFFT-based ML tree (Fig. 3) (-6493.537870 (SSBA) vs -6641.832866 (MAFFT)).
Our thorough investigation of the ITS pre-rRNA secondary structure of various taxa of Polygoneae (Figs. 7-10; Tables 3, 4) showed that MAFFT, if applied to the raw sequences of the ITS1 regions lacking either helix II, or helix III, simply aligned: the left arms of helix II with the left arms of helix III; the right arms of helix II with the right arms of helix III. Next, in the MAFFT alignment, for the taxa that have a full set of helices on the ITS1 (Reynoutria, Fallopia, Muehlenbeckia, Duma, Atraphaxis ariana), the region of central loop is partly homologized with the proximal part of the left arm of helix II (in Persepolium, Bactria ovczinnikovii, most of Atraphaxis species), or helix III (in Polygonum s. l., Bactria lazkovii, Polygonum tianschanicum). Therefore, paradoxically, the strong congruence of the ITS MAFFT-alignment based trees (Figs. 3; 4) and the plastid trees (Figs. 1; 2) (see also Yurtseva et al., 2016) may be viewed as an artifact of the MAFFT alignment strategy, if applied to the regions with extensive indels.
Table 4. Helices I—III of the pre-RNA secondary structure of the ITS2 locus in tribe Polygoneae
Taxon Species Helix I Helix II Helix III
loop loop loop
Reynoutria R. sachalinensis 1+6 2 5 4 12 2 4 4, 0/1, 3 3/4 4 5/4 9 2/0 5 1/0 4 4
Muehlenbeckia M. platyclada 6 2/3 5 4 12 1 6 2, 0/1, 6 0/1 4 5/4 8 5/0 - - 8 6
Fallopia F. baldschuanica 6 2 5 4 12 1 6 2,0/1,3 1/2 6 5/4 8 5/0 - - 8 6
Duma D.flondenta 6 2 5 4 12 2 4 2,0/1,3 1/2 6 3/2 10 2/0 4 2/1 4 3
D. hórrida 1+6 2 5 6 12 2 4 2,0/1,3 1/2 6 3/2 12 3/3 6 - - 6
Polygonella P. americana 1+6 0/1 7/6 6 12 2 4 4 0/1 4 5/4 7 4/2 5 2/1 4 3
P. polygama 1+6 0/1 6 5 12 2 4 4 0/3 6 5/4 7 4/2 5 2/1 4 3
P. basiramea 1+6 1/2 5 5 12 2 4 - - 7 1/0 7 4/2 5 2/1 4 3
P. robusta 1+6 1/2 5 4 12 2 4 2 0/1 12 1/2 9 4/2 5 2/1 4 3
Polygonum P. minimum 6 2 6 8 12 2 4 6 0/1 4 5/4 9 2/0 5 2/1 4 3
P. sect. Duravia P. douglasii 6 0/1 5 5 12 2-1 4+2 - - 4 5/4 9 2/0 5 2/1 4 3
P. sect. Pseudomollia P. mollii forme 6+5 0/1 8 5 12 7/6 4 - - 10,0/1,2 2/0 9 2/0 5 2/1 4 3
P. sect. Plebeja P. plebeium 6 1/2 6 5 12 3-1 4+2 - - 10 1/0 9 2/0 5 2/1 4 3
P. sect. Tephis P. afromontanum 6 4/4 9 5 1 + 12 3-1 4+2 2,0/1,3 1/2 7,0/1,2 2/0 9 2/0 5 2/1 4 3
P. cognatum 6 2/3 9 10 12 3-1 4+2 2,0/1,3 1/2 8,0/1,2 2/0 9 2/0 5 2/1 4 3
P. thymifolium 6 4/2 7 4 12 3 4 2,0/1,3 1/2 7,0/1,2 2/0 9 2/0 5 2/1 4 3
P. sect. Polygonum P. aviculare 6 1/2 6 4 12 3 4 2,0/1,3 1/2 7,0/1,2 2/0 9 3/1 4 2/1 4 3
P. boreale 6 1/2 7 3 12 3 4 2,0/1,3 1/2 5,0/1,2 2/0 9 3/1 4 2/1 4 3
Caelestium C. tianschanicum 1+6 1/2 6 4 12 5a 4 2 0/1 6,0/1,4 5/4 9 2/0 5 2/1 4 3
C. lazkovii (A) 1+6 1/2 6+4 4-1 12 5a 4 2 0/1 6,0/1,4 5/4 9 2/3 2 0/1 4 8
Bactria B. ovczinnikovii 6 2/3 6/7+2 4 12 4b 4 2 0/1 6,0/1,4 5/4 9 2/3 2 0/1 4 8
Persepolium P. salicornioides 6 3/3 7+2 4 2+11/12 4b 4 2 0/1 6,0/1,4 4/2 10/11 2/3 2 0/1 4 8
Atraphaxis A. ariana 6 3/3 7+2 4 12 7/8 9 3 0/1 5,0/1,4 5/4 9 2/3 2 0/1 4 8
A.fnitescens 6 2/3 7/8+2 4 12 7/8 5 3 0/1 5,0/1,4 5/4 9 2/0 5 2/1 4 3
A. pyrifolia 6 2/3 7/8+2 4 12 7/8 5 3 1 6,0/1,4 5/4 9 2/0 5 2/1 3 5
Note. The nucleotides in the terminal loop of a helix are designated in bold. The rest numbers designate the number of base pairs in the stem; 0/1 — the number of nucleotides in the left/right arm of the helix. The letters a and b in Helix II designate different substitutions in the apical part of the stem.
Fig. 10. Pre-rRNA secondary structure of the ITS2 locus for some genera of tribe Polygoneae.
