Research Article |
Corresponding author: Vadim A. Bakalin ( vabakalin@gmail.com ) Corresponding author: Seung Se Choi ( hepaticae@jbnu.ac.kr ) Academic editor: Peter de Lange
© 2023 Vadim A. Bakalin, Yulia D. Maltseva, Alfons Schäfer-Verwimp, Seung Se Choi.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Bakalin VA, Maltseva YD, Schäfer-Verwimp A, Choi SS (2023) Marsupella brasiliensis sp. nov. (Gymnomitriaceae, Marchantiophyta) from Brazil – the distribution of sect. Stolonicaulon in Neotropics is now confirmed. PhytoKeys 226: 65-77. https://doi.org/10.3897/phytokeys.226.103975
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The specimen previously identified as Marsupella microphylla from Brazil is reassessed and described as a new species, M. brasiliensis. The new species is characterized by paroicous inflorescence, bispiral elaters, scale-like, commonly unlobed leaves and very small leaf cells. Descriptions and drawings are provided along with a corresponding discussion of the morphological peculiarity of the new species. Marsupella brasiliensis belongs to sect. Stolonicaulon, and the distribution of Marsupella sect. Stolonicaulon in the New World is confirmed. The infrageneric position of M. microphylla remains unresolved, and whether it belongs to the same section is still unclear.
Distribution patterns, endemics, Latin America, Marchantiophyta, Marsupella, molecular-genetic
Marsupella sect. Stolonicaulon (N. Kitag.) Váňa is a mysterious section that includes the smallest taxa of the genus and is characterized by a disjunct range, mainly in the tropical and subtropical mountains of Asia to Melanesia, as well as in the Venezuelan Andes and the mountain range of SE Brazil of Latin America (
One of the authors (A.S.-V.) collected a specimen identified by J. Váňa as Marsupella microphylla in the Brazilian state of Rio de Janeiro. This specimen is now being investigated in our integrative research. The purpose of this account is to resolve the taxonomic position of this specimen and a review of the genetic relationships within Marsupella sect. Stolonicaulon.
The specimen identified by J. Váňa as Marsupella microphylla (referred to in the report as specimen ASV15033) was studied by traditional morphological techniques, and plant morphology was compared with other taxa of sect. Stolonicaulon (N. Kitag.) Váňa, originally described and well known in Asia, and further treatments of Marsupella microphylla by
To compile the dataset for molecular phylogenetic analysis, we sequenced two loci (ITS 1‒2 and trnL‒F) from the specimen ASV15033 and added these sequences to the dataset from
The list of voucher details and GenBank accession numbers for the specimens used in the phylogenetic analysis in the present paper. The newly obtained sequences are marked in bold.
Taxon | Specimen voucher | GenBank accession number | |
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ITS 1‒2 nrDNA | trnL‒F cpDNA | ||
Eremonotus myriocarpus (Carrington) Lindb. & Kaal. ex Pearson | Russia, Karachaevo-Cherkessian Rep., N. Konstantinova, K446-6-05, 109, 615 (KPABG) | EU791839 | EU791716 |
Gymnomitrion brevissimum (Dumort.) Warnst. | Russia, Murmansk Prov., N. Konstantinova, G 8171 (KPABG) | EU791833 | EU791711 |
