A peer-reviewed open-access journal PhytoKeys 214: 47-60 (2022) & doi: 10.3897/phytokeys.214.95125 46Ph y toKe y S https:/ / Pp hyto keys -pen soft.net Launched to accelerate biodiversity research Morphological and molecular evidence reject conspecificity of Malagasy and Mascarene Parablechnum (Polypodiopsida, Blechnaceae) Sonia Molino!, Irene Lafuente', Germinal Rouhan’, Rafael Medina! | Department of Biodiversity, Ecology and Evolution, Universidad Complutense de Madrid, Calle Jose Antonio Novais 12, 28040 Madrid, Spain 2. Institut de Systématique, Evolution, Biodiversité (ISYEB), Muséum na- tional d'Histoire naturelle, CNRS, Sorbonne Université, EPHE, Université des Antilles, Paris, France Corresponding author: Sonia Molino (sonimoli@ucm.es) Academic editor: Blanca Leén | Received 20 September 2022 | Accepted 31 October 2022 | Published 25 November 2022 Citation: Molino S, Lafuente I, Rouhan G, Medina R (2022) Morphological and molecular evidence reject conspecificity of Malagasy and Mascarene Parablechnum (Polypodiopsida, Blechnaceae). PhytoKeys 214: 47-60. https://doi.org/10.3897/phytokeys.214.95125 Abstract Under the current treatment of the Blechnaceae, only one species of the fern genus Parablechnum is recognised in the western Indian Ocean, often referred to as P marginatum. Two varieties are current- ly recognised within it: a type variety present in the Mascarene Islands of Réunion and Mauritius and P. marginatum vat. humbertii in Madagascar. Recent molecular evidence suggested that these two varieties are not closely related, questioning their conspecific status. To collect further evidence to support a taxo- nomic decision, we performed a morphological study based on 57 herbarium specimens comparing traits from general morphology, cross section of the fertile pinnae, sporangia and spores. As a result, Malagasy specimens can be distinguished morphologically from the Mascarene ones by pinna apex and pinna sec- tion, the presence of sporangiasters and spore ornamentation. Additionally, spore size analyses resulted in statistically significant differences between both varieties. Our results, aligned with the available phyloge- netic data, support that these two taxa should be recognised as separate species and, hence, we propose the necessary new combination and provide full descriptions. Keywords biogeography, Madagascar, Parablechnum humbertii, Parablechnum marginatum, Réunion, sporangiasters Copyright Sonia Molino et al. 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. 48 Sonia Molino et al. / PhytoKeys 214: 47—60 (2022) Introduction Parablechnum C.Presl is the most diverse genus within the fern family Blechnaceae, with about 65 species (Gasper et al. 2016) whose range shows two major centres of diversity, one in Meso and South America and a second one centred in Eastern Aus- tralasia. Currently, only two species are considered native outside these centres (Rako- tondrainibe et al. 2013; Gasper et al. 2016), one present in southern and south-eastern Africa, Parablechnum capense (Burm.f.) Gasper & Salino and a second one native to the western Indian Ocean (Madagascar and the Mascarenes), Parablechnum margi- natum (Kuhn) Gasper & Salino. Two varieties are currently considered within the latter (Rakotondrainibe et al. 2013; Gasper et al. 2016). The type variety, Parablech- num marginatum vat. marginatum, is present in Réunion, from where the type was originally collected and was also collected in Mauritius (Appendix 1). A second vari- ety, P marginatum vat. humbertii (Yardieu) Gasper & Salino accommodates the speci- mens found in Madagascar, originally considered as a distinct species by Tardieu-Blot (1955). The taxonomic rank of these two varieties, however, needs a reconsideration following recent molecular studies (Bauret 2017), where the specimens from Madagas- car and Réunion did not cluster together in a monophyletic group. Here, we reassess the taxonomic status of the western Indian Ocean Parablechnum after a morphological examination of herbarium specimens of the two taxa. Materials and methods We examined 57 herbarium specimens from Herb. P (Appendix 