Amplified fragment length polymorphism (AFLP)-based differentiation of selected Chara species

Charophytes (order Charales, family Characeae) are a group of highly developed green algae that grow as submerged macrophytes in standing, slow-flowing freshwater and brackish waters, on all continents except Antarctica (Wood & Imahori 1965). These green algae (known as stoneworts) have been suggested to be a sister group of embryophytes (McCourt et al. 2004). Specimens from the genus Chara L. are sometimes not easy to identify, mostly due to overlapping morphological variations, apparent intermediate forms between taxa, and the unknown extent to which phenotypic plasticity or developmental differences account for morphological variation. Identification problems such as these were addressed during the initial phase of taxonomic studies of charophyte morphology. Several researchers have attempted to characterize the degree of morphological variation in charophytes and have discovered certain traits that may be used to distinguish distinct species (Migula 1897; Groves, Bullock-Webster 1924; Olsen 1944). As a result, a narrow and monomorphic species concept has been developed for the genus Chara, which has resulted in the identification of many species (Braun, Nordstedt 1882; Corillion 1957; Urbaniak 2007; Urbaniak et al. 2008; Urbaniak 2010; Urbaniak et al. 2012). However, due to many problems concerning the phenotypic plasticity and overlapping morphological variation in many traits, Wood (1962) and Wood & Imahori (1965) adopted a wider species concept, dividing the family Characeae into two tribes: Charae (including the genera Chara, Nitellopsis, Lamprothamium, Lychnothamnus) and Nitellae (including the genera Nitella and Tolypella). This reduced the number of species and only 19 Chara species were recognized worldwide, instead of many microspecies described at that time. The existence of such different interpretations as well as taxonomic difficulties in identifying various charophytes most likely result from the lack of suitable methods to objectively determine which characteristics can be used to distinguish a species within the genus (Meiers et al. 1999).

Such classification problems are typical not only for the genus Chara (Mannschreck et al. 2002; O’Reilly et al. 2007; Urbaniak & Combik 2013), but also for the genus Nitella (Sakayama et al. 2002; Sakayama et al. 2006; Urbaniak 2011b). Recent studies of oospore dimensions and wall ornamentation morphology of the genus Chara, with the use of scanning electron microscopy (SEM), did not give clear and satisfactory results. Although oospore dimension analysis could distinguish some of the taxa classified by Wood (1962) and Wood & Imahori (1965) as varieties or forms (which should in fact be recognized as distinct species), it did not work for all the taxa. Such analysis was performed for C. globularis, C. tenuispina and C. virgata (Urbaniak 2011a; Urbaniak & Blazencic 2012). On the other hand, oospore analysis performed on sect. Hartmania (C. hispida, C. intermedia, C. polyacantha, C. rudis) indicated a very close relationship between the taxa due to a similarity in oospore wall ornamentation. Since the results based on oospore morphology do not provide a definitive answer, the presented study examines Wood & Imahori’s (1965) hypothesis concerning the taxonomic relationships within the genus Chara, using a molecular fingerprinting technique (AFLP) and morphological analysis. In particular, we investigate whether the taxa belonging to the genus sect. Grovesia (C. aspera Willd., C. globularis Thuill., C. strigosa A.Br., C. virgata Kütz. and C. tenuispina A.Br.) should be regarded as conspecific, according to Krause (1997), or they should be reduced to forms and varieties of C. globularis.

Morphological differentiation of the species studied is provided in Fig. 1a-l. C. aspera individuals had long stipulodes below the branchlet and long spine cells exceeding the axis diameter, however, these were sparse and solitary (Fig. 1a). Solitary antheridia present on the branchlet contradict that C. aspera is a dioecious species (Fig. 1b). While similar to C. aspera in the habit, C. strigosa had much longer stipulodes and dense spine cells, arranged in bunches, while oogonia were conjoined, with antheridia below (Fig. 1h). These morphological differences allow for these species to be distinguished from one another. Chara globularis and C. tenuispina were both light green with similar habit, which may account for Wood’s (1962) suggestion that C. tenuispina should be considered a variety of C. globularis. The two species can be distinguished based on the following morphological differences: C. tenuispina had spine cells mostly in the upper parts of the axis internodes, which were longer than the axis diameter, as well as two rows of stipulodes, which were elongated, acute, and longer than the axis diameter (Fig. 1i-k). Chara globularis, however, had spine cells, while stipulodes were either absent or rudimentary (Fig. 1d-e). Similarly, Urbaniak & Gąbka (2014) stated that the same features allow C. tenuispina and C. globularis to be distinguished. Additionally, both species differ in ecological preferences and grow in different conditions. Chara globularis is a cosmopolitan species that can be found in different aquatic habitats such as lakes, ponds, pools and peatland exploitation pools, and has a wide ecological range, growing in mesotrophic and eutrophic water. In contrast, C. tenuispina has a narrow ecological amplitude and can be found in freshwater peatlands or in the littoral zone of lakes, in shaded and shallow water between vascular plants.

