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Keywords:

  • cryptic species;
  • polymerase chain reaction-restriction fragment length polymorphism;
  • Pyropia yezoensis;
  • speciation

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

In a previous study on wild populations of Pyropia, the occurrence of two possible new species (Pyropia sp. 2 and Pyropia sp. 3) which are closely related to the two commercially important Pyropia species, P. yezoensis and P. tenera, was confirmed as the result of molecular phylogenetic analyses. To characterize the morphological features of the two wild Pyropia species, we collected Pyropia blades in a natural population in which Pyropia sp. 3 was known to occur, and carried out molecular identification before detailed morphological observations. Through the molecular identification we found, unexpectedly, that Pyropia sp. 2 blades grew sympatrically in the same site. Therefore, after molecular identification, we examined in detail the external morphology and anatomy of the two wild Pyropia species using more than 10 blades each. As a result, it is concluded that all of the blades of the two species are morphologically identical to P. yezoensis, but distinct from P. tenera. It is therefore considered that both of the two wild Pyropia species are cryptic species within the P. yezoensis complex. Furthermore, this study revealed that the two cryptic species grew sympatrically, even on the same rocks within the natural habitat.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The red algal order Bangiales was comprised of only two genera, blade-forming Porphyra and filamentous Bangia until three additional filamentous genera were proposed (Müller et al. 2005; Nelson et al. 2005). In Porphyra, 264 species have been reported from cold temperate to tropical waters (Guiry & Guiry 2012). Recently, Sutherland et al. (2011) proposed that Porphyra and Bangia are revised as eight and seven genera (including the abovementioned three genera) respectively on the basis of molecular phylogenetic analysis. Their study demonstrated that there is a large genetic diversity in the Bangiales despite the simple morphological features, and the two commercially important species, Porphyra yezoensis and Porphyra tenera, were transferred to the genus Pyropia, the genus that contains the largest number of species among the blade-forming genera.

On the other hand, recent molecular studies have revealed that cryptic diversity was found not only among genera of Bangiales but also in the closely related Porphyra/Pyropia species (Brodie et al. 2007; Lindstrom 2008; Niwa et al. 2009). Consequently, some new species have been described from each complex comprising several morphologically similar but genetically distinct taxa (Neefus et al. 2002; Lindstrom & Fredericq 2003; Brodie et al. 2007). In wild Pyropia populations, the presence of two possible new species Pyropia sp. 2 and Pyropia sp. 3 which are closely related to P. yezoensis and P. tenera have been confirmed by molecular phylogenetic analysis of the plastid ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene and sequence divergences of plastid RubisCO spacer and nuclear internal transcribed spacer 1 (ITS-1) rDNA regions (Niwa et al. 2009; Niwa & Kobiyama 2009). In particular, the sequence divergence of ITS-1 strongly supported that the sample (P7) of Pyropia sp. 2 and the samples (P10, TG-1) of Pyropia sp. 3 are considered as separate species, and that these two entities are clearly different species from P. yezoensis and P. tenera (see Table 1 in Niwa et al. 2009). Furthermore, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of rbcL gene was developed to distinguish among P. yezoensis, P. tenera, and the two wild Pyropia species (Pyropia sp. 2 and Pyropia sp. 3) for simple molecular discrimination (Niwa & Kobiyama 2009). However, no detailed morphological features of the two wild Pyropia species have been reported until now. Pyropia yezoensis and P. tenera themselves are extremely similar in morphology: morphological distinction between the two species, for example, with regard to the division formulas of spermatangia and zygotosporangia, is problematic with some variations occurring even within a species (Kurogi 1961; Miura 1984; Niwa et al. 2005, 2008). Therefore, in order to examine morphological differences between the two wild Pyropia species, it is necessary to make detailed morphological observations using as many blades as possible. In addition to the difficulty of morphological distinction, Pyropia sp. 3 and P. yezoensis were observed in the same habitat at Shichigahama (Niwa et al. 2009). Thus, it is also necessary to perform molecular analysis for species identification of field-collected samples prior to morphological observations with as many blades as possible in each species. These circumstances make the situation more difficult for examining morphological features of the wild Pyropia species that are closely related to P. yezoensis and P. tenera.

Table 1. Composite haplotypes classified according to Niwa and Kobiyama (2009) based on polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of rbcL gene in Pyropia sp. 2 and Pyropia sp. 3
SpeciesBglIEcoT14IHinfIComposite haplotype
  1. Values in parentheses show fragment sizes (bp) digested with restriction endonucleases.

