Research Article
Print
Research Article
Reinstatement of species rank for Grimmia limprichtii (Bryophyta, Grimmiaceae) based on molecular and morphological data
expand article infoChao Feng, Jin Kou§, Ting-Ting Wu§, Guo-Li Zhang§
‡ Inner Mongolia Agricultural University, Hohhot, China
§ Northeast Normal University, Changchun, China
Open Access

Abstract

The genus Grimmia Hedw. has been considered taxonomically difficult because of its great morphological variability, and its treatments by different specialists have led to incongruent results. One of the debates in the genus is the species status of Grimmia limprichtii Kern, an Asian-European disjunct moss species that has been considered identical to Grimmia anodon Bruch & Schimp. or Grimmia tergestina Tomm ex Bruch & Schimp. It has also been regarded as the muticous-leaved male plants of G. tergestina. Based on a detailed analysis of the type and many non-type specimens combining the molecular and morphological data, the reinstatement of species rank for G. limprichtii is proposed. The diagnostic characteristics of G. limprichtii and its distinction from some closely related species, with which it may be confused, are discussed. Grimmia obtusifolia C. Gao & T. Cao is considered a synonym of G. limprichtii based on molecular and morphological data.

Keywords

Asia-Europe disjunction, Grimmia obtusifolia, phylogenetic taxonomy

Introduction

The genus Grimmia is one of the largest genera of the moss family Grimmiaceae (Feng et al. 2013). Its species are found on all continents, and most of them prefer dry and temperate or cold environments, and all of them are saxicolous with a marked preference for acidic bedrock (Hastings and Greven 2007). Its taxonomy is reputedly difficult because of great morphological variability in most of its species and the difficulty of properly assessing some crucial characteristics (Feng et al. 2014). Therefore, its treatment by different specialists has led to incongruent results (Muñoz 1999; Ignatova and Muñoz 2004). One example is the number of species accepted in the genus, ranging from 51, according to Maier (2010), who synonymized many names of morphologically diverging taxa, to 71, as reported by Muñoz and Pando (2000), to 95, following Hastings and Greven (2007). Some of the controversial species have recently been resolved based on molecular and morphological data (Hugonnot et al. 2018; Kou et al. 2019; Feng et al. 2021).

Grimmia limprichtii Kern was described in 1897. However, since it was discovered, this species has been considered identical to Grimmia anodon Bruch & Schimp. (Loeske 1930) and this treatment was accepted by following authors (such as Wijk et al. 1962; Muñoz and Pando 2000). In recent years, it was synonymized with Grimmia tergestina Tomm. ex Bruch & Schimp. by emphasizing the cell pattern, structural characteristics of the costa, and characteristics of the perigonial leaves, as well as the occasional presence of both muticous and hair-pointed leaves in the same plant of the latter species (Maier 2002). Soon afterwards, G. limprichtii was regarded as the muticous-leaved male plant of G. tergestina, as its male plants were associated with sporulating G. tergestina in Tibet (Greven 2009).

Grimmia obtusifolia C. Gao & T. Cao was first described in Tibet, China, and later, it was discovered in many other provinces, such as Qinghai, Xinjiang, Sichuan, Tibet of China, and three locations in Mongolia (Tsegmed and Ignatova 2007; Jia and He 2013). In addition, this species may appear in Pakistan (Gruber and Peer 2010). Although G. obtusifolia was accepted by some authors (Redfearn et al. 1996; Tan and Jia 1997; Muñoz and Pando 2000; Liu et al. 2011; Jia and He 2013), soon after it was described, G. obtusifolia was synonymized by other authors with G. limprichtii (Greven and Sotiaux 1995) and G. tergestina (Maier 2002, 2010; Greven 2009). Maier (2010) synonymized G. obtusifolia with G. limprichtii due to similarities in leaf shape, laminal basal cells, and costal architecture, while Greven (2009) believed that G. obtusifolia and G. limprichtii were muticous-leaved male plants of G. tergestina. Plants with muticous leaf apices are not rare in G. tergestina and G. anodon, and the similar leaf shape, areolation of the leaf base, and costal architecture explain the synonymization with G. tergestina, and the nearly unistratose upper laminal cells may explain that with G. anodon (Maier 2002, 2010). Grimmia limprichtii and G. obtusifolia have a similar habit, concave leaves, cucullate and rounded-obtuse leaf apex, architecture of the costa, and areolation of the leaf base. The only difference between the two species is that G. obtusifolia has nearly bistratose upper laminal cells, while G. limprichtii has unistratose cells with bistratose ridges (Maier 2002; Greven 2014).

