Serpocaulon × manizalense : a new hybrid between simple-and pinnate-leaved species of Serpocaulon ( Polypodiaceae ) from

During a revision of Serpocaulon from Colombia, a new hybrid was found between S. adnatum and S. levigatum near to Manizales city, which is described and illustrated herein. Qualitative and quantitative spore and macro-morphological characters were evaluated using principal component analyses to distinguish the new taxon. Our results suggest that the perispore with leasura, lamina width, rhizome diameter, blade dissection and number of pinnae are important characters to distinguish S. × manizalense. This is the ﬁrst record of a hybrid between a simple and pinnate-leaved species in Serpocaulon, which is considered to be Critically Endangered (CR).

During a review of Serpocaulon from Colombia, a new hybrid was found co-occurring with S. adnatum (Kunze ex Klotzsch) A.R.Sm., and S. levigatum (Cav.)A.R.Sm.Considering that hybrids occur in areas that were subject to extreme changes where the parents used to be or are present (Rieseberg 1997), and that they have intermediate characters to their parental taxa (Moran & Watkins 2004), we presume that S. adnatum and S. levigatum are the putative parental taxa of the new hybrid, which also slightly resembles S. semipinnatifidum, of which no records are known from the area.Our study aims to describe the new hybrid and distinguish it from its putative parents and S. semipinnatifidum.
Serpocaulon adnatum and S. levigatum were included in the analyses as they were found co-existing with the new hybrid.Serpocaulon semipinnatifidum was not found to co-exist with them, but it was included in the analyses as it can be confused with the new hybrid.The putative parents suggested for S. semipinnatifidum, in combination with S. levigatum, S. funckii (Mett.)A.R.Sm.(Moran 1995) and S. lasiopus (Klotzsch) A.R.Sm.(Tryon & Stolze 1993), were excluded from the analysis as they were not found to co-exist with the new hybrid.Furthermore, they are not recorded in the Caldas department (Fraume et al. 1990, Sanín & Duque-Castrillón 2006, Sanín et al. 2006, 2008, Álvarez-Mejía et al. 2007, Sanín 2011).
For morphometric analyses, 38 specimens were used as operational taxonomic units (OTU), representing the entire geographical range and the morphological variability within each taxon.A total of 59 morphological characters were measured, from which 35 were qualitative and 24 were quantitative.From these quantitative characters, eight were spore characters and 16 were macro-morphological characters.
The analysed spore characters are equatorial axis, length of aperture, polar axis, endospore, exospore, central verrucae, verrucae height and width.The spore description follows Ramírez-Valencia et al. (2013).The macro-morphological characters are rhizome diameter, rhizome scale length and width, phyllopodia distance, petiole length, lamina length and width, number of pinnae, lamina scale length and width.Additionally, we measured for the medial pinna length and width, the number of areolae and sori between the principal vein and the costa, and along the principal vein and the costa.For S. levigatum, which has a simple lamina, these characters were measured Serpocaulon × manizalense: a new hybrid between simpleand pinnate-leaved species of Serpocaulon (Polypodiaceae) from Colombia for the entire lamina.The macro-morphology description follows Lellinger (2002).

Numerical and statistical analyses
Quantitative characters were analysed by range and median values.Principal component analyses (PCA) were based on quantitative values of spore and macro-morphological characters.PCA results were used to support distinctions among the taxa.These analyses were performed using R (R Development Core Team 2013).

Conservation status
The conservation status of the new hybrid was assessed by applying the IUCN Red List Categories and Criteria (IUCN 2001).