1). We observed the general morphology (i.e. shape of the frond, scales, axes etc.); anatomy of the fer- tile pinnae; morphology of the sporangia; and size and ornamentation of the spores. Microscopy work followed Gurr (1966) and Ruzin (1999). For the anatomy of the fertile pinnae, we performed cross-sections in at least two individuals of both varieties. The samples were softened for approximately 5 minutes in water and then manually cross-sectioned in the middle area of the fertile pinnae. The sections were then rinsed by immersion in a 50% solution of sodium hypochlorite for 2—5 minutes. After several washes with water, the sections were stained with 0.1% aqueous toluidine blue (TBO). All microscopic pictures were taken with a Nikon Eclipse Ci microscope with a Nikon DS-Fi2 camera. Sporangia analysis was carried out by scraping the sori of the previously softened and rinsed pinnae. The protocol and terminology followed Prada et al. (2016), Molino et al. (2020) and Wal et al. (2021). Spores and sporangia were mounted directly in water, imaged under the optical microscope and measured with the Piximétre software (Henriot and Cheype 2022). We measured at least 30 spores from three different indi- viduals of the two varieties, excluding the perispore and at least three sporangia from three different individuals of the two taxa. With the spore data, we calculated the shape and volume of each spore following the formulae of Barrington et al. (1986, 2020). Rejection Malagasy and Mascarene Parablechnum conspecificity 49 Spore and sporangium measurements were used to perform descriptive statistics and mean comparisons in R using the R Commander package (Fox 2005). Data were tested for normality using the Shapiro-Wilks normality test (Shapiro and Wilk 1965). For those that fit a normal distribution (spore volume), mean comparison was per- formed with a one-factor ANOVA and for those data that did not fit a normal distri- bution (spore length, width and shape), we performed a Mann-Whitney U test (Wil- coxon 1945; Mann and Whitney 1947). From two individuals of each variety, we studied spore ornamentation through scanning electron microscopy (SEM). The samples were mounted in a sample holder with carbon adhesive, metallised with gold and observed in a SEM JSM 6400 JEOL operating at 20 kV. ‘The observations were made at the National Center of Electronic Microscopy (CNME) of Universidad Complutense de Madrid. Photographs of details at a more macromorphological level, such as fronds or scales, were taken with a Leica Stereozoom S9i with Swing Arm Stand stereomicroscope. Results Our morphological analysis shows qualitative and quantitative differences between the two taxa that are summarised in Table 1 and the full descriptions given in the Nomenclature section. There are some differences in the sizes of the two taxa and in some characters, such as the sometimes slightly creeping rhizomes in the case of the P. marginatum vat. marginatum (vs. erect or suberect in var. humbertii). However, we believe that the most reliable characters that easily distinguish the two taxa are the obtuse or acute apices on both sterile and fertile pinnae of P marginatum var. margi- natum (Fig. 1A) vs. the long-acuminate apices in both sterile and fertile pinnae of var. humbertii (Fig 1B) and the smooth petiole surface in P marginatum vat. marginatum (Fig. 1C) vs. petioles with scars left by the scales in var. humbertii (Fig. 1D). Fertile pinnae of P marginatum vat. marginatum present a costa, grooved adaxially and prominent abaxially, with three vascular bundles, elongated receptacle in the sori, covered by a short, complex indusium (composed by more than one cell layers), which arises at approximately one third of the distance between the margin and the costa, leaving a sterile portion towards the margin. The margin of the pinna is thick (Fig. 1E). Variety humbertii presents a costa grooved adaxially and prominent abaxially, with five vascular bundles and elongated receptacle in the sori, covered by long complex indusium, which arises on the first third of the distance between the margin and the costa, leaving a very small sterile portion of the margin (Fig. 1F). We consider that the different number of vascular bundles in the cross section of the fertile pinnae is the most distinctive diagnostic character. Both taxa present monolete spores, with an ellipsoid outline in the polar view and flat-convex to concave-convex (reniform) in the equatorial longitudinal view. The spores of each taxon are described below. Sizes are rounded values; the exact values with their standard deviation can be found in Table 2, together with shape (length/width ratio) and estimated volume. 