Compared to C. intermedia (Fig. 1g), C. hispida (Fig. 1f) had spine cells longer than the axis diameter and they occurred in bunches or in the group of three. Whereas spine cells of C. intermedia were shorter and occurred in pairs. C. virgata was characterized by longer upper whorls of stipulodes below the branchlet, and short and rudimentary spine cells (Fig. 1l).

The analyzed data showed high reliability of the AFLP bands (more than 96%). Prior to analysis, we tested approximately 20 different primer combinations EcoRIxxx–MseIxxx, ranging in size from 80 to 450 bp (more bands were tested but could not be scored with confidence across gels). Out of these 20 primer combinations, four were used for further analysis (Table 3). The highest intraspecific polymorphism rate was determined for C. globularis (76%) and C. tenuispina – 67-51%. The polymorphism rate for C. aspera was 76% and for C. strigosa – 69%. The lowest polymorphism rates were determined for C. intermedia (60-58%), C. hispida (66%) and C. virgata (68%). Similarities in the AFLP profiles for pairs of samples, calculated according to Jaccard’s genetic similarity index, were 0.08-0.71. This score indicates that individuals of C. tenuispina and C. globularis showed a high degree of Jaccard’s genetic similarity (Table 4).

Table 4

Jaccard’s genetic similarity index between pairs of plants belonging to different species

Cluster analysis of the AFLP data showed the separation of several groups of species (Fig. 2). However, not all species were clearly separated and several specimens were intermixed with other species. Specimens belonging to both C. tenuispina and C. globularis formed separate clusters with high bootstrap support. C. virgata also formed separate cluster. The PCA analysis showed a separation into two distinct groups: one of them comprised mostly individuals of C. globularis (Group 1) and the second one – C. tenuispina (Group 2) (Fig. 3). A few individuals of C. virgata and C. tenuispina were intermixed with the C. globularis group. Representatives of C. virgata populations formed two subclades in one cluster, intermingled with individuals of C. globularis with high bootstrap values (Fig. 2).

Figure 2

Other specimens belonging to C. hispida and C. intermedia formed mixed clades; single individuals were intermixed or clustered together with other species. Data points representing C. aspera, C. strigosa, C. virgata, C. hispida and C. intermedia could not be distinguished using PCA analysis and formed an indistinguishable group of species (Group 3) (Fig. 3). The third group contains species with different morphological characters, which normally can be used to distinguish one species from the other. Similarly, the UPGMA method was not able to differentiate between C. aspera and C. strigosa. Within the C. aspera and C. strigosa group/clusters in the UPGMA analysis, individuals that belonged to different populations were basically grouped together, forming separate subclusters supported by medium to high bootstrap values (Fig. 2). Unfortunately, both species subgroups were intermingled and did not form separate clades for each species, which would allow for the distinction between these two taxa.

Figure 3

The primer combinations used for AFLP analysis produced a mean percentage of 96% for polymorphic bands. Similar values were obtained by Mannschreck et al. (2002), Bögle et al. (2007) and Urbaniak & Combik (2013) for other charophyte species.