Pyropia sp. 2A (856, 512)C (1205, 163)A (647, 445, 167, 56, 53)ACA
Pyropia sp. 3A (856, 512)C (1205, 163)C (1092, 167, 56, 53)ACC

Recently, a technique for simple molecular discrimination among the two wild Pyropia species, P. yezoensis and P. tenera was developed by using PCR-RFLP analysis of the rbcL gene as mentioned above (Niwa & Kobiyama 2009). This technique is powerful with the diagnostic marker to enable simple identification of a large number of samples: thus, it has become easier to collect respective blades of Pyropia sp. 2 and Pyropia sp. 3 from field-collected samples containing other Pyropia species, and to observe the morphological features of the two wild Pyropia species.

A previous study (Niwa et al. 2009) indicated that of the two wild Pyropia species, Pyropia sp. 3 occurs in a natural habitat of Togawa, Choshi, Chiba Prefecture in Japan. To observe the morphological features of Pyropia sp. 3, we collected wild Pyropia blades in Togawa, and carried out PCR-RFLP analysis of rbcL gene to identify Pyropia sp. 3 blades. Through molecular identification, it became clear that rather unexpectedly Pyropia sp. 2 blades also grew sympatrically at this site. Therefore, we decided to observe the morphological features of blades of both Pyropia species with more than 10 blades each after confirming their identification by molecular analysis. The observed features were compared with those of P. yezoensis and P. tenera.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Mature gametophytic blades of wild Pyropia were collected in Togawa, Choshi, Chiba Prefecture (Fig. 1), on 26 February 2009, 9 March 2011, 14 February 2012 and 13 March 2012. Some of the collected blades were spread on paper using a brush and were immediately photographed. The basal portions of the collected samples, including the photographed blades, were cut using a razor blade. They were used for PCR-RFLP analysis of the rbcL gene. The remaining portions were used for morphological observations. The PCR-RFLP analysis was carried out in a similar way as Niwa and Kobiyama (2009). In the previous study (Niwa & Kobiyama 2009), the two wild Pyropia species showed haplotype A for BglI and haplotype C for EcoT14I. However Pyropia sp. 2 and Pyropia sp. 3 exhibited haplotypes A and C for HinfI, respectively: therefore, in the present study, the samples that exhibited the composite haplotype ACA were identified as Pyropia sp. 2 and those exhibited the composite haplotype ACC were identified as Pyropia sp. 3, as in Table 1.

figure

Figure 1. Map showing the sampling locality, Togawa, Choshi, Chiba Prefecture, Japan, of the present study together with other sampling localities where the Pyropia sp. 2 and Pyropia sp. 3 blades were found.

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Twelve blades of Pyropia sp. 2 and 11 blades of Pyropia sp. 3, all of which were identified by PCR-RFLP analysis, were carefully observed under a microscope. In anatomical examinations, blades were sectioned by hand using a razor blade and were observed.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Molecular identification

Table 2 summarizes the number of Pyropia sp. 2 and Pyropia sp. 3 blades identified by the PCR-RFLP analysis of the rbcL gene in the wild Pyropia blades collected from seven different rocks: four different rocks (1-1, 1-2, 1-3, and 1-4 in Table 2) on 9 March 2011 and three different rocks (2-1, 2-2, and 2-3 in Table 2) on 14 February 2012 and 13 March 2012. The size of each rock was within a radius of approximately 3 m, and all the rocks were within 80 m of each other. From the results of the PCR-RFLP analysis, the two wild Pyropia species grew sympatrically on the same rocks, except only one rock on 9 March 2011. Mature blades could be observed in both of the two wild Pyropia species, but it was impossible to discriminate between the two species blades only by external morphology.

Table 2. The number of Pyropia sp. 2 and Pyropia sp. 3 blades identified by PCR-RFLP analysis of rbcL gene in the wild Pyropia blades collected from different rocks in Togawa on three different dates
DataRock No.Pyropia sp. 2Pyropia sp. 3
9 Mar. 20111-1160
1-2132
1-3132
1-4114
14 Feb. 20122-1128
2-2312
2-3127
13 Mar. 20122-1812
2-269
2-3812
Total 10268

In addition, sample blades were collected on 26 February 2009 in Togawa and were also examined by the PCR-RFLP analysis. As a result, 11 out of 12 blades were identified as Pyropia sp. 3. Although the remaining single blade was not examined by using BglI, it seemed to be Pyropia sp. 2 according to the haplotypes of EcoT14I and HinfI (data not shown).