A — Reynoutria sachalinensis; B — Atraphaxis ariana; C — A. pyrifolia; D — Bactria ovczinnikovii; E — Caelestium lazkovii; F — C. tianschanicum; G — Duma florulenta; H — Thysanella robusta; I — Polygonum afromontanum. I-IV — outer helices.
Paraphyly of Bactria on the ITS-based trees is not an artifact
Paraphyly of Bactria, which included B. ovczinniko-vii and B. lazkovii, has been shown in our previous phy-logenetic analyses of the ITS matrix, which were based on MAFFT alignment strategy (Yurtseva et al., 2016), and was confirmed in this study. Keeping in mind the monophyly of the same genus on the plastid-based tree, earlier we interpreted paraphyly of Bactria (Yurtseva et al., 2016) as a result of reticulation of one of two species, or as a putative artifact of the ITS sequence data, such as pseudogenisation (Baldwin et al., 1995; Alvarez, Wendel, 2003; Feliner, Rossello, 2007; Poczai, Hyvonen, 2010). The addition of the new non-pseudogenic ITS sequence of Polygonum tianschanicum to the ITS matrix clearly revealed the close relationship of Bactria lazkovii and Polygonum tianschanicum by the ITS and the separate position of their common clade from Bactria ovczin-nikovii in all the ITS-based trees (Figs. 3-6).
The detailed examination of the secondary structure of pre-rRNA of the ITS1 locus of Polygoneae confirmed the close relationship of Bactria lazkovii and Polygonum tianschanicum, as well as the strong contrast with Bac-tria ovczinnikovii. By the whole pre-rRNA secondary structure of the ITS1 locus, B. lazkovii and Polygonum tianschanicum are close to Polygonum s. l. (incl. Polygonella) (Fig. 7: E, F, G, H), that agrees with the results of ML analysis of the MAFFT-aligned ITS data set, but they differ from Polygonum s. l. in their unique structure of helices III and V (Fig. 7: C, D; Table 3). For the ITS2 locus, Bactria lazkovii and Polygonum tianschanicum share the structure (but not a composition) of helix I with some species of Polygonum s. l., but have a unique structure of helix II, and share the structure of helix III with Bactria ovczinnikovii, Atraphaxis and Persepolium.
In contrast, Bactria ovczinnikovii is close to Perse-polium and most of Atraphaxis species (except species having a full set of helices), by the pre-RNA secondary structure of the ITS1 and ITS2 loci and the composition of shared helices (Fig. 7: B-D, 10; Tables 3, 4).
Thus, the ITS regions of Bactria lazkovii and Polygo-num tianschanicum, on the one side, and that of Bactria ovczinnikovii, on the other, are rather different in terms of both first and secondary structures. Despite the fact that Bactria lazkovii and Polygonum tianschanicum resemble Polygonum s. l. in the entire pre-rRNA secondary structure of the ITS1, the structure of the individual helices on the ITS1 and ITS2 does not allow this group to be attributed to any subclade of Polygonum s. l. or Bactria.
The most likely explanation for the incongruent positions of Caelestium in the plastid and ITS SSBA-based trees and the difference in the pre-RNA secondary
structure of the ITS1 and ITS2 loci is that this group appeared as a result of reticulation of Bactria as a maternal taxon and an unknown taxon, which served as a putative donor of the ITS array(s) in the distant past, before its splitting into B. lazkovii and Polygonum tian-schanicum. The donor of the ITS is probably extinct or missing from the analyses. However, due to a lack of exact knowledge of the past, this explanation, even if it seems simple, remains speculative.
The pseudogenic ITS is a primary state for Bactria lazkovii?
The pseudogenic ITS1-2 of Bactria lazkovii detected in this study along with the functional one, is characterised by abundant transitions and indels, including the 5.8S gene, a low GC-content and a partly destroyed pre-rRNA secondary structure of the ITS2 locus. The pre-rRNA secondary structure of pseudogenic ITS1 locus partly matches that of its functional ITS, but has helix II. That can be considered a pseudogenic paralog of the functional ITS1-2, which preserved the primary state of pre-rRNA secondary structure of ITS1 locus with a full set of helices on the ITS1. Otherwise, it can be considered as ITS obtained as a result of hybridization of B. lazkovii with an unknown taxon that served as a donor of additional ITS. In any case, the pseudogenic ITS1-2 of B. lazkovii differs from the functional ITS1-2 of Bactria ovczinnikovii in terms of both the first and secondary structures and cannot be considered as the primary state for the latter.
A full set of helices on the pre-rRNA secondary structure of the ITS1 was apparently a primary state not only for Bactria lazkovii, but for other taxa of Polygoneae. For example, the members of Reynou-tria, Fallopia, Muehlenbeckia, Duma, some Atraphaxis (A. ariana, and also A. badgysi, A. radkanensis) have a full set of outer helices (five in total) on the ITS1. It is obvious that the same helices have been lost independently in different genera of Polygoneae. The members of Polygonum s. l., P. tianschanicum, and Bactria lazkovii have lost helix II on the functional ITS1. In contrast, most species of Atraphaxis, Persepolium, and Bactria ovczinnikovii have lost helix III on the functional ITS1 (Fig. 7; Table 3). The species of Atraphaxis with a full set of helices on the ITS1, or lacking helix III, have similar structure and composition of shared helices, that cannot be said about Bactria ovczinnikovii, on the one hand, and B. lazkovii and Polygonum tianschanicum, on the other hand.
Our study confirmed that the pre-rRNA secondary structure of the ITS1 and ITS2 is a helpful tool for optimizing alignment and for making full use of the phylo-genetic information of DNA sequences. This in itself is a
Fig. 11. Pre-rRNA secondary structure of the ITS1 (A) and ITS2 (B) loci of the pseudogenic ribotype B of Caeles-tium lazkovii.
fairly useful taxonomic character (Mai, Coleman, 1997; Gottschling et al., 2001; Wang et al., 2007; Tippery, Les, 2008; Giudicelli et al., 2017).