Throughout our continuing investigation of xerophilic mosses, which are particularly prevalent in Tibet, many Grimmia specimens were collected. Some of them belong to either G. obtusifolia or G. limprichtii. Detailed observations revealed that these samples bear archegonia, which is contradictory compared to the point of view that G. obtusifolia and G. limprichtii are muticous-leaved male plants of G. tergestina. This discovery prompted us to conduct further morphological and molecular studies to confirm their systematic position.

Materials and methods

Morphological observations

Over 2000 specimens of the genus Grimmia including types were examined during our revision of Grimmiaceae in China and these specimens were mainly from herbaria investigations (mainly IFP, KUN) and more than 50 field surveys in recent years. All specimens were studied with the typical anatomical and morphological methods applied for the Grimmiaceae (Muñoz 1999; Maier 2010). The collected specimen was deposited at NMAC. Microscopic examinations and measurements were taken with a ZEISS Primo Star light microscope, and microphotographs were obtained with a Canon EOS 70D camera mounted on the microscope. Three plants were dissected from each collection, and for each shoot every possible structure from the gametophyte and sporophyte was examined and a record kept of what was found for each individual species. Specific morphological and anatomical features of taxonomic importance were assessed mainly following Maier (2010) and Muñoz (1999). Leaves were always taken from the upper middle of the stem, and cross-sections were made in the middle part of the stem. Measurements of leaf width were taken at the base, mid- and upper leaf. Cross-sections were made mid-leaf. For comparison the morphological characters of the types of G. limprichtii, G. obtusifolia, and the sequenced Chinese G. limprichtii, the key characters including habit, leaf, laminal basal cells and the cross-sections at mid-leaf of the three specimens were shown in Fig. 1.

Figure 1. 

Grimmia limprichtii A–C habit D–F leaves G–I laminal basal cells J–L cross-sections at mid-leaf. [A, D, G, J lectotype of Grimmia limprichtii, Kern B, E, H, K Tibet, Zi Wang 20180808022 C, F, I, L holotype of Grimmia obtusifolia, Lang 1347] Photos A, D, G and J courtesy of the Farlow Herbarium of Harvard University and others by Chao Feng.

Taxon sampling, DNA amplification, and sequencing

The only recent collection record from Europe is the material collected in 1993 (Greven and Sotiaux 1995). However, the collection was nearly thirty years ago, which could not be sequenced. To investigate the phylogenetic position of G. tergestina, G. obtusifolia and G. limprichtii, three specimens collected from Tibet were sequenced. Table 1 lists the accessions of the new sequences generated in this study, and Table 2 lists the accessions of the sequences downloaded from GenBank that were used in this study. We employed the nuclear (ITS) marker, which allowed the re-use of earlier results (Streiff 2006; Hernández-Maqueda et al. 2008). DNA extraction, amplification and sequencing of the target regions followed the protocols described in Feng et al. (2021). The PCR products were purified and directly sequenced by the Invitrogen Corporation Shanghai Representative Office. Double-stranded sequencing was performed, and all sequence fragments were edited and assembled using Vector NTI (Suite 11.5) to ensure accuracy.

Table 1.

New sequences used in this study, including taxa vouchers information and GenBank accession numbers.

Species Voucher information ITS rps4 trnL-trnF
Grimmia tergestina_F China, Tibet, Zi Wang 20180809024 OL514232 OL450501 OL450510
Grimmia limprichtii_G China, Tibet, Zi Wang 20180903002 OL514233 OL450502 OL450511
Grimmia obtusifolia_H China, Tibet, Zi Wang 20180808022 OL514234 OL450503 OL450512
Table 2.