Morphological observations of Pyropia sp. 2

The 12 blades of Pyropia sp. 2, all of which were collected on 14 February 2012 and identified by the PCR-RFLP analysis, were used for morphological observations. Six of these blades are shown in Fig. 2. Their blade shape was oblanceolate, oblong-ovate, or ovate. Their color was reddish or darkish brown with greenish brown basal portions. In surface view, spermatangial portions were as stripy patches in the upper blade margins. Figure 3 shows typical morphological features of the Pyropia sp. 2 blades observed under a microscope. Table 3 summarizes the detailed morphological features of the 12 blades, except the division formulas of their reproductive cells. In sectional view, vegetative portions of the 12 blades were monostromatic, and their thickness ranged 28–57 μm (Fig. 3a, Table 3). Each cell of the vegetative portions possessed a single stellate plastid (Fig. 3a,b). The blade margin was entire (Fig. 3b). All the blades were monoecious with spermatangia, carpogonia and zygotosporangia (Fig. 3c–g). The carpogonia were elliptical or spindle-shaped possessing two conspicuous trichogynes in sectional view (Fig. 3e). Table 4 shows the division formulas of spermatangia and zygotosporangia in each of the observed 12 blades. The division formula of spermatangia was 128 (a/4, b/4, c/8) as in Fig. 3c,d, whereas the division formula of zygotosporangia was mostly 16 (a/2, b/2, c/4) as in Fig. 3f,g, or occasionally 8 (a/2, b/2, c/2).

figure

Figure 2. Wild gametophytic blades of Pyropia sp. 2 collected on 14 February 2012 in Togawa, Choshi, Chiba Prefecture, Japan. Scale bar = 5 cm.

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figure

Figure 3. Morphology of the wild blades of Pyropia sp. 2 collected on 14 February 2012 in Togawa, Choshi, Chiba Prefecture, Japan. Scale bars = 30 μm. (a) Section of vegetative cells in central portion. (b) Marginal portion, showing entire margin. (c) Surface view of spermatangia composed of 16 cells (a/4, b/4) in maximum (arrows). (d) Section of spermatangia containing eight cell layers (c/8). (e) Section of carpogonia with conspicuous trichogynes. (f) Surface view of zygotosporangia composed of four cells (a/2, b/2) (arrows). (g) Section of zygotosporangia containing four cell layers (c/4).

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Table 3. Morphological features of Pyropia sp. 2 and Pyropia sp. 3 and typical morphological features of Pyropia yezoensis and Pyropia tenera
MorphologyPyropia sp. 2Pyropia sp. 3P. yezoensisP. tenera
  1. †Features based on 12 mature blades of Pyropia sp. 2 and 11 mature blades of Pyropia sp. 3. ‡Features based on Ueda (1932), Tanaka (1952), Kurogi (1961), Fukuhara (1968), Miura (1984, 1988), and Niwa et al. (2004, 2005, 2008).

Blade shapeOblanceolate, oblong ovate, ovateLinear, oblanceolate, oblong ovate, ovateLinear, oblanceolate, elliptical, ovate, roundOblanceolate, elliptical, ovate, round
Surface view of spermatangial portionsStreaky patches at the upper marginStreaky patches at the upper marginStreaky patches at the upper marginGradational patches along margin
Blade sectionMonostromaticMonostromaticMonostromaticMonostromatic
Blade thickness (vegetative portion)28–57 μm31–44 μm25–53 μm14–35 μm
Blade plastidSingle stellateSingle stellateSingle stellateSingle stellate
Blade marginEntireEntireEntireEntire
Sex typeMonoeciousMonoeciousMonoeciousMonoecious
CarpogoniaElliptical, spindle-shapedElliptical, spindle-shapedElliptical, spindle-shapedRound, ovate, elliptical
TrichogynesConspicuousConspicuousConspicuousInconspicuous, conspicuous
Division formula of spermatangiaSee Table 4See Table 4128 (a/4, b/4, c/8)64 (a/4, b/4, c/4)
Division formula of zygotosporangiaSee Table 4See Table 416 (a/2, b/2, c/4)8 (a/2, b/2, c/2)
Table 4. Division formulas of reproductive cells of Pyropia sp. 2 (12 blades) and Pyropia sp. 3 (11 blades)
Pyropia sp. 2SpermatangiaZygotosporangiaPyropia sp. 3SpermatangiaZygotosporangia
Blade No.Blade No.
  1. †Division formulas of these blades were observed each in a small mature portion. In particular, blade 2 of Pyropia sp. 3 showed a low level of maturity. ‡ Not completely divided as c/8.