On ranks and circumscriptions
In general, we completely agree that any historical explanations (for example, the models of speciation, current reconstructions of geological histories, putative problems with the climates that may have existed in the past, even the discussions regarding the fossil records and molecular dating) that seem to be hypothetical or assumption-based by definition, should not be confounded with the criteria used to distinguish taxa (Wheeler, Platnick, 2000).
At the present stage of molecular-based studies, genera and other taxa of higher ranks can and should be congruent with a phylogenetic pattern. Many authors (Backlund, Bremer, 1998; Entwisle, Weston, 2005; Humphreys, Linder, 2009; Barraclough, Humphreys, 2015) provide the principles of phylogenetic classification and propose the criteria for distinguishing the taxa. Among them, monophyly of a taxon is a basic principle (e. g., Stevens, 1985; Backlund, Bremer, 1998; Entwisle, Weston, 2005). B. E. Pfeil and M. D. Crisp (2005) agree that taxa should be monophyletic and discuss
the problem of paraphyletic groups which perpetuate a classification, that obscures information and reduces predictive power. A. Backlund and K. Bremer (1998) also recommend minimising nomenclatural change and maximising its stability, providing phylogenetic information, and easing identification of the taxa. However, strictly speaking, there are no formal criteria that would clearly define the rank of a taxon, and the definition of a genus is sometimes even more arbitrary than the definition of a species.
Backlund and Bremer (1998) and P. F. Stevens (2001, 2002; see also Bentham, 1861) would not recommend recognising a monotypic genus if it is a sister of a larger genus. They believe that if a newly discovered taxon is the sister to an existing named genus, this does not mean that a new genus is needed for the newly described taxon. However, if the new taxon makes the genus not-monophyletic, the latter cannot be adopted in a broad circumscription (Stevens, 2001).
Thereby, the maintenance of paraphyletic Bactria, as this genus appeared on the ITS trees (Yurtseva et al., 2016 and this study) would be simply incorrect. The same is true for Polygonum s. l., which demonstrates monophyly on the plastid trees (Figs. 1; 2) but is poly-phyletic on the ITS SSBA-based trees (Figs. 5; 6).
The split of the most inclusive genus Bactria into two small monophyletic genera (monotypic Bactria with a single species Bactria ovczinnikovii and Caeles-tium including Bactria lazkovii and Polygonum tian-schanicum) is a decision alternative to the single genus Bactria Yurtseva et Mavrodiev s. l. (Yurtseva et al., 2016) with three species. As already discussed, the clear sistership of the clade (Bactria lazkovii + Polygo-num tianschanicum) and B. ovczinnikovii on the plastid trees does not contradict the separation of B. lazkovii and Polygonum tianschanicum from Bactria ovczinniko-vii as distinct genera. The sisterhood of two taxa in itself provides absolutely no analytical reason for lumping these two taxa into one taxon (for example, due to the principle of monophyly), especially where there are clear morphological and other differences between sister taxa. The flowering plants and the gymnosperms are an extreme example (Soltis et al., 2018). Another example is the recently described genus Pseudoziziphus Hauenschild (Rhamnaceae), which appeared as a sister of the genus Condalia Cav. (Hauenschild et al., 2016), which is confirmed by a clear difference in morphology and biogeography. The papers of E. V. Mavrodiev et al. (2014), M. B. Crespo et al. (2015) and S. L. Low et al. (2018) contain similar examples.
As a result of the genus-rank separation of Caeles-tium, we obtain the monotypic Bactria for B. ovczinniko-vii, which was also viewed as part of a widely treated Atraphaxis s. l. (Tavakkoli et al., 2015), including also Persepolium and Bactria s. l. The similarity of the pre-RNA secondary structure of the ITS1 and ITS2 supports the opinion by S. Tavakkoli et al. (2015). But the weak support of their common clade on the ML and BI ITS SSBA-based trees and the low support of the clade (Atraphaxis + Persepolium) in all the ITS-based trees, argue against their lumping. Sound distinctions in the morphology of the shoots, ochreas, and pollen (Yurtseva et al., 2016, 2017), as well as distinct exocarp anatomy (Yurtseva et al., 2019, in this issue) provide evidences in favor of distinguishing these genera.
Some authors consider plastid markers to be more significant than the ITS, in which case we should support the genus Bactria with three species. As we have demonstrated, Caelestium (Bactria lazkovii and Polygo-num tianschanicum) shows a maximum total difference over sequence pairs from other genera of Polygoneae (Table 2). For example, Bactria lazkovii differs from B. ovczinnikovii by one-nucleotide substitutions in 43 sites and by numerous indels in 3-loci plastid alignment 1218 bp long. These results provide strong evidence for the separation of B. lazkovii and Polygonum tianschanicum from Bactria ovczinnikovii. Strong molecular distinctions of B. lazkovii and Polygonum tianschanicum give no rea-
son for considering them a single species. They differ by substitutions in 10 sites and two indels (1 and 9 bp long) on the ITS region, and by substitutions in 7 sites and by an indel 28 bp long on the combined plastid region.
Since W. Hennig (1966), the notion that sister taxa need to have equal rank is common, as reviewed by G. Giribet et al. (2016). Thus, Bactria ovczinnikovii and Caelestium (Bactria lazkovii and Polygonum tianschanicum) can be accepted at the rank of genus. A small number of species in these genera is the result of their ancient origin and reduced diversity, which follows from their highly isolated local distribution in the old mountain systems of the Pamirs and Tien Shan.