Previously, oospore dimensions showed that Chara tenuispina A. Braun 1835 (C. globularis var. tenuispina R.D.Wood 1962) and C. globularis Thuill. 1799 ( Chara globularis var . globularis f. globularis R. D. Wood 1962) should be recognized as distinct species (Urbaniak 2011a) rather than polymorphic variations of C. globularis, as proposed by Wood & Imahori (1965). This was not confirmed by SEM images of oospore wall ornamentation (Urbaniak 2011a). Results from the presented UPGMA and PCA analyses suggest a separation of both taxa, which was further supported by high bootstrap values (Figs 2–3). Therefore, we suggest that C. tenuispina should be classified as a separate species rather than a form of the polymorphic species group C. globularis as suggested by Wood & Imahori (1965). Our study based on the AFLP fingerprinting method also confirms recent results obtained by Schneider et al. (2016), who successfully distinguished C. globularis from C. tenuispina using barcode genes. In this case, molecular analysis performed on specimens collected from various locations is consistent with morphological characteristics. Both species have a similar appearance (slender habit, no spine cells on the triplostichous cortex), but C. tenuispina has much longer stipulodes with spine cells longer than the axis diameter, especially directly below the branchlet (Fig. 1j). This shows that both characters are valid in morphological differentiation, and we agree with Krause (1997) and Urbaniak & Gabka (2013) that this feature is an important character to distinguish between the two species.

Previously, Urbaniak (2011a) found that oospores of C. globularis and C. virgata formed one aggregation and did not differ in their ornamentation, thereby suggesting a close taxonomic relationship between the taxa. Such statement can be true as long as our results do not fully separate both species. Individuals of C. globularis and C. virgata were mixed in PCA analysis with C. tenuispina (Group 1) or formed one undifferentiated group of species together with C. aspera, C. strigosa, C. virgata, C. hispida and C. intermedia (Group 3) (Fig. 3). In the UPGMA figure, C. globularis and C. virgata did not form one and clearly separated cluster supported by high bootstrap values, but almost all C. globularis individuals are located next to each other in two groups supported by high bootstrap values. Some individuals of C. virgata were also mixed with other species, however, the collected specimens of C. virgata were very similar to those described by Krause (1997), i.e. with prolonged upper rows of stipulodes below the branchlet, clearly triplostichous, with thylacanthous cortex, rudimentary spine cells on the axis and white, not spherical bulbils on the rhizoids. They differed from C. globularis specimens which had rudimentary stipulodes and lack spine cells on the cortex (Fig. 1l). This shows that AFLP results are in partial disagreement with the morphological traits that differentiate C. globularis from C. virgata and also from other species, e.g. those belonging to the sect. Hartmania that are significantly different. Based on matK and rbcL genes, Schneider et al. (2016) clearly separates C. globularis from C. virgata but accepts specimens without spines and with (usually only slightly) elongated stipulodes as C. globularis. We agree with the common meaning that C. globularis does not have elongated stipulodes and the specimens equipped with such stipulodes should be treated as transitional forms. On the other hand, stipulodes are not the only characters that may be taken into account. The observed morphological differentiation, particularly in C. globularis is huge (Migula 1897), so is the number of disputed morphological characters.

Group 3 includes individuals not only from C. virgata, but also from C. globularis and C. tenuispina. This again confirms close taxonomic/phylogenetic affinity between these species (monoecious with triplostichous cortex and delicate slender habitus). In other words, it is possible that despite the accurate identification before laboratory culturing and analysis, some specimens were wrongly identified, even though we were very careful in this particular case. Therefore, one of the likely explanations is that our molecular methods do not fully reflect the morphological differentiation.

Some individuals of C. globularis and C. virgata formed an unresolved group with triplostichous: C. strigosa, C. aspera and diplostichous C. hispida and C. intermedia – group 3 (Fig. 3). All these species are monoecious, except for C. aspera, which is a dioecious species, but this difference was again not consistent with AFLP analysis. We were not able to differentiate species in this group by PCA analysis, especially monoecious and dioecious species. In the UPGMA tree, C. aspera and C. strigosa formed a compact group of individuals, and only a few C. strigosa individuals were found among C. aspera. Unfortunately, none of the analyses show a clear acceptable differentiation between C. aspera and C. strigosa. Specimens of both species formed several subclades, instead of distinct branches (Fig. 2). The other examined specimens, for which there are doubts regarding their taxonomic differentiation, belong to the taxa: C. hispida and C. intermedia (sect. Hartmania). They differ from other individuals included in the same Group 3 in size, cortification, presence of spine cells and general appearance (Fig. 3).