50 Sonia Molino et al. / PhytoKeys 214: 47-60 (2022) Figure |. Details of the traits observed in P marginatum var. Marginatum (A, C, E, G) and P marginatum var. humbertii (B, D, EK H) A apex of a sterile pinna in the var. marginatum, adaxial surface (Cowemoy s.n., P01462834) B apex ofa sterile pinna in var. humberti, adaxial surface (Rakotondrainibe 1673, P00100192) C petiole surface in the var. marginatum (Cadet 4050B2, P01462767) D petiole surface in var. humbertii with a scar left by a scale pointed with an arrow (Rakotondrainibe 1673, P00100193) E fertile pinna cross section of the var. marginatum (Bradé 958, P00917035) F fertile pinna cross section of var. humbertii (Rakotondrainibe & Raharimalala 2519, P00904704) G sorus in the var. marginatum (Cadet 4050B1, P01462768) H sori of var. humbertii, with sporangiasters pointed with an arrow (Rakotondrainibe 2743, P00059959). Scale bar: 5 mm (A); 2.5 mm (B); 1 mm (C, D); 800 um (E, F); 2 mm (G); 500 um (H). Rejection Malagasy and Mascarene Parablechnum conspecificity 51 Table |. Summary of the most useful traits to distinguish Parablechnum marginatum vat. marginatum from P marginatum var. humbertii. Taxon Pinnae apices Petiole surface Number of bundles in Sporangiasters Spore ornamentation (Fig. 1A, B) (Fig. 1C,D)_ the costa (Fig. 1G, H) (Fig. 1E, F) (Fig. 2) P marginatum Caudate Smooth 5 Absent Perisporium forming var. marginatum defined areolae, with filaments forming a net P marginatum Long With scars left B Present Perisporium not var. humbertii attenuate by the scales forming defined areolae but a maze, filaments occasional Table 2. Characterisation of the spores of both taxa. The mean + standard deviation is presented. Taxon Spore length (um) Spore width (um) Shape Volume (m7) P marginatum 64.32 + 7.22 45.67 £ 6.83 1.42 + 0.16 7.3435.74 £ 2.6210.03 var. marginatum P marginatum 66.14 + 5.42 48.74 + 5.62 P37 20312 8.4367.01 + 2.3689.94 var. humbertii Spores of P marginatum vat. marginatum: (41—-) 64 (-81) x (27-) 46 (-60) um, perisporium folded cristate-reticulate, with protruding ridges and with large areas be- tween them (areolae), measuring approximately 30 um, covered with filamentous mi- cro-ornamentation forming a kind of net that is arranged on a nearly smooth surface; internal structure of the perisporium of spongy appearance and irregularly granular exosporium (Fig. 2A, B). Spores of P marginatum vat. humbertii: (49-) 66 (-78) x (35-) 49 (-62) um, per- ispore folded cristate-reticulate, with protruding ridges, but without large and regular areas between them, but rather irregular corridors, without filaments or with moder- ately abundant filaments distributed over the entire surface; internal structure of per- ispore spongy in appearance and exosporium regularly granular (Fig. 2C, D). After comparison of means using the tests specified above, we obtained signifi- cant differences for all characters between the two taxa (spore length W = 6268.5, p-value = 0.047; spore width W = 5588.5, p-value = 0.0014; shape W = 8.959, p- value = 0.0057; volume F = 10.56, p-value = 0.001), suggesting that the spores of P. marginatum vat. humbertii are significantly larger than those of the var. marginatum. However, we believe that the best spore character to differentiate these taxa is the per- ispore ornamentation, as, although there are significant differences in spore sizes, the ranges overlap (Fig. 2, Table 2). Spore ornamentation in P marginatum vat. margina- tum form defined areolae and present a net of filamentous processes, while in var. hum- bertii, they