Morphology and distribution of Bactria ovczinnikovii, B. lazkovii and Polygonum tianschanicum
The morphology of the taxa is given in detail in O. V. Yurtseva et al. (2019, in this issue) and is only briefly summarized here. All three species, Bactria ovczinnikovii, B. lazkovii and Polygonum tianschanicum, are dwarf shrubs 10-30 cm tall with leathery leaf blades (but of different shapes). The combinations of morphological traits that differentiate Caelestium (Bac-tria lazkovii and Polygonum tianschanicum) from Bac-tria ovczin nikovii are summarized in Table 5 (see also Yurtseva et al., 2016). The perianth of all the taxa is divided for 4/5-9/10 of its length in 5 (rarely 6) equal tepals, which bear papillae at the edges (Fig. 12). The perianth of B. ovczinnikovii resembles that of Persepolium, and the perianth of Bactria lazkovii and Polygonum tianschanicum resembles that of Polygonum s. str.: Bactria
F
Fig. 12. Perianths.
A-C — Bactria ovczinnikovii; D-E — Caelestium lazkovii; F — C. tianschanicum. Scale bars — 1 mm.
Table 5. Morphological differences of Bactria and Caelestium
Morphological traits Bactria (B. ovczinnikovii) Caelestium (C. lazkovii, C. tianschanicum)
Perianth tepals texture sepaloid petaloid
shape lanceolate broadly ovate
apex acuminate obtuse
color greenish-purple pinkish-white
Fruit ribs sharp obtuse
faces concave, matt flat, glossy
Styles shape linear below stigmas linear for the whole length
connation connate at base free from base
Stigmas shape mini-capitate inconspicuous
ovczinnikovii has sepaloid tepals of the perianth, while they are petaloid in B. lazkovii and Polygonum tian-schanicum. All three taxa have perianth glabrous outside and bearing papillae only at the tepals edges, which we have never seen in Polygonum s. str.
All three species have the microreticulate-foveolate sporoderm ornamentation (Yurtseva et al., 2014, 2016), which is quite different from any type of sporoderm ornamentation observed in Polygonum s. l. (Hedberg, 1946; Hong et al., 2005): psilate, micropunctate, micro-spinulose (palynotype Avicularia); rugulate or foveo-late with microspinules around the colpi, semitectate-reticulate at mesocolpia (palynotype Duravia); psilate around the colpi, verrucate at 1/3 of mesocolpia and poles (palynotype Pseudomollia).
Bactria ovczinnikovii and B. lazkovii demonstrate a striking contrast in the structure of the exocarp cells of their pericarp, which differ in the shape and size of the lumen and pore channels, in the details of their walls, which is combined with different shapes of fruits, styles, and tepals (Yurtseva et al., 2019, in this issue).
The distributional ranges of three species are very distant and divided by the high mountain ranges of the Pamirs and Tien Shan. Bactria ovczinnikovii is a local endemic of the Southwestern Pamir, where it grows in several populations on the right bank of the Pyandj River (Tajikistan, Khatlon reg., Shuroabad and Hama-dony districts). It is part of the Turkestanian Province of the Western Asian Subregion, the flora of which is related to the Armeno-Iranian flora in the west and to the Central Asian and northwestern Himalayan flora in the east and numbers no less than 50 endemic genera and hundreds of endemic species (Takhtajan, 1986).
Bactria lazkovii and Polygonum tianschanicum are endemics of two different regions of the Central Asian Subregion (Takhtajan, 1986). B. lazkovii was collected in a single locality in the Inner Tien Shan (Kyrgyzstan, Naryn reg., Zhumgal distr.), part of the Central Tien Shanian Province. The flora of this province includes
ca. 1800 species and only one endemic genus Tianschan-iella B. Fedtsch. ex Popov (Takhtajan, 1986), though G. A. Lazkov and A. R. Umralina (2015) listed numerous endemic or subendemic genera of flowering plants in the flora of Kyrgyzstan. Polygonum tianschanicum is a local endemic of the Dzungaro-Tien Shanian Province of the Central Asian Subregion, which contains about 50 endemic species and several endemic genera (Takhtajan, 1986).
The Pamir and Tien Shan Mountains are similar in their floristic composition, but consistently have been treated as separate biogeographical entities (e. g., Ko-rovin, 1962; Takhtajan, 1986). As V. N. Pavlov (1980) summarized, ca. 70 % of the species are common for both floras; however, the number of endemic genera is very high in the Pamir and Tien Shan regions (Nowak, Nobis, 2010; Nowak et al., 2011; Safarov, 2013; Lazkov, Umralina, 2015). In short, the floras of the Pamirs and Tien Shan are closely related, but are quite different in their composition. Similarly, Bactria ovczinnikovii and Caelestium are closely related, but differ greatly in morphology and molecular markers studied.
Morphology, distributions, and intrageneric taxa recognized in Polygonum s. l.
In our current analyses, not only Bactria, but also Polygonum s. l. are monophyletic on the plastid trees (Figs. 1; 2), but non-monophyletic on ML and BI trees based on the ITS SSBA matrix (Figs. 5; 6), that is in agreement with ITS-based topologies obtained by T. M. Schuster et al. (2015). Thus, we would like to describe a more general context of our current re-circumscription of the genus Polygonum s. l.
On the ITS SSBA-based trees, the North American clade (Polygonella + Polygonum sect. Duravia) is grouped with the members of the RFM-clade and Duma distributed in Southeast Asia, Australasia and South America, but separately from the clade Polygonum s. str. (Figs. 5; 6), which combines the species distrib-
uted mostly in Central Asia and the species distributed worldwide in the regions with temperate climate.
Our results have shown that the North American Polygonella and Polygonum sect. Duravia differ from Polygonum s. str. (as well as from the rest of Polygoneae) in the composition of helix V of the pre-rRNA secondary structure of the ITS1 (Table 3) and of the most conserved helix III (Mai, Coleman, 1997; Coleman, 2003, 2007) of the pre-rRNA secondary structure of the ITS2 (Table 4). In addition, Polygonella differs from Polygo-num sect. Duravia in the structure of helix III on the ITS1 and helix III on the ITS2.
