A New Species of the Genus Lophozia (Lophoziaceae) from the Svalbard Archipelago Новый Вид Из Рода Lophozia (Lophoziaceae) С Архипелага Шпицберген Nadezhda A
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Arctoa (2020) 29: 124–134 doi: 10.15298/arctoa.29.10 A NEW SPECIES OF THE GENUS LOPHOZIA (LOPHOZIACEAE) FROM THE SVALBARD ARCHIPELAGO НОВЫЙ ВИД ИЗ РОДА LOPHOZIA (LOPHOZIACEAE) С АРХИПЕЛАГА ШПИЦБЕРГЕН NADEZHDA A. KONSTANTINOVA1, ANNA A. VILNET1, YURIY S. MAMONTOV1,2 НАДЕЖДА А. КОНСТАНТИНОВА1, АННА А. ВИЛЬНЕТ1, ЮРИЙ С. МАМОНТОВ1,2 Abstract A new species of liverwort Lophozia svalbardensis (Marchantiophyta, Jungermanniopsida) col- lected in the Nordaustlandet (Svalbard Archipelago) is described. It is characterized by heteroicous inflorescence, presence of low perigynium, crenulate-ciliate perianth mouth, thick walled cells near perianth mouth; colourless to slightly pinkish red tinged gemmae contrasting in color with red-brown marginal cells of the uppermost leaves, two-layer thick base of leaves. A detailed description including illustrations of the species is given. The phylogenetic position of the species is considered, and mor- phological differences from similar species are discussed. Резюме Новый для науки вид Lophozia svalbardensis (Marchantiophyta, Jungermanniopsida) описан с Северо-Восточной Земли (архипелаг Шпицберген). Вид характеризуется гетерецией, наличием низкого перигиния, городчато-реснитчатым устьем периантия, толстостенными обесцвеченными клетками устья периантия, бесцветными до слегка розоватых выводковыми почками резко контрастирующими с клетками края почко-образующих листьев, двуслойными в основании листьями. Приводится детальное описание и иллюстрации вида, обсуждается его филоге- нетическое положение, даются отличия от похожих видов. KEYWORDS: molecular markers, ITS1-2, trnL–F, liverworts, taxonomy INTRODUCTION MATERIAL AND METHODS In the course of the study of the liverwort flora of The species was collected on the north-west coast of Nordaustlandet (Svalbard Archipelago) we collected a Nordvika Bay (Murchisonfjorden, Gustav V Land, Nor- species of Lophozia with a very peculiar appearance. This daustlandet, Svalbard). It covered up to 20-25% of non bright green and purple red Lophozia with numerous large sorted circles (Fig. 2) and was collected in a large num- perianth and sporophytes grows abundantly on nonsort- ber of mats since it was intended to be published in a set ed circles among crushed rocks in the arctic desert in of Hepaticae Svalbardensia Exsiccatae. The coordinates Gustav V Land (Figs. 1-2). Based on heteroicous (partly of locality were measured using GPS. just paroicous) inflorescences we provisionally assigned The samples were re-examined several times in or- it to Lophoziopsis excisa (Dicks.) Konstant. & Vilnet. der to clarify and identify the variability of different char- Subsequent thorough examination of the samples under acters. Some of them were difficult to describe e.g. it was a microscope showed that the species differs from L. ex- very hard to find and take a picture of the mouth of the cisa both in the color and shape of gemmae, as well as in perianth, since they were destroyed at most of them. Only the characters of the cell network and a number of other after a thorough study of most of the collected samples features. Molecular study has shown that the species clear- was it possible to find gemmae on smaller shoots hidden ly diverged from phylogenetically allied species. Togeth- among perianth bearing shoots. To illustrate the quite er with the significant morphological differences identi- characteristic appearance and the most important mor- fied after a thorough study of the sample, this allowed us phological features we took pictures using Nikon SMZ to describe the new species – Lophozia svalbardensis sp. 800 or they were depicted in drawings. The specimens nov. are deposited in Herbarium of Polar-Alpine Botanical 1 – Polar-Alpine Botanical Garden, Kola Sci. Centre, Russ. Acad. Sci., Kirovsk-6, Murmansk Province, 184256 Russia – Россия 184256, Мурманская область, г. Кировск-6, Полярно-альпийский ботанический сад-институт КНЦ РАН. E-mails: [email protected], [email protected] 2 – Tsitsin Main Botanical Garden, Russian Academy of Sciences, Botanicheskaya Str., 4, Moscow 127276 Russia – Россия 127276 Москва, Ботаническая 4, ГБС РАН. E-mail: [email protected] A new species of the genus Lophozia from the Svalbard Archipelago 125 Garden-Institute of the Kola Scientific Center, Russian Academy of Science, duplicates will be sent to LE, MW, MHA, VBGI. MOLECULAR DATA To reveal the affinity of collected species we imple- mented phylogenetic analyses based on ITS1-2 nrDNA and trnL-F cpDNA. Newly sequenced data were obtained for nine specimens, sequences were taken from our pre- vious studies for 30 specimens (Vilnet et al., 2008, 2010; Bakalin & Vilnet, 2019). The newly produced dataset contains the majority of molecularly studied specimens of the genera Lophozia and Lophoziopsis, a number of other key genera from family Lophoziaceae and Scapa- niaceae; the outgroup was represented by Anastrepta or- cadensis (Hook.) Schiffn. from Anastrophyllaceae. All specimens included