do not form areolae, but corridors and filamentous processes are occasional. The sporangia of both taxa are leptosporangiate, with pedicels of 2—3 rows of cells with a rosette joining them to a nearly spherical capsule with a vertical arc interrupted by a stomium. No posterior basal cells were observed. Table 3 summarises the mor- phometric variation of the sporangia. Additionally, the sporangia of P marginatum vat. humbertii are intermixed with shorter, darker, sterile sporangia that we interpret as 52 Sonia Molino et al. / PhytoKeys 214: 47-60 (2022) Figure 2. Spores of Parablechnum marginatum vat. marginatumunder SEM A spore (Cowemoy s.n., P01462832) B detail of the internal structure of the perispore and the exospore (Lorence s.n., MO2715099). Spore of P marginatum var. humbertii under SEM C spore (Rakotondrainibe 3571, P0085125) D detail of the internal structure of the perispore and the exospore (Rakotondrainibe 3571, P00085125). Scale bar: 25 um (A, C); 14 um (B); 12 um (D). sporangiasters (Fig. 1H). None of the sporangium characters showed significant dif- ferences between the two taxa (number of arc cells F = 3.237, p-value = 0.084; arc width F = 2.307, p-value = 0.142; capsule length W = 93, p-value = 0.905; capsule width F = 3.457, p-value = 0.075; number of lip cells W = 45, p-value = 0.117; up- per lip width F = 0.087, p-value = 0.771; lower lip width F = 1.493, p-value = 0.237; number of epistomium cells W = 92, p-value = 0.3; number of hypostomium cells F = 0, p-value = 0.983; pedicel length F = 1.019, p-value = 0.319; rosette length F = 0.567, p-value = 0.455). Rejection Malagasy and Mascarene Parablechnum conspecificity 53 Table 3. Characterisation of the sporangia of both taxa. The mean + standard deviation is presented, all the values are in pm. Arc = number of cells in the arch; Arc wd = thickness of the arch; Cap = size of the sporangia capsule (length x width); Lip = number of cells forming the lip (stomium); Sup = upper lip cells width; Inf = lower lip cells width; Epi = number of cells in the epistomium; Hyp = number of cells in the hypostomium; Ros = rosette length; Ped = pedicel length. Taxon Arc Arc wd Cap Lip Sup Inf Epi Hyp Ros Ped P marginatum = 22.94 7984 4414713 44 52.44 5464 354 2.54 59.34 561.72 var. marginatum 2.4 72 x258.5+ 1.1 16.5 17:3 Tel 0.8 17.7 110.7 26.6 P marginatum 214 744+ 414.8473.4 47% 5452 46+ Sl 25 a 554d SHA Soe var. humbertiii led: 7.7 x 278.14 1.1 LAS IPAS 0.9 0.8 18.8 189 2h9 Discussion To resolve the conspecificity hypothesis of Parablechnum marginatum vat. marginatum and var. humbertii, we have performed a morphological analysis using traits usually showing systematic value within the family Blechnaceae. Regarding the anatomy of fertile pinnae, the study by Prada et al. (2016) defined the characters observable in pin- nae cross-sections and showed how these have a high taxonomic value at the generic and specific level, which has been demonstrated in subsequent publications (Molino et al. 2019a, b; Bauret et al. 2020). Spores are a widely used character in fern taxonomy (Tryon and Tryon 1982; Barrington et al. 1986, 2020; Tryon and Lugardon 1990). Spore size, perisporium ornamentation and internal structure are known to be reason- ably constant within species, but with considerable variation between species (Lugar- don 1974; Tryon and Lugardon 1990). There are numerous studies on the spores of the family Blechnaceae and some of these have been used to successfully discriminate genera and species (Passarelli 2007; Passarelli et al. 2010; Moran et al. 2018; Silva et al. 2019, 2021; Molino et al. 2020; Wal et al. 2021). The genus Parablechnum is par- ticularly complicated and the study of spores could be an important element in the delimitation of infrageneric taxa, as has been shown in other studies (Wal et al. 2021). The spores observed for these two taxa present typical ornamentation of the genus: folded cristate-reticulate perispore with or without filamentous processes (Moran et al. 2018). In this case, the ornamentation of the spores serves to distinguish the two taxa. Sporangia are structures whose ontogeny and