Polygonella and Polygonum sect. Duravia have a fairly distinct palynotype Duravia (Hedberg, 1946; Hong et al., 2005). MP analysis of morphological data (Ronse De Craene et al., 2004) also combined Polygonum sect. Duravia with Polygonella in a clade sister to the rest of Polygonum s. l. In contrast to P. sect. Polygonum and P. sect. Tephis, both having petioles with 4-6 bundles and collenchyma in four strands, P. sect. Duravia and Polygonella have the petioles with 1-3 bundles and poorly developed collenchyma (Haraldson, 1978). In addition, Polygonella differs from the rest of Polygonum s. l. in having internodal branching, strongly reduced vascularization of the flower, abruptly dilated inner filaments, solitary flowers at the nodes, and conspicuous scarious bracts (Watson, 1873; Horton, 1963; Ronse De Craene et al., 2004).
The strong geographic isolation of the North American Polygonella and Polygonum sect. Duravia from Polygonum s. str., which is distributed mostly in the Old World, and clear differences in their morphology are consistent with the separate positions of these taxa on the ML and BI ITS SSBA-based trees (Figs. 5; 6), that makes the inclusion of Polygonella and Polygonum sect. Duravia into the broadly defined genus Polygonum s. l. (Ronse De Craene et al., 2004; Schuster et al., 2011b, 2015) at least questionable. Thus, our current phylo-genetic results are consistent with the narrow circumscription of Polygonella, which was previously considered as a distinct genus (Meisner, 1826, 1857; Bentham, Hooker, 1880; Dammer, 1893), and Duravia (S. Watson) Greene (1904: 23).
Two highly supported subclades of the clade Polygonum s. str. which were recognized here as P. sect. Polygonum (incl. P. aviculare) and P. sect. Tephis (incl. P. afromontanum), are special in their distribution and ecology. The subclade P. sect. Polygonum combines mostly mesophytic or mesoxerophytic species distributed worldwide in regions with a temperate climate, as pioneer species on sea or river banks, on the shores of estuaries, irrigation canals and temporary streams, as well as in disturbed steppes, urban and ruderal places.
Our results have shown that the subclades, which correspond P. sect. Polygonum (incl. P. aviculare) and P. sect. Tephis, as they appeared on the ITS-based trees, differ from each other in the composition of helix III and helix V of the pre-rRNA secondary structure of the ITS1 (Table 3) and have slight differences in the composition of the most conserved helix III (Mai, Coleman, 1997; Coleman, 2003, 2007) of the pre-rRNA secondary structure of the ITS2 (Table 4).
Our results have shown the place of P. afromonta-num, one of two members of P. sect. Tephis from Africa, in the subclade which includes the members of P. sect. Polygonum distributed mostly in Southwest and Central Asia and named here "P. sect. Tephis" (Komarov, 1936; Hara et al., 1982; Li et al., 2003).
M. Adanson (1763: 276) described the genus Tephis Adans. for Atraphaxis undulata L. (Linnaeus, 1753: 333), which presently is known as Polygonum undu-latum (L.) P. J. Bergius (1767) from South Africa and has a tetramerous perianth, dimerous ovary and short internodes. Polygonum sect. Tephis (Adans.) Meisn. was established by C. F. Meisner (1857) and accepted by G. Bentham and J. D. Hooker (1880), U. Dammer (1893), R. Jaretzky (1925), G. Roberty and S. Vautier (1964). P. afromontanum was added to this section as sharing the characteristics of stem and petiole anatomy (Haraldson, 1978), perianth, and ovary (Ronse De Craene, Akeroid, 1988). Both species have palynotype Avicularia with densely microspinulose ornametation of sporoderm (Hong et al., 2005), peculiar also for P. sect. Polygonum.
The current position of P. afromontanum does not contradict the position of P. undulatum in the clade Polygonum s. l. as a sister to the group including P. aviculare in rbcL phylogeny (Galasso et al., 2009). Also it does not contradict the results of MP analysis based on morphology (Ronse De Craene et al., 2004), which demonstrated the sisterhood of subsections Polygonum and Tephis within P. sect. Polygonum. The latter, in turn, appeared as a sister to P. sect. Duravia (incl. Polygonella). It is likely that we can apply the name "Polygonum sect. Tephis" to the whole subclade of Afro-Asian Polygonum species (Figs. 5-10), but the nomenclatural decision is premature until analyses of P. undulatum, the nomenclatural type of P. sect. Tephis.
Our results confirmed positions of Polygonum mollii-forme (the nomenclatural type of P. sect. Pseudomollia) and P. plebeium (the nomenclatural type of P. sect. Ple-beja) in the subclade "P. sect. Tephis" in the ITS-based trees, that was demonstrated earlier (Yurtseva et al., 2010; Schuster et al., 2011b). Polygonum molliiforme and P. plebeium share the structure of helices IV and V of the pre-RNA secondary structure of the ITS1, but
P. plebeium differs from the rest members of "P. sect. Tephis" by the structure of helix III. Both species have also distinct structure of helices I, II, and III of the pre-RNA secondary structure of the ITS2, P. molliiforme having the longest helices.
Our results have shown incongruent positions of P. molliiforme and P. plebeium on the plastid and ITS trees. Both species fall into the subclade "P. sect. Tephis" in our current ITS-based topologies (Figs. 3-6), but fall in the sister subclade P. sect. Polygonum on the plastid trees (Figs. 1; 2). The incongruent placement of P. mol-liiforme and P. plebeium in two sister subclades might be a result of introgression, or caused by other reasons (Soltis, Kuzoff, 1995; Rieseberg, Soltis, 1991; Rieseberg et al., 1996; Mallet et al., 2016), which makes the taxonomy of Polygonum molliiforme and P. plebeium questionable. Extensive analyses of additional plastid markers and a wider range of species are nessasary to clarify the reticulation in Polygonum s. str., which was shown also with ISSR and RAPD analyses (Yurtseva et al., 2006; Voylokova et al., 2009).