Gymnomitrion corallioides Taylor ex Carrington | Norway, Svalbard, N. Konstantinova, K155-04, 110, 103 (KPABG) | EU791826 | EU791705 |
Marsupella aleutica Mamontov, Vilnet, Konstant. & Bakalin | USA, Alaska, Schofield, 103, 958 (MO) | MH826408 | MH822632 |
Marsupella anastrophylloides Bakalin, Vilnet & Maltseva | Vietnam, Hà Giang Prov., V.A. Bakalin & K.G. Klimova, V-15-6-20 (VBGI) | OM480746 | OM489480 |
Marsupella apertifolia Steph. | Russia, Sakhalin Prov., V.A. Bakalin, K-79-2-15 (VBGI), 123, 501 (KPABG) | MH539834 | MH539891 |
Marsupella apiculata Schiffn. | Norway, Svalbard, N. Konstantinova, K93-1-06, 111, 840 (KPABG) | EU791819 | EU791699 |
Marsupella aquatica (Lindenb.) Schiffn. | Russia, Murmansk Prov., N. Konstantinova, 152/5-87, 6090 (KPABG) | EU791813 | AF519201 |
Marsupella arctica (Berggr.) Bryhn & Kaal. | Norway, Svalbard, N. Konstantinova, 128-04 (KPABG) | EU791815 | EU791695 |
Marsupella boeckii (Austin) Lindb. ex Kaal. | Russia, Murmansk Prov., N. Konstantinova, 367-2-00, 8184 (KPABG) | EU791816 | EU791696 |
Marsupella bolanderi (Austin) Underw. 2 | USA, California Monterey CO, (KPABG) | MF521464 | MF521476 |
Marsupella bolanderi (Austin) Underw. 1 | USA, Santa Yen Mts. St. Barbara, 38802 (KPABG) | MF521463 | MF521475 |
Marsupella brasiliensis Bakalin, Maltseva & Schäf.-Verw. sp. nov. | Brazil, Rio de Janeiro, Serra de Itatiaia, Schäfer-Verwimp & Verwimp, 15033, 15.10.1991 | OQ398709 | OQ408447 |
Marsupella condensata (Ångstr. ex C.Hartm.) Lindb. ex Kaal. | Russia, Kamchatka Terr., V.A. Bakalin, K-60-30-15 (VBGI) | MH539844 | MH539901 |
Marsupella disticha Steph. | Japan, Deguchi, Yamaguchi, Bryophytes of Asia 170 (2000) (KPABG) | EU791824 | EU791703 |
Marsupella emarginata (Ehrh.) Dumort. | Russia, Buryatia Rep., N. Konstantinova, 23-4-02, 104, 411 (KPABG) | EU791811 | EU791692 |
Marsupella funckii (F. Weber & D. Mohr) Dumort. | Russia, Karachaevo-Cherkessian Rep., N. Konstantinova, K516-1-05, 109, 804 (KPABG) | EU791820 | EU791700 |
Marsupella koreana Bakalin & Fedosov | Republic of Korea, KyongNam Province, V.A. Bakalin, Kor-23-18-15 (VBGI) | MH539850 | MH539907 |
Marsupella patens (N.Kitag.) Bakalin & Fedosov | Japan, Fukuoka Pref., V.A. Bakalin, J-7-26a-14 (VBGI) | MH539846 | MH539903 |
Marsupella pseudofunckii S.Hatt. | Japan, Yamanashi Pref., V.A. Bakalin, J-7-10-14 (VBGI) | MH539852 | MH539909 |
Marsupella sphacelata (Giesecke ex Lindenb.) Dumort. | Russia, Kemerovo Prov., N. Konstantinova, 65/1-00 (KPABG) | EU791821 | AF519200 |
Marsupella sprucei (Limpr.) Bernet | Russia, Kemerovo Prov., N. Konstantinova, 54-1-00, 101, 850 (KPABG) | EU791823 | HQ833031 |
Marsupella stoloniformis N.Kitag. | Vietnam, Lao Cai Prov., V.A. Bakalin & K.G. Klimova, V-11-11-17 (VBGI) | MH539859 | MH539916 |
Marsupella subemarginata Bakalin & Fedosov | Japan, Yamanashi Pref., V.A. Bakalin, J-89-31-15 (VBGI), 123, 468 (KPABG) | MH539836 | MH539893 |
Marsupella taiwanica Mamontov, Vilnet & Schäf.-Verw. 2 | China, Taiwan, Nantou Co., A. Schäfer-Verwimp 37663 (MHA, TAIE, JE, VBGI), 123, 642 (KPABG) | OM509627 | OM515126 |
Marsupella taiwanica Mamontov, Vilnet & Schäf.-Verw. 1 | China, Taiwan, Chiayi Co., A. Schäfer-Verwimp 39136 (MHA, TAIE, JE, VBGI), 123, 545 (KPABG) | OM509628 | OM515127 |