variation in characters have been studied for many groups of leptosporangiate ferns (e.g. Bower 1925; Copeland 1947; Wilson 1959). In particular, there have been specific studies in Blechnaceae where spo- rangia have been key in the separation of genera and species (Prada et al. 2016; Moli- no et al. 2020; Wal et al. 2021). Although we did not find differences in sporangial characters, their characterisation is novel and may be useful in future studies of the genus Parablechnum on a larger scale. The presence of sporangiasters as a trait with taxonomic value in Blechnaceae was recently observed for the first time in Parablechnum nesophilum (T.C.Chambers & P.A.Farrant) Gasper & Salino, a species from Papua New Guinea (Molino et al. 2021). 54 Sonia Molino et al. / PhytoKeys 214: 47—60 (2022) Their occurrence in P marginatum var. humbertii seems to also be a useful character to distinguish this taxon from the var. marginatum, suggesting that sporangiasters may be more widespread within the genus than previously thought. In line with the phylogenetic tree topology obtained by Bauret (2017), this mor- phological comparison suggests that, as stated for the first time by Tardieu-Blot (1955), Madagascar specimens are not conspecific with those of the Mascarene Islands. In said analysis, P marginatum vat. humbertii is represented by two accessions and P marginatum var. marginatum by another one. ‘The latter is resolved in a clade with 16 American spe- cies that is sister to the former. Three maximally supported internal nodes (posterior probability = 0.95, bootstrap = 95) segregate the Mascarene and Malagasy taxa. Ferns in Madagascar and the archipelagos of the western Indian Ocean may be closely related to lineages from different biogeographic regions (Bauret et al. 2017a, b, 2018; Hennequin et al. 2017; Rouhan and Gaudeul 2021). To date, phylogenetic affin- ities suggest that Parablechnum species of the western Indian Ocean Islands are nested within a clade with many Neotropical taxa, in contrast with P capense, the single con- tinental African species, nested in an Austro-Pacific clade (Gasper et al. 2016, 2017). Given the relatively recent age of the Blechnaceae (Testo and Sundue 2016) com- pared to the isolation of Madagascar and the origin of the Mascarenes, long-distance dispersal is the most likely hypothesis for explaining the presence of these taxa in these Islands (Bauret 2017; Bauret et al. 2017a, b, 2018; Rouhan and Gaudeul 2021). Given the topology of the phylogenetic tree (Bauret 2017), a single dispersal event cannot explain the occurrence of the two species in the Malagasy region and, so far, the most likely hypothesis includes two independent events. A richer sampling in an expanded phylogenetic analysis will be critical to resolving the number, origin and timing of the dispersion events to the region. From the systematic point of view, available information rejects the conspecificity of the two taxa and, hence, we propose that the Malagasy taxon should recover the species rank within Parablechnum. Nomenclature and full descriptions Parablechnum marginatum (Kuhn) Gasper & Salino Phytotaxa 275(3): 191-227, 2016. = Blechnum marginatum Kuhn, Filic. Aft.: 92, 1868; Blechnum montbrisonis C.Chr. Index Filic. 157, 1905, nom. nov. for Lomaria marginata Fée, Mém. Foug., 5. Gen. Filic.: 71, 1852, nom. illeg. hom., non L. marginata Schrad., Gott. Gel. Anz. 871. 1824 [= Lomariopsis marginata (Schrad.) Kuhn]. Type. Habitat in insula Borbonia, no date, de Montbrison s.n. (not found). Description. Plants terrestrial; rhizomes erect, sub-erect or slightly creeping, non- stoloniferous, with ovate to lanceolate scales with elongated apex, more or less filiform, concolorous, brownish, membranaceous, up to 2 cm long; fronds dimorphic; sterile Rejection Malagasy and Mascarene Parablechnum conspecificity 5 fronds with petioles light brown, grooved adaxially, smooth, up to 50 cm long, with scales in basal zone decreasing in density distally, similar to those of the rhizome, laminae 1-pinnate, elliptic-acuminate, up to 1 m long, sometimes longer, rachises light brown, smooth, adaxially