Conclusions
1. Based on the results of molecular phylogenetic analyses of combined plastid data and ITS data, on the details of the pre-rRNA secondary structure of the ITS1 and ITS2 loci, morphological and distributional data, we strongly recommend accepting the clade (Bactria lazkovii + Polygonum tianschanicum) at generic rank. This clade is presumably a result of ancient hybridization of any taxon related to Bactria and an unknown taxon, and demonstrates the role of hybridization in the generic origin in tribe Polygoneae.
2. The new genus Caelestium Yurtseva et Mavrodi-ev is established here to include two species (C. lazko-vii and C. tianschanicum). This genus is endemic to the Tien Shan Mountains (Central Asia).
3. Based on the standard molecular, as well as morphological and distributional data, the broad circumscription of the genus Polygonum as including Duravia and Polygonella seems questionable. Our current results agree with the narrow treatment of Polygonella, Duravia, and Polygonum s. str. A larger set of species and additional markers are necessary to clarify the relationships within Polygonum s. str.
Taxonomic treatment
Key to the genera Atraphaxis, Bactria, Caelestium, Duravia, Persepolium, Polygonum s. str., and Polygonella
1. Branches adnate to stems, appearing to arise internodally
................................................................................. 1. Polygonella.
+ Branches not adnate to stems, not appearing to arise inter-nodally ......................................................................................... 2.
2. Annual or perennial herbs...................................................... 3.
+ Shrubs or subshrubs ................................................................ 4.
3. Stems rounded, finely 8-16-ribbed; leaf blade venation
pinnate, secondary veins conspicuous; worldwide ...............
......................................................2. Polygonum s. str. (in part).
+ Stems quadrangular in cross section, ribs obscure or absent; leaf blade venation parallel, secondary veins not conspicuous; mostly Western North America.........3. Duravia.
4. Perianth tube long, filiform; perianth tepals 4-5, unequal;
2-3 inner tepals strongly accrescent in fruiting....................
.................................................................4. Atraphaxis (in part).
+ Perianth tube short, funnel-shaped; perianth tepals 5, equal, non accrescent in fruiting .......................................... 6.
6. Ochreas tubular, later lacerate in two aristate-subulate
lacinulas and finely serrate-incised middle portion ..............
.................................................................4. Atraphaxis (in part).
+ Ochreas tubular-lanceolate, later lacerate in two, four and more lacinulas............................................................................ 7.
7. Leaf blades linear-lanceolate, deciduous in fruiting; perianth totally shortly velutinous puberulent outside; Zagros ................................................................................ 5. Persepolium.
+ Leaf blades ovate or lanceolate, preserved in fruiting; perianth glabrous or papillate either only on the tube, or only on the tepal margins ................................................................ 8.
8. Perianth glabrous, or papillose on tube; SW and Central Asia ............................................2. Polygonum s. str. (in part).
+ Perianth glabrous, bear papillae on the tepal margins; Pamirs, Tien Shan .................................................................... 9.
9. Perianth tepals sepaloid, lanceolate or oblong-elliptical; purple with narrow pinkish margin; three outer tepals keeled and cucullate; tube funnel-shaped; fruit with distinct ribs and concave matt faces; styles fused at base in a stub; stigmas globular; SW Pamir.........................6. Bactria.
+ Perianth tepals petaloid, broadly ovate or oblong-elliptical; green with wide pinkish-white margin; three outer te-pals almost flat; tube cup-shaped or sacciform; fruit with obtuse ribs and flat glossy faces; styles free from base, linear for their whole length; stygmas invisible; Tien Shan ....
.................................................................................. 7. Caelestium.
Bactria Yurtseva et Mavrodiev, 2016, PeerJ, 4 (e1977): 42, p. p., quoad B. ovczinnikovii. = Atraphaxis sect. Ovczinnikovia Yurtseva ex S. Tavakkoli, 2015, Pl. Syst. Evol. 301 (4): 1167. — Type: Bactria ovczinniko-vii (Czukav.) Yurtseva et Mavrodiev.
Bactria ovczinnikovii (Czukav.) Yurtseva et Mavrodiev, 2016, PeerJ, 4 (e1977): 43. = Polygonum ovczinnikovii Czukav. 1962, Izv. Akad. Nauk Tadzhiksk. SSR, Otd. Est. Nauk, 2 (9): 64; ead. 1968, Fl. Tadzhiksk. SSR, 3: 250; ead. 1971, Consp. Fl. Asiae Mediae, 2: 208. = Atraphaxis ovczinnikovii (Czukav.) Yurtseva, 2014, Pl. Syst. Evol. 300 (4): 763. — Holotype: "Tajikistan [Khatlon Region, Shuroabad Distr.], the right bank of the Pyandj River to the N of the village Bag [Bog], at
gravely ridges at the Schpilau River, 1100 m a. s. l., 1 VI 1960, S. Yunusov, № 1586" (LE: LE 01065511!; isotype — TAD).
Ic.: Fig. 12: A-C. — For published figures see Czu-kavina, 1962: fig. 1; Czukavina, 1968: tab. 44, figs. 2-6; Yurtseva et al., 2014: figs. 1: A-C; 4: A-C; Yurtseva et al., 2016: figs. 3: B; 4; 5; 16: A; 19: A, B; suppl. 10; Yurtseva et al., 2017: 161, figs. 6: B, C; 9: A, B; 14: A-C; 15: K; 17: F.
