View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by RERO DOC Digital Library The Lichenologist 41(6): 649–661 (2009) © British Lichen Society, 2009 doi:10.1017/S0024282909990260 Multivariate analysis of anatomical characters confirms the differentiation of two morphologically close species, Melanohalea olivacea (L.) O. Blanco et al. and M. septentrionalis (Lynge) O. Blanco et al. Camille TRUONG, Yamama NACIRI and Philippe CLERC Abstract: This study investigates how anatomical characters can be used to differentiate two morphologically close species, Melanohalea olivacea (L.) O. Blanco et al. and M. septentrionalis (Lynge) O. Blanco et al. Although the two species are morphologically differentiated, mainly based on the position and shape of pseudocyphellae on the lobes, poorly-developed specimens can be difficult to identify based on morphology alone. For example, in Switzerland, both species are at the south-west limit of their distribution range and most specimens are small and not fully developed. In such cases, anatomical characters are particularly useful in separating the two species. Five anatomical characters were studied: spore length and width; hymenium, subhymenium and hypothecium height. A nested ANOVA showed that variation of spore length was 155 times greater between species than between individuals of the same species, itself 5 times higher than within individuals. In a Principal Component Analysis, despite a substantial variation of the anatomical characters within both species, a significant species differentiation appeared distinctly on the first axis (P < 0·001), which accounted for 62·35% of the total variance, and the type specimens nested within each species. Finally, a Discriminant Linear Analysis assigned 100% of the individuals to their species class, the best predictors to discriminate between the two species being subhymenium height and spore length. These characters proved to be powerful in identifying specimens with a poorly-developed morphology and confirmed the presence of M. olivacea in Switzerland. Key words: brown Parmelia, lichens, Melanelia, foliose, corticolous Introduction (1966) gave a detailed description of P. oli- The name ‘olivacea’ has been used in a very vacea in his monograph of the corticolous broad sense in the past, to describe various brown species of Parmelia in the Northern species of brown Parmelia, either corticolous Hemisphere. He was soon followed by or saxicolous and with or without isidia or Esslinger (1977), who confirmed the defini- soralia. Nylander (1868) was the first to tion of Ahti (1966), but also mentioned that define Parmelia olivacea s. str. as a non- the measurements of the spores and hy- isidiate and non-sorediate species, with pseu- menium of the two species overlapped. Both docyphellae on the lobes. Lynge (1912) authors took this overlap into account, but segregated P. septentrionalis from P. olivacea, stated that intermediate forms were rare by a combination of morphological and ana- when using a combination of characters. tomical characters, primarily the rarity of Nevertheless, this later contributed to some pseudocyphellae on the lobes, smaller spores confusion, and although most recent floras and a thinner subhymenium. Later, Ahti cite the two species (Poelt and Vezda 1981; Purvis et al. 1992; Krog et al. 1994; Wirth 1995; Brodo et al. 2001; Holien and Tønsberg 2004), in most of the accounts the C. Truong, Y. Naciri and P. Clerc: Conservatoire et Jardin botaniques de la Ville de Genève, ch. de anatomical characters are inaccurate or not l’Impératrice 1, CP 60, CH-1292 Chambésy/Genève, mentioned. For example, in his key of Parme- Switzerland. Email: [email protected] lia s. lat., Wirth (1995) separated the two Downloaded from https:/www.cambridge.org/core. University of Basel Library, on 11 Jul 2017 at 09:00:07, subject to the Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms. https://doi.org/10.1017/S0024282909990260 650 THE LICHENOLOGIST Vol. 41 species based on morphology only. His the taxa and, of course, the underlying measurements of spore size largely over- species concept (Arup 1994; Herrera- lapped for the two species and he did not Campos et al. 2001). In this paper we wanted mention the hymenium characters. to test, 1) the ability of the anatomical char- At the generic level, Esslinger (1978) seg- acters to segregate the two species despite the regated the brown Parmelia from Parmelia s. fact that they overlap and 2) how anatomical lat. and included P. olivacea and P. septentri- characters can be used to identify specimens onalis in a new genus named Melanelia Essl. with a poorly-developed morphology, such Ever since, the circumscription of Melanelia as those encountered in Switzerland, at the has been questioned (i.e. Lumbsch et al. limit of the distribution range of the species. 