grooved, scales similar to those of petiole, more abundant on adaxial side, pinnae up to 30 pairs, alternate or subopposite, slightly smaller at base, lanceolate to oblong, stalked, becoming basiscopically adnate towards apex of frond, ca. 11 x 2 cm, base asymmetric, subcordate to truncate, apex acute or obtuse, margins slightly serrate, with conforming terminal pinna similar to lateral ones, costae light brown, grooved adaxially, prominent abaxially, with scales at base similar to those of rachis; veinlet sim- ple or 1-furcate, patent, catadromous; fertile fronds larger than sterile ones and more erect, petioles similar to sterile fronds, laminae usually up to 50 cm long, lanceolate to oblong, rachises similar to sterile fronds, pinnae usually in more pairs than in sterile ones, linear, narrow, ca. 3.0 x 0.2 cm, slightly broader-based, asymmetrical, cordate, apex acute; aerophores present in both sterile and fertile fronds, tuberculiform, atro- purpureus; hydathodes present in both sterile and fertile fronds, rounded or ovate; sori linear, continuous, on both sides of costa forming coenosori; indusia linear, continu- ous, opening towards costa, dark brown, membranaceous, usually lacerate. Taxonomical notes. Christensen (1905) proposed Blechnum montbrisonis as a re- placement name, as the name Blechnum marginatum proposed by Kuhn (1868) was based on an illegitimate basionym, Lomaria marginata Feé. This name was proposed by Feé (1852) after this combination had already been used by Scharder (1824) for what is now known as Lomariopsis marginagta (Schard.) Kuhn. However, according to the rules of the current Code (see Art. 6.14 Ex. 18; Turland et al. 2018), Blechnum margi- natum would be a validly published replacement name for Lomaria marginata Feé and, therefore, Blechnum montbrisonis would be a superfluous name. Parablechnum humbertii (Tardieu) S.Molino & Lafuente, comb. nov. urn:lsid:ipni.org:names:77308765-1 = Blechnum humbertii Vardieu Mém. Inst. Sci. Madagascar, Sér. B, Biol. Vég. 6: 232, £.5, 1955; Blechnum montbrisonis C. Chr. var. humbertii (Tardieu) Rakontondr. Adansonia, série 3, 35(2): 178, 2013. Parablechnum marginatum var. humbertii (Tardieu) Gasper & Salino Phytotaxa 275(3): 216, 2016. Type. Mapacascar. ‘Vallée de la Lokoho, mont Beondroka, au Nord de Maroam- bihy, sylve a Lichens, sur gneiss et quartzite’, no date, Humbert 23554 (Holotype: P00483200). Description. Plants terrestrial; rhizomes erect or sub-erect, non-stoloniferous, with ovate-lanceolate scales with elongated apex, more or less filiform, concolorous, brownish, membranaceous, with entire margins, up to 20 mm long; fronds dimorphic, sterile fronds with petioles 20-30 cm long, dark brown at base, straw-greyish distally, smooth, grooved adaxially, with scales in basal zone decreasing in density distally, simi- lar to those of rhizome, leaving a black scar after falling off; laminae 1-pinnate, ellip- 56 Sonia Molino et al. / PhytoKeys 214: 47-60 (2022) tic-acuminate, up to 30 cm, sometimes longer, rachises light brown, smooth, grooved adaxially, scales similar to those of petiole but narrower, more abundant in the adaxial side, pinnae in up to 20 pairs, alternate or subopposite, slightly smaller at the base, lanceolate-oblong, stalked, becoming basiscopically adnate towards apex of the frond, 10 x 1.5 cm, base asymmetric, subcordate to truncated, apex long acuminate, margins serrate, with a conforming terminal pinnae similar to lateral ones; veinlet free, simple or 1-furcate, catadromous; fertile fronds longer than sterile and more erect, petioles similar to sterile fronds, laminae 30 cm long, sometimes longer, lanceolate-oblong, rachises similar to sterile fronds, pinnae usually in more pairs than in the sterile ones, linear, narrow, 12.0 x 0.2 cm, slightly broader-based, asymmetrical, cordate, apex long acuminate; aerophores present in both sterile and fertile fronds, tuberculiform, atro- purpureus; hydathodes present in both sterile and fertile fronds, rounded or ovate; sori linear, continuous, forming coenosori on both sides of the costa; indusia linear, continuous, open towards the costa, dark