Dwarf shrub. Woody shoots divaricately branched, covered with gray bark, fibrously disintegrated. Annual shoots leafy, shortly puberulent. Leaves thick, broadly ovate, suddenly narrowed to a petiole, joined with articulation. Ochreas lanceolate-tubular, later bilacerate or bidentate. Thyrses terminal, leafy, with 3-7 clusters of 1-2(3) flowers. Perianth campanulate, divided to 5/6-8/10 in 5(6) equal lanceolate or oblong-elliptical se-paloid tepals. Three outer tepals keeled and cucullate, papillate on the margins; tube funnel-shaped. Stamens 8(9). Styles 3, fused at base in a stub, stigmas globular. Fruit ovoid, trigonous, ribs distinct, faces concave, dull. Pollen tricolporate, oblong-spheroidal to spheroidal (P/E = 1.1-1.4), elliptical in equatorial view, rounded-trilobed in polar view, sporoderm ornamentation micro-reticulate.
Distribution and ecology. Endemic to the Southwestern Pamir (Tajikistan), grows on the right bank of the Pyandj River (Tajikistan, Khatlon reg., Shuroabad and Hamadoni distr.). Records in the adjacent part of Afghanistan can be expected. Rocky and gravelly slopes in the belt of forest-steppe, ca. 600 m a. s. l. Fl. V-VI, fr. VI.
Additional specimens examined. South Tajikistan, Khatlon Region. Shuroabad District: Mts in environs of the vill. Bag [Bog] near the Pyandj River, right bank of the Shpilau River, lower reaches, creek bed on the mountainside,
1 VI 1960, G. N. Nepli (LE!); mts in environs of the vill. Bag near the Pyandj River, red sandstones on the right bank of the Shpilau River, 1 VI 1960, V. Botschantzev, T. Egorova, № 777 (LE!); mts in environs of the vill. Bag near the Pyandj River, red and gray sandstones on the left bank of the Aarzy-Su River,
2 VI 1960, iidem, № 814 (LE!); right bank of the Pyandj River between Bog and Bakhorak, at south slope, 1100 m a. s. l., 2
VI 1960, S. Yunusov, № 1624 (LE!, TAD); right bank of the Pyandj River between Bog and Bakhorak, 29 V 1961, S. Yunusov, G. Kinzikaeva (LE!); mouth of the Surkh-Alam River, red-gray sandstone, at gravely-stone ridges, 580 m a. s. l., 25
VII 2013, U. Ukrainskaya et al., № 12 (MW!); Moskovsky District [now Hamadoni Distr.]: gorge Suleyman-Dara, near the village Sarygor, gravely slope, 31 V 1961, G. Kinzikaeva (LE!) (more in Czukavina, 1962, 1968).
Caelestium Yurtseva et Mavrodiev, gen. nov.
Type: Caelestium lazkovii (Yurtseva et Mavrodiev) Yurtseva et Mavrodiev.
Dwarf shrub. Woody shoots divaricately branched, covered with gray bark, fibrously desintegrated. Annual shoots leafy, shortly puberulent. Leaves oblong-elliptical, gradually narrowed to a petiole and articulated. Ochreas lanceolate-tubular, later bilacerate or bidentate. Thyrses terminal, leafy, with 3-7 clusters of 1-2(3) flowers. Perianth campanulate, divided to 4/5-9/10 into 5 equal petaloid tepals. Tepals oblong-elliptical or broadly ovate, almost flat, papillate on the margins; tube cup-shaped or sacciform. Stamens 8. Styles 3, free from base, linear for their whole length, stigmas inconspicuous. Fruit ovoid, trigonous, with obtuse ribs and almost flat faces, glossy. Pollen tricolporate, oblong-spheroidal to spheroidal (P/E = 1.1-1.4), elliptical in equatorial view, rounded-trilobed in polar view, sporoderm ornamentation microreticulate to foveolate-perforate.
Affinity. Caelestium resembles Bactria (in our revised circumscription) in the shrubby habit, lanceo-late-bilacerate ochreas, campanulate perianth papillate on the tepal margins, and microreticulate to foveolate-perforate ornamentation of the sporoderm (Yurtseva et al., 2016). The petaloid tepals, cup-shaped or sacciform perianth tube, fruits with obtuse ribs and flat faces, and free linear styles differentiate Caelestium from Bactria, the latter having sepaloid tepals, funnel-shaped perianth tube, fruits with distinct ribs and concave faces, and styles connate at the base forming a stub, with small capitate stigmas.
Caelestium resembles Polygonum s. str. in having a campanulate perianth with five equal-sized tepals, and in habit. However, Polygonum s. str. differs by lanceolate-tubular ochreas with 4-18 veins, deeply lacerate-fimbri-ate (Komarov, 1936; Brandbyge, 1993) and psilate, mi-cropunctate, microspinulose sporoderm ornamentation (Hedberg, 1946; Hong et al., 2005). Polygonum s. str. lacks papillae on the tepal edges, though some species (e. g., P. thymifolium Jaub. et Spach, P. biaristatum Aitch. et Hemsl.) bear papillae on the perianth tube (Yurtseva, pers. obs.).
Distribution. Locally distributed in the Inner and Eastern Tien Shan.
Etymology. The name is originated from "caeles-tis" (= heavenly, divinus).
Key to identification of species
1. Perianth divided for 4/5-5/6 of its length; tepals obtuse; leaf blades 7-10 x 2.5-3 mm, bright green, oblong-elliptical or oblanceolate, acuminate; margin revolute; fruits 2.5-3 x
1.8-2 mm; annual shoots reddish-brown...... 1. C. lazkovii.
+ Perianth divided for 8/10-9/10 of its length; tepals obtuse or slightly acuminate; leaf blades 7-8(15) x 3-5 mm, dark green, oblong-elliptical, almost spathulate, obtuse; margin flat; fruits 3-4 x 2-3 mm; annual shoots creamy or pale-gray ............................................................. 2. C. tianschanicum.