Sequences from GenBank used in this study, including taxa and GenBank accession numbers.

Species ITS rps4 trnL–trnF
Coscinodon cribrosus AJ845205 AJ847855
Dicranum muehlenbeckii AF231276 AF231245
Ditrichum flexicaule AJ845204 AJ847854
Drummondia obtusifolia AF223038 AF229895
Dryptodon anomalus EU343751
Dryptodon austrofunalis EU343752
Dryptodon decipiens EU343753
Dryptodon leibergii EU343755
Dryptodon patens EU343756
Dryptodon torquatus EU343757
Funaria hygrometrica AJ845203 AJ847853
Grimmia alpestris AJ845237 AJ847887
Grimmia anodon EU343758 AJ845209 AJ847859
Grimmia anomala AJ845210 AJ847860
Grimmia austrofunalis AJ845211 AJ847861
Grimmia bicolor EU343759
Grimmia caespiticia EU343760 AJ845212 AJ847862
Grimmia caespiticia EU343761
Grimmia capillata EU343762
Grimmia cribrosa EU343763
Grimmia crinita EU343764 AJ845213 AJ847863
Grimmia decipiens AJ845215 AJ847865
Grimmia dissimulata AJ845216 AJ847866
Grimmia donniana EU343765 AJ845217 AJ847867
Grimmia elatior EU343754 AJ845218 AJ847868
Grimmia elongata EU343766 AJ845219 AJ847869
Grimmia funalis EU343767 AJ845220 AJ847870
Grimmia funalis EU343768
Grimmia funalis EU343769
Grimmia funalis EU343770
Grimmia fuscolutea AJ845221 AJ847871
Grimmia hamulosa EU343771
Grimmia hartmanii AJ845222 AJ847872
Grimmia incrassicapsulis EU343772
Grimmia incurva EU343773 AJ845223 AJ847873
Grimmia involucrata EU343774
Grimmia involucrata EU343775
Grimmia khasiana AJ845224 AJ847874
Grimmia laevigata EU343776 AJ845225 AJ847875
Grimmia lisae AJ845226 AJ847876
Grimmia longirostris EU343777 AJ845227 AJ847877
Grimmia macroperichaetialis EU343778
Grimmia meridionalis AJ845228 AJ847878
Grimmia mollis EU343779
Grimmia montana EU343780 AJ845229 AJ847879
Grimmia montana EU343781
Grimmia muehlenbeckii AJ845230 AJ847880
Grimmia nevadensis EU343782
Grimmia orbicularis EU343783 AJ845231 AJ847881
Grimmia orbicularis EU343784
Grimmia ovalis EU343785 AJ845232 AJ847882
Grimmia pilifera EU343786 AJ845233 AJ847883
Grimmia plagiopodia EU343787 AJ845234 AJ847884
Grimmia poecilostoma EU343788
Grimmia pulvinata EU343789 AJ845235 AJ847885
Grimmia pulvinata EU343790
Grimmia ramondii AJ845214 AJ847864
Grimmia reflexidens EU343791
Grimmia serrana EU343792
Grimmia sessitana AJ845236 AJ847886
Grimmia tergestina EU343793 AJ845238 AJ847888
Grimmia torquata AJ845239 AJ847889
Grimmia trichophylla AJ845240 AJ847890
Grimmia trinervis EU343794
Grimmia ungeri EU343795
Grimmia unicolor EU343796 AJ845241 AJ847891
Grimmia wilsonii EU343797
Hydrogrimmia mollis AJ845206 AJ847856
Ptychomitrium gardneri AF231290 AF231258
Racomitrium aciculare EU343798 AJ845207 AJ847857
Racomitrium didymum EU343799
Racomitrium elongatum EU343800
Racomitrium heterostichum EU343801
Schistidium apocarpum AJ845208 AJ847858
Schistidium crassipilum EU343802
Schistidium sp. ‘lingulatum EU343750
Scouleria aquatica AF306984 AF231179