Blade 1128 (a/4, b/4, c/8)16 (a/2, b/2, c/4)Blade 1128 (a/4, b/4, c/8)16 (a/2, b/2, c/4)
Blade 2128 (a/4, b/4, c/8)16 (a/2, b/2, c/4)Blade 232 (a/4, b/2, c/4)4 (a/2, b/1, c/2)
Blade 3128 (a/4, b/4, c/8)8 (a/2, b/2, c/2)Blade 3128 (a/4, b/4, c/8)8 (a/2, b/2, c/2)
Blade 4128 (a/4, b/4, c/8)16 (a/2, b/2, c/4)Blade 4128 (a/4, b/4, c/8)8 (a/2, b/2, c/2)
Blade 5128 (a/4, b/4, c/8)16 (a/2, b/2, c/4)Blade 5128 (a/4, b/4, c/8)16 (a/2, b/2, c/4)
Blade 6128 (a/4, b/4, c/8)8 (a/2, b/2, c/2)Blade 6128 (a/4, b/4, c/8)8 (a/2, b/2, c/2)
Blade 7128 (a/4, b/4, c/8)16 (a/2, b/2, c/4)Blade 7128 (a/4, b/4, c/8)16 (a/2, b/2, c/4)
Blade 8128 (a/4, b/4, c/8)16 (a/2, b/2, c/4)Blade 8128 (a/4, b/4, c/8)16 (a/2, b/2, c/4)
Blade 9128 (a/4, b/4, c/8)16 (a/2, b/2, c/4)Blade 9128 (a/4, b/4, c/8)8 (a/2, b/2, c/2)
Blade 10128 (a/4, b/4, c/8)16 (a/2, b/2, c/4)Blade 10128 (a/4, b/4, c/8)16 (a/2, b/2, c/4)
Blade 11128 (a/4, b/4, c/8)16 (a/2, b/2, c/4)Blade 11128 (a/4, b/4, c/8)16 (a/2, b/2, c/4)
Blade 12128 (a/4, b/4, c/8)16 (a/2, b/2, c/4)   

Morphological observations of Pyropia sp. 3

The 11 blades of Pyropia sp. 3, all of which were collected on 26 February 2009 and identified by the PCR-RFLP analysis, were used for morphological observations. Six of these blades are shown in Fig. 4. Their blade shape was linear, oblanceolate, oblong-ovate, or ovate. Their color and the surface view of spermatangial portions were extremely similar to those of Pyropia sp. 2. Figure 5 shows typical morphological features of the Pyropia sp. 3 blades observed under a microscope. Table 3 summarizes the detailed morphological features of the 11 blades, except the division formulas of their reproductive cells. In sectional view, vegetative portions of the 11 blades were monostromatic, and their thickness ranged 31–44 μm (Fig. 5a, Table 3). Each cell of the vegetative portions possessed a single stellate plastid (Fig. 5a,b). The blade margin was entire (Fig. 5b). All the blades were monoecious with spermatangia, carpogonia and zygotosporangia (Fig. 5c–g). The carpogonia were elliptical or spindle-shaped possessing two conspicuous trichogynes in sectional view (Fig. 5e). The division formulas of spermatangia and zygotosporangia showed some variations in the 11 blades as summarized in Table 4. Except for the immature blade 2, the division formula of spermatangia was 128 (a/4, b/4, c/8) as in Fig. 5c,d, whereas the division formula of zygotosporangia was 8 (a/2, b/2, c/2) or 16 (a/2, b/2, c/4) as in Fig. 5f,g. Because the portion of the blade 2 that was observed was scarcely mature, it appeared that the divisions of spermatangia and zygotosporangia were not sufficiently completed (Table 4). Thus, these morphological features observed under a microscope were also almost identical to those of Pyropia sp. 2.

figure

Figure 4. Wild gametophytic blades of Pyropia sp. 3 collected on 26 February 2009 in Togawa, Choshi, Chiba Prefecture. Scale bar = 5 cm.

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figure

Figure 5. Morphology of the wild blades of Pyropia sp. 3 collected on 26 February 2009 in Togawa, Choshi, Chiba Prefecture, Japan. Scale bars = 30 μm. (a) Section of vegetative cells in central portion. (b) Marginal portion, showing entire margin. (c) Surface view of spermatangia containing 16 cells (a/4, b/4) in maximum (arrow). (d) Section of spermatangia composed of eight cell layers (c/8). (e) Section of carpogonia with conspicuous trichogynes. (f) Surface view of zygotosporangia composed of four cells (a/2, b/2) (arrows). (g) Section of zygotosporangia containing four cell layers (c/4).