brown, membranaceous, sometimes lacerate. Acknowledgements We are grateful to the Synthesys+ programme for the grant awarded to SM to visit the P herbarium. SM also received support from a Santander-UCM pre-doctoral contract (CT27/18). This work was also funded by the research grants PID2021-127118NA- 100 of the Spanish Ministry of Science and Innovation and PR44/21-29930 of the Santander-UCM research programme. Additionally, we thank Dr. Carmen Prada for her comments and suggestions. Fieldwork in Madagascar for collecting Parablech- num was supported by the ATM MNHN “Biodiversité actuelle et Fossile” and ATM MNHN “Génomique & Collections”. Collecting permits in Madagascar were grant- ed to GR by Madagascar National Parks and the “Ministére de Environnement, de PEcologie et des Foréts”. We are also grateful for field assistance to CNRE-Madagascar and MBG-Madagascar. The MNHN gives access to the collections in the framework of the RECOLNAT National Research Infrastructure. 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Con- tributions of the Gray herbarium of Harvard University 185: 97-127. https://doi. org/10.5962/p.336390 Appendix | Material examined Parablechnum marginatum (Kuhn) Gasper & Salino Mauritius. no date, Belanger 98C, (P01462824); no date, Bonpland s.n. (P01532252); ibidem (P01532253); 3 Dec 1909, Meller 6. REUNION. no date, no collector (P01462821); ibidem (P01462829); ibidem (P01462833); ibidem (P01557677); ibi- dem (P01557678); 1892, Cowemoy s.n. (P01462832); ibidem (P01462834); 1875, de Isle 606 (P01462825; P01462826; P01462827); 1842, Lépervanche-Méziére sn. (P01462830); 1898, Lépervanche-Méziére 17 (P01462771); no date, Léper- vanche-Méziére s.n. (P01462820; P01462823; P01462828); 8 Mar 1979, Lorence s.n. (MO3156135; MO3156136)3 Berge de la Riviére des Marsouins prés Coserne 60 Sonia Molino et al. / PhytoKeys 214: 47-60 (2022) des Hirondelles Nébom, 22 Jul 1973, Cadet 4050B1 (P01462768); ibidem, Cadet 4050B2 (P01462767); Bourbon, no date, Richard 99 (P01462822); cirque de Sala- zie, sentier vers La Nouvelle, 29 Nov 1973, Badré 1052 bis (P01462769); ibidem, Badré 1055 (P01462836; P01462837); Fourré 4 Philippia, sentier de la Mare a Jo- seph au coteau Kerveguen, cirque de Cilaos, 16 Nov 1973, Badré 935 (P01462835, P00917036, P00917037); ibidem, Badré 958 (P00917032; P00917033; P00917034; P00917035); Nationale 3, Bord de route, entre le Col de Bellevue et la Plaine des Palmistes, 5 Nov 2004, Rakotondrainibe & Grangaud 6910 (P00411889); Sentier de Bélouve a4 la caverne Mussard, no date, Bosser 12208 (P01625974). Parablechnum humbertii (Tardieu) S. Molino & Lafuente Mapacascar. Atsimo-Andrefana: Eboulis sableux Ambondrombe, 11 Apr 1941, Boiteau 4635 (P02284987; P01632222); Diana: Antsiranana, 24 Sep 2015, Bau- ret et al. 102 (P02435082); Antsiranana, Andapa, Parc National de Marojejy. Aux alentours du Camp 4, au bord de la riviére Andranomifototra, 25 Oct 2011, Rou- han et al. 1209 (P02432741; P02432742; P02432743; P02432744); Antsiranana, Andapa, Befingotra, RS d’Anjanaharibe-Sud, sur le versant Sud-Est, 4 12.2 km a POuest-Sud-Ouest de Befingra, 25 Nov 1994, Rakotondrainibe & Raharimalala 2519 (P00904704; P00046988); ibidem, 27 Nov 1994, Rakotondrainibe & Raha- rimalala 2534 (P00046998); Antsiranana, Andapa, RNI 12 du Marojejy. A 11 km au Nord-Ouest de Manantenina, 27 Oct 1996, Rakotondrainibe 3571(P00085122; P00085123; P00085124; P00085125); Antsiranana, Ambanja, Massif du Manonga- rivo, Mt d’Antsatrotro, berges de la riviére Ankaramihely, 19 May 1992, Rakoton- drainibe 1673 (P00100192; P00100193; P00100194); Antsiranana, Iharana (Vohé- mar), Marojejy, route vers campement 3, 13 May 2015, Rabarijaona et al. ROM1053 (P00783078; P00783098); Haute Matsiatra: Fianarantsoa, Ambalavao, Ambatom- boay, RNI d’Andringitra versant E; 4 environ 38 km au Sud d’Ambalavao, prés de la source de la riviére Sahavatoy, 30 May 1995, Rakotondrainibe 2743 (P00059958; P00059959; P00059960; P00059961); Sava: Anjanaharibe, 19 Dec 1950, Cours 3772 (P01625801); Vallée de la Lokoho (nord-est), Mont Beondroka au Nord de Maroambihy, 17—22 March 1949 H. Humbert 23554, (P00483200, holotype).