Marsupella tubulosa Steph. | Russia, Kamchatka Terr., V.A. Bakalin, K-66-7-15 (VBGI) | MH539860 | MH539917 |
Marsupella vermiformis (R.M.Schust.) Bakalin & Fedosov 2 | Republic of Korea, Jeju Prov., S.S. Choi, 120911-1 (VBGI) | MH539858 | MH539915 |
Marsupella vermiformis (R.M. Schust.) Bakalin & Fedosov 1 | Republic of Korea, Jeju Prov., S.S. Choi, 120911-2 (VBGI) | MH539857 | MH539914 |
Marsupella vietnamica Bakalin & Fedosov | Vietnam, Lao Cai Prov., V.A. Bakalin, V-2-101-16 (VBGI) | MH539862 | MH539919 |
Marsupella yakushimensis (Horik.) S.Hatt. | Republic of Korea, Gangwon Prov., S.S. Choi, 8347 (VBGI) | MH539864 | MH539921 |
Nardia compressa (Hook.) Gray | Canada, British Columbia, N. Konstantinova, A 97/1-95 (KPABG) | EU791837 | AF519188 |
Prasanthus suecicus (Gottsche) Lindb. | Norway, Svalbard, N. Konstantinova, K121-5-06, 111, 821 (KPABG) | EU791825 | EU791704 |
DNA was extracted from dried liverwort tissue using the NucleoSpin Plant II Kit (Macherey-Nagel, Germany). Amplification was performed using an Encyclo Plus PCR kit (Evrogen, Moscow, Russia) with the primers listed in Table
Locus | Sequence (5'-3') | Direction | Annealing temperature (°C) | Reference |
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ITS 1–2 nrDNA | CGGTTCGCCGCCGGTGACG | forward | 68 |
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ITS 1–2 nrDNA | CGTTGTGAGAAGTTCATTAAACC | forward | 64 |
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ITS 1–2 nrDNA | TCGTAACAAGGTTTCCGTAGGTG | forward | 68 |
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ITS 1–2 nrDNA | GATATGCTTAAACTCAGCGG | reverse | 58 |
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trnL–F cpDNA | CGAAATTGGTAGACGCTGCG | forward | 62 |
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trnL–F cpDNA | TGCCAGAAACCAGATTTGAAC | reverse | 60 |
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The polymerase chain reaction was performed in a total volume of 20 µl, including 1 µl of template DNA, 0.4 µl of Encyclo polymerase, 4 µl of Encyclo buffer, 0.4 µl of dNTP-mixture (included in Encyclo Plus PCR Kit), 13.4 µl (for trnL–F)/12.4 µl (for ITS 1–2) of double-distilled water (Evrogen, Moscow, Russia), 1 µl of dimethylsulfoxide/DMSO (for ITS 1–2) and 0.4 µl of each primer (forward and reverse, at a concentration of 5 pmol/µl). Polymerase chain reactions were carried out using the following program: 180 sec initial denaturation at 94 °C, followed by 35 cycles of 30 sec denaturation at 95 °C, 20 (for trnL–F) – 30, 35 sec (for ITS 1–2) annealing at 58 °C (trnL–F and ITS 1–2) and 60 °C (ITS 1–2) and 30 sec elongation at 72 °C. Final elongation was carried out in one 3-min step at 72 °C. Amplified fragments were visualized on 1% agarose TAE gels by EthBr staining and purified using the Cleanup Mini Kit (Evrogen, Moscow, Russia). The DNA was sequenced using the ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Waltham, MA, USA) with further analysis of the reaction products following the standard protocol on an automatic sequencer 3730 DNA Analyzer. (Applied Biosystems, Waltham, MA, USA) in the Genome Center (Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow).