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The two wild Pyropia species in this study were considered to be two possible new species closely related to P. yezoensis and P. tenera, based on the phylogenetic analysis of the plastid rbcL gene (Niwa et al. 2009), and the suggestion that each of the two wild Pyropia species was a different species from P. yezoensis and P. tenera was strongly supported by the respective sequence divergences of the plastid RubisCO spacer and the nuclear rDNA ITS-1 (Niwa et al. 2009). In this study, using more than 10 blades of each species (identified in advance by molecular analysis), external morphology and anatomy of the two wild Pyropia species were carefully observed to compare with those of P. yezoensis and P. tenera. For comparison, typical morphological and anatomical features of P. yezoensis and P. tenera are summarized in Table 3 with the morphological features of the two wild Pyropia species. Pyropia yezoensis and P. tenera differ in the surface view of the spermatangial portion, division formulas of spermatangia and zygotosporangia, and carpogonium shape (Ueda 1932; Tanaka 1952; Kurogi 1961; Fukuhara 1968; Miura 1984, 1988; Niwa et al. 2004, 2005, 2008), even though these features also show intraspecific morphological variations due mainly to changes in environmental conditions (Kurogi 1961). As shown in Table 3, these morphological comparisons indicate that both of the two wild Pyropia species are morphologically identical to P. yezoensis, but distinct from P. tenera. Therefore, at present, we suggest that both of the two wild Pyropia species are cryptic species within the P. yezoensis complex, even though molecular phylogenetic analysis and sequence divergences clearly indicate that they are two possible new species closely related to P. yezoensis and P. tenera.

This study revealed that the two cryptic species of P. yezoensis grew sympatrically, even on the same rocks in Togawa. Pyropia sp. 3 was found in two other sites as shown in Fig. 1, Shichigahama (Niwa et al. 2009) and Hakodate (unpubl. data, 2012), based on rbcL sequences. These results suggest that Pyropia sp. 3 is distributed from southern Hokkaido to northeastern Honshu in Japan. On the other hand, Pyropia sp. 2 was collected from two Japanese sites, Oosu, Miyagi Prefecture (Fig. 1, Niwa et al. 2009) and Hakodate (unpubl. data, 2012), in addition to Togawa. Furthermore, Pyropia sp. 2 was found in Yangyang, Kangwondo in Korea, based on the rbcL data (unpubl. data, 2012). These results imply that Pyropia sp. 2 may be more extensively distributed in Northeast Asia than Pyropia sp. 3.

In a previous study, Niwa et al. (2009) reported that the strain MT-1 is potentially progeny after introgression between P. yezoensis and P. tenera in the natural population. Furthermore, based on the results of the culture experiment and additional nuclear DNA markers (Niwa et al. 2010; Niwa & Sakamoto 2010), Niwa and Sakamoto (2010) confirmed that allopolyploidy occurs in the strain MT-1 that resulted from interspecific hybridization between P. yezoensis and P. tenera in the natural population. Therefore, cryptic species diversity of Porphyra/Pyropia may result from interspecific hybridization and polyploidy, one of the important mechanisms for speciation reported in land plants (Soltis & Soltis 1999; Wendel 2000). In this study, it was found that the two cryptic species blades in sympatric populations matured at the same time. Therefore, a further study is necessary to confirm whether hybridization and allopolyploidy between the two cryptic species occurred in Togawa.

Despite the simple morphology, the blade-forming genus Porphyra was revised to be comprised of eight genera (Sutherland et al. 2011). In addition, it was confirmed in this study that more than one cryptic species within the P. yezoensis complex can occur in sympatric populations. Morphological criteria for species discrimination, such as division formulas of reproductive cells, show intraspecific variations in Pyropia as mentioned above. These circumstances make the situation more essential for species discrimination among the closely related Porphyra/Pyropia species to carry out detailed morphological observations using many blades, all of which have been identified as the same species by molecular analysis. Therefore, the procedure described in this study will be a useful method to find new species and/or cryptic species in Porphyra/Pyropia and also in other algal genera especially with simple morphology.

Acknowledgments

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

We are grateful to Dr Tadao Yoshida for his valuable advice on taxonomy of Porphyra/Pyropia. We also thank Dr Wendy Nelson for providing useful comments and for correcting the English of the manuscript. This work was supported by the Nori Cultivation Promotion Grant to K. Niwa.

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  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
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