Phylogenetic analyses

The sequences were aligned using MAFFT 7.222 (Kazutaka and Daron 2013) and then edited in BioEdit 7.0.1 (Hall 1999). The concatenation of each individual rps4 and trnL-trnF fragments was performed using our custom Perl script. Phylogenetic analyses were performed using Bayesian inference (BI) and maximum likelihood (ML). MrBayes 3.2.6 (Ronquist and Huelsenbeck 2003) was used for BI analyses under the GTR substitute model. Two Markov Chain Monte Carlo (MCMC) searches were run for 1 million generations each, with a sampling frequency of 1000. The first 25% of the trees were discarded as burn-in. A posterior probability (PP) of 0.95–1.00 was considered strong support. The convergence between runs in all cases dropped below 0.01. ML analyses were executed in IQ-TREE 1.6.3 (Nguyen et al. 2014) under the TPM2u+F+G4 (for cpDNA) and TIM+F+I+G4 (for ITS) substitute models, respectively, selected by the ModelFinder program (Kalyaanamoorthy et al. 2017) based on the Bayesian information criterion (BIC), and 1000 fast bootstrapping replicates were used. Nodes with bootstrap (BS) values of 70–89% were treated as moderate and 90–100% as well supported. The final tree obtained was visualized and edited in FigTree v.1.4.0 (Rambaut 2014).

Results

Molecular data

The chloroplast (cp) and ITS alignments comprised 1149 and 1509 nucleotide sites, respectively. The BI and ML phylogenetic trees had a consistent topology, although there were different levels of support depending on the method. Hence, only the topology with branch lengths from the BI tree is presented, with added support from the ML method on the respective trees (Figs 2, 3). The inference from ITS (Fig. 2) and the chloroplast regions (Fig. 3) agree in most aspects. The topology of both ITS data and chloroplast data resolved G. limprichtii and G. obtusifolia as sister taxa in a strongly supported clade (BS = 100, PP = 1). Grimmia limprichtii and G. obtusifolia are not closely related to G. tergestina.

Figure 2. 

Phylogenetic relationships (50% majority consensus tree) from the Bayesian inference on the ITS dataset. Numbers above branches indicate posterior probability from the BI analysis, followed by bootstrap values for the ML analysis. The species investigated in this study were marked in underscore.

Figure 3. 

Phylogenetic relationships (50% majority consensus tree) from the Bayesian inference of the concatenated rps4 and trnM-trnV datasets. Numbers above branches indicate posterior probability from the BI analysis, followed by bootstrap values for the ML analysis. The species investigated in this study were marked in underscore.

Taxonomic treatment

Grimmia limprichtii Kern , Revue Bryologique 24: 56. 1897.

Figs 1, 4 Chinese name: 林氏紫萼藓

Grimmia obtusifolia C. Gao & T. Cao, Acta Botanica Yunnanica 3: 394. f. 4: 10–16. 1981. Type: Tibet, Shuanghu Xian, Lang 1347 (holotype: IFP!; paratypes: IFP!, MO).

Type

Dolomiten, Palagrouppe: Felsgallerien am limone, bei 2100m. 29.7.96 Kern (lectotype: FH!; isolectotypes: Goet!, JE, PC).

For full description and illustration, see Cao and Vitt (1986), Greven and Sotiaux (1995), and Feng (2014).

Discussion

Grimmia limprichtii is a remarkable species characterized by small and slender plants, muticous, concave to somewhat keeled and oblong-ovate leaves, somewhat cucullate and rounded-obtuse leaf apex, plane leaf margins, and a costa ending below the apex. In addition, its sexual condition is dioicous. Although the androecia of G. limprichtii were discovered in Europe and Asia (Greven and Sotiaux 1995), its archegonia were usually found in our collections from Inner Mongolia (Feng 2014) and Tibet (Fig. 4), but androecia were not found. Our findings showed that the presumption that G. limprichtii is the muticous-leaved male plant of G. tergestina (Greven 2009) is unreliable. The generation of a single generative organ in a specific area may explain why the sporophytes are not generated. The characteristic bistratose, partially bistratose or unistratose with bistratose ridges in the upper part of laminal cells is an intraspecific variation influenced by ecological factors, based on our molecular and morphological results.