in the current study are listed in Appendix 1 with GenBank accession numbers and vouch- er details. DNA isolation, amplification and sequencing DNA was extracted from dried liverwort tissue using the DNeasy Plant Mini Kit (Qiagen, Germany). The prim- Fig. 1. Map of Svalbard, collecting locality is indicated. ers given by White et al. (1990) for ITS1-2 and Taberlet et al. (1991) for trnL-F were used for amplification and tings were used for other parameters, gaps were treated sequencing reactions. as missing. The program ModelGenerator (Keane et al., PCR was carried out in 20 μml volumes with the fol- 2006) identified TN+I+Г as the best-fitting evolutionary lowing amplification cycles: 3 min at 94°C, 30 cycles model for the ITS1-2+trnL-F dataset. This model, gam- (30 s 94°C, 40 s 56°C, 60 s 72°C) and 2 min of final ma distribution with four rate categories to estimate extension time at 72°C. Amplified fragments were visu- among-site rate heterogeneity were used in the maximum alized on 1% agarose TAE gels by EthBr staining, puri- likelihood estimation. Bootstrap support (BS) for indi- fied using the QIAquick Gel Extraction Kit (Qiagen, vidual nodes was assessed using a resampling procedure Germany), and then used as a template in sequencing with 500 replicates. According to the stopping frequency reactions with the ABI Prism BigDye Terminator Cycle criterion (FC) for the bootstrap (Pattengale et al., 2010) Sequencing Ready Reaction Kit (Applied Biosystems, our dataset should require only 250 replicates to reach U.S.A.) following the standard protocol provided for 3100 convergence with Pearson average с100 = 0.994960 as Avant Genetic Analyzer (Applied Biosystems, USA). estimated by RAxML v. 7.2.6 (Stamatakis, 2006). Phylogenetic analyses For the Bayesian analysis each partition of the com- The newly obtained ITS1-2 and trnL-F nucleotide bined alignment (ITS1-2, trnL-F) was separately assigned sequences were assembled and then included in the new- the GTR+I+Г model that recommended by authors of the ly produced dataset in BioEdit 7.0.1 (Hall, 1999). The program; gamma distributions were approximated using automatic alignment procedure was done with option of four rate categories. Two independent runs of the Metrop- full multiple alignment with default settings for gaps and olis-coupled МCMC were used to sample parameter val- extension weights in the ClustalW tool. The obtained ues in proportion to their posterior probability. Each run dataset was manually corrected. The absence of incon- included three heated chains and one unheated, and two gruence between two studied loci was shown in prelimi- starting trees were chosen randomly. Chains were run for nary phylogenetic estimation. The combined ITS1- ten million generations and trees were sampled every 100th 2+trnL-F was used in subsequent analyses with inclu- generation. The software tool Tracer (Rambaut & Drum- sion of all positions, absent data at the ends of regions mond, 2007) revealed effective sample size (ESS) as were coded as missing. 29112.5555 and auto-correlation time (ACT) as 618.2968 Three criteria were implemented to the reconstruc- for our data. As determined by Tracer, the first 10 000 tion of phylogeny: maximum parsimony (MP) with TNT trees in each run were discarded as burn-in, thereafter 180 v. 1.5 (Goloboff & Catalano, 2016), maximum likelihood 000 trees were sampled from both runs. The average stan- (ML) with PhyML v. 3.0 (Guindon et al., 2010) and Baye- dard deviation of split frequencies between two runs was sian reconstruction with MrBayes v. 3.2.1 (Ronquist et 0.001357. Bayesian posterior probabilities were calculat- al., 2012). The parsimony analysis with TNT involved a ed from trees sampled after burn-in. New Technology Search for the minimal length tree by The infrageneric and infraspecific variability of ITS1- five iteration and 1000 bootstrap replicates, default set- 2 and trnL-F for the genus Lophozia was calculated as 126 N.A. KONSTANTINOVA, A.A. VILNET, YU.S. MAMONTOV A B C D Fig. 2. A: The plain with a glacial lake surrounded by a the rocky polar desert on coast of Nordvika Bay (Murchisonfjorden); B: Non sorted circle covered bryophytes, dark red-brown spots are cushions of Lophozia svalbardensis; C: Mats of Lophozia svalbardensis; D: Mats of Lophozia svalbardensis with numerous perianth with destroyed colourless perianth mouth. the average pairwise p-distances in Mega 5.1 (Tamura et The tree topologies achieved in all estimations be- al., 2011) using the pairwise deletion option for count- came highly congruent. On the Fig. 3 the tree topology ing gaps. from ML analysis is presented with ML and MP boot- RESULTS strap support values (BS) and Bayesian posterior proba- In total, ITS1-2 and trnL-F nucleotide sequences were bilities (PP) for each node. In common, tree topology is newly obtained for nine specimens and deposited into congruent with those published in our previous studies GenBank. The combined ITS1-2+trnL-F alignment for (Vilnet et al., 2008, 2010; Bakalin & Vilnet, 2019). The 39 specimens consists of 1409 sites, among them 894 tested specimen from Svalbard was found in sister rela- sites belong to ITS1-2 and 515 sites to trnL-F.