The alignments were compiled for the ITS 1–2 and trnL–F loci and aligned using MAFFT (
Phylogenetic trees were reconstructed using two approaches: maximum likelihood (ML) with IQ-tree ver. 1.6.12 (
For the ML analysis, the best fitting evolutionary model of nucleotide substitutions according to the BIC value was TIM2e+R2 determined by ModelFinder (model-selection method which is implemented in IQ-tree) (
Bayesian analyses were performed by running two parallel analyses using the GTR+I+G model. The analysis consisted of four Markov chains. Chains were run for five million generations, and trees were sampled every 500th generation. The first 2,500 trees in each run were discarded as burn-in; thereafter, 15,000 trees were sampled from both runs to produce a resulting tree. Bayesian posterior probabilities were calculated from the trees sampled after burn-in. The average standard deviation of split frequencies between two runs reached 0.0021 before the analysis was stopped.
To visualize molecular relationships within the Marsupella sect. Stolonicaulon we used TCS network inference method (
Newly obtained ITS 1‒2 and trnL‒F sequences from specimen ASV15033 were deposited in GenBank. The combined ITS 1‒2+trnL‒F alignment of the 33 specimens consisted of 1409 character sites: conservative sites ‒ 944 (67%); variable sites ‒ 420 (29.81%); and parsimony-informative sites ‒ 205 (14.55%).
The ML criterion recovered a bootstrap consensus tree with a log-likelihood = -6121.943. The arithmetic means of the log likelihoods in Bayesian analysis for each sampling run were -6145.17 and -6144.03.
On the Bayesian phylogenetic tree (Fig.
Phylogram obtained in a Bayesian analysis for the genus Marsupella based on the ITS 1‒2+trnL‒F dataset. The values of bootstrap support from the ML analysis and Bayesian posterior probabilities greater than 0.50 (50%) are indicated. The newly obtained sequences are marked in bold. Specimen voucher details and GenBank accession numbers are listed in Table
The morphological comparison confirmed many traits indicating that the studied specimen belongs to sect. Stolonicaulon. Along with the latter, the comparison showed some features that do not fit well with the morphology of M. microphylla (
Plants wiry, ascending in loose patches, densely intermixed with Cephaloziella sp. and Metasolenostoma sp., perianthous plants strongly clavate, from densely branched and rhizomatous base, sterile branches 120–200 µm wide with leaves, in perichaetium zone to 900 µm wide, rusty to brownish and grading to whitish brown in older parts, with red tint in the leaf apices in the apical part of shoot. Rhizoids virtually absent in leaved shoots, in rhizomatous base sparse and colorless, separated (not united to the fascicles). Stem 70–90 µm in diameter, branching ventral, but new branch always arose near to the ventral base of the leaf and may be regarded as deeply postical-intercalary; cross section nearly orbicular, outer cells with outer wall thin, other slightly thickened (10-)12–14 µm in diameter, inward cell walls become thicker, with large triangular trigones, while cell size become smaller, 8–10 µm in diameter. Leaves obliquely to suberect spreading, slightly narrower to twice wider than stem, 50–180×60–190 µm, reniform to widely ovate, smaller constantly entire, sometimes with obtuse apex, larger divided by U-shaped sinus descending to 1/10–1/5 of leaf length, lobes acute. Underleaves absent. Midleaf cells strongly collenchymatous with large and slightly convex trigones, 10–14 µm in diameter to shortly oblong 10–16×8–12 µm, cuticle virtually smooth. Paroicous. Perianthous branches distinctly clavate, perigynium high, ca. 1250×550 µm, with 3 pairs of leaves whose lower pair composed by strongly ventricose bracts containing antheridia; perianth hidden within bracts, conical, eroding from the mouth, and completely disappearing after sporophyte emergence. Female bracts slightly wider than long, to 500×550 µm with gamma-shaped sinus descending to 1/4–1/3 of bract length, lobes triangular, acute, with somewhat diverging apices; bracteoles absent. Androecial bracts in 1(-2) pairs in lower part of perigynium, monandrous. Capsule wall bistratose, outer cells with nodular thickenings present on vertical walls and only sometimes present in horizontal walls; inner layer of rectangular cells with small nodular (not semicircular) thickenings. Spores brownish, 8–10 µm in diameter, faintly papillose, elaters bispiral, (-75)100–130×7–8 µm brown, with narrow, but never homogenous ends.