Figure 4. 

Grimmia limprichtii archegonia. Photos: Chao Feng (Zi Wang 20180808022).

Morphologically, G. limprichtii is most similar to G. tergestina, a widely distributed species (Muñoz 1999; Ignatova and Muñoz 2004). Both species share similar leaf shapes, plane leaf margins, and indistinct costa. Additionally, some specimens of the latter species are found in leaves both with and without hair-points (Maier 2002). However, G. limprichtii can be readily distinguished from G. tergestina by its small and slender plants, costa ending below the apex, and costal guide cells in laminal parts that are distinct from laminal cells. While G. tergestina has rather stiff plants, costa percurrent and guide cells of the laminal part of the costa are hardly distinct or even indistinct from lamina cells, due to their similarity.

Grimmia crassiuscula H.C.Greven & C.Feng, a species that was recently described from the Helan mountains, China (Greven and Feng 2014), resembles G. limprichtii in the oblong-ovate and muticous leaves, cucullate leaf apex, plane leaf margins, and costa ending below the apex. Nevertheless, G. crassiuscula differs from G. limprichtii in having plants in loose and succulent mats, absence of a central strand of the stem, and costa without stereids.

Grimmia limprichtii was previously synonymized with Grimmia anodon Bruch & Schimp., a widely distributed species (Muñoz 1999; Hastings and Greven 2007). Although hair-point presence and length and the number of cell layers in leaf cross sections are variable in the latter species (Muñoz 1999), G. anodon can be separated readily from G. limprichtii by its keeled and broadly oblong-lanceolate leaves, elongate-rectangular laminal basal cells, and autoicous sexuality. G. limprichtii, by contrast, has concave and oblong-ovate leaves, quadrate to rectangular laminal basal cells, and dioicous sexuality.

Acknowledgements

We are very grateful to Dr Jesús Muñoz, Real Jardín Botánico, for his valuable advice while the authors were studying the genus Grimmia and for sending important literature to us. We really appreciate Genevieve E. Tocci of Harvard University Herbaria (FH) for providing many fine photos of the type of G. limprichtii and correcting the draft. We thank Dr Wei Li and the curator of IFP for the loan of the type and non-type specimens of G. obtusifolia to us, and to the curator of FH and Dr Marc Appelhans of GOET for the loan of the type of G. limprichtii. We are very grateful to Dr Matt von Konrat of Field Museum and an anonymous reviewer for instructive advice, comments, and corrections to draft of this paper. This work was supported by the Natural Science Foundation of China (grant no. 32060051, 42001045, 31660051), Shenzhen Key Laboratory of Southern Subtropical Plant Diversity (grant no. 99203030), the Natural Science Foundation of Inner Mongolia (grant no. 2022MS03066), and the Innovative team of China’s Ministry of Education-Research on the sustainable use of grassland resources (IRT_17R59).