1. Caelestium lazkovii (Yurtseva et Mavrodiev) Yurtseva et Mavrodiev, comb. nova. = Bactria lazkovii Yurtseva et Mavrodiev, 2016, PeerJ, 4 (e1977): 43. — Holotype: "Kyrgyzstan, Naryn Region: Zhumgal Dis-tr., Kavak-Too Ridge, 5 km N of the village Sary-Bulun, on rocks, 7 VII 2006, G. Lazkov, № 24" (MW0595509!; isotypes — B, LE).
Ic.: Fig. 12: D, E. — For published figures see Yurtseva et al., 2016: figs. 3: A; 6; 16: B; 19: K, L; suppl. 9.
Distribution and ecology. Kyrgyzstan, the Inner Tien Shan, a single location on the southern slopes of the Kavak-Too Ridge. Rocky slopes at elevation ca. 1500 m a. s. l. Fl. VI-VII, fr. VII-VIII.
2. Caelestium tianschanicum (Chang Y. Yang) Yurtseva et Mavrodiev, comb. nova. = Polygonum tianschanicum Chang Y. Yang, 1983, J. Aug. 1st Agric. Coll. Xinjiang, 4: 55; id. 2010, Sylva Xinjiangensis: 114. — Type: "China, Xinjiang, Hejing Xian, Balguntay, in promontoriis et declivibus montibus. Alt. 1400-1500 m. 15 VI 1981. Chang Y. Yang, Bing Wang, № 810295, № 810290" (syntypes — XJA).
= Polygonum popovii Borodina, 1989, Pl. As. Centr. 9: 104; A. J. Li et al. 1998, Fl. Reipubl. Popularis Sin. 25(1): 7; A. J. Li et al. 2003, Fl. China, 5: 283. = Atra-phaxis popovii (Borodina) Yurtseva, 2014, Pl. Syst. Evol. 300 (4): 763. — Polygonum vacciniifolium Popov, in sched., A. K. Skvortsov, in sched.; unpubl., non P. vacciniifolium Wall. ex Meisn. 1832. — Holotype: China, Xinjiang, "Кашгария, юго-восточная часть Тянь-Шаня, между Кучей и Курлей, горы у сел. Ишма, в скалах, 20 VIII 1929, М. Г. Попов, № 609 [Kashgaria, SE part of Tien Shan, between Kucha and Kurlya, mountains at Ishma village, in rocks, 20 VIII 1929, M. G. Popov, № 609]" (LE: LE 01013264!; isotype — LE 01013263!).
Ic.: Fig. 12: F. — For published figures see Yurtseva et al., 2014: fig. 4: L, M.
Distribution and ecology. China (Xin-jiang-Uyghur autonomous region), southern macroslope of the Eastern Tien Shan. Mountain slopes at elevations of 1400-2600 m a. s. l. (Li et al., 2003). Fl. VI-VII, fr. VII-VIII.
Additional specimen examined. China, Xinjiang, Dzungaria: declive siccum in via ab Urumczi [Urumqi] ad Karashar (на дороге от Урумчи до Карашар), 2300 m. a. s. l., 22 VII 1958, Li, Chu, № 6223 (LE 01013265! — paratype of P. popovii Borodina, Fbr. 1988). Specimens cited by A. E. Borodina (1989): Tien Shan, the upper reaches of the river Algoy, 1800-2400 m a. s. l., 12 IX 1879, A. Regel; 30 km to East of Urumqi, 1000 m a. s. l., on the slope, 21 VI 1958, Li, Chu, № 7373; the mountain road from Bartu to the timber mill in Khomot, 2160 m a. s. l., 3 VIII 1958, Li, Chu, № 6980; the Hanga river valley, 25 km NW of the village Balintoy (on
the road from Korashar [Karashar] to Yuldus), 1 VIII 1958, Yunatov, Yuan, № 175 (all in LE).
Acknowledgements
We are grateful to G. A. Lazkov (Institute for Biology and Pedology, National Academy of Sciences of the Kyrgyz Republic, Bishkek) who kindly collected for us material of Bactria lazkovii. We thank M. G. Pimenov, E. V. Klujkov, U. A. Ukrainskaja, and E. A. Zakha-rova (Lomonosov Moscow State University) for additional material of Bactria ovczinnikovii; E. Kuzmina and B. Khasanov (Severtsov Institute of Ecology and Evolution of the Russian Academy of Sciences, Moscow) collected for us material of Polygonum afromon-tanum used in this study. We thank O. V. Tscherneva and A. E. Grabovskaya-Borodina (Komarov Botanical Institute of the Russian Academy of Sciences, St. Petersburg) for their kind permission to examine the type specimens. We also thank M. V. Olonova (National Research Tomsk State University) and Chen Wenli (Institute of Botany of the Chinese Academy of Sciences, Beij ing, China) for help with some critical literature. We would like to thank V. V. Alyoshin and I. A. Milyu-tina (Lomonosov Moscow State University, Belozersky Institute of Physico-Chemical Biology) for providing primers and help in the design of experiments. We would like to thank D. E. Soltis (University of Florida (UF), Department of Biology and Florida Museum of Natural History (FLMNH)) and P. S. Soltis (UF, FLMNH) for their long-term support.
The experimental part of the work (molecular experiments) was partly carried out with the support (to OVY) of the Russian Foundation for Basic Research (project № 11-04-01300-a), and the support (to OVY) of the Russian Science Foundation (project № 14-5000029). The Russian Science Foundation supported the trip to the Herbarium of the Komarov Botanical Institute. The authors thank S. L. Mosyakin (M. G. Kholod-ny Institute of Botany of the National Academy of Sciences of Ukraine, Kiev, Ukraine) and V. S. Shneyer (Komarov Botanical Institute) for their detailed reviews of the manuscript, valuable comments and suggestions. The authors are grateful to I. V. Sokolova and D. V. Geltman (Komarov Botanical Institute) for their helpful notes and comments.
Supplementary material (Appendix) to the article is available on the journal website (www.binran.ru/ journals/novitates/).
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