Brazil, Rio de Janeiro, ca. 22°24'S, 44°41'W, Serra de Itatiaia, Hochgebirgsvegetation auf der Hochfläche bei Abrigo Rebouças, an exponierter, zeitweise sickerfeuchter Felswand [Serra de Itatiaia, high alpine vegetation on the plateau near Abrigo Rebouças, on exposed, intermittently dripping cliff], 2420 m, 15. Oct. 1991, leg. Schäfer-Verwimp & Verwimp 15033 (holotype JE!; isotype VBGI!, PRC, SP (not restudied in the preparation of the present account)).
Fig.
Marsupella brasiliensis Bakalin, Maltseva et Schäf.-Verw. sp. nov. A perianth bearing shoot, fragment B plant habit, fragment, lateral view C plant habit, fragment, ventral view D plant habit, fragment, dorsal view E, F stem cross-section G elaters H–K leaves L capsule, inner layer M capsule, outer layer. Scale bars: 1 mm (a: A); 500 µm (b: B–D, I); 100 µm (c: E–G, L, M); 100 µm (d: H–K). All from 15033 (isotype VBGI).
The newly described Marsupella brasiliensis seems to be a rather rare species, hitherto known only from the Itatiaia mountains of Southeast Brazil between 2300–2420 m.
The present study showed that this specimen identified as Marsupella microphylla previously and treated here as M. brasiliensis belongs to Marsupella sect. Stolonicaulon; therefore, the distribution of the section in Latin America is definitely confirmed. At the same time, the morphological study of the material revealed that the plants are not conspecific with M. microphylla, which has been described from the Venezuelan Andes (Estado Merida, Sierra Nevada de Merida ca 4160 m a.s.l.). On one side, the high peaks of Itatiaia and the “páramo” characteristic of the “Campos de Altitude” benefit the establishment of large numbers of Andean species, such as Aureolejeunea fulva R.M. Schust., Diplasiolejeunea pauckertii (Nees) Steph. (also present in Central America), Drepanolejeunea granatensis (J.B. Jack & Steph.) Bischler, Herbertus oblongifolius (Steph.) Gradst. et Cleef, and others (
Within Marsupella sect. Stolonicaulon, based on taxa whose position within the section is genetically confirmed, sporophytes are only known in M. stoloniformis N. Kitag. The elaters of this species are 2–3-spiral (
Therefore, the present study has confirmed the occurrence of Marsupella sect. Stolonicaulon in Latin America, but was unable to determine whether M. microphylla belongs to sect. Stolonicaulon. Taking into account that the number of elater spirals and the shape of the elaters may be a rather important taxonomic feature, there are doubts that M. microphylla can be referred to as the same section, and the subgenus Nanocaulon may require re-evaluation, at least at the rank of section. Marsupella subg. Nanocaulon was synonymized with M. sect. Boeckiorum Müll. Frib. ex R.M. Schust. (M. sect. Boeckiae Müll. Frib.) in
Marsupella brasiliensis is thus far the only known species of the sect. Stolonicaulon with paroicous inflorescence and occurring in Latin America whose position has been confirmed genetically. All other species are dioicous (with adjustment for the fact that the inflorescences are not known for M. praetermissa and M. anastrophylloides). Finally, we may assume that the mountain systems of Latin America may hide additional taxa belonging to Marsupella sect. Stolonicaulon, and purposeful research in this field should be continued.
Line-art figures were kindly prepared by Matvei Bakalin, to whom the authors are sincerely grateful. Authors are deeply indebted to Dr. Lars Söderström and Dr. Matthew Renner for critical evaluation of the manuscript and helpful comments.
The work of V.A.B. and Y.D.M. is within the frame of the institutional research project “Cryptogamic Biota of Pacific Asia” (no. 122040800088-5). The work of S.S.C. was supported by the Korean National Institute of Ecology, grant number NIE-A-2023-01.