References

  • Cao T, Vitt DH (1986) A taxonomic revision and phylogenetic analysis of Grimmia and Schistidium (Bryopsida; Grimmiaceae) in China. The Journal of the Hattori Botanical Laboratory 61: 123–247. https://doi.org/10.18968/jhbl.61.0_123
  • Feng C (2014) Grimmia Hedwig. In: Bai XL (Ed.) Illustrations of bryophyte of Helan Mountain. Ningxia People’s Publishing House, Yinchuan, 252–265.
  • Feng C, Muñoz J, Kou J, Bai X-L (2013) Grimmia ulaandamana (Grimmiaceae), a new moss species from China. Annales Botanici Fennici 50(4): 233–238. https://doi.org/10.5735/086.050.0405
  • Feng C, Bai X-L, Kou J (2014) Grimmia grevenii (Grimmiaceae), a new species from the Wudalianchi volcanoes in northeast China and its comparison with G. maido and G. longirostris. The Bryologist 117(1): 43–49. https://doi.org/10.1639/0007-2745-117.1.043
  • Greven H (2009) The end of Grimmia limprichtii. Field Bryology 99: 23.
  • Greven H, Feng C (2014) Grimmia crassiuscula sp. nov. (Grimmiaceae) from China, and its separation from Grimmia tergestina and Grimmia unicolor. Herzogia 27(1): 137–140. https://doi.org/10.13158/heia.27.1.2014.137
  • Greven H, Sotiaux A (1995) Grimmia limprichtii, a bryophyte with a disjunct distribution in the Alps and Himalayas. The Bryologist 98(2): 239–241. https://doi.org/10.2307/3243309
  • Gruber JP, Peer T (2010) A contribution to the knowledge of the bryophyte flora of the mountains of North Pakistan (Autonomous Region of Gilgit-Baltistan). Herzogia 25(2): 271–285. https://doi.org/10.13158/heia.25.2.2010.271
  • Hall T (1999) BioEdit: A user-friendly biological sequence alignment program for Windows 95/98/NT. Nucleic Acids Symposium Series 41: 95–98.
  • Hastings RI, Greven H (2007) Grimmia Hedwig. In: Flora of North America Editorial Committee (Eds) Flora of North America north of Mexico (Vol 27). Oxford University Press, New York, 204–305.
  • Hernández-Maqueda R, Quandt D, Muñoz J (2008) Testing reticulation and adaptive convergence in the Grimmiaceae (Bryophyta). Taxon 57: 500–510. https://doi.org/10.2307/25066018
  • Hugonnot V, Porley RD, Ignatov MS (2018) A taxonomic conundrum resolved: The transfer of Grimmia horrida to Coscinodon based on sporophyte discovery in France, with support from molecular data. The Bryologist 121(4): 520–528. https://doi.org/10.1639/0007-2745-121.4.520
  • Jia Y, He S (2013) Species catalogue of China. Volume 1 Plants, Bryophytes. Science Press, Beijing. [In Chinese]
  • Kalyaanamoorthy S, Minh BQ, Wong TKF, von Haeseler A, Jermiin LS (2017) ModelFinder: Fast model selection for accurate phylogenetic estimates. Nature Methods 14(6): 587–589. https://doi.org/10.1038/nmeth.4285
  • Kazutaka K, Daron MS (2013) MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability. Molecular Biology and Evolution 30(4): 772–780. https://doi.org/10.1093/molbev/mst010
  • Liu Y, Cao T, Ge X-J (2011) A case study of DNA barcoding in Chinese Grimmiaceae and a moss recorded in China for the first time. Taxon 60(1): 185–193. https://doi.org/10.1002/tax.601016
  • Loeske L (1930) Monographie der Europäischen Grimmiaceen. E. Schweizerbart’sche Verlagsbuchhandlung, Stuttgart.
  • Maier E (2002) The genus Grimmia (Musci, Grimmiaceae) in the Himalaya. Candollea 57: 143–238.
  • Maier E (2010) The genus Grimmia Hedw. (Grimmiaceae, Bryophyta): A morphological-anatomical study. Boissiera 63: 1–377.
  • Muñoz J (1999) A revision of Grimmia (Musci, Grimmiaceae) in the Americas. 1: Latin America. Annals of the Missouri Botanical Garden 86(1): 118–191. https://doi.org/10.2307/2666219
  • Muñoz J, Pando F (2000) A world synopsis of the genus Grimmia (Musci, Grimmiaceae). Monographs in Systematic Botany from the Missouri Botanical Garden 83: 1–133. https://doi.org/10.2307/2666219
  • Nguyen L-T, Schmidt HA, von Haeseler A, Minh BQ (2014) IQ-TREE: A fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Molecular Biology and Evolution 32(1): 268–274. https://doi.org/10.1093/molbev/msu300
  • Redfearn PL, Tan BC, He S (1996) A newly updated and annotated checklist of Chinese mosses. The Journal of the Hattori Botanical Laboratory 79: 163–357. https://doi.org/10.18968/jhbl.79.0_163
  • Wijk van der R, Margadant WG, Florschutz PA (1962) Index Muscorum. International Bureau for Plant Taxonomy and Nomenclature, Utrecht.