1988; Thell 1995) and recent molecular studies have shown that the genus was not monophyletic (Blanco et al. 2004; Thell et al. 2004; Blanco et al. 2006). Therefore, Blanco Materials and Methods et al. (2004, 2006) proposed the clustering Fieldwork was conducted in the cantons of Neuchâtel of the Melanelia species based on their (NE), Jura, (JU) and Bern (BE), in the Jura region of Switzerland, to verify the presence of the species, to phylogenetic clades and this resulted in the search for potential new sites and to collect more segregation of two new genera from Melane- material for the analyses. lia: Melanelixia O. Blanco et al. and Melano- A total of 48 specimens collected in the field and halea O. Blanco et al., the latter including selected from the herbarium collections at G, O, UPS M. olivacea and M. septentrionalis. and US were studied (Table 1). Morphology was inves- tigated under the stereomicroscope (Leica MS5), with Both M. olivacea and M. septentrionalis are an emphasis on the characters proposed by Ahti (1966) boreal species, being at the south-west limit and Esslinger (1977): 1) presence, shape and location of their distribution in Switzerland (Otte of pseudocyphellae on the lobes, 2) brilliancy of the et al. 2005). Specimens at the limit of the thallus, 3) lobe margins entire or eroded, 4) apothecia distribution on the thallus and 5) degree of erosion of species distribution range are often poorly- apothecial margin. developed and can therefore be difficult to To verify that the chemistry of both species was identify based solely on morphology. This consistent with previous results (Esslinger 1977), is the case in Switzerland where the status chemical analyses were performed using thin-layer of M. olivacea and M. septentrionalis has chromatography (TLC) on 8 specimens of M. olivacea and 10 specimens of M. septentrionalis, following remained unclear. Frey (1959) considered the standard method of Culberson & Ammann (1979) only the presence of M. olivacea s. lat. (in- with the solvent B modified according to Culberson & cluding M. septentrionalis) in Switzerland, Johnson (1982). whereas in the Red List of Switzerland (Scheidegger & Clerc 2002), only M. septen- Statistical tests on the anatomical dataset trionalis was included. The checklist of Sections of apothecia were prepared to study the Switzerland (Clerc 2004) indicated that the anatomical characters (spore length and width; hy- presence of M. olivacea in Switzerland menium, subhymenium and hypothecium height) under needed to be verified. Recently, Hawksworth the microscope (Leica DM2000). A total of 10–14 spores per specimen were measured, et al. (2008), in their checklist of parmelioid from a well-developed apothecium. Spores need to be of lichens in Europe, stated that records of M. good quality and at maturity and (outside the asci), so olivacea from Switzerland in fact belonged to that they had reached their full size or did not appear M. glabra or M. septentrionalis. deformed. Such parameters can greatly affect the size of Character variability among populations spores and bias the measurements. When it was not possible to obtain spores of good quality from old her- of the same species or overlapping ranges barium specimens, especially in M. olivacea, fewer among similar species is a common problem spores were measured (minimum 5) or the individual in lichen taxonomy and statistical tests, such was omitted from the analysis (3 specimens of M. oliva- as ANOVA or multivariate analysis, can help cea and2ofM. septentrionalis, see Table 1). A total of 135 M. olivacea and 285 M. septentrionalis spores were to circumscribe species. Different scenarios measured. Additionally, hymenium, subhymenium and can occur, depending on the strength of stat- hypothecium average height were measured for all 48 istical differences, the geographic situation of specimens (see Fig. 5E), by taking measurements of Downloaded from https:/www.cambridge.org/core. University of Basel Library, on 11 Jul 2017 at 09:00:07, subject to the Cambridge Core terms of use, available at https:/www.cambridge.org/core/terms. https://doi.org/10.1017/S0024282909990260 2009 Melanohalea olivacea and M. septentrionalis—Truong et al. 651 T 1. Numbers of specimens of Melanohalea olivacea and M. septentrionalis studied from the different geographical areas. A few specimens were excluded from the statistical analyses because not enough spores could be measured (less than 5 per individual) Species Number of specimens Location analysed excluded total M. olivacea Switzerland 8 – Scandinavia 4 (incl. type) 2 Asia 2 1 North America 1 – 18 M. septentrionalis Switzerland 21 1 Scandinavia 4 (incl. type) – Asia – – North America 3 1 30 th th where Yijk is the measurement of the k spore in the j individual in the ith species, µ being the whole mean (constant), (j)i the individual effect (j =15or28indi- viduals measured) nested to the species effect i,andEijk the residual (k = 5 to 14 spores measured). Similarly, species differentiation based on hy- menium, subhymenium and hypothecium mean height was tested with a two-sample t-test (Wilcoxon test when normality was not met) using the software R 2.5.0.