Similarly, significant differences in morphology, such as the habitus or the type of cortification that allow for routine identification of charophytes, also did not confirm our results as useful for distinguishing between the studied species. Urbaniak & Combik (2013), who used AFLP, and later Schneider et al. (2015), who used barcode genes, did not find differences between C. hispida and C. intermedia in a much larger data set. With the current state of knowledge, the statement of Wood & Imahori (1965) about very close taxonomic relationships between the species from sect. Hartmania seems to be acceptable.

The AFLP fingerprinting method is a useful tool for taxonomists, which can detect small genetic differences between populations of various plant species. This was successfully done in the case of C. globularis and C. tenuispina identification. Additionally in this case, morphological characters clearly correlate with the identification based on molecular methods: AFLP and rbcL+matK (Schneider et al. 2016). Sometimes, however, it is not possible to distinguish between the closely related taxa. This is the case of C. aspera and C. strigosa, C. hispida and C. intermedia or other species from the sect. Hartmania. The genetic differences between them can be reflected in morphological differences and the phylogenetic relationships can be inferred from this knowledge. Numerous examples show that this is not a rule. Another good example are C. aspera and C. strigosa, which have similar habits, but significantly differ in their reproduction system. The method of reproduction is the most important feature used to distinguish between the species. Therefore, based on Mayr’s classical definition of biological species, C. aspera and C. strigosa should be treated as separate species. Our results are only partly contradictory, however, much more data are needed to clearly define their taxonomic relationships. It is also possible that application of other methods would help. Barcode markers, matK and rbcL used by Schneider et al. (2016), were also unable to relate the morphology and reproduction system to genetic data. They successfully distinguish both species, but surprisingly C. strigosa formed one clade with C. virgata.

According to Bögle et al. (2010), who analyzed differences between populations of C. baltica and C. intermedia collected along the north-south gradient across Europe (from Sweden to Germany) using AFLPs, populations from the central part of the distribution demonstrated intermediate features, suggesting that individuals in this area might represent features of a common ancestor. One of the possible explanations is a taxonomic continuum between closely related species, especially between species from sect. Hartmania (C. baltica, C. hispida, C. horrida, C. intermedia, C. polyacantha and C. rudis). This was observed previously when using the fingerprinting method (Urbaniak & Combik 2013). The species continuum shows that only some species are completely different from the others. At the same time, species that appear to be similar, differ greatly from each other when found in nature, both in morphological and molecular terms (Sepp & Paal 1998). This is observed based on the presented results in the case of C. tenuispina but also other Chara species that can be treated as separate species: C. baueri, C. canescens, C. globularis. Most of the other species, e.g. C. baltica, C. hispida, C. intermedia, C. polyacantha and C. rudis, can be completely indistinguishable using morphological and molecular markers and convincing evidence for their distinction are still lacking. They still can be treated as variations or forms of other species (Urbaniak & Combik 2013; Schneider et al. 2016). It is possible that the continuum of species indicates that hybridization between species occurred in the past and probably occurs today.

Other authors (Schneider et al. 2015) were also unable to distinguish the species using barcode markers. Various species might represent distinct taxa but are masked by phenotypic or genotypic adaptations to different environmental conditions. Furthermore, Schneider et al. (2006) showed that changes in branching occur in C. hispida and C. intermedia as a response to different light conditions. Blindow & Schütte (2007) concluded that morphological differences between freshwater and brackish water populations of C. aspera were at least partly explained by plastic responses to salinity, rather than by genetic differences. Thus, phenotypic plasticity and genetic adaptation to different environmental conditions underlie the morphological variability observed in many charophyte species, which in turn provides the basis for natural selection to drive macroevolution (Urbaniak & Combik 2013). The close taxonomic relationship between charophyte species (and within various sections of charophytes) is commonly known and is reflected in a high degree of morphological similarity between phylogenetically closely related species (Meiers et al. 1999; Urbaniak& Combik 2013). While the results of this study do not fully support the taxonomic interpretation proposed by Wood & Imahori (1965), they do not exclude the possibility of combining separate species into macrospecies. Although taxonomic criteria for distinguishing between separate species are not clear, the idea of dividing organisms into species and macrospecies is generally acceptable, more attention should be paid to research on the observed phenotypic plasticity of charophytes in laboratory conditions and their correlation with molecular markers.