Spore characters (Table 1)
The first three components accounted for 80 % of the total variance.The first principal component had high contributing loading values from verrucae height, verrucae width, central verruca, and polar axis.The second component had high contributing loadings from the endospore, exospore, central verrucae, and verrucae height.Finally, the third component had high contributing loadings from polar axis, equatorial axis, exospore and endospore.
In the scatterplot against the first two components (Fig. 1), the OTUs are arranged in loose and slightly overlapping groups, corresponding to S. levigatum, S. semipinnatifidum and S. × manizalense.Despite the overlap, it is possible to recognize the new hybrid (S. × manizalense) between S. levigatum and S. adnatum.In addition, the spores of the other hybrid (S.semipinnatifidum) are in a cluster separate from the other taxa.(Table 2) The first three components accounted for 68.7 % of the total variance observed.The first principal component had high contributing loading values from petiole length, lamina width, rhizome diameter and number of pinnae, among the four most important.The second component had high contributing loadings from medial pinna width and lamina length.Finally, the third component had high contributing loadings from rhizome scale length, rhizome scale width, rhizome diameter, and lamina length.

Macro-morphological characters
In the scatterplot of the first two components, the OTUs are arranged in four different clusters, each representing a different taxon (Fig. 2).This result allowed us to easily characterize the four taxa and support the description of the new hybrid (S. × manizalense).Nevertheless, S. levigatum and S. semipinnatifidum are slightly overlapping.

Spore characteristics of the hybrid
The slight overlap of S. levigatum, S. semipinnatifidum and S. × manizalense in the PCA of spore morphology, could be explained by the fact that S. levigatum is a parent of S. semipinnatifidum (Tryon & Stolze 1993, Moran 1995, Sanín 2011) and with the present results we propose that it is also one of the parental species of the new hybrid.In addition, the new hybrid is arranged between S. levigatum and S. adnatum (Fig. 1), both proposed as putative parental species of S. × manizalense.
According to the PCA results, the most important spore characters to differentiate the new hybrid are the shape and orna-mentation of the spores, in particular the verrucae (Table 1).Similarly, Ramírez-Valencia et al. (2013) reported these spore characters as useful to distinguish 21 species of Serpocaulon from Colombia.
Interestingly, the described hybrid has well-formed spores (see description of the hybrid), an unusual characteristic in fern hybrids.Usually, fern hybrids have malformed spores (Haufler 2008, Sharpe et al. 2010).However, S. × manizalense is not the first described hybrid with well-formed spores.Such a characteristic has been described in other fern hybrid taxa such as Polystichum Roth.(Mullenniex et al. 1998), Asplenium L. (Morzenti 1967), Polypodium L. (Haufler et al. 1995), and also in Serpocaulon (Rojas-Alvarado & Chaves-Fallas 2013).Macro-morphology of the hybrid Some of the most frequently used characters to distinguish Serpocaulon species are: rhizome diameter, laminar length and width, petiole length, rhizome scale length and width (Lellinger 1989, Tryon & Stolze 1993, Moran 1995, Labiak & Prado 2008, Sanín 2014).However, in the present study other less frequently used characters such as number of pinnae or segments, medial pinnae length and width appeared to be important to distinguish the new hybrid.
The PCA analysis of the macro-morphology resulted in betterresolved groups than that of the spore characters, and only S. levigatum and S. semipinnatifidum are overlapping.This is not surprising, since it was reported that S. levigatum is the parental taxon of S. semipinnatifidum (Tryon & Stolze 1993, Moran 1995, Sanín 2011), while the other parental taxon remains uncertain.Moran (1995) proposed that the other putative parent may be S. funckii for the north of the Andes hybrids (Colombia and Venezuela).However, Tryon & Stolze (1993) suggested that S. lasiopus may be the other putative parental taxon of S. semipinnatifidum for the hybrids located in Peru.

Hybrid habitat
Despite the extensive botanical exploration of the Chinchiná basin river (Fraume et al. 1990, Orrego et al. 2004, Sanín & Duque-Castrillón 2006, Sanín et al. 2006, 2008, Álvarez-Mejía et al. 2007), there is only one wild population of S. × manizalensis known, and another single plant propagated by rhizome, which has been maintained for conservational purposes in the JBOUC.
Frequently, the formation of a hybrid is triggered by an extreme change in the habitat of the parental taxa (Rieseberg 1997, Rieseberg et al. 2006, Kentner & Mesler 2000).
Thus, most of the hybrids appear in highly disturbed areas such as road cuts (Barrington 1985).Probably, the generation of S. × manizalense was promoted by the conversion of the natural area to a landfill, which has been opened since 1991.
Distribution & Ecology -Serpocaulon × manizalense occurs on the western slope of the Central Andean Cordillera of Colombia, near Manizales on the road to Neira, in the locality of La Esmeralda landfill (Fig. 5a, Map 1).It was found epiphytic on Rhus striata Ruíz & Pav.(Anacardiaceae) and terrestrial in secondary forests (Fig. 5b).In addition, the only Serpocaulon species found in the area were S. adnatum and S. levigatum.Serpocaulon × manizalense was collected fertile in November 2008, and also it was seen fertile in September 2013 from cultivated plants in the Caldas University Botanical Garden (JBOUC) (Fig. 5c-e).
Notes on related taxa -The characters of S. × manizalense that are crucial to distinguish it from S. semipinnatifidum are present in the rhizome, lamina, and spores (Table 3).The rhizome in S. × manizalense is wider than in S. semipinnatifidum.The blade dissection in S. × manizalense is lobate to proximally pinnate (Fig. 3, 6), whereas S. semipinnatifidum is lobate to proximally pinnatisect (Fig. 6).Frequently, the lamina length of  S. × manizalense is larger than of S. semipinnatifidum (Fig. 6).In addition, the species differ in the number of areoles between the costae and pinna margin, S. × manizalense has 1-5 areoles and S. semipinnatifidum has 1-3 areoles.The spores of S. × manizalense show a laesura in the perispore (Fig. 4), while the spores of S. semipinnatifidum do not have a laesura in the perispore (Ramírez-Valencia et al. 2013).
The most conspicuous intermediate characters between the putative parents and the new hybrid are the blade dissection and the number of pinnae in the lamina: pinnate with 4(-7-)10 pairs in S. adnatum, lobate to pinnate with up to 4 pinnae pairs in the hybrid, and a simple lamina in S. levigatum (Fig. 6).The rhizome diameter of S. × manizalense is intermediate between S. adnatum and S. levigatum.Less conspicuous, but also intermediate were the colour and rhizome scales length.The rhizome scales in S. adnatum are dark brown to nearly black with 1-3 mm long (Fig. 7), in S. levigatum are pale orange with 1-1.7 mm long (Fig. 7), whereas in S. × manizalense are pale orange to brown with 1.3 -2.5 mm long (Fig. 7).
Conservation -IUCN Red List Category: Critically Endangered [CR B2a + D].The EOO cannot be estimated for S. × manizalense because it is known from only one location.The AOO is 9 km 2 , and it falls completely outside any protected and pristine area under the Colombian System of Protected Areas.
Acknowledgements The first author thanks L.M. Álvarez, A. Pardo, and F. Cardona for their support, as well as the staff of the Caldas University, especially the research student group (SByRFG).We thank colleagues at BR, CAUP, CHOCO, COL, CUVC, FAUC, FMB, HUA, HUQ, JAUM, LPB, MBM, MEDEL, MO, NY, PSO, QCNE, TOLI, UFP, and UPCB for allowing access to specimens.Thanks to L.F. Coca for the drawing of the habit of the plant, V.    for the SEM photographs, B. Bassuner for the IUCN threat categorization, and A. Pérez for digital design assistance.Special thanks to M. Sundue, P. Hovenkamp, and an anonymous reviewer for their kind comments that improved substantially the manuscript.This research was supported by the Caldas University (FAUC Herbarium and IIES Institute) and the Colombian Association of Herbaria (ACH).

Fig. 5
Fig. 5 Ecosystem, location, and propagated plant of the type collection.a. Ecosystem of S. × manizalense D.Sanín & Torrez; b. location of the type collection; c -e. asexual cultivated plants of S. × manizalense.

Table 1
Summary of the principal component weights for the spore characters.In bold, morphological characters showing the highest values.

Table 2
Summary of the principal component weights for the macromorphological characters.In bold, morphological characters showing the highest values.